Documenti di Didattica
Documenti di Professioni
Documenti di Cultura
Listeriosis,
and
Food Safety
FOOD SCIENCE AND TECHNOLOGY
EDITORIAL BOARD
edited by
EIIiot T. Ryser
Department of Food Science and Human Nutrition
Michigan State University
East Lansing, Michigan
EImer H. Marth
Department of Food Science
University of Wisconsin-Ma dison
Madison, Wisconsin
M A R C E L
MARCEL
DEKKER,
INC. NEWYORK BASEL
D E K K E R
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iii
iv Preface to the Second Edition
This book is useful to advanced undergraduate students, graduate students, and prac-
titioners in fooddairy microbiology, fooddairy science, bacteriology/microbiology, pub-
lic health, dietetics, meat science, poultry science, and veterinary medicine. It also will
be helpful to personnel in the fooddairy industry and in regulatory agencies and to re-
searchers in industrial, governmental, and university laboratories.
Elliot T.Ryser
Elmer H. Marth
Preface to the First Edition
V
wi Preface t o the First Edition
together with the virtual flood of Listeria-related papers that have appeared in scientific
journals, trade journals, and numerous conference proceedings prompted us to review
and summarize the current information so that food industry personnel, public health and
regulatory officials, food microbiologists, veterinarians, and academicians have a ready
source of information regarding this now fully emerged foodborne pathogen.
This book consists of 15 chapters which address the following topics: (a) L. mono-
cytogenes as the causative agent of listeriosis; (b) occurrence and survival of this pathogen
in various natural environments; (c) human and animal listeriosis; (d) characteristics of
L. monocytogenes that are important to food processors; (e) conventional and rapid meth-
ods for isolating, detecting, and identifying L. monocytogenes in food; (f ) recognition of
cases and outbreaks of foodborne listeriosis; (g) incidence and behavior of L. monocyto-
genes in fermented and unfennented dairy products, meat, poultry (including eggs), sea-
food and products of plant origin; and finally (h) incidence and control of this pathogen
within various types of food-processing facilities. It is evident that major emphasis has
been given to information that is directly applicable to food processors. Since information
concerning the bacterium and the disease has been admirably reviewed by Professor See-
liger and others, our discussion of these topics should not be considered exhaustive. Thus
the first four chapters of this book supply only pertinent background information to com-
plement our discussion of foodborne listeriosis.
While many in the scientific community must be commended for the extraordinary
progress made since 1985 toward understanding foodborne listeriosis, the continuing ex-
plosion of information concerning Listeria and foodborne listeriosis has made the 3-
year task of compiling an up-to-date review of this subject quite difficult. Therefore, to
produce as current a document as possible, we have included a bibliography of references
that have appeared since the writing of the book was completed.
We acknowledge with gratitude the many investigators whose findings made this
book both necessary and possible. Special thanks go to those individuals who shared
unpublished information with us so that we could make the book as up to date as possible.
Our thanks also go to those scientists who provided photographs or drawings; each person
is acknowledged where the appropriate figure appears in the book. We thank Barbara
Kamp, Pat Gustafson, Beverly Scullion, and Judy Grudzina for typing various parts of
the manuscript. Illustrations were prepared by Jennifer Blitz and Suzanne Smith-their
help is acknowledged and appreciated. Special thanks go to Dr. Ralston B. Read, Jr.,
formerly director of the Microbiology Division of the Food and Drug Administration and
now deceased, who in 1984 encouraged development of a research program on foodborne
Listeria at the University of Wisconsin-Madison, and to Dr. Joseph A. ODonnell, for-
merly with Dairy Research, Inc. and now director of the California Dairy Foods Research
Center, for his early interest in and support of research on behavior of L. monocytogenes
in dairy foods.
Research done in the Department of Food Science at the University of Wisconsin-
Madison and described in this book was supported by the U.S. Food and Drug Administra-
tion; National Cheese Institute; the National Dairy Promotion and Research Board; the
Wisconsin Milk Marketing Board; Kraft, Inc.; Carlin Foods; Chr. Hansens Laboratory,
Inc.; the Aristotelian University of Thessaloniki, Greece; the Cultural and Educational
Bureau of the Egyptian Embassy in the U.S.; the Malaysian Agricultural Research and
Development Institute; the Korean Professors Fund; and the College of Agricultural and
Life Sciences, the Center for Dairy Research, and the Food Research Institute, all of the
Preface to the First Edition vii
University of Wisconsin. We thank all of these agencies for their interest in and support
of research on L. rnonocytogenes.
Our book is dedicated to all persons who have contributed to a better understanding
of foodborne listeriosis so that control of this disease is facilitated.
Elliot T. Ryser
Elmer H. Marth
This page intentionally left blank
Contents
ix
X Contents
Appendix 711
Index 719
Contributors
J. Stan Bailey Russell Research Center, Agricultural Research Service, U.S. Department
of Agriculture, Athens, Georgia
Car1 A. Batt Department of Food Science, Cornell University, Ithaca, New York
Robert E. Brackett Center for Food Safety and Quality Enhancement, The University
of Georgia, Griffin, Georgia
Nelson A. Cox Russell Research Center, Agricultural Research Service, U.S. ,Depart-
ment of Agriculture, Athens, Georgia
Catherine W. Donnelly University of Vermont, Burlington, Vermont
Me1 W. Eklund" U.S. National Marine Fisheries Service, Northwest Fisheries Science
Center, Seattle, Washington
Jeffrey M. Farber Bureau of Microbial Hazards, Food Directorate, Health Canada, Ot-
tawa, Ontario, Canada
David R. Fenlon Animal Biology Division, Scottish Agricultural College, Aberdeen,
Scotland
Werner Goebel Department of Biology, University of Wiirzburg, Wurzburg, Germany
Robert Gravani Department of Food Science, Cornell University, Ithaca, New York
Lewis M. Graves Foodborne Diseases Laboratory Section, Centers for Disease Control
and Prevention, Atlanta, Georgia
Susan B. Hunter Foodborne Diseases Laboratory Section, Centers for Disease Control
and Prevention, Atlanta, Georgia
* Retired.
xi
xii Contributors
Karen C. Jinneman Seafood Products Research Center, U.S. Food and Drug Adminis-
tration, Bothell, Washington
Michael Kuhn Department of Biology, University of Wurzburg, Wurzburg, Gerrnany
Yuqian Lou Bil Mar Foods, Inc., Zeeland, Michigan
Pearl I. Peterkin Bureau of Microbial Hazards, Food Directorate, Health Canada,
Ottawa, Ontario, Canada
Jocelyne Rocourt Listeria Laboratory, Institut Pasteur, Paris, France
Elliot T. Ryser Department of Food Science and Human Nutrition, Michigan State
University, East Laming, Michigan
Anne Schuchat Respiratory Diseases Branch, National Center for Infectious Diseases,
Centers for Disease Control and Prevention, Atlanta, Georgia
Laurence Slutsker Foodborne and Diarrheal Diseases Branch, National Center for In-
fectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia
Bala Swaminathan Centers for Disease Control and Prevention, Atlanta, Georgia
Marleen M. Wekell Seafood Products Research Center, U.S. Food and Drug Adminis-
tration, Bothell, Washington
Irene V. Wesley National Animal Disease Center, U.S. Department of Agriculture,
Ames, Iowa
Ahmed E. Yousef Department of Food Science and Technology and Department of
Microbiology, The Ohio State University, Columbus, Ohio
The Genus Listeria and Listeria
monocytogenes: Bhylogenetic
Position, Taxonomy, and
Identification
JOCELYNE
ROCOURT
lnstitut Pasteur, Paris, France
HISTORY
The first published description of Listeria monocytogenes, which rapidly became the refer-
ence, was written by Murray et al. in 1926 [ ZOO]. A few earlier.reports may have described
Listeria isolation [53,141], the most plausible of which is certainly that by Hulphers [64].
However, the authors of these reports did not deposit their isolates in a permanent collec-
tion, so no subsequent investigations or comparisons with further strains were possible.
Murray et al. [loo] observed six cases of rather sudden death of young rabbits in
1924 in the animal breeding establishment of the Department of Pathology of Cambridge
University, and many more such cases occurred in the succeeding 15 months. The interest-
ing characteristics presented by the disease and the increasing mortality prompted an inves-
tigation. The authors wrote at that time [loo]:
Both the natural and the experimental disease have interesting and characteristic features and
their consideration has forced us to the conclusion that the causative organism either has not
been described previously, or has been inadequately described and so cannot be traced in the
literature. In either case, we feel justified in naming it. Its salient character is the production
2 Rocount
of a large mononuclear leucocytosis. This is far the most important and most striking character
we have discovered and we name the microorganism we shall describe in this paper Bacte-
rium monocytogenes. The question of the generic name is more difficult and we have not
succeeded in associating our organism with many other genera proposed in Bergey sManual
ofDeterminative Bacteriology ( 1925). We propose for the present to use the undefined Bacte-
rium ([. . .], for, if the present chaos is to be resolved and if the classification adopted by
the American Society of Bacteriologists is to be improved, it will be achieved only by co-
operation and with this end in view we cannot use the term Bacillus).
In 1927, during investigations of unusual deaths observed in gerbils near Johannesburg,
South Africa, Pirie [ 1 161 discovered a new microorganism, agent of what he called the
Tiger River disease. He named this new agent Listerella hepatolytica for the following
reasons:
The causative organism is a Gram-positive bacillus for which, from its most striking patho-
genic effect, I propose the specific name hepatolytica, and the generic name Listerella,
dedicating it in honour of Lord Lister, one of the most distinguished of those concerned with
bacteriology whose name has not been commemorated in bacteriological nomenclature.2
Both discoverers, Murray and Pirie, sent their strains to the National Type Collection at
the Lister Institute in London. Dr. Leningham, the director, was struck by the similarity
of the two microorganisms and put Murray and Pirie into contact. As the identity was
clear, they decided to call this bacterium Listerella monocytogenes [99,117].
However, in 1939, the Judicial Commission of the International Committee on Sys-
tematic Bacteriology rejected the generic name Listerella because it had been previ-
ously used for a mycetozoan (a slime mold) in 1906 in honor of Arthur Lister (young
brother of Lord Lister) and for a species of foraminifer (a marine protozoan) in 1933 in
honor of Joseph Jackson Lister (father of Lord Lister). As noted by Gibbons in 1972 [48],
it is certainly unique that the same name was chosen for three quite different groups of
microorganisms to honor the contributions of a father and his two sons. The next year,
in 1940, Pirie proposed the name Listeria [ 1171.
Before, and even after this date, numerous names were used to designate L. monocy-
togenes: Bacterium rnonocytogenes hominis and later Listerella hominis by Nyfeldt,
who considered that it was the agent of infectious mononucleosis [ 104,1051; Corynebac-
terium pawulum by Schultz et al. in 1934 [ 1401; Listerella ovis by Gill in 1937
[49]; Listerella bovina, L. gallinaria, L. cunniculi, and L. gerbilli by Nyfeldt
[105,106]; Erysipelothrix monocytogenes by Wilson and Miles in 1946 [ 1731; and
Corynebacterium infantisepticurn by Potel in 1951 during his first observations of fetal
and neonatal listeriosis in Germany [ 1191.
Unlike some pathogenic agents responsible for large outbreaks which have marked
the history of humans for centuries, for example, Vibrio cholerae or Yersinia pestis, the
history of L. monocytogenes and listeriosis is recent: It began officially in 1924. The first
confirmed diagnosis in a human was that of a soldier suffering from meningitis at the end
of World War I (retrospective identification of the strain [24]), and before this case, there
are no validated observations. Interestingly, however, a historian has suggested that L.
monocytogenes could have been the cause of Queen Ams 17 unsuccessful pregnancies
( 17th century) [ 1371.
Numerical Taxonomy
With development of computers for handling large amounts of data, numerical taxonomy
provided the first attempts to investigate in depth the phylogenetic position of Listeria
among gram-positive bacteria. In the first studies, Listeria was included among coryne-
form bacteria and actinomycetes and, consequently, was located either with the coryne-
bacteria [13,28] or in an indefinite position [16,62]. In contrast, since 1969, more natural
relationships were described when Listeria was compared with various representatives of
lactic acid bacteria [29,159,160]. The close relatedness with these microorganisms was
clearly demonstrated in 1975 by the broader numerical taxonomic survey of Jones, who
studied 173 characteristics of 233 strains of various genera, including both coryneform
and lactic acid bacteria 1701. The refined position of Listeria was later investigated by
Wilkinson and Jones in 1977 and Feresu and Jones in 1988 137, 1721. From these works,
it became clear that Listeria is distinct from other known genera, including Erysipelothrix
and Brochothrix thermosphacta (formerly Microbacterium ,thermosphactum), and that it
is closely related to Lactobacillus and Streptococcus. Consequently, Wilkinson and Jones
[ 1721 suggested that Listeria, Gemella, Brochothrix, Streptococcus, and Lactobacillus be
classified in the family Lactobacillaceae. Despite some imprecision concerning the exact
position of higher taxonomic relationships, especially of Brochothrix, certain lactobacilli,
and Carnobacterium [37,172], conclusions based on numerical analysis of data for large
numbers of phenotypic features were the precursors of the current phylogenetic classifica-
tion of the genus Listeria.
4 Rocourt
Chemotaxonomy
Several chemotaxonomic markers have been especially useful for solving the phylogenetic
position of the genus Listeria, reinforcing its distinctness from coryneform bacteria and
its relatedness to the lactic acid bacteria as evidenced by numerical taxonomic studies. The
G+C % DNA content of L. monocytogenes isolates ranges from 36 to 42% [37,125,160],
indicating that Listeria belongs to the low G+C % DNA content (<55%) group of gram-
positive bacteria.
Lipoteichoic acids (amphilic polymers of the cytoplasmic membranes) have been
isolated from L. monocytogenes [59,135,164]. These acids consist of hydrophilic poly-
glycerophosphate chains covalently attached to glyco- or phosphatidylglycolipids, the hy-
drophilic moieties of the molecules, and exhibit structural analogies with lipoteichoic acids
from other bacteria. Although lipoteichoic acids show a distinct structural diversity in
their hydrophilic and lipophilic portions, a given lipoteichoic acid is known to be a fairly
stable characteristic and so may be used as a taxonomic marker. For Listeria, the presence
of particular lipoteichoic acids provides further evidence that the genus is a biochemically
coherent taxon [ 1351. Furthermore, lipoteichoic acids are absent from coryneform bacteria
but are found in Bacillus, Staphylococcus, Streptococcus, and Lactobacillus, indicating
that Listeria should be grouped with these latter microorganisms [ 1351. With the exception
of one report [94], free mycolic acids, which are specific for high G+C % DNA content
gram-positive bacteria, have not been detected in Listeria [37,73]. The presence of respira-
tory menaquinones (seven isoprene units), of predominantly methyl-branched cellular fatty
acids and meso-DAP as the major peptidoglycan diamino acid support the close relat-
edness of Listeria and Brochothrix and the greater distance from lactobacilli
[2 1,22,37,40,41,12I]. Analysis of low molecular weight RNA profiles also supports the
independent identity of L. monocytogenes among other gram-positive taxa [ 1571.
rRNA Sequencing
Recent analysis of the 16s and 23s rRNA of L. monocytogenes has further clarified the
position of Listeria with regard to other genera of gram-positive bacteria. In 1986, Ludwig
et al. [9 11 unambiguously demonstrated, using the 16s rRNA cataloguing approach (partial
sequencing), that Listeria forms with Brochothrix thermosphacta one of several sublines
within the Clostridium subdivision. They did not detect any relationship with coryne-
form bacteria (except for universal and highly conserved 16s rRNA sequences that are
common to all eubacteria and those of gram-positive bacteria, respectively). Reverse tran-
scriptase sequencing of 16s rRNA data confirmed this phylogenetic position of Listeria
and indicated that the Listeria-Brochothrix subline is approximately equidistant from the
Bacillus and Enterococcus-Carnobacteriumsublines [22]. On the basis of these data and
chemotaxonomic properties, Collins et al. 1221 considered that (a) Listeria is phylogeneti-
cally remote from Lactobacillus and should not be included in the family Lactobacillaceae
and (b) the Listeria-Brochothrix subline probably merits a separate family, the Liste-
riaceae. This great distance between Lactobacillus and Listeria has been recently con-
firmed by sequencing 23s rRNA, with Listeria to be most similar to Bacillus and Staphylo-
coccus [ 1361.
Conclusion
Data accumulated during the last three decades clearly demonstrate that Listeria is a well-
defined taxon that possesses a number of features distinguishing it from neighboring taxa.
The Genus Listeria and Listeria monocytogenes 5
ica and L. innocua (not officially validated at that time), were undertaken in 1982 with
many strains of various origins [ 1291. Five DNA relatedness groups were found among
strains formerly identified as L. monocytogenes:
DNA/DNA homology experiments (optical method) in 1993 supported these results [58].
Numerical taxonomic surveys confirmed that L. monocytogenes, as defined in the eighth
edition of Bergeys Manual of Determinative Bacteriology, was not a single taxon. How-
ever, this method is of limited sensitivity for bacteria which differ by few characteristics,
with these studies also being of little help in resolving the heterogeneity [37,72]. Interest-
ingly, this new genomic classification was tested using multilocus enzyme electrophoresis
( 18 enzyme loci analyzed). Matrix cluster analysis of the genetic distances between paired
electrophoretic types revealed that L. monocytogenes, L. ivanovii, L. welshimeri, and L.
seeligeri each corresponded to a single cluster with no overlaps between them [lO].
Lists of Bacterial Names: (a) do L. grayi and L. murrayi belong to the genus Listeria?;
(b) do L. grayi and L. murrayi belong to a single species?
L. grayi, L. murrayi, and L. monocytogenes share several similarities: They cluster
in all numerical taxonomic studies [70,159,16 I , 1721, possess lipoteichoic acid [ 1351, tei-
choic acid of the polyribitol phosphate type [41], peptidoglycan of the A1 gamma variation
[40], nonhydrogenated menaquinones of the MK-7 type [21,37], and the same cyto-
chromes (albdo) [37]. However, other features support the distinctness of the L. grayi
and L. murrayi pair within the genus Listeria: A slight difference in G+C % DNA content
(42 vs 36-38) 137,1611, several biochemical reactions [146], the nature of substitution of
lipoteichoic acids [ 1351, slight differences in protein electrophoregrams [88], cellular fatty
acid composition [ 1021, antigenic structure [ 1461, and low DNA homology values [ 1601.
Finally, the 16s rRNA oligonucleotide cataloging of L. murrayi placed it close to L. mono-
cytogenes. These data, together with the substantial phenotypic similarity with L. monocy-
togenes, provided no support for exclusion of L. murrayi (and the closely related species
L. grayi) from the genus Listeria [ 1301.
The close relationship between L. grayi and L. murrayi was evidenced by various
investigations: These two species comprise a single distinct cluster in numerical taxonomic
analysis [37,172], in multilocus enzyme electrophoresis analysis [ 101, and in DNA/DNA
hybridization studies [ 1601. In addition, they share a number of chemotaxonomic proper-
ties which distinguish them from the other Listeria species: same DNA base composition
values [37,160], same substitution of lipoteichoic acids [ 1351, same cellular fatty acid and
fatty aldehyde patterns [75], and a common antigenic structure despite small differences
[145,168]. They have been distinguished from each other only on the basis of nitrate
reduction [171]. Finally, recent reexamination of the genomic relatedness of L. grayi and
L. murrayi using DNA/DNA hybridizations and multilocus enzyme electrophoresis indi-
cated that they should be considered to be members of a single species, L. grayi [132].
These data are consistent with 16s and 23s rRNA sequencing and cellular protein electro-
phoretic pattern data [22,78,136].
L, denitrificans/Jonesia denitrificans
Although only a single isolate is currently known for this species, there have been an
amazing number of papers dealing with its taxonomic position. As early as 1966, a numeri-
cal taxonomic study showed that L. denitrificans clustered with certain coryneform bacte-
ria, and this was later confirmed [ 16,70,159,160]. Results of chemotaxonomic studies and
DNA/DNA hybridization further emphasized the phenotypic differences between L. deni-
trijicans and other members of the genus Listeria [20,21,40,41,135,161]. In 1987, 16s
rRNA cataloging confirmed that this species is not a member of the genus Listeria and
belongs to the coryneform group of bacteria [ 13I]. Consequently, this species was trans-
ferred to the newly formed genus, Jonesia, and is now officially recognized as Jonesia
denitrijicans .
obviously distinct lines of descent: One contains L. grayi and the other L. monocytogenes,
L. ivanovii, L. innocua, L. welshimeri, and L. seeligeri. The species within this line can
be divided into two groups, (a) L. monocytogenes and L. innocua and (b) L. ivanovii, L.
seeligeri, and L. welshimeri [22,129,130,136].
Genus Characteristics
Morphology
Listeria is a small (0.5 pm in diameter and 1-2 pm in length), regular gram-positive rod
with rounded ends. Cells are found singly, or in short chains, or may be arranged in V
and Y forms or in palisades. Sometimes cells are coccoid, averaging about 0.5 pm in
diameter and may be confused with streptococci. In old cultures, some cells lose the ability
to retain Gram stain and may be occasionally mistaken for Haernophilus. Long, thin,
filamentous cells appear in old and rough cultures and also after osmotic shock [57,74,84].
Listeria does not produce spores and capsules are not formed [144].
Listeria is motile because of its few peritrichous flagella when cultured at 20-25C
(not or very weakly motile at 37C) [45]. Hanging-drop preparations of fresh cultures in
tryptose phosphate broth incubated at 20C show characteristic tumbling motility: cells
start with twisting and wriggling movements which increase to fast, eccentric rotations
before they suddenly move quickly in various directions. Stab cultures in semisolid motil-
ity medium produce a typical picture of umbrella or inverted pine tree growth about
one half centimeter below the surface because of the microaerophilic nature of the organ-
ism. A recent report indicates that L. rnonocytogenes and L. innocua differ markedly in
motility and flagellin production at 37C: L. monocytogenes strains are virtually nonmotile
and produce little or no detectable flagellin, whereas strains of L. innocua are frequently
motile and produce substantial amounts of flagellin [8 11.
Cu Iture
On nutrient agar, colonies are 0.2-0.8 mm in diameter, smooth, punctiform, bluish gray,
translucent, and slightly raised with a fine surface texture and entire margin after 24 h of
incubation. After 5-10 days, well-separated colonies may be 5 mm or more in diameter.
When cultures of Listeria grown for 18-24 h at 37C on a clear medium are examined
with a binocular microscope under obliquely transmitted light, the smooth colonies exhibit
a typical blue-green iridescence [52,85,97]. Even when the population of contaminants is
rather high and that of Listeria low, Listeria can still be recognized because of this charac-
teristic [52,90], Rough colonies may occasionally be observed [54]. Conversion of smooth
colonies to rough colonies is not reversible [141]. Differences in virulence between rough
and smooth colonies have been observed [61,84,87]. Petite colony formation by strains
grown on esculin-containing agar has been described [ 1531.
Listeria usually grows well on most commonly used bacteriological media. The
growth rate is increased by the presence of fermentable sugar, particularly glucose. On
plate culture, Listeria has a particularly penetrating acid odor which may be caused by
The Genus Listeria and Listeria monocytogenes 9
formation of carboxylic acids, hydroxy acids, and alcohols [27]. In broth, the medium
becomes turbid after 8-24 h of incubation at 37C. Profuse growth is always observed
slightly below the clear area near the surface of the medium, indicating the propensity
for Listeria to grow better at oxygen tensions lower than that of air [141].
The normal temperature limits for growth are + 1-2C to 45C [76,144]; however,
some multiplication has been reported to occur in chicken broth and pasteurized milk
during extended incubation at -0.1 to 0.4"C [ 1691. Growth is slow at refrigeration temper-
atures, with generation times of 30-40 h at +4"C in skim milk, for example [134]. This
property was first used by Gray [55] for selective cold enrichment of a contaminated
sample. In broth, Listeria normally grows from pH 4.4-9.6, optimally at pH 7
[ 15,47,107,112]. Growth can occur in media containing 10% (w/v) NaCl with survival at
higher concentrations [ 146,1491. Survival at low pH and high salt concentration is strongly
temperature-dependent [ 191. Listeria is one of the few foodborne pathogens that can grow
at an a, value below 0.93 [35,112].
Nutritional Requirements
According to published data, growth factors for Listeria include cystine, leucine, isoleu-
cine, arginine, methionine, valine, cysteine, riboflavin, biotin, thiamine, and thioctic acid
[ 120,151,1701. Growth is stimulated by Fe3 and phenylalanine [ 120,15I]. In some experi-
+
ments, virulent strains grew faster in the presence of iron than did avirulent strains 1251.
Glucose and glutamine are required as primary sources of carbon and nitrogen [ 146,1201.
Chemically defined media have been described for Listeria [ 120,122,1501.
Metabolism and Biochemical Characters
Listeria is aerobic, microaerophilic, facultatively anaerobic, catalase-positive (rare cata-
lase-negative strains have been observed) and oxidase-neg,ative. Although Feresu and
Jones [37] found cytochrome a,bdo, the presence of cytochrome is controversial [ 109,1631.
Listeria is homofermentative and oxidizes glycolytic intermediate compounds [27]. It pos-
sesses glucose oxidase and NADH oxidase activities [ 1091. All strains grow on glucose
forming lactate, acetate, and acetoin as main endproducts under aerobic conditions
[27,113,133]. Acetoin is not produced under anaerobic conditions. Anaerobically, only
hexoses and pentoses support growth; aerobically, maltose and lactose support growth of
some strains, but sucrose does not [ 1 131. Catabolism of glucose proceeds by the Embden-
Meyerhof pathway both aerobically and anaerobically [ 1461. L. monocytogenes imports
glucose by a high-affinity phosphoenolpyruvate-dependentphosphotransferase system and
a low-affinity proton motive force-mediated system [ 17,1081. All strains are methyl red
and Voges-Proskauer test positive. Acid is also produced from amygdalin, cellobiose,
fructose, mannose, salicin, maltose, dextrin, alpha-methyl-D-glucoside, and glycerol. Acid
production from galactose, lactose, melezitose, sorbitol, starch, sucrose, and trehalose is
variable. Acid is almost never produced from adonitol, arabinose, dulcitol, erythritol, gly-
cogen, inositol, inulin, melibiose, raffinose, or sorbose. Phenylalanine-deaminase,orni-
thine, lysine, and arginine decarboxylases are not produced. H2S is not produced. Urea is
not hydrolyzed and indole is not produced. Additional information on biochemical tests
can be found in references 37, 79, 126, 146, 148, and 172.
Species Identification
All Listeria species are phenotypically very similar, but they can be distinguished by the
following tests: hemolysis, acid production from D-xylose, L-rhamnose, alpha-methy1-D-
Rocourt
mannoside, and mannitol [ 1281 (Fig. 1). The phenotypic similarities are consistent with
the high genomic homologies between the different species [22,129,136].
Hemolysis is a key characteristic for speciation of isolates and clearly is the most
difficult characteristic to detect. During a collaborative study on Listeria identification,
Higgins and Robison [60] noted that a large percentage of errors in identification of L.
seeligeri and L. ivanovii was caused by inaccurate reading of the CAMP3-testand hemoly-
sis. Isolates of L. monocytogenes and L. seeligeri show narrow, slight clearing zones of
beta-hemolysis. L. ivanovii shows wide, clearly delineated zones of beta-hemolysis. In
contrast, L. innocua, L. welshimeri, and L. grayi are not hemolytic. L. innocua can produce
a green zone of hemolysis on certain media [ 118,1561. L. monocytogenes hemolyzes blood
from sheep, horses, cows, guinea pigs, piglets, and humans [ 138,144,154,166l.Various
methods have been developed to determine hemolytic activity, especially for weakly he-
molytic strains (L. seeligeri and some L. monocytogenes isolates) and include examination
of hemolysis underneath the colony, prolonged incubation (48 h), incubation for several
hours at +4"C, use of thin layer blood agar plates [86], several media 1441, tube tests and
microplate techniques with erythrocyte suspensions [30,31,1621, addition of an exosub-
stance from Rhodococcus equi, Staphylococcus aureus, or L. ivanovii to the blood agar
[95,154,155],and the CAMP-test with S. aureus and R. equi [ 14,42,56,65,66,97,101,128].
Positive CAMP-tests are indicated by an enhanced zone of beta-hemolysis at the intersec-
tion of the test strains, L. monocytogenes, L. ivanovii, and L. seeligeri with S. aureus and
L. ivanovii with R. equi. Conflicting readings of the CAMP-test with R. equi have been
reported, some authors considering L. monocytogenes to be positive [39,139,167] and
others negative [128,146]. The typical positive CAMP-test with R. equi, as observed with
L. ivanovii, gives a shovel-like shape. In contrast, when this test is positive with L. rnonocy-
The original CAMP-test was described by Christie, Atkins, and Munch-Peterson, who observed this lytic phe-
nomenon for Srreprococcus in 1944, and the test is named after these authors [ 141.
The Genus Listeria and Listeria monocytogenes 11
togenes, the shape is that of an onion. This could reflect either different abilities of R.
equi strains to interact with L. monocytogenes because of different amounts of listeriolysin
0 secreted by L. monocytogenes strains or variations in the capability of R. equi strains
to secrete cholesterol oxidase [38,167]. However, whether or not it is positive, this test
is not essential, as L. monocytogenes and L. ivanovii can be easily distinguished by acid
production from D-xylose, L-rhamnose, and alpha-methyl-D-mannoside. The L. monocy-
togenes exosubstance involved in the CAMP reaction with S. uureus and R. equi is listerio-
lysin 0 [ 1231. The exosubstances of S. aureus and R. equi are a sphingomyelinase C and
a cholesterol oxidase, respectively [38,96,123].
Hemolysin is a major virulence factor of L. monocytogenes. Three species, L. mono-
cytogenes, L. ivanovii, and L. seeligeri, are hemolytic and possess the virulence gene
cluster as recently demonstrated [50]; however, only two species, L. monocytogenes and
L. ivanovii, are naturally and experimentally pathogenic [93,127], L. ivanovii being mainly
responsible for abortion in animals. Therefore, pathogenicity should not be presumed on
the observation of hemolysis alone. Few nonhemolytic L. monocytogenes isolates have
been observed, with the best known example being the type strain of this species
[6 1,71,801. Dissociation between hemolytic and nonhemolytic colonies is rarely observed
[ 1 151. Nonhemolytic and several weakly hemolytic strains are non- or weakly pathogenic
[ 12,23,30,36,61,114,1621.Despite these atypical strains, routine pathogenicity testing for
L. monocytogenes is generally unnecessary [90].
Additional tests, especially to distinguish L. monocytogenes from L. iiznocua, have
been proposed and include detection of phospolipase C activity [ 18,1031, hydrolysis of
D-alanine-p-nitroanilide[79], and hydrolysis of a naphthylamide substrate (API Listeria
[81)*
Various commercial miniaturized culture or enzyme multitest assays are now used
for Listeria identification, since conventional culture procedures for identification are te-
dious and time consuming. They include API 50 CH [83,126], API-ZYM [127], API 20
STREP [92], API Listeria [8,9,44], API Coryne [82], Micro-ID Listeria [2,8,60,124], Mast
ID [83], RAPID CORYNE [46], RAPID ID 32 Strep [43], and a microtiter plate method
[ 1521. Information provided by API ZYM, API 20 STREP, and RAPID ID 32 Strep distin-
guishes isolates at the genus level, whereas API Listeria, Mast-ID, API Coryne, and API
50 CH are more appropriate for both genus and species identification.
Phenotypic markers are used for routine identification of Listeria isolates. More
sophisticated methods have been described and some can help speciate atypical isolates.
These methods include 16s rRNA sequencing [26], sequence analysis of the 16s-23s
internal transcribed spacer loci [33,5 11, ribotyping 1681, random amplification of polymor-
phic DNA [34], repetitive element sequence-based PCR 1691, multilocus enzyme electro-
phoresis [ 10I, cellular protein electrophoretic pattern analysis [78], enzymatic profiling
using fluorogenic substrates [77], analysis of fermentation products by frequency-pulsed
electon-capture gas-liquid chromatography [27], Fourier transform infrared spectroscopy
analysis [63], and thermogram determination [ 11.
CONCLUSION
Studies on the phylogenetic position of Listeria started when numerical phenetic studies
were applied to gram-positive bacteria. These first studies were of primary importance in
demonstrating that Listeria was not a coryneform bacterium. These data were confirmed
by 16s rRNA cataloging. The refined location of Listeria within the low C + C % DNA
12 Rocourt
content gram-positive bacteria was later determined by reverse transcriptase 16s and 23s
sequencing data. Numerical taxonomic studies revealed a certain heterogeneity within this
genus with the exact species content determined by DNA/DNA hybridization and rRNA
sequencing. Based on this genomic dissection, the genus contains six species which are
divided into two sublines of descent. The present state of Listeria taxonomy is the result
of more than 20 years of work done in various laboratories in different countries, using
as many methods as imaginable. Most of this work was done during the last two decades,
and the number of publications in this field is now decreasing. Fortunately, the distinction
between L. rnonocytogenes and nonpathogenic species was already defined when food-
borne transmission of listeriosis became a public health problem with a major economic
impact on the food industry. This allowed efforts to be restricted to food contaminated
with L. rnonocytogenes, since food contaminated by other Listeria species is of no public
health concern. Now, both introduction of molecular biology methods and the need to
develop new tools to understand listeriosis epidemiology are generating renewed interest
in classification of L. rnonocytogenes strains (see Chap. 9).
REFERENCES
1. Allerberger, F.J., A. Schulz, and M.P. Dierich. 1988. Microcalorimetric investigations on
Listeria. Zbl. Bakteriol. Hyg. A. 268:15-23.
2. Bannerman, E., M.N. Yersin, and J. Bille. 1992. Evaluation of the Organon-Teknika
MICRO-ID Listeria System. Appl. Environ. Microbiol. 58:2011-2015.
3. Bergeys Manual of Determinative Bacteriology. 1934. 4th edition (Bergey, D.H., ed.). Wil-
liams & Wilkins c o, Baltimore.
4. Bergeys Manual of Determinative Bacteriology. 1948.6th ed. (Breed, R.D., Murray, E.G.D.,
and Hitchens, A.P., eds.). Williams & Wilkins Co., Baltimore.
5. Bergeys Manual of Determinative Bacteriology. 1957.7th ed. (Breed, R.D., Murray, E.G.D.,
and Smith, N.R., eds.). Williams & Wilkins Co., Baltimore.
6. Bergeys Manual of Determinative Bacteriology. 1974. 8th ed. (Buchanan, R.E., and Gib-
bons, N.E., eds.). Williams & Wilkins Co., Baltimore.
7. Bergeys Manual of Systematic Bacteriology, vol. 2, 1986. (Sneath, P.H.A., Mair, N.S.,
Sharpe, N.E., and Holt, J.G., eds.). Williams & Wilkins Co., Baltimore.
8. Beumer, R.R., M.C.T. Giffel, M.T.C. Kok, and F.M. Rombouts. 1996. Confirmation and
identification of Listeria spp. Lett. Appl. Microbiol. 22:448-452.
9. Bille, J., B. Catimel, E. Bannerman, C. Jacquet, M.N. Yersin, I. Caniaux, D. Monget, and
J. Rocourt. 1992. API Listeria, a new and promising one-day system to identify Listeria
isolates. Appl. Environ. Microbiol. 58: 1857-1860.
10. Boerlin, P., J. Rocourt, and J.C. Piffaretti. 1991. Taxonomy of the genus Listeria by using
multilocus enzyme electrophoresis. Int. J. System. Bacteriol. 41 :59-64.
11. Boerlin, P., J. Rocourt, F. Grimont, P.A.D. Grimont, Ch. Jacquet, and J.C. Piffaretti. 1992.
Listeria ivanovii subsp. Londoniensis. Int. J. System. Bacteriol. 1542-46.
12. Bosgiraud, C., A. Menudier, M.J. Cornuejols, N. Hangard-Vidaud, and J.A. Nicolas. 1989.
Etude de la virulence de Listeriu monocytogenes isolies daliments de Ihomme. Microbiol.
Alim. Nut. 7:4 13-420.
13. Bousfield, I. 1972. A taxonomic study of some coryneform bacteria. J. Gen. Microbiol. 71:
441-455.
14. Brzin, B., and H.P.R. Seeliger, 1975. A brief note on the CAMP phenomenon in Listeria.
In: M. Woodbine, ed. Problems of Listeriosis. Leicester, UK: University of Leicester. pp.
34-37.
15. Buchanan, R.L., and L.A. Klawitter, 1990. Effects of temperature and oxygen on the growth
of Listeria monocytogenes at pH 4.5. J. Food Sci. 55:1754-1756.
The Genus Listeria and Listeria monocytogenes 13
16. Chatelain, R., and L. Second. 1966. Taxonomie numirique de quelques Brevibacterium. Ann.
Inst. Pasteur 111:630-644.
17. Christensen, D.P., and R.W. Hutkins, 1994. Glucose uptake by Listeria monocytogenes Scott
A and inhibition by pediocin JD. Appl. Environ. Microbiol. 60:3870-3873.
18. Coffey, A., F.M. Rombouts, and T. Abee, 1996. Influence of environmental parameters on
phosphatidylcholine phospholipase C production in Listeria monocytogenes: a convenient
method to differentiate L. monocytogenes from other Listeria species. Appl. Environ. Micro-
biol. 62: 1252-1256.
19. Cole, M.B., M.V. Jones, and C. Holyoak. 1990. The effect of pH, salt concentration and
temperature on the survival and growth of Listeria monocytogenes. J. Appl. Bacteriol. 69:
63-72.
20. Collins, M.D., S. Feresu, and D. Jones. 1983. Cell wall, DNA base composition and lipid
studies on Listeria denitriJicans (Privot). FEMS Microbiol. Lett. 18:131- 134.
21. Collins, M.D., D. Jones, M. Goodfellow, and D.E. Minnikin. 1979. Isoprenoid quinone com-
position as a guide to the classification of Listeria, Brochothrix, Erysipelothrix and Caryopha-
non. J. Gen. Microbiol. 11 1:453-457.
22. Collins, M.D., S. Wallbanks, D.J. Lane, J. Shah, R. Nietupski, J. Smida, M. Dorsch, and E.
Stackebrandt. 1991. Phylogenic analysis of the genus Listeria based on reverse transcriptase-
sequencing of 16s rRNA. Int. J. Syst. Bacteriol. 41:240-246.
23. Conner, D.E., V.N. Scott, S.S. Sumner, and D.T. Bernard. 1989. Pathogenicity of foodborne,
environmental and clinical isolates of Listeria monocytogenes in mice. J. Food Sci. 54: 1553-
1556.
24. Cotoni, L. 1942. A propos des bactCries dinommCes Listerella-Rappel dune observation
ancienne de mCningite chez Ihomme. Ann. Inst. Pasteur 68:92-95.
25. Cowart, R.E., and B.G. Foster, 1985 Differential effects of iron in the growth of Listeria
monocytogenes: minimum requirements and mechanism of acquisition. J. Infect. Dis. 151:
72 1-730.
26. Czajka, J., N. Bsat, M. Piani, W. Russ, K. Sultana, M. Wiedinann, R. Whitaker, and C.A.
Batt. 1993. Differentiation of Listeria monocytogenes and Listeria innocua by 16s rRNA
genes and intraspecies discrimination of Listeria monocytogenes strains by random amplified
polymorphic DNA polymorphisms. Appl. Environ. Microbiol. 59:304-308.
27. Daneshvar, M.I., J.B. Brooks, G.B. Malcolm, and L. Pine. 1989. Analyses of fermentation
products of Listeria species by frequency-pulsed electron-capture gas-liquid chromatography.
Can. J. Microbiol. 35:786-793.
28. Da Silva, G.A.N., and J.G. Holt, 1965. Numerical taxonomy of certain corynefonn bacteria.
J. Bacteriol. 90:921-927.
29. Davis, G.H.G., L. Fomin, E. Wilson, and K.G. Newton, 1969. Numerical taxonomy of Liste-
ria, streptococci and possibly related bacteria. J. Gen. Microbiol. 57:333-348.
30. Del Corral, F., R.L. Buchanan, M.M. Bencivengo, and P.H. Cooke, 1990. Quantitative com-
parison of selected virulence associated characteristics in food and clinical isolates of Liste-
ria. J. Food Prot. 53: 1003- 1009.
31. Dominguez Rodriguez, L., J.A. Vasquez Boland, J.F. Fernandez Garayzabal, P. Echalecu
Tranchant, E. Gomez-Lucia, E.F. Rodriguez Ferri, and G. Suarez Fernandez, 1986. Mi-
croplate technique to determine hemolytic activity for routine typing of Listeria strains. J.
Clin. Microbiol. 24:99- 103.
32. Donker-Voet, J. 1972. Listeria monocytogenes: some biochemical and serological aspects.
Acta Microbiol. Acad. Sci. Hung. 19:287-291.
33. Drebot, M., S. Neal, W. Schlech, and K. Rozee. 1996. Differentiation of Listeria isolates by
PCR amplicon profiling and sequence analysis of 16s-23s rRNA internal transcribed spacer
loci. J. Appl. Bacteriol. 80: 174- 178.
34. Farber, J.M., and C.J. Addison. 1994. RAPD typing for distinguishing species and strains
in the genus Listeria. J. Appl. Bacteriol. 77:242-250.
14 Rocourt
35. Farber, J.M., F. Coates, and E. Daley. 1992. Minimum water activity requirements for the
growth of Listeria monocytogenes. Lett. Appl. Microbiol. 15:103-105.
36. Farber, J.M., J.I. Speirs, R. Pontefract, and D.E. Conner, 1991. Characteristics of nonpatho-
genic strains of Listeria monocytogenes. Can. J. Microbiol. 37:647-650.
37. Feresu, S.B., and D. Jones. 1988. Taxonomic studies on Brochothrix, Erysipelothrix, Listeria
and atypical lactobacilli. J. Gen. Microbiol. 134:1165- 1 183.
38. Fernandez-Garayzabal, J.F., C. Delgado, M.M. Blanco, G. Suarez, and L. Dominguez. 1996.
Cholesterol oxidase from Rhodococcus equi is likely the major factor involved in the coopera-
tive lytic process (CAMP reaction) with Listeria monocytogenes. Lett. Appl. Microbiol. 22:
249-252.
39. Fernandez-Garayzabal, J.F., G. Suarez, M.M. Blanco, A. Gibello, and L. Dominguez. 1996.
Taxonomic note: a proposal for reviewing the interpretation of the CAMP reaction between
Listeria monocytogenes and Rhodococcus equi. Int. J. System. Bacteriol. 46:832-834.
40. Fiedler, F., and J. Seger. 1983. The murein types of Listeria grayi, Listeria murrayi and
Listeria denitriJicans. System. Appl. Microbiol. 4:444-450.
41. Fiedler, F., J. Seger, A. Schrettenbrunner, and H.P.R. Seeliger. 1984. The biochemistry of
murein and cell wall teichoic acids in the genus Listeria. System. Appl. Microbiol. 5:360-376.
42. Fraser, G. 1962. A plate method for the rapid identification of Listeria (Erysipelothrix) mono-
cytogenes. Vet. Rec. 74:50-5 1.
43. Freney, J., S. Bland, J. Etienne, M. Desmonceaux, J.M. Boeufgras, and J. Fleurette. 1992.
Description and evaluation of the semiautomated 4-hour rapid ID 32 Strep method for identi-
fication of streptococci and members of related genera. J. Clin. Microbiol. 30:2657-266 1.
44. Fujisawa, T., and M. Mori. 1994. Evaluation of media for determining hemolytic activity
and that of API Listeria system for identifying strains of Listeria monocytogenes. J. Clin.
Microbiol. 32: 1 127-1 129.
45. Galsworthy, S.B., S. Girdler, and S.F. Koval. 1990. Chemotaxis in Listeria monocytogenes.
Acta Microbiol. Hung. 37:8 1-85.
46. Gavin, S.E., R.B. L6onard, A.M. Briselden, and M.B. Coyle. 1992. Evaluation of the rapid
CORYNE identification system for Corynebacterium species and other coryneforms. J. Clin.
Microbiol. 30: 1692- 1695.
47. George, S.M., and B.M. Lund. 1992. The effect of culture medium and aeration on growth
of Listeria monocytogenes at pH 4.5. Lett. Appl. Microbiol. 15:49-52.
48. Gibbons, N.E. 1972. Listeria Pirie-Whom does it honor? Int. J. System. Bacteriol. 22:
1-3.
49. Gill D.A. 1937. Ovine bacterial encephalitis (circling disease) and the bacterial genus Listere-
lla. Austral. Vet. J. 13:46-56.
50. Gouin, E., J. Mengaud, and P. Cossart. 1994. The virulence gene cluster of Listeria monocyto-
genes is also present in Listeria ivanovii, an animal pathogen, and Listeria seeligeri, a non-
pathogenic species. Infect. Immun. 62:3550-3553.
51. Graham, T., E.J. Golsteyn-Thomas, V.P.J. Gannon, and J.E. Thomas. 1996. Genus- and spe-
cies-specific detection of Listeria monocytogenes using polymerase chain reaction assays
targeting the 16S/23S intergenic spacer region of the rRNA operon. Can. J. Microbiol. 42:
1155-1 162.
52. Gray, M.L. 1957. A rapid method for the detection of colonies of Listeria monocytogenes.
Zbl. Bakteriol. Parasit. Infekt. Hyg. I Orig. 169:373-377.
53. Gray, M.L., and A.H. Killinger. 1966. Listeria monocytogenes and listeric infections. Bacte-
riol. Rev. 30:309-382.
54. Gray, M.L., H.J. Stafseth, and F. Thorp, Jr. 1957. Colonial dissociation of Listeria monocyto-
genes. Zbl. Bakteriol. Parasit. Infekt. Hyg. I Orig. 169:378-392.
55. Gray, M.L., H.J. Stafseth, F. Thorp, L.B. Sholl, and W.F. Riley. 1948. A new technique for
isolating Listerellae from the bovine brain. J. Bacteriol. 55:47 1-476.
56. Groves, R.D., and H.J. Welshimer. 1977. Separation of pathogenic from apathogenic Listeria
monocytogenes by three in vitro reactions. J. Clin. Microbiol. 5559-563.
The Genus Listeria and Listeria monocytogenes 15
57. Gutekunst, K.A., L. Pine, E. White, S. Kathariou, and G.M. Carlone. 1992. A filamentous-
like rnutant of Listeria monocytogenes with reduced expression of a 60-kilodalton extracellu-
lar protein invades and grows in 3T6 and Caco-2 Cells. Can. J. Microbiol. 38:843-85 1.
58. Hartford, T., and P.H.A. Sneath. 1993. Optical DNA-DNA homology in the genus Listeria.
Int. J. Syst. Bacteriol. 43:26-3 I .
59. Hether, N.W., and L.L. Jackson. 1983. Lipoteichoic acid from Listeria monocytogenes. J.
Bacteriol. 156:809-8 17.
60. Higgins, D.L., and B. Robinson. 1993. Comparison of micro-ID Listeria method with con-
ventional biochemical methods for identification of Listeria isolated from food and environ-
mental samples: collaborative study. J. Assoc. Off. Anal. Chem. Int. 76:83 1-838.
61. Hof, H. 1984. Virulence of different strains of Listeria monocytogenes serovar 1/2. Med.
Microbiol. Immunol. 173:207-2 18.
62. Holniberg, K., and H.O. Hollander. 1973. Numerical taxonomy and laboratory identification
of Bucterionema matruchoti, Rothia dendocariosa, Actinomyces naeslundi, Actinomyces vis-
cosus, and some related bacteria. J. Gen. Microbiol. 76:43-63.
63. Holt, C., D. Hirst, A. Sutherland, and F. Macdonald. 1995. Discrimination of species in the
genus Listeria by Fourier transform infrared spectroscopy and canonical variate analysis.
Appl. Environ. Microbiol. 61 :377-378.
64. Hulphers, G. 191 1. Liver necrosis in rabbit caused by an hitherto unknown bacterium; traduc-
tion de: Lefvernekros has kanin orsakad af en ej forut beskrifven bakterie. Svensk. Vet.
Tidskrift. 2:265 -273.
65. Hunter, R. 1973. Observations on Listeria monocytogenes type 5 (Ivanov) isolated in NZ.
Med. Lab. Technol. 3 0 5 1-56.
66. Ivanov, I. 1975. Establishment of non-motile strains of Listeria monocytogenes type 5. In:
Woodbine, ed. Problems of Listeriosis. Leicester UK: University of Leicester. pp. 18-29.
67. Ivanov, I. 1962. Untersuchungen uber die Listeriose der Schafe in Bulgarien. Mh. Vet. Med.
171729-736.
68. Jacquet, C., S. Aubert, N. El Solh, and J. Rocourt. 1992. Use of rRNA gene restriction pat-
terns for the identification of Listeria species. Syst. Appl. Microbiol. 15:42-46.
69. Jersek, B., E. Tcherneva, N. Rijpens, and L. Herman. 1996. Repetitive element sequence-
based PCR for species and strain discrimination in the genus Listeria. Lett. Appl. Microbiol.
23:55-60.
70. Jones, D. 1975. A numerical taxonomic study of coryneform and related bacteria. J. Gen.
Micro bi o1. 87 :5 2 -96.
71. Jones, D., and H.P.R. Seeliger. 1983. Designation of a new type strain for Listeria monocyto-
gene,v-request for an opinion. Int. J. Syst. Bacteriol. 33:429.
72. Jones, D., S.B. Feresu, and M.D. Collins. 1986. Classification and identification of Listeria,
Brochothrix and Erysipelothrix. In: A.L. Courtieu, E.P. Espaze, and A.E. Reynaud, eds. List-
e'riose, Listeria, Listeriosis. Nantes, France. UniversitC de Nantes.
73. Jones, D., M.D. Collins, M. Goodfellow, and D.E. Minnikin. 1979. Chemical studies in the
classification of the genus Listeria and possibly related bacteria. In: I. Ivonov, ed. Problem
of Listeriosis. National Agroindustrial Union. Sofia: Center for Scientific Information. pp.
17-23.
74. Jorgensen, F., P.J. Stephens, and S. Knochel. 1995. The effect of osmotic shock and subse-
quent adaptation on the thermotolerance and cell morphology of Listeria monocytogenes. J.
Appl. Bacteriol. 79:274-28 I .
75. Julak, J., and M. Mara. 1973. Effect of glucose and glycerin in cultivation media on the fatty
acid composition of Listeria monocytogenes. J. Hyg. Epidemiol. Microbiol. Immunol. 17:
329- 338.
76. Junttila, J.R., S.I. Niemela, and J. Hirn. 1988. Minimum growth temperatures of Listeria
monocytogenes and non-haemolytic Listeria. J. Appl. Bacteriol. 65:32 1 -327.
77. Kampfer, P. 1992. Differentiation of Corynebacterium spp, Listeria spp. and related organ-
isms by using fluorogenic substrates. J. Clin. Microbiol. 30: 1067- 107 1.
16 Rocourt
78. Kampfer, P., R.U. Dastis, and W. Dott, 1994. Characterization of Listeria by standardized
cellular protein electrophoretic patterns. Syst. Appl. Microbiol. 17:211-2 15.
79. Kampfer, P., S. Bottcher, W. Dott, and H. Ruden. 1991. Physiological characterization and
identification of Listeria species. Zbl. Bakteriol. 275:423-435.
80. Kathariou, S., and L. Pine. 1991. The type strain(s) of Listeria monocytogenes: a source of
continuing difficulties. Int. J. Syst. Bacteriol. 41 :328-330.
81. Kathariou, S., R. Kanenaka, R.D. Allen, A.K. Fok, and C. Mizumoto. 1995. Repression of
motility and flagellin production at 37 degrees C is stronger in Listeria monocytogenes than
in the nonpathogenic species Listeria innocua. Can. J. Microbiol. 41 572-577.
82. Kerr, K.G., P.M. Hawkey, and R.W. Lacey. 1993. Evaluation of the API Coryne System for
identification of Listeria species. J. Clin. Microbiol. 3 1:749-750.
83. Kerr, K.G., N.A. Rotowa, P.M. Hawkey, and R.W. Lacey. 1990. Evaluation of the Mast ID
and API 50 CH systems for identification of Listeria spp. Appl. Environ. Microbiol. 56:657-
660.
84. Kuhn, M., and W. Goebel. 1989. Identification of an extracellular protein of Listeria monocy-
togenes possibly involved in intracellular uptake by mammalian cells. Infect. Immun. 57:
55-61.
85. Lachica, R.V. 1990. Simplified Henry technique for initial recognition of Listeria colonies.
Appl. Environ. Microbiol, 56: I 164- 1 165.
86. Lachica, R.V. 1996. Hemolytic activity reevaluation of putative nonpathogenic Listeria mo-
nocytogenes strains. Appl, Environ. Microbiol. 62:4293 -4295.
87. Lammerding, A.M., K.A. Glass, A. Gendrofitzpatrick, and M.P. Doyle. 1992. Determination
of virulence of different strains of Listeria monocytogenes and Listeria innocua by oral inocu-
lation of pregnant mice. Appl. Environ. Microbiol. 58:399 1-4000.
88. Lamont, R.J., D.T. Petrie, W.T. Melvin, and R. Postlethwaite. 1986. An investigation of the
taxonomy of Listeria species by comparison of electrophoretic protein patterns. In: A.L.
Courtieu, E.P. Espaze, and A.E. Reynaud, eds. ListCriose, Listeria, Listeriosis. Nantes,
France: Universitk de Nantes. pp. 41-46.
89. Larsen, H.E., and H.P.R. Seeliger. 1966. A mannitol fermenting Listeria: Listeria grayi sp.
n. In Proceedings of the Third International Symposium on Listeriosis. Bilthoven, The Neth-
erlands.
90. Lovett, J. 1988. Isolation and identification of Listeria monocytogenes in dairy products. J.
Assoc. Off. Anal. Chem. 7 1 :658-660.
91. Ludwig, W., K.-H. Schleifer, and E. Stackebrandt. 1984. 16s rRNA analysis of Listeria
monocytogenes and Brochothrix thermosphacta. FEMS Microbiol. Lett. 25: 199-204.
92. MacGowan, A.P., R.J. Marshall, and D.S. Reeves. 1989. Evaluation of API-20-STREP sys-
tem for identifying Listeria species. J. Clin. Pathol. 42548-550.
93. Mainou-Fowler, T., A.P. MacGowan, and R. Postlethwaite. 1988. Virulence of Listeria spp:
course of infection in resistant and susceptible mice. J. Med. Microbiol. 27: 131- 140.
94. Mara, M., and C. Michalec. 1977. Chromatographic study of mycolic acid-like substances
in lipids of Listeria monocytogenes. J. Chromatogr. 130:434-436.
95. McKellar, R.C. 1993. Novel mechanism for the CAMP reaction between Listeria monocyto-
genes and Corynebacterium equi. Int. J. Food Microbiol. 18:77-82.
96. McKellar, R.C. 1994. Identification of the Listeria monocytogenes virulence factors in the
CAMP reaction. Lett. Appl. Microbiol. 18:79-81.
97. McKellar, R.C. 1994. Use of the CAMP test for identification of Listeria monocytogenes.
Appl. Environ. Microbiol, 60:42 19-4225.
98. Moura, S.M., M.T. Destro, B.D.G.M. Franco, and R.M. Brancaccio. 1991. Low cost illumina-
tion system for Listeria spp. research. Rev. Microbiol. Sao Paulo. 22:75-77.
99. Murray, E.G.D. 1963. A retrospect of listeriosis. In: M.L. Gray, ed. Second Symposium on
Listeric Infection Bozeman, MT: Artcraft Printer. pp. 3-6.
100. Murray, E.G.D., R.A. Webb, and M.B.R. Swann. 1926. A disease of rabbit characterised
The Genus Listeria and Listeria monocytogenes 17
species. In: T. Bergan, and J . Norris, (eds.), Methods in Microbiology New York: Academic
Press, pp 33-48.
146. Seeliger, H.P.R., and D. Jones. 1986. Genus Listeria Pirie 1940. In: P.H.A. Sneath,
N.S. Mail, N.E. Sharpe, and J. G. Holt (eds.), Bergeys Manual of Systematic Bacteriology,
Vol. 2. Baltimore: Williams & Wilkins. pp. 788-795.
147. Seeliger, H.P.R., J. Rocourt, A. Schrettenbrunner, P.A.D. Grimont, and D. Jones. 1984. Liste-
ria ivanovii sp. nov. Int. J. Syst. Bacteriol. 34:336-337.
148. Seiler, H., and M. Busse. 1989. Biochemische Differenzierung von Listerien aus Kase. Berl.
Munch. tierarztl. Wschr. 102:166- 170.
149. Shahamat, M., A. Seaman, and M. Woodbine. 1980. Survival of Listeria monocytogenes in
high salt concentrations. Zbl. Bakteriol. Hyg., I. Abt. Orig. A. 246:506-5 1 1.
150. Siddiqi, K.,and M.A. Khan. 1982. Vitamin and nitrogen base requirements for Listeria nzono-
cytogenes and haemolysin production. Zbl. Bakteriol. Hyg. I. Abt. Orig. A. 253:225-235.
151. Siddiqi, K.,and M.A. Khan. 1989. Amino acid requirement of six strains of Listeria monocy-
togenes. Zbl. Bakteriol. 27 1 : 146-1 52.
152. Siragusa, G.R., and J.W. Nielsen. 1991. A modified microtiter plate for biochemical charac-
terization of Listeriu spp. J . Food Prot. 54: I2 I - 125.
153. Siragusa. G.R., L.A. Elphingstone, P.L. Wiese, S.M. Haefner, and M.G. Johnson. 1990. Petite
colony formation by Listeria monocytogenes and Listeria species grown on esculin-con-
taining agar. Can. J. Microbiol. 36:697-703.
154. Skalka, 13., and J. Smola. 1982. Hemolytic properties of exosubstance of serovar 5 Listeriu
monocytogenes compared with beta toxin of Stuphylococcus aureus. Zbl. Bakteriol. Hyg.,
1. Abt. Orig. A. 252:17-25.
155. Shalka, B., J. Smola, and K. Elischerova. 1982. Routine test for in vitro differentiation of
pathogenic and apathogenic Listeria monocytogenes strains. J. Clin. Microbiol. 15503-507.
156. Skalka, 13., J. Smola, and K. Elischerova. 1983. Hemolytic phenomenon under the cultivation
of Listeria innocua. Zbl. Bakteriol. Hyg., I. Abt. Orig. A. 253:559-565.
157. Slade, P.J., and D.L. Collins-Thompson. 1991. Differentiation of the genus Listeriu from
other Gram-positive species based on low molecular weight (L,MW) RNA profiles. J. Appl.
Bacteriol . 70:355 -360.
158. Sohier, I<., F. Benazet, and M. Pikhaud. 1948. Sur un germe du genre Listeriu apparemment
non pathogkne. Ann. Inst. Pasteur 7454-57,
159. Stuart, M.R., and P.E. Pease. 1972. A numerical study on the relationships of Listeria and
Erysipeiothrix. J. Gen. Microbiol. 7355 1-565.
160. Stuart, S.E., and H.J. Welshimer. 1973. Intrageneric relatedness of Listeria Pirie. Int. J. Syst.
Bacteriol. 23:8- 14.
161. Stuart, S.E., and H.J. Welshimer. 1974. Taxonomic reexamination of Listeria Pirie and tranfer
of Listeria grayi and Listeria murruyi to a new genus Murruya. Int. J. Syst. Bacteriol. 24:
177-185.
162. Tabouret, M., J. Derycke, A. Audurier, and B. Poutrel. I99 1. Pathogenicity of Listeria mono-
cytogenes isolates in immunocompromised mice in relation to listeriolysin production. J.
Med. Microbiol. 34: 13- 18.
163. Trivett, T.L., and E.A. Meyer. 1967. Effect of erythritol on the in vitro growth and respiration
of Listrria monocytogenes. J. Bacteriol. 93: 1 197- I 198.
164. Uchikawa, K.-I., I. Sekikawa, and I. Azuma. 1986. Structural studies on lipoteichoic acids
from four Listeria strains. J. Bacteriol. 168:1 15-122.
165. Validation of the publication of new names and new combinations previously effectively
published outside the IJSB. List no. 10. 1983. Int. J. Syst. Bacteriol. 33:438-440.
166. Van der Kelen, D., and J.A. Lindsay. 1990. Differential hemolytic response of Listeria mono-
cytogerres strains on various blood agars. J. Food Safety I 1 3 - 12.
167. Vazquez-Boland, J.A., L. Dominguez, J. F. Fernandez-Garayzabal, and G. Suarez. 1992.
Listeritr monocytogenes CAMP reaction. Clin. Microbiol. Rev. 5:343.
20 Rocourt
168. Vazquez-Boland, J.A., L. Dominguez Rodriguez, J.F. Fernandez Garayzabal, J.L. Blanco
Cancelo, E. Gomez-Lucia, V. Briones Dieste, and G. Suarez Fernandez. 1988. Serological
studies on Listeria grayi and Listeria murrayi. J. Appl. Microbiol. 64:371-378.
169. Walker, S.J., P. Archer, and J.G. Banks. 1990. Growth of Listeria monocytogenes at refrigera-
tion temperatures. J. Appl. Bacteriol. 68: 157- 162.
170. Welshimer, H.J. 1963. Vitamin requirements of Listeria monocytogenes. J. Bacteriol. 85:
1156-1 159.
171. Welshimer, H.J., and A.L. Meredith. 1971. Listeria murrayi: a nitrate-reducing mannitol-
fermenting Listeria. Int. J. Syst. Bacteriol. 21 :3-7.
172, Wilkinson, B.J., and D. Jones. 1975. Some serological studies on Listeria and possibly related
bacteria. In: M. Woodbine (ed.), Problems of Listeriosis. Leicester, UK: University of Leices-
ter. pp. 251-261.
173. Wilson, G.S., and A.A. Miles. 1946. Topley and Wilsons principles of bacteriology and
immunity, Vol. 1. 3rd ed., London: Edward Arnold.
Listeria monocytogenes in the
Natural Environment
DAVIDR. FENLON
Scottish Agricultural College, Aberdeen, Scotland
INTRODUCTION
Listeria monocytogenes is commonly found in soil and water and on plant material, partic-
ularly that undergoing decay, with these environments being re,gardedas the natural habitat
of the organism [86]. Decayed vegetation, such as aerobically spoiled silage, supports
development of high numbers of L. monocytogenes, and has been cited as the source
of infection in numerous cases of listeriosis in farm animals, and may be the origin of
contamination capable of spreading along the food chain. The organism can survive longer
under adverse environmental conditions than many other non-spore-forming bacteria of
importance in foodbome disease. This resistance, together with the ability to colonize,
multiply, and persist on processing equipment makes L. monocytogenes a particular threat
to the food industry.
Table 1 shows the persistence of L. monocytogenes in various natural and farm
environments and summarizes results from some of the studies mentioned in this chapter.
This ability to survive for long periods may explain why the natural environment can act
as a reservoir of contamination capable of spreading to animal and plant food products.
It is only relatively recently, with the introduction of improve:d molecular typing methods
such as multilocus enzyme electrophoresis (MEE), random fragment length polymorphism
(RFLP), random amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis
(PFGE), that the full story of listeriosis epidemiology is emerging.
21
22 Fenlon
TABLE
1 Survival of L. monocytogenes in Various Environmental Samples
Storage temperature
Sample ("C)
Soil
sterile soil (I)a Outside-winter/spring 154
clay soil (I) 24-26 225
sealed tubes 24-26 67
fertile soil (I)
sealed tubes 24-26 295
cotton-plugged tubes 24-26 67
top soil (I)
exposed to sunlight NGh 12
not exposed to sunlight NG 182
moist soil NG -497
dry soil NG >730
soil 4-12 240-3 1 I
soil 18-20 201-271
Fecal material
cattle feces (NC)' 5 182-2 190
moist horse/sheep feces (I) Outside 347
dry horse/sheep feces (I) Outside 730
sheep feces Outside 242
liquid manure Summer 36
liquid manure Winter 106
sewage
sewage sludge cake (NC)
surface 28-32 35
interior 48-56 49
sprayed on field Outside >56
Water
sterilized pond water (I) Outside 7
unsterilized pond water (I) Outside <7-63
pond water 35-37 346
pond water 15-20 299
pond water/ice 2-8 790-928
pond/river water 37 325
pond/river water 2-5 750
water Outside 140-300
distilled water (I) 4 <9
Animal feed
silage (NC) 4 450
silage (NC) 5 180-2 190
mixed feed (I) Outside 188-275
oats (I) Outside 150-300
hay (1) Outside 145-189
straw (NC/I) ca.22 365
straw (I) Outside 47-207
straw Outside-summer 23
straw Outside-winter 135
aInoculated.
Not given.
Naturally contaminated.
Source: Adapted from Refs. 3, 4, and 75.
Listeria monocytogenes in the Natural Environment 23
all positive for the organism. Fenlon et al. [36] examined five 25-g samples each of grass,
leaves, stems, and roots/stems from two crops of growing sward before harvesting. No
L. monocytogenes were detected in any samples. Only L. innocua and L. seeligeri were
isolated from 3 of 10 samples from the root/stem area. However, L. monocytogenes was
detected in 9 of 10 samples of cut grass from the same crops after wilting for 24 h before
ensiling. Other vegetable crops such as lettuce and carrots postharvest did not carry the
organism to the same extent. Farber [27] similarly found little Listeria contamination of
unprocessed vegetables. Other workers [46] have reported higher levels, with the degree
of processing and packaging having a significant effect on Listeria levels [10,16].
The higher incidence of L. monocytogenes associated with harvested (processed)
grass compared with other plant products has been attributed to the presence of a sheath
of decaying plant material at the base of the plant which might act as an inoculum at
harvest [36]. Whittenbury [ 1061 demonstrated the importance of the sheath area as a source
of lactic acid bacteria, which have a similar natural habitat to Listeria in ensiling of grass.
This may be a model for contamination of grass with Listeria during the ensiling process.
Other potential sources of L. monocytogenes in soils are from natural deposition of feces
by animals and spreading of animal waste and sewage sludge as fertilizer.
Survival of L. monocytogenes in soil depends on soil type and its moisture content.
Welshimer [ 1031, using cotton wool-plugged tubes containing either clay or fertile soils
inoculated with L. monocytogenes and stored for 67 days at 24-26"C, showed numbers
decreased in both by seven orders of magnitude as the soils dried out. Repeating the
experiment with sealed tubes to prevent water loss, the decrease was similar for the clay
soil but only two orders of magnitude for fertile soil. Watkins and Sleath [98] demonstrated
that in soil on land to which sewage sludge had been applied, there was little decrease in
Listeria spp. numbers (approximately 170 cfu per 100 g soil) over an 8-week period,
whereas salmonellae, applied at a similar rate, decreased to undetectable levels in less
than 4 weeks. This difference in rate of survival may be a reflection on the original habitat
of each species. Fenlon et al. [36] did not find L. monocytogenes in the few soils examined
that were associated with vegetable crops, but soil from fields where cattle or sheep had
been kept on silage diets did contain L. monocytogenes. In a study using autoclaved soils
inoculated with L. monocytogenes (approximately 5 X 102cfu/g) and stored at ambient
winter temperatures ranging from -15" to +18"C, Botzler [14] found an increase to 1
X 107cfu/g over a 154-day period. However, it is not known what effect the autoclaving
process had on Listeria growth by increasing availability of nutrients and eliminating
competing organisms.
From evidence in the literature to date, it does not appear that soil is a natural
reservoir in which L. monocytogenes multiplies. The widespread presence of the organism
in soil probably results from contamination by decaying plant and fecal material, with
damp surface soil providing a cool, moist protective environment and decaying vegetation
the substrate, which together enable L. monocytogenes to survive from season to season.
Fecal Material
L. monocytogenes has been found in the feces of a wide variety of healthy animal species.
Gray and Killinger [49] listed 37 mammals from whose feces the organism had been
isolated. Given that vegetation is the natural habitat of the organism, and that most reports
have involved cultivation of L. monocytogenes solely by enrichment techniques with few
quantitative data, it is not surprising that carriage in grazing animals such as healthy sheep
Listeria monocytogenes in the Natural Environment 25
[52,68,90], goats [65,96], and cattle [36,56,96] is well documented. However, L. rnonocy-
togenes excretion also has been reported in pigs [25,83], chickens [7,47,77], turkeys
[9,55,77], pheasants [89], gulls [30], rooks [30], pigeons [82], fish [6], and crustaceans [6].
As L. rnonocytogenes appears to be a food-related pathogen [73] with naturally occurring
listeriosis being recorded in many other animals, including mice [92], voles [79], rats [90],
rabbits [79,90,99], guinea pigs [84], chinchillas [38], lemmings [82], hyraxes [82], mink
[63], skunks [84], horses [102], dogs, [63,92], cats [79], foxes [79], deer [26,79], buffalo
[25], giraffes [ 181, bats [57], ducks [82], partridges [82], eagles [82], parrots [82], canaries
[82], starlings [63], frogs [13], turtles [13J,ticks [4], and flies [4], it is reasonable to suspect
these animals to also excrete the organism in their feces. Humans, both symptomatic and
asymptomatic carriers, excrete L. monocytogenes . Ralovich [841 summarized data, primar-
ily of European origin, indicating that 1.8-9.0% of healthy individuals excrete L. rnonocy-
togenes in their feces.
The influence of diet on excretion of L. monocytogenes has recently been studied,
particularly in ruminants. Low et al. [68] reported a low incidence of excretion in a flock
of 100 grazing sheep; this increased significantly ( P < .OOOOl) to between 10 and 33%
once silage feeding commenced, confirming the finding of Husu [58] that the tendency
is for excretion rates to be lower in grazing animals. Fenlon et al. [36] monitored two
groups of cattle. While grazing, none of those tested (n = 10 and 13) excreted L. monocyto-
genes. When tested after silage feeding commenced, 4 of 14 [28.6%) in one group and
4 of 13 (30.8%) in the other excreted L. rnonocytogenes.Numbers of Listeria in the excreta
were low, ranging from present in 25 g to 11 cfu/g. In the same study, examination of
feces of sheep on a hay diet showed no detectable Listeria, presumably because the mois-
ture level in hay is too low to support Listeria growth. Formulated diets for intensively
reared pigs and poultry also were free of the organism. Furthermore, of 9 samples of
broiler poultry litter tested, only 1 was positive and all 10 swabs of feces taken at the
processing plant from crates used to transport the birds were negative. Similarly, only 1
of 47 fecal samples from pigs and piglets was positive. Genigeorgis et al. [41], in a study
at a poultry processing plant, found the incidence of Listeria on carcasses increased as
processing progressed. L. monocytogenes was not isolated from composite feather samples
(n = 16) from live hanging birds or their hind gut contents (n = 16). Husu et al. [59]
noted that most 2-day-old chicks dosed orally with L. rnonocytogenes had eliminated the
organism within 9 days, indicating that chickens are unlikely reservoirs of the organism
with any carriage probably being transient. Dijkstra [21J examined the intestines of 2373
broilers from 146 farms, and showed that 4.1 % were contaminated with Listeria. Increased
excretion because of stress, as associated with salmonellosis, has not been well docu-
mented for listeriosis. Ralovich [84] observed increased excretion of L. rnonocytogenes
by sheep housed under stressful conditions. Fenlon [36] reported that in animals traveling
to three abattoirs, the level of L. rnonocytogenes in feces from cattle traveling a long
distance (>loo km) was greater than in those traveling to nearby abattoirs (<25 km)
(P = .003). 'The highest excretion rate found for L. rnonocytogenes was 800 cfu/g feces,
although L. ivanovii was found at a level of 6.4 X 104cfu/g in sheep feces. Numbers of
L. monocytogenes transferred to red meat carcasses tended to be low and rarely were
detected by swabbing, usually requiring enrichment of meat samples taken from the lower
parts of hanging carcasses. In abattoirs with a good hygienic standard, feces are only
responsible for intermittent low-level direct contamination. This may be sufficient to pro-
vide an inoculum for colonization of equipment used for further processing of the meat
and therefore may be a source of indirect contamination of foods, especially if hygiene
26 Fenlon
standards are poor. Studies have shown that more highly processed meat products are
more consistently contaminated with L. rnonocytogenes [36,87].
Sewage
One of the earliest definitive quantitative studies on L. monocytogenes was that of Watkins
and Sleath [98]. They reported levels >18,000 cfu/L in trade effluents associated with
animals and sewage sludges from treatment plants in northeast England. Levels of the
organism in primary tank settled raw sewage ranged from 700 to 18,000 cfu/L. Much
higher levels of Listeria spp, were reported by Geuenich and Muller [42] in a West German
sewage treatment plant. Both untreated waste and filtered effluent had 103- 10scfu Listeria
spp./mL. The fact that there was only a 10-fold reduction in numbers between untreated
and treated waste suggests that biological oxidation may not be an effective method for
eliminating viable Listeria in sewage. Studies in northeastern Scotland [36] showed L.
monocytogenes numbers in untreated sewage to be 120 cfu/mL and in treated effluent 2-
21 cfu/mL. Anaerobic digestion reduced numbers to 1. I cfu/g, and lime-treated sludge
had no detectable Listeria.
In Iraq, Al-Ghazali and Al-Azawi [2,3] studied survival of L. monocytogenes during
sewage treatment and in stored sewage sludge cake. A decrease of 85-97% in viable
Listeria numbers occurred during activation and digestion stages of the sewage treatment
process [2], although all steps were detrimental to the organisms survival. They recovered
<3-15 L. monocytogenes cfu/mL from final effluent and <3-7 cfu/mL from sludge cake.
The latter is frequently dried and used as a fertilizer in developing countries, although
treated sludge is increasingly subjected to land disposal in European countries as restric-
tions on sea disposal come into force. These same authors [3] demonstrated that the liste-
riae were inactivated when sludges were stored in direct sunlight for at least 8 weeks.
Inactivation was slower in the interior of the sludge piles. As reported earlier [98], studies
in the United Kingdom showed that when L. monocytogenes-contaminated sewage sludge
was spread on land, numbers of the pathogen failed to decrease over an 8-week period.
Since fecal contamination has been linked to one foodborne outbreak of human listeriosis
associated with coleslaw [SS], potentially contaminated sludges and animal wastes should
be plowed into the soil and not spread on crops which are eaten raw.
Water
The ubiquitous nature of L. monocytogenes and use of surface waterways for discharge
of sewage effluents will inevitably result in the presence of the organism in a wide range
of lakes, rivers, and streams. Dijkstra [22] found the organism in 21% of the surface
water samples in northern Netherlands, and noted that even though the lakes were used
by swimmers, no human cases were linked to this activity. A study of the course of the
River Don in northeastern Scotland [36] showed 42% of 19 samples (100 mL) were posi-
tive for L. monocytogenes in May and 53% 6 months later; numbers ranged from 10 to
350 cfu/L. Highest numbers were found at a sampling point below a sewage plant, but
this was not consistent for both sampling occasions. No factor, seasonal or otherwise,
could be related to the presence or numbers of L. monocytogenes which occurred over
the entire course of the river. In another study in the United Kingdom [39], 30 samples
(100 mL) from 21 sites showed 8 sites positive for L. seeligeri (27%), 1 for L. innocua,
Listeria monocytogenes in the Natural Environment 27
and 1 for L. welshirneri. Soil may be the source of contamination at these sites, since
McGowan [69] reported that L. seeligeri was more frequently isolated from soils than
either L. innocua or L. rnonocytogenes.
No waterborne cases of human listeriosis have been reported; however, water may
act as a source of contamination for certain foods. Soonthoranant and Garland [91] found
L. rnonocytogenes in 35- 100%of discharges from a sewage treatment pond and fish pro-
cessing factory effluents, which also contained sewage. Inshore marine waters, which
eventually received these discharges, contained L. monocytogenes in 6.6% of samples
with the contamination rate of Pacific oysters and blue mussels being 15.4%, which proba-
bly reflected their filter feeding habits. These findings confirm and extend the California
study of Colburn et al. [17] on fresh and low-salinity waters in which 81 and 62%, respec-
tively, harbored Listeria spp. including L. rnonocytogenes. One of three bay water samples
contained L. rnonocytogenes, and L. innocua was found in one of 35 oysters sampled.
Furthermore, Motes [76] isolated L. rnonocytogenes from shrimp caught off the U.S. Gulf
Coast, and Destro et al. [ 191 showed that some strains associated with shrimp can persist
in processing plants and enter the final product.
Gray et al. [50] successfully infected two sheep, four goats, and one cow by supply-
ing them with L. rnonocytogenes-contaminated water. Althoug,h current evidence on direct
infection of humans and animals with Listeria via water remains sparse, there is a legiti-
mate risk. A greater risk would appear to be contamination of foods such as marine and
freshwater fish with polluted water, especially those going for further processing [76], as
it is known that certain strains of L. rnonocytogenes can colonize equipment and contami-
nate the final product [72].
Animal Feeds
Most formulated animal feeds have low levels of available water which restricts multipli-
cation of L. monocytogenes. The same is true of hay and cereal grains, so although L.
rnonocytogenes has been reported in such materials 1401, the numbers are unlikely to reach
levels which present a serious risk to animals. Many formulated feeds are sold in a pelleted
form and will have received a degree of heat treatment capable of killing a high proportion,
if not all, Listeria present. The animal feed most closely linked with animal listeriosis
is silage, and this association is well documented. Olafson I811 noted the link between
Listerella encephalitis in ruminants and silage in 1940, and in 1960, Gray [48] reported
isolating L. monocytogenes from the fetus of a pregnant mouse fed poor-quality L. rnonocy-
togenes-contaminated silage implicated in death and abortion in cattle. Identical serotypes
of L. rnonocytogenes were isolated postmortem from the mice and cattle. Today, there
are numerous reports linking the feeding of silage with listeriosis outbreaks in sheep and
cows [32,37,44,45,52,68,78,100]. Much of this problem can be attributed to the high num-
bers of L. monocytogenes present in contaminated silage [31,321 as compared with other
animal feeds. In good-quality silage prepared from grass, maize, whole crop cereals, or
leguminous plants, which may or may not be wilted (dried to optimum moisture content)
in the field before e n d i n g , the onset of anaerobic Conditions stimulates the indigenous
or inoculated lactic acid bacteria to multiply rapidly; generally reaching a maximum of
around 10 cfu/g within 48 h. These bacteria convert the plant sugars to lactic acid, causing
a rapid decrease in pH [7 11 with well-preserved silages generally having a pH 5 4 . 5 . These
acidic conditions inhibit growth of both spoilage microorganisms and Listeria as long as
anaerobic conditions are maintained. Higher dry matter silages tend to have a higher pH,
28 Fenlon
but the lower level of available water compensates for any lessening in the preservative
effect caused by reduced acidity. Grass silages in cooler, wetter climates tend to have
lower sugar levels and higher moisture contents resulting in a poorer, slower fermentation,
and so are more susceptible to L. rnonocytogenes contamination than grass and maize
silages grown in countries with warmer climates.
L. monocytogenes Contamination is most frequently associated with poor-quality
silage. In 1979, Gr@ntsol[52] analyzed 291 grass silages from 113 farms and isolated L.
rnonocytogenes from 22% of samples with a pH <4.0; 37% with a pH of 4.0-5.0, and
56% with a pH of >5.0. In a study of clamp silage implicated in an outbreak of listeriosis
in cattle [32], levels of L. rnonocytogenes in excess of 12,000 cfu/g were found in the
surface layer (pH 8.3-8.5), whereas no Listeria spp. were detected 15 cm into the silage
mass and the pH was 4.5.
Gitter [44] noted that from 1975 to 1985, the incidence of listeriosis in sheep in
Great Britain increased from less than 50 incidents per annum to over 250. During the
same period, the rise in cattle listeriosis was much lower. He also reported that the pattern
changed from isolated single incidents to much larger flock outbreaks. This was attributed
to a change in the conserved forage used to feed sheep. In Great Britain, sheep are mainly
kept on upland areas and before this time were traditionally fed hay. The wet climate of
these hill farms is not conducive to the making of good-quality hay, and silage was not
an economic option before the mid 197Os, as it required expensive capital outlay for silos.
The invention of the big baler and the half-ton round bale made silage feeding feasible
by baling grass and sealing it in large plastic bags to make silage. Unfortunately, some
of the early attempts to make silage in this way were not very successful, and baled silage
was of poorer quality than clamp silage [30]. As L. monocytogenes is a surface problem
(Figure 1) and big bales have a much greater surface area than clamp or silo silage, the
potential for contamination to develop is much greater in bale silage if it is not made
correctly and aerobic deterioration takes place.
Fenlon [31] reproduced the problem in 500-g laboratory bales ensiled in plastic
bags. After 2-3 weeks, L. rnonocytogenes could be detected at levels of 2 1.1 X 106cfu/g
silage in moldy areas near the tie end of the bag where air infiltrated. In the center of
bags, the pH remained at 3.8, silage appeared to be of good visual quality, and it was
free of Listeria. The association between L. monocytogenes growth in silage and pH is
shown in Figure 2. The relationship between oxygen tension, pH, and L. rnonocytogenes
contamination was demonstrated by Donald et al. [24], who infused laboratory silos with
gas mixtures containing from 0.1 to 5.0% oxygen and demonstrated that the greater the
oxygen level, the more rapidly the pH rose with subsequent multiplication of Listeria.
Listeria die in well-fermented silage; however, if the pH increases before all cells
are killed, then surviving Listeria will multiply. Dijkstra [20] showed that L. rnonocyto-
genes can survive 4-6 years in naturally contaminated silage. Fenlon et al. [36] noted
that the organism could survive over 1 year in bags which had been used to wrap big
bales and stored for reuse. Fortunately, much of the L. monocytogenes contamination in
silage occurs in visibly moldy areas, and if these are removed and discarded before feeding,
the challenge to the animal is considerably reduced [33].
Inflammation of the iris (iritis) caused by L. rnonocytogenes has been increasingly
reported [97], particularly in cattle. This condition occurs when cattle and sheep burrow
their heads into bale silage in self-feeding systems. The eye becomes infected via abrasions
caused by stems of grass contaminated with the organism. Modifying feeding practices
to prevent eye contact with silage is the best preventative measure [67]. More obscure
Listeria monocytogenes in the Natural Environment 29
air
f air
tie
Bagged bale Wrapped bale
1 1 1 - 1 1 1 1 1 1 1 1 1 1 D D 1 1 1 1 1
plastic sheet
Clamp Silage 1
air
good quality silage pH4.0
6 7.5
7
5
6.5
4
6
3 5.5 PH
5
2
4.5
1
4
0.1 3.5
0 5 10 15 20 25 30 35
Days
Center bale: 43 Lmon + pH;
Tie end: Q L.mon + pH
causes of listeriosis have been reported, including silages made from orange peel and
artichokes [95], several outbreaks in Canada and the northern United States have been
caused by cattle feeding on ponderosa pine needles [ I].
TRANSMISSION
Being so widely distributed in the environment, animals and humans frequently come in
contact with L. monocytogenes through a variety of sources. What is also apparent is the
relatively low incidence of clinical listeriosis in both animals and humans. Traditionally,
the disease in both animals and humans has occurred as individual sporadic cases, and
although this is probably still the predominant form of the disease in humans [73], the
organism has since emerged as a serious foodborne pathogen, with well-documented out-
breaks being associated with processed foods, such as coleslaw [%], soft cheese [11,61],
pat6 [43], pork tongue [60], and pork rilletts [61]. These larger outbreaks have almost all
been attributable to serovar 4 strains principally serovar 4b, which, when typed by several
methods, have been shown to be closely related [15,60]. The origin of these outbreak
strains is unclear. Boerlin and Piffaretti [ 121 used MEE and showed that the electrophoretic
type (ET) which caused the soft cheese outbreak in Switzerland was also widely distributed
in bovine milk, bovine feces, minced meat, silage, and soil as well as in human and animal
clinical cases, thus suggesting an environmental origin. However, relying on one typing
method can be misleading. MEE reportedly has good discrimination for serotype 1/2 iso-
lates, which can be subdivided into a large number of ETs. However, it is less effective
for serotype 4 strains, most of which fall into relatively few ETs. Donachie et al. [23]
subjected serotype 4 isolates of the same ET from a variety of sources to analysis using
PFGE and found that all but one of the human isolates could be grouped into exclusive
human PFGE types. Multiple isolates from other sources, such as silage, also fell into a
single type. This study confirmed the need to use a combination of typing methods to
obtain maximum discrimination of strains. Further evidence for an environmental origin
for outbreak-related strains was obtained by Wesley and Ashton [ 1051, who in a retrospec-
tive subtyping study subjected clinical, environmental, and factory isolates from the Mexi-
can-style soft cheese outbreak to restriction enzyme analysis. This study showed that the
causative strain was recovered from samples of curd, the pasteurizer, cooler water, a floor
drain, and insects caught in the factory, with the widespread presence being related to
poor hygiene in the plant. McLauchlin and Nichols [74] demonstrated a direct relationship
between poor hygiene, as measured by total viable count, and the presence of Listeria
spp. in 4405 samples of seafood. A similar relationship between food processing hygiene
and Listeria was shown when pit6 samples were tested in the recent UK outbreak [43].
Sporadic and small outbreaks (<10 cases) tend to be caused by a much more diverse
range of strains than the larger outbreaks 1721, and although serovar 4b predominates over
others, serovar 1/2b is often found. In one study [72], isolates of L. monocytogenes from
cases of human listeriosis and foods were collected in the United Kingdom over a 30-year
period, and were sent to the Public Health Laboratory Service Food Hygiene Laboratory in
London for serotyping. Isolates from human listeriosis cases were of serovars 4b (60%),
1/2a (17%), 1/2b (1 l%), and 1/2c (4%) and from foods 4b (22%), 1/2a (32%), 1/2b
(15%) and 1/2c (21%), although this predominance of serovar 4b in human cases may
be the result of more virulent qualities of this serovar, the greater preponderance of serovar
112 in food isolates may be explained by the ability of these strains to adapt better to
ecological niches in the food processing environment. In a recent 12-month survey of raw
Listeria monocytogenes in the Natural Environment 31
milk from 160 farm bulk tanks, L. monocytogenes contamination was low, 4.3-9.3%, all
were serovar 1, and the maximum level was 35 cfu/mL. Most contamination was sporadic
with a diverse array of ETs present [35]. Harvey and Gilmour [54], using MEE and RFLP
analysis, compared L. monocytogenes isolates from four milk processing centers and two
dairy farms in Northern Ireland with food and clinical isolates and found the dairy-related
isolates to be quite distinct. Recurrent strains, specific to each dairy processor, colonized
plants over long periods. When isolates from a poultry processing environment were exam-
ined with RAPD analysis, Lawrence and Gilmour [64] showeld that a single RAPD type
was predominant in the raw processing environment over the 6-month period of the study,
surviving the clean-in-place schedules. In a follow-up study 12 months later, the same
RAPD type was again isolated from final cooked products.
Norrung and Skovgard [SO] found the genetic diversity of ETs isolated from cooked
meat products to be lower (0.439) than those from raw meat (0.903). They suggested that
certain clones may be better adapted to processing. An alternative explanation might be
that the diverse range of strains found on raw meat has been eliminated during heating,
and that strains on cooked products represent a more restricted range of strains present
in the postcooking environment. The extremely diverse range of strains on raw meat and
poultry products was shown by Ryser et al. [87], who tested by ribotyping up to 10 isolates
per sample from enriched samples of ground beef, pork sausage, ground turkey, and
chicken. They demonstrated that the strain selected was influenced by the enrichment
medium, but, more importantly, over half of all positive saniples had more than one L.
monocytogeizes ribotype (RT), some with as many as three. In some instances, detection
of certain clinically important ribotypes of L. monncytogenes, which were apparently over-
grown by other RTs, was only possible when 10 isolates from a sample were typed.
In animal listeriosis, where the change from hay to silage feeding for sheep has
resulted in outbreaks involving whole flocks, a similar pattern of diversity is emerging. A
temporal difficulty also exists in linking contaminated silage with cases of clinical disease,
particularly the encephalitic form, because of the long incubation period 1681. Identical
strains have been recovered from animals with clinical disease and silage fed to them
[68,105]. However, silage may contain a diverse array of L. monocytogenes strains, re-
sulting in flocks being exposed to and infected by [8] multiple strains. Nonetheless, within
a single sheep, one strain dominates during infection. Low et al. [68] noted that 6 distinct
phage types were present among 45 isolates of L. monocytogenes from baled silage. The
67 positive fecal isolates from 100 sheep being fed the silage were even more diverse
with 10 phage types other than those present in the silage being identified. The authors
commented that several strains present in feces were absent from silage. They noted that
since sheep consumed 100- 1000 times more silage than was analyzed, Listeria strains
present in excreta were more likely to be representative of the total silage population.
Two of the isolates recovered from the three clinical cases during the trial period were
serovar 4b and one was 1/2a. Identical phage types were isolated from silage and fecal
samples taken before the onset of disease, but were not necessarily the dominant strain
present, thus confirming the necessity to examine and type rriultiple isolates from individ-
ual samples. In another study 1341, the extent of Listeria contamination in silage was
directly related to its hygienic quality, as measured by the numbers of Enterobacteriaeceae
present, illustrating that management of postharvest processing of the grass can markedly
affect numbers of Listeria present.
The natural environment appears to be the initial reservoir for virulent strains of L.
monocytogenes which can enter and pass along the food chain, but this contamination is
32 Fenlon
usually of a low level and sporadic. It is significant that poultry products are more contami-
nated than beef, yet the environment in which beef cattle are reared presents a greater
risk of contact with the organism than that of the intensively reared broiler chicken. How-
ever, in processing, the latter is exposed to greater risk of contamination from other car-
casses and mechanical equipment than is in the beef carcass [85].It is at the processing
stage of food and feedstuffs that amplification of numbers and persistent contamination
occur, which in turn present a potentially more serious challenge to human and animal
health (Fig. 3).
Although discriminating power of typing methods is improving, a standardized sys-
tem is not yet in place, and it appears that a significant number of isolates must be typed
per sample, particularly with raw meats and silages, to ensure detection of the most impor-
tant strains. What is clear is that the natural environment harbors a highly diverse range
of L. monocytogenes strains, including some with the potential to cause clinical listeriosis
in humans and animals. This contamination is usually minimal and sporadic. However,
in the absence of good manufacturing and hygiene practices in both human and animal
food production, the food processing environment can become a ready source of virulent
strains with the ability to colonize equipment and contaminate food products.
0
Soil 1
f
Plants
Water
Manure Ruminants
Other
meat producing
animals and
Equipment and
Sewage ~ Environmental
Sources
FIGURE3 Spread of Listeria monocytogenes to the food chain from the natural envi-
ronment.
Listeria monocytogenes in the Natural Environment 33
ACKNOWLEDGMENTS
The author gratefully acknowledges the contribution of Professors E. T. Ryser and E. H.
Marth on which this revised chapter is based, and also the assistance and critical comments
of colleagues within and outside the Scottish Agricultural College (SAC). SAC receives
financial support from The Scottish Office Agriculture Environment and Fisheries Depart-
ment.
1. Adams, C.J., T.E. Neff, and L.L. Jackson. 1979. Induction of Listeria rnonocytogenes infec-
tion by the consumption of ponderosa pine needles. Infect. Immun. 25:117-120.
2. Al-Ghazali, M.R., and S.K. Al-Azawi. 1988. Effects of sewage treatment on the removal of
Listeria rnonocytogenes, J. Appl. Bacteriol. 55:203-208.
3. Al-Ghazali, M.R., and S.K. Al-Azawi. 1988. Storage effects of sewage sludge cake on the
survival of Listeria rnonocytogenes. J. Appl. Bacteriol. 65:209-213.
4. Amtsberg, G. von. 1979. Epidemiologie und Diagnostik der Listeriose. Dtsch. tierarztl. Wo-
chenschr. 86:253-257.
5. Anonymous. 1995. Microbiology of food and animal feeding stuffs. Horizontal method for
the detection and enumeration of Listeria rnonocytogenes. Draft International Standard JSO/
DIS 11290. Geneva: International Organization for Standardization.
6. Arrnstrong, D. 1985. Listeria rnonocytogenes. In: G.L. Mandell, R.G. Douglas Jamie Robert-
son, and J.E. Bennett, eds. Principles and Practices of Infectious Diseases. 2nd ed. New York:
Wiley, pp. I 177-1 182.
7. Basher. H.A., D.R. Fowler, F.G. Rodgers, A. Seaman, and M. Woodbine. 1984. Pathogenicity
of natural and experimental listeriosis in newly hatched chicks. Res. Vet. Sci. 36:76-80.
8. Baxter, F., F. Wright, R.M. Chalmers, J.C. Low, and W. Donachie. 1993. Characterization
by mu1tilocus enzyme electrophoresis of Listeria monocytogmes isolates involved in ovine
listeriosis outbreaks in Scotland from 1989 to 1991. Appl. Environ. Microbiol. 59:3 126-
3129.
9. Belding, R.C., and M.L. Mayer. 1959. Listeriosis in the turkey-two case reports. J. Am.
Vet. Med. Assoc. 131:296-297.
10. Beuchat, L.R., and R.E. Brackett. 1990. Survival and growth of Listeria rnonocytogenes on
lettuce as influenced by shredding, chlorine treatment, modified atmosphere packaging, and
temperature. J. Food Prot. 55:755-758, 890.
11. Bille, J. 1990. Epidemiology of human listeriosis in Europe, with special reference to the
Swiss outbreak. In: A.J. Miller, J.L. Smith, and G.A. Somkuti eds. Foodborne Listeriosis
New York: Elsevier.
12. Boerlin, P., and J.C. Piffaretti. 1991. Typing of human, animal, food and environmental
isolates of Listeria rnonocytogenes by multilocus enzyme electrophoresis. Appl. Environ.
Microbiol. 57: 1624-1629.
13. Botzler, R.G. 1973. Listeria in aquatic animals. J. Wildl. Dis. 9:163-170.
14. Botzler, R.G. 1974. Survival of Listeria monocytogenes in soil and water. J. Wildl. Dis. 10:
204-2 12.
15. Buchreiser, C.R., R. Brosch, B. Catimel, and J. Rocourt. 1993. A new view of human listeri-
osis epidemiology: pulsed-field gel electrophoresis applied far comparing Listeria rnonocyto-
genes strains involved in outbreaks. Can. J. Microbiol. 36395-401.
16. Carlin, F., C. Nguyen-the, and A. Abreu de Silva. 1995. Factors affecting the growth of
Listeria rnonocytogenes on minimally processed endive. J. Appl. Bacteriol. 78:636-646.
17. Colburn, K.G., C.A. Kaysner, C. Abeyta, and M.M. Wekell. 1990. Listeria species in a Cali-
fornia coast estuarine environment. Appl. Environ. Microbiol. 56:2007-2011,
34 Fenlon
18. Cranfield, M., M.A. Eckhaus, B.A. Valentine, and J.D. Strandberg. 1985. Listeriosis in Ango-
lan giraffes. J. Am. Vet. Med. Assoc. 187:1238-1240.
19. Destro, M.T., M.F.F. Leitio, and J.M. Faber. 1996. Use of molecular typing methods to trace
the dissemination of Listeria rnonocytogenes in a shrimp processing plant. Appl. Environ.
Microbiol. 62:705-7 1 1.
20. Dijkstra, R.G. 1971. Investigations on the survival times of Listeria bacteria in suspensions
of brain tissue, silage and faeces and in milk. Zbl. Bakteriol. I. Abt. Orig. 216:92-95.
21. Dijkstra, R.G. 1979. Listeria monocytogenes in intestinal contents and faeces from healthy
broilers of different ages, in the litter and its potential danger for other animals including
cattle. 1. Ivanov, ed. Proceeding of VII International Symposium of Problems in Listeriosis.
National Agroindustrial Union, Center for Scientific Information, Sofia, pp. 289-294.
22. Dijkstra, R.G. 1982. The occurrence of Listeria rnonocytogenes in surface water of canals
and lakes, in ditches of one big polder and in the effluents of canals of a sewage treatment
plant. Zbl. Bakteriol. Hyg. 1. Abt. Orig. B 176:202-205.
23. Donachie, W., J.C. Low, F. Baxter, and F. Thompson-Carter. 1995. Typing of Listeria mono-
cytogenes isolates with Multilocus Enzyme Electrophoresis (MEE) and Pulsed Field Gel
Electrophoresis (PFGE). Proceedings of XI1 International Symposium on Problems of Liste-
riosis, Perth, W. Australia. Publ. Promaco Conventions Pty. Ltd. pp. 4 17-420.
24. Donald, A.S., D.R. Fenlon, and B. Seddon. 1995. The relationship between ecophysiology,
indigenous microflora and growth of Listeria rnonocytogems in grass silage. J. Appl. Bacter-
iol. 79:141-148.
25. Dutta, P.K., and B.S. Malik. 1981. Isolation and characterization of Listeria monocytogenes
from animals and human beings. Indian J. Animal Sci. 5 1 : 1045- 1052.
26. Eriksen, L., H.E. Larson, T. Christiansen, M.M. Jensen, and E. Eriksen. 1989. An outbreak
of meningio-encephalitis in fallow deer caused by Listeria monocytogenes. Vet. Rec. 19:
274-276.
27. Farber, J.M., G.W. Sanders, and M.A. Johnston. 1989. A study of various foods for the
presence of Listeria species. J. Food Prot. 52:456-458.
28. Farber, J.M. 1992. Current research on Listeria monocytogenes in foods: an overview. Elev-
enth International Symposium on Problems of Listeriosis, Copenhagen, pp. 112-1 14.
29. Farber, J.M. 1993. Current research on Listeria monocytogenes in foods: an overview. J.
Food Prot. 56:640-643.
30. Fenlon, D.R. 1985. Wild birds and silage as reservoirs of Listeria in the agricultural environ-
ment. J. Appl. Bacteriol. 59:537-543.
31. Fenlon, D.R. 1986. Growth of naturally occurring Listeria spp. in silage: a comparative study
of laboratory and farm ensiled grass. Grass Forage Sci. 41:375-378.
32. Fenlon, D.R. 1986. Rapid quantitative assessment of the distribution of Listeria in silage
implicated in a suspected outbreak of listeriosis in calves. Vet. Rec. 1 18:240-242.
33. Fenlon, D.R. 1988. Listeriosis. In: B.A. Stark and J.M. Wilkinson, eds. Silage and Health.
Marlow, England: Chalcombe Publications, pp. 7- 18.
34. Fenlon, D.R., and J. Wilson. 1996. Enterobacteria as indicators of poor fermentation and
listeria contamination of silage, Proceedings of XI International Silage Conference, IGER,
Aberystwyth, UK, pp. 102- 103.
35. Fenlon, D.R., J. Wilson, and W. Donachie. 1994. The incidence, numbers and types of
Listeria monocytogenes isolated from farm bulk tank milks. Lett. Appl. Microbiol. 2057-
60.
36. Fenlon. D.R., J. Wilson, and W. Donachie. 1996. The incidence and level of Listeria rnonocy-
togenes contamination of food sources at primary production and initial processing. J. Appl.
Bacteriol. 8 1 :64 1-650.
37. Fensterbank, R., A. Audurier, J. Godu, P. Guerrault, and N. Malo. 1984. Study of Listeria
strains isolated from sick animals and from the silage consumed. Ann. Rech. Vet. 15:113-
118.
Listeria monocytogenes in the Natural Environment 35
38. Finley, G.G., and J.R. Long. 1977. An epizootic of listeriosis in chinchillas. Can. Vet. J. 18:
164- 167.
39. Frances, N., H. Hornby, and P.R. Hunter. 1991. The isolation of Listeria species from fresh-
water sites in Cheshire and North Wales. Epidemiol. Infect. 107:235-238.
40. Garcia, E., M. De Paz, J.L. Rodrigez, P. Gaya, M. Medina, and M. Nuiiez. 1996. Exogenous
sources of Listeria contamination of ewes milk. J. Food Pro1. 59:950-954.
41. Genigeorgis, C.A., D. Dutelescu, and J.F. Garayzabal. 1989. Prevalence of Listeria spp. in
poultry meat at the supermarket and slaughterhouse level. J. Food Prot. 52:618-624, 630.
42. Geuenich, H.-H., and H.E. Miiller. 1984. Isolation and quantilative determination of Listeria
monoc,vtogenes in raw and biologically treated waste water. Zbl. Bakteriol. Hyg. 1. Abt. Orig.
1791266-273.
43. Gilbert, J., J. McLauchlin, and S.K. Velani. 1993. The contamination of pat6 by Listeria
monocytogenes in England and Wales in 1989 and 1990. Epidemiol. Infect. I 10543-55 1.
44. Gitter, M. 1986. A changing pattern of ovine listeriosis in Great Britain. Proceedings of IX
International Symposium on Problems of Listeriosis. A.L. Courtieu, E.P. Espage, and A.E.
Reynaud, eds. University of Nantes, Nantes, France, pp. 294-299.
45. Gitter, M.R., StJ. Stebbings, J.A. Morris, D. Hannam, and C. Harris. 1986. Relationship
between ovine listeriosis and silage feeding. Vet. Rec. 1 18:207-208.
46. Gras, M.H., C. Druet-Mechaud, and 0. Cerf. 1994. La flore bacterienne des feveilles de
salade fraiche. Sci. Alim. 14:173-188.
47. Gray, M.L. 1958. Listeriosis in fowls-a review. Avian. Dis. 2:296-314.
48. Gray, M.L. 1960. Silage feeding and listeriosis. J. Am. Vet. Med. Assoc. 136:205-208.
49. Gray, M.L., and A.H. Killinger. 1966. Listeria monocytogenes and listeric infections. Bacte-
riol. Rev. 30:308-382.
50. Gray, M.L., C. Singh, and F. Thorp. 1956. Abortion and pre- or post-natal death of young
due to Listeria monocytogenes. 111. Studies in ruminants. Am. J. Vet. Res. 17:510-516.
51. Gray, M.L., H.J. Stafseth, F. Thorp, L.B. Scholl, and W.F. Riley. 1948. A new technique
for isolating listerellae from the bovine brain. J. Bacteriol. 55:443-444.
52. Grmstol, H. 1979. Listeriosis in sheep-Listeria monocytogenes from grass silage. Acta
Vet. Scand. 20:492-497.
53. Gunasinghe, C.P.G.L., C. Henderson, and M.A. Rutter. 1094. Comparative study of two
plating media (PALCAM and Oxford) for detection of Lisferia species in a range of meat
products following a variety of enrichment procedures. Lett. Appl. Microbiol. 18: 156- 158.
54. Harvey, J., and A. Gilmour. 1994. Application of multilocus enzyme electrophoresis and
restriction fragment length polymorphism analysis to the typing of Listeriu monocytogenes
strains isolated from raw milk, non-dairy foods and clinical and veterinary sources. Appl.
Environ. Microbiol. 60: 1547- 1553.
55. Hatkin, J.M., W.E. Phillips, and J. Robertson. 1986. Isolation of Listeria monocytogenes
from an eastern wild turkey. J. Wildl. Dis. 22:llO-112.
56. Hofer, E. 1983. Bacteriologic and epidemiologic studies on the occurrence of Listeria mono-
cytogerzes in healthy cattle. Zbl. Bakteriol. Hyg. A 256: 175- 183.
57. Hohne, K., B. Loose, and H.P.R. Seeliger. 1975. Isolation of Listeria monocytogenes in
slaughter animals and bats of Togo (West Africa). Ann. Inst. Pasteur Microbiol. 126A:501-
507.
58. Husu, J.R. 1990. Epidemiological studies on the occurrence of Listeria monocytogenes in
the faeces of dairy cattle. J. Vet. Med. B. 37:276-282.
59. Husu, J.R., J.T. Beery, E. Nurmi, and M.P. Doyle. 1990. Fate of Listeria monocytogenes in
orally dosed chicks. Intern. J. Food Microbiol. 11:259-269.
60. Jacquet, C., B. Catimel, R Brosch, C. Buchreiser, P. Dehaumont, V. Goulet, A. Lepoutre,
P. Veit, and J. Rocourt. 1995. Investigations related to the epidemic strain involved in the
French listeriosis outbreak in 1992. Appl. Environ. Microbiol. 6 1 :2242-2246.
61. Jacquet, C., B. Catimel, V. Goulet, A. Lepoutre, P. Veit, P. Dehaumont, and J. Rocourt.
36 Fenlon
82. Plagemann, O., and A. Weber. 1988. Listeria monocytogenes als Abortursache bei
Klippschliefern (Procavia capensis). Kleintierpraxis 33:3 17-3 18.
83. Rahman, T., D.K. Sarma, B.K. Goswami, T.N. Upadhyaya, and B. Choudhury. 1985. Occur-
rence of listerial meningoencephalitis in pigs. Ind. Vet. J. 62:7-9.
84. Ralovich, B. 1984. Listeriosis Research-Present Situation anti Perspective. Budapest: Aka-
demiai Kiado.
85. Richmond, M. 1990. The Microbiological Safety of Food, Parts I & 11. Report of the Commit-
tee on the Micribiological Safety of Food, London, HMSO.
86. Rocourt, J., and H.P.R. Seeliger. 1985. Distribution des especes du genre Listeria. Zentral.
Bakteriol. Mikrobiol. Hyg. A. 259:3 17-330.
87. Ryser, E.T., S.M. Arimi, M.M. Bunduki, and C.W. Donnelly. 1996. Recovery of different
Listeria ribotypes from naturally contaminated raw refrigerated meat and poultry with two
primary enrichment media. Appl. Environ. Microbiol. 62: 1781- 1787.
88. Schlech, W.F., P.M. Lavigne, R.A. Bortolussi, A.C. Allen, C.V. Haldane, A.J. Wort, A.W.
Hightower, S.E. Johnson, S.H. King, E.S. Nicholls, and C.V. Broome. 1993. Epidemic
listeriosis-evidence for transmission by food. N. Engl. J. Med. 308:203-206.
89. Schwartz, J.C. 1969. Attempted isolations of Listeria monocytogenes from diagnostic acces-
sions. Am. J. Vet. Res. 30:483-484.
90. Seeliger, H.P.R. 1961. Listeriosis. New York: Hafner Publishing.
91. Soontharanont, S., and C.D. Garland. 1995. The occurrence of Listeria in temperate aquatic
habitats. Proceedings of XI1 International Symposium on Problems of Listeriosis. Perth,
Western Australia, Publ. Promaco Conventions, pp. 145- 146.
92. Sturgess, C.P. 1989. Listerial abortion in the bitch. Vet. Rec. 124:177-87.
93. Van Netten, P., I. Perales, A. Van de Moosdijk, G.D.W. Curtis, and D.A.A. Mossel. 1989.
Liquid and solid selective differential media for the detection and enumeration of Listeria
monoc-ytogenesand other Listeria spp. Int. J. Food Microbiol. 6: 187- 198.
94. Van Renterghem, B., F. Huysman, R. Rygole, and W. Verstraete. 1991. Detection and preva-
lence of Listeria rnonocytogenes in the agricultural ecosystem. J. Appl. Bacteriol. 7 1:211-
217.
95. Vizcaino, L.L., M.-J. Cubero, and A. Contreras. 1988. Listeric abortions in ewes and cows
associated to orange peel and artichoke silage feeding. Proceedings of X International Sym-
posium on Listeriosis, Pecs, Hungary, Abst. P29.
96. Von Winkenwerder, W. 1967. Das Vorkommen von Listeria monocytogenes bei Rindern in
Neidersachsen. Berl. Munch. tieriirztl. Wschr. 23:445-449.
97. Walker, J.K., and J.H. Morgan. 1993. Ovine ophthalmitis associated with Listeria monocyto-
genes. Vet. Rec. 132:636.
98. Watkins, J., and K.P. Sleath. 1981. Isolation and enumeration of Listeria monocytogenes
from sewage, sewage sludge and river water. J. Appl. Bacteriol. 50: 1-9.
99. Watson, G.L., and M.G. Evans. 1985. Listeriosis in a rabbit. Vet. Pathol. 22:191-193.
100. Weidmann, M., J. Czajka, N. Bsat, M. Bodia, M.C. Smith, T.J. Divers, and C.A. Batt.
1994. Diagnosis and epidemiological association of Lis#teria monocytogenes strains in
two outbreaks of listerial encephalitis in small ruminants. J. Clin. Microbiol. 32:99 1-996.
101. Weis, J., and H.P.R. Seeliger. 1975. Incidence of Listeria monocytogenes in nature. Appl.
Microbiol. 30:29-32.
102. Welsh, R.D. 1983. Equine abortion caused by Listeria monocytogenes serotype 4. J. Am.
Vet. Med. Assoc. 182:291.
103. Welshimer, H.J. 1960. Survival of Listeria monocytogenes in soil. J. Bacteriol. 80:3 16-320.
104. Welshimer, H.J., and J. Donker-Voet. 1971. Listeria rnonocytogenes in nature. Appl. Micro-
biol. 21516-519.
105. Wesley, I.V., and F. Ashton. 1991. Restriction enzyme analysis of Listeria monocytogenes
strains associated with food-borne epidemics. Appl. Environ. Microbiol. 57:969-975.
106. Wittenbury, R. 1968. Microbiology of grass silage. Process. Biochem. 3:27-3 1.
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Listeriosis in Animals
IRENE
V. WESLEY
National Animal Disease Center, U.S. Department of Agriculture,
Ames, Iowa
INTRODUCTION
Numerous animal species are susceptible to listerial infection, with a large proportion of
healthy asymptomatic animals shedding Listeria monocytogenes in their feces. Although
most infections are subclinical, listeriosis in animals can occur either sporadically or as
epidemics and often leads to fatal forms of encephalitis. Virtually all domestic animals
are susceptible to listeriosis [ 1781, with sheep [7,109,205,207,239,285,302], cattle [ 109,
205,209,2 17,226,255,2991, goats [ 169,170,2431, and less frequently chickens [ 106,200,
205,2231 succumbing to infection. Several comprehensive reviews have detailed the distri-
bution and pathology of L. mnnocytrogenes in food animals [7,108,127,160,176,2191.
INCIDENCE
Listeriosis in domestic livestock is being recognized with increasing frequency around
the world [107,279]. Since listeriosis is not a reportable disease in animals, the exact
incidence of listerial infections in domestic livestock remains unknown. According to
Ralovich [221], the annual number of cases in animals has increased substantially since
1966 with about 2200, 1000, and 900 cases being reported in Bulgaria, eastern Ger-
many, and Hungary in 1976, 1972, and 1980, respectively. Reports of listeriosis in do-
mestic animals also have increased in New Zealand, Germany, Greece, and England.
This may reflect a natural emergence, increased awareness, and/or improved detection
methods.
39
40 Wesley
disease of veterinarians and farmers who have attended deliveries of stillborn or aborting
bovine fetuses [4,44,191,207,211,2751. A case of septicemia and ultimately fatal meningi-
tis was reported in a Dutch farmer who developed cutaneous lesions after assisting in the
delivery of a stillborn calf [199].
Apart from human infections acquired from contact with infected animals or from
food directly contaminated by an infected animal, the connections, if any, between human
and animal listeriosis are unclear. The springtime peaks of animal listeriosis and the au-
tumn seasonality of human cases suggest that cases are not causally related. The source
of contamination for human food and animal feed is usually environmental [ 176,1901.
The rise in cases of human listeriosis is probably the result, in part, of changes in food
manufacturing and postprocessing contamination [ 1761. The World Health Organization
(WHO) [294] concluded that foodborne listeriosis is predominantly transmitted by non-
zoonotic means, and that L. rnonocytogenes is an environmental organism whose primary
route of transmission to humans is via foods contaminated during production. Several
strain-specific typing methods such as multilocus enzyme electrophoresis and pulsed field
gel electrophoresis have shown that L. rnonocytogenes strains isolated from meat or raw
milk mainly originated from the processing environment rather than from animals
[32,119]. Although an animal origin of contamination was inferred for the three major
epidemics of listeriosis occurring in North America, in the absence of documented evi-
dence, the role of direct animal involvement in human foodborne outbreaks of the disease
remains speculative [290].
Sheep
Ovine listeriosis is commonly caused by L. rnonocytogenes serotypes 1/2,3, and 4 as well
as by L. ivanovii [ 1741. Although listeric-like infections were previously observed in
sheep [239], Gill is credited with the first isolation of L. rnonocytogenes from domestic
farm animals [loo]. In 1929, he observed an illness in sheep in New Zealand which he
called circling disease. This name is still used today to describe listerial encephalitis,
encephalomyelitis, and meningioencephalitis [239].
Clinical manifestations of ovine listeriosis are (a) encephalitis, (b) placentitis with
abortions occurring in the last trimester [ 109,I8 1,2801, and (c) gastrointestinal septicemia
with hepatitis, splenitis, and pneumonitis [ 1541. Encephalitis is the most common form
diagnosed in sheep [154]. Lambs as young as 5 weeks of age may develop septicemia
with older feedlot lambs (4-8 months) manifesting encephalitis. All sheep are probably
exposed to the same contaminated feed, indicating a high natural resistance with 5-10%
of exposed animals exhibiting clinical signs [ 1541.
L. rnonocytogenes usually enters the animal via ingestion. Following entry into the
intestinal epithelium via either M cells in Peyers patches or epithelia1 cells [229], a bacter-
emic or septicemic phase or latent infection may develop, depending on the immune status
of the host. L. monocytogenes subsequently colonizes the viscera, gravid uterus, or medulla
oblongata [ 1541. In pregnant animals, the organism can localize in the placentomes and
enter the amniotic fluid. The fetus aspirates the pathogen, which multiplies and kills the
fetus late in gestation [267]. A single flock may experience abortion, septicemia, and
encephalitis [ 1791.
Direct entry via abrasions or lesions of the buccal mucosa, lips and nostrils, or
conjunctiva may lead to encephalitis. Because entry into the dental terminals of the trigem-
inal nerve in sheep can cause an ascending neuritis and encephalitis [49,50], listerial en-
42 Wesley
cephalitis is most common in winter and early spring in sheep that are losing and cutting
teeth [ 171. L. monocytogenes penetrates the buccal epithelium, accesses the endings of
the trigeminal (V) and hypoglossal (XII) cranial nerves, and enters the brain stem where
replication and dissemination to the medulla and pons occur. In severe cases, respiratory
failure and death follow within 1 month of infection [174,154]. Abrasions of the eye by
contaminated silage leads to ophthalmitis without any other clinical manifestations [283].
Meningoencephalitis caused by L. monocytogenes is the most common bacterial
infection of the central nervous system of adult sheep [237]. After an incubation period
of not more than 3 weeks, clinical symptoms of ovine encephalitis appear. These include
elevated temperature and refusal to eat or drink followed by neurological disturbances,
which include grinding of teeth, paralysis of masticatory muscles, and a stiff walk. At
this point, the animal moves in circles to the right or left, depending on the direction in
which the head is bent. This characteristic movement accounts for the name circling dis-
ease. Excessive salivation often occurs because of the animal's inability to swallow. In
advanced stages, muscular incoordination develops and is followed by inability of the
animal to walk. Death usually occurs within 2-3 days after onset of clinical symptoms,
with the illness seldom lingering beyond 10 days [239]. In the brain stem, Listeria antigens
are characteristically variable but always sparse. Perivascular microabscesses with L. mon-
ocytogenes and microgranulomas in histopathological specimens of brain stems are char-
acteristic. T lymphocytes (CD8' and CD4') and B lymphocytes contribute to the inflam-
matory process [ 1591.
After ingestion and hematogenous spread to the gravid uterus, L. monocytogenes
appears in the amniotic fluid and fetus within 48 h [ 1741. Listerial infections in pregnant
sheep often result in premature birth and infectious abortions [ 109,I 8 1,2801. This illness
seldom occurs concurrently with encephalitis [ 1351. Initially, pregnant ewes contract puru-
lent metritis, from which most recover. Intrauterine transmission of L. monocytogenes via
the placenta leads to a septic infection of the fetus, which in turn gives rise to abortion
or premature delivery with most fatalities occurring as stillbirths. Clinical symptoms are
resolved following expulsion of the fetus, after which the ewes recover. Morbidity in ewes
ranges from 1 to 20% [272] with mortality of lambs usually being high [154]. Injecting
L. monocytogenes into pregnant ewes caused 10% of those animals to abort. Significant
retardation of bone growth is seen in lambs born to experimentally infected ewes [ 1041.
Septicemia is most frequent in neonates and lambs and appears 2-3 days after oral
infection, although congenital and navel infection can also occur [ 1741. Septicemia is
characterized by an elevated temperature, loss of appetite, and diarrhea. Although death
may eventually occur as a result of extensive liver damage and focal pneumonia, the
mortality rate is much lower for the septicemic than for the encephalitic form of listeriosis.
Listeriosis is not a reportable disease in animals, thus precluding comparisons be-
tween countries. In addition, most infections in livestock are subclinical and therefore go
undiagnosed [ 1541. In Hungary, 14% of sheep excreted Listeria [222], but the distinction
between L. monocytogenes and L. ivanovii was not made. Although the incidence of L.
monocytogenes in sheep and cattle has decreased in The Netherlands, which has been
attributed in part to the change of silage production, in Great Britain modifications in
silage production may have caused an increase in ovine listeriosis [ 1021. To illustrate, in
1975, listeriosis was reported in a modest number of cattle (n = 37) and sheep (n = 44).
By 1984, sheep listeriosis cases had increased (n = 269) with little change in the number
of bovine listeriosis cases [ 1021. In parallel with the rise in the incidence in the United
Listeriosis in Animals 43
Kingdom, there was also a change in the disease pattern with both encephalitis and abor-
tion occurring in the same flock 102,179j.
Listeriosis occurs most frequently in late autumn, winter, and early spring. Stress
factors, such as abrupt changes in feed, concurrent disease, changes in dentition, and physi-
cal or viral damage to the epithelia] lining of the digestive tract may predispose to infection
11541. Introduction of new animals into the flock and overcrowding also are contributing
fdctors [ 1531. Climatic changes, such as heavy rains [302], especially following a drought,
which may spoil feed [228], or periods of extremely cold weather, which may cause ani-
mals to be housed indoors resulting in overcrowding [ 193,2851, are also determinants. A
prospective study conducted on early lambing flocks in southwest England ( 1989- 1991)
monitored 44 1 3 lambs from birth to slaughter for listerial meningoencephalitis. Two of
three flocks developed clinical disease attributed to L. monocytogenes serotype 1/2. Six
weeks before lambing, ewes on the two affected premises were bedded in straw; ewes on
the farm without clinical listeriosis were on softwood slats [ I 101. In the single flock with
no cases, preventive measures after lambing included replacing bedding and silage daily
and regular cleaning of the silage feeders which were on concrete floors. In the affected
flocks, silage was replaced on alternate days and the silage feeders were on soil-based
floors and thus easily contaminated. Before weaning, lambs were eating more silage, since
ewes were producing less milk. Although all lambs were exposed to these risk factors,
only an estimated 1.3% developed clinical infection with death occurring in 0.56% (21
of 4413) lambs from 4 to 32 days postweaning [109].
Quality of silage, as measured by digestibility ( D value), may influence the incidence
of ovine listeriosis [88]. In an outbreak of ovine listeriosis in the United Kingdom, the D
value of silage was below the optimum of 65-70% 12971. In Scotland, listeriosis caused
by L. monocytogenes serotype 1/2 occurred in sheep which were reluctant to eat poor-
quality silage [ 1791. Silage analysis indicated pH > 4 and a high ash content, reflecting
soil contamination. Despite antibiotic treatment, 19 ewes died, more than 60 developed
vaginal discharges, and 94 were barren at lambing [ 179).
The role of silage feeding in an epizootic of encephalitic listeriosis has been investi-
gated. A British study found a significant association between silage feeding and develop-
ment of ovine listerial encephalitis (relative risk of 3.8). In another report, excretion of
L. monocytogenes by sheep was linked to diet, with animals being fed entirely on hay or
manufactured diets not excreting detectable levels of L. monocytogenes. However, animals
fed on silage commonly excreted the organism [297]. The feeding of poor-quality silage
(pH 7.8) which was highly contaminated (10' L. monocytogeneslg) was the cause of an
outbreak in Spain [272]. In this outbreak, the flock consisted of 450 animals (attack rate
11.8%) with a case fatality rate of 94.3%.
Multiple strains of L. monocytogenes of the same or different serotype may be in-
volved in an outbreak in the same flock. When isolates of the same serovar are recovered
from a single outbreak, they may be further differentiated by DNA fingerprinting, phage
typing [ 101, or pyrolysis mass spectrometry [ 1751. In the Spanish outbreak just mentioned,
the serovar and phagovar of the L. monocytogenes strains isolated from two silage samples
and the brains of 3 of the 53 affected sheep were indistinguishable [272]. In addition,
DNA fingerprinting by random amplified polymorphic DNA (RAPD) analysis and ribotyp-
ing have been used to affirm that L. monocytogenes strains from silage, farm equipment,
and sheep brains were identical [295,296]. In contrast, examination of outbreaks of ovine
listeriosis in Scotland indicated multiple strains of L. monocytogenes serovar I /2 could
44 Wesley
be recovered from the silage incriminated in the outbreak. By multilocus enzyme electro-
phoresis (MEE), the L. rnonocytogenes strains for each of the affected animals in an out-
break were identical. Yet by MEE, none of the strains from the silage matched those
recovered from the brains. This could reflect bias during sampling from the bales, thus
missing a small virulent population of L. monocytogenes in the vegetation which, upon
entry into the ovine host, preferentially replicated to become the dominant strain and thus
cause disease [20].
Reports on the incidence of L. monocytogenes in ewes milk are limited. In Spain,
L. monocytogenes was present in 2.2% of 1052 ewes milk samples representing 283
farms. Yet, L. monocytogenes was recovered from 18% of milk tanker samples in Spain.
Tests of farm ewes milk samples indicated contamination by L. rnonocytogenes was sig-
nificantly higher on premises where cows were also reared than on farms where only ewes
were maintained [230]. The data suggest environmental contamination on farms resulting
from either L. monocytogenes excretion in cows feces or common exposure to contami-
nated ensilage on premises shared by sheep and cattle [230]. Interestingly, no seasonal
variation in milk contamination rates was evident.
Although not widely practiced in the United States, vaccination with live attenuated
strains of L. rnonocytogenes can effectively reduce ovine listeriosis [ 116,168,202,2821.In
Norway, immunization of sheep with a commercially available live attenuated vaccine of
serovars 1/2a and 4b reduced the incidence of listeriosis and abortions when compared
with unvaccinated control farms [115,116]. In this study [115], half of the sheep in 70
flocks (total of 3 130 sheep) with a history of listeriosis received two attenuated strains of L.
rnonocytogenes serotypes 12 and 4b, whereas the remaining sheep served as unvaccinated
controls. Both groups of animals were then housed together in the same pens. Results of
this study showed listeriosis incidences of 1 and 3% in vaccinated and unvaccinated sheep,
respectively. In 1984, a special license was issued to allow limited use of this vaccine in
a 2-year field study [116]. After vaccinating approximately 8% of all Norwegian sheep
(about 145,000 head), the incidence of listeriosis decreased from approximately 4% before
introduction of the vaccine to 1.5% after vaccination began. The incidence of abortions
was 0.7% in vaccinated compared with 1.1% in unvaccinated flocks. In another study, an
experimental vaccine, consisting of L. rnonocytogenes serovars 1/2a and 4b which were
attenuated via metabolic drift mutations, was tested in sheep, lambs, and ewes [168]. The
results of field tests indicated that vaccinated ewes delivered more lambs free of listeriosis
(93.4 vs 69.7%) and of higher birth weight (2.2 kg vs 1.8 kg) than lambs from control
unvaccinated ewes. In addition, L. rnonocytogenes was not isolated from milk samples of
vaccinated ewes in contrast to controls in which 32% of milk samples yielded Listeria
[ 1681. Although vaccination produced few adverse side effects, economic constraints sug-
gest that vaccination of sheep should be confined to flocks that have exhibited recurrent
listerial infections.
The epidemiology and economics of clinical listeriosis were described for a flock
of sheep in southern Illinois [202]. In that study, in which consumption of contaminated
silage was a key factor, unvaccinated Rambouillet ewes were more at risk (odds ratio 4.6)
than other ewes and yearling ewes were more at risk than older animals (odds ratio 4.1).
Interestingly, use of a bacterin did not decrease the risk of L. rnonocytogenes in Rambouil-
lets (odds ratio 0.8) but did among ewes of other breeds (odds radio 0.1). This indicates that
the inherent susceptibility of Rambouillet ewes to L. rnonocytogenes cannot be modified by
vaccination [202].
Serosurveys have been used to estimate the occurrence of listeriosis in sheep [250].
Listeriosis in Animals 45
However, antibodies to other gram-positive microbes cross react with Listeria antigens,
thus giving rise to false-positive results. In 1990, Berche and coworkers [26j developed
a more specific test for L. rnonocytogenes based on detection of antibodies to listeriolysin
0 (LLO) an antigenic protein of 58 kD. LLO is a virulence factor unique to L. monocyto-
genes that is required for the organism to escape from vacuoles and grow intracellularly.
It is not found in other Listeria species, including L. ivanovii. Antibodies to LLO which
are specific for L. monocytogenes and do not cross react with other Listeria species have
been detected in lambs experimentally infected with L. monocytogenes [ 13,166,1771.Puri-
fied LLO was evaluated as a specific antigen to detect both humoral and cell-mediated
immune responses of sheep infected with L. rnonocytogenes, L. innocua, and L. ivanovii
[ 131. LLO antibodies were seen only in sheep infected with L. rnonocytogenes. Further-
more, in a blastogenesis assay and skin test, two indicators of cell-mediated immunity,
only L. rnonocytogenes-infected sheep responded to LLO [ 131.
Listeria ivanovii (formerly L. monocytogenes, serotype 5 ; [240]) was first described
in association with ovine abortion and accounts for up to 8% of all animal listeriosis cases
[ 101. Listeria ivanovii is a recognized cause of ovine abortions and stillbirths and unthrifty
lambs [37,63,130,180,1811. Goats are not susceptible to L. ivanovii. To illustrate, L. iva-
novii and L. rrzonocytogeneswere both reported in two migratory flocks of sheep and goats
in Himachal Kadesh, India. Whereas L. monocytogenes was isolated from both sheep and
goats, L. ivanovii was cultured only from sheep [244]. Experimental infections of sheep
with L. ivanovii indicate that, unlike L. rnonocytogenes, abortions occur without encephali-
tis [ 1371. Factors which predispose to L. ivanovii abortions in livestock are similar to
those described for L. monocytogenes: a lowering of ewes resktance to infection by nutri-
tional stress, periods of cold and wet weather, feeding of poor-quality silage, and exposure
to carrier animals [75,101,219].
Outbreaks of listeriosis caused by L. ivanovii have been reported to affect up to
45% of pregnant ewes [ 1371. One outbreak in New South Wales involved a total of 1 10
animals which aborted or died shortly after birth. Heavy grazing by sheep (1 20 sheep per
hectare for 18 days) on pastures which had been cut for hay which had not been baled
and which had spoiled as a result of heavy rains was presumed to be the source of initial
contamination by L. ivanovii [242]. Multiple hepatic foci were seen in aborted lambs. L.
ivanovii was cultured from liver, lung, and stomach contents [242]. A report of bovine
abortions caused by L. ivanovii was associated with the grazing of cattle on pastures previ-
ously used by sheep [3]. Although rarely causing infections in humans [189], L. ivanovii
has been reported as a cause of abortion [75] and septicemia in acquired immunodeficiency
syndrome (AIDS) patients [56,164]. Even though L. monocytogenes is regarded as being
more pathogenic, both L. ivanovii and L. monocytogenes invade mammalian cells in tissue
culture, use actin filaments for intercellular spread, induce myometrial contractions in an
in vitro uterine strip model, and elicit conjunctivitis after ocular inoculation into rabbits
[ 1621.
The manifestations of clinical listeriosis in goats and sheep are essentially the same: en-
cephalitis, septicemia, and abortion. As is true for other livestock species, asymptomatic
infections also have been noted in goats [239].
After ingestion, L. rnonocytogenes penetrates the intestinal tract and sets up a tran-
sient bacteremia, which leads to dissemination to the central nervous system, viscera, or
46 Wesley
placenta. Depression, loss of appetite, a drop in milk yield, and elevated body temperature
(up to 41C) are the first indications of septicemia. The animals also may have diarrhea
[ 1131. In the pregnant doe, L. rnonocytogenes may penetrate the placenta, enter the fetus
where it replicates, and cause late-term abortion. In experimental studies with pregnant
goats, localization of L. monocytogenes in the placenta led to an elevation in prostaglandin
F2 and a decrease in progesterone levels. A slight decrease in secretion of estrone sulfate
by the fetal-placental unit prompted myometrial contraction and abortion [80].
Meningoencephalitis is the most frequently reported form of listeriosis in goats with
fatalities reaching 60% [ 1 131. The early signs of listerial encephalitis mirror the lesions
to the respective cranial nerves indicated by roman numerals in parenthesis: drooped ears
(VII), marked drooling (IX and X), protrusion of the tongue (XII), and cud retention from
difficulty in swallowing (IX and X). Goats may be more susceptible than sheep to listerial
encephalitis based on the severity of brain damage in goats [ 161. In an outbreak of encepha-
litis and abortion in a mixed herd of goats and sheep in Iraq, the morbidity (30 vs 17%)
and mortality (21 vs 15%) was higher in goats than in sheep [302]. Heavy rains, cold
weather, and susceptibility of pregnant animals may have contributed to the high numbers
of encephalitis cases [302]. More frequent recovery of L. monocytogenes serotype 1/2b
from goats than from sheep also has been documented in Sudan [246].
Listeriosis is linked to feeding silage [ 170,1741. However, in a study of 355 goat
herds in Missouri, encephalitic listeriosis was correlated with browsing on woody plants
and location of the herd in areas with a preponderance of alkaline soil [ 1441. Heavy browse
consumption may have led to oral lesions followed by penetration by L. monocytogenes
into the dental pulp or buccal cavity that may have led to encephalitis [ 1441.
Although not practiced in the United States, vaccination has limited the number of
goat listeriosis cases [66,93,202]. Before vaccinating goats in Norway with live attenuated
strains, the abortion rate in the test herd was 20-25%. However, after vaccination, the
incidence rate of abortions decreased to 3% [157]. A live attenuated L. monocytogenes
strain (strain Aer), obtained by three successive mutations in regard to streptomycin and
erythromycin resistance, afforded some protection against abortion in vaccinated goats
[93]. The optimal time for vaccinating goats and sheep may be shortly before the mating
season [ 1571.
As with other species of livestock, goats excrete Listeria in feces and milk during
and after septicemia and may contaminate the environment. Thus newborn kids housed
with the does may be infected through the navel or through sucking on soiled teats [ 1141.
Few studies describe the distribution of L. monocytogenes in raw goats milk. In one
report [l 1 I], L. monocytogenes was detected in 3.6% of raw milk samples from cows
with considerably lower recovery of L. monocytogenes from samples from goats (1%)
and ewes (2%) milk.
Serum antibody titers to L. monocytogenes may not indicate the immune status of
the host [ 1951. Animals with septicemia develop high antibody titers, whereas animals
with encephalitis have low titers, presumably because the brain is an immunologically
privileged site. More recently, antibodies to LLO have been proposed as a specific gauge
of antibody status. In experimentally infected goats, an increase in LLO antibodies is
correlated with rapid clearance of L. rnonocytogenes from the gastrointestinal tract and
thus predicts a favorable course of clinical infection [ 1961.
DNA fingerprinting methods are clarifying the role of animals in human infection.
To illustrate, an isolate of L. monocytogenes serovar 1/2b from the brain of a goat with
listeriosis exhibited the identical DNA profile by ribotyping as an isolate from cheese
Listeriosis in Animals 47
made from that goats milk and an isolate from the refrigerator in which the cheese was
kept. It is suggested that the cheese made from the infected goats milk may have contami-
nated the refrigerator shelves, thus serving as a reservoir for L. monocytogenes (741. In
contrast, a human endocarditis fatality with a recent history of exposure to goats showed
that the isolates from humans and animals were of the same serogroup. However, the
goat and human strains were clearly different via DNA analysis, thus making zoonotic
transmission less likely [%I.
[239]. Histopathological lesions of the brain stem consist of foci of necrosis infiltrated
with neutrophils, macrophages, and bacteria [ 1421. Perivascular cuffing with mononuclear
cells is evident [267]. Unlike listerial encephalitis in sheep and goats, most cattle survive
at least 4-14 days after the initial onset of symptoms, with a few reports of spontaneous
recovery [22 11.
Listeriosis in cattle is frequently associated with abortion [ 105,209,221,2391,which
generally occurs during the last trimester of pregnancy. However, as demonstrated by
Dora, an 1l-year-old cow with atypical mastitis, healthy calves can be born to chronic
carriers that shed the pathogen in milk [72]. As was true for sheep, L. monocytogenes is
transmitted to the placenta, and then into the fetus. Meningitis in neonates may follow
intrauterine infection with the septicemic young animal dying shortly after birth [241].
In the US Midwest, Listeria spp. are the third most frequently encountered bacteria
from bovine late-trimester abortion cases. L. rnonocytogenes accounted for 1.35% of bo-
vine abortion and stillbirth submissions received by the South Dakota Animal Disease
Research and Diagnostic Laboratory from 1980 to 1989 [ 1551. Ten years earlier, of 2544
bovine abortion cases examined in the Northern Plains region of the United States, 2.2%
were attributed to listeriosis [ 1561. Similarly, in Germany, from 1984 to 1989, L. monocy-
togenes serotype 1/2 was recovered from 1.2% (122 of 993 1) and 1.8% (122 of 993 1) of
bovine abortions [31] in the Erfurt and Cottbus regions, respectively.
L. ivanovii is most often associated with sheep [20,37,63,130,137,180,181,2421 and
is sometimes recovered from cattle [3,101]. In California, during a 3-year period, five cases
of listerial bovine abortion were diagnosed among 243 fetuses submitted for evaluation. L.
ivanovii was recovered from four cases, whereas L. monocytogenes was isolated from
only a single bovine abortion. The pathological findings in these five listeriosis cases were
similar [3].
L. rnonocytogenes may enter a herd through contaminated feeds, introduction of
new stock, and rodents. Bovine abortions and stillbirths occur shortly after contaminated
silage is fed [5]. Improvement in silage production and hay making resulted in a decrease
(from 8.7 to 1.2%) in the number of listerial bovine abortion cases in The Netherlands
[64]. A change in silage production also was linked to a reduction in the percentage of
carrier animals (from 15 to 0.8%) based on fecal sampling [69]. An outbreak in Nigeria
resulted in 35 cases of bovine encephalitis and four abortions in the same herd. Although
the source of infection was unknown, the authors proposed that L. monocytogenes was
introduced into the herd by introducing new animals [2]. As expected, the number of
healthy carriers is lower on farms without overt disease than on farms with clinical listeri-
osis. To illustrate, L. monocytogenes was cultured from 2.0% of cows on farms without
L. monocytogenes and from 6.7% of healthy animals on farms on which listeriosis had
occurred [64]. This may indicate exposure to a common source of infection. Rodents are
known carriers of L. monocytogenes, and fecal contamination of animal feed is a potential
source of contamination [5,107,153].
Although not particularly common, generalized listerial infections can give rise to
mastitis [41,59,60,72,103,148,216,258,281].Beginning in 1938, Schmidt and Nyfeldt pos-
tulated that a small outbreak of human listeriosis in Denmark may have been caused by
drinking milk from mastitic cows. However, the role of Listeria in mastitic infections was
not clearly identified until 1944 when Wramby [300] isolated L. rnonocytogenes from milk
and udders of mastitic cows in Sweden. In 1956, de Vries and Strikwerda [60] described
another case of bovine mastitis in which a penicillin-resistant strain of L. monocytogenes
was cultured from one quarter of a 6-year-old dairy cow. Following acute onset, the condi-
Listeriosis in Animals 49
tion soon became chronic with shedding of L. monocytogenes in milk for 3 months. Pro-
longed excretion of L. monocytogenes in milk [65,66,135,209,2 10,2211, the apparently
normal appearance of the milk, and consumption of raw milk on farms could be important
factors in the transmission and epidemiology of milkborne listerial infections [ 1031. From
a public health aspect, culling of L. monocytogenes-infected cows with clinical mastitis
which do not respond to treatment is recommended [245]. After slaughter, cross contami-
nation of the carcass with bacteria from the infected udder is possible through evisceration,
meat inspection, or other manipulations [274].
L. monocytogenes strain Scott A (serotype 4b), a clinical isolate recovered from
the New England outbreak, has produced experimental mastitis in dairy cows [38,291].
Following repeated intramammary inoculation of 34 Holstein cows with I 03- 107L. mono-
cytogenes cells, 75% of the animals became chronically infected and shed Listeria in milk
(about IO3-1Os L. monocytogenes cfu/mL of milk) intermittently for up to 8 months. The
intramammary route of inoculation was used to simulate infection from contaminated
bedding directly into the teat canal. Interestingly, one of these experimentally infected
cows delivered a normal healthy bull calf. Bourry et al. [4] also induced mastitis via
intramammary inoculation with a single dose of 300 cells of L. monocytogenes serotypes
4b and 112. L. monocytogenes was recovered from the supramammary lymph node but
not from the spleen or liver, indicating clearance in the affected region. DNA profiles
of isolates recovered throughout experimental infection confirmed the persistence of the
inoculated strain [4].
As with sheep [7] and goats [ 1691, L. monocytogenes also is shed in milk by healthy
dairy cattle with no indication of mastitis [64,73,86,103,127,134,135,234,278].Schultz
[234] collected milk samples from 1004 cows and isolated L. monocytogenes from the
milk of 10 animals (0.1%), 7 of which appeared perfectly healthy. Shedding of Listeria
in milk by these animals was intermittent but continued for up to 12 months. Examination
of dairy herds in Yugoslavia [247] also has demonstrated that clinically healthy cows can
act as asymptomatic carriers of L. monocytogenes and secrete the organism in their milk
for months over several lactation periods. In one such survey, L. monocytogenes was
detected in milk from 3.2% of 845 clinically normal cows on seven farms on which listeri-
osis had been previously diagnosed [ 15 81. In addition, Kampelmacher [ 1481 reported that
dairy cattle shed L. monocytogenes at levels of 10,000-20,000 cfu/mL of milk.
Surveys of raw milk prompted by dairy-related outbreaks of human listeriosis in
1983, 1985, and 1987 confirmed that asymptomatic cattle are carriers of L. monocytogenes.
Indirect contamination of bulk milk occurs from unhygienic rnilking practices, if L. mono-
cytogenes is present in feeds, feces, udder surface, or bedding [87], or if an animal is
recovering from a recent infection [ 104,1951. L. rnonocytogenes has been reported in raw
cows milk with a distribution ranging from 0.1 to 45% [22,7 I ,86,87,94,111,122,173,234,
254,260,2651. Recoveries varied depending on whether individual cows, bulk tanks main-
tained on the farm, or milk tankers serving multiple premises were sampled. When avail-
able, data indicate that the incidence of L. monocytogenes from individual farms may be
lower than that reported for processing centers or tanker trucks [120,230]. A 23-year sur-
vey involving 36,200 dairy herds in Denmark indicated that the incidence of L. monocyto-
genes-infected cows varied from 0.01 to 0.1% and of herds with an infected cow from
0.2 to 4.270. However, 8.5% of bulk milk samples (n = 4451 were contaminated with L.
monocytogenes [6]. Regional differences in the recovery of L. rnonocytogenes from milk
have been documented. For example, Dominguez Rodriguez et al. [7 I ] found L. monocyto-
genes in 45.3% of 95 raw milk samples from a single bulk tank (80,000-L capacity). The
50 Wesley
dairy received raw milk from several small farms in western and central Spain over a 16-
month interval. In the northeastern United States, Hayes et al. [122] found L. monocyto-
genes in 12% of raw milk samples. L. monocytogenes was found in 4.4% of raw milk
samples (n = 137) obtained from the Utrecht region of The Netherlands [22] and in 3.8%
of raw milk samples in Scotland 1911. This parallels the recovery of L. monocytogenes
in 4.1% of raw milk samples from Tennessee [231]. Interestingly, in that US study, con-
sumption of raw bulk milk was reported by 35% of dairy producers [231].
A seasonal distribution of L. rnonocytogenes in raw milk, mirroring numerous deter-
minants including a change of diet or weather-related stress, has been observed. Lovett
et al. [ 1733 surveyed raw milk at various times from three different regions in the United
States and found L. monocytogenes in 4.2% of the overall samples. However, recovery
of L. monocytogenes from Massachusetts samples was seasonal with the incidence being
highest during cooler months and lowest in hot weather months. Interestingly, no seasonal
trend was exhibited by samples collected from Ohio, Kentucky, and Indiana [173]. L.
monocytogenes was cultured from 4.9% of raw milk samples obtained from 70 Irish farms
with the incidence being higher in the winter when cows were housed indoors than in the
summer [224]. A study of L. monocytogenes in raw milk in Nebraska indicated a seasonal
distribution with 6% of raw milk samples harboring L. monocytogenes in February; 2%
of samples were positive in July [ 1671. Seasonal variation may be related to silage feeding
during the winter. In Scotland, a seasonal distribution was indicated for L. monocytogenes,
which was present on 25 of the 160 farms surveyed (16%). Contamination was sporadic,
with bacterial titers generally < 1 L. monocytogenes cfu per mL. Although more raw milk
samples were positive for L. monocytogenes in January than at other sampling times
throughout the year, the authors caution that no link to farm management practices was
evident [90]. In Ontario, Canada, some geographical differences were observed, with the
incidence of L. monocytogenes in raw milk being higher in the eastern region [86]. Despite
the modest recovery of L. monocytogenes from raw milk in Ontario (1.3%, 6 of 455
samples), the incidence was lower during the winter and autumn than at other times [86].
In another report, L. monocytogenes was found in 5.14% of raw milk samples screened
monthly in Ontario with no indication of seasonal differences [254].
Normal healthy cattle may intermittently shed Listeria in their feces, with prevalence
rates ranging from a few percent to 52%, with some seasonality [87,131,271,2881. Fecal
shedding may reflect levels of L. monocytogenes in feed. In one study, shedding of L.
monocytogenes (52%) was related to feeding wet feed (e.g., silage of beet tops, oat, and
pea straw); 67% of the samples were contaminated 12521. L. monocytogenes was isolated
from the feces of 8.7% of nearly 4000 randomly selected dairy cows in Finland over a
2-year period [ 1331. Again, L. monocytogenes was recovered more frequently in bovine
feces on farms where L. monocytogenes was in feed than on premises where feed (silage
or pasture grass) was negative for L. monocytogenes [ 13 1,1331. Switching cattle from
grazing to a diet of silage increased fecal shedding of L. monocytogenes. The distribution
was seasonal with L. monocytogenes recovered from feces more frequently during the
indoor season (9.2%) than when animals were on pasture (3.I %). Expectedly, the seasonal
occurrence of L. monocytogenes in milk reflected the frequency of this pathogen in feces
but not in grass silage or pasture grass, thus inferring fecal contamination during milking
[131].
Serosurveys have been used to monitor distribution of L. monocytogenes infections
in dairy cows. Infection rates in cattle can be estimated by measuring antibody levels to
whole cells as well as antibodies specifically targeting LLO [ 12,331. Agglutination titers
Listeriosis in Animals 57
of serum and whey were evaluated in experimentally infected dairy cattle ( n = 34). By
the eighth week postinfection, 80% of the cows exhibited serum titers of > I :20,480.
Whey titers, which reflected the local mucosal immunity to L. monocytogenes in the mam-
mary gland, rarely exceeded I :256, because of the lower immunoglobulin concentration
in milk versus blood [72,293]. Since up to 33% of dairy cattle continued to shed L. monocy-
togenes despite high serum titers, antibody levels did not accurately predict the presence
of L. monocytogenes in milk [72,257,276,293]. As with humans [26], sheep 1 13,166,1771,
and goats [ 1061, antibodies to LLO have been used to evaluate the immune response of
experimentally infected cattle. [ 12,331. A positive response to ILL0 specifically confirmed
previous or current infection with L. rnonocytogenes in dairy cows [ 12,331.
Stress-related immunosuppression associated with change of diet, weather, transpor-
tation [89], pregnancy, parturition, and lactation may lower resistance to bovine listeriosis
[239]. Dexamethasone mimics the stress-related release of glucocorticoids. In cattle, dexa-
methasone elevates total white blood neutrophil counts and decreases eosinophil and lym-
phocyte populations. When administrated to cows experimentally infected with L. monocy-
togenes, dexamethasone increased shedding of the pathogen in milk by up to 100-fold
[291]. Increased levels of L. monocytogenes in milk may reflect impairment of cell-medi-
ated immune mechanisms and phagocytic cell functions that underlie listerial immunity
[269]. Likewise, transport of live animals over long distance!; significantly increased the
level of fecal excretion of L. monocytogenes. However, contamination of the resultant
cattle and sheep carcasses was minimal [92].
A major concern of bovine listeriosis is the potential risk posed to humans. In Den-
mark, a case-control study indicated that human listeriosis was frequently linked to con-
sumption of unpasteurized milk (risk factor of 8.6), although other factors, such as immu-
nosuppression and underlying diseases, were regarded as more significant [ 1401.
Furthermore, a comparison of 33 isolates from bovine mastitis and 27 human clinical
isolates recovered in Denmark during 1993 was made by sero- and ribotyping. Serotyping
showed that all bovine and 63% of human isolates belonged to serogroup 1, whereas 37%
of the human isolates were of serogroup 4. DNA fingerprinting by ribotyping indicated
that a low but constant percentage of Danish dairy herds had cows infected with L. monocy-
togenes strains which were similar to human clinical strains 11411. L. monocytogenes
ribotypes common to both dairy processing and the farm environment (dairy cattle, raw
milk, silage) were also reported in the United States, thus suggesting that the farm may
serve as a reservoir for L. monncytogenes strains capable of entering the dairy processing
facility [9]. The findings also verify the ubiquitous distribution of the pathogen.
Although foodborne listeriosis in humans is more frequently linked to consumption
of contaminated dairy products than to beef, L. monocytogenes was recovered from 3% of
composite fecal samples representing 224 feedlot beef cattle [249]. In a limited study
of experimentally infected Holstein cows (n = 4), L. monocytogenes was cultured from
muscle, organ, and lymphoid tissues at 2 days postinfection; none was recovered at 6 or
54 days after inoculation [ 1451. Thus culled dairy cattle may be an insignificant source
of L. monocytogenes contamination in meat. Epizootics have been observed in both feedlot
and beef cattle herds (5,299). Transport of cattle over long distances increased the level
of fecal excretion of L. monocytogenes, but contamination of carcasses was not high [89].
Yet in this study, L. monocytogenes was detected in 9 1 % (2 1 of 23) of minced beef sam-
ples, demonstrating that processing significantly increases the level of Contamination com-
pared with that of the whole carcass 1891. This hypothesis is further strengthened by
tracking L. monocytogenes strains by multilocus enzyme dectrophoresis. L. monocyto-
52 Wesley
genes strains of electrophoretic type (ET) 1 have been implicated in major human food-
borne epidemics and are found coincidentally in livestock. L. monocytogenes strains of ET
1 predominate in cattle at the beginning of slaughter but are not detected on the carcasses at
the end of processing or in the environment of the abattoir [32]. In contrast, environmental
strains such as ET 19 contaminate the carcass during processing. ET 19 strains were found
on the carcasses of pigs at the end of processing in two slaughterhouses but not on live
animals or at the beginning of slaughter. Overall these findings indicate that contamination
of meat occurs during processing by L. monocytogenes strains which are resident in the
packing plant rather than by strains indigenous to animals [32]
in healthy pigs, its distribution can be estimated from surveys of fecal excretors and recov-
eries from tonsils and carcass swabs collected at slaughter. L. monocytogenes was cultured
from 5% of rectal swabs and from 1.9% of hog carcasses in Trinidad [ 11, indicating mini-
mal carcass contamination during processing. L. monocytogenes was present in 13% of
hog tonsils and in 2% of lymph tissues in pigs in Togo, West Africa [ 1291. In Belgium,
16% of fresh pig feces (n = 25 samples) harbored L. monocytogenes [271]; 5.9% of swine
fecal samples were positive in Germany [288]. In Yugoslavia, L. monocytogenes was
cultured more frequently from hog tonsils (25%) than from fecal samples ( 5 % ) taken from
the same animals [277]. In Scotland, L. monocytogenes was not found in either pig feces
before slaughter or on swine carcasses [92]. Likewise, L. monocytogenes was not recov-
ered from hog carcasses in Norway or Sweden [204].
Asymptomatic carriers of Listeria may be more prevalent in eastern Europe. To
illustrate, L. monocytogenes was recovered from 25.6% of swine feces in Hungary [222].
Ralovich [221] reviewed studies in which a fecal recovery rate of 47% in individual ani-
mals and in 11 of 12 among farms was described. Yet in Yugoslavia, 45% of all pigs
examined harbored L. monocytogenes in the tonsils, whereas only 3% were fecal excretors
[40]. A high infection rate in pigs has led to speculation that swine may be important
reservoirs of L. monocytogenes [ 1081.
Husbandry practices such as feeding pigs dry feed or silage, rearing in closed houses,
and maintaining specific pathogen free (SPF) herds as well as differences in sampling
sites (tonsils versus feces) may account for the variation in the incidence of healthy porcine
carriers reported. For example, in Yugoslavia, L. monocytogenes was recovered more
frequently from tonsils of pigs raised on silage (61%) than from animals raised on dry
feed (29%). Interestingly, in that study, L. monocytogenes was also recovered from more
than 19% of pork meat products tested [40]. Although not found in fecal samples in SPF
herds, L. rnonocytogenes was cultured from 2.2% of fecal samples from non-SPF herds
in Denmark [252]. Norrung et al. [206] detected Listeria spp. in 29% of tonsils removed
from market weight hogs and in 75% of pig feed samples tested in Denmark. Unfortu-
nately, data on the specific distribution of L. monocytogenes in this study were not pro-
vided. L. monocytogenes was recovered from the lymph nodes of 5% of slaughtered pigs
and from 8% of pork samples in Bosnia and Hercegovina [ 1711. Of L. monocytogenes
recovered in that survey, 76% were from hog carcasses sampled during the autumn and
winter months [ 1711.
Skovgaard et al. [252] reported that only 1.7% of pig fecal samples yielded L. mono-
cytogenes; however, the pathogen was detected in 12% of ground pork samples tested in
Denmark indicating dissemination of Listeria during processing. In Yugoslavia, where L.
monocytogenes was isolated more frequently from ground pork (69%) than from deep
muscle (O%), contamination occurs during pork processing [40]. In France, an outbreak
involving 279 human cases incriminated pickled pork tongue as a major vehicle of trans-
mission, although other highly processed, ready-to-eat delicatessen items subject to envi-
ronmental contamination were also implicated [ 1381.
Although limited epidemiological data are provided, two cases of human neonatal
listeriosis may have been linked indirectly to contact with pigs [256]. Alternatively, they
may reflect exposure to a common source of contamination.
Avian listeriosis was first described in 1935 [238], 3 years after TenBroeck isolated L.
monocytogenes (then Bacterium monocytogenes) from diseased chickens. Both wild [227]
54 Wesley
and domestic avians, including turkeys [24,12 1,2051, ducks [ 106,2391, geese [ 106,2391,
and pheasants [ 1061, are the largest group of asymptomatic carriers [ 106,1081. A survey
of healthy urban rooks indicated carriage with L. monocytogenes (33%), L. innocua (24%),
and L. seeligeri (8%) [35]. Likewise, L. monocytogenes was detected in 9.5% of fecal
samples from apparently healthy, ring-billed gulls (Larus delawarensis) in Montreal. In
contrast, samples from pigeons in Barcelona were negative [46], whereas L. monocyto-
genes was found in 1% of pigeons examined in Germany [2881.
Up to 33% of all healthy chickens may asymptomatically shed L. monocytogenes
in fecal material [67,68,252]. Birds most likely become infected by pecking Listeria-con-
taminated soil, feces, or dead animals; however, contaminated fecal material also may
pose a hazard to other livestock. Bovine encephalitic listeriosis developed in four cows
which were housed in stables where chicken litter was used as bedding. L. monocytogenes
serogroup 4b was recovered from the bovine brains, litter, and the intestinal contents of
4.1% of the donor birds 1671. In another study, the increased incidence of L. monocyto-
genes in rooks coincided with the nesting season and the peak of ovine listeriosis, which
in turn was linked to consumption of contaminated silage [88]. Thus, although the true
incidence of listeriosis in birds and other forms of domestic livestock is undoubtedly much
higher than published reports, clinical listeriosis is uncommon in domestic fowl.
Despite the many sporadic cases of avian listeriosis that have been documented over
the last 60 years, this disease is far less common in birds than in sheep, goats, and cattle
[ 106,107,108]. For example, listerial infections were discovered in only 13 of more than
38,000 chickens submitted for examination in Pennsylvania between 1960 and 1965 [236].
Furthermore, large-scale outbreaks of listeriosis in chickens appear to be uncommon
[200,212]. In an outbreak in India involving young chicks, death (mortality rate of 60%)
was sudden and usually with no prior symptoms. Some birds exhibited symptoms of weak-
ness and lassitude and a tendency to stand in an isolated dark place [200].
Listeriosis in birds may be a secondary infection associated with viral infections
[57] as well as salmonellosis, Newcastle disease, fowl pest, coryza, coccidiosis, worm
infestations, mites, enteritis, lymphomatosis, ovarian tumors, and other immunocom-
promising conditions [108]. In 1988, an encephalitic form of listeriosis was reported in
broiler chickens in California [54]. Predisposing conditions which may have precipitated
the outbreak included recent debeaking and vaccination with a modified live viral arthritic
vaccine which was given subcutaneously in the neck. L. monocytogenes serotype 4b was
recovered from a liver and multiple brain samples. Three years later, a second outbreak
occurred in breeder replacement birds and affected 0.3% of the 54,000 birds in the flock.
L. monocytogenes was recovered from soil samples collected near an adjacent dairy but
not from other sites on the premises. The stress associated with the unusually cold climate
described in the report coupled with vaccine stress of the 7- to 10-day-old chicks may
have precipitated this outbreak [54].
Septicemia, the most frequent manifestation of listeriosis in chickens and other do-
mestic fowl, is characterized by focal necrosis within the viscera, particularly the liver
and spleen [ 1081. Although not present in all cases [200], cardiac lesions frequently de-
velop, which in turn lead to engorgement of cardiac vessels, pericarditis, and increased
amounts of pericardial fluid [ 108,2231. Other conditions produced by the septicemic form
of avian listeriosis have included splenomeglia, nephritis, peritonitis, enteritis, ulcers in
the ileum and ceca, necrosis of the oviduct, generalized or pulmonary edema, inflammation
of the air sacs, and conjunctivitis. In acute cases, lesions resulting from these conditions
may be partially obscured by congestion and hemorrhages throughout the viscera [ 1081.
Listeriosis in Animals 55
Unfortunately, chickens and other domestic fowl that suffer from listerial septicemia nor-
mally exhibit few overt signs of disease other than progressive emaciation and usually
die within 5-9 days of infection.
Although far less common than the septicemic form of listeriosis, L. monocytogenes
also can produce meningoencephalitis in domestic fowl. Domestic birds suffering from
listerial meningoencephalitis exhibit several striking behavioral changes, including incoor-
dination, tremors, torticollis, unilateraUbilatera1 toe paralysis, and dropped wings, all of
which directly relate to disturbances of the central nervous system [ 181. Such infections
are virtually always fatal. Postmortem examination often reveals congestion and necrotic
foci in the brain [ 181 along with many of the aforementioned conditions that are character-
istic of listerial septicemia. Microscopically, gliosis, and satellitosis in the cerebellum and
microabscesses containing gram-positive bacteria are found in the midbrain and medulla
of birds with encephalitic listeriosis [55].
L. monocytogenes can colonize both chick embryos and young birds with older birds
appearing more resistant [ 108,117]. Following oral challenge of chickens with 102or 106
L. monocytogenes cells, Bailey et al. [ 151 detected the pathogen more frequently in ceca,
spleen, liver, and cloacal swab samples from 1-day-old chicks rather than 14- or 35-day-
old chickens. Diarrhea and emaciation have been noted in experimental infection, thus
facilitating spread via feces and nasal secretions. In a later study, 2-day-old chicks were
experimentally infected with L. monocytogenes. Although most of the inoculated chicks
appeared healthy, depression, ruffled feathers, dullness, and diarrhea followed by death
were noted 2-5 days postinoculation. Milder symptoms such as anorexia and drowsiness
were also observed in several animals. At 5 days postinfection, 100% of the cecal samples
yielded L. monocytogenes. However, the percentage of L. monocytogenes-positive birds
decreased, and by day 28, L. monocytogenes was recovered from the ceca of only 10%
of experimentally infected birds [132]. In another report, with tests on a smaller num-
ber of birds, L. monocytogenes was only found on the first day following infection in 15%
of fecal samples [186]. These data suggest that L. rnonocytogenes is cleared rapidly from
infected birds, indicating that chicks are transiently infected and unlikely reservoirs of L.
monocytogenes. Interestingly, following artificial infection, Pustovaia [2 I 81 found that
Columbiformes (pigeons, doves), Passeriformes (perching birds), and Galliformes (tur-
keys, pheasants) were susceptible, whereas Falconiformes (falcons, hawks) and Strigi-
formes (owls) were resistant [ 1281.
L. monocytogenes has been used to investigate macrophage function in retroviral
infection and the cell-mediated immune response in susceptible and resistant chickens
exposed to Mareks disease virus 145,571. Viral infection depressed the resistance of 10-
day-old chickens to experimental infection with an avian osteopetrosis virus [57]. When
compared with virus-free chickens, the dual infected birds were less efficient in clearing
L. monocytogenes from their spleens [57).
L. monocytogenes can be recovered from infected chicks by inoculation of 1O-day-
old embryos [62]. Thus, egg inoculation has been suggested as an assay to replace the
mouse test to gauge the virulence of L. monocytogenes [ 1 11. For example, L. monocyto-
genes and L. ivanovii are fatal to experimentally inoculated 10-day-old chick embryos.
In contrast, embryos infected with the nonpathogenic species, L. innocua, L. seeligeri,
and L. welshimeri, generally survived [266].
L. monocytogenes is present in 0-33% of healthy birds [67,68,76,99,127,136,288],
with contamination in retail poultry ranging from 17 to 70% [ 14,32,76,92,99,186,208].A
link between transport stress and fecal shedding of L. rnonocytogenes has been suggested.
56 Wesley
In one study, L. monocytogenes was found in 33% of pooled fecal samples collected from
cages suggesting recrudescence of L. monocytogenes because of transport stress. However,
no data on the status of L. monocytogenes in these birds before shipment are provided
[252].
The presence of Listeria on retail poultry probably results from contamination during
processing rather than from the bird. In an effort to trace the source of L. monocytogenes
in retail poultry, low levels of natural carriage ( 5 % ) were reported in cecal samples from
parent flocks providing broilers. L. monocytogenes was not cultured from cecal samples
from over 2000 broilers (90 flocks) in Denmark [208]. However, L. monocytogenes was
found in processed poultry. Comparison of DNA fingerprinting patterns by pulsed-field
gel electrophoresis (PFGE) indicated that live birds contributed little to the total contami-
nation of the product [208].
Once processing is initiated, the numbers (and percentage) of Listeria-positive sam-
ples increase [92]. Studies in poultry slaughterhouses failed to detect L. monocytogenes
in several organs, including the intestinal tract and ceca. Yet it was found in processing
water, in mechanically deboned meats, and on the hands and gloves of 34% of the meat
cutters, indicating cross contamination during processing [99]. Later studies used DNA
profiles of L. monocytogenes collected during processing to indicate significant environ-
mental contamination during processing [32]
Sporadic human cases of listeriosis have been epidemiologically linked to consump-
tion of undercooked poultry products [235]. Analysis of risk factors associated with spo-
radic human listeriosis in the United States indicated that cancer and immunocompromised
patients, in whom 69% of listeriosis cases occur, were more likely than controls to have
eaten undercooked poultry (odds ratio = 3.3) [233].
Minor Species
Listeriosis has been diagnosed in several minor livestock species, such as horses, llamas,
animals raised commercially for pelts, companion animals, deer, and primates. The routes
of transmission and symptoms parallel those of cattle, sheep, and goats.
As in other livestock species, Listeria infection in horses can cause abortion [289],
septicemia [25,51,79,112], and encephalitis [ 1821. In contrast to cattle and sheep, few
cases of equine listeriosis are reported [ 160,182,185,239,263,264,2681.A survey of fecal
samples from 400 German horses indicated a carrier rate of 4.8% for L. monocytogenes,
6% for L. innocua, and 1.5% for L. seeligeri with less than 1% harboring L. welshimeri
[288]. The few surveys describing L. monocytogenes-seropositive horses should be inter-
preted cautiously in the light of possible cross reactivity between antibodies of other bacte-
rial species and Listeria.
Prior contact with cattle and feeding on silage may explain sporadic cases of equine
listeriosis [ 1871. L. monocytogenes was reported from four Welsh and two Shetland ponies
housed together with cattle, one of which was diagnosed with listeriosis, and given poor-
quality silage [79]. At necropsy, L. monocytogenes was cultured from the equine liver,
spleen, heart, kidneys, and lungs [79]. In Tasmania, abortions occurred in two mares which
were allowed to graze on a pasture which had previously been a sheep farm but had most
recently served as a dairy farm. L. monocytogenes serogroup 1 was cultured from the lung
and stomach of one fetus. Following antibiotic therapy, the mare was bred and later gave
birth to a normal live foal, indicating that L. monocytogenes infection does not lead to
permanent infertility [ 1831. Equine abortion, preceded by mild respiratory tract infection
Listeriosis in Animals 57
caused by L. rnonocytogenes serotype 4, was reported for a mare which had wintered with
cattle and had consumed ensilage [289]. L. monocytogenes was cultured from the fetal
liver, lung, spleen, and stomach. Neonatal septicemia was documented in a 3-day-old foal
whose mare was housed indoors and fed poor-quality contaminated hay [126].
As with other species, the origin of infection in equine listeriosis cases may be
unknown. To illustrate, L. monocytogenes was recovered from the brain stem of a 16-
year-old Welsh pony gelding with signs of ataxia, weakness, and deficits of cranial nerves.
No immunological deficit was detected and there was no history of contact with ruminants
or access to silage [182]. A 21-day-old Appaloosa filly was examined because of diarrhea
of 2 weeks duration [284]. As the animals condition deteriorated, gentamicin sulfate and
procaine penicillin G were administered. Septicemia was diagnosed based on the presence
of L. monocyrogenes cultured from the blood. No sources of infection were evident, thus
leading to speculation that L. monocytogenes was transmitted to the foal via contaminated
mares milk.
As is true for humans, listeriosis also occurs more frequently in immunocompro-
mised livestock with defects in both the humoral and cell-mediated immune systems [269].
Listeriosis was described in an Arabian foal with combined irnmunodeficiency [ 5 I]. The
I-month-old foal was ataxic, lethargic, failed to nurse, and spent most of the time with its
head down. Most strikingly, hoofs were dragged when the animal exercised. At necropsy,
widespread lesions were present in the viscera and central nervous system.
In the llama, listeriosis occurs as a septicemia with meningitis in neonates, but more
commonly causes asymmetrical vestibular disease in adults [ 191. Most affected animals
are weaned and grazing or consuming roughage but not silage. Multifocal suppurative
encephalitic listeriosis was diagnosed in two adult llamas, both of which were pregnant.
L. monocytogenes was cultured from one of two animals and was observed in brain stem
lesions of both llamas by fluorescein-conjugatedantibody to L. monocytogenes [43]. In an-
other report, L. monocytogenes caused fatal meningoencephalomyelitis in a 3- to 5-month-
old llama. The animal displayed unilateral peripheral disease progressing to encepha-
litis 12701. The source of infection for cases detailed in these two reports is unknown [ 19,431.
Listeriosis can have an economic impact on commerciall pelt farms. An outbreak of
disseminated visceral listeriosis in chinchillas in Nova Scotia [95] was associated with
consumption of contaminated sugar beet pulp, although L. monocytogenes was not isolated
from the feed. This outbreak occurred in a colony with a 2390 mortality rate of breeding
chinchillas [298]. Approximately 4 days before death, animals were anorexic and hunched
and some had torticollis (twisted necks). However, many animals were found dead without
clinical signs (2981. Hay contaminated with rodent, bird, or ruminant feces has been impli-
cated in previous outbreaks of listeriosis among chinchillas with removal of contaminated
feed often interrupting the cycle of transmission [47,95]. L. monocytogenes could have
been transmitted by coprophagia, since animals defecated in dust bath pans and the pans
were transferred from cage to cage [298]. In an enzootic outbreak of listeriosis in a rab-
bitry, L. monocytogenes 1/2a was cultured from feed samples and from a doe which had
died of septic metritis 12141.
The early literature describes L. monocytogenes in nondomesticated ruminants, in-
cluding reindeer, roe deer, and a Grants gazelle that had previous contact with Listeria-
infected sheep [23,81,83,151,205,2861. L. monocytogenes has been recovered at necropsy
from ruminants housed in zoological parks [8 133,2861. Meningoencephalitis occurred
during the winter and early spring in 42 of 1800 deer in a Danish park. This was preceded,
in the previous spring, by death of six deer which exhibited circling and appeared to be
58 Wesley
blind. The following year, the first sign of illness was a drooping ear, caused by paralysis
of the facial nerve, and a slight inability to follow the herd. No external source of L.
monocytogenes was evident and stress resulting from a poor beech-mast crop, an increased
stocking rate of animals resulting in overcrowding with possible introduction of asymp-
tomatic carriers, and a sudden change in the weather were all potential contributing factors
[811.
Listeriosis is rarely reported in dogs and can cause encephalitis, including circling
[232], and abortion [261]. In a survey of domestic animals, L. monocytogenes was detected
in 1.3% of dog fecal (n = 300) samples and 0.4% of cat fecal (n = 275) samples [288].
The low recovery may indicate that companion animals are not important in the epidemiol-
ogy of listeriosis in humans [287] or that shedding is sporadic. In contrast, serosurveys
which indicate that up to 90% of dogs may be seropositive [48,25 11 should be interpreted
cautiously because of the cross reactivity inherent in agglutination tests. A single report
on possible transmission of L. monocytogenes from humans to dogs [262] warrants reeval-
uation, since the isolates were later identified as L. innocua. Nevertheless, recovery of a
single species of Listeria from both humans and dogs in close proximity could reflect
substantial environmental contamination rather than human-to-dog passage.
Listeriosis also can occur in non-human primates where it manifests itself as septice-
mia [303], meningoencephalitis [61], and stillbirths [ 188,2731, as documented in a large
outdoor breeding colony in California [2 131. Transmission may occur by consumption of
contaminated foods [273]. Attempts have been made to experimentally infect Cynomolgus
monkeys (Macacafascicularis) through feeding [85] and exposure to aerosols [ 1491. Al-
though these monkeys shed L. monocytogenes in their feces for up to 21 days after ingest-
ing I O9 Listeria, neither septicemia nor encephalitis were reported indicating that normal
healthy primates are resistant to L. monocytogenes.
log cfu/g wet weight). L. monocytogenes was not detected in the water or feed. Unfortu-
nately, the authors provided no data on the percentage of L. monocytogenes-positive fish,
but they concluded that bacterial concentrations in the viscera suggest cross contamination
is possible during evisceration [ 1651. A survey of three rainbow trout farms in Switzerland
showed that L. monocytogenes was present in the feces (40%) and on the skin (33%; 5
of 15) of fish from one of the farms, yet L. monocytogenes was detected on the finished
product in only 6% of the fish from this farm. In contrast, L. monocytogenes was not
found in feces, skin of fish, or the finished product of two of the farms where fish were
raised in concrete ponds and starved 3-7 days before harvest [ 1391. Similarly, L. monocy-
togenes was not recovered from the skin, gills, intestines, tank water, diet of striped bass
grown in recirculating water tanks [203]. Experimental infection of zebrafish (Bruchy-
dunio rerio) indicated the LDSowas higher in fish than in mice and that L. monocytogenes
did not multiply in fish [ 1921. Experimental infection increased production of granulocytes
and monocytes. In contrast to L. monocytogenes, strains of L. welshimeri, L. innocuu, and
L. seeligeri killed more than 50% of the fish 7 days postinfection [ 1921
Brackett [36] proposed that contamination of fish and shellfish through their ambient
waters may influence distribution of L. monocytogenes. Surface waters, sewage effluents,
and agricultural runoff all may potentially contribute Listeriu spp. to the aquatic environ-
ment. In addition, the presence of L. monocytogenes in sea gulls may be another source
of shellfish contamination [88]. In 1959, L. monocytogenes was detected in crustaceans
gathered from a Russian stream [248]. A more recent survey conducted on the Gulf Coast
of the United States examined shrimp, oysters, and estuarine waters for L. monocytogenes
[198]. The pathogen was detected in 11% of unprocessed shrimp (n = 74) but not in
oysters (n = 7 9 , although some of the oysters were harvested from prohibited shellfish-
growing sites. In a parallel study conducted in freshwater tributaries off the Humboldt-
Arcata Bay in Northern California, L. monocytogenes was detected in freshwater samples
(61%), some of which received runoff from nearby farms. However, L. monocytogenes
was not found in oysters in that study [52] nor in oysters kept in live holding tanks in
seafood markets in Seattle [53]. Albeit a modest number of shellfish were examined, L.
monocytogenes was not detected in fresh shellfish (shrimp, cuttlefish, clams) tested in
Cochin, India [98].
Overall, these data indicate that live fish and shellfish are not likely carriers of L.
monocytogenes. Polymerase chain reaction (PCR) tests have been used to detect L. mono-
cytogenes in experimentally contaminated marinated rainbow trout [82]. With the in-
creased interest in L. monocytogenes in fish and seafoods 1771, this sensitive technique
may be useful for rapid screening of live shellfish and fish for L. monocytogenes. Neverthe-
less, the paucity of reports documenting L. monocytogenes in live freshwater fish and
shellfish suggests that Listeriu spp. detected in the retail product most likely resulted from
postharvest contamination.
TREATMENT
Poor animal husbandry, consumption of contaminated feed, and stress are important fac-
tors in precipating listeriosis. Thus identifying and eliminating these problems are critical
to preventing reoccurrences. In general, since antemortem diagnosis is rarely made, treat-
ment is seldom attempted.
Since listerial encephalitis is a rapidly debilitating disease in ruminants, treatment
must be initiated early during the course of infection if there is to be any reasonable hope
60 Wesley
REFERENCES
I. Adesiyun, A.A., and C. Krishnan. 1995. Occurrence of Yersinia enterocolitica 0:3, Listeria
monocytogenes 0:4 and thermophilic Campylobacter spp. in slaughter pigs and carcasses in
Trinidad. Food Microbiol. I2:99- 107.
2. Akpavie, S.O., and J.O. Uikheloa. 1992. An outbreak of listeriosis in cattle in Nigeria. Revue.
Elev. Med. Vet. Pays. Trop. 45:3-4:263-264.
3. Alexander, A.V., R.L. Walker, B.J. Johnson, B.R. Charlton, and L.W. Woods. 1992. Bovine
abortions attributable to Listeria ivanovii: four cases (1988- 1990). J. Am. Vet. Med. Assoc.
200:7 11-7 14.
4. Allcock, J.C. 1992. Cutaneous listeriosis. Vet. Rec. 130:18- 19.
5. Amstutz, H.E. 1980. Listeriosis. In: Bovine Medicine and Surgery. Vol. I. Santa Barbara,
CA: American Veterinary Publications, pp. 252-255.
6. Anonymous. 1990. Pilotprojekt vedrorende integreret kontrol of maelk og mejeriprodukter.
Report No. 20 1. Danish Dairy Organization.
7. Anonymous. 1991. The ecology of Listeria monocytogenes. Int. J. Food Microbiol 14:194-
199.
8. Anonymous. 1994. Veterinary Investigation Diagnosis and Analysis 111. Weybridge, UK:
Ministry of Agriculture Fisheries and Food.
9. Arimi, S.N.M., E.T. Ryser, T.J. Pritchard, and C.W. Donnelly. 1997. Diversity of Listeria
ribotypes recovered from dairy cattle, silage, and dairy processing environments. J. Food
Prot. 60:s 1 1-8 16.
10. Audurier, A., M. Gitter, and A. Raoult. 1986. Phage typing of Listeria strains isolated by
the Veterinary Investigation Centres in Great Britain from 198 I - 1984. In: A.L. Courtieu, E.P.
Espaze, A.E. Reynaud, eds., Proceedings of 9th International Symposium on the Problems of
Listeriosis, Nantes, France: University of Nantes, pp. 410.
Listeriosis in Animals 61
11. Avery, S.M., and S. Buncic. 1997. Differences in pathogenicity for chick embryos and growth
kinetics at 37C between clinical and meat isolates of Listeria monocytogenes previously
stored at 4C. Int. J. Food Microbiol. 34:319-327.
12. Baetz, A.L., and I.V. Wesley. 1995. Detection of anti-listeriolysin 0 in dairy cattle experi-
mentally infected with Listeria monocytogenes. J. Vet. Diag. Invest. 7:82-86.
13. Baetz, A.L., I.V. Wesley, and M.G. Stevens. 1996. The use of listeriolysin 0 in an ELISA, a
skin test and a lymphocyte blastogenesis assay on sheep experimentally infected with Listeria
monocvtogenes, Listeria ivanovii or Listeria innocua. Vet. Microbiol. 5 1 :15 I - 159.
14. Bailey, J.S., D.L. Fletcher, and N.A. Cox. 1989. Recovery and serotype distribution of Liste-
ria monocytogenes from broiler chickens in the southeastern United States. J. Food Prot. 52:
148- 150.
15. Bailey, J.S., D.L. Fletcher, and N.A. Cox. 1990. Listeria inonocytogenes colonization of
broiler chickens. Poult. Sci. 6:457-461.
16. Barlej, J. 1989. Listeria and listeriosis. Goats Today 82:145.
17. Barlow, R.M., and B. McGorum. 1995. Ovine listerial encephalitis: analysis, hypothesis and
synthesis. Vet. Rec. 1 I6:233-236.
18. Basher, H.A., D.R. Fowler, F.G. Rodgers, A. Seaman, and M. Woodbine. 1984. Pathogenicity
of natural and experimental listeriosis in newly hatched chicks. Res. Vet. Sci. 36:76-80.
19. Baum, K. 1994. Neurologic diseases. In: Update on Llama Medicine. Vet. Clin. North Am.
101384-385.
20. Baxter, F., F. Wright, R.M. Chalmers, J.C. Low, and W. Donachie. 1993. Characterization
by multilocus enzyme electrophoresis of Listeria moncytogenes isolates involved in ovine
listeriosis outbreaks in Scotland from 1989 to 1991. Appl. Environ. Microbiol. 59:3 126-
3129.
21. Beauregard, M., and K.L. Malkin. 1971. Isolation of Listcvia monocytogenes from brain
specimens of domestic animals in Ontario. Can. Vet. J. 12:221-223.
22. Beckers, H.J., P.S.S. Soentoro, and E.H.M. Delfgou van ,4sch. 1987. The occurrence of
Listeria monocytogenes in soft cheeses and raw milk and its resistance to heat. Int. J. Food
Microbiol. 4:249-256.
23. Beer, J., W. Seffner, and J. Potel. 1957. Listerienfunde bei Tieren und ihre Bedeutung fur
die Epidemiologie der Listeriose. Arch. Exp. Vet. Med. 11 550-577.
24. Belding, R.C., and M.L. Mayer. 1957. Listeriosis in the turkey-two case reports. J. Am.
Vet. Med. Assoc. 13I :296-297.
25. Belin, M. 1946. La listerellose equine. Bull. Acad. Vet. Fr 19:176-181.
26. Berche, P., K.A. Reich, M. Bonnichon, J.L. Beretti, C. Geoffroy, J. Faveneau, P. Cossart,
J.L. Gaillard, P. Geslin, K. Kreis, and M. Veron. 1990. Detection of anti-listeriolysin 0 for
serodiagnosis of human listeriosis. Lancet 335:624-627.
27. Bhunia, A.K. 1997. Antibodies to Listeria monocytogenes. Crit. Rev. Microbiol. 23:77- 107.
28. Biester, H.E., and L.H. Schwarte. 1940. Listerella infection in swine. J. Am. Vet. Med. Assoc.
96:339-342.
29. Blenden, D.C. 1986. Listeriosis. In: A.D. Leman, B. Straw, R.D. Glock, W.I. Mengeling,
R.H.C. Penny, and E. Scholl, eds. Diseases of Swine. 6th ed. Ames, IA: Iowa State Univer-
sity, pp. 584-590.
30. Bochdalek, R., S. Lewandowska, J. Nowacki, Z. Staroniewicz, and Z. Walchnick. 1980.
Course of experimental listeriosis in cattle, sheep and pigs. Panstwowe wydawncitwo rol-
nicze i lesne 36:609-615.
31. Bocklisch, H., D. Wilhelms, C. Mirle, S. Lange, and U. Kucken. 1991. Listeria abortions
in cattle: bacteriology, serology and epizootiology. Berl. Munch. tieriirztl. Wochenschr. 104:
307-3 13.
32. Boerlin, P., and J.C. Piffaretti. 1991. Typing of human, animal food and environmental iso-
lates of Listeria monocytogens by multilocus enzyme electrophoresis. Appl. Environ. Micro-
biol. 57: 1624- 1629.
62 Wesley
33. Bourry, A., and B. Poutrel. 1996. Bovine mastitis caused by L. monocytogenes: Kinetics of
antibody responses in serum and milk after experimental infection. J. Dairy Sci. 79:2 189-
2195.
34. Bourry, A., B. Poutrel, and J. Rocourt. 1995. Bovine mastitis caused by Listeria rnonocyto-
genes: characteristics of natural and experimental infections. J. Med. Microbiol. 43: 125-
132.
35. Bouttefroy, A., J.P. Lemaitre, and A.L. Roussset. 1997. Prevalence of Listeria sp. in drop-
pings from urban rooks (Cowus frugilelgus). J. Appl. Bacteriol. 82:641-647.
36. Brackett, R.E., 1988. Presence and persistence of Listeria rnonocytogenes in food and water.
Food Technol. 42: 162- 164.
37. Broadbent, D.W. 1972. Listeria as a cause of abortion and neonatal mortality in sheep. Aust.
Vet. J. 48:39 1-394.
38. Bryner, J., R. Thornhill, I. Wesley, and M. van der Maaten. 1988. Experimental intramam-
mary infection of dairy cows with Listeria rnonocytogenes. Abstr. Annu. Mtg. Amer. Soc.
Microbiol., Miami Beach, FL, May 8-13, Abstr. P-20.
39, Bryner, J., I. Wesley, and M. van der Maaten. 1989. Research on listeriosis in milk cows
with intramammary inoculation of Listeriu monocytogenes. Acta Microbiol. Hung. 36: 137-
140.
40. Buncic, S. 1991. The incidence of Listeria monocytogenes in slaughtered animals, in meat
and in meat products in Yugoslavia. Int. J. Food Microbiol. 112:173-180.
41. Bunning, V.K., R.G. Crawford, J.G. Bradshaw, J.T. Peeler, J.T. Tierney, and R.M. Twedt.
1986. Thermal resistance of intracellular Listeria monocytogenes cells suspended in raw bo-
vine milk. Appl. Environ. Microbiol. 52: 1398- 1402.
42. Busch, R.H., D.M. Barnes, and J.H. Sautter. 1971. Pathogenesis and pathologic changes
of experimentally induced listeriosis in newborn pigs. Am. J. Vet. Res. 32: 1313-
1320.
43. Butt, M., A. Weldon, D. Step, L.A. De, and C. Huxtable. 1991. Encephalitic listeriosis in
two adult llamas (Lama glama): clinical presentations, lesions and immunofluorescence of
Listeria monocytogenes in brainstem lesions. Cornell Vet. 3 1:25 1-258.
44. Cain, D.B., and V. McCann. 1986. An unusual case of cutaneous listeriosis. J. Clin. Micro-
biol. 23:976-977.
45. Carpenter, S.L. and M. Sevoian. 1983. Cellular immune response to Mareks Disease: listeri-
osis as a model of study. Avian Dis. 27:344-356.
46. Casanovas, L., M. de Simon, M.D. Ferrer, J. Arques, and G. Monzon. 1995. Intestinal car-
riage of campylobacters, salmonellas, yersinias and listerias in pigeons in the city of Barce-
lona. J. Appl. Bacteriol. 78: 1 I - 13.
47. Cavil], J.P. 1967. Listeriosis in chinchillas (Chinchilla laaniger). Vet. Rec. 80592-594.
48. Chambouris, R., W. Sixl, D. Stunzner, and M. Kock. 1989. Serological studies of listeriosis
antibodies in dogs in Greece. Georgr. Med. 3(suppl): 15-18.
49. Charlton, K.M. 1977. Spontaneous listeric encephalitis in sheep. Electron microscopic stud-
ies. Vet. Pathol. 14:429-434.
50. Charlton, K.M., and M.M. Garcia. 1977. Spontaneous listeric encephalitis in sheep. Light
microscopic studies. Vet. Pathol. 14:297-3 13.
51. Clark, E.G., A.S. Turner, B.G. Boysen, and B.T. Rouse. 1978. Listeriosis in an Arabian foal
with combined immunodeficiency. J. Am. Vet. Med. Assoc. 172:363-366.
52. Colburn, K.G., C.A. Kaysner, C. Abeyta, and M.M. Wekell. 1990. Listeria species in a Cali-
fornia coast estuarine environment. Appl. Environ. Microbiol. 56:2007-20 1 1.
53. Colburn, K.G., C.A. Kaysner, M.M. Wekell, J.R. Matches, C. Abeyta, and R. Stott. 1989.
Microbiological quality of oysters (Crussostrea gigas) and water of live holding tanks in
Seattle, WA markets. J. Food Prot. 52:lOO-104.
54. Cooper, G.L. 1989. A encephalitic form of listeriosis in broiler chickens. Avian Dis. 33:
182- 185.
Listeriosis in Animals 63
76. Elischerova, K., S. Stupalova, R. Helbichova, and J. Stepanek. 1979. Incidence of Listeria
monocytogenes in faeces of employees of meat processing plants and meat shops. Cesk.
Epidemiol. Mikrobiol. Imunol. 28:97- 102.
77. Embarek, P.K.B. 1994. Presence, detection and growth of Listeria monocytogenes in sea-
foods: a review. Int. J. Food Microbiol. 23:17-34.
78. Embarek, P.K.B., L.T. Hanson, 0. Enger, and H.H. Huss. 1997. Occurrence of Listeria spp.
in farmed salmon and during subsequent slaughter: comparison of ListertestB lift and the
USDA method. Food. Microbiol. 14:39-46.
79. Emerson, F.G., and A.A. Jarvis. 1968. Listeriosis in ponies J. Am. Vet. Med. Assoc. 152:
1645- 1646.
80. Engeland, I.V., H. Waldeland, E. Ropstad, H. Kindahl, and 0. Andresen. 1997. Effect of
experimental infection with Listeria monocytogenes on the development of pregnancy and
on concentrations of progesterone, oestrone sulphate and 15-ketodihydro-PGF2, in the goat.
Anim. Reprod. Sci. 45:3 11-327.
81. Ericksen, L., H.E. Larsen, T. Christiansen, M. M. Jensen, and E. Erisksen. 1988. An outbreak
of meningoencephalitis in fallow deer caused by Listeria monocytogenes. Vet. Rec. 122:
274-276.
82. Ericsson, H., and P. Stalhandske. 1997. PCR detection of Listeria monocytogenes in gra-
vad rainbow trout, Int. J. Food Microbiol. 34:28 1-285.
83. Evans, M., and G. Watson. 1987. Septicemic listeriosis in a reindeer calf. J. Wildl. Dis. 23:
3 14-3 17.
84. Facinelli, B., and P.E. Varaldo. 1989. Ignorance about Listeria. Br. Med. J. 299:738.
85. Farber, J.M., E. Daley, F. Coates, N. Beausoleil, and J. Fournier. 1991. Feeding trials of
Listeria monocytogenes with a nonhuman primate model. J. Clin. Microbiol. 29:2606-
2608.
86. Farber, J.M., G.W. Sanders, and S.A. Malcom. 1988. The presence of Listeria spp. in raw
milk in Ontario. Can. J. Microbiol. 34:95-100.
87. Fedio, W.M., and H. Jackson. 1992. On the origin of Listeria monocytogenes in raw bulk-
tank milk. Int. Dairy J. 2:197-208.
88. Fenlon, D. 1985. Wild birds and silage as reservoirs of Listeria in the agricultural environ-
ment. J . Appl. Bacteriol. 59:537-543.
89. Fenlon, D.R. 1986. Rapid quantitative assessment of the distribution of Listeria in
silage implicated in a suspected outbreak of listeriosis in calves. Vet. Rec. 118:240-
242.
90. Fenlon, D.R., T. Stewart, and W. Donachie. 1995. The incidence, numbers and types of
Listeria monocytogenes isolated from farm bulk tank milks. Lett. Appl. Microbiol. 20:57-
60.
91. Fenlon, D.R., and J. Wilson. 1989. The incidence of Listeria monocytogenes in raw milk
from farm bulk tanks in north-east Scotland. J. Appl. Bacteriol. 66:191-196.
92. Fenlon, D.R., J. Wilson, and W. Donachie. 1996. The incidence and level of Listeria monocy-
togenes contamination of food sources at primary production and initial processing. J. Appl.
Bacteriol. 8 1 :64 1-650.
93. Fensterbank, R. 1987. Vaccination with a Listeria strain of reduced virulence against experi-
mental Listeria abortion in goats. Ann. Rech. Vet. 18:415-419.
94. Fernandez-Garayzabal, J.F., L. Dominguez, J.A. Vazquez, E. Gomez-Lucia, E.R. Rodriguez-
Ferri, and G. Suarez. 1987. Occurrence of Listeria monocytogenes in raw milk. Vet. Rec.
120~258-259.
95. Finley, G.G., and J.R. Long. 1977. An epizootic of listeriosis in chinchillas. Can. Vet. J. 18:
164- 167.
96. Fraser, C.M., J.A. Bergeron, A. Mays, and S.E. Aiello, eds. 1991. The Merck Veterinary
Manual: A handbook of diagnosis, therapy, and disease prevention and control for the veteri-
narian. 7th ed. Rahway, NJ: Merck, pp. 358.
Listeriosis in Animals 65
97. Fraser. C.M., J.A. Bergeron, A. Mays, and S.E. Aiello, eds. 1991. The Merck Veterinary
Manual: A handbook of diagnosis, therapy, and disease prevention and control for the veteri-
narian. 7th ed. Rahway, NJ: Merck, pp. 1579.
98. Fuchs, R.S., and P.K. Surendran. 1989. Incidence of Listeria in tropical fish and fishery
products. Lett. Appl. Microbiol. 9:49-5 1.
99. Genigeorgis, C.A., D. Dutulescu, and J.F. Garayzabal. 1989. Prevalence of Listeria spp. in
poultry meat at the supermarket and slaughterhouse level. J. Food Prot. 52:618-624.
100. Gill, D.A. 1931. Circling disease of sheep in New Zealand. Vet. J. 87:60-74.
101. Gill, P.A., J.G. Boulton, G.C. Fraser, A.E. Stevenson, and L.A. Reddacliff. 1997. Bovine
abortion caused by Listeria ivanovii.75:2 14.
102. Gitter, M. 1985. Listeriosis in farm animals in Great Britain. In: C.H. Collins and J.M.
Grange, eds. Isolation and Identification of Microorganisms of Medical and Veterinary Im-
portance. London: Academic Press,
103. Gitter, M., R. Bradley, and P.H. Blampied. 1980. Listeria monocytogenes infection in bovine
mastilis. Vet. Rec. 107:390-393.
104. Gitter, M., C. Richardson, and E. Boughton. 1986. Experimental infection of pregnant ewes
with Listeria monocytogenes. Vet. Kec. 1 18575-578.
105. Graham, R. 1939. Listerella from a premature bovine fetus. Science 90:336-337.
106. Gray, M.L. 1958. Listeriosis in fowls-A review. Avian Dis. 2:296-314.
107. Gray, M.L. 1963. Epidemiological aspects of listeriosis. Am. J. Pub. Health 53:554-563.
108. Gray, M.L., and A.H. Killinger. 1966. Listeria monocytogenes and listeric infections. Bacte-
riol. Kev. 30:309-382.
109. Gray. M.L., C. Singh, and F. Thorp. 1956. Abortion and pre- or postnatal death of young
due to Listeria monocytogenes. 111. Studies in ruminants. Am. J . Vet. Res. 17:510-516.
110. Green, L.E., and K.L. Morgan. 1994. Descriptive epidemiology of listerial meningoencepha-
litis in housed lambs. Prev. Vet. Med. 18:79-87.
111. Greenwood, M.H., D. Roberts, and P. Burden. 1991. The occurrence of Listeria species in
milk and dairy products: A national survey in England and Wales. Int. J. Food Microbiol.
12:107-206.
112. Grini, 0. 1943. Listerella monocytogenes as a cause of septicemia in foals. Nord. Vet.
Tidsskr. 55:97- 104.
113. Grginstol, H. 1979. Listeriosis in sheep-Listeria monocytogenes excretion and immunological
state in healthy sheep. Acta Vet. Scand. 20:168-179.
114. Grginstol, H. 1984. Listeriosis in goats. Les maladies de la chevre. Nirot (France) INRA 28:
189- 192.
115. Gudding, R., H. Gronstol, and H.J. Larsen. 1985. Vaccination against listeriosis in sheep.
Vet. Rec. 117:89-90.
116. Gudding, R., L.L. Nesse, and H. Granstol. 1989. Immunisation against infections caused by
Listcria monocytogenes in sheep. Vet. Rec. 125:1 1 1- I 14.
117. Guerden, L.M.G., and A. Devos. 1952. Listerellose bijpluimvee. Vlaams. Diergeneesk.
Tschr. 2 1:165- 175.
118. Harcourt, R.A. 1966. Listeria monocytogenes in a piglet. Vet. Rec. 78:735.
119. Harvey, J., and A. Gilmour. 1992. Occurrence of Listeria spp. in raw milk and dairy products
produced in Northern Ireland. J. Appl. Bacteriol. 72: 119- 125.
120. Harvey, J., and A. Gilmour. 1994. Application of multilocus enzyme electrophoresis and
restriction fragment length polymorphism analysis to the typing of Listeria monocytogenes
strains isolated from raw milk, nondairy foods, and clinical and veterinary sources. Appl.
Environ. Microbiol. 60: 1547-1 553.
121. Hatkin, J.M., and W.E. Phillips, Jr. 1986. Isolation of Listeria rnonocytogenes from an eastern
wild turkey. J. Wildlife Dis. 22:llO-112.
122. Hayes, P.S., J.C. Feeley, L.M. Graves, G.W. Ajello, and D.W. Fleming. 1986. Isolation of
Listeria monocytogenes from raw milk. Appl. Environ. Microbiol. 5 1 :438-440.
66 Wesley
123. Hefnawy, Y., S. Moustafa, and R.S. Refai. 1989. Occurrence of Yersinia enterocolitica and
Listeria monocytogenes in fresh water fish. Assiut. Vet. Med. J. 21:135-139.
124. Heim, D., R. Fatzer, B. Hornlimann, and M. Vandevelde. 1997. Frequency of neurological
disease in cattle. Schweiz. Arch. Tierheilkd. I39:354-362.
125. Hessen, L. 1957. Listeriose hos gris. Nord. Vet. Med. 9:951-958.
126. Higgins, R., G. Goyette, R. Sauvageau, and T. Lemaire. 1987. Septicemia due to Listeria
monocytogenes in a newborn foal. Can. Vet. J. 28:63.
127. Hird, D., and C. Genigeorgis. 1990. Listeriosis in food animals: Clinical signs and livestock
as a potential source of direct (nonfoodborne) infections in humans. In: A.J. Miller, J.L.
Smith, and G.A. Somkuti, eds. Foodborne Listeriosis. Amsterdam: Elsevier, pp. 3 1-39.
128. Hofstad, M.S. 1984. Listeriosis. In: B.W. Calnek, H.J. Barnes, C.W. Beard, W.M. Reid and
H.W. Yoder, Jr., eds. Disease of Poultry. 9th ed. Ames, IA: Iowa State University Press, pp.
26 1-263.
129. Hohne, K., B. Loose, and H.P. Seeliger. 1975. Isolation of Listeria monocytogenes in slaugh-
ter animals and bats of Togo (West Africa). Ann. Microbiol. Paris 126A501-507.
130. Hunter, R. 1973. Observations on Listeria monocytogenes type 5 (Iwanow) isolated in New
Zealand. Med. Lab. Technol. 3 0 5 1-56.
131. Husu, J.R. 1990. Epidemiological studies on the occurrence of Listeria monocytogenes in
the faeces of dairy cattle. Zentralbl. Veterinarmed. B 37:276-282.
132. Husu, J.R., J.T. Beery, E. Nurmi, and M.P. Doyle. 1990. Fate of Listeria monocytogenes in
orally dosed chicks. Int. J. Food Microbiol. 1 1 :259-270.
133. Husu, J.R., J.T. Seppanen, S.K. Sivela, and A.O.L. Raurama. 1990. Contamination of raw
milk by Listeria monocytogenes on dairy farms. Zentralbl. Veteriniirmed. B 37:268-
275.
134. Hyslop, N.St.G. 1975. Epdemiologic and immunologic factors in listeriosis. In: M. Wood-
bine, ed. Problems of Listeriosis. Leicester, UK: Leicester University Press, pp. 94- 105.
135. Hyslop, N.St.G., and A.D. Osborne. 1959. Listeriosis: a potential danger to public health.
Vet. Rec. 71:1082-1091.
136. Iida, T., M. Kanzaki, T. Maruyama, S. Inoue, and C. Kaneuchi. 1991. Prevalence of Listeria
monocytogenes in intestinal contents of healthy animals in Japan. J. Vet. Med. Sci. 53:873-
875.
137. Ivanov, I. 1957. La listeriose chez les ovins et les caprins. Bull. Off. Int. Epizoot. 57571-
583.
138. Jacquet, C., B. Catimel, R. Brosch, C. Buchrieser, P. Dehaumont, V. Goulet, A. Lepoutre,
P. Veit, and J. Rocourt. 1995. Investigations related to the epidemic strain involved in the
French listeriosis outbreak in 1992. Appl. Environ. Microbiol. 61 :2242-2246.
139. Jemmi, T., and A. Keusch. 1994. Occurrence of Listeria monocytogenes in freshwater fish
farms and fish-smoking plants. Food Microbiol. 11:309-3 16.
140. Jensen, A., W. Frederiksen, and P. Gerner-Smidt. 1994. Risk factors for listeriosis in Den-
mark, 1989-1990. Scand. J. Infect. Dis. 26:171-178.
141. Jensen, N.E., F.M. Aarestrup, J. Jensen, and H.C. Wegener. 1996. Listeria monocytogenes
in bovine mastitis. Possible implication for human health. Int. J. Food Microbiol. 32:209-
216.
142. Jensen, R., and D.R. Mackey. 1979. Diseases of Feedlot Cattle, 3rd ed. Philadelphia: Lea &
Febiger, pp. 71-75.
143. Johnson, G.C., W.H. Fales, C.W. Maddox, and J.A. Ramos-Vara. 1995. Evaluation of labora-
tory tests for confirming the diagnosis of encephalitic listeriosis in ruminants. J. Vet. Diag.
Invest. 7:223-228.
144. Johnson, G.C., C.W. Maddox, W.H. Fales, W.A. Wolff, R.F. Randle, J.A. Ramos, H.
Schwartz, K.M. Heise, A.L. Beetz, and I.V. Wesley. 1996. Epidemiologic evaluation of en-
cephalitic listeriosis in goats. J. Am. Vet. Med. Assoc. 208: 1695-1696.
145. Johnson, J.L., M.P. Doyle, R.G. Cassens, and J.L. Schoeni. 1988. Fate of Listeria monocyto-
Listeriosis in Animals 67
genes in tissues of experimentally infected cattle and in hard salami. Appl. Environ. Micro-
biol. 54:497-501.
146. Jones, F.S., and R.B. Linle. 1934. Sporadic encephalitis in cows. Arch. Pathol. 18:580-581.
147. Jubb, K.V.F., and C.R. Huxtable. 1993. Listeriosis. In: K.V.F. Jubb, P.C. Kennedy, and N.
Palmer, eds. Pathology of Domestic Animals, 4th ed. San Diego, CA: Academic Press, pp.
393-397.
148. Kampelmacher, E.H. 1962. Animal products as a source of listeric infection in man. In: M.L.
Gray, ed. Second Symposium on Listeric Infection. Bozeman, MT: Montana State College,
pp. 146-156.
149. Kautter, D.A., S.J. Silverman, W.G. Roessler, and J.F. Drawdy. 1963. Virulence of Listeria
monocytogenes for experimental animals. J. Infect. Dis. 112: 167-180.
150. Kemenes, F., T. Antal, and F. Vetesi. 1971. Experimental listeriosis in pigs. Magyar Allatorv.
Lapja. 26:39-42.
151. Kemenes, F., R. Glavits, E. Ivanics, G. Kovacs, and A. Vanyi. 1983. Listeriosis of roe-deer
in Hungary. Zentralbl. Veterinarmed. B 30:258.
152. Kerlin, D.I., and R. Graham. 1945. Studies of listerellosis. VI. Isolation of Listerella mono-
ctyogerzes from the liver of a pig. Proc. Soc. Exp. Med. 58:35 1 .
153. Killinger, A.H., and M.E. Mansfield. 1970. Epizootiology of listeric infection in sheep. J.
Am. Vet. Med. Assoc. 157:1318- 1324.
154. Kimberling, C.V. 1988. Diseases of the central nervous system. In: Jensen and Swifts Dis-
eases of Sheep, 3rd ed. Philadelphia: Lea & Febiger, pp. 195-199.
155. Kirkbride, C.A. 1993. Bacterial agents detected in a 10-year study of bovine abortions and
stillbirths. J. Vet. Diag. Invest. 5:64-68.
156. Kirkbride, C.A., E.J. Bicknell, D.E. Reed, M.G. Robl, W.U. Knudtson, and K. Wohlgemuth.
1973. A diagnostic survey of bovine abortion and stillbirth in the northern plains states. J.
Am. Vet. Med. Assoc. 16236-560.
157. Kloster, O., and R. Guidding. 1987. Prevention of listeric abortion in goats by vaccination.
Vet. Rec. 120563.
158. Kovincic, I., B. Stajner, S. Zakula, and M. Galic. 1979. The finding of L. monocytogenes in
the milk of cows from infected herds. In: I. Ivanov, ed. Proceedings of Seventh International
Symposium on Listeriosis. National Agroindustrial Union, Center for Scientific Information,
Sofia, pp. 221-224.
159. Krueger, N., C. Low, and W. Donachie. 1995. Phenotypic characterization of the cells of
the inflammatory response in ovine encephalitic listeriosis. J. Comp. Pathol. 1 13:263-
275.
160. Ladds, P.W., S.M. Dennis, and C.O. Njoku. 1974. Pathology of listeric infection in domestic
animals. Vet. Bull. 44:67-74.
161. Larson, D.J. 1997. Personal communication.
162. Lechner, W., F. Allerberger, A. Bergant, E. Solder, and M. P. Dierich. 1993. Effect of Listeria
on contractibility of human uterine muscle. Z. Geburtshilfe-Perinatol. 197:179- 183.
163. Lennon, D., B. Lewis, C. Mantell, D. Becroft, B. Dove, K. Farmer, S. Tonkin, N. Yeates,
R. Stamp, and K. Mickleson. 1984. Epidemic perinatal listeriosis. Pediatr. Infect. Dis. 3:30-
34.
164. Lessing, M.P.A., G.D.W. Curtis, and I.C.F. Bowler. 1994. Listeria ivanovii infection. J. In-
fect. 29:230-23 1.
165. Leung, C., Y. Huang, and O.C. Pancorbo. 1992. Bacterial pathogens and indicators in catfish
and pond environments. J. Food Prot. 55:424-427.
166. Lhopital, S., J. Marly, P. Pardon, and P. Berche. 1993. Kinetics of antibody production
against listeriolysin 0 in sheep with listeriosis. J. Clin. Microbiol. 3 1:1537- 1540.
167. Liewen, M.B., and M.W. Plautz. 1988. Occurrence of Listeria monocytogenes in raw milk
in Nebraska. J. Food Prot. 5 1:840-841.
168. Linde, K., G.C. Fthenakis, R. Lippmann, J. Kinne, and A. Abraham. 1995. The efficacy of
68 Wesley
a live Listeria monocytogenes combined serotype 12 and serotype 4b vaccine. Vaccine 13:
923-926.
169. Loken, T., E. Aspoy, and H. Grgnstol. 1982. Listeria monocytogenes excretion and humoral
immunity in goats in a herd with outbreaks of listeriosis and in a dairy herd. Acta Vet. Scand.
23~392-399.
170. Loken, T., and H. Gronstol. 1982. Clinical investigations in a goat herd with outbreaks of
listeriosis. Acta Vet. Scand. 23:380-391.
171. Loncarevic, S., A. Milanovic, F. Caklovica, W. Tham, and M-L. Danielsson-Tham. 1994.
Occurrence of Listeria species in an abattoir for cattle and pigs in Bosnia and Hercegovina.
Acta Vet. Scand. 34: 11-15.
172. Lopez, A., and R. Bildfell. 1989. Neonatal porcine listeriosis. Can. Vet. J. 30:828-829.
173. Lovett, J., D.W. Francis, and J.M. Hunt. 1987. Listeria monocytogenes in raw milk: detection,
incidence and pathogenicity. J. Food Prot. 50: 188- 192.
174. Low, C., and K. Linklater. 1985. Listeriosis in sheep. In Practice 7:66-67.
175. Low, J.C., R.M. Chalmers, W. Donachie, R. Freeman, J. McLauchlin, and P.R. Sisson. 1992.
Pyrolysis mass spectrometry of Listeria monocytogenes isolates from sheep. Res. Vet. Sci.
53~64-67.
176. Low, J.C., and W. Donachie, 1989. Listeria in food: a veterinary perspective. Lancet 1 :322.
177. Low, J.C., and W. Donachie. 1991. Clinical and serum antibody responses of lambs to infec-
tion by Listeria monocytogenes. Res. Vet. Sci. 5 I :185- 192.
178. Low, J.C., and W. Donachie. 1997. A review of Listeria rnonocytogenes and listeriosis. Vet.
J. 15319-29.
179. Low, J.C., and C.P. Renton 1985. Septicaemia, encephalitis and abortions in a housed flock
of sheep caused by Listeria monocytogenes type 1/2. Vet. Rec. I 16:147- 150.
180. Low, J.C., F. Wright, J. McLauchlin, and W. Donachie. 1993. Serotyping and distribution
of Listeria isolates from cases of ovine listeriosis. Vet. Rec. 133:165- 166.
181. Macleod, N.S.M., and J.A. Watt. 1974. Listeria monocytogenes type 5 as a cause of abortion
in sheep. Vet. Rec. 95:365-367.
182. Mansmann, R.A., E.S. McAllister, and P. W. Pratt. 1982. Equine Medicine and Survey, 3rd
ed. Santa Barbara, CA: American Veterinary Publications, pp. 1246.
183. Mason, R.W., R.G. Brennan, and A. Corbould. 1980. Listeria monocytogenes abortion in a
mare. Aust. Vet. J. 56:613.
184. Mathews, F.P. 1928. Encephalitis in calves. J. Am. Vet. Assoc. 73513-516.
185. Mayer, H., M. Kinzler, and E. Sickel. 1976. Listeriosis in a herd of saddle horses. Berl.
Munch. tierarztl. Wochenschr. 89:209-2 1 1.
186. Mazzette, R., E. Sanna, E.P. De Santis, S. Pisanu, and A. Leoni. 1991. Experimental listeri-
osis in chickens: microbiological and anatomo-histpathological studies and health and hy-
giene considerations. Boll. Soc. Ital. Biol. Sper. 67569-76.
187. McCain, C.S., and M. Robinson. 1976. Listeria monocytogenes in the equine. Proc. Am.
Assoc. Vet. Lab. Diagn. I8:257-261.
188. McClure, H.M., and L.M. Strozier. 1975. Perinatal listeric septicemia in a Celebese black
ape. Am. J. Vet. Res. 167:637-638.
189. McLauchlin, J. 1987. Listeria monocytogenes, recent advances in the taxonomy and epidemi-
ology of listeriosis in humans. J. Appl. Bacteriol. 63: 1- 1 1.
190. McLauchlin, J. 1997. Animal and human listeriosis: a shared problem? Vet. J. 153:3-5.
191. McLauchlin, J., and J.C. Low. 1994. Primary cutaneous listeriosis in adults: an occupational
disease of veterinarians and farmers. Vet. Rec. 135615-6 17.
192. Menudier, A., F.P. Rougier, and C. Bosgiraud. 1996. Comparative virulence between differ-
ent stains of Listeria in zebrafish (Brachydonio rerio) and mice. Pathol. Biol. 44:783-789.
193. Meredith, C., and D. Schneider. 1984. An outbreak of ovine listeriosis associated with poor
flock management practices. J. S. Afr. Vet. Med. Assoc. 5555-56.
194. Meyer, E.P., and J.M. Gardner. 1970. A case of listeriosis in piglets. Aust. Vet. J. 46514.
Listeriosis in Animals 69
195. Miettinen, A., J. Husu, and J. Tuomi. 1990. Serum antibody response to Listeria monocyto-
genes, listerial excretion and clinical characteristics in experimentally infected goats, J. Clin.
Microhiol. 28:340-343.
196. Miettinen, A., and J. Husu. 1991. Antibodies to listeriolysin 0 reflect the acquired resistance
of Listcria rnonocytogenes in experimentally infected goats. FEMS Microbiol. Lett. 77: 18I -
186.
197. Morgan, J.H. 1977. Infectious keratoconjunctivitis in cattle associated with Listeria monocy-
togenes. Vet. Rec. 100:1 13- 114.
198. Motes, M.L. 1991. Incidence of Listeria spp. in shrimp, oysters and estuarine waters. J. Food
Prot. 54:17-173.
199. Mouton, R.P., and E.H. Kampelmacher. 1966. Proceedings of the 3rd International Sympo-
sium on Listeriosis, Bilthoven, Netherlands, pp. 425.
200. Nagi, M.S., and J.D. Verma. 1967. An outbreak of listeriosis in chickens. Indian J. Vet. 44:
539-543.
201. Narucka, V., and J.F. Westendorp. 1973. Het voorkomen van Listeria monocytogenes by
slachtvarkens. Tijdschr. Diergeneesk. 98: 1208.
202. Nash, M.L., L.L. Hungerford, T.G. Nash, and G.M. Zinn. 1995. Epidemiology and economics
of clinical listeriosis in a sheep flock. Prev. Vet. Med. 24:147-156.
203. Nedoluha, P.C., and D. Westhoff. 1997. Microbiological analysis of striped bass (Morone
saxatilis) grown in a recirculating system. J. Food Prot. 60:948-953.
204. Nesbakken, T., E. Nerbrink, O.J. Rotterud, and E. Borch. 1994. Reduction of Yersinia entero-
colitica and Listeria spp. on carcasses by enclosure of the rectum during slaughter. Int. J.
Food Microbiol. 23: 197-208.
205. Nilsson, A., and K.A. Karlsson. 1959. Listeria monocytogenes isolations from animals in
Sweden during 1948 to 1957. Nord. Vet. Med. 11:305-315.
206. Norrung, B., M. Solve, M. Ovesen, and N. Skogaard. 199 1. Evaluation of an ELISA test
for the detection of Listeria spp. J. Food Prot. 54:752-755.
207. Odegaard, B., R. Grelland, and S.D. Henricksen. 1952. A case of Listeria-infection in man,
transmitted from sheep. Acta Med. Scand. 67:23 1-238.
208. Ojeniyi, B., H.C. Wegener, N.E. Hjensen, and M. Bisgaard. 1996. Listeria monocytogenes
in poultry and poultry products: epidemiological investigations in seven Danish abattoirs. J.
Appl. Bacteriol. 80:395-401.
209. Osebold, J.W., J.W. Kendrick, and A. Njoku-Obi. 1960. Abortion in cattle-experimentally
with Listeria monocytogenes. J. Am. Vet. Med. Assoc. 137:227-233.
210. Osebold, J.W., J.W. Kendrick, and A. Njoku-Obi. 1960. Cattle abortion associated with natu-
ral Listeria monocytogenes infections. J. Am. Vet. Med. Assoc. 137:221-226.
21 1. Owen, C.R., A. Meis, J.W. Jackson, and H.G. Stoenner. 1960. A case of primary cutaneous
listeriosis. N. Engl. J. Med. 262: 1026-1028.
212. Paterson, J.S. 1937. Listerella infection in fowls-preliminary note on its occurrence in East
Anglia. Vet. Rec. 49: 1533- 1534.
213. Paul-Murphy, J., J.E. Markovitz, I.V. Wesley, and J.A. Roberts. 1990. Listeriosis causing
stillbirths and neonatal septicemia in outdoor housed macaques. 41 st Ann. Mtg. Am. Assoc.
Lab. Anim. Sci. 40547.
214. Peters, M., and G . Scheele. 1996. Listeriosis in a rabbitry. Dtsch. tierarztl. Wochenschr. 103:
460--462.
215. Pohjanvirta, R., and T. Huttunen. 1985. Some aspects of murine experimental listeriosis.
Acta Vet. Scand. 26563-580.
216. Potel, J. 1953/ 1954. Atiologie der Granulomatosis Infantiseptica. Wiss. Z. Martin Luther
Univ.-Halle, Wittenberg 3:341.
217. Price, H.H. 198 1. Outbreak of septicemic listeriosis in a dairy herd. Vet. Med. Small Anim.
Clin. 76:73-74.
21 8. Pustovaia, L.F. 1970. Susceptibility of wild fowl to listeriosis. Vet. Bull. 41 533-534.
70 Wesley
219. Radostits, O.M., D.C. Blood, and E.E. Gay, eds. 1994. Diseases caused by Listeria spp. In:
Veterinary Medicine: A Textbook of the Diseases of Cattle, Sheep, Pigs, Goats and Horses.
Bailliire Tindall, London, pp. 660-666.
220. Rahman, T., D.K. Sarma, B.K. Goswami, T.N. Upadlhyaya, and B. Choudhury. 1985. Occur-
rence of listerial meningoencephalitis in pigs. Indian Vet. J. 62:7-9.
221. Ralovich, B. 1984. Listeriosis Research-Present Situation and Perspective. Budapest: Akade-
miai Kiado.
222. Ralovich, B., and H. Domjiin-Kovacs. 1996. Occurrence of Listeria and listeriosis in Hun-
gary. Acta Vet. Hung. 44:277-285.
223. Ramos, J.A., M. Domingo, L. Dominguez, L. Ferrer, and A. Marco. 1988. Immunohistologic
diagnosis of avian listeriosis. Avian Pathol. 17:227-233.
224. Rea, M.C., T.M. Cogan, and S. Tobin. 1992. Incidence of pathogenic bacteria in raw milk
in Ireland. J. Appl. Bacteriol. 73:33 1-336.
225. Rebhun, W.C. 1987. Listeriosis. Vet. Clin. North Am. Food Anim. Pract. 3:75-83.
226. Rebhun, W.C., and A. delahunta. 1982. Diagnosis and treatment of bovine listeriosis. J.
Am. Vet. Med. Assoc. 180:395-398.
227. Reece, R.L., P.C. Scott, and D.A. Barr. 1992. Some unusual diseases in the birds of Victoria,
Australia. Vet. Rec. 130:178-85.
228. Reuter, R., M. Bowden, and M. Palmer. 1989. Ovine listeriosis in south coastal Western
Australia. Aust. Vet. J. 66:223-224.
229. Rocourt, J., and P. Cossart. 1997. Listeria monocytogenes. In: M.P. Doyle, L.R. Beuchat,
and T.J. Montville, eds. Food Microbiology Fundamentals and Frontiers. Washington, DC,
ASM Press: pp. 337-352.
230. Rodriguez, J.L., P. Gaya, M. Medina, and M. Nunez. 1994. Incidence of Listeria monocyto-
genes and other Listeria spp. in ewes raw milk. J. Food Prot. 57:571-575.
231. Rohrbach, B.W., F.A. Draughon, P.M. Davidson, and S.P. Oliver. 1992. Prevalence of Liste-
ria monocytogenes, Campylobacter jejuni, Yersinia enterocolitica and Salmonella in bulk
tank milk: risk factors and risk of human exposure. J. Food Prot. 55:93-97.
232. Schroeder, H., and I.B. van Rensburg. 1993. Generalized Listeria monocytogenes infection
in a dog. J. S. Afr. Vet. Assoc. 64:133-136.
233. Schuchat, A., K.A. Deaver, J.D. Wenger, B.D. Plikaytis, L. Mascola, R.W. Pinner,
A.L. Reingold, and C.V. Broome. 1992. Role of foods in sporadic listeriosis. I. Case-control
study of dietary risk factors. J.A.M.A. 267:2041-2045.
234. Schultz, G. 1967. Untersuchungen uber das Vorkommen von Listerien in Rohmilch. Mo-
natsh. Veterinaermed. 22:766-768.
235. Schwartz, B., C.A. Ciesielski, C.V. Broome, S. Gaventa, G.R. Brown, B.G. Gellin,
A.W. Hightower, and L. Mascola. 1988. Association of sporadic listeriosis with consumption
of uncooked hot dogs and undercooked chicken. Lancet 2:779-782.
236. Schwartz, J.C. 1967. Incidence of listeriosis in Pennsylvania livestock. J. Am. Vet. Med.
ASSOC.15111435-1437.
237. Scott, P.R. 1993. A field study of ovine listerial meningo-encephalitis with particular refer-
ence to cerebrospinal fluid analysis as an aid to diagnosis and prognosis. Br. Vet. J. 149:
165- 170.
238. Seastone, C.V. 1935. Pathogenic organisms of the genus Listerella. J. Exp. Med. 62:203-
212.
239. Seeliger, H.P.R. 1961. Listeriosis. New York: Hafner.
240. Seeliger, H.P. 1984. Modern taxonomy of the Listeria group relationship to its pathogenicity.
Clin. Invest. Med. 7:217-22 1.
241. Seimiya, Y., K. Ohshima, H. Itoh, and R. Murakami. 1992. Listeria septicemia with meningi-
tis in a neontal calf. J. Vet. Med. Sci. 54:1205-1207.
242. Sergeant, E.S.G., S.C.J. Love, and A. McInnes. 1991. Abortions in sheep due to Listeria
ivanovii. Aust. Vet. J. 68:39.
Listeriosis in Animals 71
243. Sharma, K.N., P.K. Mehrotra, and P.N. Mehrotra. 1983. Characterization of L. monocyto-
genes strains causing occulo-encephalitis in goats. Ind. J. Anim. Sci. 545 14-5 15.
244. Sharma, M., M.K. Batta, and R.C. Kataoch. 1996. Listeria monocytogenes abortions among
migratory sheep and goats in Himachal Pradesh. Ind. J. Anim. Sci. 66: 1 I 17- 1 1 19.
245. Sharp, M.W. 1989. Bovine mastitis and Listeria monocytogenes. Vet. Rec. 1255 12- 1513.
246. Shigidi, M.T.A. 1979. Isolation of Listeria monocytogenes from animals in the Sudan. Br.
Vet. J. 135:297-298.
247. Sindoni, L., V. Ciano, I. Picemo, A. Di Pietro, and W. Farina. 1983. Ricerche sulla epidemio-
logia della listeriosi-Nota 11: Ulteriori risultati di un indagine sierologica sulla frequenza di
anticorpi anti-Listeria in diverse specie animali dimoranti in alcune zone della Sicilia e della
Calabria. Arch. Vet. Ital. 34:103-109.
248. Shlygina, K.N. 1959. Studies of variation in the causative organism of listeriosis. Zh. Mikrob-
iol. Epidemiol. Immunobiol. 30:68-75.
249. Siragusa, G.R., J.S. Dickson, and E.K. Daniels. 1993. Isolation of Listeria spp. from the
feces of feedlot cattle. J. Food Prot. 56: 102-105.
250. Sixl, W., Z. Sebek, M. Kock, E. Marth, and H. Withalm. 1980. Serological studies of domes-
tic animals for listeriosis, Q-fever and brucellosis in Cairo. Georgr. Med. 3(suppl): 127-
128.
251. Sixl, W., E. Wisidagama, D. Stunzner, H. Withalm, and 3. Sixl-Vigt. 1988. Serological
examinations of dogs in Colombo/Sri Lanka. Georgr. Med. 1(suppl):89-92.
252. Skovgaard, N., and B. Norrung. 1989. The incidence of Li,rteria spp. in faeces of Danish
pigs and in minced pork meat. Int. J. Food Mirobiol. 859-63.
253. Slabospitskii, T.P. 1938. Pro novii mikroorganizm, vidilenii vid porosyat. (New microorgan-
ism isolated from piglets) Nauk. Zap. Kiev. Vet. Inst. 1:39. Vet. Bull. 12:367.
254. Slade, P.J., D.L. Collins-Thompson, and F. Fletcher. 1988. Incidence of Listeria species in
Ontario raw milk. Can. Inst. Food Sci. Technol. 21:425-429.
255. Smith, R.E., I.M. Reynolds, and R.A. Bennett. 1955. Listeria monocytogenes and abortion
in a cow. J. Am. Vet. Med. Assoc. 126:106-110.
256. Smyth, R.L., and M.F.M. Bamford. 1988. Neonatal listeriosis: experience in Suffolk. J. In-
fect. 17:65-70.
257. Srivastava, N.C., and S.S. Khera. 1980. The prevalence of Listeria antibodies in farm stock.
Indian Vet. J. 57:270-272.
258. Stajner, B. 1975. Excretion of Listeria through milk of infected cows. Dairy Sci. Abstr. 37:
180.
259. Stamatin, N., C. Ungureanu, E. Constantinescu, A. Solnitzky, and E. Vasilescu. 1957. Infectia
naturala cu Listeria monocytogenes la pastravul curcubeu Salmo irideus. Annuar. Inst. Ani-
mal Pathol. Hyg. Bucuresti 7: 163- 180.
260. Stone, D.L. 1987. A survey of raw whole milk for Campylohacter jejuni, Listeria monocyto-
genes, and Yersinia enterocolitica. N. Z. J. Dairy Sci. 22:257.
261. Sturgess, C.P. 1989. Listerial abortion in the bitch. Vet. Rec. 124:177.
262. Svabic-Vlahovic, M., N.D. Pantic, M. Pavicic, and J.H. Bryner. 1988. Transmission of Liste-
ria rnonocytogenes from mothers milk to her baby and to puppies. Lancet 2:2101.
263. Svenkerud, R.R. 1948. Listerella (erysipelothrix) monocytogenes infection, especially in
horses. Nord. Vet. Tidsskr. 60:321-340.
264. Svenning, M., and V. Baverud. 1992. Listeriosis diagnosed in a live foal. First recorded case
in Sweden. Svensk Veterinartiddning 44% 1-554.
265. Terplan, G., R. Schoen, W. Springmeyer, I. Degle, and H. Becker. 1986. Occurrence, behav-
iour and significance of Listeria in milk and dairy products. Arch. Lebensmittel. Hyg. 36:
131-137.
266. Terplan, G., and S. Steinmeyer. 1989. Investigations on the pathogenicity of Listeria spp.
by experimental infection of the chick embryo. Int. J . Food Microbiol. 8:277-280.
267. Timoney, J.F., J.H. Gillespie, F.W. Scott, and J.E. Barlough. 1988. Hagan and Brunners
72 Wesley
Microbiology and Infectious Diseases of Domestic Animals. 8th ed. Ithaca, NY: Comstock
Publishing Associates, pp. 241 -246.
268. Ugorski, L., J. Kaminski, and S. Strojna. 1959. Listeriosis in horses. Medycyna Wet. 15:
153- 156.
269. Unanue, E.R. 1997. Studies in listeriosis show the strong symbiosis between the innate cellu-
lar system and the T-cell response. Immun. Rev. 158: 11-25.
270. van Metre, D., G. Barrington, S. Parish, and D. Tumas. 1991. Otitis media and suppurative
meningoencephalomyelitis associated with L. monocytogenes infection in a llama. J. Am.
Vet. Med. Assoc. 199:236-240.
271. van Renterghem, B., F. Huysman, R. Rygole, and W. Verstraete. 1991. Detection and preva-
lence of Listeria monocytogenes in the agricultural ecosystem. J. Appl. Bacteriol. 7 1:211-
217.
272. Vazquez-Boland, H.J.A., L. Dominguez, M. Blanco, J. Rocourt, J.F. Fernandez-Garayzabal,
C.B. Gutierrez, R.I. Tascon, and E.F. Rodriquez-Ferri. 1992. Epidemiologic investigation of
a silage-associated epizootic of ovine listeric encephalitis, using a new Listeria selective
enumeration medium and phage typing. Am. J. Vet. Res. 3:368-371.
273. Vetesi, F., A. Balsai, and F. Kemenes. 1972. Abortion in Grays monkey (Cercopitkecus
mona) associated with Listeria monocytogenes. Acta Microbiol. Acad. Sci., Hung. 19:441-
443.
274. Vishinsky, Y., A. Grinberg, and R. Ozery. 1993. Listeria monocytogenes udder infection and
carcase contamination. Vet. Rec. 133:484.
275. Visser, I.J. 1996. Pustular dermatitis in veterinarians following delivery in domestic animals:
an occupational disease. Ned. Tijdschr. Geneeskd. 140: 1 186-1 190.
276. Vizcaino, L.L., and M.A. Garcia. 1975. A note on Listeria milk excretion in sero-positive
apparently healthy cows. In: M. Woodbine, ed. Problems of Listeriosis. Surrey, UK: Leicester
University Press, p, 74.
277. Vojinovic, G. 1992. The incidence of Listeria monocytogenes in slaughtered healthy animals
and minced meat. Acta Vet. (Beograd) 42:329-336.
278. von Amtsberg, G., A. Elsner, H.A. Grabbar, and W. Winkenwerder. 1969 Die epidemiolog-
ische und lebensmittelhygienische Bedeutung der Listerieninfektion des Rindes. Dtsch. tier-
arztl. Wschr. 76:497-50 I .
279. von Amtsberg, G., A. Elsner, H.A. Grabbar, and W. Winkenwerder. 1969. Animal Health
Yearbook. 1986. Food and Agriculture Organization of the United Nations, World Health
Organization and the International Office of Epizootics, Rome.
280. von Arda, M., W. Bisping, N, Aydin, E. Istanbulluoglu, 0. Akay, M. Izgur, Z. Karaer, S.
Diker, and G. Kirpal. 1987. Atiologische Untersuchungen uber den Abort bei Schafen unter
besonderer Beriicksichtigung des Nachweises von Brucellen, Campylobacter, Salmonellen,
Listerien, Leptospiren und Chlamydien. Berl. Munch. tierirztl. Wochschr. 100:405-408.
281. von Hartwigk, H. 1958. Zum Nachweis von Listerien in der Kuhmilch. Berlin Munch. tier-
arztl. Wochschr. 7 1 :82-85.
282. von Selbitz, H.-J. 1986. Immunological principles for control of listeriosis. Monatsh. Veteri-
naermed. 4 1 :2 17-2 19.
283. Walker, J.K., and J.H. Morgan. 1993. Ovine ophthalmitis associated with Listeria monocyto-
genes. Vet. Rec. 132:636.
284. Wallace, S., and T. Hathcock. 1995. Listeria monocytogenes septicemia in a foal. J. Am.
Vet. Med. Assoc. 207: 1325- 1326.
285. Wardrope, D.D., and N.S.M. Macleod. 1983. Outbreak of Listeria meningioenceplalitis in
young lambs. Vet. Rec. 113:213-214.
286. Webb, D., and A. Rebar. 1987. Listeriosis in an immature black buck antelope (Antilope
cewicapra) J. Wild]. Dis. 23:3 18-320.
287. Weber, A., C. Datzmann, and J. Potel. 1993. Prevalence of Listeria monocytogenes in fecal
samples from dogs and cats. Tierarztl. Umsch. 48:727-730.
Listeriosis in Animals 73
288. Weber, A., J. Potel, R. Schafer-Schmidt, A. Prell, and C. Datzmann. 1995. Investigations
on the occurrence of Listeria rnonocytogenes in fecal samples of domestic and companion
animals. Zentralbl. Hyg. Umweltmed. 198: 1 17- 123.
289. Welsh, A. 1983. Equine abortion caused by Listeria monocytogenes serotype 4. J. Am. Vet.
Med. Assoc. 182:29 1 .
290. Wesley, I.V., and F. Ashton. 1991. Restriction enzyme analysis of Listeria monocytogenes
strains associated with food-borne epidemics. Appl. Environ. Microbiol. 57:969-975.
291. Wesley, I.V., J.H. Bryner, and M.J. van der Maaten. 1989. Effects of dexamethasone on
shedding of Listeria monocytogenes in dairy cattle. Am. J. Vet. Res. 50:2009-2 1 13.
292. Wesley, I.V., M. van der Maaten, and J. Bryner. 1990. Antibody response of dairy cattle
experimentally infected with Listeria monocytogenes. Acta Microbiol. Hung. 37: 105- 1 1 1.
293. Wesley, I., J. Warg, J. Bryner, and M. van der Maaten. 1988. Agglutination titers of serum
and whey obtained from Listeria monocytogerzes-infected dairy cattle. Abstr. Ann. Mtg.
Amer. Soc. Microbiol., Miami Beach, FL, May 8-13, Abstr. E-91.
294. WHO Working Group. 1988. Foodborne listeriosis. Bull. WHO 66:42 1-428.
295. Wiedmann, M., T. Arvik, J. Bruce, J. Neubauer, F. Pierro, M.C. Smith, J. Hurley, H.O.
Mohammed, and C.A. Batt. 1997. Investigation of a listeriosis epizootic in sheep in New
York State. Am. J. Vet. Res. 58:733-737.
296. Wiedmann, M., J.L. Bruce, R. Knorr, M. Bodis, E.M. Cole, C.I. McDowell, P.L. McDo-
nough, and C.A. Batt. 1996. Ribotype diversity of Listeria monocytogenes strains associated
with outbreaks of listeriosis in ruminants. J. Clin. Microbiol. 34: 1086- 1090.
297. Wilesmith, J.W., and M. Gitter. 1986. Epidemiology of ovine listeriosis in Great Britain.
Vet. Rec. 1 19:467-470.
298. Wilkerson, M.J., A. Melendy, and E. Stauber. 1997. An outbreak of listeriosis in a breeding
colony of chinchillas. J. Vet. Diag. Invest. 9:320-323.
299. Wohler, W.H., and C.L. Baugh. 1983. Pulmonary listeriosis in feeder cattle. Med. Vet. Pract.
64:736-739.
300. Wramby, G.O. 1944. Om Listerella monocytogenes bakteriologi och om forekomst av Lister-
ella infectioner has djur. Skand. Vet. Tskr. 34:278-290.
301. Yndestad, M. 1987. Personal communication.
302. Yousif, Y.A., B.P. Joshi, and H.A. Ali. 1984. Ovine and caprine listeric encephalitis in Iraq.
Trop. Anim. Health Prod. 16:27-28.
303. Zwart, P., and J. Donker-Voet. 1959. Listeriosis bij in gevangenschap gehouden dieren.
Tijdschr. Diergeneesk. 84:7 12-7 16.
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Listeriosis in Humans
INTRODUCTION
Listeria monocytogenes has been recognized as a human pathogen since 1929 [74]. This
organism is found in multiple ecological sites throughout the environment, including soil
[ 1051, water, and decaying vegetation [ 104,1061. Recent studies of epidemic and sporadic
cases of listeriosis have increased our knowledge of important sources of L. monocyto-
genes in human illness. Epidemiological investigations during,the last 15 years have shown
that epidemic listeriosis is a foodborne disease. [ 10,17,23,27,38,44,56,66,80,85]. Simi-
larly, recent studies have suggested that a substantial proportion of sporadic cases of listeri-
osis are also caused by consumption of the organism in foods [72,89,92]. Development
of improved laboratory techniques to detect and subtype L. monocytogenes has also con-
tributed to an improved understanding of human listeriosis [7,9,11,82,83].
Human disease caused by L. monocytogenes usually occurs in certain well-defined
high-risk groups, including pregnant women, neonates, and immunocompromised adults
but may occasionally occur in persons who have no predisposing underlying condition
(Table 1). The ongoing epidemic of acquired immunodeficiency syndrome (AIDS), as
well as widespread use of immunosuppressive medications for treatment of malignancy
and management of organ transplantation, has expanded the immunocompromised popula-
tion at increased risk of listeriosis. Unlike infection with other common foodborne patho-
gens such as Salmonella, which rarely result in fatalities, listeriosis is associated with a
mortality rate of approximately 20% [34]. This high case-fatality rate, along with the
heightened awareness of listeriosis as a foodborne disease and increasing clinical concern
75
76 Slutsker and Schuchat
TABLE
1 Clinical S y n d r o m e s Associated with Infection with L isteria
rnonocytogenes
Predisposing conditions
Population Clinical presentation Diagnosis or circumstances
Pregnant women Fever, _t myalgias, & Blood culture 5
diarrhea Amniotic fluid
Preterm delivery culture
Abortion
Stillbirth
Newborn s
<7 days old Sepsis, pneumonia Blood culture Prematurity
2 7 days old Meningitis, sepsis Cerebrospinal
fluid culture
Nonpregnant adults Sepsis, meningitis, focal Culture of blood, Immunosuppression,
infections cerebrospinal advanced age
fluid, or other
normally sterile
site
Healthy adults Diarrhea and fever Stool culture in se- Possibly large inoc-
lective enrich- ulum
ment broth
about the importance of illness caused by this organism in the expanding population of
highly susceptible persons, has resulted in increased attention to the importance of L.
monocytogenes as a human pathogen.
In this chapter, we will consider various aspects of listeriosis in humans, including
infection and clinical manifestations of disease, epidemiological patterns of disease, diag-
nosis, treatment, and prevention. Information on the microbiology, ecology, pathogenesis,
detection, subtyping, manifestations of infection in other animals, and occurrence of L.
monocytogenes in various foods is presented elsewhere in this book. Although some
foodborne outbreaks of listeriosis will be discussed here as examples of epidemic disease
among humans, a more exhaustive treatment of foodborne listeriosis appears in Chap-
ter 10.
TABLE
2 Continued
No. % with
Year Population studied L. monocytogenes Method Reference
Age-, race-, and 7 0 Point preva-
hospital- lence
matched controls
Cheese plant em- 31 9.7 Point preva-
ployees lence
Household contacts 94 10.6 Point preva-
of cheese plant lence
employees
1993 Household contacts 82 21 .o Point preva- 88
of patients with lence
listeriosis (per-
sons)
Households of pa- 28 21.0 Point preva-
tients with listeri- lence
osis with at least
one carrier
1993 Healthy pregnant 147 2.7 Cumulative 40
women prevalence
(mean 2.4
specimens
per patient)
4.8% of 1147 healthy slaughterhouse workers had stool cultures yielding the organism
[ 131. Similar surveys by the same researchers documented L. monocytogenes fecal carriage
rates of 1.2% among 1034 hospitalized adults, none of 195 hospitalized children, and 1%
of 595 hospitalized adults with diarrhea. Kampelmacher et al. reported high rates of fecal
carriage among laboratory workers having daily contact with L. monocytogenes (77% of
26) as well as among office workers who had no contact with the organism (62% of 26).
Because stool cultures were collected weekly for 8 weeks, figures from this study represent
cumulative rather than point prevalence estimates [52]. Subjects had L. monocytogenes
isolated an average of 1.3 times out of the eight serial specimens collected.
Among other populations, a large stool survey conducted in Germany found that 6
of 1000 (0.6%) fecal specimens collected from persons with diarrhea yielded L. monocyto-
genes, giving a point prevalence similar to the 0.8% found in 2000 healthy food handlers
from the same area during the same time period [68]. In 1987, MacGowan et al. surveyed
177 renal transplant recipients with multiple fecal samples obtained over a 1-year period.
Overall, 10 (5.7%) individuals had positive stool cultures, and a positive culture was asso-
ciated with ranitidine use or consumption of three or more types of cheese since the begin-
ning of the year [59]. The same investigators reported fecal carriage rates of 1.8% among
171 patients with gastroenteritis attending a general practice, and 2.5% of 80 home hemo-
dialysis patients. Among all three patient groups, Listeria isolations were highest during
the months of July and August.
Among pregnant women, Lamont and Postlethwaite reported a fecal carriage rate
of L. monocytogenes of 2% among 51 women early in pregnancy (10-16 weeks), similar
to the 3.4% rate observed among 59 nonpregnant women attending the same clinic [54].
Listeriosis in Humans 79
Timing of carriage was examined among 147 women attending an antenatal clinic in the
United Kingdom [40]. One fecal specimen was obtained during each trimester. Among
the four (2.7%) women whose fecal specimens yielded L. monocytogenes, one was in the
first, two in the second, and one in the third trimester. During a large foodborne listeriosis
epidemic in Los Angeles, Mascola et al. compared fecal carriage rates in pregnant women
with listeriosis to age-, sex-, and hospital-matched controls; carriage rates were not sig-
nificantly different between the two groups (1 of 18 vs 0 of 7, respectively, P = NS) [62].
Household contacts of patients with listeriosis also have been surveyed. In Denmark,
fecal specimens from 26% of 34 household contacts of persons with listeriosis yielded L.
monocytogenes [13]. In this study, among 14 households sampled, 5 (36%) had at least
1 household member with a positive stool culture; however, only two family members
had the same serotype of L. monocytogenes as the patient. IJp to eight specimens were
collected from each household contact, suggesting that the carriage rate in this study may
not be directly comparable to others. In the United States, 82 household contacts of 28
patients with invasive listeriosis were identified through active surveillance and investi-
gated [88]. Twenty-one percent of these individuals (and households) were positive for
L. monocytogenes; 88% of 17 isolates were of the same serotype and enzyme type as the
strain from the index patient. The rate of carriage was significantly higher among persons
less than 30 years of age than among older persons. The prevalence rate among 60 house-
hold contacts of 18 pregnant women with listeriosis in Los Angeles was 8.3%, whereas
no Listeria were isolated from 30 household contacts of age-, sex-, and hospital-matched
controls [62].
occurred in persons with at least one underlying illness, although some were conditions
such as heart disease that are not traditionally considered immunosuppressive [89]. The
most frequently identified diseases or conditions were heart disease (33%), corticosteroid
therapy (3 1%), cancer (29%), renal disease (24%), and diabetes (24%); several patients
had more than one underlying condition. Malignancy, corticosteroid use, and HIV infec-
tion or AIDS were the most common immunosuppressive conditions, and at least one of
these three conditions was present in 69% of nonpregnant adult patients. In an earlier
report, 30% of patients with meningitis and 11 % of those with bacteremia caused by L.
rnonocytogenes had no recognized predisposing condition [72]. In this latter study, how-
ever, cases were largely identified through a literature review, and thus may not be compa-
rable to those identified through population-based surveillance.
There have been many reports of patients with HIV infection or AIDS who coun-
tracted listerial meningitis or bacteremia [ 12,22,24,42,611. Although listeriosis does not
appear to be a common opportunistic infection among persons with HIV infection or
AIDS, it nonetheless occurs far more frequently among these persons than among the
general population. In 1989, in a prospective population-based 2-year study in San Fran-
cisco, the incidence of listeriosis among AIDS patients was estimated to be 280 times the
baseline incidence of listeriosis in the general population [89]. Using similar methodology,
a prospective, population-based 2-year study in metropolitan Atlanta estimated that the
annual incidence of listeriosis was 52 and 115 cases per 100,000 patients for those with
HIV infection and AIDS, respectively; these rates were 62 and 145 times the rate among
nonpregnant adults not known to be infected with HIV [51]. A 1995 prospective study
in Los Angeles estimated the annual incidence to be 9 and 96 cases of listeriosis per
100,000 patients in persons with HIV infection and AIDS, respectively, compared with
a rate of 1 per 100,000 in the total population [25]. Differences in risk estimates between
these last two studies likely resulted from use of different methods to estimate the total
numbers of HIV-infected persons in the study areas.
Nonpregnant adults with listeriosis most frequently present with sepsis, meningitis,
or meningoencephalitis. Although meningitis is usually reported to be the most common
form of listeriosis in adults, recent reports have documented bacteremia to be even more
common. In the United States, active surveillance in an aggregate population of 34 million
persons in 1986 found that 66% of 179 nonpregnant adults had bacteremia without menin-
gitis, 19% had meningitis with concurrent bacteremia, and 12% had meningitis without
documented bacteremia [35]. Presenting symptoms in nonpregnant adults with central
nervous system listeriosis may include fever, malaise, ataxia, seizures, and altered mental
status. Listerial brain stem encephalitis (rhombencephalitis) occurs infrequently and is
characterized by asymmetrical cranial nerve deficits, cerebellar signs, and hemiparesis or
hemisensory deficits [5,97,101]. Fever is generally present in patients with bacteremia;
other nonspecific symptoms such as malaise, fatigue, and abdominal pain may also occur.
In meningitis caused by L. monocytogenes, the cerebrospinal fluid may exhibit a pleo-
cytosis; the Gram stain may show gram-positive bacilli but is often unrevealing. Because
the spinal fluid white cell count and differential, glucose, and protein levels can vary
widely, the spinal fluid profile cannot be used to differentiate listerial meningitis from
meningitis caused by other bacteria.
In addition to sepsis, meningitis, and meningoencephalitis, a variety of other clinical
manifestations of infection with L. rnonocytogenes have been described. Endocarditis from
L. monocytogenes occurs primarily in patients with an underlying cardiac lesion, including
prosthetic or porcine valves, and is clinically indistinguishable from other causes of endo-
Listeriosis in Humans 81
carditis [8,33]. Focal infections are rare and usually result from seeding during a preceding
bacteremic phase. Several different sites of involvement have been reported, including
endophthalmitis [6], septic arthritis [70], osteomyelitis [45], pleural infection [63], and
peritonitis [72]. Cutaneous infections without bacteremia have been reported in persons
handling infected animals [7S] and in accidentally exposed laboratory workers [2].
life. Between 45 and 70% of neonatal listeriosis is of early onset [31,64]; this disease
often presents with sepsis rather than meningitis [35]. Less frequently, infants with early-
onset disease may present with granulomatosis infantiseptica, a syndrome characterized
by disseminated abscesses or granulomas in multiple internal organs, including the liver,
spleen, lungs, kidney, and brain [39]. In this syndrome, evidence of amnionitis or meco-
nium-stained fluid may be present, and the infant may appear obviously ill; in some in-
stances, however, the infant may merely appear weak and may develop respiratory or
circulatory insufficiency. Early-onset disease in the neonate may be complicated by aspira-
tion of meconium fluid with resultant respiratory complications, including cyanosis, apnea,
and pneumonia.
same subtype of L. monocytogenes was isolated from the stools of ill persons and the
chocolate milk. I11 persons were more likely than well persons to have elevated antilisterio-
lysin 0 levels and to have stool cultures that yielded L. monocytogenes. None of the
individuals involved in this outbreak had a chronic illness or immunodeficiency. Of partic-
ular note, three cases of sporadic invasive listeriosis in two other states were also linked
to consumption of the implicated chocolate milk; the same subtype of L. monocytogenes
as the outbreak strain was isolated from these patients.
Other outbreak investigations also support the concept of febrile gastroenteritis being
caused by L. rnonocytogenes. In an outbreak of invasive listeriosis in Philadelphia in 1986
and 1987, case-patients were significantly more likely than controls to have reported fever,
vomiting, or diarrhea in the week before the cases positive culture [93]. In another out-
break investigation, two pregnant women who attended a catered party each delivered
infants infected with the same strain of L. monocytogenes [80]. Diarrhea or fever was
reported by 22% of the 36 party attendees. However, stool cultures were not obtained
until several weeks after the party, and the outbreak strain of L. monocytogenes was iso-
lated from only one other party attendee, so a correlation between L. monocytogenes stool
carriage and gastrointestinal symptoms could not be definitively established. Finally, in
an outbreak of gastroenteritis among immunocompetent adults attending a supper party
in Italy, diarrhea and fever occurred in over 70% of the ill party-goers, and two developed
bacteremia caused by L. rnonocytogenes [84]. The median incubation period from time
of the supper to onset of gastrointestinal symptoms was 18 hours. Although the same
strain of L. rnonocytogenes that was isolated from the patients was also cultured from
several foods leftover from the supper, no stool specimens collected from ill persons
yielded L. rnonocytogenes.
The frequency of febrile gastroenteritis caused by L. monocytogenes remains unde-
termined, as does the infectious dose and characteristics ofthe host that are associated
with this syndrome. Clinicians and public health officials should consider examining stool
cultures for L. rnonocytogenes in outbreaks of illness characterized by fever, diarrhea,
headaches, and myalgia, if stool cultures for other more common enteric pathogens have
been negative. When such an outbreak is suspected, care should be taken to notify the
laboratory that L. monocytogenes is suspected, so that appropriate special culture media
are used.
Epidemic Listeriosis
The first convincing evidence that listeriosis can be a foodborne disease comes from a
1981 outbreak in Nova Scotia [85]. Thirty-four pregnancy-associated cases and seven
cases in nonpregnant adults occurred over a 6-month period in the Maritime Provinces.
Twenty-seven percent of the infants who were born alive died. No patient had evidence
of underlying immunosuppression. Case-patients were significantly more likely than con-
trols to have consumed locally produced coleslaw in the 3 months before illness onset.
The epidemic strain was subsequently isolated from coleslaw in the refrigerator of one
84 Slutsker and Schuchat
TABLE
3 Foodborne Outbreaks of Invasive Listeriosis
No. of Implicated % of
cases (or likely) perinatal L. monoctogenes
Reference Year Place (deaths) vehicle cases se rotype
patient, and later from two unopened packages of the product. On review of the production
process, it was determined that cabbage used in the coleslaw came from a farm where
cases of listeriosis in sheep had occurred, and that the cabbage fields had been fertilized
with raw sheep manure. Harvested cabbage was stored over the winter and spring in an
unheated shed, potentially enhancing growth of L. monocytogenes. As well as establishing
listeriosis as a foodborne disease, this outbreak also highlighted the potential for uncooked
vegetables to be a source of infection.
Another outbreak of listeriosis, in Massachusetts in 1979, may also have involved
raw produce (441. Twenty patients with serotype 4b infection were hospitalized during a
2-month period during 1979; only nine cases had been detected in the previous 26 months.
Ten of the patients were immunosuppressed adults and five died. Fifteen patients were
thought to have acquired their infection in the hospital. Patients were more likely than
controls to have consumed tuna fish, chicken salad, or cheese, but no one brand was
implicated. It was postulated that raw celery and lettuce, served as a garnish with these
foods, may have been the vehicle of infection. Although a definitive source of infection
was not identified in this outbreak, information suggested that gastrointestinal tract condi-
tions might be important in acquiring infection. Case-patients were significantly more
likely than controls to have taken cimetidine or antacids, raising the possibility that, like
salmonellosis, decreased gastric acidity might increase the chance that L. monocytogenes
could survive passage through the stomach.
Pasteurized milk was identified as the most likely vehicle of infection in another
large outbreak of listeriosis in Massachusetts in 1983 [27]. Forty-nine cases occurred over
a 2-month period, 42 in immunosuppressed adults and 7 in pregnant women; the overall
Listeriosis in Humans 85
case-fatality rate was 29%. Case-patients were more likely than neighborhood-matched
controls to have consumed a specific brand of 2% fat pasteurized milk; other evidence
supporting this milk as the vehicle of infection included a dose-response effect, a protective
effect of drinking low-fat milk (1 % or skim), an association between the implicated brand
of milk and cases of listeriosis in another state, and the linking of a specific phage type
of L. monocytogenes with infection in the 2% milk drinkers. The 2% milk came from
farms where cows were known to have had listeriosis, and multiple serotypes (but not
the epidemic strain) of L. monocytogenes were isolated from raw milk at the implicated
dairy. No defects in the pasteurization process were noted at the dairy. Although this
outbreak initially raised the question of whether pasteurization was adequate to eliminate
L. monocytogenes from milk, subsequent investigations have shown that L. monocytogenes
is inactivated by proper pasteurization [ 181. In this outbreak, contamination likely occurred
during post-pasteurization handling.
The largest North American outbreak of listeriosis occurred in Los Angeles County,
California, in 1985; 142 cases were detected over an 8-month period (561. Pregnant women
accounted for 93 cases, and nonpregnant adults 49 cases; 48 of the nonpregnant adult
cases had predisposing conditions for listeriosis. Among the pregnancy-associated cases,
87% occurred in Hispanic women. The case-fatality rate was 32% among the perinatal
cases (all were fetal or neonatal deaths) and 37% in the nonpregnant adults. A case-control
study implicated a particular brand of Mexican-style soft cheese produced locally in Cali-
fornia as the vehicle, and the epidemic serotypes and phage type were isolated from un-
opened packages of this product. Inadequate pasteurization and mixing of pasteurized and
unpasteurized milk both likely contributed to the contamination.
This outbreak provided valuable data on the incubation period of invasive listeriosis,
since food histories were available for four patients who had a single known exposure to
the implicated cheese. The median incubation period in these patients of 31 days (range
11-70 days) is far longer than that observed for most common foodborne pathogens, and
it highlights the difficulty in obtaining a relevant food history when investigating cases
of sporadic listeriosis.
This outbreak was detected quickly through public health surveillance, because
many infections occurred in an ethnic minority group that sought care primarily at one
medical facility. However, it is likely that the outbreak would not have been as readily
detected had the product been distributed over a larger geographical area or eaten by
a more diverse group of consumers. Detection of outbreaks can be improved by active
surveillance and timely reporting and by serotyping and subtyping L. monocytogenes iso-
lates.
Another soft cheese-related outbreak occurred in Switzerland during 1983 to 1987
with 122 cases of listeriosis affecting 65 pregnant women (and their infants) and 57 non-
pregnant adults [10]. Over one-half of the nonpregnant adult cases had no underlying
predisposirig condition; the case-fatality rate for nonpregnant patients was 32% [ 171. In-
creasing age and clinical presentation with meningoencephalitis were independently asso-
ciated with an increased risk of death. Neurological sequelae were present in 30% of
survivors at follow-up 5 months to 3 years later. Although early case-control studies failed
to incriminate a particular food, in 1987 investigators implicated a locally produced soft
cheese as the vehicle of infection. Two epidemic strains of L. monocytogenes serotype
4b with a particular phage type were isolated from the product and led to an international
recall.
Two recent outbreaks in Europe were associated with ready-to-eat meats. In En-
86 Slutsker and Schuchat
gland, Wales, and Northern Ireland, the annual total of listeriosis cases approximately
doubled from 1987 to 1989 compared with the annual totals for the 3 previous years. Of
the 823 isolates reported during 1987-1989,30-54% were of two subtypes of L. monocy-
togenes serovar 4b (4bX and 4b phage type 6,7) that had occurred less commonly before
and after this period [66]. A microbiological survey showed that L. monocytogenes con-
tamination in meat pit6 from one manufacturer was more frequent (48% of 107 samples
of pit6 from the implicated manufacturer vs 4% of 781 samples of pit6 from other manu-
facturers) and heavy (1 1% of samples of pit6 from manufacturer A with 21000
organismdg vs 0.6% of samples of pit6 from other manufacturers with 2 1000 organisms/
g). Ninety-six percent of pgt6 isolates from manufacturer A were 4bX or 4b phage type
6,7 compared with 19% of isolates from other manufacturers. Patients infected with either
of these two subtypes were significantly more likely to have eaten pit6 than patients in-
fected with other strains. Warning about pit6 consumption and removal of manufacturer
As pit6 from sale in late 1989 resulted in a dramatic decrease in the incidence of listeri-
osis. This investigation illustrated how subtyping can help clarify surveillance data and
ultimately lead to public health action.
In 1992, a different ready-to-eat meat product was implicated in an outbreak of 279
cases of listeriosis in France [38]. Ninety-two cases (33%) were pregnancy related and
187 occurred in nonpregnant adults; 73 (39%) of the nonpregnant adults had no known
predisposing condition for listeriosis. A case-control study implicated one brand of pork
tongue in jelly as the major vehicle in the outbreak; however, other ready-to-eat meats,
cross contaminated by the implicated meat at retail stores where the implicated brand of
pork tongue in jelly was sold, were thought to have been responsible for some infections.
The epidemic strain was isolated from samples of food, and subtyping analysis helped to
confirm the findings of the epidemiological investigation that implicated the pork tongue
in jelly [47].
Heightened surveillance efforts in France have also led to detection of two smaller
outbreaks of listeriosis. In 1993, an outbreak of 39 cases was associated with rilletes (pork
pit&)[37]. In 1995, 20 cases were traced to Brie de Meaux cheese made from raw milk.
Implicated cheese was removed from sale based on results of the epidemiological investi-
gation [37].
Sporadic Disease-Incidence
Although much has been learned about epidemic listeriosis, most cases of human listeriosis
occur sporadically. In the United States, voluntary disease reporting and hospital discharge
data have been used to estimate the number of sporadic listeriosis cases [21]. However,
such methods are generally insensitive and result in underestimates of the true incidence
of listeriosis in the population.
Beginning in 1986, active surveillance for listeriosis in the United States has been
done by the Centers for Disease Control and Prevention (CDC) in several well-defined
populations representing different geographical areas. Surveillance officers systematically
contacted infection control practitioners at all acute-care hospitals and clinical microbiol-
ogy laboratories in the study areas to collect information on all patients from whom L.
monocytogenes was isolated from a normally sterile site [35,89]. The study population
ranged from 19 to 34 million depending on the number of study sites participating each
year [99].
In 1986, in an aggregate population of 34 million persons, the annual incidence of
Listeriosis in Humans 87
listeriosis was 7 cases per million; of the 246 cases that occurred, 67 (27%) were perinatal
and 179 (73%) were nonperinatal [35]. Perinatal listeriosis rates were highest in Los
Angeles (24.3 per 100,000 live births), whereas nonperinatal rates did not vary signifi-
cantly among the study sites. The estimated sensitivity of the surveillance system was
93%.
From 1988 to 1990, in an aggregate population of 19 million, the annualized inci-
dence of listeriosis was 7.4 per million. Incidence rates varied by geographical site, with
the highest rate being observed in San Francisco (9.3 per million) and the lowest rate in
Oklahoma (4.8 per million) [89]. No clear seasonal trends were noted.
In the most recent analysis of combined data from 1989 to 1993 for a study popula-
tion of 19 million, the annualized incidence of listeriosis decreased from 7.9 per million
in 1989 to 4.4 per million in 1993; this decrease was distributed uniformly throughout the
different geographical areas [99]. Based on these data, projected estimates of the number of
cases and deaths from listeriosis in the entire United States population were 1965 cases
and 489 deaths in 1989, decreasing to 1092 cases and 248 deaths in 1993. Case-fatality
rates (23-25%) did not vary significantly by year. Pregnancy-associated cases accounted
for about one third of all cases each year. Both perinatal and nonperinatal case rates
showed similar decreases over the 4-year period. Serotypes 4b (43%), 1/2b (35%), and
1/2a (20%) accounted for almost all infections. The observed decrease in incidence may
have resulted from enhanced listeriosis prevention efforts by the US food industry, includ-
ing enforcement by regulatory agencies of a zero-tolerance policy for processed meat, and
intensified clean-up programs in meat-processing facilities. Published dietary recommen-
dations for consumers may also have contributed to the decreased disease incidence
[20,28,29].
Numerous reports of listeriosis incidence rates in other cities and countries have
been published. For example, based on a search of hospital records, the estimated incidence
of listeriosis in the English city of Bristol from 1983 through 1992 was 3.5 per million
[50]. In England, Wales, and Northern Ireland in 1991, the estimated annual incidence
based on passive case reporting was 1.8 per million [71]. In Denmark, monitoring of
laboratory-diagnosed cases during the 1980s resulted in an estimate of six to seven cases
per million per year [49]. Listeriosis incidence estimates from a number of countries have
been recently summarized [81]. These rates should be interpreted in the context of the
methods used for case ascertainment and reporting in each country.
and unopened packages of turkey frankfurters at a retail store [ 191. Subsequent investiga-
tion of the turkey frankfurter production facility found that cultures from a conveyor belt
transporting finished frankfurters yielded the case strain of L. monocytogenes [ 1071. Sys-
tematic culturing at various points in the production process identified likely points where
L. monocytogenes was being introduced into the product and suggested appropriate control
points for reducing contamination in such food processing facilities.
From 1988 to 1990, a larger case-control study of 165 patients and 376 controls
was conducted that included microbiological assessment of foods eaten by patients [89].
Case-patients were significantly more likely than controls to have eaten soft cheeses or
delicatessen counter foods. In a separate analysis examining dietary risks among a subset
of patients defined as highly immunosuppressed (persons with malignancy, AIDS, or organ
transplants or who had received corticosteroids or chemotherapy), consumption of un-
dercooked chicken was associated with a threefold increased risk of listeriosis. Other expo-
sures associated with an increased risk of sporadic disease included recent use of antacids,
laxatives, or H2-blocking agents.
In the microbiological component of this study, foods were collected from the refrig-
erators of 123 patients [78]. L. monocytogenes was isolated from at least one food in the
refrigerators of 64% of patients. Highest contamination rates among the 20 13 food speci-
mens were seen in beef (36%) and poultry (31%) with 7.6% of ready-to-eat foods (pro-
cessed meats, raw vegetables, leftovers, and cheeses) also yielding L. monocytogenes.
One-third of refrigerators contained food isolates of L. monocytogenes that were the same
enzyme type as those isolated from the patient. In multivariate analysis, foods that were
ready-to-eat, foods that contained high numbers of L. monocytogenes, and foods that
yielded serovar 4b were associated with disease.
Dietary risk factors for sporadic listeriosis were also examined in a recent study in
Denmark; drinking unpasteurized milk or eating pgt6 were the only risk factors identified
[49]. However, one-third of cases reported during the study period could not be included
in the risk analysis for sporadic disease, because the ill persons were infected with an
outbreak strain epidemiologically linked to Danish blue-mold cheeses.
5% sheep, horse, or rabbit blood. The organism is usually identified within 36 h. Isolation
of the organism from other sources such as stool specimens that contain large numbers
of competing microorganisms is more difficult; these specimens should be selectively
enriched for Listeria spp. before being plated, on Listeria-selective media. Identification
of L. monocytogenes by use of fluorescent antibody methods or approaches that use DNA
probes coupled with PCR technology may prove useful for some specimens. Experimental
assays for antibody to listeriolysin 0 have been useful in some epidemiological investiga-
tions [23] and have been used to support the diagnosis in culture-negative listeriosis of
the central nervous system [32].
TREATMENT
Controlled trials to determine the optimal antibiotic therapy for listeriosis have not been
done. Bacteriostatic drugs such as chloramphenicol or tetracycline have been associated
with high treatment failure rates, and they are not recommended [98]. Generally, ampicillin
or penicillin has been recommended as the drug of choice. However, relapses have been
reported in immunosuppressed patients after 2 weeks of penicillin therapy [ 1031. The
ability of L. monocytogenes to grow and survive within cells probably explains the poor
response to bacteriostatic drugs and the slow response to penicillin [98]. Intracellular con-
centrations of penicillin may be insufficient for complete eradication. Since many immuno-
suppressed patients have a decreased ability to clear infected cells, antibiotic treatment
for 3 to 6 weeks may be prudent [4]. Optimal length of therapy for other groups of patients
has not been established. A prudent treatment course may be 2 weeks for listeriosis in
pregnancy; 2-3 weeks for neonatal listeriosis; 2-4 weeks for nonimmunosuppressed
adults with meningitis and bacteremia; and longer for complicated infections such as endo-
carditis.
Although experimental evidence suggests that aminoglycosides are synergistic with
ampicillin or penicillin in vitro, they penetrate cells poorly and may be ineffective in
the living host. L. monocytogenes continues to grow in cells despite high extracelluar
concentrations of aminoglycosides [43].
Trimethoprim-sulfamethoxazolereadily enters cells and kills L. monocytogenes, and
it may be the most effective treatment. This drug combination has proved effective in
patients with listeriosis who have hypersensitivity to penicillin [ 5 ] .
addition, ready-to-eat foods such as frankfurters and leftover foods should be cooked until
steaming hot before being eaten. These persons may also choose to avoid delicatessen
foods or thoroughly reheat cold cuts before eating.
In addition to individual advice for consumers, control of listeriosis requires action
from public health agencies and the food industry. Important control strategies from public
health agencies include developing and maintaining timely and effective disease surveil-
lance programs, promptly investigating clusters of listeriosis cases, and enforcing current
regulations designed to minimize L. monocytogenes in foods that are consumed without
further cooking. The food industry should continue to develop and implement hazard
analysis critical control point programs (HACCP) to minimize the presence of L. monocy-
togenes at important points in the processing, distribution, and marketing of processed
foods [ 11.
REFERENCES
1. Anonymous. 1991. Listeria monocytogenes: recommendations by the National Advisory
Committee on microbiological criteria for foods. Int. J. Food. Microbiol. 14:185-246.
2. Anspacher, R., K.A. Borchardt, M.W. Hannegan, and W.A. Boyson. 1966. Clinical investiga-
tion of Listeria monocytogenes as a possible cause of human fetal wastage. Am. J. Obstet.
Gynecol. 94:386-390.
3. Anton, W. 1934. Kritisch-experimentaller Beitrag zur Biologie des Bakterium monocyto-
genes. Zentralb. Bakteriol. Mikrobiol. Hyg. A 131:89-103.
4. Armstrong, D. 1995. Listeria monocytogenes, In G.L. Mandell, J.E. Bennett, R. D o h , eds.
Mandell, Douglas, and Bennetts Principles and Practice of Infectious Diseases. New York,
NY: Churchill Livingstone, pp. 1880- 1885.
5. Armstrong, R.W., and P.C. Fung. 1993. Brainstem encephalitis (rhombenephalitis) due to
Listeria monocytogenes: case report and review. Clin. Infect. Dis. 16:689-702.
6. Ballen, P.H., F.R. Loffredo, and B. Painter. 1979. Listeria endophthalmitis. Arch. Ophthal-
mol. 97:lOl-102.
7. Baloga, A.O., and S.K. Harlander. 1991. Comparison of methods for discrimination between
strains of Listeria monocytogenes from epidemiological surveys. Appl. Environ. Microbiol.
57:2324-2331.
8. Bassan, R. 1986. Bacterial endocarditis produced by Listeria monocytogenes: case presenta-
tion and review of the literature. Am. J. Clin. Pathol. 63522-527.
9. Bassler, H.A., S.J.A. Flood, K.J. Livak, J. Marmaro, R. Knorr, and C.A. Batt. 1995. Use of
a fluorogenic probe in a PCR-based assay for the detection of Listeria monocytogenes. Appl.
Environ. Microbiol. 61 :3724-3728.
10. Bille, J. 1990. Epidemiology of human listeriosis in Europe, with special reference to the
Swiss outbreak. In: A.J. Miller, J.L. Smith., and G.A. Somkuti, eds. Foodborne Listeriosis.
Amsterdam: Elsevier, pp. 7 1-74.
11. Bille, J., and J. Rocourt. 1996. WHO international multicenter Listeria monocytogenes sub-
typing study-rationale and set-up of the study. Int. J. Food Microbiol. 32:25 1-262.
12. Bizet, C., D. Mechali, J. Rocourt, and F. Fraisse. 1989. Listeria monocytogenes bacteremia
in AIDS. Lancet 1:501.
13. Bojsen-MQller, J. 1972. Human listeriosis: diagnostic, epidemiological, and clinical studies.
Acta Pathol. Microbiol. Scand. 229 (Sect B. suppl):72-92.
14. Bortolussi, R. 1990. Neonatal listeriosis. Semin. Perinatol. 14(suppl.):44-48.
15. Boucher, M., and M.L. Yonekura. 1984. Listeria meningitis during pregnancy. Am. J. Perina-
tol. 1:312-318.
16. Buchner, L.H., and S.S. Schneier. 1968. Clinical and laboratory aspects of Listeria monocyto-
genes infection, with a report of ten cases. Am. J. Med. 45:904-921.
Listeriosis in Humans 91
17. Biila, C.J., J. Bille, and M.P. Glauser. 1995. An epidemic of food-borne listeriosis in Western
Switzerland: description of 57 cases involving adults. Clin. Infect. Dis. 20:66-72.
18. Centers for Disease Control. 1988. Update-listeriosis and pasteurized milk. M.M.W.R. 37:
764-766.
19. Centers for Disease Control. 1989. Listeriosis associated with consumption of turkey franks.
M.M.W.R. 381267-268.
20. Centers for Disease Control. 1992. Preventing Foodborne Illness: Listeriosis. Division of
Bacterial and Mycotic Diseases, National Center for Infectious Diseases, US Centers for
Disease Control and Prevention, Atlanta.
21. Ciesielski, C.A., A.W. Hightower, S.K. Parsons, and C.V. Eh-oome. 1988. Listeriosis in the
United States:1980-1982. Arch. Intern. Med. 148:1416-1419.
22. Coffey T., M. Nelson, M. Bower, and B.G. Gazzard. 1989. Listeria monocytogenes meningi-
tis in an HIV-infected patient. AIDS 3:614-615.
23. Dalton, C.B., C.C. Austin, J. Sobel, P.S. Hayes, W.F. Bibb, L.M. Graves, B. Swaminathan,
M.E. Proctor, and P.M. Griffin. 1997. Listeriosis from chocolate milk: linking of an outbreak
of febrile gastroenteritis and sporadic invasive disease. N. Engl. J. Med. 336: 100-
105.
24. Decker, C.F., G.L. Simon, R.A. DiGioia, and C.U. Tuazon. 1991. Listeria monocytogenes
infections in patients with AIDS: report of five cases and review. Rev. Infect. Dis. 13:413-
417.
25. Ewert, D.P., L. Lieb, P.S. Hayes, M.W. Reeves, and L. Mascola. 1995. Listeria monocyto-
genes infection and serotype distribution among HIV-infected persons in Los Angeles
County, 1985-1992. J. Acquir. Immune Defic. Syndr. Hum. Retrovirol. 8:461-465.
26. Farber, J.M., and P.I. Peterkin. 1991. Listeria rnonocytogenes, a food-borne pathogen. Micro-
biol. Rev. 55:476-5 1 1.
27. Fleming D.W., S.L. Cochi, K.L. MacDonald, J. Brondum, P. S. Hayes, B.D. Plikaytis, M.B.
Holmes, A. Audurier, C.V. Broome, and A.L. Reingold. 1985. Pasteurized milk as a vehicle
of infection in an outbreak of listeriosis. N. Engl. J. Med. 312:404-407.
28. Food and Drug Administration. 1992. Eating Defensively: Food Safety Advice for Persons
with AIDS. Food and Drug Administration. FDA publication 92-2232, Washington, D.C.
29. Food Safety and Inspection Service. 1992. Backgrounder: Li,steria rnonocytogenes. Washing-
ton, DC: U.S. Department of Agriculture.
30. Frederiksen, B. 1992. Maternal septicemia with Listeria rnonocytogenes in second trimester
without infection of the fetus. Acta Obstet. Gynecol. Scand. 7 1:3 13-3 15.
31. Frederiksen, B. 1992. Feto-maternal listeriosis in Denmark. 1981-1988. J. Infect. 24:277-
287.
32. Gaillard, J.L., J.L. Beretti, M. Boulot-Tolle, J.M. Wilhelm, J.L. Bertrand, T. Herbelleau, and
P. Berche. 1992. Serological evidence for culture negative listeriosis of the central nervous
system. Lancet 340560.
33. Gallagher, P.C., C.A. Amedia, and C. Watanakunakorn. 1986. Listeria rnonocytognes endo-
carditis in a patient on chronic hemodialysis, successfully treated with vancomycin-gentami-
cin: case report. Infection 14:125- 128.
34. Gellin, B.G., and C.V. Broome. 1989. Listeriosis. J.A.M.A. 261:1313-1320.
35. Gellin, B.G., C.V. Broome, W.F. Bibb, R.E. Weaver, S. Gaventa, L. Mascola, and the Listeri-
osis Study Group. 1991. The epidemiology of listeriosis in the United States-1986. Am.
J. Epidemiol. 133:392-401.
36. Giraud, J.R., F. Denis, F. Gargot, T. Fizazi, P. Babin, R.Y. Rautlin, A. Hoppeler, J. Brisou,
and 1.Tourris. 1973. La listeriose. Incidence dans les interruptions spontanees de la grossese.
Nouv. Presse Med. 2:215-218.
37. Goulet, V., C. Jacquet, V. Vaillant, I. Rebikre, E. Mouret, C. Lorente, E. Maillot, F. Stainer,
and J. Rocourt. 1995. Listeriosis from consumption of raw-milk cheese. Lancet 345: 1581-
1582.
92 Slutsker and Schuchat
38. Goulet, V., A. Lepoutre, J. Rocourt, A. L. Courtieu, P. Dehaumont, and P. Veit. 1993. Epi-
dkmie de listkriose en France: bilan final et rksultats de Ienqu2te kpidkmiologique. Bull.
Epidimiol. Hebdom. 4: 13- 14.
39. Gray, M.L., and A.H. Killinger. 1966. Listeria monocytogenes and listeric infections. Bacte-
riol. Rev. 30:309-382.
40. Gray, J.W., J.F.R. Barrett, S.J. Pedler, and T. Lind. 1993. Faecal carriage of Listeria during
pregnancy. Br. J. Obstet. Gynaecol. 100:873-874.
41. Harisdangkul, V., S. Songcharoen, and A.C. Lin. 1992. Listerial infections in patients with
systemic lupus erythematosus. South. Med. J. 85:957-960.
42. Harvey, R.L., and P.H. Chandreskar. 1988. Chronic meningitis caused by Listeria in a patient
infected with the human immunodeficiency virus. J. Infect. Dis. 157:1091- 1092.
43. Havell, E.A. 1986. Synthesis and secretion of interferon by murine fibroblasts in response
to intracellular Listeria monocytogenes. Infect. Tmmun. 54:787-92.
44. Ho, J.L., K.N. Shands, G. Friedland, P. Eckind, and D. W. Fraser. 1986. An outbreak of
type 4b Listeria monocytogenes infection involving patients from eight Boston hospitals.
Arch. Intern. Med. 146520-524.
45. Houang, E.T., C.J. Williams, and P.F.M. Wrigley. 1976. Acute Listeria monocytogenes os-
teomyelitis. Infection 4: 1 13- 1 14.
46. Hume, O.S. 1976. Maternal L. monocytogenes septicemia with sparing of the fetus. Obstet.
Gynecol. 48(suppl):33S-34S.
47. Jacquet, C., B. Catimel, R. Brosch, C. Buchreiser, P. Dehaumont, V. Goulet, A. Lepoutre,
P. Veit, and J. Rocourt. 1995. Investigations related to the epidemic strain involved in the
French listeriosis outbreak in 1992. Appl. Environ. Microbiol. 6 1 :2242-2246.
48. Jean, D., J. Croize, P. Hirtz, C. Legeais, I. Pelloux, M. Favier, M.R. Mallaret, P. Le Noc,
and P. Rambaud. I99 1. Infection nosocomiale 2 Listeria monocytogenes en maternitk. Arch.
Fr. Pediatr. 48:4 19-422.
49. Jensen, A., W. Frederiksen, and P. Gerner-Smidt. 1994. Risk factors for listeriosis in Den-
mark, 1989-1990. Scan. J. Infect. Dis. 26:171-178.
50. Jones, E.M., S.Y. McCulloch, D. S. Reeves, and A. P. MacGowan. 1994. A 10 year survey
of the epidemiology and clinical aspects of listeriosis in a provinical English city. J. Infect.
29:9 I - 103.
51. Jurado, R.L., M.M. Farley, E. Pereira, R.C. Harvey, A. Schuchat, J.D. Wenger, and D.S.
Stephens. 1993. Increased risk of meningitis and bacteremia due to Listeria monocytogenes
in patients with human immunodeficiency virus infection. Clin. Infect. Dis. I7:224-227.
52. Kampelmacher, E.H., and L.M. van Noorle Jansen. 1972. Further studies on the isolation of
Listeria monocytogenes in clinically healthy individuals. Zentralb. Bakteriol. Mikrobiol. Hyg.
Abt. I. Orig. Reihe A 2221:70-77.
53. Kraus A., A.R. Cabral, J. Sifuentes-Osornio, and D. Alarcon-Segovia. 1994. Listeriosis in
patients with connective tissue diseases. J. Rheumatol. 2 1 :635-638.
54. Lamont, R.J., and R. Postlethwaite. 1986. Carriage of Listeria monocytogenes and related
species in pregnant and non-pregnant women in Aberdeen, Scotland. J. Infect. 13:187-193.
55. Larsson S., A. Cederberg, S. Ivarsson, L. Svanberg, and S. Cronberg. 1978. Listeria monocy-
togenes causing hospital acquired enterocolitis and meningitis in newborn infants. Br. Med.
J. 2:473-474.
56. Linnan, M.J., L. Mascola, X.D. Lou, V. Goulet, S. May, C. Salminen, D.W. Hird, M.L.
Yonkura, P. Hayes, R. Weaver, A. Audurier, B.D. Plikaytis, S.L. Fannin, A. Kleks, and C.V.
Broome. 1988. Epidemic listeriosis associated with Mexican-style cheese. N. Engl. J. Med.
3 19:823-828.
57. Lorber, B. 1997. Listeriosis. Clin. Infect. Dis. 24: I - 1 I .
58. Louria, D.B., T. Hensle, D. Armstrong, H.S. Collins, A. Blevins, D. Krugman, and M. Buse.
1967. Listeriosis complicating malignant disease: a new association. Ann. Intern. Med. 67:
26 1-268.
Listeriosis in Humans 93
59. MacGowan, A.P., R.J. Marshall, I.M. MacKay, and D.S. Reeves. 1991. Listeria faecal car-
riage by renal transplant recipients, haemodialysis patients and patients in general practice:
its relation to season, drug therapy, foreign travel, animal exposure, and diet. Epidemiol.
Infect. 106:157- 166.
60. Mascola, L., D.P. Ewert, and A. Eller. 1994. Listeriosis: a previously unreported medical
complication in women with multiple gestations. Am. J. Obstet. Gynecol. 170:1328- 1332.
61. Mascola L., L. Lieb, J. Chiu, S.L. Fannin, and M.J. Linnan. 1988. Listeriosis: an uncommon
opportunistic infection in patients with the acquired immuriodeficiency syndrome. Am. J.
Med. 84: 162- 164.
62. Mascola, L., F. Sorvillo, V. Goulet, B. Hall, R. Weaver, and M. Linnan. 1992. Fecal carriage
of Lisreria rnonocytoRenes-observations during a community-wide, common-source out-
break. Clin. Infect. Dis. 15557-558.
63. Mazzuli T., and I.E. Salit. I99 I . Pleural fluid infection caused by Listeria rnonocytogenes:
case report and review. Rev. Infect. Dis. 13564-570.
64. McLauchlin, J.S. 1990. Human listeriosis in Britian, 1967-85, a summary of 722 cases: 1 .
Listeriosis during pregnancy and in the newborn. Epidemiol. Infect. 104:181- 189.
65. McLauchlin, J.S. 1990. Human listeriosis in Britian, 1967-85, a summary of 722 cases: 2.
Listeriosis in non-pregnant individuals, a changing pattern of infection and seasonal inci-
dence. Epidemiol. Infect. 104:19 1-201.
66. McLauchlin, J., S.M. Hall, S.K. Velani, and R.J. Gilbert. 1991. Human listeriosis and pate:
a possible association. Br. Med. J. 303:773-775.
67. Mossey, R.T., and J. Sondheimer. 1985. Listeriosis in patients with long-term hemodialysis
and transfusional iron overload. Am. J. Med. 79:397-400.
68. Muller, H.E. 1990. Listeria isolations from feces of patients with diarrhea and from healthy
food handlers. Infection. 18:97- 100.
69. Nelson, K.E., D. Warren, A. M. Tomasi, T.N. Raju, and D. Vidyasagar. 1985. Transmission
of neonatal listeriosis in a delivery room. Am. J. Dis. Child. 139:903-905.
70. Newman, J.H., S. Waycott, and L.M. Cooney Jr.. 1979. Arthritis due to Listeria rnonocyto-
genes Arthritis Rheum. 22: 1 139- 1 140.
71. Newton, L., S.M. Hall, M. Perlerin, and J. McLauchin. 1992. Listeriosis surveillance: 1991.
Comniun. Dis. Rep. 2:R 142-R 144.
72. Nguyen, M.H., and V.L. Yu. 1994. Listeria monocytogenes peritonitis in cirrhotic patients.
Dig. 13s. Sci. 39:215-218.
73. Niemnn, R.E., and B. Lorber. 1980. Listeriosis in adults: a changing pattern. Rev. Infect.
Dis. 2:207-227.
74. Nyfeldt, A. 1929. Etiologie de la mononucleose infectieuse. Soc. Biol. I0 I 590-592.
75. Owen C.R., A. Meis, J.W. Jackson, and H.G. Stoenner. 1960. A case of primary cutaneous
listeriosis. N. Engl. J . Med. 262: 1026-1028.
76. Paul, M.L., D.E. Dwyer, C. Chow, J. Robson, I. Chambers, G. Eagles, and V. Ackerman.
1994. Listeriosis-a review of eighty-four cases. Med. J. Austral. I60:489-493.
77. Pinner, R.W., and C.V. Broome. 1992. Lisferia monocytogenes. In: Gorbach, Bartlett, and
Blacklow, eds. Infectious Diseases. Philadelphia: Saunders. pp. 1437- 1440.
78. Pinner, R.W., A. Schuchat, B. Swaminathan, P.S. Hayes, K. Deaver, R.E. Weaver, B.D.
Plikaytis, M. Reeves, C.V. Broome, J.D. Wenger, and the Listeria Study Group. 1992. Role
of foods in sporadic listeriosis, 11: Microbiologic and epiderniologic investigation. J.A.M.A.
267: 2046-2050.
79. Rappaport, F., M. Rabinovitz, R. Toaff, and N. Krochic. 1960. Genital listeriosis as a cause
of repeated abortions. Lancet 1 : 1273- 1275.
80. Riedo, F.X., R.W. Pinner, M.L. Tosca, M.L. Cartter, L.M. Graves, M.W. Reeves,
R. E. Weaver, B. D. Plikaytis, and C. V. Broome. 1994. A point-source foodborne listeriosis
outbreak: documented incubation period and possible mild illness. J. Infect. Dis. 17O:693-
696.
94 Slutsker and Schuchat
81. Ryser, E.T., and E.H. Marth. 1991. Listeriosis in humans. In: Listeria, Listeriosis, and Food
Safety. New York: Marcel Dekker, pp. 45-65.
82. Ryser, E.T., and E.H. Marth, 199 1. Conventional methods to detect and isolate Listeria mono-
cytogenes. In: Listeria, Listeriosis, and Food Safety. New York: Marcel Dekker, pp. 120-
193.
83. Ryser, E.T., and E.H. Marth, 1991. Rapid methods to detect Listeria rnonocytogenes in food
and environmental samples. In: Listeria, Listeriosis, and Food Safety. New York: Marcel
Dekker, pp. 194-239.
84. Salamina, G., E.D. Donne, A. Niccolini, G. Poda, D. Cesaroni, M. Bucci, R. Fini, M. Maldini,
A. Schuchat, B. Swaminathan, W. Bibb, J. Rocourt, N. Binkin, and S. Salmoso. 1996. A
foodborne outbreak of gastroenteritis involving Listeria monocytogenes. Epidemiol. Infect.
1 17:429-436.
85. Schlech, W.F., P.M. Lavigne, R.A. Bortolussi, A.C. Allen, E.V. Haldane, A.J. Wort,
A.W. Hightower, S.E. Johnson, S.H. King, E.S. Nicholls, and C.V. Broome. 1983. Epidemic
listeriosis-evidence for transmission by food. N. Engl. J. Med. 308:203-206.
86. Schlech, W.F. 1997. Listeria gastroenteritis-old syndrome, new pathogen. N. Engl. J. Med.
336:130-132.
87. Schuchat, A. 1997. Listeriosis and pregnancy: Food for thought. Obstet. Gynecol. Surv. 52:
72 1-722.
88. Schuchat, A., K.A. Deaver, P.S. Hayes, L. Graves, L. Mascola, and J.D. Wenger. 1993.
Gastrointestinal carriage of Listeria monocytogenes in household contacts of patients with
listeriosis. J. Infect. Dis. 167:1261-1262.
89. Schuchat, A., K. Deaver, J.D. Wenger, B.D. Plikaytis, L. Mascola, R.W. Pinner, A.L. Rein-
gold, C.V. Broome, and the Listeria Study Group. 1992. Role of foods in sporadic listeriosis,
I: Case-control study of dietary risk factors. J.A.M.A. 267:2041-2045.
90. Schuchat, A., C. Lizano, C.V. Broome, B. Swaminathan, C. Kim, and K. Winn. 1991. Out-
break of neonatal listeriosis associated with mineral oil. Pediatr. Infect. Dis. 10:183-189.
91. Schuchat, A., B. Swaminathan, and C.V. Broome. 1991. Epidemiology of human listeriosis.
Clin. Microbiol. Rev. 4: 169- 183.
92. Schwartz, B., C.A. Ciesielski, C.V. Broome, S. Gaventa, G.R. Brown, B.G. Gellin,
A.W. Hightower, L. Mascola, and the Listeria Study Group. 1988. Association of sporadic
listeriosis with consumption of uncooked hotdogs and undercooked chicken. Lancet 2:779-
782.
93. Schwartz, B., D. Hexter, C.V. Broome, A.W. Hightower, R.B. Hirschhorn, J.D. Porter, P.
S. Hayes, W. F. Bibb, B. Lorber, and D. G. Faris. 1989. Investigation of an outbreak of
listeriosis: new hypotheses for the etiology of epidemic Listeria rnonocytogenes infections.
J. Infect. Dis. 159:680-685.
94. Simmons, M.D., P.M. Cockroft, and O.A. Okubadejo. 1986. Neonatal listeriosis due to cross-
infection in an obstetric theatre. J. Infect. I3:235-239.
95. Simpson, J.F., J.P. Leddy, and J.D. Hare. 1967. Listeriosis complicating lymphoma. Am. J.
Med. 43:39-49.
96. Skogberg, K., J. Syrjanen, M. Jahkola, O.V. Renkonen, J. Paavonen, J. Ahonen, J Kontiainen,
S. Ruutu, and V. Valtonen. 1992. Clinical presentation and outcome of listeriosis in patients
with and without immunosuppressive therapy. Clin. Infect. Dis. 14:815-821.
97. Soo, M.S., R.D. Tien, L. Gray, P. I. Andrews, and H. Friedman. 1993. Mesenrhombencepha-
litis: MR findings in nine patients. Am. J. Radiol. 160:1089- 1093.
98. Southwick, F.S., and D.L. Purich. 1996. Intracellular pathogenesis of listeriosis. N. Engl. J.
Med. 334:770-776.
99. Tappero, J.W., A. Schuchat, K.A. Deaver, L. Mascola, J.D. Wenger, and the Listeriosis Study
Group. 1995. Reduction in the incidence of human listeriosis in the United States: effective-
ness of prevention efforts? J.A.M.A. 273: 1 1 18- 1 122.
100. Tipple, M.A., L.A. Bland, J.J. Murphy, M.J. Arduino, A.L. Panlilio, J.J. Farmer, M.A. Tour-
Listeriosis in Humans 95
MICHAEL
KUHNAND WERNER
GOEBEL
University of Wurzburg, Wurzburg, Germany
INTRODUCTION
Studies which aimed to unravel the pathogenicity of Listeria monocytogenrs and its inter-
action with host cells on the cellular, molecular, and genetic levels were initiated only 10
years ago. The early studies used transposon mutagenesis and infection of primary and
established cell lines (epithelia] cell, fibroblast, and macrophage) to obtain insights into
the interaction of L. monocytogenes with eukaryotic host cells (reviewed in refs. 1 1 1 and
174). Development of new genetic tools now allows manipulation of L. monocytogenes,
which has, together with the cell culture models, greatly broadened our understanding of
the molecular and cell biology of L. monocytogenes infections.
Most studies on the cell biology of L. monocytogenes infections used epithelia-like
and macrophage-like cell lines [55,143,194]. Macrophages actively ingest L. monocyto-
genes, but internalization of the bacterium by normally nonphagocytic cells is triggered
by L. moncicytogenes-specific products. Besides the internalization step, the intracellular
life cycle of listeriae in phagocytes or normally nonphagocytic mammalian cells is, how-
ever, very similar. The pathogen first appears in a vacuole, which is subsequently lysed
by most of the ingested bacteria allowing L. monocytogenes to escape into the cytoplasm.
Whereas listeriae begin to replicate in the cytoplasm, cells remaining in the phagosome
are killed and digested. Concomitant with the onset of intracellular replication, L. monocy-
togenes induces nucleation of host actin filaments which form a cloud around the bacterial
cell. The actin filaments are then rearranged to a polar tail which consists of short actin
97
98 Kuhn and Goebel
FIGURE1 The intracellular life cycle o f Listeria monocytogenes. See text for details.
(Adapted from Refs. 14 and 194 and kindly provided by J. Kreft.)
filaments and other host actin binding proteins which stabilize this structure. Formation
of the actin tail at one pole of the bacterial cell produces the propulsive force which moves
the listeriae through the cytoplasm of the host cell. This bacterial movement requires
continuous de novo actin polymerization. Listeriae which reach the surface of the infected
host cell induce formation of pseudopod-like structures with the bacterium at the tip and
the actin tail behind. These pseudopods are taken up by neighboring cells. The bacteria
thus entering the neighboring cells are within a vacuole that is surrounded by a double
membrane which is subsequently lysed to release the listeriae into the cytoplasm of the
new host cell (Fig. 1).
Most of the known virulence genes whose products are involved in the intracellular
life cycle of L. monocytogenes are clustered on the chromosome in the so-called PrfA-
dependent virulence gene cluster. The cluster comprises six well-characterized genes,
p$A, plcA, hly, mpl, actA, and plcB (Fig. 2 and Table 1) and three small open reading
frames (ORFs) of unknown functions downstream of plcB, called X, Y, and Z. The ends
of the gene cluster are defined by genes coding for housekeeping enzymes. Distal from
prfA, defining the left border of the gene cluster, is located the prs gene encoding a
phosphoribosyl-pyrophosphate synthetase [72,115]. The Zdh gene coding for lactate dehy-
FIGURE2 The virulence gene cluster from L. monocytogenes. Black boxes represent
PrfA-boxes and arrows represent transcripts. (Kindly provided b y F. Engelbrecht.)
Pathogenesis of Listeria monocytogenes 99
TABLE
1 Features of the L. rnonocytogenes Virulence Determinantsa
mRNA Sig. PrfA Temp.
Gene Protein ORF bp (kb) AAh MWcal' MWd PI' Seq.' Reg' Reg .g
drogenase together with the orfs A and B [72,196] mark the right border of the gene
cluster downstream from plcB and the small orfs X, Y, and Z. The products of these
virulence genes are: listeriolysin (encoded by hly), a phosphatidylinositol-specific phos-
pholipase C (pZcA),a phosphatidylcholine-specificphospholipase C (pZcB),a metallopro-
tease (mpl), ActA, a protein involved in actin polymerization (actA), and the positive
regulatory factor PrfA (prfA). The internalin genes inlA, inlB, and inlC coding for inter-
nalin, InlB, and InlC, respectively, the iap gene coding for p60, and other genes suggested
to play a role in virulence are located outside the virulence gene cluster. Most of these
are, however, connected to the virulence cluster genes, as they are also regulated by the
transcriptional activater PrfA (see below).
SS 8 SR LR
InlB N C*
I I I I 1 I
30 63 238 399 466 559
SS 8 SR
InlC N C
I I I
34 62 234
FIGURE
3 Schematic structure of the members of the internalin family: InlA, InlB, and
InlC. SS, signal peptide; SR, short leucine-rich repeat; LR, long repeat; MA, membrane
anchor.
tions suggesting the basolateral membrane as an entry site for L. monocytogenes [ 1901.
Antibodies directed against the leucine-rich repeat region of internalin block entry of L.
monocytogenes into cells expressing E-cadherin, thereby uriderlining the importance of
the repeat regions of internalin for its function as an invasin [ 1361.
InlB, a 630-amino acid protein, also carries an N-terminal transport signal sequence
and repeat domains, but, in contrast to InlA, has no obvious membrane anchor and no
cell wall-spanning region (Fig. 3) [41]. Nevertheless, InlB is a listerial surface protein,
but the mechanism(s) which target it to the bacterial surface are unknown [40]. Recently
it was shown that inlB mutants still expressing inlA are invasive for human enterocyte-
like Caco-2 cells. InlB is required but is obviously not sufficient to promote entry of L.
monocytogmes into hepatocytes 1401, but results concerning its role in the entry of epithe-
lial cells are controversial [40,125]. Invasion of fibroblasts by L. monocytogenes seems
to be independent of either inlA or inlB and even double mutants are still invasive for
fibroblasts, suggesting that different cell type-specific adhesion and invasion systems are
present in I,. monocytogenes [41,125]. The high sequence sirnilarity of inlA and inlB indi-
cates that the two genes originate from a common ancestral gene and represent members
of a gene family in L. monocytogenes. One additional member of this gene family, called
inlC, was cloned and characterized and encodes a small (297 amino acids), secreted protein
(Fig. 3) which is mainly expressed at later stages of the intracellular life cycle and obvi-
ously not involved in the entry process into epithelia1 cells [48]. InlC was identified inde-
pendently and called Irp (the gene irpA), and it also was found present in the supernatant
liquid of the closely related species L. ivanovii [39,124].
Protein p60
Rough mutants of L. monocytogenes expressing reduced amounts of a 60-kD extracellular
protein, termed p60, show appreciably reduced uptake by 3T6 fibroblast cells [ 1091. These
p60 mutants (also referred to as R-mutants because of their rough colony appearance)
from long cell chains which possess double septa between the individual cells. Treatment
of L. monocytogenes R-mutants with partially purified p60 protein disassociates these cell
chains into normal-sized single bacteria which are again invasive for fibroblasts. Ultrason-
ication, which leads to physical disruption of the cell chains, produces similar single cells
102 Kuhn and Goebel
which are, however, noninvasive. On treatment with wild-type p60, these ultrasonicated
mutant cells are again able to invade fibroblasts [16,109]. Reduced invasiveness of p60
mutants is only observed with certain mammalian host cells. Cell chains of p60 mutants
adhere normally to Caco-2 epithelial cells and are perfectly invasive on disruption of the
bacterial cell chains by ultrasonication without addition of p60 [ 161. Protein p60 is a major
secreted protein of all L. monocytogenes isolates [16,109], but it is also found on the cell
surface of L. monocytogenes [163]. In contrast to other virulence factors, p60 is also an
essential metabolic enzyme of L. rnonocytogenes, since it possesses murein hydrolase
activity which appears to be involved in a late step of cell division [202]. The gene coding
for this obviously bifunctional protein, called iap (invasion associated protein), was cloned
from a L. monocytogenes gene bank using an anti-p60 antiserum and sequenced [103].
Its expression is controlled on the posttranscriptional level by a yet unknown mechanism
[102]. The amino acid sequence of p60 predicts an extremely basic protein of 484 amino
acids with a 27-amino acid signal sequence and an extended repeat domain consisting
of 19 threonine-asparagine units which are separated by a proline-serine-lysine motif. A
single cysteine found in the C-terminal part of p60 is probably essential for its enzymatic
activity [103,202]. A stretch of 50 amino acids in the N-terminal part of the protein which
is also present in p60 proteins of the other Listeria species shows homology to sequences
found in an autolysin of Streptococcusfaecalis (Enterococcusfaecalis). In this species, the
sequence is thought to represent a possible murein binding site. Interestingly, the sequence
motive occurs twice in the p60 protein of L. monocytogenes [202].
Protein ActA
The listerial surface protein ActA (Fig. 4), a major virulence factor primarily involved in
actin-based motility [38,99] (see below for details), was recently suggested also to play
a role in internalin-independent uptake of L. monocytogenes by epithelial cells [3,108].
Analysis of the invasive capacity of an inZA deletion mutant and mutant PKP-1 without
the virulence gene cluster genes [48] complemented with multiple copies of PrfA strongly
suggest that PrfA-dependent proteins from the virulence gene cluster may cause invasion
of Caco-2 cells in the absence of InlA [ 1081. Such an ActA-promoted attachment and
invasion of Chinese hamster ovary (CHO) epithelia-like cells as well as IC-21 murine
macrophages was mediated by interaction of the listerial surface protein ActA with a
heparan-sulfate proteoglycan receptor [3]. Electrostatic interactions between heparan sul-
fate and positively charged residues in the N-terminal part of ActA could presumably
result in low-stringency binding to cell surface proteoglycan receptors which are widely
distributed in mammalian cells [3]. Whether the proposed low-stringency binding of L.
monocytogenes to heparan sulfate proteoglycan receptors triggers uptake directly or results
in adequate presentation of other bacterial factors to the host cell membrane which ulti-
mately lead to phagocytosis remains to be clarified.
FIGURE4 Schematic structure of the bifunctional protein ActA. SS, signal peptide;
AP, region critical for actin polymerization; PRR, proline-rich repeat; TA, transmem-
brane domain.
Pathogenesis of Listeria monocytogenes 103
entry by the apical surface of polarized Caco-2 cells was also observed [91]. Attachment
of wild-type L. monocytogenes to these host cells induces structural modification(s) in
microvilli which are not observed with less invasive p$A mutants of L. monocytogenes
(see below) [66,91]. Whether the apical route of infection of polarized cells is also inter-
nalin-dependent is not known, and the overall significance of each of these two proposed
entry sites of epithelial cells is still under debate.
The picture of listerial invasion is becoming more and more complex, since afore-
mentioned data point to different invasion sites on the same cell as well as to different
mechanisms involved in invasion of different cell types. These observations are in line
with the idea of tissue tropism, with different known and unknown bacterial factors like
internalins and p60 being responsible for invasion of different tissues during infection.
Presently the data can be summarized as follows: Protein p60 is clearly not involved in
epithelial cell invasion, but it may play a role in fibroblast invasion [16,109]. SalrnoneZZa
strains expressing p60 are taken up significantly better as the control strains by macro-
phages and hepatocytes, also indicating a role of p60 for invasion of these cell types [85].
Internalin is an important factor in epithelial cell and hepatocyte infection, but the capacity
of InlA alone to promote epithelial cell entry is still under debate, since conflicting results
have been published [40,54,125]. InlB, originally proposed as a specific factor for hepato-
cyte invasion [40], is now suggested also to be critical for epithelial cell invasion, but
both internalins play no role in fibroblast invasion [ 1251. The eukaryotic receptor for InlB
is not known, but the recent report of InlB-dependent stimulation of phosphoinositide-3-
kinase being required for efficient L. monocytogenes invasion of nonprofessional phago-
cytic cells [87] underlines the importance of InlB in the invasion process. High-efficiency
binding to and invasion of human endothelial cells by L. monocytogenes is dependent on
one or both inZAB gene products. However, low-level invasion in the absence of both
internalins was also observed [45]. Internalin-independent invasion was also reported for
the dentritic cell line CB1 [76] and at low levels also for Caco-2 epithelial cells [57]. The
role of the small internalin family member InlC (Irp), if any, in invasion is unclear [48].
L. monocytogenes can spread from macrophages to endothelial cells by direct transfer of
the bacteria from one cell to the other [45]. Because of its expression in the late stages
of the infectious cycle, InlC was thought to be involved in this type of heterologous cell-
to-cell spreading event [48].
The in vivo significance of these listerial proteins is even less clear. InZAB as well
as iap mutants are clearly impaired in virulence in the mouse model [40,85]. However,
an inlAB mutant was only transiently impaired in persistence in the liver and behaved like
the wild-type in spleens and lymph nodes of infected mice [40]. In a different study,
however, inZAB mutants were only rarely found inside hepatocytes, compared with the
wild-type strain, indicating a role for the inlAB locus in hepatocyte invasion in vivo [58].
In contrast, Gregory et al. [74] found that the inlAB operon of L. monocytogenes is not
required for entry into hepatic cells in vivo.
sults from the action of a cytolysin, called listeriolysin 0 (LLO). In experimental infec-
tions, all virulent strains were hemolytic, whereas nonhemolytic strains were avirulent.
Nonhemolytic mutants which were obtained after transposon rnutagenesis using the conju-
gative transposons Tn1545 or Tn916 [56,92,152] always proved to be avirulent in the
mouse model. Virulence is restored in hemolytic revertants, which have lost the transposon
insertion or by introduction of the cloned hly gene into a nonhemolytic L. monocytogenes
transposon mutant [25]. Despite the clear correlation between hemolysis and virulence,
the level of hemolysin production in vitro is not directly proportional to virulence of
producing strains in the mouse [94], suggesting that synthesis of LLO under intracellular
conditions is different from that observed under extracellular growth conditions.
Listeriolysin 0, a secreted protein of 58-60 kD, belongs to a family of pore-forming,
sulfhydryl-activated cytolysins for which streptolysin 0 is the prototype [ 1801. All mem-
bers of this family are inhibited by low concentrations of cholesterol and oxygen and
activated by reducing agents like DTT. Cholesterol is considered as a receptor for these
cytolysins, since this component inhibits pore formation and toxicity. On addition to eryth-
rocytes, toxin monomers oligomerise in the target cell membrane to form stable pores
which can be visualized by electron microscopy [ 1481. Listeriolysin 0 has been purified
to homogeneity and its toxicity, as determined by intraperitoneal injection in the mouse,
shows a LDSoof 1.7 pg per mouse. Optimal hemolytic activity is found at pH 5.5, a pH
value which is much lower than that determined for the other SH-activated cytolysins
[60], a property which is in accord with the function of LLO in the acidified phagosome
(see below).
The gene encoding LLO, hly, was cloned from strains of different serovars of L.
monocytogvnes and sequenced [35,132,137]. The deduced amino acid sequence for LLO
yielded 529 amino acids, including a N-terminal signal sequence of 25 amino acids. As
expected, the sequence shows extended homologies with the protein sequences of other
SH-activated cytolysins. The highest homology is observed in the C-terminal part and
includes a highly conserved undecapeptide containing the unique cysteine which was
thought to be essential for cytolytic activity. Site-directed mutagenesis revealed, however,
that cysteine is not essential for hemolytic activity. In contrast, a tryptophan residue, in
close vicinity to cysteine, appears to be required for both hemolytic activity and virulence
[139].
The role of LLO in virulence was determined by injection, intravenous and intraperi-
toneal, of wild-type and nonhemolytic mutants of L. monocytogenes into mice and follow-
ing the fate of the listeriae in liver and spleen. In contrast to the wild-type strain, nonhemo-
lytic mutants are eliminated from these organs within a few hours without eliciting
protective immunity [25,56,92,152]. The role of LLO in intracellular survival was deter-
mined using different mouse and human cell lines. In the human enterocyte-like cell line
Caco-2 [55], mouse 3T6 fibroblasts [ I 131, and mouse CL.7 fibroblasts [152], nonhemolytic
L. monocytogenes mutants were as invasive as isogenic wild-type strains. The nonhemo-
lytic mutants are, however, incapable of intracellular growth and survival within these
host cells and also in mouse peritonea1 macrophages [ 1 131, mouse bone marrow-derived
macrophages [ 1521, and the mouse macrophage-like cell line 5774 [ 1521. Electron micros-
copy of infected macrophages and epithelia1 cells reveals that nonhemolytic L,
monocytogenes mutants which are found inside cells are unable to open the phagosome
to escape into the cytoplasm of the host cells [55,194]. Bafilomycin treatment inhibits
vacuolar acidification and prevents L. monocytogenes from escaping phagosomes of in-
fected Caco-2 cells. These findings further support the importance of the low pH activity
106 Kuhn and Goebel
optimum of LLO for its role as a vacuole opener [24]. Taken together, these results suggest
that hemolytic activity is indispensable for lysis of the phagosomal membrane. Additional
evidence for LLO being essential for lysis of the phagosomal membrane and for intracellu-
lar growth was obtained by infection of macrophages with a Bacillus subtilis strain ex-
pressing LLO [7]. This engineered strain escapes from the phagosome into the cytoplasm,
whereas the nonhemolytic B. subtilis parental strain stays in the phagosome, as do the
nonhemolytic L. monocytogenes mutants.
Recently, Portnoy and coworkers [ 88,891 analyzed the role of LLO by constructing
L. monocytogenes strains which secrete the closely related extracellular cytolysin perfrin-
golysin instead of LLO. Such a strain escaped from the vacuole but damaged the host
cell. Using an elegant selection procedure, mutants were isolated which did not damage
the host cell on perfringolysin expression in the cytoplasm. The mutated perfringolysins
were either less hemolytic at neutral pH, generally less active, or had a shorter half-life
in the cytoplasm. Thus the low activity of LLO at neutral pH values and its short half-life
in the cytoplasm are critical parameters of its suitability as a phagosome opener without
concomitant cytotoxicity. Strains expressing mutated perfringolysins which allow intracel-
Mar growth without cell damage are, however, totally avirulent. This unexpected finding
points to additional functions for LLO in converting the host cell cytoplasm into a suitable
growth compartment [ 64,891.
Fusion of L. monocytogenes-containing phagosomes with endosomes has been ob-
served in electron microscopy studies [ 1941.However, it is not known whether such an
event is necessary for L. monocytogenes to progress through its intracellular life cycle.
The recent description of rab5-regulated fusion of L. manocytogenes-containing phago-
somes with endosomes and the observation that live L. monocytogenes upregulates this
process by recruiting rab5 to the membrane strongly argues for phagogosome-lysosome
fusion as being an important step in the life cycle of L. monocytogenes [l].
Listeriolysin 0-independent escape of L. monocytogenes from primary vacuoles in
human epithelia1 cells [ 1521 is mediated by the phosphatidylcholine-specific phospholip-
ase C (PC-PLC) and a metalloprotease [ 1 291. The phosphatidylinositol-specific phospholi-
pase C (PI-PLC) contributes to vacuole escape in other cells like bone marrow-derived
macrophages [ 181. Phospholipase activity of L. monocytogenes cultures was first observed
as a zone of opacity surrounding colonies on egg yolk agar [53]. Transposon mutants of
L. monocytogenes lacking phospholipase activity were identified by formation of small
plaques on fibroblast cell monolayers [ 1871 and by reduced hemolysis on blood-agar plates
[93], indicating a participation of phospholipase activity in hemolysis. One transposon
insertion was mapped in an ORF located adjacent to the hly gene on the chromosome of
L. monocytogenes [ 138,1871. The gene, plcA, was cloned 17,119,13I ] and it encoded a
protein of 34 kD which exhibits high homology to several gram-positive phospholipases
and contains a typical transport signal sequence. The enzyme called phosphatidylinositol-
specific phospholipase C (PI-PLC) was purified from culture supernatant liquids of an
overexpressing L. monocytogenes strain [ 681 and was highly specific for phosphatidyl-
inositol with no detectable activity on phosphatidylethanolamine, phosphatidylcholine, or
phosphatidylserine. It also does not cleave phosphatidy linositol-4-phosphate or phosphati-
dylinositol-4,5-bisphosphate,but it is active, albeit with low specific activity, on glyco-
sylated phosphatidylinositol-anchored proteins [59].
Besides the highly specific PI-PLC, L. monocytogenes produces a second phopholip-
ase C which hydrolyzes phosphatidylcholine (lecithin), and it is thus a phosphatidylcho-
Pafhogenesis of Listeria monocytogenes 107
way in which Mpl contributes to lysis of the vacuole in Henle 407 cells is not known,
but the most favorable hypothesis suggests that Mpl is necessary to activate PC-PLC as
shown in broth culture [ 1541.
Located immediately downstream of the hly gene, the mpl gene [36,134] encoding
a zinc-dependent metalloprotease, is the first gene of the lecithinase operon [36,134,196].
The deduced amino acid sequence of this protease shows high homology to several met-
alloproteases from Bacillus species and yields 5 10 amino acids with a typical N-terminal
signal sequence and a putative internal cleavage site. Like other metalloproteases, the
enzyme is activated by proteolytic maturation resulting in a 35-kD mature protein [36,134].
A 60-kD protein is detected with an antiserum raised against Bacillus stearothermophilus
thermolysin which probably represents the proform of the metalloprotease. Only small
amounts of the postulated 35-kD mature form of the protein were detected in the superna-
tant liquid of a L. monocytogenes culture [36].
The role of the virulence gene cluster products LLO, PI-PLC, PC-PLC, and Mpl in
escape from the phagocytic vacuole and in intracellular growth has been analyzed in some
detail during the last decade, as just described. A ClpC ATPase of L. monocytogenes
was recently identified as a new type of virulence factor being involved in intracellular
multiplication. The gene encoding the ClpC ATPase, called clpC, was identified by Tn917
mutagenesis with selection of mutants dependent on iron [161]. The clpC mutants are
highly susceptible to stress from iron limitation, elevated temperatures, and high osmolar-
ity. Virulence of these mutants is severely impaired in the mouse with restricted capacity
to grow in bone marrow-derived macrophages [ 1621. Molecular mechanisms by which
the ClpC ATPase of L. rnonocytogenes protects against stress and promotes intracellular
multiplication are unknown, but obviously the PrfA-dependent virulence machinery
(e.g., the virulence gene cluster products) is not significantly affected in clpC mutants
[162].
actin polymerization around bacteria, and lost intracellular motility [38,99]. Inside host
cells, the actA mutant forms microcolonies which are located near the nucleus [38].
To elucidate the role of ActA in actin filament assembly, actA was transfected into
mammalian cells [52,150,1511. Expression of the complete ActA protein (including the
membrane anchor) results in targeting of the protein to mitochondria, which subsequently
recruits actin to these organelles, suggesting that ActA alone is sufficient to polymerize
actin. However, the mitochondria did not move intracellularly [ 150,1511. Expression of
ActA lacking its signal sequence and its membrane anchor resulted in increased amounts
of F-actin in the transfected cells. ActA fused to a plasma membrane anchor which targeted
the fusion protein to the plasma membrane resulted in actin polymerization and formation
of aberrant protuberances on the cell surface [52]. From these assays, it appears that ActA
is sufficient to induce actin assembly.
To prove that ActA is also sufficient to promote intracellular movement, the nonmo-
tile species L. innocua was engineered to express ActA. The recombinant bacterium pro-
duced actin tails and moved in cytoplasmic extracts as did the wild-type L. monocytogenes
strain. In all parameters tested, the recombinant L. innocua strain expressing ActA was
indistinguishable from L. monocytogenes [ 10I].
The ActA protein is distributed asymmetrically on the surface of L. monocytogenes
but is not found within the actin tail [145]. After cell division, it is not present at the new
bacterial pole but is concentrated at the old pole [100,190). Using streptococci coated
asymmetrically with genetically engineered ActA protein, this asymmetrical distribution
of the ActA protein was shown to be required and sufficient to direct actin-based motility
[178]. In a cell-free system, these streptococci, but not uniformly coated ones, moved
efficiently in cytoplasmic extracts [ 1781.
The precise mechanisms by which ActA allows actin recruitment and intracellular
movement are still unknown. However, expression of mutated forms of ActA either in
mammalian cells [ 1513 or in L. rnonocytogenes [ 1 16,1791made it possible to define regions
of the ActA protein with specific functions in actin polymerization and movement. Dele-
tion of the N-terminal domain of ActA was followed in both systems by total abolishment
of actin polymerization and intracellular movement, thus showing the absolute necessity
of this domain in ActA function. In contrast, deletion of neither the proline-rich repeat
domain nor the C-terminal domain prevented actin assembly. However, the actin tails
produced by L. rnonocytogenes strains expressing ActA without proline-rich repeats were
appreciably shorter, and the number of repeats deleted corresponded with reduction in
speed, pointing to a stimulatory function for this region. Earlier work suggested a more
prominent function of the proline-rich repeats, since polyproline peptides, peptides repre-
senting one internal ActA repeat, or a naturally occuring polyproline peptide, blocked
actin assembly and motility after microinjection into L. nzonocytogenes-infected cells
[ 185,1861. It was speculated that the polyproline peptides would bind profilin [ 1861, which
in turn was suspected to be directly involved in actin-mediated motility of L. rnonocyto-
genes [ 1921, thereby inhibiting movement by inhibiting the association of profilin with
the repeat region.
Among actin binding proteins localized on actin tails--a-actinin, tropomyosin, vin-
culin, talin, fimbrin, villin, ezrinkadixin, profilin, the vasodilator-stimulated phosphopro-
tein (VASP), Mena, and Arp3 [20,28,34,63,98,190,192,200]-only profilin, Mena, and
VASP are associated with the surface of moving bacteria and colocalize with ActA. VASP,
a natural ligand of profilin [156], recently was shown to bind directly to the proline-rich
repeats of ActA [20] and can stimulate actin assembly by binding to ActA and enhancing
110 Kuhn and Goebel
the profilin concentration near the bacterium. Mena, which is closely related to VASP,
also binds ActA and profilin directly and might function in concert with VASP to recruit
profilin-actin complexes to the site of actin polymerization [63]. However, this model is
questioned by results of studies in which profilin was depleted from cytoplasmic extracts.
In such experiments, profilin-depleted extracts still supported actin assembly and bacterial
movement [ 1281. The recently described Arp2/3 complex consisting of eight host cell
proteins found in actin tails of moving bacteria may represent the host-cell actin polymer-
ization machinery [200]. The pure complex is sufficient to initiate ActA-dependent actin
polymerization on the surface of L. monocytogenes in a cell-free system and is thought
to interact at least transiently with ActA. Identification and purification of the Arp2/3
complex as a constituent of actin tails represents a great step forward toward the full in
vitro reconstitution of L. monocytogenes motility with purified components.
As recently shown, the 92-kD ActA surface protein is cleaved by the listerial me-
talloprotease (see above) resulting in a major 72-kD degradation product and, dependent
on the strains, additional smaller degradation products [66,145]. These products are either
found on the bacterial surface or in the supernatant fluid as 65- and 30-kD fragments
[ 1451. Whether degradation also occurs inside the cytoplasm of the infected host cell is
unknown. Additionally, the ActA protein is phosphorylated inside host cells, which yields
three distinct forms of this protein with slightly different sizes in SDS-PAGE [15]. How-
ever, a genetically engineered ActA variant which was fully functional but lacking the
C-terminal region is no longer phosphorylated inside host cells, suggesting that phosphory-
lation may not be necessary for movement [ 1 161.
As just mentioned, L. monocytogenes can spread from cell to cell without leaving
the cytoplasm by forming microvilli-like protrusions on the host cell surface which are
phagocytized by neighboring cells. The mechanism of microvilli formation and of induc-
tion of phagocytosis by neighboring cells are totally unknown. In cells infected with Shi-
gella flexneri, which uses a similar mechanism of cell-to-cell spread, proteins of the
cadherin family are critical for the spreading mechanism [ 1651. Whether this is also true
for L. monocytogenes remains to be clarified. Once inside the double membrane-bound
vacuole, bacteria again have to escape into the cytoplasm.
On monolayers of 3T3 fibroblasts, pZcB mutants form only small plaques, suggesting
that the cell-to-cell spread is impaired in these mutants. Electron micrographs of pZcB
mutants inside mammalian cells [ 1961 show numerous bacteria possessing actin tails
which are trapped in vacoules surrounded by a double membrane. This indicates that these
pZcB mutants cannot lyse the double membrane of the vacuole which is formed when
listeriae spread from cell to cell. Plaque formation capacity (which is thought to be a
strong indicator of intercellular spread) of different mutants revealed that in addition to
the broad-spectrum phospholipase PC-PLC, PI-PLC and the metalloprotease also contrib-
uted to plaque formation, most likely by supporting lysis of the double-membrane vacuole
[ 1771. The importance of LLO in this step has not yet been revealed.
L. ivanovii, a species pathogenic only to animals, is also invasive to most mammalian
cells tested, and the intracellular life cycle of this bacterium is similar to that of L. monocy-
togenes. Inside host cells, L. ivanovii polymerizes F-actin-like L. monocytogenes albeit
at a reduced rate, with actin tail formation and cell-to-cell spread also being observed
[90]. Recent cloning and sequencing of the actA-related gene from L. ivanovii [72,105]
showed surprisingly little sequence homology with the actA gene of L. monocytogenes.
On the protein level, some homology exists at the N- and C-termini and in the proline-
rich repeat sequences between the two proteins which are both active in actin polymeriza-
Pathogenesis of Listeria monocytogenes 111
tion [105]. The ActA-related protein of L. ivanovii is larger in size (1044 amino acids)
than ActA of L. monocytogenes (639 amino acids) because of two insertions which are
missing in ActA of L. monocytogenes and an increased number of proline-rich repeats
[72,105]. Despite the overall low-sequence similarity of the two ActA proteins, the mecha-
nism of actin polymerization seems to be similar, since host microfilament proteins that
bind to L. monocytogenes ActA also bind to L. ivanovii ActA [20,62]. Additionally, L.
ivanovii actA can replace L. monocytogenes actA in an L. rnonocytogenes actA mutant
[711.
PrfA Nm HTH
I I
HTH LZ
I I
C
7 30 169 194
one is PrfA dependent and harbors a rather incomplete palindromic PrfA-box [ 10,421.
The inZC gene is, however, transcribed from a single PrfA-dependent promoter which
contains a conserved PrfA-box at position -40 from the transcriptional start point. A second
possible PrfA binding site is located downstream from the transcriptional start site in a
position different from those of all other known PrfA-boxes in the promoter regions of
the PrfA-dependent genes [48]. The significance of this second PrfA-box for regulation
of inlC expression, however, is unknown.
release of preformed proteins and de novo synthesized proteins. More recent studies deter-
mined expression of the affected host genes more specifically by semiquantitatively mea-
suring expression on the transcriptional level using the highly sensitive reverse tran-
scriptase-polymerase chain reaction (RT-PCR) method.
The early reports showed that primary mouse embryonic fibroblasts infected with
L. monocytogenes released interferon-a@ (IFN-a@) into the culture medium [83]. In-
terleukin-1 (IL- 1) production by mouse peritoneal macrophages was observed with viable,
virulent L. rnonocytogenes strains but not with killed or avirulent Listeria. Northern blot
analysis further showed that the increase in IL-1 secretion correlates with an increase
in IL- 1a-specific mRNA after infection of macrophages with L. monocytogenes [ 1411.
Appreciable amounts of tumor necrosis factor (TNF) and IL-6 are secreted in alveolar
macrophages after infection with viable L. monocytogenes [84]. IL-6 is induced in embry-
onic fibroblasts even by heat-killed L. monocytogenes albeit to a lesser extent than by
infection with viable bacteria. TNF is secreted only after treatment with killed but not
with viable L. monocytogenes [841. Differences between killed and viable Listeria in
induction of TNF-a also were observed after infection of mouse peritoneal cell prepara-
tions consisting mostly of macrophages [203). Release of the proinflammatory cytokines,
IL-lp, IL-6, and TNF-a, occurs in human polymorphonuclear granulocytes and in the
human epithelial cell line HEp-2 after L. monocytogenes infection [4]. The granulocytes
secrete all three cytokines in response to infection, whereas the HEp-2 epithelial cells
secrete IL-6 and small amounts of TNF-a but no IL-lp. Transcription of the respective
genes also is induced in these host cells. Using the human epithelial cell line Caco-2, we
could only detect IL-6-specific mRNA expression on L. monocytogenes infection which
was, however, already induced by adherent L. monocytogenes, since cytochalasin D treat-
ment did not inhibit IL-6 expression [ 1081. Mouse peritoneal macrophages also secrete
IL-6 after L. monocytogenes infection [ 1061. Hemolytic L. monocytogenes strains are less
efficient in IL-6 induction than are nonhemolytic mutants, probably because of cell damage
caused by the high level of secreted listeriolysin. Inhibited maturation of IL-lp by L.
monocytogcnes in mouse peritoneal macrophages was recently proposed as a novel mecha-
nism of how L. monocytogenes may escape the host cell response, since infection of the
macrophages by L. monocytogenes results in intracellular accumulation of unprocessed
IL- 1 p precursor [61.
In a recent study using the mouse macrophage-like cell line P388DI and different
well-defined mutants of L. monocytogenes to analyze cytokine induction after infection,
we showed [ I 101 that viable L. monocytogenes rapidly induced IL- 1 a, IL- 1p, IL-6, and
TNF-a mRNAs in these host cells, whereas killed L. monocytogenes only induced IL- 1 p
mRNA. Nonhemolytic mutants which cannot escape into the cytoplasm and which do not
multiply are unable to induce I L - l a , IL-6, and TNF-a but still induce IL-lp mRNA.
In most instances, the amount of cytokines in the culture supernatant liquid of infected
macrophages correlates well with levels of induced mRNAs. The exception is IL- 1a, of
which only low levels are found in the supernatant liquid despite an appreciable induction
of IL- l a l p mRNA. Mouse bone marrow-derived macrophages infected with L. monocyto-
genes also induce proinflammatory cytokines [ 1 101. However, in these cells, a nonhemo-
lytic L. monocytogenes mutant induces the same types and amount of cytokines as the
wild-type strain, indicating that intracellular growth is not necessary for transcriptional
induction of these cytokines in bone marrow-derived macrophages [33].IL-6 is produced
in the bone marrow-derived macrophages independently of IL-1 and TNF [33]. The im-
munomodulating cytokines, IL- 10, IL- 12, and the IL- 1 receptor antagonist, also are in-
116 Kuhn and Goebel
sion [ 1471. MHC I and I1 expression also was repressed by L. rnonocytogenes infection in
P388D1 macrophages previously activated by IFN-y treatment. This suppression of MHC
expression in activated macrophages was, however, only detectable on infection with wild-
type L. rnonocytogenes but not with a p$A mutant unable to escape efficiently from the
vacuole into the cytoplasm [ 1671. Suppression of MHC gene transcription may represent
an important mechanism allowing L. rnonocytogenes to reduce macrophage-mediated anti-
gen presentation followed by T-lymphocyte activation.
Using a modification of the previously described procedure of differential PCR
[ 123,1701, induction or repression of host genes after infection by L. rnonocytogenes was
determined in a more general way. This method allows isolation of cDNAs representing
fragments of genes which are expressed differently in infected and uninfected macro-
phages. By this procedure we obtained several cDNA clones derived from macrophage
genes that were either transcriptionally activated or downregulated after infection by L.
monocytogenes [ 123,1701. Some of the cloned cDNAs were sequenced and subsequent
homology searches revealed that some of the sequences did not show any significant ho-
mologies to known genes [ 1081. One of the cloned cDNA fragments showed more than
99% homology to murine mitogen-activated protein kinase phosphatase (MKP-1) [ 1701
which was earlier described as being upregulated in macrophages on L. rnonocytogenes
infection [ 1681.
KB requires only adhesion of L. monocytogenes to P38SDI cells. This induction also occurs
with avirulent mutants of L. monocytogenes and the avirulent L. innocua with similar
efficiency. This event likely involves lipoteichoic acid, the cell wall component of L.
monocytogenes, since purified lipoteichoic acid shows the same transient triggering of
NF-KB. A second, but permanent, induction of NF-KB occurs after release of listeriae into
the cytoplasm of the host cell. This event occurs exclusively with virulent L. monocyto-
genes strains and requires the bacterial phospholipases PI-PLC and PC-PLC that are pro-
duced in the infected host cells cytoplasm [Sl]. Activation of NF-KB also occurs in
Caco-2 cells after infection with L. monocytogenes but with slower kinetics than seen in
the macrophages [SO]. The DNA binding activity of two other transcription factors, AP-
1 (activator protein-]) and NF-IL6, is not changed after infecting the P388D1 cell line
with L. monocytogenes, indicating that the observed activation of NF-KB by L. monocyto-
genes is a specific event [82].
Apoptosis
Programmed cell death, or apoptosis, induced by pathogenic bacteria was first documented
for Shigella jexneri using the mouse macrophage-like cell line 5774 [204]. Induction of
apoptosis was later shown for several facultative intracellular bacteria, including Salmo-
nella typhimurium [ 1421, Bordetella pertussis [96], Legionella pneumophila [ 1441, and
L. monocytogenes [75,159]. L. monocytogenes, originally described as being unable to
induce apoptosis in 5774 macrophages [204], was recently shown to induce apoptosis in
hepatocytes [159] and in dendritic cells with listeriolysin 0 being thought to trigger
apoptosis [75]. L. monocytogenes-infected hepatocytes undergo apoptosis in vitro as well
as in infected mice, and it was suggested that events of hepatocyte apoptosis which are
linked to neutrophil recruitment eliminate infected cells rapidly and thereby inhibit L.
monocytogenes spread [ 1591. In most instances, the mechanisms of apoptosis induction
by bacteria are totally unknown. However, for S. Jexneri, IpaB invasin was reported to
bind directly to an interleukin-converting-enzyme (ICE) protease and thereby interfere
with the apoptosis-controlling network of the host cell [22].
CONCLUSIONS
The last years have seen an enormous increase in our understanding of the molecular basis
of infectious diseases. Our knowledge of the genes determining virulence of L. monocyto-
genes and the role which the virulence gene products play in the infectious process is
rapidly expanding. However, many problems concerning virulence of L. monocytogenes
still remain unsolved.
For instance, L. ivanovii, a species largely nonpathogenic for humans [ 1711, resem-
bles L. monocytogenes in its intracellular life cycle [90]. Genes homologous to most of
the known virulence genes of L. monocytogenes are also detectable in this species
[72,77,105], and the complete PrfA-regulated gene cluster identified in L. monocytogenes
apparently also is present in L. ivanovii 1721. However, L. ivanovii is only virulent for
animals and avirulent for humans with an experimental L. ivanovii infection in mice yield-
ing a different outcome than that by L. rnonocytogenes [86]. What is the molecular explana-
tion for this obvious difference in the pathogenic potential of these two Listeria species?
Is it the result of a different mechanism in regulation of known virulence genes inside
infected cells or differences in specific activity of known virulence gene products? Are
Pathogenesis of Listeria monocytogenes 7 79
there as yet unknown virulence factors in L. monocytogenes which are absent in L. ivanovii
or vice versa?
Expression of L. monocytogenes virulence genes inside infected mammalian host
cells and tissues is another important but unsolved problem. The expression pattern of
known L. monocytogenes virulence determinants is already complex under in vitro growth
conditions and regulated by PrfA-dependent and PrfA-independent mechanisms. Very lit-
tle is known about how PrfA and other putative regulatory factors control these genes
while the bacteria reside inside host cells and tissues. Preferential synthesis of listeriolysin
inside the phagosome and of ActA and InlC inside the cytoplasm has been described
[ 1 1,481. However, the precise timing in expression of virulence genes as well as cellular
signals and bacterial sensors which may control their intracellular expression are largely
unknown. Most analyses of listerial virulence factors were done with commonly used
laboratory strains such as serotype 1/2a strain EGD or serotype I /2c strain L028. How-
ever, many human infections and most foodborne outbreaks have been associated with
serotype 4b strains [ 1491. In the future, differences in structure, function, and especially
regulation of virulence factors of different L. monocytogenes serotypes [ 1841 and clinical
isolates will likely gain much more interest.
Analysis of host cell responses to a L. monocytogenes infection is now becoming
a topic of major interest, as it represents a suitable model system for studying the molecular
basis of pathogen-host cell interactions. Research now concentrates on identification of
new host genes which are differentially expressed during various steps of a L. monocyto-
genes infection. Characterization of such host genes may help us to understand better the
strategies which these two partners are using in their intimate and sometimes very severe
cross talks. The molecular mechanisms of this intimate cross talk which require signal
transduction from pathogens to their host cells and vice versa are now being analyzed.
Elucidation of the interaction of bacterial and host cell proteins also will shed new light
on the coevolution of bacteria and their hosts. These central questions of pathogenesis of
facultative intracellular bacteria pertain not only to L. monocytogenes but also to several
gram-negative bacteria, such as Shigella, Yersinia, and Salmonella, and may lead to excit-
ing answers in the near future.
ACKNOWLEDGMENTS
We thank A. Demuth for carefully and critically reading this manuscript, J. Kreft and F.
Engelbrecht for providing figures, and all members of our laboratory for allowing us to
quote their unpublished results. We apologize to all who contributed to our current knowl-
edge on the infection biology of L. monocytogenes but were not mentioned in this chapter.
Work from the group at the University of Wiirzburg was supported by the Deutsche
Forschungsgemeinschaft through the grant SFB 165-B4.
REFERENCES
1.Alvarez-Dominguez, C., A.M. Barbieri, W. Beron, A. Wandinger-Ness, and P.D. Stahl. 1996.
Phagocytosed live Listeria rnonocytogenes influences Rab5-regulated in vitro phagosome-
endosome fusion. J. Biol. Chem. 27 1 : 13834-1 3843.
2. Alvarez-Dominguez, C., E. Carrasco-Marin, and F. Leyva-Cobian. 1993. Role of comple-
ment component C 1q in phagocytosis of Listeria monocytogenes by murine macrophage-
like cell lines. Infect. Immun. 61 :3664-3672.
120 Kuhn and Goebel
Seganti, P. Visca, and P. Valenti. 1996. Iron availability affects entry of Listeria monocyto-
genes into the enterocytelike cell line Caco-2. Infect. Immun. 64:3925-3939.
24. Conte, M.P., G. Petrone, C. Longhi, P. Valenti, R. Morelli, F. Superti, and L. Seganti. 1996.
The effects of inhibitors of vacuolar acidification on the release of Listeria rnonocytogenes
from phagosomes of Caco-2 cells. J. Med. Microbiol. 44:41X-424.
25. Cossart, P., M.F. Vicente, J. Mengaud, F. Baquero, J.C. Perez-Diaz, and P. Berche. 1989.
Listeriolysin 0 is essential for virulence of Listeria monocytogenes: direct evidence obtained
by gene complementation. Infect. Immun. 57:3629-3636.
26. Cowart, R.E., and B.G. Forster. 1981. The role of iron in the production of hemolysin by
Listeria monocytogenes. Curr. Microbiol. 6:287-290.
27. Croize, J., J. Arvieux, P. Berche, and M.G. Colomb. 1993. Activation of the human comple-
ment alternative pathway by Listeria rnonocytogenes: evidence for direct binding and proteol-
ysis of the C3 component on bacteria. Infect. Immun. 615134-5139.
28. Dabiri, G.A., J.M. Sanger, D.A. Portnoy, and F.S. Southwick. 1990. Listeria monocytogenes
moves rapidly through the host-cell cytoplasm by inducing directional actin assembly. Proc.
Natl. Acad. Sci. USA 87:6068-6072.
29. Datta, A.R., and M.H. Kothary. 1993. Effects of glucose, growth temperature, and pH on
listeriolysin 0 production in Listeria monocytogenes. Appl. Environ. Microbiol. 59:3495-
3497.
30. Davies, W.A. 1983. Kinetics of killing of Listeria monocytogenes by macrophages: rapid
killing accompanying phagocytosis. J. Reticuloendothel. Soc. 34: I3 1- 141 .
31. De Chastellier, C., and P. Berche. 1994. Fate of Listeria rnonocytogenes in murine macro-
phages: evidence for simultaneous killing and survival of intracellular bacteria. Infect. Im-
mun. 62543-553.
32. Demuth, A., T. Chakraborty, G. Krohne, and W. Goebel. 1994. Mammalian cells transfected
with the listeriolysin gene exhibit enhanced proliferation and focus formation. Infect. Immun.
6 2 5 102-5 111.
33. Demuth, A., W. Goebel, H.U. Beuscher, and M. Kuhn. 1996. Differential regulation of cytok-
ine arid cytokine receptor mRNA expression upon infection of bone marrow-derived macro-
phages with Listeria monocytogenes. Infect. Immun. 64:3475-3483.
34. Dold. F.G., J.M. Sanger, and J.W. Sanger. 1994. Intact a-actinin molecules are needed for
both the assembly of actin into tails and the locomotion of Listeria monocytogenes inside
infected cells. Cell Motil. Cytoskel. 28:97- 107.
35. Domann, E., and T. Chakraborty. 1989. Nucleotide sequence of the listeriolysin gene from
a Lister-ia monocytogenes serotype 1l 2 a strain. Nucleic Acids Res. 17:6406.
36. Domann, E., M. Leimeister-Wachter, W. Goebel, and T. Chakraborty. 1991 . Molecular clon-
ing, sequencing, and identification of a metalloprotease gene from Listeria monocytogenes
that is species specific and physically linked to the listeriolysin gene. Infect. Immun. 59:65-
72.
37. Domann, E,, J. Wehland, K. Niebuhr, C. Haffner, M. Leimeister-Wachter, and T. Chakra-
borty . 1993. Detection of a p$A-independent promoter responsible for listeriolysin gene ex-
pression in mutant Listeria monocytogenes strains lacking the PrfA regulator. Infect. Immun.
61~3073-3075.
38. Domann, E., J. Wehland, M. Rohde, S. Pistor, M. Hartl, W. Goebel, M. Leimeister-Wachter,
M. Wuenscher, and T. Chakraborty. 1992. A novel bacterial virulence gene in Listeria mono-
cytogenes required for host cell microfilament interaction with homology to the proline-rich
region of vinculin. EMBO J. 11:1981-1990.
39. Domann, E., S. Zechel, A. Lingnau, T. Hain, A. Darji, T. Nichterlein, J. Wehland, and T.
Chakraborty. 1997. Identification and characterization of a novel PrfA-regulated gene in Lis-
teriu rnonocytogenes whose product, IrpA, is highly homologous to internalin proteins, which
contain leucine-rich repeats. Infect. Immun. 65: 101- 109.
40. Dramsi, S., I. Biswas, E. Maguin, L. Braun, P. Mastroeni, and P. Cossart. 1995. Entry of
122 Kuhn and Goebel
from Listeria ivanovii and of Is0 from Listeria seeligeri. Biochim. Biophys. Acta 1130:81-
84.
78. Haas, A., and W. Goebel. 1992. Microbial strategies to prevent oxygen-dependent killing
by phagocytes. Free Radic. Res. Commun. 16:137- 157.
79. Hanawa, T., T. Yamamoto, and S. Kamiya. 1995. Listeria monocytogenes can grow in macro-
phages without the aid of proteins induced by environmental stresses. Infect. Immun. 63:
4595-4599.
80. Hauf, N., W. Goebel, F. Fiedler, R. Bockmann, and M. Kuhn. Listeria monocytogenes infec-
tion of Caco-2 human epithelial cells induces transient activation of transcription factor NF-
KB/Rel-like DNA binding activities (submitted for publication).
81. Hauf, N., W. Goebel, F. Fiedler, Z. Sokolovic, and M. Kuhn. 1997. Listeria monocytogenes
infection of P388D1macrophages results in a biphasic NF-KB (RelA/pSO) activation induced
by lipoteichoic acid and bacterial phospholipases and mediated by IKBa and IKBP degrada-
tion. Proc. Natl. Acad. Sci. 94:9394-9399.
82. Hauf, N., W. Goebel, E. Serfling, and M. Kuhn. 1994. Listeria monocytogenes infection
enhances transcription factor NF-KB in P388D, macrophage-like cells. Infect. Immun. 62:
2740-2747.
83. Havell, E.A. 1986. Synthesis and secretion of interferon by murine fibroblasts in response
to intracellular Listeria rnonocytogenes. Infect. Immun. 54:787-792.
84. Havell, E.A., and P.B. Sehgal. 1991. Tumor necrosis factor-independent IL-6 production
during murine listeriosis. J. Immunol. 146:756-76 1.
85. Hess, J., I. Gentschev, G. Szalay, C. Ladel, A. Bubert, W. Goebel, and S.H.E. Kaufmann.
1995. Listerin rnonocytogenes p60 supports host cell invasion by and in vivo survival of
attenuated Salmonella typhimurium. Infect. Immun. 63:2047 -2053.
86. Hof, H., and P. Hefner. 1988. Pathogenicity of Listeria monocytogenes in comparison to
other Listeria species. Infection 16 (suppl 2):141-144.
87. Ireton, K., B. Payrastre, H. Chap, W. Ogawa, H. Sakaue, M. Kasuga, and P. Cossart. 1996.
A role for phosphoinositide 3-kinase in bacterial invasion. Science 274:780-782.
88. Jones, S., and D.A. Portnoy. 1994. Characterization of Listeria monocytogenes pathogenesis
in a strain expressing perfringolysin 0 instead of listeriolysin 0. Infect. Immun. 62:5608-
5613.
89. Jones, S., K. Preiter, and D.A. Portnoy. 1996. Conversion of an extracellular cytolysin into
a phagosome-specific lysin which supports the growth of an intracellular pathogen. Mol.
Microbiol. 21: 1219-1225.
90. Karunasagar, I., G. Krohne, and W. Goebel. 1993. Listeria ivanovii is capable of cell-to-cell
spread involving actin polymerization. Infect. Immun. 61 :162- 169.
91. Karunasagar, I., B. Senghaas, G. Krohne, and W. Goebel. 1994. Ultrastructural study of
Listeria monocytogenes entry into cultured human colonic epithelial cells. Infect. Immun.
6213554-3558.
92. Kathariou, S., P. Metz, H. Hof, and W. Goebel. 1987. Tn926-induced mutations in the
hemolysin determinant affecting virulence of Listeria rnonocytogenes. J. Bacteriol. 169:
1291- 1297.
93. Kathariou, S., L. Pine. V. George, G.M. Carlone, and B.P. Holloway. 1990. Nonhemolytic
Listeria monocytogenes mutants that are also noninvasive for mammalian cells in culture:
evidence for coordinate regulation of virulence. Infect. Immun. 58:3988-3995.
94. Kathariou, S., J. Rocourt, H. Hof, and W. Goebel. 1988. Levels of Listeria rnonocytogenes
hemolysin are not directly proprtional to virulence in experimental infections of mice. Infect.
Immun. 56:534-536.
95. Kaufmann, S.H.E. 1993. Immunity to intracellular bacteria. In: W.E. Paul (ed.). Fundamental
Immunology. 3rd ed. New York: Raven Press, pp 1251- 1286.
96. Khelef, N., A. Zychlinsky, and N. Guiso. 1993. Bordetella pertussis induces apoptosis in
macrophages: role of adenylate cyclase-hemolysin. Infect. Immun. 6 1:4064-407 1.
Pathogenesis of Listeria monocytogenes 125
97. Klarsfeld, A.D., P.L. Goossens, and P. Cossart. 1994. Five Listeria rnonocytogenes genes
preferentially expressed in infected mammalian cells: plcA, purH, purl), pyrE and an arginine
ABC transporter gene arpJ. Mol. Microbiol. 13:585-597.
98. Kocks, C. 1994. Directional actin assembly by Listeria rnonocytogenes at the site of polar
surface expression of the actA gene product involving the actin-binding protein plastin (fim-
brin). Infect. Agents Dis. 2:207-209.
99. Kocks, C., E. Gouin, M. Tabouret, P. Berche, H. Ohayon, and P. Cossart. 1992. Listeria
rnonoc.ytogenes-inducedactin assembly requires the actA gene product, a surface protein.
Cell 68521 -53 1.
100. Kocks, C., R. Hellio, P. Gounon, H. Ohayon, and P. Cossart. 1993. Polarized distribution
of Listeria rnonocytogenes surface protein ActA at the site of directional actin assembly. J.
Cell. Sci. 3:699-710.
101. Kocks, C., J.-B. Marchand, E. Gouin, H. d'Hauteville, P.J. Sansonetti, M.-F. Carlier, and P.
Cossart. 1995. The unrelated surface proteins ActA of Listeria rnonocytogenes and IcsA of
Shigella Jiexneri are sufficient to confer actin-based motility on Listeria innocua and Esche-
richia coli, respectively. Mol. Microbiol. 18:413-423.
102. Kohler, S., A. Bubert, M. Vogel, and W. Goebel. 1991. Expression of the iap gene coding
for protein p60 in Listeria rnonocytogenes is controlled on the posttranscriptional level. J.
Bacteriol. 173:4668-4674.
103. Kohler, S., M. Leimeister-Wachter, T. Chakraborty, F. Lottspeich, and W. Goebel. 1990.
The gene coding for protein p60 of Listeria rnonocytogenes and its use as a species specific
probe for Listeria rnonocytogenes. Infect. Immun. 58: 1943- 1950.
104. Kreft, J., J. Bohne, R. Gross, H. Kestler, Z. Sokolovic, and W. Goebel. 1995. Control of
Listeria rnonocytogenes virulence by the transcriptional regulator PrfA. In: R. Rappuoli, V.
Scarlato, and B. Arico (eds.). Signal Transduction and Bacterial Virulence. Austin, TX: R.G.
Landes, pp 129-142.
105. Kreft, J., M. Dumbsky, and S. TheiB. 1995. The actin-polymerization protein from Listeria
ivanovii is a large repeat protein which shows only limited amino acid sequence homology
to ActA from Listeria rnonocytogenes. FEMS Microbiol. L,ett. 126:113- 122.
106. Kretschmar, M., T. Nichterlein, T. Chakraborty, J. Aufenanger, and H. Hof. 1993. Evidence
that listeriolysin is a major inducer of the early IL-6 production in Listeriu rnonocytogenes
infected mice. Med. Microbiol. Lett. 2:95-101.
107. Kiigler, S., S. Schuller, and W. Goebel. 1997. Involvement of MAP-kinases and phosphatases
in uptake and intracellular replication of Listeria monocytogenes in 5774 macrophage cells.
FEMS Microbiol. Lett. 157: 131- 136.
108. Kuhn, M., F. Engelbrecht, Z. Sokolovic, S. Kugler, S. Schuller, A. Bubert, I. Karunasagar,
R. Bockmann, N. Hauf, A. Demuth, J. Kreft, and W. Goebel. 1997. Interaction of intracellular
bacteria with mammalian host cells and host cell responses. Nova Acta Leopoldina N775,
301 :207-221.
109. Kuhn, M., and W. Goebel. 1989. Identification of an extracellular protein of Listeria rnonocy-
togenes possibly involved in the intracellular uptake by mammalian cells. Infect. Immun.
57155-61.
110. Kuhn, M., and W. Goebel. 1994. Induction of cytokines in phagocytic mammalian cells
infected with virulent and avirulent Listeria strains. Infect. Immun. 62348-356.
111. Kuhn, M., and W. Goebel. 1995. Molecular studies on the virulence of Listeria rnonocyto-
genes. Gen. Engr. 17:31-51.
112. Kuhn, M., N. Hauf, A. Demuth, W.R. Schwan, S. Kiigler, S. Schuller, and W. Goebel. 1995.
Host cell responses to Listeria rnonocytogenes infections. In: 12" International Symposium
on Problems of Listeriosis. Perth, WA: Promaco Con, pp 227-236.
113. Kuhn, M., S. Kathariou, and W. Goebel. 1988. Hemolysin supports survival but not entry
of the intracellular bacterium Listeria rnonocytogenes. Infect. Immun. 56:79-82.
114. Kuhn, M., M.-C. Privost, J. Mounier, and P.J. Sansonetti. 1990. A nonvirulent mutant of
126 Kuhn and Goebel
Listeria monocytogenes does not move intracellularly but still induces polymerization of
actin. Infect. Immun. 58:3477-3486.
115. Lampidis, R., R. Gross, Z . Sokolovic, W. Goebel, and J. Kreft. 1994. The virulence regulator
protein of Listeria ivanovii is highly homologous to PrfA from Listeria monocytogenes and
both belong to the Crp-Fnr family of transcriptional regulators. Mol. Microbiol. 13:141- 151.
116. Lasa, I., V. David, E. Gouin, J.-B. Marchand, and P. Cossart. 1995. The amino-terminal part
of ActA is critical for the actin-based motility of Listeria monocytogenes; the central proline-
rich region acts as a stimulator. Mol. Microbiol. 18:425-436.
117. Leblond-Francillard, M., J.L. Gaillard, and P. Berche. 1989. Loss of catalase activity in
TnZ545-induced mutants does not reduce growth of Listeria monocytogenes in vivo. Infect.
Immun. 57:2569-2573.
118. Lebrun, M., J. Mengaud, H. Ohayon, F. Nato, and P. Cossart. 1996. Internalin must be on
the bacterial surface to mediate entry of Listeria monocytogenes into epithelial cells. Mol.
Microbiol. 21 :579-592.
119. Leimeister-Wachter, M., E. Domann, and T. Chakraborty. 1991. Detection of a gene encoding
a phosphatidylinositol-specific phospholipase C that is co-ordinately expressed with listeri-
olysin in Listeria monocytogenes. Mol. Microbiol. 5:36 1-366.
120. Leimeister-Wachter, M., E. Domann, and T. Chakraborty. 1992. The expression of virulence
genes in Listeria monocytogenes is thermoregulated. J. Bacteriol. 174:947-952.
121. Leimeister-Wachter, M., W. Goebel, and T. Chakraborty. 1989. Mutations affecting hemoly-
sin production located outside the listeriolysin gene. FEMS Microbiol. Lett. 65:23-30.
122. Leimeister-Wachter, M., C. Haffner, E. Domann, W. Goebel, and T. Chakraborty. 1990.
Identification of a gene that positively regulates listeriolysin, the major virulence factor of
Listeria monocytogenes. Proc. Natl. Acad. Sci. USA 87:8336-8340.
123. Liang, P., and A.B. Pardee. 1992. Differential display of eukaryotic messenger RNA by
means of the polymerase chain reaction. Science 257:967-97 I .
124. Lingnau, A., T. Chakraborty, K. Niebuhr, E. Domann, and J. Wehland. 1996. Identification
and purification of novel internalin-related proteins in Listeria monocytogenes and Listeria
ivanovii. Infect. Immun. 64: 1002-1006.
125. Lingnau, A., E. Domann, M. Hudel, M. Bock, T. Nichterlein, J. Wehland, and T. Chakra-
borty. 1995. Expression of the Listeria monocytogenes EGD inlA and inlB genes, whose
products mediate bacterial entry into tissue culture cell lines, by PrfA-dependent and -inde-
pendent mechanisms. Infect. Immun. 63:3896-3903.
126. Ly, T.M.C., and H.E. Muller. 1990. Ingested Listeria monocytogenes survive and multiply
in protozoa. J. Med. Microbiol. 3 3 5 1-54.
127. Mackaness, G.B. 1962. Cellular resistance to infection. J . Exp. Med. I16:381-406.
128. Marchand, J.-B., P. Moreau, A. Paoletti, P. Cossart, M.-F. Carlier, and D. Pantaloni. 1995.
Actin-based movement of Listeria monocytogenes: actin assembly results from the local
maintenance of uncapped filament barbed ends at the bacterium surface. J. Cell Biol. 130:
33 1-343.
129. Marquis, H., V. Doshi, and D.A. Portnoy. 1995. The broad-range phospholipase C and a
metalloprotease mediate listeriolysin 0-independent escape of Listeria monocytogenes from
a primary vacuole in human epithelia1 cells. Infect. Immun. 63:453 1-4534.
130. Mason, 1. 1994. Do adhesion molecules signal via FGF receptors? Curr. Biol. 4: 1158-1 161.
131. Mengaud, J., C. Braun-Breton, and P. Cossart. 1991. Identification of phosphatidylinsitol-
specific phospholipase C activity in Listeria monocytogenes: a novel type of virulence factor?
Mol. Microbiol. 5:367-372.
132. Mengaud, J., J. Chenevert, C. Geoffroy, J.L. Gaillard, and P. Cossart. 1987. Identification
of the structural gene encoding the SH-activated hemolysin in Listeria monocytogenes: list-
eriolysin 0 is homologous with streptolysin 0 and pneumolysin. Infect. Immun. 55:3225-
3227.
133. Mengaud, J., S. Dramsi, E. Gouin, J.A. Vazquez-Boland, G. Milon, and P. Cossart. 1991.
Pathogenesis of Listeria monocytogenes 127
151. Pistor, S., T. Chakraborty, U. Walter, and J. Wehland. 1995. The bacterial actin nucleator
protein ActA of Listeria monocytogenes contains multiple binding sites for host microfila-
ment proteins. Curr. Biol. 5:517-525.
152. Portnoy, D.A., P.S. Jacks, and D.J. Hinrichs. 1988. Role of hemolysin for the intracellular
growth of Listeria monocytogenes. J. Exp. Med. 167:1459- 1471 .
153. Poyart, C., E. Abachin, I. Razfimanantsoa, and P. Berche. 1993. The zinc metallopro-
tease of Listeria monocytogenes is required for maturation of the phosphatidylcholine
phospholipase C: direct evidence obtained by gene complementation. Infect. Immun. 6 1 :
1576- 1580.
154. Raveneau, J., C. Geoffroy, J.L. Beretti, J.L. Gaillard, J.E. Alouf, and P. Berche. 1992. Re-
duced virulence of a Listeria monocytogenes phospholipase-deficient mutant obtained by
transposon insertion into the zinc metalloprotease gene. Infect. Immun. 60:9 16-921.
155. Raybourne, R.B., and V.K. Bunning. 1994. Bacterium-host cell interaction on the cellular
level: fluorescent labeling of the bacteria and analysis of short-term bacterium-phagocyte
interaction by flow cytometry. Infect. Immun. 62:665-672.
156. Reinhard, M., K. Giehl, K. Abel, C. Haffner, T. Jarchau, V. Hoppe, B.M. Jockusch, and U.
Walter. 1995. The proline-rich focal adhesion and microfilament protein VASP is a ligand
for profilins. EMBO J. 14: 1583- 1589.
157. Ripio, M.-T., G. Dominguez-Bernal, M. Suarez, K. Brehm, P. Berche, and J.-A. Vazquez-
Boland. 1996. Transcriptional activation of virulence genes in wild-type strains of Listeria
monocytogenes in response to a change in the extracellular medium composition. Res. Micro-
biol. 147:371-384.
158. Robbins, D.J., E. Zhen, M. Cheng, S. Xu, D. Ebert, and M.H. Cobb. 1994. MAP kinases
ERKl and ERK2: pleiotropic enzymes in a ubiquitous signaling network. Adv. Cancer Res.
63193-1 16.
159. Rogers, H.W., M.P. Callery, B. Deck, and E.R. Unanue. 1996. Listeria rnonocytogenes in-
duces apoptosis of infected hepatocytes. J. Immunol. 156:679-684.
160. Rosenshine, I., V. Duronio, and B.B. Finlay. 1992. Protein tyrosine kinase inhibitors block
invasin-promoted bacterial uptake by epithelial cells. Infect. Immun. 60:2211-2217.
161. Rouquette, C., J.M. Bolla, and P. Berche. 1995. An iron-dependent mutant of Listeria mono-
cytogenes of attenuated virulence. FEMS Microbiol. Lett. 133:77-83.
162. Rouquette, C., M.-T. Ripio, E. Pellegrini, J.M. Bolla, R.I. Tascon, J.-A. Vazquez-Boland,
and P. Berche. 1996. Identification of a ClpC ATPase required for stress tolerance and in
vivo survival of Listeria monocytogenes. Mol. Microbiol. 2 1:977-987.
163. Ruhland, G.J., M. Hellwig, G. Wanner, and F. Fiedler. 1993. Cell-surface location of Listeria-
specific protein p60-detection of Listeria cells by indirect immunofluorescence. J. Gen.
Microbiol. 139:609-6 16.
164. Sanger, J.M., J.W. Sanger, and F.S. Southwick. 1992. Host cell actin assembly is necessary
and likely to provide the propulsive force for intracellular movement of Listeria monocyto-
genes. Infect. Immun. 60:3609-3619.
165. Sansonetti, P.J., J. Mounier, M.-C. Privost, and R.-M. Mkge. 1994. Cadherin expression is
required for the spread of Shigella Jlexneri between epithelial cells. Cell 76:829-839.
166. Sawyer, R.T., D.A. Drevets, P.A. Campbell, and T.A. Potter. 1996. Internalin A can mediate
phagocytosis of Listeria monocytogenes by mouse macrophage cell lines. J. Leukoc. Biol.
60~603-610.
167. Schuller, S., S. Kugler, and W. Goebel. 1998. Suppression of major histocompatibility com-
plex class I and class IT gene expression in Listeria monocytogenes-infected murine macro-
phages. FEMS Immunol. Med. Microbiol. 20:289-299.
168. Schwan, W.R., and W. Goebel. 1994. Host cell responses to Listeria rnonocytogenes infection
include differential transcription of host stress genes involved in signal transduction. Proc.
Natl. Acad. Sci. USA 91:6428-6432.
169. Schwan, W.R., A. Demuth, M. Kuhn, and W. Goebel. 1994. Phosphatidylinositol-specific
Pathogenesis of Listeria monocytogenes 729
oostatic factor, a natural oligoproline peptide uncoupler of profilin action. Infect. Immun.
63: 182- 190.
187. Sun, A.N., A. Camilli, and D.A. Portnoy. 1990. Isolation of Listeria monocytogenes small-
plaque mutants defective for intracellular growth and cell-to-cell spread. Infect. Immun. 58:
3770-3778.
188. Tang, P., I. Rosenshine, P. Cossart, and B.B. Finlay. 1996. Listeriolysin 0 activates mitogen-
activated protein kinase in eucaryotic cells. Infect. Immun. 64:2359-236 I .
189. Tang, P., I. Rosenshine, and B.B. Finlay. 1994. Listeria monocytogenes, an invasive bacte-
rium, stimulates MAP kinase upon attachment to epithelia1 cells. Mol. Biol. Cell 5:455-464.
190. Temm-Grove, C.T., B. Jokusch, M. Rohde, K. Niebuhr, T. Chakraborty, and J. Wehland.
1994. Exploitation of microfilament proteins by Listeria monocytogenes: microvillus-like
composition of the comet tails and vectorial spreading in polarized epithelial sheets. J. Cell.
Sci 107:2951-2960.
191. Theriot, J.A., T.J. Mitchison, L.G. Tilney, and D.A. Portnoy. 1992. The rate of actin-based
motility of intracellular Listeria monocytogenes equals the rate of actin polymerization. Na-
ture 357:257-260.
192. Theriot, J.A., J. Rosenblatt, D.A. Portnoy, P.J. Goldschmidt-Clermont, and T.J. Mitchison.
1994. Involvement of profilin in the actin-based motility of Listeria monocytogenes in cells
and cell free extracts. Cell 76505-517.
193. Tilney, L.G., D.J. DeRosier, A. Weber, and M.S. Tilney. 1992. How Listeria exploits host
cell actin to form its own cytoskeleton: 11. Nucleation, actin filament polarity, filament assem-
bly, and evidence for a pointed end capper. J. Cell Biol. 118:83-93.
194. Tilney, L.G., and D.A. Portnoy. 1989. Actin filaments and the growth, movement, and spread
of the intracellular bacterial parasite, Listeria monocytogenes. J. Cell Biol. 109:1597-1608.
195. Van Dissel, J.T., J.J.M. Stikkelbroeck, and R. van Furth. 1993. Differences in the rate of
intracellular killing of catalase-negative and catalase-positive Listeria monocytogenes by nor-
mal and interferon-y activated macrophages. Scand. J. Immunol. 37:443-446.
196. Vazquez-Boland, J.-A., C. Kocks, S. Dramsi, H. Ohayon, C. Geoffroy, J. Mengaud, and P.
Cossart. 1992. Nucleotide sequence of the lecithinase operon in Listeria monocytogenes and
possible role of lecithinase in cell-to-cell spread. Infect. Immun. 60:2 19-230.
197. Velge, P., E. Bottreau, B. Kaeffer, N. Yurdusev, P. Pardon, and N. Van Langendonck. 1994.
Protein tyrosine kinase inhibitors block the entries of Listeria monocytogenes and Listeria
ivanovii into epithelial cells. Microbial Pathol. 17:37-50.
198. Weiglein, I., W. Goebel, J. Troppmair, U.R. Rapp, A. Demuth, and M. Kuhn. 1997. Listeria
monocytogenes infection of HeLa cells results in LLO mediated transient activation of the
Raf-MEK-MAP kinase pathway. FEMS Microbiol. Lett. 148:189-195.
199. Welch, D.F. 1987. Role of catalase and superoxide dismutase in the virulence of Listeria
monocytogenes. Ann. Inst. Pasteur/Microbiol. 138:265-268.
200. Welch, M.D., A. Iwamatsu, and T.J. Mitchison. 1997. Actin polymerization is induced by
Arp2/3 protein complex at the surface of Listeria monocytogenes. Nature 385:265-269.
201. Wood, S., N. Maroushek, and C.J. Czuprynski. 1993. Multiplication of Listeria monocyto-
genes in a murine hepatocyte cell line. Infect. Immun. 61:3068-3072.
202. Wuenscher, M.D., S. Kohler, A. Bubert, U. Gerike, and W. Goebel. 1993. The iap gene of
Listeria monocytogenes is essential for cell viability and its gene product, p60, has bacterio-
lytic activity. J. Bacteriol. 175:3491-3501.
203. Zhan, Y., and C. Cheers. 1995. Differential induction of macrophage-derived cytokines by
live and dead intracellular bacteria in vitro. Infect. Immun. 63:720-723.
204. Zychlinsky, A., M.-C. Prkvost, and P.J. Sansonetti. 1992. Shigella Jexneri induces apoptosis
in infected macrophages. Nature 358: 167- 169.
Characteristics of Listeria
monocytogenes Important to
Food Processors
YUQIANLou
Bil Mar Foods, Inc., Zeeland, Michigan
AHMEDE. YOUSEF
The Ohio State University, Columbus, Ohio
INTRODUCTION
Todays food manufacturer relies on a variety of processing and preservation techniques
to produce a safe and wholesome product with a suitable shelf life. Preservation methods
ensure the safety and stability of food by inactivating or inhibiting growth of foodborne
spoilage and pathogenic microorganisms. Methods currently used in food preservation
involve physical, chemical, and biological factors. Physical preservation includes heating,
cooling, freezing, and irradiation. Chemical treatments include addition of antimicrobial
agents such as benzoates, propionates, and sorbates, acidifying agents such as acetic, and
lactic acids or curing agents such as sodium chloride and sodium nitrite. Preservation
by biological means (biopreservation) includes fermentations which control spoilage and
pathogenic microorganisms through gradual lowering of pH. Combinations of these pres-
ervation factors also are applied simultaneously or sequentially in food processing. In
addition to these conventional preservation methods, novel nonthermal processing tech-
nologies are being investigated to meet increasing consumer demands for minimally
processed food with fresh-like taste and texture. Ultra-high pressure, pulsed light or
electric fields, and oscillating magnetic fields are examples of these novel techniques.
131
132 Lou and Yousef
TEMPERATURE
Temperatures to which food is exposed may have lethal, growth-conductive, or preserving
effects on microorganisms in the product. In general, temperatures greater than 50C are
lethal to L. monocytogenes. At -0 to 45OC, the pathogen grows to various degrees when
present in a suitable medium. Temperatures below 0C freeze the culture or food and
preserve or moderately inactivate the pathogen. These three ranges of temperatures will
be addressed separately.
Growth Temperatures
Microorganisms grown at optimum incubation conditions exhibit short initial lag periods,
short generation times during exponential growth, and high cell counts or densities at the
stationary phase. Incubation at temperatures different than the optimum extends the lag
period and/or the generation time and may decrease the maximum attainable population.
In this chapter, growth parameters of Listeria monocytogenes as a function of incubation
temperature will be described.
The temperature range within which L. monocytogenes grows is of particular interest
to food processors, since this pathogen has common features of both psychrotrophic and
mesophilic bacteria. Under laboratory conditions, L. monocytogenes was originally re-
ported to grow at temperatures between 3 and 45C [160], with optimal growth occuring
between 30 and 37C [300,340]. In 1972, Wilkins et al. [394] examined the temperature
range for growth of L. monocytogenes in a medium containing 1% tryptone, 1% yeast
Characteristics of Listeria monocytogenes 133
extract, 0.3% K2HP04, and 0.1% glucose. Extrapolation of data from an Arrhenius plot
of exponential growth rates collected at various temperatures indicated that the bacterium
had maximum, optimum, and minimum growth temperatures of 45-5OoC, 38"C, and 3"C,
respectively, which generally agree with the growth temperatures most frequently men-
tioned in earlier textbooks. However, Bergey 's Manual of Systematic Bacteriology [341]
gives the minimum growth temperature as 1C. In support of this change from 3 to lC,
Junttila et al. [ 1911 found that growth of 78 L. monocytogenes strains on tryptose soy agar
occurred at a mean minimum temperature of l.l(t0.3)"C, with 10 and 2 strains of L.
monocytogenes serotype 1/2 growing at 0.8 and OSOC, respectively. In contrast, L. innocua
(19 strains), L. murrayi (1 strain), and L. grayi (1 strain) failed to grow at temperatures
below 1.7 (tr0.4), 2.8, and 3.OoC, respectively. Although researchers in Florida also ob-
served slight growth of some L. monocytogenes strains in laboratory media at 1"C, Walker
et al. [382] confirmed the ability of this pathogen to multiply at even lower temperatures,
with three L. monocytogenes strains exhibiting generation times of 62- 131 h in chicken
broth and pasteurized milk, respectively, during extended incubation at -0.1 to -0.4"C.
Lowest growth temperature was reported by Hudson et al. [ 1781. L. rnonocytogenes and
two other ps ychrotrophic pathogens, Aeromonas hydrophila and Yersinia enterocolitica,
grew at - 1.5"C in vacuum-packaged sliced roast beef with calculated lag times of 174,
110, and 49 h and generation times of 100, 33, and 32 h, respectively.
Growth of L. monocytogenes in laboratory media at 1C is very slow. However,
when incubated at 3-6"C, the growth rate of the pathogen increases with final populations
of approximately 1OS CFU/mL attained after several weeks of incubation [ 1 151. In a study
by Bojsen-Moller [39] in 1972, flasks containing Tryptose Phosphate Broth (TPB) were
inoculated with one of several L. monocytogenes strains and incubated at different temper-
atures. L. monocytogenes exhibited average generation or doubling times of 12.0, 5.0, and
2.6 h during incubation at 4, 10, and 15"C, respectively. In a more recent study [27], the
average generation times for 39 L. monocytogenes strains growing in Tryptic Soy Broth
(TSB) supplemented with 0.6% yeast extract were 43, 6.6, and 1.1 h at 4, 10, and 37"C,
respectively, whereas the respective average lag times were 151, 48, and 7.3 h. In the
latter study, L. monocytogenes strains reached maximum OD600-valuesof 0.74, 0.92, and
0.97 after incubation at 4, 10, and 37C for 336, 113, and 16 h, respectively. Since many
other bacterial species fail to grow at refrigeration temperatures, extended cold storage
of clinical, environmental, and food samples previously diluted in a nonselective medium
such as Tryptose Broth (TB) often was successful for isolating L. monocytogenes. This
Listeria-detection procedure, which forms the basis for cold-enrichment, was widely used
until the mid 1980s.
Significant growth variation among 39 strains of L. monocytogenes, especially at
refrigeration temperature, also was observed by Barbosa et al. [27]. The lag phase for 39
strains varied from 69.8 to 270 h at 4C and from 36.5 to 68.9 h at 10C. Scott A, the
strain most extensively used in Listeria-related research, had the longest (209 h) and the
second longest (62.8 h) average lag periods at 4 and 10C, respectively. However, when
strains of I;. rnonocytogenes were grouped according to serotypes, few differences in
growth parameters among serotypes were noticed. Therefore, the choice of L. munocytu-
genes strains for use in challenge studies, particularly at refrigeration temperatures, may
affect the results and conclusions regarding food safety. Greater safety margins will be
obtained if the hardiest L. monocytogenes strains are used in such studies.
Because of the safety concerns, researchers attempted to find nonpathogenic indica-
tor microorganisms which can replace L. monocytogenes, particularly for studies done in
pilot plants or food processing facilities. L. innocua, which is nonpathogenic and has
134 Lou and Yousef
similar or higher growth rates and resistance to common food preservation methods than
L. monocytogenes, was considered a suitable substitute for L. monocytogenes
[97,143,299]. L. innocua PFEI, a strain with antibiotic resistance which aids its enumera-
tion in foods, is reported to be a good thermal-resistance indicator of L. monocytogenes
[143]. When incubated in Brain Heart Infusion (BHI) broth at 2-46"C, this L. innocua
strain grew faster below 42"C, slower above 42"C, and had a shorter lag phase below
8C than L. monocytogenes, and thus was generally considered a good indicator of the
growth behavior of L. monocytogenes [97]. Similar or faster growth of L. innocua in a
laboratory broth and a cheese sauce, compared with that of L. monocytogenes, was also
noted by Petran and Swanson [299].
Prior treatment of a microorganism affects its behavior during subsequent growth.
Gay et al. [149] found that the lag phase of L. monocytogenes (Scott A and V7) and L.
innocua increased by a low inoculum ( 10' vs 1O3 CFU/mL) cold storage and preincubation
at 30" rather than 14C. Listeria strains tested by these investigators exhibited a slower
first logarithmic growth phase and a faster second phase under most of the conditions
tested.
The food medium can also influence growth and calculated growth parameters of
L. monocytogenes. Rosenow and Marth [322] measured growth parameters of four L.
monocytogenes strains in autoclaved samples of skim, whole, and chocolate milk and
whipping cream that were stored at 4-35C. Listeria growth rates were generally similar
in all four products at a given temperature and increased with an increase in incubation
temperature. Generation times in hours for listeriae in all four products were 29.7-45.6
at 4OC, 8.7- 14.6 at 8OC, 4.5-6.9 at 13OC, 1.7-1.9 at 2 1 OC, and a uniform 0.68 at 35C.
L. monocytogenes reached maximum populations of 1 07- 1O9 CFU/mL in all products that
were incubated 30-45 days at 4"C, 11-14 days at 8"C, 5.0-5.8 days at 13"C, 2.1 days
at 21 "C, and 1 day at 35C. In addition, numbers of listeriae failed to decrease substantially
in the four products during extended storage. Donnelly and Briggs [92] also reported
rapid growth of five L. monocytogenes strains in inoculated samples of whole, skim, and
reconstituted nonfat dry milk (1 1% total solids) during incubation at 4, 10, 22, and 37C.
Growth of L. monocytogenes at low temperatures is also stimulated by presence of
certain solutes in growth media. Such compounds include glycine betaine or carnitine
[209,352], which are known as osmoprotectants, osmolytes, or compatible solutes. Similar
osmolytes may exist in foods at measurable levels. Such osmolytes usually accumulate
in microbial cells during periods of osmotic stress. Compatible osmolytes may stabilize
the otherwise unstable physiological functions of cytoplasmic proteins or other structures
under osmotic stress [398]. KO et al. [209] found that osmolytes accumulated in cells of
L. monocytogenes at low temperatures and during chill stress. When L. monocytogenes
was surface-plated on a defined medium containing 130 pM glycine betaine, colonies
were observed after 32 days at 7"C, whereas no colonies were visible without this osmo-
protectant. When L. monocytogenes was grown in the defined liquid medium at 4"C, addi-
tion of 130 pM glycine betaine nearly doubled the specific growth rate [209].
Virulence of Listeria is increased when the bacterium is grown at low rather than
high temperatures. Durst [99] reported that 7 of 36 weakly virulent L. monocytogenes
strains became markedly virulent to mice by intraperitoneal injection after the cultures
were maintained on agar slants for 6 months at 4C. Similarly, Wood and Woodbine [397]
found that one strain of L. monocytogenes was more virulent to chick embryos when
grown at 4 rather than 37C. Thus the possibility exists that cold storage may enhance
virulence of some L. monocytogenes strains isolated from refrigerated foods.
Characteristics of Listeria mon ocytogen es 735
Freezing
Numerous reports of Listeria-contaminated frozen foods in the United States and else-
where have prompted investigators to examine viability of L. monocytogenes during frozen
storage. Survival of L. monocytogenes in laboratory media, buffers, and milk during freez-
ing and frozen storage was assessed by Hof et al. [ 17 I], El-Kest and Marth [ 107,108,109]
and El-Kest et al. [ I 121.
cytogenes to sublethal levels of certain environmental stresses, such as low pH, ethanol,
NaCl, heat shock, or starvation can increase survival of this pathogen during freezing,
frozen storage, and freezelthaw cycles [237].
Viability in Frozen Foods
Oscroft [282] found that frozen storage of three L. rnonocytogenes strains in carrot or
chicken homogenate at - 18C for 29-84 days did not appreciably reduce the viable cell
counts. Results from other studies [ 164,284,2881demonstrated that L. rnonocytogenes pop-
ulations decreased only <1-3 logs in inoculated samples of packaged fish and shrimp,
canned milk, 10%Karo corn syrup, ground beef, ground turkey, frankfurters, carined corn,
and ice cream mix during 2-3 months of frozen storage at - 18 to -20C. A somewhat
greater decrease in viability was observed in samples of frozen tomato soup, possibly
because of the lower pH of the product. Gianfranceschi and Aureli [155] investigated
survival of two L. rnonocytogenes strains in five foods (spinach, mozzarella cheese, cod
fish, chicken breast, and beef hamburger) during initial quick freezing at -50C for 57 min
and subsequent frozen storage at - 18C for 240-300 days. Quick freezing and subsequent
storage reduced viable Listeria populations by only 0.1- 1.6 and 0- 1.O log, respectively.
Injury among survivors ranged from a nondetectable level to 90%. In contrast, Palumbo
and Williams [284] did not recover injured listeriae from a variety of frozen foods tested
except for tomato soup.
Thermal Inactivation
Thermal processing is the most widely used method to preserve food and to destroy harm-
ful microorganisms, thus rendering food safe for human consumption. The established
association of L. monocytogenes with raw milk in the 1950s gave rise to several early
studies dealing with the possible resistance of this organism to pasteurization. In 1983,
interest in this topic was revived as a result of a listeriosis outbreak in Massachusetts
that was epidemiologically linked to consumption of pasteurized milk. The literature now
contains a wealth of information on thermal resistance of L. rnonocytogenes in a wide
variety of foods. Although data concerning heat resistance of L. monocytogenes in fluid
milk will be presented now, the discussion regarding thermal inactivation of listeriae in
other foods has been reserved for other chapters which deal with the incidence and behav-
ior of Listeria spp. in meat, poultry, seafood, and products of plant origin.
Early studies on the thermotolerance of L. rnonocytogenes gave controversial results
because of the methods used in measuring heat resistance. The 1983 outbreak suggested
that L. rnonocytogenes, at levels that may exist in milk, can survive minimal high-tempera-
ture short-time (HTST) pasteurization. Subsequent studies on freely suspended cells
showed that minimal HTST pasteurization is adequate; however, results from investiga-
tions on resistance of intracellular L. rnonocytogenes were in conflict. Further efforts by
the US Food and Drug Administration (FDA) [44,63,240], Centers for Disease Control
and Prevention (CDC) [ 13,14,18], and the World Health Organization (WHO) [393] sup-
port HTST pasteurization as a safe process. The following is a discussion of these aspects
of thermal inactivation of L. monocytogenes given in the sequence just outlined.
Conflicting Results in Early Literature
Numerous conflicting reports concerning the unusual heat resistance of L. rnonocytogenes
in milk can be found in the early literature. In 1951, Potel [307] demonstrated that L.
Characteristics of Listeria monocytogenes 137
monocytogenes died rapidly in milk held at 80C. However, the following year, Ozgen
[283] reported that L. monocytogenes survived 15 s at 100C. In 1955, Stenberg and
Hammainen [364] published results of a study which examined the heat resistance of five
L. monocytogenes strains in milk at different pasteurization temperatures. Using small-
diameter capillary tubes filled with inoculated milk, these researchers demonstrated that
L. monocytogenes was not completely inactivated until the milk was held at 65C for 5
min, 75C for 2 min, or 80C for 3-5 min. Thermal resistance of L. monocytogenes also
was studied by Stajner et al. [362]. When milk contained approximately 5 X 105L. mono-
cytogenes CFU/mL, the organism survived heat treatments of 71 and 74C for 42 s but
did not survive heating at 85 and 95C for 15 and 5 s, respectively. In 1957, Dedie and
Schulze [82] examined thermal resistance of 54 strains of L. nzonocytogenes in milk using
0.2- to 0.3-mm diameter capillary tubes. According to their results, L. rnonocytogenes
survived 30--40 s at 65"C, 10 s at 75OC, and -1 s at 85C. Ikonomov and Todorov [182]
used a pilot plant-sized tubular glass pasteurizer to examine heat resistance of Listeria
in milk obtained from ewes and cows. The milk was pasteurized (63-65"C/30 min), inocu-
-
lated to contain 107- 108L. monocytogenes CFU/ml, and then repasteurized at tempera-
tures between 63 and 74C. They found that the pathogen survived 20 min at 63"C, 10
rnin at 65OC1, 3 rnin at 68"C, 1 rnin at 7OoC, 20 s at 72"C, and <20 s at 74C. Thus
results of virtually all these early studies indicate that L. monocytogenes can survive HTST
pasteurization at 71.6"C for 15 s.
Variability in Results Caused by Thermal Inactivation
Methods
Several different approaches have been used to determine thermal resistance and have
given rise to conflicting results. Findings from the early pasteurization study of Bearns
and Girard [28], which included an "open-tube" heating procedure became highly suspect
during the 1980s. Their experimental approach involved inoculating 20 X 150-mm screw-
capped test tubes of sterile skim milk with approximately 5 X: 10' to 5 X 107L. monocyto-
genes CFU/ml. All tubes were placed in a water bath at 61.7"C so that the milk surface
was 3-4 cm below the water level in the water bath. Tubes were held in a wire test tube
rack attached to a mechanical shaker and were allowed to bounce in the rack to aid in
mixing. Results obtained from direct plating of milk on Tryptose Agar (TA) indicate that
L. monocytogenes survived 35 min at 61.7"C provided that the organism was present at
an initial level 2 5 X 104CFU/mL. From these data, the authors calculated a D61.70C value
(i.e., the time necessary to decrease the population 90% at 61.7"C) of 10.9 min, which
indicates that L. monocytogenes, if present at populations > 103CFU/mL, can survive vat
pasteurization at 61.7"C for 30 min.
Using the method of Beams and Girard [28], Donnelly et al. [94] demonstrated that
complete inactivation of L. monocytogenes in milk with an initial population of 106-1O7
Listeria CFU/mL cannot be accomplished within 30 rnin at 62, 72, 82, or even 92C.
Extensive tailing of survivor curves was observed after an initial 3- to 4-log decrease
during the first 5 rnin of heating. These investigators concluded that the open-tube method
of Bearns and Girard [28] is unreliable to determine thermal inactivation rates of microor-
ganisms, and they offered several explanations for their conclusion. One explanation is
that condensate and splashed cells accumulated on the test tube cap, which was above
the level of water in the water bath and, therefore, not exposed to thermal-inactivation
temperatures. Condensate containing listeriae would be expected to drip back into the
heating menstruum, thus eventually establishing a constant low population of survivors.
138 Lou and Yousef
A more likely explanation is that the test tube walls were coated with cells of Listeria
during initial mixing. The test tubes were not completely submerged in the water bath;
therefore, cells on the test tube wall would not be exposed to thermal-inactivation tempera-
tures. Since a constant surface area is presumably coated with listeriae, low levels of
survivors likely would be detected throughout the inactivation process. Concurrent studies
by Donnelly et al. 1941 using a sealed-tube method demonstrated that L. monocytogenes
was rapidly inactivated in milk at 62C. The sealed-tube method involved adding I .5 ml
-
of sterile whole milk inoculated to contain 107L. monocytogenes CFU/mL to a 2-ml
vial, sealing it, and then submerging the vial in a water bath at the desired temperature
for various times. In contrast to results of Bearns and Girard [28], thermal-inactivation
profiles obtained by the sealed-tube method were linear for three strains of L. monocyto-
genes during the entire inactivation period and gave rise to D620C values between 0.1 and
0.4 min depending on the strain of bacterium. From the aforementioned results, it is appar-
ent that the inactivation rate for L. monocytogenes at pasteurization temperature depends
on the method used to study heat resistance of the bacterium. In 1987, Beckers et al. [29]
compared thermal resistance of L. manmytogenes in TB using an open-tube and sealed-
bag method and obtained results similar to those of Donnelly et al. [94] just described.
Heating in sealed tubes may produce anaerobic conditions in the heating menstruum.
It is well known that anaerobic recovery of heat-injured cells of many pathogens, including
L. monocytogenes, results in higher D-values [208,234]. Knabel et al. [208] found that
after heating a Listeria-broth mixture in thermal death time (TDT) tubes, the broth at the
bottom of the tubes became anaerobic, as indicated by the color change of the redox
potential indicator, resazurin. Although no color change was found in milk in TDT tubes,
some degree of anaerobiosis may also exist.
The shortcomings of the open-tube procedure can be eliminated by using the capil-
lary tube method [ 122,238,356,357,3661. In this method, a small sample of culture or
inoculated liquid food (e.g., 40 1 L ) is introduced into a sterile capillary tube (e.g., 0.8-
1.10 X 100 mm) and both ends are carefully heat sealed. The sample is then heat treated
for a specified time after which the capillary tube is rapidly cooled, sanitized, and crushed
inside a large test tube. The released sample is then diluted appropriately and plated to
enumerate survivors. Compared with oiher methods, the capillary tube procedure allows
uniform heating of the sample and minimizes come-up and cooling-down times. When
compared with the capillary tube, other methods require a larger sample size and thus a
significant part of the heat treatment occurs during come-up and cooling-down times. The
amount of heat the sample receives during the coming-up and cooling-down times must
be calculated to avoid inaccurate D-values. The capillary tube method was used by several
investigators to determine the thermal resistance of L. monocytogenes in liquid media
and foods [ 122,2381. Overall, thermal inactivation rates for L. rnonocytogenes were linear
throughout the entire course of heating in the range of 50-75C.
Thermal Inactivation of Freely Suspended
L. monocytogenes
As a result of the 1983 listeriosis outbreak in Massachusetts that was epidemiologically
linked to consumption of pasteurized milk, Bradshaw et al. [42] investigated thermal resis-
tance of freely suspended L. monocytogenes in raw milk. A culture of L. rnonocytogenes
strain Scott A (serotype 4b, clinical isolate associated with the outbreak in Massachusetts)
-
was diluted in phosphate-buffered water and inoculated into raw milk to yield 105CFU/
mL. Portions of 1.5 mL were dispensed into 13 X 100-mm borosilicate glass tubes, which
Characteristics of Listeria monocyt ogenes 139
were sealed and immersed in a water bath at temperatures ranging between 52.2 and
71.7"C. Inoculated samples of raw milk also were heated in a slug flow heat exchanger
at 7 1.7 and 74.4"C. Thermal processing of inoculated milk samples at seven temperatures
between 52.2 and 74.4"C led to D-values ranging between 28. I min and 0.7 s, respectively,
including a I)717C of 0.9 s and a Dh33oc of 19.9 s. These investigators also noted that the
thermal resistance of strain Scott A remained unchanged over a 2-year period. Survivors
from some heating trials also were tested and were no more heat resistant than the parent
culture, which suggests that the extensive tailing observed by Bearns and Girard [28]
cannot be explained on the basis of heat-resistant spontaneous mutants.
Working in France in 1988, Lemaire et al. [227] used open vessels and sealed capil-
lary tubes to assess resistance of L. monocytogenes strains to vat and HTST pasteurization,
respectively. When samples of inoculated milk were held at 60C in open vessels, DhOoC
values for 38 different L. monocytogenes strains ranged from 1.3 to 6.5 s. In contrast,
D7yC values of 0.06-1.5 s were obtained when L. monocytogenes was heated in sealed
capillary tubes, with strains of serotype 1 being generally more heat resistant than those
of serotype 4. These findings along with those of Bradshaw et al. [42] indicate that current
minimum vat (61.7"C/30 min) and HTST (7 1.6"C/ 15 s) pasteurization requirements estab-
lished by the FDA are probably adequate to destroy expected levels of L. monocytogenes
in raw milk.
In 1986, Donnelly and Briggs [92) reported results of a study that examined the
influence of milk composition and incubation temperature on thermal resistance of L.
monocytogenes. The researchers inoculated five L. monocytogenes strains into sterile
whole milk, skim milk, and reconstituted nonfat milk containing 11% solids. Following
-
incubation, they heat-treated milk containing I Ox L. monocytogenes CFU/mL in sealed
glass vials at temperatures between 55 and 65C. Thermal resistance of L. monocytogenes
was not significantly affected by prior growth in skim, whole, or reconstituted nonfat
milk with I I % solids. Additionally, thermal inactivation experiments using the most heat-
resistant strain resulted in D550C and D6,0cvalues of 24.0 and 0.1 min, respectively, and a
z-value of 4.3"C. After extrapolating the linear thermal inactivation plot through 7 I .7"C,
the authors concluded that the most heat-resistant strain used in their study would be
unable to survive in whole milk during HTST pasteurization.
Going one step further, Bradshaw et al. [43], in 1987, examined the thermal resis-
tance of L. monocytogenes strain Scott A in raw, autoclaved, and commercially sterile
whole milk (-3.25% milk fat) and raw and autoclaved skim milk (<0.5% milk fat).
-
Products were inoculated at 105L. monocytogenes CFU/mL, and thermal resistance
was determined by the sealed-tube method. Listeria-inactivation studies done with raw,
autoclaved, and commercially sterile whole milk yielded I371 7 c cvalues of 0.9, 2.0, and
2.7 s, respectively, indicating significantly ( P 5 .05) greater survival in presterilized than
in other samples of whole milk. When heated in presterilized skim milk, the D7,70cvalue
for strain Scott A was 1.7 s. Although their data indicate that L. monocytogenes should
not survive in properly pasteurized raw whole milk, their other findings raise questions
concerning the adequacy of pasteurization to inactivate L. monocytogenes in reprocessed
products.
Working in Canada, Farber et al. [ I361 inoculated 1200 L of raw whole milk to
contain 105L. monocytogenes (a mixture of 10 strains including Scott A) CFU/mL. After
heating milk at 60-72C for 16.2 s in a pilot plant-sized regenerative plate pasteurizer,
L. monocytogenes was recovered from milk heated up to 67.5"C but was not recovered
from milk processed at 69 or 72C. Scientists from the FDA [240] also found that L.
140 Lou and Yousef
monocytogenes cells (2.6 X 105CFU/mL) freely suspended in raw milk were inactivated
by the minimum HTST pasteurization process.
Thermal inactivation of L. monocytogenes in reconstituted nonfat dry milk (NFDM)
was investigated by El-Shenawy et al. [122] in 1989. Suspensions of L. monocytogenes
cells in reconstituted NFDM (10% solids) were placed in capillary tubes which were
heated in a water bath at 50, 55, 60, 65, 70, and 75C for various times. Overall, thermal
inactivation rates for L. monocytogenes were linear throughout the entire course of heating
with estimated D62.80C- and D7,,70C-valuesof 20 and 0.94 s, thus reaffirming that pasteuriza-
tion as defined by the FDA should inactivate freely suspended cells of L. monocytogenes.
In 1991, Bradshaw et al. [44] reported that, in raw and sterile milk, other Listeria species
were no more heat resistant than L. monocytogenes. Therefore, properly pasteurized milk
should be free of all Listeria spp.
Although L. monocytogenes was more resistant in presterilized or repasteurized milk
than in raw milk, all investigations described thus far showed that HTST pasteurization
will inactivate L. monocytogenes when the pathogen is freely suspended in milk at levels
up to 105CFU/mL. In contrast, Fernandez Garayzabal et al. [141] reported, in 1987, that
the pathogen, when inoculated at high levels into raw whole milk, could survive minimum
pasteurization. The Spanish researchers inoculated milk to contain 3 X 106, 1 X 107,or
2 X 10 L. monocytogenes CFU/mL and pasteurized the milk at 72 or 73C for 15 s
(HTST method) in a pilot plant-sized pasteurizer. Using cold enrichment, Listeria was
detected in five of seven batches of milk treated at 72C for 15 s, with an estimated
D720c-valueof 1.8-2.1 s. Listeria, however, was not detected in the three pasteurization
trials at 73C for 15 s. This experiment was criticized by Lovett et al. [240] for possible
overloading of the pasteurizer. Large initial populations may have been a factor in promot-
ing Listeria survival during pasteurization in the study of Fernandez Garayzabal et al.
[ 1411. Additionally, since numbers of L. monocytogenes in naturally contaminated raw
milk are typically very low, these findings do not discount the adequacy of minimum
HTST pasteurization.
The heat resistance of L. monocytogenes Scott A in heavy cream (38% milk fat)
and pasteurized ice cream mix (-10.6% milk fat) was investigated by Bradshaw et al.
[43] using the sealed tube method. The organism had D6x,90c-values of 6.0 and 7.8 s in
raw and autoclaved cream, respectively. Thermal processing of pasteurized ice cream mix
at 68.3, 73.9, and 79.4C resulted in D-values of 231.0, 31.5, and 2.6 s, respectively.
Again their data indicate that L. monocytogenes should not survive in properly pasteurized
ice cream mix and that the pathogen had increased heat resistance in reprocessed products.
Holsinger et al. [173] investigated the effect of components of ice cream mix on thermal
resistance of L. monocytogenes. The D600C of L. monocytogenes in the mix correlated more
closely with the level of high-fructose corn syrup solids (HFCSS) or stabilizer (guar gum
and carrageenan) than with that of milk fat. Therefore, higher thermal resistance was
conferred by higher levels of HFCSS or stabilizer.
Although the aforementioned studies were all done with cows milk, MacDonald
and Sutherland [243] compared the thermal resistance of L. monocytogenes in sheeps
and cows milk using the sealed-tube method. The authors found that sheeps milk had
a protective effect on L. monocytogenes during heating at 65C when compared with cows
milk. When Listeria was initially present in milk at 106-107 CFU/mL, a count of s103
Listeria CFU/mL was observed after heating the sheeps milk ( 5 and 10% fat) for 45 min
at 65C; however, when cows milk and sheeps skim milk were treated similarly, no
survivors were detected by direct plating. Thus milk fat in sheeps milk protected Listeria
Characteristics of Listeria monocytogenes 74 7
during heating, whereas milk fat in cow's milk did not provide similar protection. When
whole sheep's milk was inoculated to contain 1O6 L. monocytogenes CFU/mL and pasteur-
ized at 68, 70, 72, and 74C for 15 s in an APV plate pasteurizer, the pathogen was only
detected in milk processed at 68OC, which indicates the adequacy of minimum HTST milk
pasteurization. However, caution should be exercised when interpreting these data, since
detection of Listeria in this study was done by direct plating of pasteurized milk on a
selective agar rather than using a preenrichment procedure for enhanced detection of suble-
thally injured cells.
Therma I Inactivat ion of Intrace1Iular L. rnonocytogenes
Studies discussed thus far have dealt with thermal inactivation of freely suspended cells
of L. rnonocytogenes in milk and other fluid dairy products. However, in cases of naturally
acquired listerial mastitis, the pathogen is normally not freely suspended in milk but rather
exists as a facultative intracellular bacterium within phagocytic leukocytes (neutrophils
and macrophages) typically present in milk. The facultative intracellular nature of L. rnono-
cytogenes has led some investigators to speculate that cells of the pathogen inside leuko-
cytes may be partially protected from thermal inactivation and thus are more able to sur-
vive pasteurization than are freely suspended cells of the bacterium in milk.
Intracellular L. rnonocytogenes cells induced by in vitro methods [45,93] were used
in early studies [61,64]. In 1986, Bunning et al. [61] determined thermal resistance of
L. rnonocytogenes in parallel experiments using freely suspended bacteria in raw milk as
well as L. rnonocytogenes cells that were inside of mouse peritonea1 phagocytes. Phago-
cytes were elicited in mice by injecting 107heat-killed L. monocytogenes (strain Scott A)
cells into the peritoneum and then were harvested by peritonea1 lavage. Differential stain-
ing indicated that the cell preparation was made up of 70% macrophages, 25% neutrophils,
and 5% lymphocytes. Opsonized cells of L. monocytogenes (i.e., incubated in normal
mouse serum at 37C for 30 min) were incubated in the phagocytic suspension for 60
min to allow phagocytosis. Phagocytes containing listeriae (average of 2.7-19.1
organisms/cell) were washed thrice by centrifugation and suspended in raw milk to obtain
- l O5 intracellular ListerialmL. Thermal resistance determinations were done using the
sealed-tube method of Bradshaw et al. [42,43] described earlier in this chapter. Mean
D-values for suspensions of intracellular L. rnonocytogenes in raw milk held at 52.2, 57.8,
63.3, and 68.9"C were 3170.0, 490.0, 33.3, and 7.0 s, respectively, as compared with
D-values of 2290.0, 445.0, 33.4, and 7.2 s when freely suspended cells were heat treated
at the same temperatures. Extrapolation of the data led to 1 ~ 7 1 . 7 0 C -of~ a1.9 e ~ 1.6 s
l ~and
for phagocytized and freely suspended listeriae, respectively. Under these experimental
conditions, the intracellular position did not appreciably protect L. rnonocytogenes from
thermal inactivation during pasteurization.
Subsequently, several methods were developed to obtain bovine phagocytes con-
taining internalized cells of L. monocytogenes, and such phagocytes have proven useful
in evaluating thermal resistance of intracellular listeriae. Briggs et al. [45] enhanced pro-
duction of bovine phagocytes (93% neutrophils, 5% macrophages, and 2% lymphocytes)
by infusing Escherichia coli endotoxin into the mammary gland. This procedure produced
an average of 2.4 X 106phagocytes/mL of milk, of which 89% were viable. Although
only 39% of the endotoxin-induced phagocytes ingested 1,. monocytogenes (average of
27 listeriadphagocyte) as compared with 64% of normal bovine phagocytes, no difference
in bactericidal activity was observed between endotoxin-induced and normal phagocytes.
In another study, Donnelly et al. [93] developed an in vitro assay to analyze uptake of
142 Lou and Yousef
L. monocytogenes cells by bovine phagocytes. Somatic cells harvested from fresh mastitic
milk were composed of 6 1% neutrophils, 20% macrophages, and 19% lymphocytes. Al-
though 75% of the neutrophils ingested opsonized L. monocytogenes cells as compared
with only 41% of the macrophages, both cell types contained an average of 19 listeriae
per phagocyte. Maximum Listeria uptake by phagocytes occurred within 30 min of incuba-
tion at 37C. Following ingestion, listeriae were resistant to the bactericidal activity of
phagocytes.
In 1988, Bunning et al. [64] reported results of a study comparing thermal resistance
of freely suspended and phagocytized cells of L. monocytogenes, the latter having been
prepared as previously described using endotoxin-induced bovine phagocytes [45]. Sterile
-
whole milk was inoculated to contain 1 Oh intracellular (average of 26 bacterialphago-
cyte) or freely suspended (obtained by sonicating phagocytes) L. monocytogenes cells/
mL and heated at 57.8, 62.8, 66.1, and 68.9"C using the sealed-tube method or at 66.1,
68.9, 7 1.7, and 74.4"C using a slug flow heat exchanger. Using the sealed-tube method,
the predicted D,,,80c-valuefor intracellular L. monocytogenes was 53.8 s, indicating a safe
33.4-D margin of inactivation for vat pasteurization (62.8"C/30 min). Using the slug flow
heat exchanger, D 7 1 , 7 0 C - ~ apredicted
lue~ from linear regression analysis were 4.1 s for intra-
cellular and 2.7 s for freely suspended listeriae. Hence, the intracellular position of
L. monocytogenes did not significantly (statistically) increase heat resistance under the
defined parameters of this study. More important, these results indicate potentially unsafe
3.7- and 5.6-D margins of inactivation for intracellular and freely suspended listeriae,
respectively, using the present minimum HTST pasteurization requirements (7 1.7"C/ I5 s).
The aforementioned data on heat resistance of intracellular L. monocytogenes were
obtained using phagocytes that were artificially induced to engulf listeriae. Heat resistance
of intracellular L. monncytogenes cells in milk from naturally or artificially infected cows
was investigated by several groups of researchers [9 1,96,136,240].A study that examined
heat resistance of L. rnonocytogenes in milk from a naturally infected cow was reported
in 1962 by Donker-Voet [91]. Milk from this cow contained 2 X 103-2X 104extracellular
listeriae and >I O6 leukocytes/mL but otherwise appeared completely normal. Although
no attempt was made to examine bovine phagocytes for intracellular listeriae, the organism
was presumably present in some of the leukocytes. After pooling the milk for a week and
holding it at 4"C, milk was heated in a plate-pasteurizer at 54-76.5"C for 15 s and then
examined for surviving Listeria cells. Unfortunately, by the time enough milk was ob-
tained for a pasteurization trial, the milk was heavily contaminated with other microorgan-
isms, making isolation of listeriae from milk extremely difficult. Furthermore, leukocytes
may have disintegrated, and the bacterial cells they may have contained were liberated
and became freely suspended cells in the milk. I n this study, L. monocytogenes survived
a heat treatment of 59.0"C for 15 s but did not survive in milk heated at 262.3"C for
15 s. This experiment was repeated using naturally contaminated milk from the same cow
that was held for only 2 days at 4C. Pasteurized milk was added to the contaminated milk
to increase the volume of milk available for pasteurization. Although the initial Listeria
population was not determined in the diluted milk before heating, L. monocytogenes was
detected in milk processed at 63.7"C for 15 s. However, L. monocytogenes was not found
in milk heated at 66.3, 68.0, 70.0, or 72.8"C for 15 s.
Pasteurization studies using L. monocytogenes-contaminated milk obtained from
cows artificially infected with the bacterium were conducted in 1987 by Doyle et al. 1961.
A laboratory culture of L. monocytogenes strain Scott A was inoculated into the udder of
each of four Holstein cows. Once listerial mastitis had developed, milk from these animals
Characteristics of Listeria monocytoge nes 743
was pooled and held 2 days (and in one instance 4 days) at 4C until sufficient quantities
were available to process in a pilot-scale plate pasteurizer (Cherry Burrell, model 217SB-
1) at 71.7-73.9"C for 16.4 s (nine trials) or 76.4-77.8"C for 15.4 s (three trials). Before
pasteurization, milk contained < 102- 1.9 X 10'' free Listeria cells and 4.5 X 105-2.4 X
1O6 somatic cells/mL. In addition, the milk generally contained 103- 1O4 L. monocytogenes
cells within polymorphonuclear leukocytes (PMNLs) per milliliter (average of 1.5-9.2
listeriae/PMNL). During pasteurization, 1 00-mL samples of milk were taken after 2, 4,
and 6 min of operation and analyzed for L. monocytogenes using two direct-plating and
three enrichrnent procedures. L. monocytogenes was isolated from milk in six of nine trials
in which the milk was heated to 71.7-73.9"C for 16.4 s. In contrast, L. morzocvtogenes
was not detected in milk from the remaining three trials in which the milk was processed
at 76.4-773C for 15.4 s. Additional studies on the fate of L. monocytogenes within
PMNLs indicated that the organism was no longer detectable in PMNLs after 3 days of
storage at 4C. Disappearance of listeriae after 3 days was accompanied by partial degrada-
tion of PMNLs, with complete breakdown occurring after 4 days. These findings suggest
that holding raw milk at 4C for 4 or more days would elirninate any thermoprotective
effect for listeriae that might result from their engulfment by PMNLs.
In the study just described, Doyle et al. [96] contended that phagocytized L. monocy-
togenes (<U. 1% infectivity with 1.5-9.2 listeriae/PMNL) cells were protected during pas-
teurization of milk at 71.7-73.9"C for 16.4 s. In contrast, using an in vitro method of
phagocytosis, Bunning et al. [64] reported that the intracellular position of L. monocyto-
genes (42% infectivity with 26 Listerialphagocyte) did not augment heat resistance of the
organism despite much larger numbers of engulfed listeriae in this study than in that of
Doyle et al. [96]. Lack of agreement between these two studies might be the result of the
bacterium being in different physiological states. It is well known that bacteria are gener-
ally more heat resistant during the stationary than the logarithmic phase of growth. Thus
nongrowing listeriae within bovine phagocytes may be more heat resistant than actively
growing cells that are engulfed by phagocytes or are freely suspended in milk.
In 1988, Farber et al. [ 1361, in Canada, tested raw milk containing 103-104L. mono-
cytogenes CFU/mL (- 105 somatic cells/mL with 10-50% of macrophages containing
- 1-20 listeriae/macrophage) which was obtained from a naturally infected cow, pooled
for 2.0-2.5 days, held at 4C and then heated in a pasteurizer. According to these authors,
L. monocytogenes survived heat treatments of 64 and 66C for 16.2 s but failed to survive
processing at 2 67C for 16.2 s.
Knabel et al. [208] realized that if L. monocytogenes is shed from infected cows
which have developed fever, the pathogen may have grown at elevated temperatures. They
also believed that Listeria cells inside macrophages are exposed to anaerobic conditions.
Therefore, a study was initiated to investigate the heat resistance of L. rntinocytogenes in
relation to its growth at elevated temperatures before heat treatment and anaerobic recov-
ery of heat-treated cells. The investigators heat treated L. monocytogenes (previously
grown at 37 or 43C) in sterile, whole, and homogenized milk and compared thermal
resistance data obtained when the heat-treated cells were recovered by incubation under
aerobic and anaerobic conditions. When Listeria was grown at 43C before heating and
recovered by anaerobic incubation after the treatment, the D62suc-value was 243 s as com-
pared with 36 s when the pathogen was grown at 37C and plated aerobically after the
heat inactivation. The researchers concluded that if L. monocytogenes is present at high
levels in milk, it could survive the minimum low-temperature, long-time milk pasteuriza-
tion process. Their results about the effect of anaerobic recovery also suggested that a few
144 Lou and Yousef
heat-injured cells remaining after minimum HTST milk pasteurization may grow under
anaerobic conditions that may exist in phagocytes. The investigators suggested that previ-
ous studies included (a) sample preparation practices such as sonication or mechanical
agitation in the presence of glass beads that may have disrupted phagocytes in milk, and
(b) aerobic plating may not have permitted recovery of heat-injured cells, thus D-values
for such studies were underestimated. Bunning et al. [63] argued that the homogenization
of raw milk at all milk processing plants will disrupt the phagocytes and that anaerobic
conditions did not exist in heat-treated milk as suggested by Knabel et al. [208].
In response to the previous study [208], Farber et al. [ 1331 investigated the impact
of growth temperature (30, 39, and 43C) and anaerobic incubation on recovery of L.
monocytogenes (a mixture of 10 strains) during milk pasteurization in a regenerative plate
pasteurizer at 63, 66, 69, and 72C for a minimum holding time of 16.3 s. The milk was
preheated at 85C for 1 h and cooled before inoculation and then held at 4C overnight
to simulate commercial holding practice. Four detection procedures, direct plating, a three-
tube most probable number (MPN) method, cold enrichment, and a warm enrichment
procedure were used and combined with both aerobic and anaerobic incubation. The milk
was inoculated to contain 5.0 X 104L. rnonocytogenes CFU/mL, which possibly represents
a worst-case situation. Consistent with the study of Knabel et al. [208], Listeria grown
at higher temperatures were more heat resistant. When the milk was pasteurized at 72"C,
L. monocytogenes was detected in four of four, two of five, one of four, and zero of four
trials when the cells were grown at 43, 39 (with 3 days of holding at 4"C), and 3OoC,
respectively. Therefore, L. monocytogenes cells grown at higher temperatures can survive
the minimum HTST milk pasteurization process. Although anaerobic incubation did not
appreciably enhance recovery of Listeria by direct plating, survivors in five of seven trails
at 72C were only detected under anaerobic conditions. An approximate D720c-valueof
8.1 s was calculated for Listeria grown at 43C. Increasing the holding time at 4C from
overnight to 3 days decreased the heat resistance of this pathogen.
Lovett et al. [240] investigated inactivation of both freely suspended and intracellu-
lar L. monocytogenes Scott A during the minimum HTST pasteurization (71.7"C, 15 s)
in a two-phase slug flow heat exchanger. Freely suspended listeriae were obtained by
inoculating raw milk to contain 2.6 X 105L. rnonocytogenes CFU/mL. Raw milk was
also inoculated to contain 5 X 104Listeria CFU/mL, of which 3-91% (average of 54%)
were intracellular, obtained through an in vitro internalization process. Raw milk for heat
treatment was also obtained from experimentally infected cows; the milk contained 3.4 X
103L. monocytogenes CFU/mL, with 53% being internalized. Three different enrichment
procedures were followed for detection of L. monocytogenes in pasteurized milks. The
researchers did not detect L. monocytogenes in any of the 23 minimum HTST milk pasteur-
ization trials.
Recognizing the impact of heat shock on thermotolerance, Bunning and coworkers
[63] studied the effect of heat shock on inactivation of L. monocytogenes during minimum
HTST pasteurization of whole milk. Heat shocking (48"C, 15 min) of Listeria in milk
increased the D71.70C from 3.0_+ 1.0 to 4.6 2 0.5 s; the latter value is comparable to that
for intracellular L. monocytogenes as measured in a previous study [64]. The authors
considered D71.70c-values after heat shock and those obtained with intracellular Listeria as
representing the upper limit of heat resistance in Listeria. However, after assessing the
data through risk analysis, Bunning et al. [63] believed that this increase in D71.7"C is
not a convincing reason to raise the minimum HTST milk pasteurization temperature
(71.7"C, 15 s).
Characteristics of Listeria monocytogenes 145
L. monocytogenes cells had a D550c-valueof 18.7 min and 26.4 min when the enumeration
plates were incubated aerobically and anaerobically, respectively [234]. Incubation at 25C
improved recovery of injured L. monocytogenes cells [64].
Conditions similar to heat shock exist in food processing. Slow heating or cooking,
preheating, hot water washing, mild thermal processes, and holding food in warm trays
(as occurs in food service establishments) are examples of heat shock that may happen
during food processing and handling. Heat shock may result, as suggested by Farber and
Brown [ 131 1, when foods are minimally processed or when the food is too bulky to allow
rapid heating. Heat shock may occur during vat pasteurization of dairy products or produc-
tion of "sous-vide" processed refrigerated foods, both of which involve a long-time tem-
perature coming-up and low-temperature heating/cooking [ 2331. The thermotolerance of
L. monocytogenes, Salmonella typhimurium, and Enterococcus faecium was increased by
low heating rates [203,244,309,3 10,3651. Quintavalla and Campanini [309] found that L.
monocytogenes became more heat resistant during slow (OS"C/min) rather than fast heat-
ing. A nearly twofold increase in the D-value for L. monocytogenes was noted by Kim
et al. [203] when the pathogen was heated in ground pork at I.3"C/min compared with
8.OoC/min. Stephens et al. [365] investigated heat inactivation of a 17-h-old culture of L.
monocytogenes (Scott A) in Tryptic Phosphate Broth at 50--64"C by both instantaneous
heating (adding a small portion of concentrated cells into a large volume of preheated
medium) and slow heating (0.7- 1 1 "C/min). Compared with instantaneous heating, slow
heating at a rate between 0.7 and 5"C/min significantly increased the heat resistance of
L. monocytogenes. This increase was maximal at a heating rate of I0.7"Clmin with a
population 1.7 X 105-foldhigher than that after instantaneous heating.
The heat-shock-induced thermotolerance of L. monocytogenes persists for a variable
time. Acquired thermotolerance of stationary-phase L. monocytogenes lasted at least 24
h at 4C in a sausage mix [131], < 1 h at 35"C, and 2 4 h at the heat-shock temperature
(42C) [62].
Besides heat shock, adaptation to other environmental stresses may also increase
the thermotolerance of pathogens. Farber and Pagotto [ 1341 found that exposing a station-
ary-phase culture of L. monocytogenes to a laboratory broth at pH 4.0 for 1 h rather than
2 or 4 h increased the DSROc-value in sterile whole milk from 2.75 to 3.90 min. A gradual
decrease of pH to 4 during 4 or 24 h also significantly increased heat resistance. Replacing
HC1 with acetic acid failed to increase heat resistance [ 1341. Lou and Yousef [238] found
that starvation and adaptation of L. monocytogenes to sublethal levels of HCl, ethanol,
and hydrogen peroxide significantly increased the thermotolerance. Maximum thermotol-
erance was observed in cells exposed to 4-8% (v/v) ethanol, pH 4.5, and 500 ppm hydro-
gen peroxide; the corresponding averages of Ds60Cin a phosphate buffer (pH 7.0) were
4.1, 8.8, and 2.9 min, whereas nonadapted L. monocytogenes cells had a D560C of 1.O min.
In phosphate buffer, starvation at 30C for up to 163 h increased the D-value of the re-
maining viable cells to 13.6 min (2381. Sudden osmotic shock (holding cells in a Tryptic
Phosphate Broth with 3.0-9.0% [w/v] NaC1) and osmotic adaptation (growth at high NaCl
concentrations) significantly increased the thermotolerance of L. monocytogenes Scott A
[ 1901. Thermotolerance of L. monocytogenes at 60C increased during osmotic up-shift
until the cells became almost as heat resistant as the culture grown for 48 h at the same
high osmotic conditions. Increased thermotolerance was rapidly lost (<5 min) during an
osmotic down-shock [ 1901.
Heat-shock and other environmental stresses also affect the virulence of L. monocy-
togenes. Environmental stresses are sensed by pathogens as signals for expression of viru-
148 Lou and Yousef
lence factors to enhance survival [20,235,257,28 I]. Heat shocking may increase the viru-
lence of L. rnonocytogenes. When L. rnonocytogenes was heat shocked at 48C for 2 h,
listeriolysin 0 was almost totally lost; however, subsequent growth of the heat-shocked
cells at 37C resulted in a 40-fold increase in production of the listeriolysin, whereas
unshocked cells exhibited only a 2-fold increase [202].
ACIDITY
Growth a t Low pH
According to Bergey 's Manual of Systematic Bacteriology [34 11, L. monocytogenes can
only grow at pH values from 5.6 to 9.6, with optimal growth occurring at neutral to slightly
alkaline pH values; the latter was verified by Petran and Zottola [300]. The minimum pH
value for growth is based on the work of Seeliger [340], who, in 1961, reported that L.
monocytogenes failed to grow in dextrose (glucose) broth at pH <5.6 after 2-3 days of
incubation at 37C. In addition, subcultures from the medium were no longer routinely
successful.
Listeriosis outbreaks linked to consumption of fermented dairy products have re-
opened the issue of a minimum pH requirement for growth which has now been revised
downward. During 1987, Lang et al. [219] examined growth at 13C of L. monocytogenes
(strain Ohio, isolated from recalled Liederkranz cheese) in TB adjusted to pH 5.0 and 5.6.
Following lag periods of 2.0 days at pH 5.0 and 0.5 day at pH 5.6, the pathogen grew
and reached maximum populations of 1.5 X 108and 4 X 10' CFU/mL in TB adjusted
to pH 5.0 and 5.6, respectively. During logarithmic growth, the organism exhibited genera-
tion times of 13.1 and 4.4 h in media adjusted to pH 5.0 and 5.6, respectively. Thus,
although Listeria failed to grow in TB at pH 5.0 during the initial 2 days of incubation,
further incubation led to growth of the organism with maximum populations being reached
after approximately 21 days at 13C.
Subsequent investigations have shown that L. rnonocytogenes can proliferate in labo-
ratory media adjusted to even lower pH values. When inoculated into Trypticase Soy
Broth acidified with hydrochloric acid, according to George et al. [153], all 16 L. monocy-
togenes strains tested initiated growth at pH values as low as 4.39-4.63 during extended
incubation at 20 or 30C. Although results from other independent studies
[40,15 1,288,3591confirm the ability of L. monocytogenes to multiply in similar laboratory
media adjusted to pH 4.4-4.6 with hydrochloric, citric, or malic acid, Farber et al. [135]
observed growth of L. monocytogenes at 30C in double-strength BHI broth acidified with
hydrochloric acid to a pH value as low as 4.3. Furthermore, L. innocua, L. seeligeri, and
L. ivanovii also were reported to grow in BHI broth acidified with hydrochloric acid to
pH values as low as 4.53, 4.88, and 5.16, respectively [151]. Thus the minimum pH at
which L. monocytogenes and most other Listeria spp. can grow is well below pH 5 pro-
vided that these organisms are incubated at near-optimum temperatures and allowed suffi-
cient time to overcome an extended lag phase.
As might be expected, growth of Listeria at low pH values is markedly influenced
by incubation temperature and the type of acid added to the medium; the latter will be
discussed in some detail later in this chapter. In one study [397], TB previously adjusted
to pH 5.0 and 5.6 was inoculated to contain -1 X 103L. monocytogenes CFU/mL and
then incubated at 4 and 13C. Listeriae not only failed to grow when incubated in TB
Characteristics of Listeria monocytogenes 149
(pH 5) at 4"C, but populations of the bacterium decreased clO-fold during 67 days of
storage. In contrast, increasing the incubation temperature to 13C led to growth at pH
5, with the organism attaining a final population of -1 X 10' CFU/mL. George et al.
[ 1531 found that minimum pH values for growth of 16 L. monocytogenes strains in Trypti-
case Soy Broth increased in the range of 4.39-5.45 as the temperature of incubation de-
creased from 30 to 4C. Sorrells et al. [359] reported that four different L. monocytogenes
strains grew in TB acidified to pH values as low as 4.40 following 7-28 days of incubation
at 10C, whereas growth of this organism at pH 4.4 was previously only observed at
220C. Hence, some L. monocytogenes strains may be able to grow, albeit slowly, in
laboratory media adjusted to pH 4.4 and incubated at near-refrigeration temperatures. Bu-
chanan and Klawitter [54] also reported a similar effect of incubation temperature on
growth of Listeria in TPB acidified to pH 4.5 with HCl. At 37"C, L. monocytogenes Scott
A was completely inactivated after 50 h of incubation, with populations, remaining stable
at 10 and 5C. However, at 28 and 19"C, the organism grew to -107 and -10' CFU/mL
in -100 and -500 h, respectively.
Growth of L. monocytogenes in acid or acidified foods confirms the findings in
laboratory media. In a study prompted by the listeriosis outbreak in Canada linked to
consumption of contaminated coleslaw [337], Conner et al. [74] demonstrated that L.
monocytogenes can tolerate and, in some instances, grow in cabbage juice at pH values
<5.6. Juice expressed from fresh cabbage was adjusted with lactic acid to pH values of
3.8-5.6, inoculated with L. rnonocytogenes at 104CFU/mL, and incubated at either 5 or
30C. After 3 days at 3OoC, Listeria reached maximum populations of -109 CFU/mL in
cabbage juice which had an initial pH 2 5.2. Rapid growth of listeriae during this period
was followed by equally rapid destruction, with the organism being no longer detectable
after -15 days at 30C. In cabbage juice adjusted to pH 5 and incubated at 3OoC, Listeria
exhibited a 3-day lag period and then grew to maximum populations 2 10' CFU/mL after
7 days of incubation before numbers decreased. At pH 5 4.8, L. monocytogenes was
inactivated in samples incubated at 30C. Although incubation at 5C prevented growth
of L. monocytogenes in cabbage juice adjusted to pH 5 5.6, listeriae populations remained
constant in samples at pH 2 5.2 during 22 days of storage. Interesting findings were
reported by Parrish and Higgins [288] on potential growth of' L. monocytogenes in orange
juice with modified pH. Initial Listeria populations of 106 CFU/mL increased approxi-
mately 1 and 2 logs in orange serum adjusted to pH values of 4.8 and 5.0, respectively,
during the first 2 days of incubation at 30C before decreasing to nondetectable levels 6
days later.
In 1988, Ryser and Marth [331J examined growth of L. monocytogenes at different
pH values in whey collected during manufacture of Camembert cheese. Samples of whey
were adjusted to pH values between 5.0 and 6.8, filter sterilized, inoculated to contain
5 X 10'-1 X 103L. monocytogens (four strains) CFU/mL, and incubated at 6C. Although
no growth occurred in whey at pH 5 5.4, small numbers of the organism survived during
the entire 35-day storage period. In contrast to the study involving cabbage juice [74], all
four strains grew in whey at pH 5.6 after 3 days of incubation at 6C. Under these condi-
tions, the four Listeria strains had generation times ranging between 25.3 and 3 1.6 h and
-
attained maximum populations of 1 X 107CFU/mL after 24 days at 6C. As expected,
L. monocytogenes had significantly (P < .OS) shorter generation times in whey samples
at pH 6.2 (14.8-21.1 h) and pH 6.8 (14.0-19.4 h) than at pH 5.6. The organism also
attained higher final populations in whey at pH 6.2 and 6.8 than at pH 5.6.
150 Lou and Yousef
Survival at Low pH
Although growth of L. monocytogenes at pH < 4.3 has not yet been documented, this
organism appears to be fairly acid tolerant. According to Reimer et al. [314], L. monocyto-
genes was recovered from inoculated samples of citrate/phosphate buffer that were acidi-
fied to pH 3.3 and held 4 h at 37C. However, the pathogen survived < I h in a similar
buffer adjusted to pH 1.4. Resistance of L. monocytogenes to acid was measured in D-
values by Ahamad and Marth (41. The bacterium exhibited average D-values of 13.3 and
11.3 days when held at 7C in TB previously adjusted with citric acid to pH values of
4.0-4.1 and 3.6-3.7, respectively. Since these authors obtained average D-values of only
2.2 and 1.4 days for corresponding cultures incubated at 35C L. monocytogenes can
clearly tolerate exposure to acid far better at near-refrigeration than at ambient tempera-
tures. Such behavior raises concerns about the safety of certain refrigerated acid and low-
acid foods that are often subjected to postprocessing contamination. Growth temperature
and growth rate before acid challenge also affect acid resistance of L. rnonocytogenes.
Patchett et al. [290] measured the acid tolerance of continuous cultures of L. monocyto-
genes that were grown at different growth rates or temperatures (10 and 30C). At the
same growth rate, L. monocytogenes grown at the higher temperature was more acid resis-
tant, whereas at the same temperature (30"C), cultures grown at a slower growth rate were
more acid tolerant.
Survival of Listeria in acid foods also varies with pH and temperature of storage,
as previously observed with laboratory media. Conner et al. [74) demonstrated that inacti-
vation rates for L. monocytogenes in acidified cabbage juice were inversely related to pH
with the organism surviving 49 days at pH 5.0-4.8 as compared with <2 I days in samples
of cabbage juice adjusted to pH 4.6 and 4.4. Parrish and Higgins [288] investigated behav-
ior of L. monocytogenes at 4C in inoculated (- 10' CFU/mL) samples of orange serum
adjusted to pH values of 3.6-5.0. Survival of the pathogen ranged between 21 days at
pH 3.6 and >90 days at pH 4.8 and 5.0 with slight growth of listeriae during storage
limited to orange serum adjusted to pH 5.0. Listeria was inactivated faster at higher rather
than lower incubation temperatures, with the pathogen being eliminated after 5 and 8 days
from orange serum at 30C and adjusted to pH values of 3.6-4.0 and 4.2-5.0, respectively.
Although acidic fruit juices appear to be unlikely sources of L. monocytogenes, the fact
that this pathogen survived well beyond the normal shelf life of nonsterile orange juice
(orange serum) suggests that such products should not automatically be eliminated as
possible vehicles of infection in future epidemiological investigations of human listeriosis.
Proper acid development is critical to the safety and quality of fermented foods.
Behavior of L. monocytogenes in these foods depends on numerous extrinsic and intrinsic
factors, including the pH. Camembert [329] (a mold-ripened cheese), Brick cheese [332],
and white pickled cheese [ 1) supported growth of L. monocytogenes, with the pH of these
cheeses being 5.9-7.2, 6.9-7.3, and >6.0, respectively. In contrast, the bacterium was
inactivated rapidly in Parmesan [403], mozzarella [50], and water-buffalo mozzarella
cheese [378], with final pH values of these cheeses being 5.0-5.1 , 5.2-5.3, and 4.0, respec-
tively. In most other cheeses investigated, L. monocytogenes survived to various degrees.
The bacterium persisted at least 28 days in creamed and uncreamed cottage cheese at pH
5.02-5.68 [327], 70 to 2434 days in Cheddar cheese at pH 5.0-5.15 13281, > 1 15 days
in Colby cheese at pH 5.0-5.18 [401], 270 days in semihard Manchego-type cheese at
pH 5.10-5.80 [90], 290 days in Trappist cheese at pH 4.70-5.42 [214] and feta cheese
at pH 4.6 [287], <66-80 days in Swiss cheese [49], >50 days in blue cheese [286], and
Characteristics of Listeria monocytogenes 75 7
2180 days in cold-pack cheese food without preservatives at pH 5.21-5.45 [330]. Viable
counts of L. monocytogenes decreased in cottage cheese stored at 4- 12C [ 170,3021.Simi-
lar studies concerned with behavior of L. monocytogenes in fermented meats have shown
that this bacterium can survive in hard salami at pH 4.3-4.5 during refrigerated storage
[ 1881. When cows milk was inoculated to contain 103and 107Listeria CFU/mL, made
into yogurt, and stored at 4OC, the pathogen survived for 2 and 7 days, respectively, at
pH 4.2-5.0 [25 I]. Other investigators, however, reported that L. monocytogenes remained
viable 13-27 days in yogurt stored at 4C [70]. Survival of the pathogen in yogurt was
reduced when milk was fermented at 42C with thermophilic starters compared with fer-
mentations that were done at 37C with mesophilic starters [335].Although these and
other studies will be discussed in greater detail in later chapters of this book dealing with
behavior of Listeria in dairy and meat products, it may be concluded that L. monocytogenes
is unlikely to initiate growth in food products which have a pH 5 5.2.
tures and unadapted cultures of an acid-tolerant mutant showed enhanced survival during
storage of cottage cheese (pH 4.71) for 15 days at 4OC, ripening of Cheddar cheese (pH
5.16-5.25) for 70 days at 8OC, storage of yogurt (pH 3.9) for 48 h at 4OC, active milk
fermentation (pH <4.8 or <5.5), and storage in acidic foods such as salad dressing (pH
3.0) and orange juice (pH 3.76) for 7 h at 4C. However, no significant differences were
seen in survival of both types of cells in mozzarella cheese (pH 5.6). The acid-adapted
L. monocytogenes culture and its acid-tolerant mutant ( 105CFU/mL) were partially inacti-
vated in yogurt during 48 h of storage at 4C with -3 and -5 log reductions for these
two types of cells, respectively, whereas the unadapted control was inactivated within
24 h. When salad dressing (pH 3, attained by adding acetic acid) was inoculated to contain
106L. monocytogenes CFU/ml and stored at 4OC, the unadapted cells were completely
inactivated in 15 min, whereas both types of acid-adapted cells survived up to 90 min.
L. monocytogenes (105 CFU/mL) also was added to milk being actively fermented with
S. thermophilus at 37C when the pH decreased to 4.8 or 5.5. During an additional 7 h
of fermentation (pH was reduced to 4.15), populations of unadapted L. monocytogenes
cells decreased >3 logs, whereas numbers of adapted and mutant cells decreased 5 1 log
[147].
O'Driscoll et al. [275] found that long-time acid challenge selected for acid-tolerant
mutants of L. monocytogenes. This is contrary to findings of Buchanan et al. [53]that no
subpopulation of acid-tolerant L. monocytogenes developed after treatment with a combi-
nation of 1.0% lactic acid, 6.3% NaCl, and 100 pg/mL NaN02at 19OC, a condition known
to cause tailing of inactivation curves. This discrepancy may have resulted from differ-
ences in methods used in these two studies to select mutants.
According to O'Driscoll et al. [275], acid-tolerant mutants have increased virulence
compared with that of the parental cells. Intraperitoneal injection of 105CFU mutant cells/
mL into mice resulted in death of three of four infected mice, whereas none of the mice
infected with parental cells showed any signs of infection. Additionally, higher counts of
mutant rather than parental L. monocytogenes were found in the spleen after injection
[275]. Virulent and avirulent Listeria strains also respond differently to stress [268]. Aviru-
lent strains of L. monocytogenes did not multiply [78] or were completely inactivated
[ 1691 inside macrophages, whereas virulent strains survived and multiplied inside the mac-
rophage [78,169]. Therefore, considering that the increased acid tolerance of acid-adapted
or acid-tolerant mutants of L. monocytogenes may help the pathogen to survive inside
macrophages, and that L. monocytogenes can produce hemolysin over a wide range of
pH values (55-29) [200], it would not be surprising to observe the increased virulence
of acid-adapted or constitutively acid-tolerant cells as demonstrated by O'Driscoll et al.
[275].
Environmental stresses presumably are used by L. monocytogenes and other patho-
gens as signals for expressing virulence factors and enhancing survival [20,211,212,235].
Although weak organic acids and their salts inhibit growth of L. monocytogenes, these
compounds may enhance the virulence of this pathogen, and so should not be over-
looked in assessing the safety of preserved foods. Kouassi and Shelef [211] tested the
effect of salts of five weak acids on growth of L. monocytogenes and associated secretion
of listeriolysin 0, the exotoxin important for the spread of the pathogen, in TSB (pH
7.2-7.4) at 35 and 20C. Citrate, acetate, lactate, and propionate increased secretion of
listeriolysin 0, with only sorbate inhibiting secretion of this toxin. The inhibitory effect
of sorbate was later confirmed by the same authors [212]. McKellar [255] found that
Characteristics of Listeria monocytogenes 153
listeriolysin 0 is stable at >pH 5.3 (lactic acid), and has maximum activity at pH
4.0-5.0.
WATER ACTIVITY
The moisture requirement for microbial growth can best be expressed in terms of water
activity (a,), which is defined as the ratio of the water vapor pressure of a food substrate
to the vapor pressure of pure water at the same temperature. Like most bacterial species,
L. monocytogenes grows optimally at a, -0.97 [300]. However, when compared with
most foodborne pathogens, this bacterium has a rather unique ability to multiply at a,
values as low as 0.90.
L. monocytogenes can grow in complex laboratory media containing up to 10%
NaCl [341]. Skovgaard [351] estimated the a, of such a medium at -0.93 and therefore
predicted that L. monocytogenes would not grow at a, < 0.93. The minimum a, for growth
of L. monocytogenes estimated by Skovgaard was confirmed by Sperber [360]. Using
liquid laboratory media adjusted with NaCl to various a, values, growth of L. monocyto-
genes at 35C was observed at an a, of 0.943 but not at 0.935. Similarly, adjustment of
a, using sucrose and glycerol allowed growth of L. monocytogenes at minimum a, values
of 0.941 and 0.932, respectively. More recently, growth of L. monocytogenes at lower a,
was observed by Petran and Zottola [300], Sorrells and Enigl [358], and Miller [260].
The bacterium grew in TSB containing 39.4% sucrose (a, = 0.92) when incubated at
30C for 24 h [300] or in BHI broth containing 12% NaCl (a, -0.92) during incubation
at 10 and 25C [358]. Tapia de Daza et al. [370] found that when the a, of TSB was
adjusted with glycerol, sucrose, or NaCl, two strains of L. monocytogenes grew minimally
(determined as detectable turbidity of the culture in 20 days) at a, values of 0.90, 0.92,
and 0.93 at 3OoC, and 0.92, 0.93, and 0.94 at 4"C, respectively. Nolan et al. [274] found
the minimum growth (at least 1 log increase in 22 days at 2 1 "C) a, of L. monocytogenes
in TSBYE to be 0.90, 0.92, and 0.92 when water activity was adjusted with glycerol,
NaC1, and sucrose, respectively. When L. monocytogenes Scott A was grown at 28C in
BHI broth adjusted to different a, values (0.99-0.80) with glycerol, NaC1, or propylene
glycol; the minimum a, values for growth at 28C were 0.90, 0.92, and 0.97, respectively
[260].
Although L. monocytogenes does not appear to grow at a, < 0.90, the bacterium
can survive for extended periods at lower a, values. Shaharnat et al. [343] reported that
the bacterium survived at least 132 days at 4C in Trypticase Soy Broth containing 25.5%
NaCl, which would be expected to have an a, of -0.83. Survival of L. monocytogenes
under reduced moisture depends on both the a, and the dominant solute in the medium.
In BHI broth adjusted to the same a, values, the pathogen survived longest with glycerol
and shortest with propylene glycol and NaCl yielded intermediate survival [260]. Nolan
et al. [274] also reported generally shorter survival of L. monocytogenes in NaC1-adjusted
than in sucrose-or glycerol-adjusted TSYEB.
Survival of L. monocytogenes during processing and storage of food may depend
on a, of the medium. When sucrose/phosphate buffer solutions were inoculated to contain
- 104-105 L. monocytogenes CFU/mL and held at 140"C, Sumner et al. [369] found that
the pathogen was about four times more heat resistant in buffer having an a, value of
0.90 as compared with 0.98. Thus, given the inverse relationship between a, and thermal
resistance along with the ability of L. monocytogenes to grow at an a, value of 0.92 and
754 Lou and Yousef
ferment concentrated sucrose solutions, this organism also may be important to companies
that manufacture foods containing high levels of sugar, as has already been demonstrated
for Karo corn syrup stored at refrigeration temperatures 12841.
Of more practical importance to food processors, Johnson et al. [ 1881 found that L.
monocytogenes survived at least 84 days at 4C in fermented hard salami which had an
a, between 0.79 and 0.86. Extended survival of listeriae in sausage occurred despite the
presence of 5.0-7.8% NaCI, 156 ppm sodium nitrite, and a pH of 4.3-4.5. The authors
suggested that L. monocytogenes might survive longer at an a, of 0.9 1, which is occasion-
ally found in commercial hard salami. However, they also predicted that growth of the
bacterium in such sausage would be unlikely given the combination of salt, sodium nitrite,
low pH, and low storage temperature.
Additional information concerning the relationship between a, and growth/survival
of L. monocytogenes can be obtained from several dairy-related studies. Using pasteurized
whole milk inoculated to contain -500 L. monocytogenes CFU/mL, Ryser and Marth
[328] manufactured Cheddar cheese which, according to Marcos and Esteban [250], had
a, values between 0.972 and 0.979. The organism survived as long as 224 and >434
days in Cheddar cheese (pH 5.0-5.1) ripened at 13 and 6OC, respectively. Since Listeria
reportedly grows well within this a, range, the combined effects of low pH and low-
ripening temperature probably played a dominant role in preventing growth of listeriae.
Camembert cheese, also prepared by Ryser and Marth [329], had a, values between 0.959
and 0.984 [250], which should have allowed growth of L. monocytogenes. However, lister-
iae populations remained constant or decreased in cheese at pH 4.6 to -5.5 during the
first 20-30 days of ripening. Initiation of rapid Listeria growth in cheese at a pH between
-5.5 and 6.0 illustrates that pH rather than a, is primarily responsible for determining
growth characteristics of listeriae in Camembert cheese. Parmesan cheese was made of
pasteurized milk inoculated with 104- 10sListeria CFU/mL [4031. Listera was inactivated
rapidly in this cheese and was not detectable after 2- 16 weeks of ripening. A combination
of low moisture (30.1-3 1.4%), low pH (5.0-5. I), and heat treatment during curd cooking
(51C for -45 min) likely contributed to the rapid demise of Listera in this cheese.
Salt (i.e., sodium chloride or NaCl), an important ingredient in defining the water activity
(a,) of many foods, affects microbial growth and survival in such foods. Salt, however,
also exerts antimicrobial effects that can not be explained by its ability to lower a food's
a,. Therefore, resistance of L. monocytogenes to salt and interaction of this important
ingredient with the pathogen are described here in some detail.
Characteristics of Listeria monocytogenes 755
Salt Tolerance
According to Bergey 's Manual of Systematic Bacteriology [34 11, L. monocytogenes can
grow in Nutrient Broth (NB) supplemented with up to 10% !w/v) NaCl. Although this
viewpoint concerning tolerance of Listeriu to NaCl is apparently based on results from
Larsen [224], preliminary data from one investigative team [lSl], reported in 1988, indi-
cate that one strain of L. monocytogenes grew at 8-30C during extended incubation in
-
BHI broth (pH 5.0) that contained up to 12% NaCl. Under identical conditions, single
strains of L. ivanovii, L. seeligeri, and L. innocua were only slightly less halotolerant,
with growth ceasing in the presence of >10% NaCl. In agreement with these findings,
Sorrells and Enigl [358] found that two strains of L. monocytogenes grew in TSB con-
taining 10% NaCl at 35C or 12% NaCl at 10 and 25C. Listeria may grow to high
numbers in the presence of moderate amounts of salt. Hudson [ 1771 inoculated BHI broth,
which contained 6.5% NaCl, with 106 CFU L. monocytogeizeslml. The bacterium in-
creased by at least 3 logs after 15 and 26 days at 10 and 0-4"C, respectively.
Lang et al. [219] found that growth of L. monocytogenes in TB containing 6% NaCl
was markedly influenced by pH. In their study, TB containing either 0 or 6% NaCl (w/
v) was adjusted to pH 5.0, 5.6, 6.2, and 6.8 with HCI, inoculated to contain -5 X 102
L. monocytogenes CFU/mL, and incubated at 13C. When grown in salt-free media at
pH 5.0, 5.6, 6.2, and 6.8, this organism had generation times of 13.1, 4.4, 3.5, and 2.9 h,
as compared with 77.8, 7.2, 5.0, and 6.3 h in the same medium containing 6% NaCl,
respectively. Thus the combination of pH 5 and 6% NaCl was most effective in inhibiting
growth of Listeria. Borovian 1401 also reported that L. monocytogenes grew at 10C in
culture media adjusted to pH 4.5 and 6.0 and containing 5 4 and 5 7 % NaCl, respectively.
These findings agree with those of Lang et al. [219].
Extended survival of listeriae occurs at a wide range of salt concentrations. Studies
at ambient temperatures demonstrated that L. monocytogenes can persist at least 150 days
in pure salt 13481 and 545 days in 0.85% NaCl 13051. In 1955, Stenberg and Hammainen
[364] reported that 10 L. monocytogenes strains survived > 1 year at 20-24C in NB
containing I % glucose and 10% NaCl. Listeriae also survived 34-68 days and 24 days
in the same medium containing 12 and 24% NaCI, respectively. When Stenberg and Ham-
mainen [364] stored organs (liver, heart, kidney) from Listeria-infected mice in salt solu-
tions at 4"C, L. monocytogenes remained viable for 238-246, 88-1 12, and 27 days in
solutions containing 3, 6, and 12% NaCI, respectively.
Survival of Listeria in the presence of salt varies with the storage temperature. In
experiments by Shahamat et al. [343], L. monocytogenes was inactivated in Trypticase
Soy Broth containing 10.5, 13.0, and 25.5% NaCl after 14, 9, and 4 days of incubation
at 37"C, respectively. Survival times in media containing 25.5% NaCl increased from 3
days at 37C to 24 days at 22C and to > 132 days at 4C. Sorrells and Enigl [358] found
that -106 CFU/mL of L. monocytogenes (two strains) in TSB, which contained 12 and
14% NaCI, decreased to a nondetectable level in 14-21, and 36 days at 35 and 25"C,
respectively, whereas reductions of only -2 logs occurred when listeriae were kept in
14% NaCl for 36 days at 10C. In a study by Hudson [ 1771, L.monocytogenes populations
(106 CFU/mL) in BHI broth containing 26.5% NaCI, decreased 4, 2, and 0 logs at 10,
0-4, and -- 18"C, respectively, after 33 days of storage with D-values of 6 and 19 days
at 10 and 0-4"C, respectively. Presence of 16.5% NaCl in the same medium did not affect
the Listeriii count after 33 days of storage at all three temperatures. These data indicate
156 Lou and Yousef
minimal medium containing 3% NaCl, with final numbers reaching 109CFU/mL. A popu-
lation of only 10' CFU/mL was reached in the absence of osmoprotectants [35]. In accor-
dance with these findings, KO et al. [209] and Smith [352] reported that exogenous addition
of betaine stimulated growth of L. monocytogenes in a defined medium with a high content
of NaC1. Foods usually contain enough osmoprotectants to support microbial growth. Plant
foods are rich in betaine, whereas foods of animal origin are high in choline (the precursor
of betaine) and carnitine [35]. Processed meats (bologna, frankfurters, wieners, ham, brat-
wurst, salami) contain betaine at 0.34-0.48 nmol/mg and carnitine at 0.23-0.95 nmol/
mg [352]. Processed and ready-to-eat meats, which are high in salt and low in a,, can
contain L. monocytogenes. The capacity of L. rnonocytogenes to grow on the surface of
processed meats is reportedly related to the organism's ability to accumulate high levels
of betaine and carnitine (200- 1000 nmol/mg cell protein), with salami supporting neither
good growth nor the accumulation of betaine or carnitine [352].
did not grow at 4C. However, at pH 6 and 4C Listeria growth occurred at the three
lower concentrations (0,0.03, and 0.003 M) of lactic acid. At pH 7, listeriae grew at 35"C,
regardless of lactic acid concentration; however, at 4C and the same pH, the pathogen
only grew in media containing 10.03 M lactic acid with a 7-day lag time.
Using an experimental design similar to that of Bojsen-MQIler [39], Ahamad and
Marth [4] examined the ability of acetic, citric, and lactic acid to prevent growth of L.
monocytogenes in TB during extended incubation at 7-35C. As expected, the pathogen
was markedly affected by type and concentration of acid as well as incubation temperature.
The presence of as little as 0.05% acetic acid (pH 5.8-5.9) caused noticeable inhibition
of Listeria, with deleterious effects of acetic as well as citric and lactic acid being more
evident at low rather than high incubation temperatures. Increasing the concentration of
acetic, citric, or lactic acid to 0.2% (pH 4.4-4.6) completely suppressed growth of the
organism at all incubation temperatures, with death of the pathogen occurring in the pres-
ence of 10.3% (pH < 4.2-4.3) acetic, citric, or lactic acid. In contrast, citric acid was
less inhibitory than acetic acid, with growth of listeriae occurring in all samples with 0.1%
citric acid regardless of temperature. The relationship between incubation temperature
and inhibition of L. monocytogenes was most pronounced with lactic acid; the pathogen
proliferated in the presence of 0.1% lactic acid at all temperatures except 7C. Results
from a follow-up study [ 5 ] showed that during extended incubation at both 13 and 35"C,
the presence of 0.3 and 0.5% citric acid in TB was most injurious to L. monocytogenes
followed in order by similar concentrations of lactic and acetic acid. Acid-injured listeriae
survived approximately nine times longer at 13" than 35C.
In 1989, Sorrells et al. [359] published results of a study that examined the effect
of pH, acidulant, time, and temperature on growth and survival of L. monocytogenes in
TSB acidified to pH values of 4.4-5.2 with hydrochloric, acetic, lactic, malic, or citric
acid. Based on average minimum pH values permitting growth of four L. monocytogenes
strains at 10, 20, and 35"C, acetic acid was again most inhibitory (pH 5.04) followed by
lactic (pH 4.73), citric (pH 4.53), malic (pH 4.46), and hydrochloric acids (pH 4.46).
These findings generally agree with those of Ahamad and Marth [4] and several other
investigators [33,116,135]. As in the previous study by Ahamad and Marth [4], longest
survival of listeriae occurred at lower rather than higher incubation temperatures. How-
ever, since the inhibitory activity of the various acids tested was markedly different when
based on equal molar concentrations of acid rather than pH, these data again indicate that
differences in antilisterial activity of acidulants depend on both type and concentration of
acid rather than on pH alone.
According to results just described and those from subsequent studies [4,184,213,
359,4001, acetic acid is most listericidal and listeriostatic followed by citric acid when
used on an equal weight (% w/w) or molar concentration basis at the same pH. However,
based on equal molar concentrations of undissociated organic acid at the same pH, the
order was reversed (i.e., citric >lactic ?acetic acid) for growth inhibition at 2 5 3 7 C
[51,400]. Differences in listericidal activity among the three acids were much greater at
low rather than at high concentrations, and diminished as the concentration of undissoci-
ated acid increased to 3 mM [5I].
Citric and lactic acids have different effects on survival of listeriae. Although high
levels of both acids inactivated listeriae in a similar pattern, low levels (0.1-0.5 M) of
citric acid, especially at pH 5-6, protected listeriae from death [51,521. Low concentrations
(50 mmol/mL) of citric acid were also noted by Young and Foegeding [400] to enhance
growth of L. monocytogenes at pH 4.7-5.0.
Characteristics of Listeria monocytogenes 159
and stored for 40-50 days at 5C. Similar inhibition for up to 35 days at 10C was observed
in the presence of a combination of 3% lactate and 3% water-phase NaCl or of 2% lactate,
3% NaC1, and 125 ppm NaN02. L. monocytogenes grew appreciably in control samples
that contained 3% water-phase NaCl or 3% NaCl plus 125 ppm NaNO, [298]. The antilis-
terial activity of lactate in broth and bologna-type sausages was modeled recently by Hout-
sma et al. [175]; the model can be applied to predict Listeria growth in the presence of
lactates in this product.
In 1995, Buncic et al. [60] reported on the antilisterial activity of sodium lactate
and other antimicrobial agents in a buffered BHI broth (pH 5.5) held at 4C. When used
alone or in combination with sodium nitrite ( I 25 ppm) and/or polyphosphate (0.5%) so-
dium lactate (4%) prevented growth of L. monocytogenes (initial population 107CFUI
mL) during 7 weeks of incubation at 4C; however, no bactericidal effect was observed.
The antilisterial activity of sodium lactate was improved by addition of nisin (400 IU/
mL) but not 0.3% sorbate. Lactate and nisin had a synergistic effect against L. monocyto-
genes which was further enhanced by addition of polyphosphate (0.5%). Combinations
of lactatehisin and lactate/nisin/polyphosphatedecreased Listeria population by 2.2-2.4
and 4.2 logs after 28 and 20 days, respectively. In contrast, nisin alone only resulted in
an initial 1.1 -log decrease, but listeriae grew during prolonged refrigerated storage.
Sodium Diacetate
Sodium diacetate (CH,COOH CH,COONa), which contains acetic acid (about 40%) and
sodium acetate, is considered a generally recognized as safe (GRAS) additive by the
FDA. It is used as an acidulant, flavoring agent, and antimicrobial agent in foods [67,346].
Shelef and Addala [346] investigated the antilisterial activity of sodium diacetate in BHI
broth at 35, 20, and 5C. After adding sodium diacetate (18-35 mM) to BHI broth, the
resulting mixture of pH 6.3-5.25 was inoculated to contain about 103L. monocytogens
CFU/mL. Inhibition of L. monocytogenes increased with increasing diacetate levels and
decreasing incubation temperatures. The minimum inhibitory concentrations (MICs) of
diacetate in BHI broth were 35, 32, and 28 mM at 35, 20, and 5OC, respectively. Based
on equal levels of undissociated acetic acid at different pH values, sodium diacetate was
more effective and had lower MICs at 35C than did acetic acid; the MICs were 5 , 20,
30, 40, and > 100 mM for sodium diacetate, and 5 , 20, >50, >100, and > 150 mM for
acetic acid, at pH 4.7, 5.0, 5.5, 6.0, and 6.5, respectively.
The same study also assessed the antimicrobial activity of diacetate in meat [346].
Sodium diacetate added to ground beef (pH 5.6) or beef slurry (pH 5.6) greatly inhibited
growth of aerobic microflora during storage at 5C. Addition of 21 and 28 mM sodium
diacetate decreased the pH of ground beef from 5.6 to 5.17 and 5.10, respectively. Growth
of aerobes was measured with and without adjusting the sodium diacetate-containing
ground beef to pH 5.6. Populations of aerobes, after storage at 5C for 8 days, reached
7.12 and 6.21 logs and 5.98 and 5.1 1 logs in pH-adjusted and pH-unadjusted ground beef
containing 2 1 and 28 mM sodium diacetate, respectively. However, these organism, also
increased from 3.38 to 9.72 logs in the sodium acetate-free control. Similar trends were
seen in beef slurry. When 15 different aerobic bacteria were tested, sodium diacetate gener-
ally was more inhibitory to gram-negative than to gram-positive bacteria, although some
exceptions were noted [3461.
Schlyter et al. [338] investigated the antibacterial activity of sodium diacetate alone
or in combination with the commercial shelf-life extender, ATLA 2341 (a fermentation
product from lactic acid bacteria, Quest International Bioproducts, Sarasota, FL) in turkey
Characteristics of Listeria monocytogenes 161
slurry (pH 6.2) at 25C. During 7 days of incubation, the L. monocytogenes population,
initially present at 3.7 logloCFU/g, increased to 8.6 logs in the control and 6.8 logs in
the slurry, which was treated with 0.3% (21 mM) sodium diacetate. Additionally, sodium
diacetate at 05% (35 mM) inhibited growth and caused a slight decrease in the viable
Listeria population. The presence of ATLA 2341 at 0.25-0.75% did not appreciably affect
growth of listeriae; however, a synergistic inhibitory effect against Listeria was observed
when the additive, at the levels just indicated, was used in combination with 0.3 and 0.5%
diacetate. In a subsequent study by the same group [339], adding 0.3 and 0.5% sodium
diacetate to turkey slurry made the product listericidal when held at 4 and 2SC, respec-
tively. Antilisterial activity of sodium diacetate was synergistically enhanced by 2.5%
sodium lactate or 5000 units pediocin/mL but not by 30 ppm of sodium nitrite. The count
of Listeria in turkey slurry, which contained pediocin only, decreased by 0.9 log and then
increased and reached a final population (8.0 logs) similar to that of the control. Combined
use of pediocin and 0.3% diacetate at 4C or pediocin and 0.5% diacetate at 25C gave
counts of Listeria in the product that were -7 logs lower than those in the additive-free
controls.
Sodium Propionate
In 1987, Lang et al. [219] found that L. monocytogenes grew at 13C in TB at pH 5
supplemented with 0 and 6% NaC1; however, growth was prevented by addition of 5000
ppm propionic acid at both salt concentrations as well as by the combination of 6% NaCl
and 0.1% propionic acid. Using TB at pH 5.6 and containing 0, 1000, or 5000 ppm propi-
onic acid, L. monocytogenes grew to final populations of 107--10*and 104- 105CFU/mL
in the presence of 0 and 6% NaC1, respectively. Generation times calculated for listeriae
in the salt-free medium at pH 5.6 and containing 0, 1000, and 5000 ppm propionic acid
were 4.4, 10.3, and 16.1 h, respectively, rather than 7.2, 18.1, and 42.1 h, respectively,
in the same medium containing 6% NaCl. Similar behavior by L. monocytogenes was
subsequently noted during extended incubation at 4-30C in BHI broth (pH 5.9) con-
taining 4.0% NaCl and 0.15% potassium sorbate [151].
El-Shenawy and Marth [ 1 171 demonstrated that >2000 ppm sodium propionate can
inhibit growth of L. monocytogenes in TB at pH 5. Generation times for L. monocytogenes
in TB at pH 5.6 and without sodium propionate decreased from 68 to 49 min as the
incubation temperature increased from 4 to 35C. In TB at pH 5.6 and containing 3000
ppm sodium propionate, generation times decreased from 3.0 days to 4.5 h as the incuba-
tion temperature increased from 4 to 35C. Using TB at pH 5 and containing 3000 ppm
sodium propionate, Listeria populations decreased 1 log during 67 days of incubation at
4C. When the same medium was incubated at 35"C, numbers of L. monocytogenes de-
creased -3 logs, with the organism no longer being detected after 78 days,
In a follow-up study, El-Shenawy and Marth [ 1201 investigated the antilisterial activ-
ity at 13 and 35C of sodium propionate in combination with common organic acids. TB
was prepared to contain 0, 500, 1500, or 3000 ppm sodium propionate and the pH of the
medium was adjusted to 5.0 or 5.6 with HC1 or one of four common organic acids (acetic,
tartaric, lactic, and citric). Decreasing the pH from 5.6 to 5.0 enhanced the antilisterial
activity of propionate. Organic acids, when compared with HCl, greatly enhanced the
antilisterial activity of propionate, with acetic acid being the most effective followed by
tartaric, lactic, and citric acids. Lowering the incubation temperature from 35 to 13C not
only diminished the growth rate of L. rnonocytogenes, but also decreased the maximum
population of the bacterium for all combinations of propionate and organic acids.
162 Lou and Yousef
When used at 3000 ppm, Ryser and Marth [330] found that sodium propionate was
less effective than sorbic acid in eliminating four strains of L. monocytogenes from cold-
pack cheese food at pH 5.20-5.45. Cheese food was inoculated to contain -5 X 102L.
monocytogenes CFU/g and stored at 4C; the pathogen survived an average of 142 and
130 days in product that contained sodium propionate and sorbic acid, respectively. In
contrast, the pathogen was present in cheese food made without preservatives at levels
-
of I X 102CFU/g after 6 months of refrigerated storage.
Potassium Sorbate
Since receiving GRAS status in the United States during the 1950s, potassium sorbate
and sorbic acid have been widely used to extend the shelf life of many foods, including
butter, cheese, meat, cereals, and bakery items. Although most effective against yeasts
and molds, these antimicrobial agents also inhibit a wide range of bacteria, particularly
aerobic catalase-positive organisms. Consequently, the ability of potassium sorbate and
sorbic acid to inhibit L. monocytogenes has been assessed in laboratory media and several
foods. Moir and Eyles [265]measured the minimum inhibitory concentrations (MICs) of
sorbate against L. monocytogenes Scott A in buffered BHI broth. These authors reported
MICs of 400-600 and >5000 mg/L at pH 5 and 6, respectively, when the culture was
incubated at 35C and 1500 mg/L in broth of pH 6 which was refrigerated at 5C.
According to data collected by El-Shenawy and Marth [ 1 1.51, the ability of potassium
sorbate to prevent growth of L. monocytogenes is related to temperature and pH. In the
absence of potassium sorbate, generation times for L. monocytogenes in TB at pH 5.6
decreased from I . 13 days to 49 min as the incubation temperature increased from 4 to
35C. Addition of 2500 ppm potassium sorbate prevented growth of Listeria at 4C and
led to complete demise of the organism after 66 days, whereas listeriae grew with a genera-
tion time of 9 h in the same sorbate-containing medium incubated at 35C. The lower the
storage temperature and pH of the medium, the greater was the effectiveness of sorbates
against L. monocytogenes. In a subsequent study, El-Shenawy and Marth [ 1 191 investi-
gated the antibacterial activity of sorbate in the presence of other organic acids. TB was
prepared to contain 500, 1500, and 3000 ppm potassium sorbate and pH of the medium
was adjusted to 5.0 or 5.6 using HCI or organic acids (acetic, tartaric, lactic, or citric),
inoculated with L. monocytogenes, and incubated at 13 or 35C. When compared with
HCl as an acidulant, the antilisterial activity of sorbate was enhanced more by organic
acids, with acetic and tartaric acids being more effective than lactic and citric acids.
Working with food, Ryser and Marth [330]found that four strains of L. monocyto-
genes were eliminated faster from cold-pack cheese food at pH 5.45 that contained 3000
ppm sorbic acid (4100 ppm potassium sorbate) rather than from the same product at pH
5.2 manufactured without preservative. After inoculating cheese food containing sorbic
acid with one of four L. monocytogenes strains at a level of -5 X 1O2 CFU/g, the pathogen
survived an average of 142 days at 4C. Although L. monocytogenes failed to grow in
cheese food with a pH of 5.21 prepared without sorbic acid, the pathogen survived during
-
the normal 6-month shelf life of the product at potentially hazardous levels of 1 X 1 O2
CFU/g. Since potassium sorbate works best at pH < 6.0 and is generally ineffective at
pH > 6.5, it is not surprising that Dje et al. [SS] found potassium sorbate to be of little
use in inactivating L. monocytogenes in samples of reconstituted nonfat dry milk.
More recently, the antilisterial activity of potassium sorbate and other antimicrobial
agents was investigated by Buncic et al. [60] in buffered BHI broth (pH 5.5) at 4C.
Potassium sorbate (0.3%) prevented growth of L. monocytogenes (initially 1O7 CFU/mL)
Characteristics of Listeria monocyt ogenes 163
during 6 weeks of incubation; however, no bactericidal effect was observed. Strong listeri-
cidal effects were observed when sorbate was used in combination with sodium nitrite
(125 ppm), polyphosphate (0.5%),or nisin (400 IU/mL). Nitrite alone was only inhibitory
to L. monocytogenes, whereas nisin alone only caused an initial 1.1 -log decrease, with
Listeria survivors subsequently growing during prolonged incubation. Combined use of
sorbate and nitrite caused a 6.7-log reduction in 6 weeks. Adding polyphosphate to this
sorbatehitrite combination resulted in 6.4-log reduction in about 4 weeks. The combina-
tion of sorbate and nisin caused a 4.5-log reduction in 5 weeks, and this synergistic effect
was further increased by addition of nitrite (125 ppm). No antilisterial interaction was
observed between sorbate and 4% sodium lactate. A combination of lactate, sorbate, and
nisin prevented growth of Listeria but did not cause significant inactivation. Adding nitrite
to the three agent combination reduced populations by 3.7 logs after 37 days, with the
same reduction being achieved in I2 days by further incorporation of 0.5% polyphosphate.
Sodium Benzoate
El-Shenawy and Marth [ 1 141 reported that sodium benzoate is more inhibitory to L. mono-
cytogenes than is either potassium sorbate or sodium propionate. In this study, TB was
supplemented with 0-3000 ppm sodium benzoate in increments of 500 ppm, adjusted to
-
pH 5.0 and 5.6 with hydrochloric acid, inoculated to contain 103L. monocytogenes CFU/
mL, and incubated at 4, 13, 21, or 35C. At pH 5.6, L. monocytogenes was inactivated
in the presence of 22000 ppm sodium benzoate after 60 days of incubation at 4C. At
pH 5 , the organism was completely nonviable in TB containing 2 1500 ppm sodium benzo-
ate after 24-30 days of incubation at 4"C, whereas lower concentrations of sodium benzo-
ate led to gradual decreases in numbers of listeriae during 66 days at 4C. Inhibition of
Listeria by benzoate decreased at incubation temperatures above 4C and at pH values
higher than 5.
Inhibition and inactivation of L. monocytogenes in the presence of sodium benzoate
is affected by (a) temperature (i.e., more rapid at higher than lower incubation tempera-
tures), (b) concentration of benzoic acid (i.e., more rapid at higher than lower concentra-
tions), and (c) pH (i.e., more rapid at lower than higher pH values) as well as the type
of acid used to adjust the growth medium. When TB was acidified to pH values of 5.0
and 5.6 with acetic, tartaric, lactic, or citric acid rather than hydrochloric acid, El-Shenawy
and Marth [ I 161 found that the antilisterial activity of sodium benzoate was greatly en-
hanced. For example, 1500 ppm sodium benzoate led to complete inactivation of L. mono-
cytogenes after 96 h at 35C when acetic or tartaric acid was used to adjust the pH of the
medium to 5 ; under the same conditions, the pathogen remained viable at least 78 h longer
when the pH of the medium was adjusted with hydrochloric acid. The authors concluded
that acetic and tartaric acid were most effective in enhancing the antilisterial effects of
sodium benzoate followed by lactic and citric acid.
Using a minimal glucose-citrate medium (lacking a nitrogen source) adjusted to
pH 5.5 with sodium hydroxide, Yousef et al. [404] demonstrated that death rates for L.
monocytogenes were affected far more by incubation temperature than by the presence
of 1000-3000 ppm benzoic acid in the medium with D-values decreasing - 100-fold as
the incubation temperature was increased from 4 to 35C. Injured listeriae also were de-
tected after plating samples on restrictive and nonrestrictive media. The extent of cell
injury was somewhat greater at lower than higher incubation temperatures. These data
along with the isolation of an apparent sodium benzoate-resistant strain of L. monocyto-
genes from an animal-based dairy ingredient [ 1021 suggest that use of benzoic acid alone
164 Lou and Yousef
to control Listeria in food is questionable. However, Emme et al. [ 1251 found that repeated
exposure of L. monocytogenes to 1000 ppm benzoic acid did not increase resistance of
the organism to this widely used food additive.
Parabens and Other Benzoic Acid Derivatives
Parabens are esters of p-hydroxybenzoic acids. Of these esters, methyl, propyl, and heptyl
parabens are approved in several countries for direct addition to food. Since the pK, of
these derivatives is higher than that of benzoic acid, the molecules remain undissociated
at pH values up to 8.5. Although benzoic acid is effective as an antimicrobial agent only
in acidic foods, parabens retain activity over a wide range of pH values [81].
Payne et al. [294] examined the ability of methyl and propyl paraben to inhibit
growth of L. monocytogenes on Tryptose Phosphate Agar plates during 18 h of incubation
at 35C. Using eight strains of the pathogen, propyl and methyl paraben yielded minimum
inhibitory concentrations of 5 12 and >5 12 ppm, respectively. Moir and Eyles [265] treated
a culture of L. monocytogenes at 35 and 5C with methyl paraben in buffered BHI broth
and measured the MICs. Overall, L monocytogenes was more resistant to this paraben
than were other psychrotrophic bacteria, namely, Pseudomonas putida, Yersinia enterocol-
itica, and Aeromonas hydrophila. When Listeria in broth of pH 6 was incubated at 5C
in the presence of 1000 ppm methyl paraben, the count decreased only -2 logs in 4
months, with 86-99.9% of the viable cells being injured after 2 months. The MIC, defined
as the lowest concentration preventing visible growth in buffered BHI broth after 10 days
of incubation at 30C or 3 months at 5"C, was 300-700 ppm at pH 5 and 3OoC, 1300-
1600 ppm at pH 6 and 3OoC,and 600 ppm, at pH 6 and 5C. Therefore, the MIC decreased
as incubation temperature or pH decreased.
Parabens also exhibited antilisterial activity against Listeria when the additive was
tested in food. Using reconstituted nonfat dry milk (10% solids) inoculated to contain 10'
or 103L. monocytogenes CFU/mL, Payne et al. [294] found that populations of listeriae
were approximately three to four orders of magnitude lower in samples containing 1000
rather than 0 ppm propyl paraben following 24 h of incubation at 35C. When these
experiments were repeated at refrigeration temperatures [881, Listeria counts in milk sam-
ples containing 1000 ppm propyl paraben remained constant during 10 days at 4C.
Dje et al. [88] also investigated behavior of L. monocytogenes in 10% (w/v) aqueous
suspensions of raw chicken meat and frankfurters to which 1000 ppm propyl paraben was
-
added. Although L. monocytogenes attained maximum populations of 1O8 CFU/mL in
propyl paraben-free chicken suspensions following 24 h at 35"C, numbers of listeriae
increased only approximately 10-fold to a maximum of 105CFU/mL in similar suspen-
sions containing 1000 ppm propyl paraben. However, addition of 1000 ppm propyl para-
ben to frankfurter suspensions failed to prevent growth of L. monocytogenes, with similar
-
growth rates and maximum populations of 108CFU/mL appearing in samples prepared
both with and without propyl paraben after 24 h of incubation at 35C. Since L. monocyto-
genes and propyl paraben are primarily present in the water and lipid phases of these
suspensions, respectively, the higher percentage of fat in frankfurter than in chicken sus-
pensions likely accounts for increased growth of listeriae in the former.
Antilisterial activity of p-aminobenzoic acid (pK, of 4.8) relative to common or-
ganic acids (formic, propionic, acetic, lactic, and citric) was investigated by Richards et
al. [3 181. The authors reported that p-aminobenzoic acid had greater inhibitory activity
against L. monocytogenes, S. enteritidis, and E. coli than did other organic acids. The
MIC of p-aminobenzoic acid, measured against L. monocytogenes in BHI broth after 24
Characteristics uf Lister i a mon ocytogenes 165
h of incubation at 37OC, was 9-12 mmol/L at pH 6.7-6.8 compared with MICs of 18-
20 mmol/L for formic acid at pH 6.1-6.2, 18-25 mmol/L for propionic acid at pH 6.0-
6.3, 18-30 mmol/L for acetic acid at pH 5.5-6.2, 35-40 mmol/L for lactic acid at pH
5.2-5.4, and 12-14 mmol/L for citric acid at pH 5.2-5.4. When BHI broth was adjusted
to pH 6.5 with the acids included in the study, their antilisterial potency was as follows:
p-aminobenzoic >propionic >formic >acetic >citric >lactic acid. Antilisterial activity
of p-aminobenzoic acid was pH dependent, with higher activity being observed at lower
pH values; however, this acid showed some inhibitory activity even at near neutral pH
values near neutral.
<0.861 mmol/L at pH 5.0 and 5.5, respectively. Therefore, some strains of L. innocua
appear to be more resistant to hexanoic acid than L. rnonocytogenes. Free fatty acids are
present in certain cheeses, with blue cheese being reported to contain 4.6 and 1.9 mmol/
kg of hexanoic and octanoic acids, respectively [246]. Therefore, concentrations of these
two fatty acids comparable to those found in cheese may have marked antilisterial activity
in acidic foods. However, the antilisterial activity of these two fatty acids would be ex-
pected to decrease dramatically in blue cheese during ripening as the pH increases from
about 4.6 to 6.2 [286].
Fatty Acid Monoesters
Some fatty acid monoesters of glycerol (i.e., monoacylglycerols, which are commonly
known as monoglycerides) and sucrose are potentially useful as antimicrobial food addi-
tives. Monolaurin, the lauryl glycerol monoester, and sucrose laureate were studied exten-
sively and will be discussed in some detail below.
The antilisterial activity of monolaurin was first reported in 1992 by Oh and Marshall
[276] and Wang and Johnson [385]. Oh and Marshall [276] noted that monolaurin was
more antilisterial than other common antimicrobial agents (sorbate, propyl paraben, ter-
tiary butyl hydroxyquione [TBHQ], propyl gallate, and butylated hydroxyanisol [BHA])
and inhibited four L. rnonocytogenes strains (- 10' CFU/mL) in a laboratory broth at 35C
with MICs of 3-4, 7, 9, and 10 pg/mL at pH 5.0, 5.5, 6.0, and 7.0, respectively. Thus
different L. rnonocytogenes strains had similar sensitivity to monolaurin. Wang and John-
son [385] found that L. rnonocytogenes was inhibited by 10 pg/mL monolaurin in BHI
broth at pH 5 and 6. When L. monocytogenes was cultured on BHI agar plates, the MICs
for monolaurin were 96, 14, 7, and 5 pg/mL at pH 7.0, 6.0, 5.5, and 5.0, respectively
[3 121. A monolaurin MIC of 16 pg/mL for L. monocytogenes on Tryptic Soy Agar (TSA)
also was reported by Bal'a and Marshall [25].
Inhibition of microorganisms, including L. rnonocytogenes, is more pronounced us-
ing monolaurin than other fatty acid monoesters. The minimum bactericidal concentrations
at which 103-10" L. rnonocytogenes CFU/mL was completely inactivated in BHI broth
(pH 6) after 24 h at 37C were 25,50, and 75 yg/mL for monolaurin (MC,,), monocaprin
(MC,,), and monomyristin (MC,,), respectively, whereas a concentration of 300 pg/mL
monocaprylin (MC,) was only inhibitory [387]. According to these authors, L. rnonocyto-
genes was not inhibited by 300 pg/mL of monopalmitin (MC,,), monostearin (MC,,),
monoolein (MC18:,),or monolinolein (MCIX:?).
Gram-positive bacteria are more sensitive to monolaurin than are gram-negative
organisms. L. monocytogenes was the most resistant among the gram-positive bacteria
that were tested by Razavi-Rohani and Griffiths [3 121. These investigators used a spiral
gradient method and found that growth of L. monocytogenes on BHI agar was completely
inhibited by a minimum of 96 pg/mL monolaurin, whereas complete inhibition of six
other gram-positive bacteria (Bacillus, Stuphylococcus, and Lactococcus) required a mini-
mum of 8-24 lg/mL monolaurin. In contrast, concentrations as high as 3170 pg/mL
failed to inhibit nine gram-negative bacteria [3 121.
Monolaurin in combination with other treatments or antimicrobial agents, such as
low temperature, low pH, organic acids, chelating agents, and antioxidants, exhibited en-
hanced antimicrobial activity. Gram-negative bacteria were inhibited by some of these
combinations. In the presence of 5 4 % NaCl, growth of gram-negative bacteria on BHI
agar was inhibited by <2 pg/mL monolaurin [312]. As described previously, the MICs
of monolaurin decreased as the pH decreased [276]. Oh and Marshall [277] investigated
Characteristics of Listeria monocyt ogen es 167
the antilisterial activity of monolaurin (5-9 pg/mL) at different temperatures (7, 15, and
35C) and pH values (5.0,5.5, and 7.0) in TSBYE. Although monolaurin was listeriostatic
at most temperature/pH combinations, listericidal activity was detected using 8-9 pg/mL
of monolaurin at pH 5.0 with L. monocytogenes strain Scott A (initially inoculated at
- 1O3 CFU/mL) reportedly undetectable at this concentration after 20.0-22.0, 6.0, and 0.5
days at 7, 15, and 35"C, respectively. The study just described provides evidence that
inhibition of L, monocytogenes by monolaurin is highly temperature-dependent. Although
Wang and Johnson [385] found that monolaurin at 200 pg/mL was listericidal in skim
milk at 4"C, no antilisterial activity was observed at 30C.
Oh and Marshall [278] reported a greater listeriostatic effect in TSBYE at 35C
when 18 pM monolaurin was used in combination with sublethal concentrations of organic
acids (acetic, benzoic, lactic, or citric) than when monolaurin or the acids were used sepa-
rately. When tested in crawfish tail meat at 4OC, 336 mM lactic acid decreased L. rnonocy-
togenes populations from about 103CFU/g to nondetectable levels in 10 days; however,
similar inactivation could be achieved using a combination of 0.72 mM monolaurin and
224 mM lactic acid. When used alone, 224 mM lactic acid resulted in complete inhibition
12781. Bal'a and Marshall [25] investigated the combined effect of NaCl (2.5-7.8%), pH
(5.4-7.8), temperature ( 5 , 15, 25, and 35"C), and sublethal levels of monolaurin (2-8 pg/
L) on growth of L. monocytogenes on double (salt-pH) gradient plates and found signifi-
cant interactions between these factors. More recently, Razavi-Rohani and Griffiths
[3 I2,3 131 detected enhanced antimicrobial activity of monolaurin when this compound
was used in combination with ethylenediaminetetraacetic acid (EDTA), BHA, low pH,
or NaC1; however, no marked enhancement was observed in the presence of lysozyme.
The presence of EDTA not only reduced the MICs for all gram-positive bacteria (70 pg/
mL for L. rnonocytogenes), but also sensitized gram-negative bacteria to monolaurin
(MICs of 90-1500 pg/mL). However, no decrease in monolaurin MICs was caused by
the presence of EDTA at pH 1 6 . Sodium citrate and monoglyceride citrate, two other
chelating agents tested, decreased the antimicrobial effect of monolaurin [3 121.
Antilisterial activity of monolaurin increased in the presence of organic acids and
antioxidants [386]. According to Wang and Johnson, inhibitory activity of monolaurin ( 5
pg/g) against L. monocytogenes in BHI broth was enhanced by other antilisterial agents
such as BHA (100 pg/g), TBHQ (30 pg/g), propyl gallate (200 p/g), acidulants (0.1%
acetic or lactic acid), and potassium sorbate (0.1%) [386]. Propyl gallate (200 p/g) and
lactic acid ( 0.2%) enhanced the bacteriostatic activity of monolaurin and monocaprin
against L. rnonocytogenes in seafood (imitation crab meat and cooked shrimp) and Cam-
embert cheese, respectively, which were stored at 4C. However, when tested in foods,
0.1% glycine, sodium citrate, propylene glycol, or Tween 20,O. 1-0.2% potassium sorbate,
200 pg/mL lysozyrne, or 200 pg/mL BHA did not enhance activity of monoacylglycerols
[386]. Oh and Marshall [280] reported that combined use of 200 pg/g rnonolaurin and
0.5% lactic acid significantly inhibited growth of Listeriu in crawfish meat homogenate
stored at 4"C, whereas these additives had little or no effect on growth of Listeria when
used separately.
Antilisterial interactions occur not only between monolaurin and other factors but
also among different monoacylglycerols as well. Wang et al. [387] reported an additive
effect between monolaurin ( 100 pg/mL) and monocaprin ( 100 pg/mL) and a synergistic
effect between monomyristin (200 pg/mL) and monocaprin (200 pg/mL) when these com-
pounds were tested in skim milk at 4C. The authors also noted strong antilisterial activities
by monoacylglycerols synthesized from coconut oil. These monoacylglycerols had a MIC
168 Lou and Yousef
(10 pg/mL), which was lower than that of monolaurin (25 pg/mL) when both agents
were tested in BHI broth (pH 6) with incubation at 37C. Compared to milk fat-derived
monoacylglycerols, which were not inhibitory to L. monocytogenes at 300 pg/mL, coconut
monoacylglycerols are rich in lauric (C12), myristic (C14), and capric (C10) acids, and
thus may contain higher levels of monolaurin, monomyristin, and monocaprin. Demon-
strated antilisterial activity of these three dominant monoacylglycerols and the synergistic
interaction between them may account for the high antilisterial activity of coconut mono-
acylglycerols. When tested in refrigerated skim milk, 2% milk, and whole milk, monoca-
prin, monolaurin, and coconut oil-derived monoacylglycerols showed strong antilisterial
activity. At >200 pg/mL, monocaprin was more effective than monolaurin in inactivating
L. monocytogenes in all three refrigerated milks, possibly because of the higher water
solubility of monocaprin.
High temperatures of incubation and high fat content of the growth medium de-
creased the effectiveness of monoacylglycerols. In skim milk containing 250 pg/mL of
the coconut-derived monoacylglycerols, L. monocytogenes (- 1O3 CFU/mL) was inacti-
vated after storage at 4C for 7 days but grew to 107- 1 Ox CFU/mL at I 3 and 23C. Concen-
trations of 250-400,500-750, and 750- 1000 pg/mL of coconut-derived monoacylglycer-
01s were required to inactivate L. monocytogenes in refrigerated skim milk, 2% milk, and
whole milk, respectively [387]. Compared with 1000 pg/g monolaurin or monocaprin,
coconut monoacylglycerols ( I000 pg/g) and a combination of monolaurin (500 pg/g) plus
monocaprin (500 pg/g) exhibited greater listericidal activity in beef frankfurter slurries
(pH 5.0 and 5.5) and seafood salad (pH 4.9) stored at 4C. However, at 12OC, Listeria
grew rapidly in beef frankfurter slurries (pH 5.5) with or without such levels of these
monoacylglycerols. When present in turkey frankfurters (pH 5.6 and 6.1) stored at 4"C,
the monoacylglycerols ( 1000 pg/g) were bacteriostatic, with half this concentration being
listeriostatic in cooked shrimp (pH 7.1), imitation crab meat (pH 6.5), and Camembert
cheese (pH 6.2) [386]. Although 10-96 pg of monolaurin/mL can inhibit Listeria growth
in culture media [25,276,277,312,385,3871, much higher concentrations are required to
elicit similar levels of inhibition in foods. Monoacylglycerols may interact with food com-
ponents such as lipids and protein, and thus become less available for contact with microor-
ganisms and so less capable of exerting an antimicrobial effect. Wang and Johnson [385]
found that antilisterial activity of monolaurin decreased dramatically as the fat content of
milk increased. Although 100 pg/mL and 2200 pg/mL monolaurin was listeriostatic and
listericidal, respectively, in skim milk at 4OC, 200 pg/mL monolaurin showed no such
activity in whole milk.
Levels of monolaurin required to inhibit L. monocytogenes vary with the type of
food. Oh and Marshall [280] found that 200 pg/g monolaurin did not significantly inhibit
growth of L. monocytogenes in refrigerated crawfish tail meat homogenate packaged in
air or a modified atmosphere. However, this level of monolaurin significantly decreased
the growth of Listeria in vacuum-packaged meat samples. Earlier these authors [278]
found that 0.72 and 1.44 pM monolaurin extended the generation times of L. monocyto-
genes in crawfish tail meat stored at 4C from 16.7 (for untreated control) to 28 and 48
h, respectively. In 1997, Wang and Johnson [386] reported that antilisterial activity of
monoacylglycerols was much greater in beef frankfurter slurries and seafood salad than
in turkey frankfurter slurries, summer sausage, cooked shrimp, imitation crabmeat, yogurt,
and cottage and Camembert cheeses. Monolaurin had greater antilisterial activity in 2%
chocolate milk than in 2% milk, possibly because of the enhancing effect of cocoa.
Since monolaurin is poorly soluble in water at high concentrations and has a soapy
Characteristics of Listeria monocytogenes 169
Sodium Nitrite
Studies undertaken by Shahamat et al. [342] during the 1970s examined effects of various
concentrations of sodium nitrite and sodium chloride on growth of L. monocytogenes in
TSB at different temperatures and pH values. When incubated at 37, 22, and 4C in broth
at pH 7.4, L. rnonocytogenes grew at nitrite concentrations as high as 25,000, 30,000, and
10,000ppm, respectively. Inhibitory effects of nitrite were enhanced at pH 6.5, particularly
at lower incubation temperatures, with complete inhibition being caused by 1500 ppm
nitrite at 4C. MICs of nitrite were further reduced at all three incubation temperatures
and at pH 5.5, with 600 ppm nitrite being sufficient to inhibit growth at 4C. The bacterio-
static activity of nitrite was greatest at pH 5 and at 22 and 3 7 T , with no growth reported
at nitrite concentrations >800 and 400 ppm, respectively. Addition of 3% sodium chloride
to TSB failed to increase the bacteriostatic action of sodium nitrite. Although MICs for
170 Lou and Yousef
nitrite were only slightly lower with 5.5 or 8.0% sodium chloride at pH 7.4 or 6.5, the
combination of 5.5 or 8.0% sodium chloride and pH values of 5.0 or 5.5 led to MICs for
nitrite that were generally 8- to 20-fold lower than controls prepared without sodium chlo-
ride. Inhibitory effects of nitrite were again most pronounced at 4C when the chemical
was combined with sodium chloride.
The study by Shahamat et al. [342] was criticized by McClure et al. [254], who
found that autoclaving caused nitrite to decompose. After autoclaving nitrite-containing
TSB at pH 5, no nitrite was detected. Since only 10-72% nitrite was detected after auto-
claving nitrite-containing TSB at pH 5.3-6.7 [254], McClure et al. [254], together with
Buchanan et al. [%I, believed that the antimicrobial activity of nitrite was seriously under-
estimated by Shahamat et al. [342]. Using filter-sterilized rather than autoclaved sodium
nitrite, McClure et al. [254] reported much greater antilisterial activity of nitrite in TSB.
Filter-sterilized nitrite, at 50 pg/mL and pH 5 , prevented visible growth for 48 h; however,
200 pg/mL autoclaved nitrite under the same conditions did not retard Listeria growth.
As mentioned earlier, behavior of L. monocytogenes in food and culture media de-
pends on the interactive effects of temperature, pH, type of acidulant, salt content, a,, and
types and concentrations of food additives that may be present in the system. The effective-
ness of sodium nitrite as an antilisterial agent also is strongly influenced by these same
factors. Buchanan et al. (581 used a factorial design to determine the effect of sodium
nitrite (0- 1000 ppm) in combination with incubation temperature (5-37"C), initial pH
(4.5-7.5), sodium chloride (0.5-4.5%), and atmosphere (aerobic vs anaerobic) on growth
of L. monocytogenes in TPB. Although lag periods, generation times, and maximum popu-
lations were all affected by these five interacting variables, sodium nitrite was most
listeriostatic when used in conjunction with low pH, increased sodium chloride, refrigera-
tion temperatures, and anaerobic conditions that simulated vacuum packaging. McClure
et al. [254] reported that antilisterial activity of nitrite strongly depended on pH. At pH
2 6, nitrite, even at 400 pg/mL, had little antilisterial activity. However, below pH 6,
nitrite, even at 50 pg/mL, exhibited antilisterial activity. Visible growth (defined as 0.03-
unit increase in ODhO0 in 21 days at 5-30C) of this pathogen in TSB at 20C was prevented
by 50 pg/mL sodium nitrite and pH 5 5.3. Autoclaving nitrite dramatically decreased its
antilisterial activity.
Buchanan et al. [53] found that inactivation of L. monocytogenes by NaNO, was
affected by several factors, with lactic acid being the most influential. At high levels of
lactic acid (low pH), the listericidal action of nitrite increased, whereas low levels of lactic
acid had little effect on nitrite activity. The antilisterial activity of sodium nitrite and other
antimicrobial agents also was studied by Buncic et al. [60]. Growth of L. monocytogenes
(initially 107CFU/mL) in buffered BHI broth (pH 5.5) incubated at 4C for up to 7 weeks
was prevented by addition of 125 ppm sodium nitrite or a combination of 125 ppm sodium
nitrite and 0.5% polyphosphate. Polyphosphate (0.5%) alone did not prevent growth of
Listeria. This is consistent with results from several research groups [58,254,342], who
noted that nitrite inhibited L. monocytogenes at low pH values. Nitrite and nisin also had
a synergistic effect against L. monocytogenes. When used alone, nisin (400 IU/mL), re-
sulted in an initial 1.1 -log reduction, with Listeria survivors eventually growing during
extended incubation. However, a combination of nisin and nitrite (125 ppm) not only
prevented Listeria regrowth but caused a further (1.4 log) reduction when compared with
nisin alone.
Working with meat products, Johnson et al. [ 1881 found that growth of L. monocyto-
genes was suppressed at 4C in hard salami (pH 4.3-4.5) that contained 5.0-7.8% NaCl
Characteristics of Listeria monocytogenes 171
plus 156 ppm sodium nitrite. Although their findings agree with those of Shahamat et al.
[342], the combination of low water activity (a, 0.79-0.86) and low pH were probably
more important in preventing growth of listeriae than was the addition of 156 ppm sodium
nitrite. Findings of Glass and Doyle [ 1561 also indicate that 3.5% sodium chloride plus
156 ppm sodium nitrite in sausage batter at pH 6.2 controlled growth of L. monocytogenes
in the product during fermentation at 90F. Under these conditions, additional acid devel-
opment through fermentation is essential to prevent growth of listeriae.
Antioxidants
Antioxidants such as BHA, butylated hydroxytoluene (BHT), TBHQ, and propyl gallate
comprise an important category of food additives. Although primarily used to prevent
oxidation of fat, some of these antioxidants also possess antimicrobial activity.
Limited trials on destruction of L. monocytogenes by BHA were initiated by Al-
Issa et al. 161 during the early 1980s. Tryptone Soy Broth containing 50 ppm BHA was
-
inoculated to contain 105L. monocytogenes CFU/mL and examined for numbers of the
bacterium. The Listeria population decreased -3 logs during the first 12 h at 37C and
remained at a level of -102 CFU/mL after 24 h of incubation. The same authors also
found that successive subculturing of L. monocytogenes in a medium containing glycerol
followed by inoculation into Tryptone Soy Broth containing 50 ppm BHA led to rapid
Listeria growth with populations reaching 10'- 1O9 CFU/mL after 24 h at 37C. Develop-
ment of BHA resistance correlated with a high lipid content in the cell wall and membrane
from prior growth in a medium containing glycerol. Payne et al. [294] investigated the
potential of BHA, BHT, TBHQ, and propyl gallate to inhibit growth of L. monocytogenes
on Tryptose Phosphate Agar during 18 h of incubation at 35C. Using an agar dilution
method, TBHQ was the most effective antioxidant tested with a MIC of 64 ppm followed
by BHA, propyl gallate, and BHT with MICs of 128, 256, and 5 12 ppm, respectively.
Although these findings may at first appear promising for the food industry, L. mono-
cytogenes is far more likely to encounter sublethal levels rather than MICs of antioxidants
in food. Consequently, Yousef et al. [405] examined the growth kinetics of L. monocyto-
genes strain Scott A in TB containing BHA (100-300 ppm), BHT (300-700 ppm), and
TBHQ (10-30 ppm) during 54 h of incubation at 35C. Overall, these findings agreed
with those of Payne et al. [294] in that TBHQ again was most inhibitory to L. monocyto-
genes followed by BHA and BHT. According to the authors, L. monocytogenes exhibited
increasingly longer lag periods and generation times as well as lower maximum popula-
tions in the presence of BHA at 100-200 ppm, with concentrations 2300 ppm proving
to be lethal. Since all three growth parameters were increasingly affected as the sublethal
concentrations of BHA increased, the organism was probably unable to detoxify this anti-
oxidant metabolically. Therefore, addition of up to 200 ppm BHA to food, as permitted
by the FDA, will likely contribute to overall keeping quality and safety of some products.
Unlike BHA, L. monocytogenes was unaffected by (-300 ppm BHT; however, poor solu-
bility of BHT in TB prevented critical analysis of this antioxidant at concentrations >300
ppm. Interestingly, increasing the concentration of TBHQ from 10 to 30 ppm led to an
exponentially longer lag period for L. monocytogenes, but it did not appreciably affect
generation times or maximum populations. Hence, unlike BHA, these observations suggest
that L. monocytogenes can metabolically detoxify sublethal concentrations of TBHQ to
safe levels and initiate growth thereafter. From this, it appears that BHA may be of greater
benefit than TBHQ for inhibiting growth of listeriae in food.
172 Lou and Yousef
Liquid Smoke
Many commercially available liquid smoke products used in processed meats and sausages
can inactivate common foodborne organisms, including E. coli, S. aureus, and Lactobacil-
lus viridescens. These artificial liquid smoke flavorants owe their activity to the presence
of phenolic compounds and acetic acid, both of which are bactericidal at relatively low
concentrations.
After L. monocytogenes was recognized as a possible health hazard in ready-to-eat
meat and sausage products, several investigators examined the potential of various liquid
smoke compounds to inactivate L. monocytogenes in phosphate buffer and culture media
commonly used to isolate this pathogen from meat products. Using sterile phosphate buffer
at pH 5.64 and inoculated to contain 1 X 105L. monocytogenes CFU/mL [259], three of
five liquid smoke compounds (Charsol- 10, Aro-Smoke P-50, and CharDex Hickory, Red
Arrow Products, Manitowoc, WI) tested at a concentration of 0.5% reduced numbers of
listeriae to nondetectable levels after 4 h at ambient temperature. When the concentration
of liquid smoke products was decreased to 0.25%, numbers of listeriae were still reduced
to nondetectable levels within 4 h using either CharSol- I0 or Aro-Smoke P-50. However,
CharDex Hickory was far less effective at the lower concentration with 24 h of incubation
required to inactivate the pathogen completely. Listericidal activity of these liquid smoke
compounds also appeared to be at least partially related to levels of acetic acid present
in the various preparations.
Subsequently, Wendorff 13921 found that the same liquid smoke compounds were
Characteristics of Lister ia monocyt ogenes 173
far less listericidal when added to USDA-recommended Listeria Enrichment Broth rather
than phosphate buffer. Interactions between liquid smoke constituents and protein in the
enrichment broth were probably at least partially responsible for the observed decrease
in listericidal activity. Although three of five liquid smoke compounds were effective
against L. monocytogenes in buffer and to a lesser extent in culture media, later work
by Wendorff [392] showed that concentrations of liquid sinoke needed to inactivate L.
monocytogenes in processed meats were well above organoleptically acceptable limits.
In 1992, Faith et al. [128] reported that adding 0.2 and 0.6% (v/v) of liquid smoke
(CharSol Supreme, Red Arrow Products, Manitowoc, WI) to wiener exudate inactivated
L. monocytogenes with D-values of 36 and 4.5 h, respectively, whereas Listeria in the
-
smoke-free exudate grew from initial levels of 10sto 108CFU/mL after 3 days at 25C.
The authors further investigated the antilisterial activity of selected smoke components
in TB at pM 7. When the culture was incubated at 37"C, they found that among 11 phenol
compounds tested, only isoeugenol retarded growth of Listeria. Although final maximum
populations were similar, lag-phase duration increased linearly from -3 to 21 h as isoeu-
genol levels increased from 0 to 200 ppm. It was estimated that an isoeugenol concentra-
tion of 236 ppm was required to increase the lag phase by one order of magnitude. L.
monocytogenes in the presence of 100 ppm isoeugenol L. monocytogenes also was inhib-
ited to a greater extent when the pH of TB was adjusted to 5.8 compared with 7.0.
cinnamon was somewhat inhibitory to L. monocytogenes, with the pathogen attaining max-
imum populations that were 1.5-2.6 orders of magnitude lower than in controls. Growth
of listeriae at 35C also was partly suppressed by 0.5%cloves, with the pathogen attaining
a maximum population 1.6 logs lower than that observed in controls. However, Listeria
populations decreased steadily in TB containing 0.5% cloves during extended incubation
at 4C.
Working in Thailand, Stonsaovapak and Chareonthamawat [367a] used a similar
experimental design to test nine dried native Thai spices, namely, cinnamon, black pepper,
white pepper, cloves, cardamom, coriander, star anise, nutmeg, and cumin seed at concen-
trations of 1,3, and 5% for activity against L. rnonocytogenes in TSB containing 10' CFU/
mL. As in the previous two studies [24,375], cloves exhibited strong listeriocidal activity
with concentrations 2 1%, reducing Listeria populations >8 logs and >6 logs after 7 days
of incubation at 35 and 4OC, respectively. When exposed to concentrations 2 1 % for 7
days, nutmeg and star anise also reduced numbers of listeriae 5-8 logs at 35C and
2-6 logs at 4C. Using 2 1 % white pepper, black pepper, or coriander, Listeria population
decreased 2-5 logs and 1-2 logs at 35 and 4OC, respectively, with these and the other
spices being most effective at concentrations of 5%. However, little if any inhibition was
observed using cardamom regardless of concentration or incubation temperature.
Antilisterial activity of 32 plant essential oils was investigated by Aureli et al. [22]
in Italy. The essential oils were dissolved in ethanol at a concentration of 1 5 (v/v) with
antilisterial activity assessed on TSA plates using the disc diffusion method. Five essential
oils showed activity against four strains of L. rnonocytogenes. Essential oils of origanum
and thyme were most active against Listeria, followed by oils of cinnamon, clove, and
pimento. Further tests with thyme, origanum, and cloves showed that the three oils main-
tained antilisterial activity at a 150 (v/v) dilution. Although nutmeg, rosemary, and sage
were found to the antimicrobially active by Ting and Deibel [375], the essential oils of
the three spices failed to inhibit listeriae. Essential oils that did not inhibit listeriae were
those of basil, camomile, celery, coriander, cumin, estragon, fennel, garlic, ginger, laurel,
lemon, mandarin, marjoram, neroly, onion, orange, parsley, pepper, peppermint, petti-
grain, saffron, and vanilla. Survival of L. rnonocytogenes ( IO5-1O6 CFU/mL) in a saline
solution containing 0.1% (v/v) of essential oils of origanum, thyme, cinnamon, cloves,
or pimento was similar among five strains of the pathogen. Oil of pimento had the greatest
activity and that of cinnamon the least. Essential oil of pimento decreased the population
of Listeria from > 103CFU/mL to an undetectable level in 1 h. All five oils reduced the
viable count to undetectable levels after 4 h of incubation at room temperature. In minced
pork stored for 8 days at 4 and 8OC, adding 100 pL of the diluted (15, vol/vol) essential
oil of thyme to 25 g of product, decreased the population of L. rnonocytogenes by -1
-
log, whereas the organism grew from 1 X 107to 6 X 107and 2 X 108CFU/g in untreated
controls at 4 and 8OC, respectively.
In 1993, Hefnawy et al. [ 1661 reported on the sensitivity of L. rnonocytogenes strains
Scott A and V7 to 10 spices in TSB at 4C and found the latter strain to be generally
more resistant. L. monocytogenes Scott A decreased from an initial population of 105to
<1 CFU/mL by 1% sage in 1 day; I % allspice in 4 days; 1% red pepper, paprika, garlic
powder, or cumin in 7 days; 5% black pepper in 4 days; or 5% mace in 7 days, whereas
5% nutmeg partially inactivated the organism and 1-5% white pepper enhanced growth.
For strain V7, only 1, 3, and 5% sage reduced the initial population (-107 CFU/mL) to
< I CFU/mL in 7, 4, and 4 days, respectively, whereas 3-5% allspice, mace, or nutmeg
showed partial inactivation.
Characteristics of Listeria monocytogenes 175
Pandit and Shelef [285] investigated the antilisterial activity of 18 spices and herbs
by measuring growth of L. monocytogenes on BHI agar with (0.1%) and without spices/
herbs. The authors found that only rosemary and cloves showed antilisterial activity, and
contrary to previous studies [22,375], cinnamon, nutmeg, oregano, and sage had no activ-
ity. Other noninhibitory spices and herbs were ajowan, allspice, asafoetida, cardamom,
cumin, fenugreek, ginger, marjoram, black mustard, red hot pepper, and turmeric. Growth
of Listeria on agar plates was completely inhibited by 0.5% rosemary or 1.0% cloves.
An aqueous extract of rosemary only prevented growth of Listeria in BHI broth at 35C
for the first 24 h, whereas the same levels of rosemary or its ethanol extract had significant
listericidal activity. The authors further investigated the antilisterial activity of rosemary
oil and its four major components (cineole, borneole, camphor, and pinene), rosemary
oleoresin, rosemary oil encapsulated in modified starch, and an antioxidant fraction ex-
tracted from rosemary by liquid carbon dioxide. Rosemary oil ( 10 pL/ 100 mL) was listeri-
ostatic during 48 h of incubation, whereas oleoresin (up to 100 mg/ 100 mL) did not inhibit
listeriae. Encapsulated oil was listeriostatic at 1 pg/lOO mL, and listericidal at 5 pL/lOO
mL. The antioxidant extract at 0.02 g/lOO mL was listericidal. Of the four rosemary oil
components, only pinene at 0.1 pL/ 100 mL suppressed growth of Listeria, whereas the
other three components had no activity at concentrations up to 1 pL/lOO mL. When added
to refrigerated (5C) pork liver sausage, 1% rosemary, 0.5% rosemary oil, 0.3% antioxi-
dant extract, or 5% encapsulated oil suppressed growth of Listeria, with maximal popula-
tions of -log, -108, -105, and -105 CFU/mL being attained after 25, 25, 50, and 50
-
days, respectively, whereas 1O3 CFU/mL of Listeria in the control reached 109CFU/
mL after 25 days.
Tassou et al. [372], working in Greece, investigated the antimicrobial effect of the
essential oil of mint on L. monocytogenes and S. enteritidis in a broth culture and in three
foods; tzatzilu (cucumber and yogurt salad, pH 4.3), taramosalata (fish roe salad, pH 4.9),
and piit6 (pH 6.8). Concentrations of mint oil tested in this study were 0.5, 1.0, 1.5, and
2.0%, v/w, and incubation temperatures were 4 and 10C. Although gram-positive bacteria
generally are more sensitive to essential oils than gram-negative microbes, the authors
found that L. monocytogenes was more resistant to mint essential oil than S. enteritidis.
Antibacterial activity varied between foods and pH values. The presence of 1-2% essential
oil in tzatziki decreased salmonellae numbers to undetectable levels after 3-6 h, whereas
>2 logs of the organism remained viable after 6 days in the untreated control that initially
contained l O7 L. monocytogenes CFU/g. However, in the same treated food, populations
of L. rnonocytogenes remained unchanged for the first 4 days and decreased gradually
during subsequent storage, with >4 logs being detected after 8 days at both temperatures.
Compared with the control, the presence of mint oil decreased the rate of death of L.
monocytogvnes in the product. Populations of L. rnonocytogenes and S. enteritidis de-
creased slightly in taramosalata made with and without mint essential oil during 9 days
of incubation at 4 and 10C. The essential oil was without antimicrobial activity in pit&,
which had an almost neutral pH. In all pgt6 samples, salmonellae populations decreased
at 4C or decreased and then increased at 10C, whereas listeriae increased > 1 and >3
logs at 4 and IOOC, respectively, after 6 days of incubation. Greater antimicrobial activity
of mint oil was observed in broth than in food, with both organisms being inhibited by
0.5-2.0% mint oil in broth at pH 6.6.
Because of the potential link between consumption of chocolate milk and listeriosis,
Pearson and Marth [297] studied the effect of caffeine and theobromine, two methylxan-
thine compounds in cocoa, against L. monocytogenes strain V7 (initially 103CFU/rnL)
176 Lou and Yousef
in a modified TPB and skim milk at 30C. L. monocytogenes grew similarly in both media.
Although limited antilisterial activity was observed with 2.5% theobromine, the authors
found that 0.5% caffeine had antilisterial activity in both substrates and increased the lag
phase from <3 (control) to 6-9 h, increased generation times from 1.2 to 2.17 h, and
decreased the final maximum population from 8.6 to 7.2 logs. A combination of 2.5%
theobromine and 0.5% caffeine had slightly more antilisterial activity than did 0.5% caf-
feine alone.
In conclusion, essential oils are more effective in broth than in foods. Because of
their hydrophobic nature, antilisterial activity of essential oils is adversely affected by
high fat and protein contents in food. Antimicrobial activity of essential oils is enhanced
by low pH, high salt content, and low storage temperatures.
Lysozyme
Lysozyme is an important natural enzyme which prevents bacterial growth (particularly
gram-positive organisms) in foods of animal origin, including hens eggs and milk. Lyso-
zyme is particularly attractive as a food preservative, since the enzyme is active between
4 and 95OC, stable over a wide range of pH values, specific for bacterial cell walls, and
is not harmful to humans. Although not yet approved as a food additive in the United
States, lysozyme has been used successfully in Europe to prevent blowing caused by
Clostridium spp. in Gouda, Edam, and other brine-salted cheeses.
In 1987, Hughey and Johnson [ 1791evaluated four L. monocytogenes strains isolated
during foodborne listeriosis outbreaks for their susceptibility to lysozyme. After nongrow-
ing cells of L. monocytogenes in the stationary growth phase were suspended in phosphate
buffer containing 10 mg of lysozyme/L, 70-80% of the cells were lysed after 6 h, as
determined by optical density measurement. In 1994, Johansen et al. [ 1871 tested the ability
of egg white lysozyme to lyse five L. monocytogenes strains, which were suspended in
phosphate buffer of pH 7. At 200 U/mL, lysozyme caused lysis of the five strains, and
the rate of lysis was maximum at a lysozyme concentration of 1000 U/mL. Sensitivity
of Listeria to lysozyme varied with the temperature at which the bacterium was grown
before the treatment. Cells grown at 5C were the most and those grown at 37C the least
sensitive, with cells grown at 25C showing intermediate sensitivity. A similar effect of
Listeria growth temperature on lysozyme sensitivity was noted by Smith et al. [355]; the
authors found that L. rnonocytogenes grown at 37C was 1.8-2.5 times more resistant to
lysis by lysozyme than were cells grown at 5, 12, and 19C.
In contrast to the listericidal effect of lysozyme in nutrient-poor buffers, Listeria
can grow in nutrient-rich media containing lysozyme. Growth inhibition detected in such
media depends on the concentration of lysozyme, pH of the medium, incubation tempera-
ture, and the presence of various growth modifiers. According to Hughey and Johnson
[179], four strains of Listeria grew in BHI broth containing 20-200 mg of lysozyme/L.
Similarly, cells of L. monocytogenes in the logarithmic growth phase were not lysed after
12 h of exposure to 100 mg of lysozyme/L. In a more recent investigation [187], the
presence of 10,000 U/mL lysozyme in TSB at 5C extended the lag phase from 0 to 8
days at pH 7.0, and from 9 to 60-70 days at pH 5.5. A similar pattern was seen at 25C
with a lag phase at pH 5.5 of 4 h without lysozyme and 37 h with 50,000 U/mL lysozyme.
The authors attributed the pH-dependent increase in bacteriostatic activity of lysozyme
to the growth-retarding effect of low pH, which allowed cell lysis to proceed faster than
Characteristics of Listeria monocytogenes 177
cell multiplication. The lytic action of lysozyme was not affected as the pH was lowered
from 7.0 to 5.5.
Various chemical treatments also have been examined for their ability to potentiate
lysis of growing cells of L. monocytogenes in the presence of lysozyme [ 1791. Addition
of 0.85% lactic acid stopped growth of L. monocytogenes and led to gradual lysis of
cells by lysozyme. EDTA was the most effective potentiator of cell lysis, whereas 0.05%
potassium sorbate, 0.25% glycine, 5 mM sodium acetate, 0.95% ethanol, 0.01% sodium
dodecyl sulfate, 5 mM thioglycolate, 5 mM dithiothreitol, and 10 mM ascorbic acid were
relatively ineffective. The inhibitory action of EDTA and lysozyme toward Listeria also
was tested using BHI agar. Although 100 mg of lysozyme/L or 1 mM EDTA failed to in-
hibit growth of two of four L. monocytogenes strains, the combination of EDTA and lyso-
zyme led to substantial growth inhibition as compared with the additive-free control [ 1791.
The antimicrobial activity of lysozyme against L. monocytogenes suspended in water
was enhanced by prior treatment with lipase, an enzyme naturally existing in milk, or fro-
zen storage for up to 6 weeks [ I 10,1111. Enhancement of lysozyme activity against L. mono-
cytogenes suspended in buffer or broth by lipase also was observed by Liberti et al. [23 11.
When compared with results using laboratory media, the antilisterial activity of lyso-
zyme is variably reduced in foods. Hughey et al. [I801 found that lysozyme was more
effective in controlling L. monocytogenes in vegetables than in meat products. A combina-
tion of lysozyme and EDTA inactivated L. monocytogenes (at 10" CFU/g) in fresh green
beans, fresh corn, shredded cabbage, shredded lettuce, and carrots during storage at 5OC,
whereas Listeria in control samples grew to 106-107CFU/g. In fresh pork sausage (brat-
wurst), lysozyme was only listeriostatic for 2-3 weeks and did not prevent growth during
extended storage of the food. During ripening of Camembert cheese, lysozyme alone or
in combination with EDTA decreased listeriae by about 1 log during the first 3-4 weeks,
and then the Listeria count increased slowly and reached 106- 10' CFU/g after 55 days
of ripening. Carminati and Carini [66] found that lysozyme at 25 and 1000 ppm only
caused 22 and 57% reductions in count, respectively, for two of four L. monocytogenes
strains inoculated into sterile skim milk. Addition of lysozyme to heat-treated skim milk
that contained Listeria did not inhibit outgrowth of survivors.
Kihm et al. [201] found that minerals in milk protected L. monocytogenes against
lysozyme. Hen's egg white lysozyme (100 mg/L) had no antilisterial activity in whole
milk. However, removal of minerals from milk by cation exchange slightly enhanced
activity of lysozyme at 4C. Prior heating (62.5"C for 15 s) of L. monocytogenes in a
phosphate buffer sensitized the pathogen to lysozyme in MES (2-[N-morpholino] ethane-
sulfonic acid) buffer or demineralized milk. Furthermore, heating the pathogen at 55C
in the presence of lysozyme greatly increased inactivation of the pathogen in demineralized
rather than undemineralized milk. Although lysozyme alone did not inhibit Listeria growth
in milk [20 I ] , Payne et al. [295] found that lysozyme plus EDTA had an interactive antimi-
crobial effect against L. monocytogenes growing in ultrahigh-temperature (UHT) milk.
Although lysozyme activity is affected by many food components, adding lysozyme
to foods is potentially beneficial to control this pathogen. As discussed previously, lyso-
zyme alone or in combination with EDTA inactivated Listeria in vegetables and retarded
growth of the pathogen in fresh pork sausage and Camembert cheese [ 1SO]. Lysozyme
(500 ppm), when added to acidified sterile skim milk (pH 5.3) that was stored at 4C for
6 weeks (i.e., simulation of cheese making), enhanced inhibition of Listeria [66]. Egg
white lysozyme contributed to the high antilisterial activity of some mayonnaise products
178 Lou and Yousef
[126]. Consistent with this finding, Wang and Shelef [383] found that raw egg albumin
at levels >15% was bactericidal to L. monocytogenes in TSB at 35"C, with this activity
being primarily attributed to lysozyme.
Wang and Shelef [384] later found that L. monocytogenes growth in raw cod fish
fillets could be retarded by lysozyme alone or in combination with EDTA. The fish fillets
were dipped for 10 min at 20C in solutions of lysozyme (3 mg/mL), EDTA (5-25 mM),
or a combination of lysozyme (3 mg/mL) and EDTA (25 mM), inoculated with about 103
CFU/g L. monocytogenes, and then monitored for Listeria growth during storage at 20C
for 3 days or at 5C for I7 days. The authors found that lysozyme plus EDTA had substan-
tial antilisterial activity at both storage temperatures. Listeria populations in the control
and in samples pretreated with 5-10 mM EDTA increased to about 108CFU/g after stor-
age at 2OoC, whereas the pathogen only increased <2 logs CFU/g in samples pretreated
with 15 and 25 mM EDTA. In lysozyme-treated samples stored at 2OoC, final Listeria
populations were about 10-fold lower than in the control. Lysozyme and EDTA (25 mM)
interacted synergistically and resulted in > 1-log decrease of listeriae in the first 18 h; the
pathogen never grew to a level exceeding the initial population during the entire storage
period. When control and treated samples were stored at 5C for 17 days, Listeria popula-
tions increased 1 log in the control, remained almost unchanged in EDTA-treated samples,
and decreased up to 1 log in samples treated with lysozyme or with the combination of
lysozyme and EDTA.
Hydrogen Peroxide
Although hydrogen peroxide is used as a preservative, particularly for raw milk in some
parts of the world, use of this antimicrobial agent in the United States is very limited.
The FDA permits adding up to 0.05% (w/w) hydrogen peroxide to raw milk intended to
be made into certain kinds of cheese. It has also been approved by the FDA for sterilizing
multilayer packaging materials used in aseptic processing systems.
The antilisterial effect of hydrogen peroxide at levels permitted by the FDA was
investigated in milk by Dominguez et al. [89]. These investigators found that L. monocyto-
genes was eliminated from autoclaved milk that had been inoculated to contain 9.5 X 107
L. monocytogenes CFU/mL, treated with 0.0495% hydrogen peroxide, and held for 24 h
at 15C. However, when raw milk was treated with 0.0495% hydrogen peroxide, inocu-
lated to contain -2 X 105L. monocytogenes CFU/mL, and incubated at 4OC, numbers
of listeriae increased slightly as compared with the natural microflora. In another experi-
ment by the same researchers, samples of autoclaved milk containing 50.0495% hydrogen
peroxide were inoculated to contain a mixed culture of L. monocytogenes, S. aureus, and
Enterococcus faecalis (each organism at --I X 107CFU/mL) and incubated for 48 h at
4, 15, and 22C. Although L. monocytogenes populations decreased approximately 10-,
16-, and 40-fold during the first 24 h of incubation at 4, 15, and 22OC, respectively, the
organism grew during the second 24 h and reached populations 2 1 X 107CFU/mL. In
a subsequent study, Kamau et al. [ 1951 observed that 0.6 mM (i.e., 0.002%) H202slightly
inhibited growth of L. monocytogenes in bovine milk that was mildly heated (57"C, 20
min), cooled, and stored at 10C compared with the control without H202.Thus hydrogen
peroxide was relatively ineffective in decreasing numbers of listeriae in raw milk or milk
containing equal numbers of S. aureus and E. faecalis.
The impact of hydrogen peroxide on destruction of L. monocytogenes by heat was
investigated by two research groups. Kamau et al. [ 1961 reported that the presence of 0.6
Characteristics of Listeria monocytogenes 179
mM H202in milk did not enhance inactivation of L. monocytogenes by heat. Lou and
Yousef [238] found that adaptation of L. monocytogenes to H202increased the resistance
of this pathogen to heat. The authors added 500 ppm hydrogen peroxide into a culture of
L. monocytogenes in the exponential growth phase, incubated the culture for an addi-
tional 1-2 h at 35"'-,, and then determined heat resistance of treated Listeria cells in a
H20,-free phosptidte buffer. Compared with the unadapted culture, H202adaptation in-
creased DS(,oC-values by 2.9-fold. The same authors 12391 also reported an increase in
resistance of L. monocytogenes to a lethal level (i.e., 0.1%, w/w) of H202after the bacte-
rium was adapted to pH 4.5-5.0, 500 ppm H202,5% ethanol, 7% NaCI, or heat shocked
at 45C for 1 h.
Lactoperoxidase System
The lactoperoxidase (LP) system, a naturally occurring antimicrobial system in milk, has
been proposed as a means for extending the shelf life of raw milk when extended refriger-
ated storage is not possible, as in certain developing countries. Proper functioning of this
system depends on adequate levels of lactoperoxidase, thiocyanate, and hydrogen perox-
ide. Lactoperoxidase in milk represents 1 % of whey proteins [3 151, which is an adequate
amount for functioning of the lactoperoxidase system. Thiocyanate, however, is present
in bovine milk at only 1-7 pprn [36,37] and H202needs to be added exogenously or
generated by exogenous enzymes, such as glucose oxidase. In the LP system, lactoperoxi-
dase catalyzes the oxidation of thiocyanate (SCN-) by hydrogen peroxide to hypothiocya-
nous acid (HOSCN) and hypothiocyanate (OSCN-); these endproducts are responsible for
inactivating the microflora common to milk, including S. aureus, Salmonella typhimurium,
psychrotrophic pseudomonads, and some lactic acid bacteria.
Siragusa and Johnson [350] reported results of a study which examined inhibition
of L. monocytogenes by the LP system. Their model LP system contained equimolar con-
centrations (0.3 mM) of potassium thiocyanate and hydrogen peroxide in TSB fortified
with 0.5% yeast extract. After addition of 0.37 U lactoperoxidase/mL, flasks were inocu-
lated with 1,. monocytogenes in the late logarithmic growth phase. L. monocytogenes had
lag periods of 147.3-159.6, 46.6-55.5, 16.4-17.1, and 7.1 h in the presence of the LP
system and 61.4-77.4,23.5-32.5,7.5-10.3, and 4.3-5.7 h in the control (with or without
0.3 mM H202)when the pathogen was incubated at 5, 10, 20, and 3OoC, respectively.
Although the LP system appreciably extended the lag phase, maximum specific growth
rates were not affected. When the LP system was tested in sterile reconstituted skim milk
at 2OoC, the lag phase of L. monocytogenes was extended from 9 h (control) to 12-36 h.
Maximum Listeria populations also were lower with the LP system than in controls. Thus,
in this particular study, the LP system was bacteriostatic rather than bactericidal to L.
monocytogenes, and it was more effective at low than at high incubation temperatures.
In a subsequent study, Kamau et al. [ 1951 activated the lactoperoxidase system by
adding 2.4 mM SCN- and 0.6 mM H202to preheated (57"C, 20 min) bovine milk, which
contained adequate residual lactoperoxidase (9.2 mg/mL). Concentrations of SCN- and
H202used in this study did not have measurable antimicrobial activity against L. monocyto-
genes. When the LP system was activated at 35"C, L. monocytogenes (initially 104CFU/
mL,) decreased slightly in the first 2 h and began to grow after 8 h. At 10C, the LP
system inhibited Listeria for 96 h before appreciable growth was observed. The times
required to achieve half of the maximum growth were 16.9, 11.7, and 10.6 h at 35C and
436, 170, arid 137 h at 10C in milk (a) with activated LP systems, (b) with 0.6 mM H202,
180 Lou and Yousef
and (c) without additives, respectively. At 35OC, Listeria grew in all milk samples at a
similar maximum specific growth rate (0.162-0.221 h-I), whereas at IOOC, the pathogen
had a lower specific growth rate (0.0047 h-I) in the LP system-activated milk than in
that of the control (0.0103-0.0123 h-I).
Although Kamau et al. [195] and Siragusa and Johnson [350] reported that the LP
system was mainly bacteriostatic toward L. monocytogenes in a laboratory medium and
preheated milk, several other research groups noted appreciable bactericidal activity of
the system. El-Shenawy et al. [I211 found that initial L. monocytogenes populations of
30-50 CFU/mL decreased to nondetectable levels following 2 h of exposure to the LP
system at 35C. Using selective and nonselective plating media, these researchers also
demonstrated that the pathogen was not sublethally injured during exposure to the LP
system. Denis and Ramet [86] reported that the LP system completely eliminated L. mono-
cytogenes (initial populations - 10'-10' CFU/mL) from TSB with 0.65% yeast extract
following 5 1 , 2-6, and 4-10 days of incubation at 30, 15, and 4OC, respectively, de-
pending on the initial inoculum. However, unlike the previously described model broth
systems, these authors added glucose oxidase to their LP system. Since this enzyme oxi-
dizes glucose to gluconic acid, the resulting lowering of pH likely increased the bacteri-
cidal effect of the LP system beyond what would have been observed in similar model
systems having pH values near neutrality. Furthermore, L. monocytogenes is also inhibited
and/or inactivated in TB and milk containing 20.75% gluconic acid during extended
incubation at 13 and 35C [ 1181. Hence, these findings likely reflect the combined effects
of the LP system, pH, and gluconic acid rather than that of the LP system alone.
Additional investigations dealing with antilisterial activity of the LP system in UHT
milk rather than in culture media appeared in the scientific literature. Using two different
UHT milk-based LP systems containing lactoperoxidase (30 mg/L), potassium thiocya-
nate (84 mg/L), glucose (10 g/L), and glucose oxidase (2 mg/L) both with and without
urea peroxide (376 mg/L) as a hydrogen peroxide-generating mechanism, Earnshaw and
-
Banks [ 1011 found that initial L. monocytogenes populations of 104CFU/mL decreased
to 102CFU/mL in both LP systems during 6 days of incubation at 10C. Denis and Ramet
[86] also found that L. monocytogenes populations decreased in a similar UHT milk-
based LP system containing lactoperoxidase, potassium thiocyanate, and glucose oxidase.
However, unlike the previous study, their LP system completely eliminated the pathogen
(initial populations of 101-104CFU/mL) from UHT milk following 6-21 and 7-30 days
of incubation at 15 and 4OC, respectively, with estimated D-values of approximately 5
and 8 days at these same temperatures. Thus, as expected, the LP system was more detri-
mental to listeriae at higher rather than lower temperatures. In contrast, without the LP
-
system, the pathogen attained populations of 1OS and 1O4 CFU/mL following 7 days of
incubation at 15 and 4OC, respectively.
Several groups investigated antilisterial activity of the LP system in raw milk con-
taining naturally occurring levels of lactoperoxidase. El-Shenawy et al. [121] used an LP
system in raw milk containing naturally occurring levels of lactoperoxidase along with
0.25 mM thiocyanate anion and 0.25 mM hydrogen peroxide, and they found that L.
monocytogenes was often only slightly inhibited. In samples inoculated to contain 104-
and 107L. monocytogenes CFU/mL, the pathogen attained maximum populations of 2 108
CFU/mL after overcoming an extended lag phase. However, this LP system was far more
effective in raw milk inoculated to contain Listeria populations (i.e., 1O2 CFU/mL) similar
to those that have been observed in cases of naturally occurring listerial mastitis. Under
these conditions, the pathogen was completely inactivated after 2-4 and 12-24 h of incu-
Characteristics of Listeria monocytogenes 181
bation at 35 and 4C. Thus, as was true for microbiological media and UHT milk, the LP
system was again more effective in raw milk stored at higher rather than lower tempera-
tures. In a subsequent study, Gaya et al. [ 1501 investigated antilisterial activity of the LP
system activated by adding equal concentrations (0.25 mM) of sodium thiocyanate and
H 2 0 2to raw bovine milk stored at 4 and 8C. The authors reported D-values of 4.1- 1 1.2
days at 4C and 4.4-9.7 days at 8C for four L. monocytogenes strains added to the LP
system-activated milk. Lactoperoxidase activity decreased during incubation, with the
loss being more rapid at 8C than at 4C. In a more recent study, Zapico et al. [407]
reported that the activated LP system in goat's milk remained bactericidal against three
L. monocytogenes strains for 3-9 days at 4C and 1-7 days at 8C. Bacteriostatic activity
against Listeria was observed at 20C.
The LP system can be used in conjunction with thermal processing to increase de-
struction of listeriae in raw milk. Kamau et al. [ 1961 reported that the LP system (0.24
mM SCN- and 0.6 mM H202)enhanced thermal inactivation of L. monocytogenes in
preheated (57"C, 20 min) bovine milk containing 9.2 pg/mL of lactoperoxidase. Biphasic
heat inactivation curves were observed when the LP system was activated, with most of
the population being heat sensitive and inactivated rapidly during heating. The D-values
(based on the heat-resistant fraction of the population if biphasic inactivation curves oc-
curred) in milk (a) with the activated LP system, (b) with 0.6 mM H202,and (c) without
any additives (control) were 10.7, 29.4, and 30.2 min at 52.2"C, 1.6, 11.1, and 8.2 min
at 55.2"C, and 0.5,2.6, and 2.3 min at 57.8"C, respectively. When the LP system-activated
milk was held at 35C for different periods before heating, thermotolerance of L. monocy-
togenes decreased as the holding time increased, with the D 55.2"C being only 6.8 min after
16 h of holding time.
In summary, L. monocytogenes is susceptible to the LP system, especially at low
incubation temperatures. The LP system also can be used in combination with other treat-
ments, such as heat to increase inactivation of listeriae. This system will likely prove to
be useful for decreasing numbers of naturally occurring listeriae in raw milk before milk
processing facilities receive the product.
Lactoferr in
The presence of iron in culture media stimulates growth of some microorganisms. Lacto-
ferrin, a glycoprotein found in mammalian milk, exerts its antimicrobial activity through
binding of iron. Thus the antimicrobial activity of lactoferrin is affected by its degree of
iron saturation and iron availability in the medium. The degree of saturation of lactoferrin
with iron can be reduced by dialysis, and the resulting product is known as apo-lactoferrin.
Both lactoferrin and apo-lactoferrin exhibit antilisterial activity. Recently, lactoferrin was
found to inhibit invasion of L. monocytogenes into cultured intestinal cells [ 191.
Payne et al. [296] studied the effect of bovine lactoferrin and apo-lactoferrin, with
52% and 18% iron saturation, respectively, on growth of L. inonocytogenes in UHT milk
with 2% fat. After 18 h of incubation at 35"C, two strains of L. monocytogenes grew in
the presence or absence of lactoferrin (46 mg/mL), but the count of Listeria was 1.6- 1.8
logs lower in treated milk than in the control. Compared with lactoferrin, apo-lactoferrin
had greater antilisterial activity. When added to milk incubated at 35"C, apo-lactoferrin
was strongly listeriostatic at 15 mg/mL and listericidal at 30 mg/mL. Addition of 0.125
M ferric ammonium citrate eliminated the inhibitory effect of 30 mg/mL apo-lactoferrin
against Listeriu.
182 Lou and Yousef
BIOPRESERVATION
The terms biological preservation, biopreservation, and biocontrol all refer to the use of
microorganisms or their metabolic products to inhibit or inactivate undesired microorgan-
isms in foods. Biopreservation, as a means of naturally controlling pathogens and spoilage
microorganisms, especially in minimally processed foods, has been extensively studied
and excellent reviews are available [2,162,174,256,272,31 1,3361. Lactic acid bacteria
(LAB) and their metabolic products are commonly used in biopreservation, since these
bacteria are used in many traditional foods and are GRAS. Biopreservation by LAB occurs
because these bacteria compete with other microorganisms for nutrients and/or because
they produce antimicrobial compounds, such as weak acids, hydrogen peroxide, diacetyl,
and bacteriocins. The discussion in this section will focus on biopreservation with bacterio-
cins or bacteriocin-producing LAB which target L. monocytogenes in food.
Bacteriocins are antimicrobial substances that have a peptide or protein component
essential for their activity. Although most bacteriocins have a narrow spectrum of inhibi-
tion and only inhibit closely related species, some bacteriocins, such as nisin and pediocin,
have a relatively broad spectrum and can inhibit some less closely related organisms. A
large number of LAB bacteriocins are active against L. monocytogenes. Although their
modes of action vary, bacteriocins usually destabilize the cytoplasmic membrane of sensi-
tive cells, increase membrane permeability, and dissipate the proton motive force by form-
Characteristics of Listeria monocytogenes 183
Nisin
Nisin is a bacteriocin produced by certain strains of L. lactis subsp. lactis and has proven
to be extremely useful in preventing outgrowth of Clostridium spp., including Clostridium
botulinum, in fermented dairy and meat products. In 1980, Mohamed et al. [264] reported
results from a series of experiments that were designed to determine the effectiveness of
nisin against Listeria. When NB at pH 7.4 contained 4-16 International Units (IU) of
nisin per milliliter, populations of L. monocytogenes decreased >5 logs during 28 h at
37C. After this initial decrease, Listeria grew rapidly and attained final populations of
184 Lou and Yousef
- 108CFU/mL. Decreasing the pH of the medium from 7.4 to 5.5 led to a 16-fold decrease
in the level of nisin required to inhibit the bacterium.
Strains of L. monocytogenes may vary in resistance to nisin. Using Trypticase Soy
Agar, Benkerroum and Sandine [311 found that six L. monocytogenes strains were variably
resistant to nisin with MICs ranging from 1.4 X 102to 1.18 X 105IU/mL. Several addi-
tional studies also have demonstrated various degrees of nisin resistance for L. monocyto-
genes. Although Tatini [373] found that 512-1024 ppm nisin was required to inhibit
growth of 12 L. rnonocytogenes strains in laboratory media, S. typhimuriurn and E. coli
remained viable in the presence of up to 10,000ppm nisin. Although these findings suggest
that L. monocytogenes may be less resistant to nisin than some other potentially hazardous
microorganisms, one must keep in mind that some unusually resistant strains of L. monocy-
togenes do exist [ 163,262,263,2371.In 1989, Harris et al. [ 1631 examined sensitivity and
resistance of L. monocytogenes to nisin. According to these authors, populations of lister-
iae decreased 6-7 logs when nisin levels in BHI agar were increased from 0 to 10 pg/
mL. However, a relatively stable population of nisin-resistant mutants (- 100- 1000 CFU/
mL) developed on agar plates containing 1-50 pg nisin/mL with nisin-resistant mutants
occurring at a frequency of 10-6-10-x in media containing 50 pg nisin/mL. Although all
nisin-resistant mutants selected from agar plates were more resistant than their parent
strains, further testing revealed that nisin resistance was related to ability of nisin-resistant
strains to bind nisin rather than to specific genes coding for nisin resistance in plasmid
DNA. Similar nisin-resistant mutants also were obtained by Ming and Daeschel [262,
2631. Besides nisin resistance observed in spontaneous mutants, Lou [237] found that
acid adaptation or starvation increased resistance of L. monocytogenes to nisin and
pediocin.
As indicated by the earlier findings of Mohamed et al. [264], antilisterial activity
of nisin is strongly influenced by various environmental factors, including pH. Benker-
roum and Sandine [3I] determined the sensitivity of one L. monocytogenes strain to nisin
in Tryptose Soy Broth adjusted to pH values of 3.5-7.0. Populations increased -1 log
in broth cultures at pH 7.0 and 6.48 during the first 12 h of incubation, but no increase
in count was observed in similar samples adjusted to pH 5 5.94. Enhanced activity of
nisin against Listeria at lower pH values also has been observed by Harris et al. [ 1631.
Furthermore, data from Tatini [3731 indicate that average minimum nisin concentrations
of 5 12, 1365,2560, and 2496 ppm were required to inhibit growth of several L. monocyto-
genes strains on Trypticase Soy-Yeast Extract Agar adjusted to pH values of 5.0, 5.5,
6.0 and 6.5, respectively. Thus increased susceptibility of L. monocytogenes to nisin at
pH values <6 appears to be fairly well established.
The antilisterial action of nisin is further complicated by incubation temperature and
the presence of sodium chloride. According to Tatini [373], minimum concentrations of
nisin necessary to inhibit growth of L. monocytogenes were typically two to four times
greater at 35 than at 4C. In addition, when L. monocytogenes was incubated at 4C in
broth containing 1400 pprn nisin, lag periods for the various strains tested increased from
16 to 69 days as the nisin concentration increased from 0 to 400 ppm. In 1989, Harris et al.
[ 1631 also reported that addition of 2% sodium chloride enhanced the listericidal activity of
nisin in laboratory media, particularly at levels of <10 pg/mL.
Although many European countries have allowed direct addition of nisin to food
for some time, this practice was not permitted in the United States until 1989, when FDA
officials amended the food standard for pasteurized process cheese to allow addition of
not more than 250 pprn nisin to the finished product [ 1 1,16,17]. However, since allowable
Characteristics of Lister ia monocyt ogenes 185
levels of nisin may not completely inhibit L. monocytogenes in pasteurized process cheese
spreads that have been subjected to postpasteurization contamination, addition of nisin to
such products should not preclude use of proper sanitary practices.
Nisin-producing starter cultures provide some protection against L. monocytogenes
during cheese manufacture. Mainsnier-Patin et al. [248] inoculated L. monocytogenes into
milk which was used to make Camembert cheese. Nisin-producing or nonproducing starter
cultures were used in making the cheese. Counts of L. monocytogenes in the final product
were 2.4 log lower in cheese made with the nisin-producer than that made with the nonpro-
ducing strain. In another study, Zottola et al. [409] made Cheddar cheese with a nisin-
producing L. lactis starter and then prepared pasteurized processed cheese using this Ched-
dar cheese as an ingredient. Over 56 days of storage, populations of L. rnonocytogenes
decreased more rapidly in processed cheese made with rather than without the nisin-pro-
ducing culture.
The psychrotrophic and facultative nature of L. monocytogenes makes this patho-
gen a potential safety hazard for many minimally processed and vacuum/modified
atmosphere-packaged foods which require refrigeration. Incorporating bacteriocin or
bacteriocin-producing LAB into these products could be an effective way of minimizing
L. monocytogrnes growth and survival. However, sensory changes caused by addition of
biopreservatives must also be considered to ensure consumer acceptance [256].
Adding 10,000IU/mL nisin to cooked pork tenderloin that was packaged in air and
refrigerated prevented growth of inoculated L. monocytogenes but not Pseudomonas fragi.
However, use of nisin (1000 and 10,000IU/mL) in combination with modified atmosphere
packaging ( 100% CO2 or 80% CO2 + 20% air) inhibited growth of both bacteria, and
this inhibition was more pronounced at refrigeration than at room temperature [ 1291. Lac-
tic acid bacteria, such as Leuconostoc spp., often spoil minimally heat-processed vacuum-
packaged meat products. Some of the spoilage LAB are sensitive to nisin and nisin-produc-
ing Lactococcus spp. Thus Yang and Ray [399] suggested that biocontrol of these spoilage
LAB with nisin or nisin-producing strains could be an effective solution.
Pediocin
Pediocin, a wide-spectrum bacteriocin produced by certain strains of Pedicoccus acidilac-
tici, is a potent inhibitor of L. monocytogenes. Several research groups have reported
that P. acidilactici strains H, PAC1.O, and PO2 produced pediocin AcH, PA-1, and P02,
respectively. Luchansky et al. [24 I ] later reported that the restriction enzyme fragments
generated from plasmids encoding for these three pediocins were identical, with the three
producer strains also yielding identical genomic DNA fingerprints. These findings, in com-
bination with the DNA sequences for pediocin AcH and PA-1 (250a, 270a) indicate that
the three bacteriocins are similar in structure.
Control of L. monocytogenes by pediocin in several foods was explored. In a series
of studies on meat and meat products, Luchansky and his coworkers investigated the
antilisterial effect of pediocin AcH in wiener exudate, wiener packages, and turkey sum-
mer sausage [8524 1,4061. In refrigerated exudate from beef wieners, pediocin AcH de-
creased L. nzonocytogenes numbers by 0.74 log within 2 h. When the exudate was inocu-
lated with L,. nzonocytogenes and a pediocin-producing strain of P. acidilactici and kept
at 25OC, the count of L. monocytogenes increased initially and then markedly decreased
during extended incubation compared to the control. Pediocin activity in wiener exudate
was detected during the late stages of P. acidilactici growth [406]. Degnan et al. [85]
186 Lou and Yousef
groups. Pucci et al. [308] inoculated commercial samples of cottage cheese (pH 5.1),
cheese sauce (pH 6.0), and half-and-half (pH 6.6) to contain 102-104L. monocytogenes
CFU/g and then added a crude extract of pediocin PA-1 to these products. According to
these authors, viable numbers of L. monocytogenes decreased rapidly in all foods during
the first day of refrigerated storage. Although the pathogen attained populations of 103- 105
CFU/mL or CFU/g in cheese sauce and half-and-half following 7- 14 days of refrigerated
storage, these levels were still approximately 2-5 logs lower than those observed in corre-
sponding samples prepared without PA-1 powder. Motlagh et al. [270] studied the effec-
tiveness of pediocin AcH (up to 1350 AU/mL), produced by P. acidilactici H, in control-
ling L. monocytogenes in reconstituted dry milk, ice cream, and cottage cheese at 4 and
10C. After 1 h of storage at 4"C, this treatment decreased numbers of L. monocytogenes
- - -
by 1 log. When milk was inoculated to contain 102or 104L. monocytogenes CFU/
mL and incubated at 4C for 28 days and at 10C for 12 days, 1350 AU/mL pediocin
reduced Listeria populations about 2- to 4-logs during the first day of storage but did not
inhibit growth of Listeria survivors. Liao et al. 12301 prepared a pediocin P02-containing
powder through fermentation of whey permeate with P. acidilactici P02. When the pow-
der was added to Listeria-contaminated whole milk, the antilisterial activity of this prepa-
ration was clearly demonstrated. Addition of pediocin 5, produced by P. acidilactici UL5,
to 1% fat milk reduced the viable L. monocytogenes by -3 logs after one day of storage
at 4C [176].
In addition to meat and dairy products, other foods may benefit from biopreservation
by pediocin and pediocin-producing strains. Choi and Beuchat [69J added a crude bacterio-
cin extract from P. acidilactici M to kimchi during fermentation. This treatment immedi-
ately reduced numbers of L. monocytogenes in the inoculated product and inhibited growth
by the organism during 16 days of fermentation. Adding pediocin PA- 1, curvaticin FS47,
or lacticin FS56 to liquid whole egg dramatically reduced the heating time required to
inactivate L. monocytogenes in this product [272].
Other Bacteriocins
In addition to nisin and pediocin, several other bacteriocins also are effective against
L. monocytogenes. Lactobacillus bavaricus MN, a meat isolate, inhibited growth of L.
monocytogenes in a model beef gravy at 10C, with this inhibition being attributed to a
bacteriocin [395]. In a subsequent study, Winkowski et al. [396] found that L. bavaricus
MN, when coinoculated with L. monocytogenes into three model beef systems, beef cubes
and beef cubes with gravy andlor 0.5% glucose, significantly inhibited Listeria growth
at 4 and 10C The inhibition was greater at 4C than at 1O"C, and increased with addition
of glucose to gravy or with use of a higher inoculum of this Lactobacillus strain. Other
bacteriocin-producing strains of L. bavaricus were reported [222,223]. Some of the many
other bacteria that produce antilisterial bacteriocins are Lactobacillus salivarius M7 [46],
Lactobacillus curvatus FS47 and LTH 1174 [148,374,379], L. sake [3 191, L. sake Lb674
and LTH673 [ 172,3741, Lactobacillus plantarum MCS [65], Leuconostoc carnosum LA54
[ 1981, Leuconostoc mesenteroides [771, Propionibacterium thoenii P 127 [ 2421, Carno-
bacterium piscicola [55,56,252], and Enterococcus spp. [2 1,1971.
MODIFIED ATMOSPHERE
Buchanan and Klawitter [54] investigated aerobic versus anaerobic incubation in relation
to growth and survival of Listeria in TPB at pH 4.5. Under aerobic conditions, L. monocy-
188 Lou and Yousef
togenes Scott A was undetectable after -50 h at 37"C, survived without change in numbers
- - -
at 10 and 5"C, and grew to 107and 10' CFU/mL at 28 and 19C in 100 and -500
h, respectively. When the experiments were repeated under anaerobic conditions, a similar
trend in Listeria growth and survival as related to incubation temperature was observed.
However, anaerobic incubation was more conducive to Listeria growth or survival than
aerobic incubation. Anaerobic incubation at 19C decreased the length of the lag phase
from 80.6 (aerobic) to 27.3 h and the generation time from 19.1 (aerobic) to 6.8 h. Al-
though anaerobic incubation at 37C initially decreased the count by -2 logs, the popula-
tion gradually increased to a level close to that of the initial inoculum. The authors sug-
gested that anaerobiosis improved recovery of acid-injured cells at 37C and led to
subsequent long-term survival of repaired cells. Using similar experimental conditions,
George and Lund [ 1521 examined the effect of anaerobiosis on growth of two L. monocyto-
genes strains when incubated at 20C in TPB and Tryptone Soya Broth which was supple-
mented with yeast extract (3 g/L) and glucose (10 g/L) (TSYGB) and adjusted to pH 4.5
with HCI. Contrary to findings of Buchanan and Klawitter [54], the authors noted that
the anaerobic condition (generated through flushing with nitrogen), when compared with
aerobic incubation, inhibited growth in both media. When the investigators changed incu-
bation conditions for TPB from aerobic to anaerobic, the generation time of Listeria in-
creased from 4.22-4.98 to 6.12-6.7 1 h, increased the lag-phase duration from 45.8-47.5
to 73.8-80.1 h, and decreased the maximal population from 9.55-10.09 to 8.74-8.86 logs.
Although these results are conflicting, it is generally believed that Listeria grows
well under both aerobic and anaerobic conditions [54,58]. The capacity for anaerobic
growth at refrigeration temperatures makes L. monocytogenes a potential threat to the
safety of foods packaged under vacuum or modified atmosphere. Sous vide-processed
foods also fall into this category. Such foods are vacuum packaged, then cooked, chilled,
and finally stored refrigerated. Two gases, N2 and CO2, are commonly used in modified
atmosphere packaging. For packaging of fresh meats, vegetables, and fruits, limited levels
of O2 or air may be incorporated to maintain food quality. Aerobic microflora are greatly
inhibited by modified atmosphere packaging; however, some psychrotrophic or anaerobic
pathogens, such as L. monocytogenes, A. hydrophila, Y. enterocolitica, and Clostridium
botulinum are potentially capable of growing under these conditions.
L. monocytogenes can grow in food which has been packaged under vacuum or N2
gas. However, incorporation of CO2 improves the antilisterial activity of the packaging
atmosphere [23,130,140,178,2151. Growth of L. innocua was not seen in cottage cheese
packaged under 100% CO2during 28 days of storage at 5C; however, the organism grew
after 7 days in containers packaged under air and 100% N2 [140]. Fang and Lin [130]
reported that growth of L. monocytogenes was inhibited in raw pork tenderloin packaged
under 100% CO2 when stored for 10 days at 20C or 20 days at 4C. Avery et al. [23]
also found that when L. monocytogenes was inoculated into packaged fresh beef striploin
steaks (pH 5.3-5.5) counts of the pathogen decreased slightly under saturated CO2 atmo-
sphere during storage at 5 to 10C but increased by 3 logs in vacuum-packaged steaks
[23]. Kraemer and Baumgart [215] investigated growth of L. monocytogenes at 4, 7, or
10C in sliced frankfurter-type sausage that was packaged under 0, 20, 30, 50, or 80%
CO2,with the remainder being under N2packaging Growth of L. monocytogenes decreased
as levels of CO2 increased, and complete inhibition occurred under 80% CO2. Packaging
under 50% CO2 resulted in only partial inhibition. Concurrent with this work, Farber and
Daley [132] observed that L. monocytogenes was inhibited by 270% CO2 in modified
Characteristics of Listeria monocytoge nes 189
atmosphere--packaged turkey roll slices stored at 4 and 8C; however, the pathogen grew
in packages containing 30 and 50% CO2.
Because of the threat from some foodborne pathogens, especially C. botulinum,
modified atmosphere-packaged foods rely heavily on refrigerated storage to prevent out-
growth of C. botulinum and toxin production. However, refrigeration alone can not guaran-
tee food safety [317]. Growth of L. monocytogenes, A. hydrophila, and Y. enterocolitica
was observed in vacuum-packaged sliced roast beef stored at - 1.5"C [ 1781. Adding addi-
tional microbiological hurdles should increase the safety of modified atmosphere-pack-
aged foods. Wederquist et al. [390] found that inhibition of L. rnonocytogenes increased
when 0.5% oodium acetate, 2% sodium lactate, or 0.26% potassium sorbate were added
to vacuum-packaged bologna stored at 4C. Safety of raw pork tenderloin packaged under
a modified atmosphere was improved by incorporation of nisin [ 129,1301. Furthermore,
Degnan et al. [ 851 observed that inoculation of pediocin-producing P. acidilactici into
vacuum-packaged beef wieners decreased numbers of the coinoculated L. monocytogenes
during storage at abusive temperatures.
Irradiation
The entire electromagnetic spectrum consists of at least six distinct forms of radiation that
differ in wavelength, frequency, and penetrating power, of these forms, microwaves and
ultraviolet and gamma radiation are of primary interest to food manufacturers. In food
processing, microwave radiation is mainly used for its heating properties; thus discussion
of effects of this form of radiant energy on L. monocytogenes are not covered in this
section. Ultraviolet (UV) radiation, which is nonionizing, ranges in wavelength from 136
A
to 4000 and has some application in food processing. The poor penetrating power of
UV radiation restricts its use to a few specialized beverage applications, eradication of
190 Lou and Yousef
airborne contaminants and treatment of food contact and ?on-food contact surfaces.
Gamma radiation, which has a shorter wavelength (0.1- 1.4 A) than UV, is better suited
for external and internal decontamination of foods. Information concerning the ability of
gamma and ultraviolet radiation to inactivate L. monocytogenes in laboratory media will
be reviewed now. Findings from similar food-related studies are discussed elsewhere in
this book.
Gamma Irradiation
Literature on sensitivity of L. monocytogenes to gamma irradiation is less controversial
than that addressing thermal inactivation. Results from gamma irradiation studies con-
ducted in the United States [72,123,181] and Hungary [371] are strikingly similar, with
reported D-values ranging from 0.28 to 0.6 1 kGy for 12 different L. monocytogenes strains.
-
In addition, exposure to a gamma radiation dose of 1.7-4.0 kGy was generally sufficient
to reduce numbers of L. monocytogenes, L. ivanovii, and L. seeligeri by six to seven orders
of magnitude. Overall, these findings suggest that Listeria spp. are likely to be at least
equally, if not slightly more, resistant to gamma radiation in culture media than are other
commonly encountered non-spore-forming foodborne pathogens such as S. typhimurium
(D-value = 0.28 kGy) [377], S. aureus (D-value = 0.24 kGy) [377], and Y. enterocolitica
(D-value = 0.1 1 kGy) [ 1241. Although differences between L. monocytogenes strains
likely account for most of the observed variation in D-values, radiation sensitivity of L.
monocytogenes is also affected by age of the culture, irradiation menstruum, and the type
of medium used to enumerate the pathogen after irradiation. According to Huhtanen et
al. [ 1811, 1.5- and 2.5-h-old cultures of L. monocytogenes were somewhat more resistant
to gamma radiation than those incubated 5 and 18 h before exposure. Furthermore, surviv-
ing cells previously exposed to high radiation doses were no more resistant than the parent
culture. Consequently, observed differences between sensitivity of young and old cultures
probably resulted from innate differences between strains rather than from development
of radiation-resistant mutants. These authors also reported that 12-h-old centrifuged cul-
tures of L. monocytogenes were most resistant to 1.0 kGy gamma radiation when resus-
pended in fresh culture media or the original culture supernatant liquid followed in order
by phosphate buffer and distilled water. Inability of distilled water effectively to scavenge
cell-damaging free radicals produced during irradiation is likely responsible for decreased
resistance of the pathogen in water than in culture media that contain high concentrations
of free radical-quenching organic compounds. It is not surprising that L. monocytogenes
is more resistant to gamma radiation when present in foods than in culture media. Two
independent investigations [ 123,2921 have shown that D-values for radiation resistance
are markedly affected by the type of plating media used to enumerate the pathogen after
irradiation. In both studies, a significantly higher ( P < .OS) D-value resulted from in-
creased recovery of the pathogen with nonselective or semiselective rather than highly
selective plating media. These findings indicate that substantial numbers of listeriae were
sublethally injured during exposure to gamma irradiation. Since repair and subsequent
growth of injured cells is frequently inhibited by some of the selective agents used in
highly selective media, D-values for organisms exposed to irradiation or any other poten-
tially injurious treatment always should be determined using a plating medium with low
selectivity.
Andrews et al. [9] found that sensitivity of L. monocytogenes in TSB to gamma
radiation was affected by broth temperature (-80, 4, or 20C) during treatment and the
initial count (103,106,and 109CFU/mL). Under this wide range of conditions, the organ-
Characteristics of Listeria monocytogenes 191
ism exhibited D-values of 0.4 1-0.62 kGy. L. monocytogenes was significantly more resis-
tant to irradiation at room temperature (20C) than at refrigeration (4C) or freezing
(-80C) temperatures. D-values obtained with an initial count of 109CFU/mL were sig-
nificantly lower than those with 106CFU/mL.
Andrews and Grodner [8] later reported that gamma radiation was more effective
in inactivating L. monocytogenes when split into two equal doses than when the same
dose was applied as a single treatment. At 2OoC, the split dose of gamma radiation with
1-2 h between treatment decreased D-values from 0.50-0.58 kGy (single irradiation) to
0.41-0.42 kGy. However, a similar trend was not observed at refrigeration (4C) or sub-
freezing (- 80C) temperatures.
Ultraviolet Radiation
In 1971, Collins [73] determined the susceptibility of L. monocytogenes to UV radiation
emitted from a 14-W cold cathode mercury vapor lamp. Tryptone Soy Agar plates con-
-
taining 109 L. monocytogenes were exposed to a radiation output of 40 W/cm2 at 40
cm from the source for 30, 60, 90, and 120 s and then incubated for 3 days at 37C.
Populations of L. monocytogenes decreased 10-fold during the first 60 s of irradiation (D-
value of 60 s) after which the rate of inactivation increased sharply with a D-value of
- 15 s. L. monocytogenes was much more resistant to radiation than E. coli or Serratia
marcescens, which are commonly used to test the effectiveness of UV lamps.
Yousef and Marth [402] also reported that L. monocytogenes was inactivated by
exposing the bacterium to UV energy. Following 4 min of exposure to short-wave (254
nm) ultraviolet energy (100 pW/cm2), numbers of L. monocytogenes (strain Scott A) de-
creased approximately 7 logs on Tryptose Agar plates that were previously spread with
a 24- or 48-h-old culture of the test organism. In contrast, L. monocytogenes numbers
remained constant after 10 min of exposure to long-wave (364 nm) UV energy. Increasing
the intensity of short-wave UV radiation to 550 pW/cm2 nearly doubled the rate at which
L. monocytogenes was inactivated. These investigators also found that dry rather than
moist Listeria cells were more resistant to radiation. Exposing a dried film of L. monocyto-
genes cells in a Petri plate to short-wave UV energy (100 pW/cm2) decreased the popula-
tion by 2 rather than 7 logs for moist cells on Tryptose Agar. Fortunately, when present
in food processing environments, numbers of listeriae appear to be relatively low. Hence,
results from the aforementioned study suggest that UV energy may be of some practical
importance in reducing airborne contaminants, including listeriae, in food production and
storage areas.
High-Intensity Pulsed Light
Pulsed light, which has wavelengths ranging from -200 nm (UV) to 1 mm (near-infrared)
with peak emissions at 400-500 nm, inactivates microorganisms with flashes of intense
sunlight-like radiation. Currently employed pulsed light has intensities about 20,000 times
that of sunlight. Literature currently available on the effect of pulsed light on L. monocyto-
genes is limited to one 1995 report by Dunn et al. [98]. They found that L. monocytogenes
present on surfaces was inactivated to a greater extent by pulsed light than by UV energy.
-
Treatment with a single flash of pulsed light at 0.5-1.0 J/cm2 inactivated 10s CFU/cm2
of various microorganisms, including L. monocytogenes, on an agar surface, and several
-
such flashes inactivated up to 10' CFU/cm2. Although pulsed light is much more effec-
tive in inactivating organisms than UV light, both types of radiation have limited penetrat-
ing power and thus are only useful for inactivating microorganisms on surfaces of foods
192 Lou and Yousef
and packaging materials or in transparent food ingredients, such as water and some bever-
ages. When these authors used pulsed light to treat wieners that were previously surface
inoculated with L. innocua, populations decreased about 100-fold, with similar pulsed
light treatments being shown to be effective in extending the shelf life of baked foods,
seafood, and meat.
COMBINED TREATMENTS
Since total reliance on any single preservation method (e.g., heat, acidity, salt) usually
causes quality deterioration, many food processors use several treatments in combination
to process and preserve food. The well-known hurdle concept emphasizes the combined
use of antimicrobial factors to inhibit growth or eliminate microorganisms from food.
When preservation factors (hurdles) are combined, an additive antimicrobial effect often is
observed. However, combined hurdles sometimes act synergistically to enhance microbial
inhibition and inactivation beyond the additive effect. In other circumstances, however,
one hurdle may negate the antimicrobial effect of another hurdle. Further complications
may arise when hurdles are applied in sequence, with time gaps, rather than simulta-
neously. When used intermittently, a mild hurdle may stress an organism and elicit an
adaptive response which will in turn protect the microorganism against subsequent expo-
sure to more severe hurdles. This phenomenon of adaptation and protection is receiving
great attention in relation to efficacy of preservation by multiple hurdles and microbial
safety of the resulting food. In this section, examples illustrating the interaction between
hurdles will be presented in relation to control of L. monocytogenes in food. It should be
cautioned, however, that the outcome of interaction between hurdles depends heavily on
the conditions under which these hurdles are applied.
A two-hurdle interaction was demonstrated by Johansen et al. [ 1871, who found that
antilisterial activity of lysozyme was synergistically enhanced by low pH values. Another
example of a two-hurdle interaction was presented by Mainsnier-Patin et al. 12491, who
found that adding nisin to skim milk dramatically reduced the heating time required to
inactivate L. monocytogenes. Results of a study by Conner et al. [75]illustrates the nega-
tive interaction between two hurdles, refrigeration and high acidity. The investigators
observed that at maximum growth-limiting pH values, L. rnonocytogenes populations
decreased from -104 to < 10 CFU/mL in 1-3 weeks at 35C; whereas at 10C, listeriae
survived for 6- 12 weeks.
Interaction between multiple hurdles was presented by Bala and Marshal1 [25] who
investigated the combined effect of NaCl (2.5-7.8%), pH (5.4-7.8), temperature (5, 15,
25, and 35C), and sublethal levels of monolaurin (2-8 pg/L) against L. monocytogenes
grown on double (salt-pH) gradient plates. Addition of monolaurin to the gradient plates
reduced salt and pH tolerance of the pathogen. Complicated interactions between preserva-
tion factors (hurdles) were evident in a recent study by Lou [2371, who noted that antiliste-
rial activity of nisin was affected by pH and the presence of NaCl. Addition of NaCl
194 Lou and Yousef
(3.5-7.5%), to Trypticase Soy Broth decreased the bactericidal action of nisin against L.
monocytogenes. However, the presence of 3.5-5.5% NaCl interacted synergistically with
nisin to inhibit outgrowth of the pathogen on Trypticase Soy Agar plates.
Inhibition of L. monocytogenes by multiple hurdles was studied by Buchanan and
coworkers [58]. A factorial design was used to determine the combined effect of incuba-
tion temperature (5-37C), initial pH (6.0-7.5), sodium chloride (0.5 vs 4.5%), sodium
nitrite (0-1000 ppm), and atmosphere (aerobic vs anaerobic) on growth of L. monocyto-
genes in TPB. Although lag periods, generation times, and maximum populations were
all affected by these five interacting variables, sodium nitrite was most listeriostatic when
used in conjunction with low pH, increased sodium chloride, refrigeration temperatures,
and anaerobic conditions that simulated vacuum packaging.
Research using predictive microbiological modeling is likely to be valuable in as-
sessing the safety of foods preserved by multiple hurdles. Additive, nullifying, or syner-
gistic antimicrobial effects of multiple hurdles can be estimated by predictive models.
Consistent with these objectives, Buchanan et al. [5 1,52,57] attempted to predict behavior
of L. monocytogenes in response to an array of extrinsic factors. Buchanan et al. [57] used
a factoriallsupplemental central composite design to assess quantitatively the effects of
temperature (5, 10, 19,28, 37C), pH (4.50, .5.25, 6.00, 6.75, 7.50), sodium chloride (0.5,
1.5, 2.5, 3.5, 4.5%), sodium nitrite (0, 50, 100, 150, 200, 1000 ppm), and atmosphere
(aerobic vs anaerobic) on the growth kinetics of L. monocytogenes strain Scott A in TPB.
After growth curves were constructed from each experiment using regression analysis to
obtain best fit Gompertz equation curves, results were analyzed by response surface
analysis to generate a polynomial model that could mathematically predict lag periods,
exponential growth rates, generation times, and maximum populations for L. monocyto-
genes in association with any of the five variables examined. Overall, changes in response
of the organism to the five environmental factors were most evident as altered specific
growth rates and lag periods. L. monocytogenes also achieved similar maximum popula-
tions in all instances except those that involved growth of the pathogen under environmen-
tal extremes in the presence of high concentrations of sodium nitrite.
As a result of these and other studies, Buchanans group developed useful mathemat-
ical models to quantify behavior of L, monocytogenes in response to multiple environmen-
tal factors or hurdles [53]. These models were incorporated into a computer program called
the Pathogen Modeling Program. As of 1997, the program is available as version 5.0 for
Microsoft Windows and can be downloaded from a USDA site on the internet or requested
from the developers. The L. monocytogenes module of this program can be used to predict
lag time, growth rate, maximum population, and time required to attain a given count of
Listeria under a wide range of environmental conditions.
Interaction between hurdles becomes even more complicated when the history of
Listeria cells to be inactivated by the multiple hurdles is considered. Adaptation of L.
monocytogenes during sublethal exposure to various preservation techniques (or stress)
may protect the pathogen against subsequent exposure to the the same, different, or any
combination of stresses at normally lethal levels. Kroll and Patchett [216] reported that
adaptation to pH 5 greatly increased survival of L. monocytogenes at pH 3 as compared
with the unadapted cultures. According to Lou and Yousef [238,239], adaptation of L.
monocytogenes to sublethal levels of acid, ethanol, and hydrogen peroxide and starvation
increased resistance of L. monocytogenes to lethal levels of these factors and heat. This
stress adaptation, or hardening, complements the hurdle concept, since such hurdles
in foods can be applied simultaneously or sequentially. When applied sequentially, hurdles
Characteristics of Listeria monocytogenes 795
may not deliver the desired effect. Stress adaptation to the first encountered hurdle, which
' 'hardens' ' pathogens and increases their resistance to subsequent preservation factors,
may counteract hurdle build-up.
Ronner and Wong [320], buna-n rubber was strongly bacteriostatic to L. monocytogenes
growing in Peptone Glucose Phosphate (PGP) broth (a low nutrient medium) and slightly
bacteriostatic in TSB. Four of seven strains of L. monocytogenes growing in PGP formed
less dense biofilm populations on buna-n rubber than on stainless steel.
Temperature and moisture also affect survival of L. monocytogenes on surfaces.
Palumbo and Williams (284a) suspended a mixture of seven L. monocytogenes strains in
seven menstrua (distilled water, tryptone broth, nonfat dry milk, canned milk, glycerol,
light Karo syrup, and beef extract), and then dried these cell suspensions on glass plates
which were stored at 5 or 25C and I-75% RH. Enhanced survival was observed at 5C
rather than at 25C and at lower rather than higher RH. In a subsequent study, Helke and
Wong [ 1671 investigated survival of L. monocytogenes on surfaces under 32.5 and 75.5%
RH and temperatures of 6 and 25C. The authors found that survival of Listeria was higher
at 6C than at 25C and, in contrast to the previous study, survival was greater under
humid (75.5% RH) rather than dry (32.5% RH) conditions.
Microorganisms embedded in biofilms are more resistant to heat, sanitizers, and
other antimicrobial agents than are freely suspended (planktonic) cells. Frank and Koffi
[ 1461 prepared L. monocytogenes in three states: (a) planktonic cells, obtained by growing
the bacterium in TSB for 38 h at 21"C, (b) adherent single cells, prepared by immersing
glass slides in a planktonic cell culture for 4 h at 2 I "C; the attached Listeria was mostly
single cells, and (c) adherent microcolony cells, made by incubating the glass slides with
attached L. monocytogenes cells for 14 days at 2loC, during which the slides were periodi-
cally washed with a saline solution and incubated in fresh media; the attached cells in
this instance were mostly in microcolonies. The investigators found that Listeria was more
sensitive to banzalkonium chloride (n-alkyl dimethyl dichlorobenzyl ammonium chloride,
a quaternary ammonia sanitizer) and dodecyl benzene sulfonic acid, an anionic acid sani-
tizer (DBSA) when present in the planktonic rather than in the adherent single cell or
microcolony state. Contact with either sanitizer ( 100-800 ppm) at ambient temperature
immediately reduced populations of the planktonic cells from 1Oh CFU/ml to undetectable
levels. In contrast, adherent single cells (initially 105- 106CFU/cm2) and adherent micro-
colony cells (initially 106- 107CFU/cm2) decreased 3-5 and 2-3 logs, respectively. The
few remaining adherent single cells became undetectable after 16 min of sanitizer expo-
sure, with adherent microcolonies surviving a maximum of 20 min. Survival of these
adherent cells was not caused by depletion of sanitizers, since the remaining sanitizers
produced similar inactivation when new microcolony slides were treated. When planktonic
cells were heat-treated ( 5 min at 55 or 70C) in the presence of 400 ppm benzalkonium
chloride or 200 ppm DBSA, the combined treatment, regardless of temperature, decreased
populations -5 logs from an initial - 106CFU/mL, whereas similar treatments decreased
counts of microcolonies only -2 and >5 logs, respectively.
In a subsequent study, Lee and Frank [226] investigated the sensitivity of stainless
-
steel-adhering single and microcolony cells (initially 1O5 L. monocytogenes CFU/cm2)
on stainless steel to hypochlorite and heat. They found that adherent single cells on stain-
less steel were more sensitive to hypochlorite and heat inactivation than adherent microcol-
ony cells. Exposure to a hypochlorite solution, which contained 200 ppm residual chlorine,
for 30 s decreased the population of adherent single and microcolony cells by 4.8 and 2.6
log units, respectively, with microcolony cells surviving up to 5 min of exposure to this
agent. Although heating at 65C for 30 s resulted in a 3.8-log reduction of both types of
adherent cells, only microcolony cells were detectable after 3 min of heating. Adherent
single and microcolony cells became undetectable after 30 and 60 s of heating at 72"C,
198 Lou and Yousef
respectively. Combined exposure to 65C for 30 s and 200 ppm chlorine decreased the
number of microcolony cells to undetectable levels.
Krysinski et al. [217] obtained adherent L. monocytogenes cells by growing the
organism in a culture medium for 24 h at 25C that contained pieces of stainless steel,
polyester belt, or polyester-polyurethane conveyer belt and then tested the effectiveness
of 10 sanitizers and 6 cleaners in inactivating or removing adhering L. monocytogenes
cells after 10 min of exposure. Although L. monocytogenes attached to these surfaces at
similar levels (-2 X 104CFU/cm2), protection provided by these surfaces against sani-
tizers or cleaning agents varied with the substrate, with polyester belt being most protective
followed by polyester-polyurethane belt and stainless steel. Although most of the sanitizers
and cleaners that were tested inactivated Listeria attached to stainless steel, none of these
agents could effectively eliminate cells attached to polyester-polyurethane belt. However,
detergent cleaning followed by sanitizing, a practice commonly followed in industry, was
more effective in controlling adherent L. monocytogenes cells than when either cleaning
or sanitizing was used alone.
Besides sanitizers, effectiveness of listeriaphages in inactivating surface-adherent
L. monocytogenes was also investigated [326]. Adherent cells were obtained by immersing
chips of stainless steel or polypropylene in a L. monocytogenes culture for 1 h at 26C.
Treatment of adherent L. monocytogenes cells with a phage suspension (3.5 X 108PFU/
mL) reduced populations of the pathogen by -3.4 logs, with mixtures of three different
phages proving to be most effective. Although use of QUATAL (containing 10.5% N-
alkyldimethyl-benzylammonium HCl and 5.5% glutaraldehyde as active ingredients,
Ecochimie LtLe, Quebec, Canada) at 50 pprn destroyed the adherent Listeria flora, a com-
bination of 108PFU/mL phage and 30 pprn QUATAL resulted in similar destruction.
SANITIZERS
Sanitizers have been widely used in the food industry to decrease populations of patho-
genic and spoilage organisms in food production and processing facilities. Most of these
sanitizing agents belong to one of four categories: (a) chlorine-containing compounds, (b)
iodophors, (c) quaternary ammonium compounds frequently called quats, or (d) acid
sanitizers. Additionally, ozone has been used for decades in some European counties, and
application of this sanitizer in the US food industry is likely to increase.
When in aqueous solutions, chlorine-containing compounds release hypochlorous
acid which accounts for their bactericidal action. Iodophors, water-soluble complexes of
elemental iodine, and nonionic surface-active agents owe their bactericidal activity to re-
lease of free elemental iodine and hypoiodous acid, which is enhanced under acidic condi-
tions. In contrast, quaternary ammonium compounds are best classified as noncorrosive
germicidal cationic detergents that remain active at relatively high pH values. Finally,
acid sanitizers such as phosphoric and citric acid-containing compounds are frequently
used in conjunction with rinsing agents in automated cleaning systems better known as
clean-in-place (CIP) systems. Unlike iodophors, acid sanitizers are nonvolatile and retain
their bactericidal activity at temperatures below 100C. Sanitizing agents must reduce
populations of a given test organism at least 5 logs during 30 s of exposure at ambient
temperatures before the particular agent is deemed to be effective.
Chlorine Compounds
In the absence of organic debris, chlorine rapidly inactivates most non-spore-forming
bacteria even when used at the very low concentrations found in chlorinated drinking
Characteristics of Listeria monocyto genes 199
water. Although the actual mechanism of disinfection is not fully understood, germicidal
activity of chlorine has generally been attributed to hypochlorous acid (HOCI), which
is generated in aqueous solutions of sodium hypochlorite and other chlorine-containing
compounds. Although HOCl can in turn dissociate to form the hypochlorite ion (OC1-)
and hydrogen ion (H'), depending on the pH of the solution, the neutral electric charge
of the former suggests that HOCl can more easily penetrate the bacterial cell membrane
than OCI-. Thus it is not surprising that the germicidal activity of HOCl is 80 times that
of OC1-. After diffusing into the cell, HOCl is thought to inactivate the organism by
inducing formation of toxic oxygen species or combining with proteins, which may in
turn inhibit key enzymatic reactions and alter cell membrane permeability.
Numerous studies have dealt with the lethal effects of various chlorinated sanitizing
agents on L. monocytogenes. Beginning in 1969, Baranenkov [26] found that hypochlorite
could effectively control L. monocytogenes on the surface of hen's eggs. Chloramine also
was later shown to be listericidal when used under acidic conditions at concentrations of
0.1-0.2% [ 2581. Subsequently, Lopes [236] reported that two solutions of chlorine-based
sanitizers (one containing 8.5% sodium hypochlorite with 8%)active chlorine and the other
containing 25.8% sodium dichloro-s-triazinetrione) containing I00 pprn active chlorine
both reduced L. monocytogenes populations by more than 5 logs after 30 s of exposure.
These findings were subsequently confirmed by Rossmoore and Drenzek [324]. Further
tests by Lopes [236] revealed that the organic chlorine-based sanitizer was slightly more
effective against L. monocytogenes than the sodium hypochlorite-based sanitizer; the for-
mer had a lower pH which would in turn lead to higher concentrations of HOCI, the most
bactericidal form of chlorine. A chlorine dioxide-based sanitizing agent also has been
approved by the FDA for use in the food industry. According to its manufacturer [ 121, the
unusual effectiveness of this formula against L. monocytogenes and other microorganisms
results from a special activator which converts large quantities of stabilized chlorine diox-
ide to the free form.
Following the published report by Lopes [236], Brackett [41] determined the germi-
cidal effect of reagent-grade sodium hypochlorite and household bleach on two L. monocy-
togenes strains (Scott A and LCDC 8 1-861 ) previously associated with outbreaks of food-
borne listeriosis. After 20 s of exposure to 2 5 0 ppm available chlorine, both compounds
led to substantial reductions in numbers of viable L. monocytogenes in phosphate buffer.
However, Listeria populations remained relatively stable for an additional 4.6 min, and
in several instances listeriae survived 1 5 min with free residual chlorine levels that ap-
proached 40 ppm. Since 10 ppm available chlorine was ineffective, results of this study
indicate that the minimum chlorine concentration needed to kill L. monocytogenes lies
between 10 and 40 ppm.
Effectiveness of chlorine against L. monocytogenes also was examined in depth and
later reviewed by El-Kest and Marth [103-1051. Cells of L. monocytogenes strain Scott
A were harvested from 24- and 48-h-old slants or broth cultures, washed by centrifugation
in 20 mM phosphate buffer solution or 0.3 12 mM phosphate buffer dilution water, and
then exposed at 25C to sodium hypochlorite solutions at pH 7 (25C) that contained 0.5-
10.0 ppm available chlorine. Using a solution containing 5 ppm available chlorine, num-
bers of survivors decreased -6 logs after only 30 s, with the organism no longer being
detectable by direct plating on TA after 1 h. (Results from Rosales et al. [32 I ] also showed
that populations of L. monocytogenes, L. ivanovii, and L. seeligeri decreased >5 logs
-
following 30 s of exposure to distilled water (pH 7) containing 2 2 5 pprn hypochlorite
[i.e., 223.8 ppm available chlorine]). Exposing L. monocytogenes to 0.5, 1.0, 2.0, 5.0,
and 10.0 ppm available chlorine resulted in corresponding D-values of 61.7, I 1.3, 6.7,
200 Lou and Yousef
4.9, and 4.7 s. Although disinfecting activity clearly increased with increasing concentra-
tions of available chlorine, the effectiveness of sodium hypochlorite also was affected by
several additional factors. Increased resistance of L. monocytogenes to chlorine was ob-
served using (a) 24- rather than 48-h-old cultures, (b) cells harvested from broth rather
than agar slants, and (c) cultures exposed to solutions containing 20 mM rather than 0.3 12
mM phosphate. Five and 10 ppm of available chlorine was partially neutralized in the
presence of 0.05 and 0.1% peptone (nitrogenous compound) [103]. Given the findings
indicating that hypochlorite concentrations of up to 400 ppm were of little use against
L. monocytogenes, L. ivanovii, or L. seeligeri when these organisms were suspended in
reconstituted NFDM (10% solids) [321], it is clear that antimicrobial activity of chlorine
can only be maintained if organic material is effectively removed before exposure.
In addition to the factors just discussed, Lee and Frank [225] found that resistance
of L. monocytogenes cells in late exponential phase to hypochlorite solution (1-5 ppm
available chlorine) was greater when the organism was grown at 35C than at 6 or 21C.
Exposure to 1 ppm available chlorine for 5 min at ambient temperature decreased popula-
tions of the organism previously grown at 6, 21, and 35C by 3.4, 3.1, and 2.1 logs,
respectively. Furthermore, L. monocytogenes grown in a nutrient-poor medium ( 15-fold
diluted TSB) was 10-times more resistant to chlorine than when grown in regular TSB
[225].
Additional work by El-Kest and Marth [105] demonstrated that populations of L.
monocytogenes decreased most rapidly in sodium hypochlorite solutions at 5C followed
by 35 and 25C. Marked variation in chlorine sensitivity also was observed among the
three L. monocytogenes strains tested. However, since dissociation of HOCl to OC1- and
Hi increases with increasing pH, resistance and/or survival of L. monocytogenes in the
presence of chlorine compounds ultimately depends on the pH of the suspending medium.
For example, exposing the pathogen to 1 ppm available chlorine for 30 s led to population
decreases of -4.0, 3.0, and 0.7 logs at pH 5, 7, and 9, respectively. Hence, for chlorine
to be effective against listeriae and other microorganisms, it is imperative that such solu-
tions have pH values <7.
Although the work of El-Kest and Marth [103-1051 clearly indicates that the mini-
mum listericidal concentration of free chlorine lies between 1 and 5 ppm (similar to that
observed for many other non-spore-forming bacteria) depending on pH, temperature, the
presence of organic material, and bacterial strain, earlier studies [41,10I] conducted under
less controlled conditions showed minimum listericidal concentrations of free chlorine
that were markedly higher. Similar problems also were probably encountered by Mustapha
and Liewen [273], who found that a minimum of -100 ppm sodium hypochlorite was
required to reduce L. monocytogenes populations >4 logs in sterile distilled water during
2-5 min of exposure.
Chlorine is used extensively in fresh vegetable processing. Therefore, Zhang and
Farber [408] investigated the efficacy of several chlorine-based compounds against a cock-
tail of five L. monocytogenes strains on the surface of freshly cut lettuce and cabbage at
refrigeration and ambient temperatures. Sanitizers tested by these investigators included
chlorine from a hypochlorite-containing bleach, chlorine dioxide and a sodium chlorite-
based oxy-halogen compound. Immersing Listeria-contaminated vegetables in solutions
containing 200 ppm chlorine, 5 ppm chlorine dioxide, or 200 ppm Salmide for 10 min
resulted in maximum reductions of 1.3- 1.7,0.8-1.1, and 0.6 logs, respectively, for lettuce,
and 0.9-1.2, 0.4-0.8, and 1.8 logs for cabbage. The presence of surfactants reduced the
effect of chlorine. The authors also tested trisodium phosphate and lactic acid on lettuce
Characteristics of Listeria monocytogenes 201
and cabbage. Trisodium phosphate (0.1 and 0.2%) failed to inactivate listeriae, whereas
0.1% lactic or acetic acid reduced populations by only 0.5 and 0.2 log, respectively.
Many researchers have investigated the ability of commonly used sanitizers to inacti-
vate L. rnonoc.ytogenes on various types of food contact surfaces. Mustapha and Liewen
[273] found that destruction of L. rnonocytogenes was greater on smooth rather than pitted
stainless steel surfaces. However, cells incubated on either surface for 1 h were more
resistant to the lethal action of sodium hypochlorite than those remaining on such surfaces
24 h before exposure. Lower moisture levels on stainless steel surfaces incubated 24 rather
than 1 h may have enhanced the listericidal effect of sodium hypochlorite. In contrast to
these findings, Rossmoore and Drenzek [324] reported that L. rnonocytogenes populations
decreased 5 logs on relatively moist surfaces of glazed and unglazed ceramic tile as well
as stainless steel chips following exposure to 100 ppm sodium hypochlorite as directed
by the manufacturer. Furthermore, in no instance was L. monocytogenes more resistant
than single cultures of Pseudornonas or Serratia. However, when the same three surfaces
were treated with 1 and 10% solutions of milk and blood, Listeria populations decreased
1-4 logs in the presence of 100 ppm sodium hypochlorite.
As mentioned earlier, commonly used sanitizers are generally less effective against
L. rnonocytogenes in biofilms than when the cells are freely suspended. Lee and Frank
[226] reported that microcolonies of L. rnonocytogenes adhering to stainless steel
(-105CFU/cm2) decreased 2.6 logs after 30 s of exposure to 200 ppm chlorine (from a
hypochlorite solution), with some cells surviving a 5-min treatment. Mosteller and Bishop
[269] found that 200 ppm chlorine was sufficient to inactivate more than 5 logs of freely
suspended L. rnonocytogenes cells. However, a similar treatment failed to inactivate 3
logs of L. rnonocytogenes when a milk biofilm (initially 1O4-1O5CFU/cm2)was formed
on surfaces of Teflon and buna-n rubber. Resistance to sanitizers, including chlorine, in-
creased when L. rnonocytogenes biofilms were prepared on surfaces of polyester or polyes-
ter-polyurethane instead of stainless steel [2 171. Therefore, although freely suspended L.
rnonocytogenes can be controlled by 100 ppm chlorine, a higher level of chlorine is re-
quired to eliminate L. rnonocytogenes from biofilms.
Ozone
Ozone, a powerful sanitizing gas, is a better alternative to chlorine in many food processing
applications. Although used in European countries for decades, ozone is only approved
in the United States for treatment of bottled drinking water. Recently, a panel of experts
representing academia, food processors, and utility companies self-affirmed the GRAS
status of ozone, thus permitting its use in food processing applications [ 1591.
Ozone can be applied as a sanitizer in its gaseous form or as ozonated water. Ozo-
nated water is bactericidal to various microorganisms, with vegetative cells being more
sensitive to ozone than molds or bacterial spores. Use of ozone, in the form of ozonated
water, in food preservation and for decreasing microbial loads of meat and poultry and
of food plant effluents has been investigated [ 127,192,3441. Several factors affect the
bactericidal activity of ozonated water; organic matter such as food components quickly
react with ozone and reduce its effectivity.
Restaino et al. [316] investigated the lethality of ozonated water with and without
20 ppm organic matter, soluble starch (SS), or bovine serum albumin (BSA), against four
gram-positive (including L. rnonocytogenes) and four gram-negative (E. coli, S. typhirnu-
riurn, Y. enterocolitica, and P. aeruginosa) bacteria, two yeasts (Candida albicans and
202 Lou and Yousef
Zygosaccharomyces bailii), and mold spores (Aspergillus niger). Initial ozone concentra-
tions of 0.15-0.20 ppm produced by the ozone generator were higher in deionized water
with or without 20 ppm SS than in BSA-containing deionized water. Biphasic inactivation
curves were observed for bacteria and yeasts. Vegetative cells were inactivated instantly
(decreased >4 logs) after contact with ozone, with a much slower decrease in microbial
counts occurring during extended incubation. Gram-positive bacteria were generally more
resistant to ozone than gram-negative organisms, with the four gram-negative species hav-
ing similar sensitivity to ozone; however, L. monocytogenes was an exception. Contact
with ozone instantly inactivated >5 logs of L. monocytogenes but only -3 logs of the
other gram-positive species. The presence of organic matter during ozonation decreased
the lethality of ozone; however, the type of organic matter was more important than the
concentration. Incorporating 20 ppm SS had little effect on lethality of ozone toward
Listeria, whereas 20 ppm BSA significantly decreased the inactivation rate.
Antiseptic Soaps
Cross contamination of foods by food handlers or raw products in food service facilities
is a potential threat to public safety. Kerr et al. [ 1991 found that 12 and 7% of food workers
carried Listeria spp. and L. monocytogenes on their hands, respectively. Therefore, elimi-
nating L. monocytogenes from hands should decrease the incidence of Listeria in many
foods and enhance overall food safety. According to one report [32 I], full-strength solu-
tions of three commercially available antiseptic soaps, namely Mikro-x, Isoderm, and Zer-
obac were strongly listericidal, with populations of L. monocytogenes, L. ivanovii, and L.
seeligeri decreasing 7 logs following 30 s of exposure. Isoderm (a chlorine/quaternary
ammonium compound-based soap) remained almost equally effective when diluted 1 :4,
whereas Zerobac (an iodophor-based soap) retained strong listericidal activity at a dilution
of 1:8.
In another study [2 101, fingers of human volunteers were inoculated to contain 105
or 109L. monocytogenes CFU/finger to test the effectiveness of moist soap and a commer-
cially produced finger wipe containing isopropyl alcohol and citric acid as active ingredi-
ents. Overall, numbers of listeriae on fingers were generally reduced no more than 2-4
logs after 5 s of rubbing in phosphate buffer and moist soap, respectively. Therefore, a
population decrease of approximately 2 logs can be attributed to physical removal of the
pathogen during rubbing. In contrast, L. monocytogenes populations consistently de-
creased 2 4 logs after rubbing fingers with finger wipes for 5 s. Thus, strong listericidal
activity of these particular finger wipes and the ease with which they can be used should
make such products beneficial for food handlers in the food service industry.
In 1996, McCarthy 12531 checked inactivation of L. monocytogenes on latex gloves
by five commercially available hand-washing sanitizers. The latex gloves were artificially
contaminated by dipping them for 30 s into a PBS solution or crab cooking water that
contained - 1 O5 Listeria CFU/mL and then were treated with various hand-washing sani-
tizers. Dipping contaminated gloves into PBS containing a commercial chlorine bleach
solution (50 and 100 pprn chlorine), Zepamine A ( I95 ppm active quaternaries) or Ultra-
Kleen (a peroxide-based powder at 56 g/3.8 L) decreased counts of L. monocytogenes on
surfaces of gloves to undetectable levels, whereas treatment with Zep-i-dine (25 ppm
titratable iodine) and Zep Instant Hand Sanitizer that contains 60% ethanol reduced popu-
lations only 2 logs. Using nutrient-rich crab cooking water instead of PBS dramatically
Characteristics of Lister ia mon ocyt ogen es 203
decreased the effectiveness of both 50 ppm chlorine and Zep-i-dine and slightly decreased
the effectiveness of 100 ppm chlorine and Zepamine A. According to this study, only
Ultra-Kleen maintained the same effectiveness in cooking water.
onus and other microbial contaminants. Hence, if L. rnonocytogenes was present initially,
this bacterium was likely to be eliminated during exposure to glutaraldehyde.
As is true for steel and tile surfaces, conveyor lubricants also come in contact with
various organic materials in food processing facilities. Hence, Rossmoore and Drenzek
[324] examined behavior of L. rnonocytogenes in lubricants containing 25-50 ppm glutar-
aldehyde, 5- 10 ppm MCI, and 500- 1000 ppm parachlorometaxylenol in combination
with 1% added milk and blood. In the presence of 1% milk, 50 ppm glutaraldehyde was
most effective, with numbers of listeriae decreasing >5 logs following 3 h of exposure.
However, in samples containing 1% blood rather than 1% milk, only 1000 ppm parachloro-
metaxylenol retained sufficient bactericidal activity to reduce Listeriu populations >5 logs
within 24 h. Although parachlorometaxylenol exhibited similar activity in the presence
of milk, addition of 5-10 ppm MCI was of little value in decreasing numbers of listeriae
in lubricant containing 1% milk or blood.
In addition to lubricants, several water-based cooling system fluids used in the dairy
and meat industry also are subject to sporadic contamination with pathogenic microorgan-
isms, including L. rnonocytogenes. Consequently, Rossmoore and Drenzek [324] also ex-
amined the potential benefit of adding low concentrations of glutaraldehyde, parachloro-
metaxylenol, and MCI to sweet water (i.e., potable refrigerated water containing a
corrosion inhibitor) and an aqueous solution of 35% propylene glycol, both of which are
commonly used in the cooling section of pasteurizers and other types of heat exchangers.
According to this report, L. rnonocytogenes populations in inoculated samples of sweet
-
water (pH 9.3) and 35% propylene glycol (pH 8.8) decreased only 1 and 3 logs, respec-
tively following 14 days of storage at 3.5"C. In sharp contrast, addition of 25 ppm glutaral-
dehyde to sweet water and propylene glycol containing 1% milk completely inactivated
L. rnonocytogenes populations of 105 CFU/mL in less than 1 h, as did addition of 100
ppm parachlorometaxylenol to propylene glycol. Inclusion of 100 ppm parachlorometaxy-
lenol in sweet water and 10 ppm MCI in both coolants was at best only marginally effec-
tive, with the pathogen surviving at least 48 h in several instances. Thus, although a low
concentration of glutaraldehyde will inactivate L. rnonocytogenes in sweet water and pro-
pylene glycol, use of parachlorometaxylenol for such a purpose should be limited to solu-
tions of propylene glycol.
REFERENCES
1. Abdalla, O.M., G.L. Christen, and P.M. Davidson. 1993. Chemical composition of and Liste-
ria rnonocytogenes survival in white pickled cheese. J. Food Prot. 56941 -846.
2. Abee, T., L. Krockel, and C. Hill. 1995. Bacteriocins: mode of action and potentials in food
preservation and control of food poisoning. Int. J. Food Microbiol. 28: 169- 185.
3. Agard, D.A. 1993. To fold or not to fold. Science 260:1903-1904.
4. Ahamad, N., and E.H. Marth. 1989. Behavior of Listeria rnonocytogenes at 7 , 13, 21, and
35OC in tryptose broth acidified with acetic, citric or lactic acid. J. Food Prot. 52:688-695.
5. Ahamad, N., and E.H. Marth. 1990. Acid injury of Listeria rnonocytogenes. J. Food Prot.
53126-29.
6. Al-Issa, M., D.R. Fowler, A. Seaman, and M. Woodbine. 1984. Role of lipid in butylated
hydroxyanisole (BHA) resistance of Listeria rnonocytogenes. Zbl. Bakteriol. Hyg. A 258:
42-50.
7. Al-Makhlafi, H., J. McGuire, and M. Daeschel. 1994. Influence of preabsorbed milk protein
206 Lou and Yousef
spectrum of lactoferricin B, a potent bactericidal peptide derived from the N-terminal region
of bovine lactoferrin. J. Appl. Bacteriol. 73:472-479.
31. Benkerroum, N., and W.E. Sandine. 1988. Inhibitory action of nisin against Listeriu monocy-
togenes. J. Dairy Sci. 71 :3237-3245.
32. Berry, E.D., R.W. Hutkins, and R.W. Mandigo. 1991. The use of bacteriocin-producing Pedi-
ococ(~iisacidilactici to control postprocessing Listeria monocytogenes contamination of
frankfurters. J. Food Prot. 54:68 1-686.
33. Berry, E.D., and M.B. Liewen. 1988. Survival and growth of Listeria monocytogenes in the
presence of food preservatives and acids. Annual Meeting of the Institute of Food Technol-
nology. New Orleans, June 19-22, Abstr. 3 17.
34. Berrq, E.D., M.B. Liewen, R.W. Mandigo, and R.W. Hutkins. 1990. Inhibition of Listeria
monocytogenes by bacteriocin-producing Pediococcus during the manufacture of fermented
semidry sausage. J. Food Prot. 53: 194- 197.
35. Beumer, R.R., M.C. Te Giffel, L.J. Cox, F.M. Rombouts, and T. Abee. 1994. Effect of exoge-
nous proline, betaine, and carnitine on growth of Listeriu monocytogenes in a minimal me-
dium Appl. Environ. Microbiol. 60: 1359- 1363.
36. Bjorck, L., 0. Claesson, and W. Schulthess. 1979. The lactoperoxidase/thiocyanate/hydro-
gen peroxide system as a temporary preservative for raw milk in developing countries. Milch-
wissenschaft 34:726-729.
37. Bjorck, L,., C.G. Roskn, V. Marshall, and B. Reiter. 1975. Antimicrobial activity of the lactop-
eroxitlase system in milk against pseudomonads and other Gram-negative bacteria. Appl.
Environ. Microbiol. 30: 199-204.
38. Blackman, I.C., and J.F. Frank. 1996. Growth of Listeria rnonocytogenes as a biofilm on
various food-processing surfaces. J. Food Prot. 59:827-83 1.
39. Bojsen-MQIler, J. 1972. Human listeriosis-diagnostic, epidemiological and clinical studies.
Acta Pathol. Microbiol. Scand. Sec. B. (Suppl.) 229: 1- 157.
40. Borovian, G.E. 1989. Control of Listeria monocytogenes in comparison to other food patho-
gens using food preservatives. Proc. Annual Meeting of the American Society for Microbiol-
ogy, New Orleans, May 14-18, Abstr. 167.
41. Brackett, R.E. 1987. Antimicrobial effect of chlorine on Listeria rnonocytogenes. J. Food
Prot. 50:999- 1003.
42. Bradshaw, J.G., J.T. Peeler, J.J. Corwin, J.M. Hunt, J.T. Tierney, E.P. Larkin, and R.M.
Twedt. 1985. Thermal resistance of Listeria monocytogenes in milk. J. Food Prot. 48:743-
745.
43. Bradshaw, J.G., J.T. Peeler, J.J. Corwin, J.M. Hunt, and R.M. Twedt. 1987. Thermal resis-
tance of Listeriu rnonocytogenes in dairy products. J. Food Prot. 50543-544,546.
44. Bradshaw, J.G., J.T. Peeler, and J. Lovett. 1991. Thermal resistance of Listeria species in
whole milk. J. Food Prot. 54: 12- 14.
45. Briggs, E.H., C.W. Donnelly, C.M. Beliveau, and W.L. Beeken. 1987. Comparison of uptake
J rnonocytogenes by normal and endotoxin-induced phagocytes of bovine origin.
of L ~feria
Proc. of Annual Meeting of the American Society for Microbiology, Atlanta, March 1-6,
Abstr. P-26.
46. Brink, B., M. Minekus, J.M.B.M. van der Vossen, R.J. Leer, J.H.J. int Veld Huis. 1994.
Antimicrobial activity of lactobacilli: preliminary characterization and optimization of pro-
duction of acidocin B, a novel bacteriocin produced by Lactobacillus ucidophilus M46. J.
Appl. Bacteriol. 77: 140-148.
47. Brzin. B. 1973. The effect of NaCl on the morphology of Listeriu monocytogenes. Zbl. Bakt-
eriol. Hyg., I Abt. Orig. A 225:80-84.
48. Brzin. B. 1975. Further observations of changed growth of Listeria monocytogenes on salt
agar. Zbl. Bakteriol. I. Abt. Orig. A 232:287-293.
49. Buazzi, M.M., M.E. Johnson, and E.H. Marth. 1992. Survival of Listeria monocytogenes
during the manufacture and ripening of Swiss cheese. J. Dairy Sci. 75:380-386.
208 Lou and Yousef
50. Buazzi, M.M., M.E. Johnson, and E.H. Marth. 1992. Fate of Listeria monocytogenes during
the manufacture of Mozzarella cheese. J. Food Prot. 5590-83.
51. Buchanan, R.L., and M.H. Golden. 1994. Interaction of citric acid concentration and pH on
the kinetics of Listeria monocytogenes inactivation. J. Food Prot. 57567-570.
52. Buchanan, R.L., M.H. Golden, and R.C. Whiting. 1993. Differentiation of the effects of pH
and lactic or acetic acid concentration on the kinetics of Listeria monocytogenes inactivation.
J. Food Prot. 56:474-478.
53. Buchanan, R.L., M.H. Golden, R.C. Whiting, J.G. Philips, and J.L. Smith. 1994. Nonthermal
inactivation models for Listeria monocytogenes. J. Food Sci. 59: 179- 188.
54. Buchanan, R.L., and L.A. Klawitter. 1990. Effects of temperature and oxygen on the growth
of Listeria monocytogenes at pH 4.5. J. Food Sci. 55:1754-1756.
55. Buchanan, R.L., and L.A. Klawitter. 1992. Characterization of a lactic acid bacterium, Carno-
bacterium piscicola LK5, with activity against Listeria monocytogenes at refrigeration tem-
peratures. J. Food Safety 12:199-217.
56. Buchanan, R.L., and L.A. Klawitter. 1992. Effectiveness of Carnobacterium piscicola LK5
for controlling the growth of Listeria monocytogenes Scott A in refrigerated foods. J. Food
Safety 12:219-236.
57. Buchanan, R.L., and J.G. Philips. 1990. Response surface model for predicting the effects
of temperature, pH, sodium chloride content, sodium nitrite concentration and atmosphere
on the growth of Listeria monocytogenes. J. Food Prot. 53:370-376.
58. Buchanan, R.L., H.G. Stahl, and R.C. Whiting. 1989. Effects and interactions of temperature,
pH, atmosphere, sodium chloride and sodium nitrite on growth of Listeria monocytogenes.
J. Food Prot. 52:844-851.
59. Buchnev, K.N., and T.F. Omarov. 1972. Resistance of Listeria to some physical and chemical
factors. Tr. Alma-At. Zoovet. Inst. 20:142- 145.
60. Buncic, S., C.M. Fitzgerald, R.G. Bell., and J.A. Hudson. 1995. Individual and combined
listericidal effects of sodium lactate, potassium sorbate, nisin, and curing salts at refrigeration
temperature. J. Food Safety 15:247-264.
61. Bunning, V.K., R.G. Crawford, J.G. Bradshaw, J.T. Peeler, J.T. Tierney, and R.M. Twedt.
1986. Thermal resistance of intracellular Listeria monocytogenes cells suspended in raw bo-
vine milk. Appl. Environ. Microbiol. 52: 1398-1402.
62. Bunning, V.K., R.G. Crawford, J.T. Tierney, and J.T. Peeler. 1990. Thermotolerance of Liste-
ria monocytogenes and Salmonella typhimuriurn after sublethal heat shock. Appl. Environ.
Microbiol. 56:3216-3219.
63. Bunning, V.K., R.G. Crawford, J.T. Tierney, and J.T. Peeler. 1992. Thermotolerance of heat-
shocked Listeria monocytogenes in milk exposed to high-temperature, short-time pasteuriza-
tion. Appl. Environ. Microbiol. 58:2096-2098.
64. Bunning, V.K., C.W. Donnelly, J.T. Peeler, E.H. Briggs, J.G. Bradshaw, R.G. Crawford,
C.M. Beliveau, and J.T. Tierney. 1988. Thermal inactivation of Listeria monocytogenes
within bovine milk phagocytes. Appl. Environ. Microbiol. 54:364-370.
65. Campanini, M., I. Pedrazzoni, S. Barbuti, and P. Baldini. 1993. Behavior of Listeria monocy-
togenes during the maturation of naturally and artificially contaminated salami: effect of
lactic acid bacteria starter cultures. Int. J. Food Microbiol. 20: 169-175.
66. Carminati, D., and S. Carini. 1989. Antimicrobial activity of lysozyme against Listeria mono-
cytogenes in milk. Microbiol. Aliments Nutr. 7:49-56.
67. CFR. 1991. Sodium diacetate (18.1754). Code of Federal Regulations, Title 21. Office of
Federal Register, US Government Printing Office, Washington, D.C.
68. Chen, N., and L.A. Shelef. 1992. Relationship between water activity, salts of lactic acids,
and growth of Listeria monocytogenes in a meat system. J. Food Prot. 55574-578.
69. Choi, S.-Y., and L.R. Beuchat. 1994. Growth inhibition of Listeria monocytogenes by a
bacteriocin of Pediococcus acidilactici M during fermentation of kimchi. Food Microbiol.
11:301-307.
Characteristics of Listeria monocytogenes 209
70. Choi, H.K., M.M. Schaack, and E.H. Marth. 1988. Survival of Listeria monocytogenes in
cultured buttermilk and yogurt. Milchwissenschaft 43:790-792.
71. Chung, K.-T., S.E. Stevens, Jr., W.-F. Lin, and C.I. Wei. 1993. Growth inhibition of selected
food-borne bacteria by tannic acid, propyl gallate and related compounds. Lett. Appl. Micro-
biol. I7:29-32.
72. Cirigliano, M.C., and C.L. Hartman. 1989. Sensitivity of Listeria monocytogenes to gamma
irradiation. Proc. Annual Meeting of the American Society for Microbiology, New Orleans,
May 14-18, Abstr. P-2.
73. Collins, F.M. 1971. Relative susceptibility of acid-fast and non-acid-fast bacteria to ultravio-
let light. Appl. Microbiol. 21:411-413.
74. Conner, D.E., R.E. Brackett, and L.R. Beuchat. 1986. Effect of temperature, sodium chloride,
and pH on growth of Listeria monocytogenes in cabbage juice. Appl. Environ. Microbiol.
52159 -63.
75. Conner, D.E., V.N. Scott, and D.T. Bernard. 1990. Growth, inhibition, and survival of Liste-
ria monocytogenes as affected by acidic conditions. J. Food Prot. 53:652-655.
76. Craig. E.A., B.D. Gambill, and R.J. Nelson. 1993. Heat shock proteins: molecular chaperones
of protein biogenesis. Microbiol. Rev. 57:402-414.
77. Daba, H., S. Pandian, J.F. Gossenlin, R.E. Simard, J. Huang, and L. Lacroix. 1991. Detection
and activity of a bacteriocin produced by Leuconostoc mesenteroides. Appl. Environ. Micro-
biol. 57:3450-3455.
78. Dallas, H.L., D.P. Thomas, and A.D. Hutkins. 1996. Virulence of Listeria monocytogenes,
Listeria seeligeri, and Listeria innocua assayed with in vitro murine macrophagocytosis. J.
Food Prot. 59:24-27.
79. Dallmier, A.W., and S.T. Martin. 1988. Catalase and superoxide dismutase activities after
heat injury of Listeria monocytogenes. Appl. Environ. Microbiol. 5458 1-582.
80. Darie, P., and I. Constantina. 1988. Studies on Listeria monocytogenes resistance under labo-
ratory conditions. Proc. of Xth International Symposium on Listeriosis, Pecs, Hungary, Au-
gust 22-26, Abstr. 50.
81. Davidson, P.M. 1993. Parabens and phenolic compounds. In P.M. Davidson and A.L. Branen,
eds., Antimicrobials in Foods. 2nd ed. New York: Marcel Dekker, pp. 263-306.
82. Dedie, K., and D. Schulze. 1957. Die Hitzeresistenz. von Listeria monocytogenes in Milch.
Berl. Miinchener tierarztl. Wochenscher. 70:23 1-232.
83. Degnan, A.J., N. Buyong, and J.B. Luchansky. 1993. Antilisterial activity of pediocin AcH
in model systems in the presence of an emulsifier or encapsulated within liposomes. Int. J.
Food Microbiol. 18:127-138.
84. Degnan, A.J., C.W. Kaspar, W.S. Otwell, M.L. Tamplin, and J.B. Luchansky. 1994. Evalua-
tion of lactic acid bacterium fermentation products and food-grade chemicals to control Liste-
ria monocytogenes in blue crab (Callinectes sapidus) meat. Appl. Environ. Microbiol. 60:
3198-3203.
85. Degnan, A.J., A.E. Yousef, and J.B. Luchansky. 1992. Use of Pediococcus acidilactici to
control Listeria monocytogenes in temperature-abused vacuum-packaged wieners. J. Food
Prot. 55:98-103.
86. Denis. F., and J.-P. Ramet. 1989. Antibacterial activity of the lactoperoxidase system on
Listeria monocytogenes in trypticase soy broth, UHT milk and French soft cheese. J. Food
Prot. S2:706-7 11.
87. Dickgiesser, N. 1980. Listeria monocytogenes as a cause of nosocomial infections: A study
of the survival of pathogenic microorganisms in the environment. Infection 8: 199-201.
88. Dje, Y., K.D. Payne, and P.M. Davidson. 1989. Unpublished data.
89. Dominguez, L., J.F.F. Garayzabal. E.R. Ferri, J.A. Vazquez, E. Gomez-Lucia, C. Ambrosio,
and G. Suarez. 1987. Viability of Listeria monocytogenes in milk treated with hydrogen
peroxide. J. Food Prot. 50:636-639.
90. Dominguez, L., J.F.F. Garayzabal, J.A. Vazquez, J.L. Blanco, and G. Suarez. 1987. Fate
210 Lou and Yousef
of Listeria monocytogenes during manufacture and ripening semi-hard cheese. Lett. Appl.
Microbiol. 4: 125- 127.
91. Donker-Voet, J. 1962. My view on the epidemiology of Listeria infections. In: M.L. Gray,
ed. Second Symposium on Listeric Infection, Montana State College, Bozeman, MT, pp.
133-139.
92. Donnelly, C.W., and E.H. Briggs. 1986. Psychrotrophic growth and thermal inactivation of
Listeria monocytogenes as a function of milk composition. J. Food Prot. 49:994-998.
93. Donnelly, C.W., E.H. Briggs, C.M. Beliveau, and W.L. Beeken. 1987. In vitro phagocytosis
of Listeria monocytogenes by neutrophils and macrophages of bovine origin. Proc. Annual
Meeting of the American Society for Microbiology, Atlanta, March 1-6.
94. Donnelly, C.W., E.H. Briggs, and L.S. Donnelly. 1987. Comparison of heat resistance of
Listeria monocytogenes by neutrophils and macrophages of bovine origin. Proc. Annual
Meeting of the American Society for Microbiology, Atlanta, March 1-6.
95. Doores, S. 1993. Organic acids. In: P.M. Davidson and A.L. Branen, eds. Antimicrobials in
Foods. 2nd ed. New York: Marcel Dekker, pp. 95-136.
96. Doyle, M.P., K.A. Glass, J.T. Beery, G.A. Garcia, D.J. Pollard, and R.D. Schultz. 1987.
Survival of Listeria monocytogenes in milk during high-temperature, short-time pasteuriza-
tion. Appl. Environ. Microbial. 53: 1433- 1438.
97 Duh, Y.-H., and D.W. Schaffner. 1993. Modeling the effect of temperature on the growth
rate and lag time of Listeria innocua and Listeria monocytogenes. J. Food Prot. 56:205-
210.
98. Dunn, J., T. Ott, and W. Clark. 1995. Pulsed-light treatment of food and packaging. Food
Technol. 49(9):95 -98.
99. Durst, J. 1975. The role of temperature factors in the epidemiology of listeriosis. Zbl. Bakter-
iol. Hyg., I. Abt. Orig. A 233:72-74.
100. Durst, J., and A. Sawinsky. 1972. Beitrage zur Untersuchung der Ausbreitungsmoglichkeit
der Listeria monocytogenes. Z. Gesamte Hyg. Grenzgeb. 18:1 17- I 18.
101. Earnshaw, R.G., and J.G. Banks. 1989. A note on the inhibition of Listeria monocytogenes
NCTC 11994 in milk by an activated lactoperoxidase system. Lett. Appl. Microbiol. 8:203-
205.
102. El-Gazzar, F.E., and E.H. Marth. 1991. An apparent benzoate-resistant strain of Listeria
monocytogenes recovered from a milk clotting agent of animal origin. Milchwissenschaft
461350-354.
103. El-Kest, S.E., and E.H. Marth. 1988. Inactivation of Listeria monocytogenes by chlorine. J.
Food Prot. 5 1520-524.
104. El-Kest, S.E., and E.H. Marth. 1988. Listeria monocytogenes and its inactivation by chlorine:
A review. Lebensm. Wiss. Technol. 2 1 :346-35 1.
105. El-Kest, S.E., and E.H. Marth. 1988. Temperature, pH and strain of pathogen as factors
affecting inactivation of Listeria monocytogenes by chlorine. J. Food Prot. 5 1:622-625.
106. El-Kest, S.E., and E.H. Marth. 1989. Unpublished data.
107. El-Kest, S.E., and E.H. Marth. 1991. Injury and death of frozen Listeria monocytogenes as
affected by glycerol and milk components. J. Dairy Sci. 74: 1201-1208.
108. El-Kest, S.E., and E.H. Marth. 1991. Strains and suspending menstrua as factors affecting,
death and injury of Listeria monocytogenes during freezing and frozen storage. J. Dairy Sci.
74: 1209- 1213.
109. El-Kest, S.E., and E.H. Marth. 1992. Freezing of Listeria monocytogenes and other microor-
ganisms: a review. J. Food Prot. 55:639-648.
110. El-Kest, S.E., and E.H. Marth. 1992. Transmission electron microscopy of unfrozen and
frozedthawed cells of L. monocytogenes treated with lipase and lysozyme. J. Food Prot. 55:
687-696.
111 El-Kest, S.E., and E.H. Marth. 1992. Lysozyme and lipase alter unfrozen and frozedthawed
cells of L. monocytogenes. J. Food Prot. 55:777-78 1.
Characteristics of Listeria monocytogenes 21 1
112. El-Kest, S.E., A.E. Yousef, and E.H. Marth. 1991. Fate of Listeria monocytogenes during
freezing and frozen storage. J. Food Sci. 56: 1068- I07 1.
113. El-Khateib, T., A.E. Yousef, and H.W. Ockerman. 1993. Inactivation and attachment of Liste-
ria rnonocytogenes on beef muscle treated with lactic acid and selected bacteriocins. J. Food
Prot. 56:29-33.
114. El-Shenawy, M.A., and E.H. Marth. 1988. Sodium benzoate inhibits growth of or inactivates
Listcria monocytogenes. J. Food Prot. 5 1 :525-530.
115. El-Shenawy, M.A., and E.H. Marth, 1988. Inhibition and inactivation of Listeriu monocyto-
genc0.s by sorbic acid. J. Food Prot. 51:842-847.
116. El-Shenawy, M.A., and E.H. Marth, 1989. Inhibition or inactivation of Listeria monocyto-
genes by sodium benzoate together with some organic acids. J. Food Prot. 52:77 1-776.
117. El-Shenawy, M.A., and E.H. Marth, 1989. Behavior of Listeria monocytogenes in the pres-
ence of sodium propionate. Int. J. Food Microbiol. 8:85-94.
118. El-Shenawy, M.A., and E.H. Marth. 1990. Behavior of Listeriu monocytogenes in the pres-
ence of gluconic acid and during preparation of cottage cheese curd using gluconic acid. J.
Dairy Sci. 73: 1429- 1438.
119. El-Shenawy, M.A., and E.H. Marth. 199 1. Organic acids enhance the antilisterial activity of
potassium sorbate. J. Food Prot. 54:593-597.
120. El-Shenawy, M.A., and E.H. Marth. 1992. Behavior of Listeria monocytogenes in the pres-
ence of sodium propionate together with food acids. J. Food Prot. 55:241-245.
121. El-Shenawy, M.A., H.S. Garcia, and E.H. Marth. 1990. Inhibition and inactivation of Listeria
monocytogenes by the lactoperoxidase system in raw milk buffer or a semisynthetic medium.
Milchwissenschaft 45638-64 I .
122. El-Shenawy, M.A., A.E. Yousef, and E.H. Marth. 1989. Heat injury and inactivation of Liste-
ria monocytogenes in reconstituted nonfat dry milk. Milchwissenschaft 44:74 1-745.
123. El-Shenawy, M.A., A.E. Yousef, and E.H. Marth. 1989. Inactivation and injury of Listeria
monocytogenes in tryptic soy broth or ground beef treated with gamma irradiation. Lebensm.
Wiss. Technol. 22:387-390.
124. El-Zawahry, Y.A., and D.B. Rowley. 1979. Radiation resistance and injury of Yersinia enter-
ocolitica. Appl. Environ. Microbiol. 37:50-54.
125. Emme, A.E., A.E. Yousef, and E.H. Marth. 1989. Unpublished data.
126. Erickson, J.P., and P. Jenkins. 1991. Comparative Salmonella spp. and Listeria monocyto-
genes inactivation rates in four commercial mayonnaise products. J. Food Prot. 54:9 13-
916.
127. Ewell, A.W. 1950. Ozone and its application in food preservation. Refrig. Eng. 58: 1-4.
128. Faith, N.G., A.E. Yousef, and J.B. Luchansky. 1992. Inhibition of Listeria monocytogenes
by liquid smoke and isoeugenol, a phenolic component found in smoke. J . Food Safety 12:
303-3 14.
129, Fang. T.J., and L.-W. Lin. 1994. Growth of Listeria monocytogenes and Pseudomonas fragi
on cooked pork in a modified atmosphere packaging/nisin combination system. J. Food Prot.
57:479-485.
130. Fang, T.J., and L.-W. Lin. 1994. Inactivation of Listeria monocytogenes on raw pork treated
with modified atmosphere packaging and nisin. J. Food Drug Anal. 2: 189-200.
131. Farber, J.M., and B.E. Brown. 1990. Effect of prior heat shock on heat resistance of Listeria
moizocytogenes in meat. Appl. Environ. Microbiol. 56: 1584- 1587.
132. Farber, J.M., and E. Daley. 1994. Fate of Listeria monocytogenes on modified-atmosphere
packaged turkey roll slices. J. Food Prot. 57: 1098- 1 100.
133. Farber, J.M., E. Daley, F. Coates, D.B. Emmons, and R. McKellar. 1992. Factors influencing
survival of Listeria monocytogenes in milk in a high-temperature short-time pasteurizer. J.
Food Prot. 55:946-95 1.
134. Farber, J.M., and F. Pagotto. 1992. The effect of acid shock on the heat resistance of Listeria
monocytogenes. Lett. Appl. Microbiol. 15: 197-201.
212 Lou and Yousef
135. Farber, J.M., G.W. Sanders, S. Dunfield, and R. Prescott. 1989. The effect of various acidu-
lants on the growth of Listeria monocytogenes. Lett. Appl. Microbiol. 9: 181-1 83.
136. Farber, J.M., G.W. Sanders, J.I. Speirs, J.-Y. DAoust, D.B. Emmons, and R. McKellar.
1988. Thermal resistance of Listeria monocytogenes in inoculated and naturally contaminated
raw milk. Int. J. Food Microbiol. 7:277-286.
137. FDA, 1988. Nisin preparation: affirmation of GRAS status as a direct human food ingredient.
Fed. Regist. 54:6120-6123.
138. FDA, 1990. Food Additives. Code of Federal Regulations. Titile 21, Part 170, p. 5-23. U.S.
Government Printing Office, Washington, DC.
139. Feido, W.M., and H. Jackson. 1989. Effect of tempering on the heat resistance of Listeria
rnonocytogenes. Lett. Appl. Microbiol. 9: 157- 160.
140. Feido, W.M., A. Macleod, and L. Ozimek. 1994. The effect of modified atmosphere packag-
ing on the growth of microorganisms in cottage cheese. Milchwissenschaft 49:622-629.
141. Fernandez Garayzabal, J.F., L. Dominguez Rodriguez, J.A. Vazquez Boland, E.F. Rodriguez
Ferri, V. Briones Dieste, J.L. Blanco Cancelo, and G. Suarez Fernandez. 1987. Survival of
Listeria monocytogenes in raw milk treated in a pilot plant size pasteurizer. J. Appl. Bacteriol.
63~533-537.
142. Fields, F.O. 1996. Use of bacteriocins in food: regulatory considerations. J. Food Prot.
59(Supplement):72-77.
143. Foegeding, P.M., and N.W. Stanley. 1991. Listeria innocua transformed with an antibiotic
resistance plasmid as a thermal-resistance indicator for Listeria monocytogenes. J. Food Prot.
54:5 19-523.
144. Foegeding, P.M., A.B. Thomas, D.H. Pilkington, and T.R. Klaenhammer. 1992. Enhanced
control of Listeria monocytogenes by in situ-produced pediocin during dry fermented sausage
production. Appl. Environ. Microbiol. 58:884-890.
145. Foster, J.W., and M.P. Spector. 1995. How Salmonella survive against the odds. Annu. Rev.
Microbiol. 49: 145- 174.
146. Frank, J.F., and R.A. Koffi. 1990. Surface-adherent growth of Listeria rnonocytogenes is
associated with increased resistance to surfactant sanitizers and heat. J. Food Prot. 53550-
554.
147. Gahan, C.G.M., B. ODriscoll, and C. Hill. 1996. Acid adaptation of Listeria monocytogenes
can enhance survival in acidic foods and during milk fermentation. Appl. Environ. Microbiol.
6213128-3 132.
148. Garver, K.I., and P.M. Muriana. 1994. Purification and partial amino acid sequence of curvat-
icin FS47, a heat-stable bacteriocin produced by Lactobacillus cuwatus FS47. Appl. Environ.
Microbiol. 60:2 191-2 195.
149. Gay, M., 0. Cerf, and K.R. Davey. 1996. Significance of pre-incubation temperature and
inoculum concentration on subsequent growth of Listeria rnonocytogenes at 14C. J. Appl.
Bacteriol. 8 1:433-438.
150. Gaya, P., M. Medina, and M. Nunez. 1993. Effect of the lactoperoxidase system on Listeria
monocytogenes behavior in raw milk at refrigeration temperatures. Appl. Environ. Microbiol.
57:3355-3360.
151. Genigeorgis, C . 1989. Personal communication.
152. George, S.M., and B.M. Lund. 1992. The effect of culture medium and aeration on growth
of Listeria rnonocytogenes at pH 4.5. Lett. Appl. Microbiol. 15:49-52.
153. George, S.M., B.M. Lund, and T.F. Brocklehurst. 1988. The effect of pH and temperature
on initiation of growth of Listeria monocytogenes. Lett. Appl. Microbiol. 6: 153-156.
154. Gervilla, R., M. Capellas, V. Ferragut, and B. Guamis. 1997. Effect of high hydrostatic
pressure on Listeria innocua 910 CECT inoculated into ewes milk. J. Food Prot. 60:33-
37.
155. Gianfranceschi, M., and P. Aureli. 1996. Freezing and frozen storage on the survival of
Listeria rnonocytogenes in different foods. Ital. J. Food Sci. 8:303-309.
Characteristics of Listeria monocytogenes 213
156. Glass, K.A., and M.P. Doyle. 1989. Fate and thermal inactivation of Listeria monocytogenes
in beaker sausage and pepperoni. J. Food Prot. 52:226-23 1,235.
157. Goff, J.F., A.K. Bhunia, and M.G. Johnson. 1996. Complete inhibition of low levels of Liste-
ria rnonocytogenes on refrigerated chicken meat with pediocin AcH bound to heat-killed
Pediococcus acidilactici cells. Appl. Environ. Microbiol. 59: 1 187- 1 192.
158. Golden, D.A., L.R. Beuchat, and R.E. Brackett. 1988. Inactivation and injury of Listeria
rnonocytogenes as affected by heating and freezing. Food Microbiol. 5: 17-23.
159. Graham, D.M. 1997. Use of ozone for food processing. Food Technol. 51(6):72-75.
160. Gray, M.L., and A.H. Killinger. 1966. Listeria monocytogenes and listeric infections. Bacte-
riol. Rev. 30:309-382.
161. Hanlin, M.B., N. Kalchayanand, P. Ray, and B. Ray. 1993. Bacteriocins of lactic acid bacteria
in combination have greater antimicrobial activity. J. Food Prot. 56:252-255.
162. Hansen, J.N. 1994. Nisin as a model preservative. Crit. Rev. Food Sci. Nutr. 34:69-93.
163. Harris, L.J., H.P. Fleming, and T.R. Klaenhammer. 1991. Sensitivity and resistance of Liste-
ria monocytogenes ATCC 19115, Scott A, and UALSOO to nisin. J. Food Prot. 54:836-840.
164. Harrison, M.A., Y.-W. Huang, C.-H. Chao, and T. Shineman. 1991. Fate of Listeria monocy-
togenes on packaged, refrigerated, and frozen seafood, J. Food Prot. 54524-527.
165. Hauben, K.J.A., E.Y. Wuytack, C.C.F. Soontjens, and C.W. Michiels. 1996. High-pressure
transient sensitization of Escherichia coli to lysozyme and nisin by disruption of outer-mem-
brane permeability. J. Food Prot. 59:350-355.
166. Hefnawy, Y.A., S.I. Moustafa, and E.H. Marth. 1993. Sensitivity of Listeria monocytogenes
to selected spices. J. Food Prot. 56:876-878.
167. Helke, D.M., and A.C.L. Wong. 1994. Survival and growth characteristics of Listeria mono-
cytogenes and Salmonella typhimurium on stainless steel and buna-n rubber. J. Food Prot.
571963-968.
168. Herald, P.J., and E.A. Zottola. 1988. Attachment of Listeria monocytogenes to stainless steel
surfaces at various temperatures and pH values. J. Food Sci. 53:1549-1552, 1562.
169. Hevin, B., M. Morange, and R.M. Fauve. 1993. Absence of an early increase in heat-shock
protein synthesis by Listeria monocytogenes within mouse mononuclear phagocytes. Res.
Immunol. 144:679-689.
170. Hicks, S.J., and B.M. Lund. 1991. The survival of Listeria inonocytogenes in cottage cheese.
J. Appl. Bacteriol. 70:308-3 14.
171. Hof, H., H.P.R. Seeliger, A. Schrettenbrunner, and S. Chatzipanagiotou. 1986. The role of
Listeria monocytogenes and other Listeria spp. in foodborne infections. In Proc. 2nd World
Congress, Foodborne Infections and Intoxications, Berlin, Germany, pp. 220-223.
172. Holck, A.L., L. Axelsson, K. Huehne, and L. Kroeckel. 1994. Purification and cloning of
sakacin 674, a bacteriocin from Lactobacillus sake Lb674. FEMS Microbiol. Lett. 1 15:143-
149.
173. Holsinger, V.H., P.W. Smith, J.L. Smith, and S.A. Palumbo. 1992. Thermal destruction of
Listeria monocytogenes in ice cream mix. J. Food Prot. 55:234-237.
174. Holzapfel, W.H., R. Geisen., and U. Schillinger. 1995. Biological preservation of foods with
reference to protective cultures, bacteriocins and food-grade enzymes. Int. J. Food Microbiol.
24~343-362.
175. Houtsma, P.C., M.L. Kant-Muermans, F.M. Rombouts, and M.H. Zwietering. 1996. Model
for the combined effects of temperature, pH, and sodium lactate on growth rates of Listeria
monocytogenes in broth and bologna-type sausages. Appl. Environ. Microbiol. 62: 1616-
1622.
176. Huang, J., C. Lacroix, H. Daba, and R.E. Simard. 1994. Growth of Listeria monocytogenes
in milk and its control by pediocin 5 produced by Pediococcus acidilactici UL5. Int. J. Food
Microbiol. 4:429-443.
177. Hudson, J.A. 1992. Efficacy of high sodium chloride concentrations for the destruction of
Listeria monocytogenes. Lett. Appl. Microbiol. 14:178- 180.
214 Lou and Yousef
178. Hudson, J.A., S.J. Mott, and N. Penney. 1994. Growth of Listeria monocytogenes, Aeromonas
hydrophila, and Yersinia enterocolitica on vacuum and saturated carbon dioxide controlled
atmosphere-packaged sliced roast beef. J. Food Prot. 57:204-208.
179. Hughey, J.L., and E.A. Johnson. 1987. Antimicrobial activity of lysozyme against bac-
teria involved in food spoilage and food-borne disease. Appl. Environ. Microbiol. 53:2 165-
2170.
180. Hughey, J.L., P.A. Wilger, and E.A. Johnson. 1989. Antibacterial activity of hen egg white
lysozyme against Listeria rnonocytogenes Scott A in foods. Appl. Environ. Microbiol. 55:
63 1-638.
181. Huhtanen, C.N., R.K. Jenkins, and D.W. Thayer. 1989. Gamma radiation sensitivity of Liste-
ria rnonocytogenes. J. Food Prot. 52:610-613.
182. Ikonomov, L., and D. Todorov. 1967. Microbiological studies on the pasteurization of ewes
milk. 111. Resistance of some pathogenic bacteria. Vet. Med. Nauki, Sof. 4:99-108.
183. Isom, L.L., Z.S. Khambatta, J.L. Moluf, D.F. Akers, and S.E. Martin. 1995. Filament forma-
tion of Listeria rnonocytogenes. J. Food Prot. 58: 1031-1033.
184. Ita, P.S., and R. W. Hutkins. 199I . Intracellular pH and survival of Listeria rnonocytogenes
Scott A in tryptic soy broth containing acetic, lactic, citric, and hydrochloric acids. J. Food
Prot. 54: 15- 19.
185. Jack, R.W., J.R. Tagg, and B. Ray. 1995. Bacteriocins of Gram-positive bacteria. Microbiol.
Rev. 59: I7 1-200.
186. Jeong, D.K., and J.F. Frank. 1994. Growth of Listeria rnonocytogenes at 10C in biofilms
with microorganisms isolated from meat and dairy processing environments. J. Food Prot.
571576-586.
187. Johansen, C., L. Gram, and A.S. Meyer. 1994. The combined inhibitory effect of lysozyme
and low pH on growth of Listeria monocytogenes. J. Food Prot. 57561-566.
188. Johnson, J.L., M.P. Doyle, R.G. Cassens, and J.L. Schoeni. 1988. Fate of Listeria monocyto-
genes in tissues of experimentally infected cattle and in hard salami. Appl. Environ. Micro-
biol. 54:497-501.
189. Jorgensen, F., B. Panaretou, P.J. Stephens, and S. Knochel. 1996. Effect of pre-and post-heat
shock temperature on the persistence of thermotolerance and heat shock-induced proteins in
Listeria rnonocytogenes. J. Appl. Bacteriol. 80:216-224.
190. Jorgensen, F., P.J. Stephens, and S. Knochel. 1995. The effect of osmotic shock and subse-
quent adaptation on the thermotolerance and cell morphology of Listeria monocytogenes. J.
Appl. Bacteriol. 79:274-28 1.
191. Junttila, J.R., S.I. Niemela, and J. Him. 1988. Minimum growth temperatures of Listeria
monocytogenes and non-haemolytic Listeria. J. Appl. Bacterial. 65:32 1-327.
192. Kaess, G., and J.F. Weidemann, 1968. Ozone treatment of chilled beef. I. Effect of low
concentrations of ozone on microbial spoilage and surface colour of beef. J. Food Technol.
3:325-334.
193. Kalchayanand, N., M.B. Hanlin, and B. Ray. 1992. Sublethal injury makes Gram-negative
and resistant Gram-positive bacteria sensitive to the bacteriocins, pediocin AcH and nisin.
Lett. Appl. Microbiol. 15:239-243.
194. Kalchayanand, N., T. Sikes, C.P. Dunnne, and B. Ray. 1994. Hydrostatic pressure and elec-
troporation have increased bactericidal efficiency in combination with bacteriocins. Appl.
Environ. Microbiol. 60:4174-4177.
195. Kamau, D.N., S. Doores, and K.M. Pruitt. 1990. Antimicrobial activity of the lactoperoxidase
system against Listeria rnonocytogenes and Staphylococcus aureus. J. Food Prot. 53: I0 10-
1014.
196. Kamau, D.N., S. Doores, and K.M. Pruitt. 1990. Enhanced thermal destruction of Listeria
rnonocytogenes and Staphylococcus uureus by the lactoperoxidase system. Appl. Environ.
Microbiol. 56:27 1 1-27 16.
197. Kato, T., T. Matsuda, Y. Yoneyama, H . Kato, and R. Nakamura. 1993. Isolation of Entero-
Characteristics of Listeria monocytogenes 215
coccus faeciurn with antimicrobial activity and characterization of its bacteriocin. Biosci.,
Biotechnol. Biochem. 5 7 5 5 1-556.
198. Keppler, K., R. Geisen, and W.H. Holzapfel. 1994. An alpha-amylase sensitive bacteriocin
of Lruconostoc carnosurn. Food Microbiol. 1 1:39-45.
199. Kerr, K.G., D. Birkenhead, K. Seale, J. Major, and P.M. Hawkey. 1993. Prevalence of Liste-
ria spp. on the hands of food workers. J. Food Prot. 56525-527.
200. Khan, S.A., S.M. Khalid, and R. Siddiqui. 1993. The effect of pH and temperature on haemol-
ysin production by Listeria species. Lett. Appl. Microbiol. 17: 14- 16.
201. Kihm, D.J., G.J. Leyer, G.-H. An, and E.A. Johnson. 1994. Sensitization of heat-treated
Listeria rnonocytogenes to added lysozyme in milk. Appl. Environ. Microbiol. 60:3854-
3861.
202. Kim. K.-T., E.A. Murano, and D.G. Olson. 1994. Effect of heat shock on production of
listeriolysin 0 by Listeria rnonocytogenes. J. Food Safety 14:273-279.
203. Kim. K.-T., E.A. Murano, and D.G. Olson. 1994. Heating and storage conditions affect the
survival and recovery of Listeria rnonocytogenes in ground pork. J. Food Sci. 59:30-32, 59.
204. Kim. K.Y., and J.F. Frank. 1994. Effect of growth nutrients on attachment of Listeria mono-
cytogenes to stainless steel. J. Food Prot. 57:720-726.
205. Kinderlerer, J.L., and B.M. Lund. 1992. Inhibition of Listeria rnonocytogenes and Listeria
innocua by hexanoic and octanoic acids. Lett. Appl. Microbiol. 14:27 1-274.
206. Klaenhammer, T.R. 1993. Genetics of bacteriocins produced by lactic acid bacteria. FEMS
Microbiol. Rev. 12:39-86.
207. Knabel, S.J., and S.A. Thielen. 1995. Enhanced recovery of severely heat-injured, thermotol-
erant Listeria monocytogenes from USDA and FDA primary enrichment media using a novel,
simple, strictly anaerobic method. J. Food Prot. 58:29-34.
208. Knabel, S.J., H.W. Walker, P.A. Hartman, and A.F. Mendonca. 1990. Effects of growth
temperature and strictly anaerobic recovery on survival of Listeria rnonocytogenes during
pasteurization. Appl. Environ. Microbiol. 56:370-376.
209. KO, li., L.T. Smith, and G.M. Smith. 1994. Glycine betaine confers enhanced osmotolerance
and cryotolerance on Listeriu rnonocytogenes. J. Bacteriol. 176:426-43 I .
210. Kostenbader, K.D., and D.O. Cliver. 1989. Unpublished data.
21 1. Kouassi, Y., and L.A. Shelef. 1995. Listeriolysin 0 secretion by Listeria rnonocytogenes in
broth containing salts of organic acids. J. Food Prot. 58: 1314- 1319.
212. Kouassi, Y., and L.A. Shelef. 1995. Listeriolysin 0 secretion by Listeria rnonocytogenes in
the presence of cysteine and sorbate. Lett. Appl. Microbiol. 20:295-299.
213. Kouassi, Y., and L.A. Shelef. 1996. Metabolic activities of Listeria rnonocytogenes in the
presence of sodium propionate, acetate, lactate and citrate. J. Appl. Bacteriol. 8 1 :147-153.
214. Kovincic, I., I.F. Vujicic, M. Svabic-Vlahovic, M. Vuluc, M. Gagic, and I.V. Wesley. 1991.
Survival of Listeria rnonocytogenes during the manufacture and ripening of Trappist cheese.
J. Food Prot. 54:418-420.
215. Kraemer, K.H., and J. Baumgart. 1993. Sliced frankfurter-type sausage. Inhibiting Listeria
rnonocytogenes by means of a modified atmosphere. Fleischwirtschaft 73: 1279- 1280.
216. Kroll, R.G., and R.A. Patchett. 1992. Induced acid tolerance in Listeria rnonocytogenes. Lett.
Appl. Microbiol. 14:224-227.
217. Krysinski, E.P., L.J. Brown, and T.J. Marchisello. 1992. Effect of cleaners and sanitizers on
Listeria rnonocytogenes attached to product contact surfaces. J. Food Prot. 55:246-25 1.
218. Kukharkova, L.L., P.K. Boyarshinov, V.A. Adutskevich, and P.B. Perova. 1960. Data on
the hygienic judgement of meat in case of listeriosis. Veterinariya 37:74-79.
219. Lang, D.M., E.T. Ryser, and E.H. Marth. 1987. Unpublished data.
220. Lanciotti, R., F. Gardini, M. Sinigaglia, and M.E. Guerzoni. 1996. Effects of growth condi-
tions on the resistance of some pathogenic and spoilage species to high pressure homogeniza-
tion. Lett. Appl. Microbiol. 22: 165- 168.
221. Lanciotti, R., M. Sinigaglia, P. Angelini, and M.E. Guerzoni. 1994. Effects of homogeniza-
216 Lou and Yousef
tion pressure on the survival and growth of some food spoilage and pathogenic microorgan-
isms. Lett. Appl. Microbiol. 18:319-322.
222. Larsen, A.G., and B. Normng. 1993. Inhibition of Listeria monocytogenes by bavaricin A,
a bacteriocin produced by Lactobacillus bavaricus MI401. Lett. Appl. Microbiol. 17:132-
134.
223. Larsen, A.G., F.K. Vogensen, and J. Josephsen. 1993. Antimicrobial activity of lactic acid
bacteria isolated from sour doughs: purification and characterization of bavaricin A, a bacte-
riocin produced by Lactobacillus bavaricus MI401. J. Appl. Bacteriol. 75: 113- 122.
224. Larsen, H.E. 1969. Listeria monocytogenes. Studies on Isolation Techniques and Epidemiol-
ogy. Copenhagen: Car1 Fr. Mortensen.
225. Lee, S.-H., and J.F. Frank. 1990. Effect of growth temperature and media on inactivation of
Listeria monocytogenes by chlorine. J. Food Safety 11:65-7 I .
226. Lee, S.-H., and J.F. Frank. 1991. Inactivation of surface-adherent Listeria monocytogenes.
Hypochlorite and heat. J. Food Prot. 54:4-6.
227. Lemaire, V., 0. Cerf, and A. Audurier. 1988. Heat resistance of Listeria monocytogenes.
Proceedings of Xth International Symposium on Listeriosis. Pecs. Hungary, Aug. 22-26,
Abstr. 54.
228. Leyer, G.J., and E.A. Johnson. 1992. Acid adaptation promotes survival of Salmonella spp.
in cheese. Appl. Environ. Microbiol. 58:2075-2080.
229. Leyer, G.J., L.-H. Wang, and E.A. Johnson. 1995. Acid adaptation of Escherichia coli 0157:
H7 increases survival in acidic foods. Appl. Environ. Microbiol. 61 :3752-3755.
230. Liao, C.-C., A.E. Yousef, E.R. Richter, and G.W. Chism. 1993. Pediococcus acidilactici
PO2 bacteriocin production in whey permeate and inhibition of Listeria monocytogenes in
foods. J. Food Sci. 58:430-434.
231. Liberti, R., G. Fanciosa, M. Gianfranceschi, and P. Aureli. 1996. Effect of combined lyso-
zyme and lipase treatment on the survival of Listeria monocytogenes. Int. J. Food Microbiol.
32:235-242.
232. Lindquist, S. 1986. The heat shock response. Annu. Rev. Biochem. 55:1151-1191.
233. Linton, R.H., M.D. Pierson, and J.R. Bishop. 1990. Increase in heat resistance of Listeria
monocytogenes during pasteurization. J. Food Prot. 53:370-376.
234. Linton, R.H., J.B. Webster, M.D. Pierson, J.R. Bishop, and C.R. Hackney. 1992. The effect
of sublethal heat shock and growth atmosphere on the heat resistance of Listeria monocyto-
genes Scott A. J. Food Prot. 55:84-87.
235. Loewen, P.C., and R. Hengge-Aronis. 1994. The role of the sigma factor 0(KatF) in bacterial
global regulation. Annu. Rev. Microbiol. 48:53-80.
236. Lopes, J.A. 1986. Evaluation of dairy and food plant sanitizers against Salmonella typhimu-
rium and Listeria monocytogenes. J. Dairy Sci. 69:279 1-2796.
237. Lou, Y. 1997. Environmental stress adaptation and stress protection in Listeria monocyto-
genes. PhD. dissertation, Ohio State University, Columbus, OH.
238. Lou, Y., and A.E. Yousef. 1996. Resistance of Listeria monocytogenes to heat after adapta-
tion to environmental stresses. J. Food Prot. 59:465-47 1.
239. Lou, Y., and A.E. Yousef. 1997. Adaptation to sublethal environmental stresses protects
Listeria monocytogenes against lethal preservation factors. Appl. Environ. Microbiol. 63:
1252- 1255.
240. Lovett, J., I.V. Wesley, M.J. Vandermaaten, J.G. Bradshaw, D.W. Francis, R.G. Crawford,
C.W. Donnelly., and J.W. Wesser. 1990. High-temperature short-time pasteurization inacti-
vates Listeria monocytogenes. J. Food Prot. 53:734-738.
241. Luchansky, J.B., K.A. Glass, K.D. Harsono, A.J. Degnan, N.G. Faith, B. Cauvin, G. Baccus-
Taylor, K. Arihara, B. Bater, A.J. Maurer, and R.G. Cassens. 1992. Genomic analysis of
Pediococcus starter cultures used to control Listeria monocytogenes in turkey summer sau-
sage. Appl. Environ. Microbiol. 58:3053-3059.
242. Lyon, W.J., J.K. Sethi, and B.A. Glatz. 1993. Inhibition of psychrotrophic organisms by
Characteristics of Listeria monocytogenes 217
263. Ming, X., and M.A. Daeschel, 1995. Correlation of cellular phospholipid content with nisin
resistance of Listeria monocytogenes Scott A. J. Food Prot. 58:4 16-420.
264. Mohamed, G.E.E., A. Seaman, and A. Woodbine. 1980. Food antibiotic nisin: Comparative
effects on Erysipelothrix and Listeria. In: M. Woodbine, ed. Antimicrobials and Agriculture,
London: Butterworths, pp. 435-442.
265. Moir, C.J., and M.J. Eyles. 1992. Inhibition, injury, and inactivation of four psychrotrophic
foodborne bacteria by the preservatives methyl p-hydroxybenzoate and potassium sorbate.
J. Food Prot. 55:360-366.
266. Monk, J.D., and L.R. Beuchat. 1995. Viability of Listeria monocytogenes, Staphylococcus
aureus and psychrotrophic spoilage micro-organisms in refrigerated ground beef supple-
mented with sucrose esters of fatty acids. Food Microbiol. I2:397-404.
267. Monk, J.D., L.R. Beuchat, and A.K. Hathcox. 1996. Inhibitory effects of sucrose monolaur-
ate, alone and in combination with organic acids, on Listeria monocytogenes and Staphylo-
coccus aureus. J. Appl. Bacteriol. 8 1:7- 18.
268. Morange, M., B. Hevin, and R.M. Fauve. 1993. Differential heat-shock protein synthesis
and response to stress in three avirulent and virulent Listeria species. Res. Immunol. 144:
667-677.
269. Mosteller, T.M., and J.R. Bishop. 1993. Sanitizer efficacy against attached bacteria in a milk
biofilm. J. Food Prot. 56:34-41.
270. Motlagh, A.M., S. Holla, M.C. Johnson, B. Ray, and R.A. Field. 1992. Inhibition of Listeria
spp. in sterile food systems by pediocin AcH, a bacteriocin produced by Pediococcus acidi-
Zactici H. J. Food Prot. 55:337-343.
270a. Motlagh, A.M., A.K. Bhunia, F. Szostek, T.R. Hansen, M.C. Johnson, and B. Ray. 1992.
Nucleotide and amino acid sequence of pap-gene (pediocin AcH production) in Pediococcus
acidilactici H. Lett. Appl. Microbiol. 15:45-48.
271. Murano, E.A., and M.D. Pierson. 1992. Effect of heat shock and growth atmosphere on the
heat resistance of Escherichia coli 0157:H7. J. Food Prot. 55: 17 I - 175.
272. Muriana, P.M. 1996. Bacteriocins for control of Listeria spp. in food. J. Food Prot.
59(Suppl.):54-63.
273. Mustapha, A., and M.B. Liewen. 1989. Destruction of Listeria monocytogenes by sodium
hypochlorite and quaternary ammonium sanitizers. J. Food Prot. 52:306-3 1 1.
274. Nolan, D.A., D.C. Chamblin, and J.A. Troller. 1992. Minimal water activity levels for growth
and survival of Listeria monocytogenes and Listeria innocua. Int. J. Food Microbiol. 16:
323-335.
275. ODriscoll, B., C.G.M. Gahan, and C. Hill. 1996. Adaptive acid tolerance response in Listeria
monocytogenes: isolation of an acid-tolerant mutant which demonstrates increased virulence.
Appl. Environ. Microbiol. 62: 1693- 1698.
276. Oh, D.-H., and D.L. Marshall. 1992. Effect of pH on the minimum inhibitory concentration
of monolaurin against Listeria monocytogenes. J. Food Prot. 55:449-450.
277. Oh, D.-H., and D.L. Marshall. 1993. Influence of temperature, pH and glycerol monolaurate
on growth and survival of Listeria monocytogenes. J. Food Prot. 56:744-749.
278. Oh, D.-H., and D.L. Marshall. 1994. Enhanced inhibition of Listeria monocytogenes by glyc-
erol monolaurate with organic acids. J. Food Sci. 59: 1258- 1261.
279. Oh, D.-H., and D.L. Marshall. 1995. Destruction of Listeria monocytogenes biofilms on stain-
less steel using monolaurin and heat. J. Food Prot. 57:251-255.
280. Oh, D.-H., and D.L. Marshall. 1995. Influence of packaging method, lactic acid and mono-
laurin on Listeria monocytogenes in crawfish tail meat homogenate. Food Microbiol. 12:
159-163.
281. Olson, E.R. 1993. Influence of pH on bacterial gene expression. Mol. Microbiol. 8:5-14.
282. Oscroft, C.A. 1989. Effects of freezing on the survival of Li.steria rnonocytogenes. Technical
Memorandum, Campden Food & Drink Research Association. No. 535.
283. Ozgen, H. 1952. Zur Serologic der Listeria rnonocytogenes. Z. Tropenmed. 4:40-45.
Characteristics of Listeria monocytogenes 219
284. Palumbo, S., and A.C. Williams. 1991. Resistance of Listeria monocytogenes to freezing in
foods. Food Microbiol. 8:63-68.
284a. Palumbo, S., and A.C. Williams. 1990. Effect of temperature, relative humidity, and sus-
pending menstrua on the resistance of Listeria monocytogenes to drying. J. Food Prot. 53:
377-38 1 .
285. Pandit, V.A., and L.A. Shelef. 1994. Sensitivity of Listeria monocytogenes to rosemary
(RoJmarinus ofJlcinulis L.). Food Microbiol. 1 1 57-63.
286. Papageorgiou, D.K., and E.H. Marth. 1989. Fate of Listeria monocytogenes during the manu-
facture and ripening of blue cheese. J. Food Prot. 52:459--465.
287. Papageorgiou, D.K., and E.H. Marth. 1989. Fate of Listeria monocytogenes during the manu-
facture, ripening and storage of Feta cheese. J. Food Prot. 52:82-87.
288. Parish, M.E., and D.P. Higgins. 1989. Survival of Listeria monocytogenes in low pH model
broth systems J. Food Prot. 52: 144- 147.
289. Patchett, R.A., A.F. Kelly, and R.G. Kroll. 1992. Effect of sodium chloride on the intracellu-
lar pools of Listeriu monocytogenes. Appl. Environ. Microbiol. 58:3959-3963.
290. Patchett, R.A., N.Watson, P.S. Fernandez, and R.G. Kroll. 1996. The effect of temperature
and growth rate on the susceptibility of Listeria monocytogenes to environmental stress con-
ditions. Lett. Appl. Microbiol. 22: 12 1 - 124.
291. Patel, J.R., C.-A. Hwang, L.R. Beuchat, M.P. Doyle, and R.E. Brackett. 1995. Comparison
of oxygen scavengers for their ability to enhance resuscitation of heat-injured Listeria mono-
cytogenes. J. Food Prot. 58:244-250.
292. Patterson, M. 1989. Sensitivity of Listeria monocytogenes to irradiation on poultry meat and
in phosphate-buffered saline. Lett. Appl. Microbiol. 8: 181- 184.
293. Patterson, M.F., M. Quinn, R. Simpson, and A. Gilmour. 1995. Sensitivity of vegetative
pathogens to high hydrostatic pressure treatment in phosphate-buffered saline and foods. J.
Food Prot. 58524-529.
294. Payne, K.D., E. Rico-Munoz, and P.M. Davidson. 1989. The antimicrobial activity of pheno-
lic compounds against Listeria monocytogenes and their effectiveness in a model milk sys-
tem. J. Food Prot. 52: 15 1 - 153.
295. Payne, K.D., S.P. Oliver, and P.M. Davidson. 1994. Comparison of EDTA and apolactoferrin
with lysozyme on the growth of foodborne pathogenic and spoilage bacteria. J. Food Prot.
57:62-65.
296. Payne, K.D., P.M. Davidson, S.P. Oliver, and G.L. Christen. 1990. Influence of bovine lacto-
ferriri on the growth of Listeria monocytogenes. J. Food Prot. 53:468-472.
297. Pearson. L.J., and E.H. Marth. 1990. Behavior of Listeria monocytogenes in the presence of
methylxanthines-caffeine and theobromine. J. Food Prot. 53:47-50.
298. Pelroy, G.A., M.E. Peterson, P.J. Holland, and M.W. Eklund. 1994. Inhibition of Listeria
monocytogenes in cold-process (smoked) salmon by sodium lactate. J. Food Prot. 57: 108-
113.
299. Petran, R.L., and K.M.J. Swanson. 1993. Simultaneous growth of Listeriu monocytogenes
and Listeria innocua. J. Food Prot. 56:6 16-6 18.
300. Petran, R.L., and E.A. Zottola. 1989. A study of factors affecting growth and recovery of
Listeria monocytogenes Scott A. J. Food Sci. 54:458-460.
301. Pfeiffer, J., E.T. Ryser, and E.H. Marth. 1988. Unpublished data.
302. Piccinin, D.M., and L.A. Shelef. 1995. Survival of Listeria monocytogenes in cottage cheese.
J. Food Prot. 58:128-131.
303. Polyakov, A.A., and M.A. Baranenkov. 1973. Enzymic activity of listeriae under normal
conditions and after the action of chemical disinfectants. Tr. Vses. Nauchno-Issled. Inst. Vet.
Sanit. 45:235-25 1.
304. Polyakov, A.A., M.A. Baranenkov, and V.P. Andreev. 1972. Structural changes in Listeria
monocytogenes after the action of disinfecting solutions on them. Dokl. Veses. Akad. Se1-
skokhoz. Nauk. 3:32-34.
220 Lou and Yousef
305. Pomanskaya, L.A. 1961. Polymorphism of Listeria. Zh. Microbiol. Epidemiol. Immunobiol.
38: 124- 128.
306. Post, R.C. 1996. Regulatory perspective of the USDA on the use of antimicrobials and inhibi-
tors in foods. J. Food Prot. 59(Suppl.):78-8 1.
307. Potel, J. 1951. The morphology, culture and pathogenicity of C. infantisepticum. Zbl. Bakter-
iol. Parasitol. 156:490-496.
308. Pucci, M.J., E.R. Vedamuthu, B.S. Kunka, and P.A. Vandebergh. 1988. Inhibition of Listeria
monocytogenes by using bacteriocin PA-I produced by Pediococcus acidilactici PAC 1.O.
Appl. Environ. Microbiol. 54:2349-2353.
309. Quintavalla, S., and M. Campanini. 1991. Effect of rising temperature on the heat resistance
of Listeria monocytogenes in meat emulsion. Lett. Appl. Microbiol. 12:184- 187.
310. Quintavalla, S., M. Campanini, and L. Miglioli. 1988. Effect of heating rate on the heat
resistance of Streptococcus faecium. Ind. Conserve 63:252-256.
31 1. Ray, B., and M. Daeschel. 1992. Food Biopreservatives of Microbial Origin. Boca-Raton,
FL: CRC Press.
312. Razavi-Rohani, S.M., and M.W. Griffiths. 1994. The effect of mono and polyglycerol
laurate on spoilage and pathogenic bacteria associated with foods. J. Food Safety 14:131-
151.
313. Razavi-Rohani, S.M., and M.W. Griffiths. 1996. Inhibition of spoilage and pathogenic bacte-
ria associated with foods by combinations of antimicrobial agents. J. Food Safety 16:87-
104.
3 14. Reimer, L., S. Mottice, and D. Andrews. 1988. The effect of pH on survival of Listeria
monocytogenes. Proc. Annual Meeting of the American Society for Microbiology, Miami
Beach, May 8-13, Abstr. C-175.
315. Reiter, B. 1985. Lactoperoxidase system of bovine milk. In: K.M. Pruitt and J.O. Tenovuo,
eds. The Lactoperoxidase System: Chemistry and Biological Significance. Immunology se-
ries no. 27. New York: Marcel Dekker, pp. 123-141.
3 16. Restaino, L., E.W. Frampton, J.B. Hemphill, and P. Palnikar. 1995. Efficacy of ozonated
water against various food-related microorganisms. Appl. Environ. Microbiol. 6 1:347 1-
3475.
317. Rhodehamel, E.J. 1992. FDAs concerns with sous vide processing. Food Technol. 46(12):
73-76.
318. Richards, R.M.E., D.K.L. Xing, and T.P. King. 1995. Activity of p-aminobenzoic acid
compared with other organic acids against selected bacteria. J. Appl. Bacteriol. 78:209-2 15.
319. Rodriguez, J.M., O.J. Sobrino, W.L. Moreira, M.F. Fernandez, L.M. Cintas, P. Casaus, B.
Sanz, and P.E. Hernandez. 1994. Inhibition of Listeria monocytogenes by Luctobacillus sake
strains of meat origin. Meat Sci. 38:17-26.
320. Ronner, A.B., and A.C.L. Wong. 1993. Biofilm development and sanitizer inactivation of
Listeria monocytogenes and Salmonella typhimurium on stainless steel and buna-n rubber.
J. Food Prot. 56:750-758.
321. Rosales, J., M. Verder-Elepano, C.E. Franti, and C. Genigeorgis. 1988. Antimicrobial activity
of selected food plant sanitizers, cleaners and soaps on Listeria species, Salmonella typhimu-
rium, Escherichia coli and Pseudomonas aeruginosa in the presence or absence of organic
matter. Vet. Med. Res. Rep. University of California, Davis, CA.
322. Rosenow, E.M., and E.H. Marth. 1987. Growth of Listeria monocytogenes in skim, whole
and chocolate milk, and in whipping cream during incubation at 4, 8, 13, 21 and 35C. J.
Food Prot. 50:452-459.
323. Rossmoore, K. 1988. The microbial activity of glutaraldehyde in chain conveyor lubricant
formulations. In: D.R. Houghton, R.N. Smith, and H.O.W. Eggins, eds. Proc. 7th Interna-
tional Biodeterioration Symposium, Cambridge, UK: Elsevier, pp. 242-247.
324. Rossmoore, K., and C. Drenzek. 1989. Unpublished data.
325. Rowbury, R.J. 1995. An assessment of environmental factors influencing acid tolerance and
Characteristics of Listeria monocytogenes 221
sensitivity in Escherichia coli, Salmonella spp. and other enterobacteria. Lett. Appl. Micro-
biol. 20:333-337.
326. Roy, B., H. W. Ackermann, S. Pandian, G. Picard, and J. Goulet. 1993. Biological inactiva-
tion of adhering Listeria monocytogenes by listeriaphages and a quaternary ammonium com-
pound. Appl. Environ. Microbiol. 59:2914-2917.
327. Ryser, E.T., and E.H. Marth. 1985. Survival of Listeria monocytogenes during manufacture
and storage of cottage cheese. J. Food Prot. 48:74-750.
328. Ryser, E.T., and E.H. Marth. 1987. Behavior of Listeria monocytogenes during the manufac-
ture and ripening of Cheddar cheese. J. Food Prot. 50:7-13.
329. Ryser, E.T., and E.H. Marth. 1987. Fate of Listeria monocytogenes during manufacture and
ripening of Camembert cheese. J. Food Prot. 50:372-378.
330. Ryser, E.T., and E.H. Marth. 1988. Survival of Listeria monocytogenes in cold-pack cheese
food during refrigerated storage. J. Food Prot. 51 :615-621,625.
331. Ryser, E.T., and E.H. Marth. 1988. Growth of Listeria monocytogenes at different pH values
in uncultured whey or whey cultured with Penicillium camemberti. Can. J. Microbiol. 34:
730--735.
332. Ryser, E.T., and E.H. Marth. 1989. Behavior of Listeria monocytogenes during manufacture
and ripening of brick cheese. J. Dairy Sci. 72:838-853.
333. Ryser, E.T., and E.H. Marth. 1991. Characteristics of Listeria monocytogenes important to
food processors. In: Listeria, Listeriosis and Food Safety. New York: Marcel Dekker, pp. 66-
119.
334. Sasahara, K.C., and E.A. Zottola. 1993. Biofilm formation by Listeria monocytogenes util-
izes a primary colonizing microorganism in flowing systems. J. Food Prot. 56:1022-
1028.
335. Schaack, M.M., and E.H. Marth. 1988. Survival of Listeria monocytogenes in refrigerated
cultured milks and yogurt. J. Food Prot. 51:848-853.
336. Schillinger, U., R. Geisen, and W.H. Holzapfel. 1996. Potential of antagonistic microorgan-
isms and bacteriocins for the biological preservation of foods. Trends Food Sci. Technol. 7:
158- 164.
337. Schlech, W.F., P.M. Lavigne, R.A. Bortolussi, A.C. Allen, E.V. Haldane, A.J. Won, A.W.
Hightower, S.E. Johnson, S.H. King, E.S. Nicholls, and C.V. Broome. 1985. Epidemic listeri-
osis--evidence for transmission by food. N. Engl. J. Med. 303:203-206.
338. Schlyter, J.H., A.L. Degnan, J. Loeffelholz, K.A. Glass, and J.B. Luchansky. 1993. Evalua-
tion of sodium diacetate and ALTATM2341 on viability of Listeria monocytogenes slurries.
J. Food Prot. 56:808-810.
339. Schlyter, J.H., K.A. Glass, J. Loeffelholz, A.J. Degnan, J.B. Luchansky. 1993. The effects
of diacetate with nitrite, lactate, or pediocin on the viability of Listeria monocytogenes in
turkey slurries. Int. J. Food Microbiol. 19:271-281.
340. Seeliger, H.P.R. 1961. Listeriosis. New York: Hafner.
341. Seeliger, H.P.R., and D. Jones. 1986. Listeria. In Bergeys Manual of Systematic Bacteriol-
ogy. Baltimore: Williams and Wilkins, pp. 1235- 1245.
342. Shahamat, M., A. Seaman, and M. Woodbine. 1980. Influence of sodium chloride, pH and
temperature on the inhibitory activity of sodium nitrite on Listeria monocytogenes. In: G.W.
Gould and J.E.L. Corry, eds. Microbial Growth and Survival in Extremes of Environment.
New York: Academic Press, pp. 227-237.
343. Shahamat, M., A. Seaman, and M. Woodbine. 1980. Survival of Listeria monocytogenes in
high salt concentrations. Zbl. Bakteriol. Hyg., I. Abt. Orig. A 246:506-511.
344. Sheldon, B.W., and A.L. Brown. 1986. Efficacy of ozone as a disinfectant for poultry car-
casses and chill water. J. Food Sci. 51:305-309.
345. Shelef, L.A. 1994. Antimicrobial effects of lactates: a review. J. Food Prot. 57:445-450.
346. Shelef, L.A., and L. Addala. 1994. Inhibition of Listeria rnonocytogenes and other bacteria
by sodium diacetate. J. Food Safety 14:103-115.
222 Lou and Yousef
347. Shelef, L.A., and Q. Yang. 1991. Growth suppression of Listeria monocytogenes by lactates
in broth, chicken, and beef. J. Food Prot. 54:283-287.
348. Sielaff, H. von. 1968. Die lebensmittelhygienische Bedeutung der Listeriose. Monatsh. Veter-
inarmed. 21 :750-758.
349. Sikes, A., and S. Whitfield. 1992. Antimicrobial activity of sucrose laurate, EDTA, and BHA
alone and in combination. Proc. of 4th Science Symposium, Natick, US Army Research,
Development and Engineering Center. Natick, MA, pp. 237-250.
350. Siragusa, G.R., and M.G. Johnson. 1989. Inhibition of Listeria monocytogenes by the lactop-
eroxidase-thiocyanate-hydrogen peroxide antibacterial system. Appl. Environ. Microbiol. 55:
2802-2805.
35 I Skovgaard, N. 1987. Listeria: Major sources and routes of human infection-environment and
plants. In: A. Schonberg, ed. Listeriosis-Joint WHO/ROI Consultation on Prevention and
Control, West Berlin, December 10- 12, 1986, Institut fur Veterinarmedizin des Bundesge-
sundheitsamtes, Berlin, pp. 86-97.
352. Smith, L.T. 1996. Role of osmolytes in adaptation of osmotically stressed and chill-stressed
Listeria monocytogenes grown in liquid media and on processed meat surfaces. Appl. Envi-
ron. Microbiol. 62:3088-3093.
353. Smith, J.L., and B.S. Marmer. 199I . Temperature shift effects on injury and death in Listeria
monocytogenes. J. Food Safety 1 1 :73-80.
354. Smith, J.L., B.S. Marmer, and R.C. Benedict. 1991. Influence of growth temperature on
injury and death of Listeria monocytogenes Scott A during a mild heat treatment. J. Food
Prot. 54: 166- 169.
355. Smith, J.L., C. McColgan, and B.S. Marmer. 1991. Growth temperature and action of lyso-
zyme on Listeria monocytogenes. J. Food Sci. 56: I 101, 1 103.
356. Sorqvist, S. 1989. Heat resistance of Campylohacter and Yersinia strains by three methods.
J. Appl. Bacteriol. 67543-549.
357. Sorqvist, S. 1994. Heat resistance of different serovars of Listeria monocytogenes. J. Appl.
Bacteriol. 76:383-388.
358. Sorrells, K.M., and D.C. Enigl. 1990. Effect of pH, acidulant, sodium chloride, and tempera-
ture on the growth of Listeria monocytogenes. J. Food Safety 11:31-37.
359. Sorrells, K.M., D.C. Enigl, and J.R. Hatfield. 1989. Effect of pH, acidulant, time and
temperature on the growth and survival of Listeria monocytogenes. J. Food Prot. 52:571-
573.
360. Sperber, W. 1987. Personal communication.
361. Spurlock, A.T., and E.A. Zottola. 1991 . Growth and attachment of Listeria monocytogenes
to cast iron. J. Food Prot. 54:925-929.
362. Stajner, B., S. Zakula, I. Kovincic, and M. Galic. 1979. Heat resistance of Listeria monocyto-
genes and its survival in raw milk products. Veterinarski Glasnik 33:109-112.
363. Stanfield, J.T., C.R. Wilson, W.H. Andrews, and G.J. Jackson. 1987. Potential role of refrig-
erated milk packaging in the transmission of listeriosis and salmonellosis. J. Food Prot. 50:
730-732.
364. Stenberg, H., and T. Hammainen. 1955. On determination in vitro of the resistance of Listeria
monocytogenes to sodium chloride and heat and on experimental monocytosis in albino mice.
Nord. Vet. Med. 7:853-868.
365. Stephens, P.J., M.B. Cole, and M.V. Jones. 1994. Effect of heating rate on the thermal inacti-
vation of Listeria monocytogenes. J. Appl. Bacteriol. 77:702-708.
366. Stern, J.A., and B.E. Proctor. 1954. A micro-method and apparatus for the multiple determi-
nation of rates of destruction of bacteria and bacterial spores subjected to heat. Food Technol.
8~139-143.
367. Stevens, K.A., B.W. Sheldon, N.A. Klapes, and T.R. Klaenhammer. 1992. Effect of treatment
conditions on nisin inactivation of Gram-negative bacteria. J. Food Prot. 55:763-766.
367a. Stansaovapak, S., and P. Chareonthamawat. 1995. Sensitivity of Listeria monocytogenes to
Characteristics of Lister ia mo n ocytogen es 223
natike Thai spices. Proc. of XIIth International Symposium on Problems of Listeriosis, Perth,
Western Australia, Oct. 2-6, pp. 129- 134.
368. Styles, M.F., D.G. Hoover, and D.F. Farkas. 1991. Response of Listeria monocytogenes and
Vibrio parahaemolyticus to high hydrostatic pressure. J. Food Sci. 56: 1404- 1407.
369. Sumner, S.S., T.M. Sandros, M. Harmon, V.N. Scott, and D.T. Bernard. I99 1. Heat resistance
of Sulmonella typhimurium and Listeria monocytogenes in sucrose solutions of various water
activities. J. Food Sci. 56: 1741- 1743.
370. Tapia de Daza, M.S., Y. Villegas, and A. Martinez. 1991. Minimal water activity for growth
of Listeria rnonocytogenes as affected by solute and temperature. Int. J. Food Microbiol. 14:
333- 337.
371. Tarjan, V. 1988. The sensitivity of Listeria monocytogenes to gamma radiation. Proc. of Xth
International Symposium on Listeriosis, Pecs. Hungary, Aug. 22-26, Abstr. P57.
372. Tassou, C.C., E.H. Drosinos, and G.J.E. Nychas. 1995. Effects of essential oils from mint
(Mentha peperita) on Salmonella enteritidis and Listeria monocytogenes in model food sys-
tems at 4 and 10C. J. Appl. Bacteriol. 78593-600.
373. Tatini, S.R. 1990. Personal communication.
374. Tichnczek, P.S., M.J. Nissen, I.F. Nes, R.F. Vogel, and W.P. Hammes. 1992. Characterization
of the bacteriocins curvicin A from Lactobacillus curvatus LTHll74 and sakacin P from L.
sake LTH673. System. Appl. Microbiol. 15:460-468.
375. Ting, W.T.E., and K.E. Deibel. 1992. Sensitivity of Listeria monocytogenes to spices at two
temperatures. J. Food Safety 12:129- 137.
376. Urbach, H., and G. Schabinski. 1955. Zur Listeriose des Menschen. Z. Hyg. Infektionskr.
141:239-248.
377. Urbain, W.M. 1986. Food Irradiation. New York: Academic Press.
378. Villani, F., 0. Pepe, G. Mauriello, G. Moschetti, L. Sannino, and S. Coppola. 1996. Behav-
iour of Listeria monocytogenes during the traditional manufacture of water-buffalo Mozzar-
ela cheese. Lett. Appl. Microbiol. 22:357-360.
379. Vogel, R.F., B.S. Pohle, P.S. Tichaczek, and W.P. Hammes. 1993. The competitive advantage
of Lactobacillus cumatus LTH 1 174 in sausage fermentations is caused by formation of cur-
vicin A. System. Appl. Microbiol. 16:457-462.
380. Vranchen, Z.E., O.N. Shuvaeva, and Y.I. Andryunin. 1974. Soil decontamination in some
infectious diseases of animals. Tr. Vses. Nauchno-Issled. Inst. Vet. Sanit. 49:226-229.
381. Wakabayashi, H., W. Bellamy, M. Takase, and M. Tomita. 1992. Inactivation of Listeria
monocytogenes by lactoferricin, a potent antimicrobial peptide derived from cows milk. J.
Food Prot. 55:238-240.
382. Walker, S.J., P. Archer, and J.G. Banks. 1990. Growth of Listeria monocytogenes at refrigera-
tion temperatures. J. Appl. Bacteriol. 68: 157-162.
383. Wang, C., and L.A. Shelef. 1991. Factors contributing to antilisterial effects of raw egg
albumin. J. Food Sci. 56: 1251 - 1254.
384. Wang, C., and L.A. Shelef. 1992. Behavior of Listeria monocytogenes and the spoilage mi-
croflora in fresh cod fish treated with lysozyme and EDTA. Food Microbiol. 9:207-213.
385. Wang. L.-L., and E.A. Johnson. 1992. Inhibition of Listeria rnonocytogenes by fatty acids
and nionoglycerides. Appl. Environ. Microbiol. 58:624-629.
386. Wang, L.-L., and E.A. Johnson. 1997. Control of Listeria monocytogenes by monoglycerides
in foods. J. Food Prot. 60: 131 - 138.
387. Wang, L.-L., B.-K. Yang, K.L. Parkin, and E.A. Johnson. 1993. Inhibition of Listeria mono-
cytogcnes by monoacylglycerols synthesized from coconut oil and milkfat by lipase-cata-
lyzed glycerolysis. J. Agric. Food Chem. 4 I :1000- 1005.
388. Watscm, K . 1990. Microbial stress proteins. Adv. Microbial. Physiol. 3 1 :184-223.
389. Weaver, R.A., and L.A. Shelef. 1993. Antilisterial activity of sodium, potassium or calcium
lactate in pork liver sausage. J. Food Safety 13: 133- 146.
390. Wederquist, H.J., J.N. Sofos, and G.R. Schmidt. 1994. Listeria monocytogenes inhibition in
224 Lou and Yousef
refrigerated vacuum packaged turkey bologna by chemical additives. J. Food Sci. 59:498-
500.
391. Welshimer, H.J. 1960. Survival of Listeria monocytogenes in soil. J. Bacteriol. 80:3 16-320.
392. Wendorff, W.L. 1989. Effect of smoke flavorings on Listeria monocytogenes in skinless
franks. Seminar presentation, Department of Food Science, University of Wisconsin-Madi-
son, Jan. 13.
393. WHO Working Group. 1988. Foodborne listeriosis. Bull. WHO 66:421-428.
394. Wilkins, P.O., R. Bourgeois, and R.G.E. Murray. 1972. Psychrotrophic properties of Listeria
monocytogenes. Can. J. Microbiol. 18:543-55 1.
395. Winkowski, K., and T.J. Montville. 1992. Use of meat isolate, Lactobacillus bavaricus MN,
to inhibit Listeria monocytogenes growth in a model meat gravy system. J. Food Safety 13:
19-31.
396. Winkowski, K., A.D. Crandall, and T.J. Montville. 1993. Inhibition of Listeria monocyto-
genes by Lactobacillus bavaricus MN in beef systems at refrigeration temperatures. Appl.
Environ. Microbiol. 59:2552-2557.
397. Wood, L.V., and M. Woodbine. 1979. Low temperature virulence of Listeria monocytogenes
in the avian embryo. Zbl. Bakteriol. Hyg., I. Abt. Orig. A 243:74-81.
398. Yancey, P.H., M.E. Clark, S.C. Hand, R.D. Bowlus, and G.N. Somero. 1982. Living with
water stress: evolution of osmolyte system. Science 217: 1214-1222.
399. Yang, R., and B. Ray. 1994. Prevalence and biological control of bacteriocin-producing
psychrotrophic leuconostocs associated with spoilage of vacuum-packaged processed meats.
J. Food Prot. 57:209-217.
400. Young, K.M., and P.M. Foegeding. 1993. Acetic, lactic and citric acids and pH inhibition
of Listeria monocytogenes Scott A and the effect on intracellular pH. J. Appl. Bacteriol. 74:
5 15-520.
401. Yousef, A.E., and E.H. Marth. 1988. Behavior of Listeria monocytogenes during manufacture
and storage of Colby cheese. J. Food Prot. 51:12-15.
402. Yousef, A.E., and E.H. Marth. 1988. Inactivation of Listeria monocytogenes by ultraviolet
energy. J. Food Sci. 52:57 1-573.
403. Yousef, A.E., and E.H. Marth. 1990. Fate of Listeria rnonocytogenes during the manufacture
and ripening of Parmesan cheese. J. Dairy Sci. 73:335 1-3356.
404. Yousef, A.E., M.A. El-Shenawy, and E.H. Marth. 1989. Inactivation and injury of Listeria
monocytogenes in a minimal medium as affected by benzoic acid and incubation temperature.
J. Food Sci. 54:650-652.
405. Yousef, A.E., R.J. Gajewski 11, and E.H. Marth. 1991. Kinetics of growth and inhibition of
Listeria monocytogenes in the presence of antioxidant food additives. J. Food Sci. 56: 10-
13.
406. Yousef, A.E., J.B. Luchansky, A.J. Degnan, and M.P. Doyle. 1991. Behavior of Listeria
monocytogenes in wiener exudates in the presence of Pediococcus acidilactici H or pediocin
AcH during storage at 4 and 25C. Appl. Environ. Microbiol. 57:1461-1467.
407. Zapico, P., P. Gara, M. Nunez, and M. Medina. 1993. Goats milk lactoperoxidase system
against Listeria monocytogenes. J. Food Prot. 56:988-990.
408. Zhang, S., and J.M. Farber. 1996. The effects of various disinfectants against Listeria mono-
cytogenes on fresh-cut vegetables. Food Microbiol. 13:31 1-32 1.
409. Zottola, E.A., T.L. Yezzi, D.B. Ajao, and R.F. Roberts. 1994. Utilization of Cheddar cheese
containing nisin as an antimicrobial agent in other foods. Int. J. Food Microbiol. 24:227-
238.
Conventional Methods
to Detect and Isolate Listeria
monocytogenes
W. DONNELLV
CATHERINE
University of Vermont, Burlington, Vermont
INTRODUCTION
Listeria monocytogenes is a nonfastidious organism that can be subcultured on most com-
mon bacteriological media (i.e., Tryptose Agar, Nutrient Agar, and Blood Agar); however,
attempted isolation or reisolation of Listeria from inoculated or naturally contaminated
food and clinical specimens by use of nonselective media is often unsuccessful. Difficulties
encountered in isolating L. monocytogenes date back to initial characterization of this
pathogen in 1926 when Murray and his coworkers [ 1061 stated, The isolation of the
infecting organism is not easy and we found this to remain true even after we had estab-
lished the cause of the disease. Although efforts to isolate L. monocytogenes from blood
and cerebrospinal fluid of infected patients have met with considerable success mainly
because of the presence of Listeria in pure culture, obvious difficulties arise when food
and clinical specimens (tissue biopsies and autopsy specimens) contain small populations
of L. monocytogenes in combination with large numbers of other organisms.
Direct plating, cold enrichment, selective enrichment, and several rapid methods all
225
226 Donnelly
COLD ENRICHMENT
Difficulties in isolating L. monocytogenes typically arise when small numbers of Listeria
are present in environmental and clinical food samples containing large numbers of indige-
nous microorganisms. Hence, numbers of Listeria must be increased, relative to that of
the background flora, before the bacterium can be detected. Thirteen years after the first
description of L. monocytogenes by Murray et al. [106], Biester and Schwarte 1131 ob-
served that Listerella (Listeriu) could be frequently isolated from naturally infected sheep
organs that were held refrigerated in 50% glycerol for several months. Although the organ-
ism was only rarely isolated after initial plating of diluted specimens, these authors failed
to comment on the significance of cold storage. Following similar chance observations,
a young graduate student, M. L. Gray, recognized the benefits of low-temperature incuba-
tion for recovering L. monocytogenes from clinical specimens. In 1948, Gray et al. [64]
reported that in three of five bovine listeriosis cases, L. monocytogenes was only isolated
Methods to Detect and lsolate L. monocytogenes 227
after brain tissue diluted in Tryptose Broth, was stored for 5-13 weeks at 4C and then
plated on Tryptose Agar. Although a few Listeria colonies were observed after directly
plating the remaining two brain tissue samples on Tryptose Agar, the bacterium was more
readily isolated following cold enrichment. These results clearly showed the ability of L.
monocytogerzesto multiply to detectable levels in the presence of other microbial contami-
nants during extended storage at 4C.
Gray\ cold enrichment method, in which samples hornogenized in Tryptose Broth
were incubated at 4C and plated weekly or biweekly on Tryptose Agar during 3 months of
storage, was soon adopted as the standard procedure for recovering L. monocytogenes.
Normally only a few weeks of cold enrichment are required before Listeria can be de-
tected; however, in one instance 1621, 6 months of refrigerated storage was necessary
before L. monocytogenes could be isolated from calf brains. Although the cold enrichment
procedure is clearly slow and laborious, this method greatly enhances the likelihood of
isolating Listeria from a variety of specimens, including food.
In 13 studies summarized by Bojsen-Mgller 1171, Listeria was identified in 995
tissue and organ specimens from naturally and experimentally infected domestic animals.
Using both direct plating and cold enrichment procedures, Lipteria was isolated from 684
of 995 (68.7%) specimens, whereas 307 of 995 (30.8%) specimens required cold enrich-
ment before the bacterium could be detected. Furthermore, cold enrichment failed to detect
Listeria in only 4 of 684 (0.6%) samples that were previously positive by direct plating.
A study by Ryser et al. [ I3 I ] stressed the importance of colcl enrichment for recovery of
L. monocytoqenes from cottage cheese manufactured from milk inoculated with this patho-
gen. Using direct plating, L. monocytogenes was recovered from 43 of 1 12 (38.4%) cottage
cheese samples stored at 3C for up to 28 days, whereas cold enrichment of the same
samples in Tryptose Broth for up to 8 weeks yielded Listeriu in 59 of 1 12 (52.7%) samples.
Thus, cold enrichment was necessary to detect this pathogen in 16 of 1 12 ( 14.3%) cheese
samples. Ryser and Marth also found cold enrichment to be of great value in detecting
low levels of L. monocytogenes in Cheddar [ 1321, Camembert [ 1331, and brick cheese
[ 1351 manufactured from pasteurized milk inoculated with the bacterium.
Despite the proven success of cold enrichment, the mechanism by which numbers
of L. monocytogenes are enhanced during prolonged incubation at 4C is not fully under-
stood. Although cold enrichment exploits the psychrotrophic nature of L. monocytogenes
and simultaneously suppresses growth of indigenous nonpsyc hrotrophic organisms, Gray
and Killinger [62] indicated that, at times, growth of Listerig was too rapid to attribute
enhanced growth of this pathogen to mere multiplication. When this procedure was first
described in 1948, Gray et al. [64] suggested possible involvement of an inhibitory factor
in bovine brain tissue that suppressed growth of competing organisms. However, this
theory has been dispelled by subsequent studies which demonstrated enhanced growth of
Listeria during cold enrichment of such diverse samples as mouse liver [ 1441, oat silage
[61], feces [ 1171, sewage [46], cabbage [66], raw milk 11441, and cheese 1131-1351. A
more plausible explanation is that in many clinical specimens, Listeria may exist within
monocytes, rnacrophages, or other phagocytic cells, with colcl storage facilitating release
of the intracellular organism. More recent research on the role of cold-shock proteins,
cold-acclimating proteins, and other mechanisms which enable psychrotrophic growth of
L. monocytogenes may help further explain the preferential growth of Listeria during cold
enrichment [K,8I]. For instance, anteiso-C15 fatty acid reportedly plays a critical role in
adaptation of L. monocytogenes to cold temperatures [4], with mutants deficient in this
fatty acid being shown to be cold sensitive.
228 Donnelly
As previously reviewed by Ryser and Marth [ 1361, over 20 media formulations have
been successfully used to cold enrich a diverse group of samples that were either naturally
or artificially contaminated with L. monocytogenes. Since incubation at 4C is in itself
partially selective for growth of L. monocytogenes, nonselective broths such as Tryptose
Broth and Oxoid Nutrient Broth No. 2 (ONB2) rapidly emerged as media of choice, with
Tryptose Broth generally recognized as being superior. In earlier studies, cold enrichment
was used as the sole enrichment procedure and was followed by plating a portion of
the enriched sample on Tryptose Agar at intervals during 2-12 months [139]. Following
incubation, plates were examined under oblique lighting for typical bluish green, Listeria-
like colonies.
Although growth of L. rnonocytogenes is favored at 4OC, other organisms, including
Proteus, Hafiia, Pseudornonas, enterococci, and certain lactic acid bacteria, also can mul-
tiply in nonselective media at refrigeration temperatures [2], thus making detection of
Listeria more difficult. To prevent overgrowth by non-Listeria organisms, investigators
began adding inhibitory agents to various nonselective cold enrichment broths. In 1972,
Bojsen-MQller [ 171 recognized that supplementing Tryptose Phosphate Broth with poly-
myxin B substantially reduced populations of gram-negative rods (i.e., Escherichia coli,
Pseudomonas aeruginosa, and Proteus spp.) and enterococci while at the same time
allowing rapid growth of L. monocytogenes. Unfortunately, certain species of lactic acid
bacteria resistant to polymyxin B can ferment lactose to lactic acid and reduce the pH to
the point where L. rnonocytogenes fails to grow at 4C. Attempts at maintaining a pH of
7.2 by adding 0.1 M MOPS (3-N-morpholino propane sulfonic acid) to cold-enriched raw
milk samples were unsuccessful [68].
Recovery of L. monocytogenes also is enhanced when cold enrichment is used as
a secondary enrichment preceded by a selective primary enrichment at 30-37C. Ban-
nerman and Bille [7] subjected numerous cheese and cheese factory environmental sam-
ples to secondary cold enrichment in FDA Enrichment Broth (Listeria Enrichment Broth
[LEB]) that were previously incubated at 30C for 48 h (primary warm enrichment). After
plating enrichments on two selective agars, 34 and 62 of 96 isolates were obtained using
warm and cold enrichment, respectively. Thus cold enrichment for 28 days resulted in a
29.2% (28 of 96) increase in recovery of L. monocytogenes from cheese and cheese factory
samples. However, with the advent of improved selective media and methods, most inves-
tigators have concluded that cold enrichment offers no advantages over selective enrich-
ment [70]. In addition, the lengthy incubation period necessary for cold enrichment makes
this procedure impractical for routine regulatory analysis of foods.
the first warm enrichment media for selective isolation of L. rnonocytogenes. Since 1950,
various combinations of selective agents have been added to basal media (i.e., Tryptose
Broth, ONB2, and Tryptose Phosphate Broth) to obtain media suitable for selective enrich-
ment of Listeria at 30-37C. Mavrothalassitis [99] reported an optimum incubation tem-
perature of 30C for enrichment of L. rnonocytogenes from heavily contaminated samples.
Results from at least two additional studies [33,109] also showed that laboratory cultures
of L. rnonocytogenes, L. seeligeri, and/or L. ivanovii were more susceptible to commonly
used Listeria selective agents (i.e., ceftazidime, cefotetan, laxamoxef, and fosfomycin)
when incubated at 37 rather than 30C. Hence, most Listeria enrichments are done at
30C. Ryser and Marth [ 1361 previously reviewed the wide range of media formulations
that have been developed for selective enrichment of L. rnonocytogenes from environmen-
tal and clinical food specimens.
Selective Agents
Modest, nonspecific nutritional requirements of L. rnonocytogenes have led to difficulties
in formulating media that enhance growth of this pathogen. Consequently, efforts have
primarily focused on inhibition of the indigenous bacterial fIora by taking advantage of
the resistance of L. monocytogenes to various selective agents and antibiotics. The major
advances that have contributed to our present-day ability to isolate Listeria from heavily
contaminated environments are shown in Table 1. Although tnany inhibitory agents have
proven to be at least somewhat useful for selective isolation of L. rnonocytogenes from
TABLE
1 Recognition of Selective Agents Useful in Isolation of Listeria
Year Compound Role in selective media References
1950 Potassium tellurite Selective/differential for Liste- 20,63,80,83,89,100,111,122,145
ria, which reduces tellurite to
tellurium, producing black
colonies
1960 Lithium chloride/ Amplification of Listeria in the 38,49,65,66,68,87,92,100,132,
phen ylethanol presence of gram-negative 133,144
bacteria
1966 Nalidixic acid Inhibitory to gram-negative bac- 1,16,42,45,60,77,80,112,113,
teria through interference 124,141
with DNA gyrase
1971 Acriflavin(e)/ Inhibitory to gram-positive 3,15,36,41,42,47,65,75,78,79,
trypaflavin(e) cocci 1 12,113,122,123,125,126,
127
1971 Polyniyxin B Prevents growth of gram- 17,35,38,92,112,127,142
negative rods and strepto-
cocci
1986 Moxalactam Broad spectrum; inhibitory to 74,87,103,112
many gram-positive and
gram-negative contaminants,
including Staphy Lococcus,
Proteus, and Pseudomonas
1988 Ceftazidime Broad-spectrum cephalosporin 7,03,96,109
antibiotic
230 Donnelly
Nalidixic Acid
Beerens and Tahon-Caste1 [ 111 were first to report the usefulness of nalidixic acid in
isolating L. rnonocytogenes from heavily contaminated pathological specimens. Increased
isolation of Listeria using media containing nalidixic acid primarily resulted from inhibi-
tion of indigenous gram-negative bacteria [60]. The benefits of adding nalidixic acid to
otherwise noninhibitory media were soon confirmed in many laboratories [ 16,42,77,80,
1 12,113,1411. After discovering the benefits of adding nalidixic acid to enrichment broth
[ 1 11, Ralovich et al. [ 1241 effectively used serum agar containing nalidixic acid to isolate
L. monocytogenes from feces, organs, and other clinical specimens. Although the micro-
bial background flora was largely inhibited on this medium, streptococci and other nali-
dixic acid-resistant organisms occasionally persisted. Nalidixic acid was eventually recog-
nized as one of the most important selective agents, and it is now used alone or more
commonly in combination with other selective agents for isolating L. monocytogenes from
food and clinical specimens. Farber et al. [45] developed an improved Listeria-selective
plating medium by combining the positive attributes of McBride Listeria Agar and LPM
Agar. In their formula for Farber Listeria Agar, oxolinic acid was substituted for nali-
dixic acid. Both agents function by interfering with the activity of DNA gyrase, an enzyme
needed to maintain proper DNA structure and resealing of chromosomal nicks [60].
Trypaf lavine/Acriflavi ne
Despite successful use of nalidixic acid, Ralovich et al. [125,126] found that growth of
certain gram-positive cocci and gram-negative rods in the presence of this selective agent
complicated the isolation of Listeria. Such difficulties led to inclusion of trypaflavine, a
known inhi bitor of gram-positive cocci, in media containing nalidixic acid. This medium
soon became known as Trypaflavine Nalidixic Acid Serum ,4gar (TNSA). The end result
was the selective inhibition of virtually all other bacteria, whereas growth of L. monocyto-
genes was only slightly decreased [ 15,1111. Following successful use of this medium in
many European studies [ 15,78,112,113,125], Ralovich et al. [ 1221 endorsed TNSA as the
plating medium of choice for isolating L. monocytogenes from contaminated materials.
Additional work revealed that contaminating organisms, predominantly streptococci, grew
infrequently on clear media containing both antibiotics and were generally discernible
from L. rnonocytogenes with the naked eye. In 1972, Seeliger [140] reported that the
combined use of acriflavine and nalidixic acid greatly suppressed gram-negative organisms
and fecal streptococci without apparently affecting recovery of L. rnonocytogenes. These
findings were subsequently confirmed by Bockemuhl et al. [ 161, who reported easy recov-
ery of L. monocytogenes from enriched fecal samples using an agar medium that contained
nalidixic acid and acridine dye. Confirmation of these findings in other European labora-
tories [3,42,47,65,79] led to widespread use of trypaflavinehalidixic acid as Listeria-
selective agents. In 1974, Hofer [75] proposed using a medium prepared from Tryptose
Agar containing nalidixic acid, trypaflavine, and thallous acetate. Trypaflavine can be
replaced by other acridine dyes, including xanthacridine, acriflavine, or proflavinehemi-
sulfate [123]. According to Gregario et al. [65], use of nalidixic acid together with either
acriflavine or trypaflavine gave rise to media that were equally inhibitory to background
microflora, suggesting that similar results can be obtained by substituting acriflavine for
trypaflavine. Based on results from European laboratories [36,41,78,123], a Serum Agar-
or Blood Agar-based medium containing trypaflavine, acriflavine, and nalidixic acid ap-
232 Donnelly
any benefit was gained by adding polymyxin B to media already containing nalidixic acid
and acriflavine. Doyle and Schoeni [38] successfully isolated L. monocytogenes from milk
and clinical and fecal samples after enrichment in a selective broth containing polymyxin
B, acriflavine, and nalidixic acid that resembled Isolation Medium I1 developed by Rodri-
guez et al. [ 1271. Although the selective enrichment broth developed by Doyle and Schoeni
gained some attention [92], the necessity for polymyxin B in this medium remains some-
what questionable. Siragusa and Johnson [ 1421 successfully isolated L. monocytogenes
from yogurt using a medium containing polymyxin B, nalidkic acid, and acriflavine. Their
medium reportedly prevented growth of Streptococcus thermophilus and Lactobacillus
delbrueckii subsp. bulgaricus, and thus making it particularly suitable for isolating L.
monocytogenes from certain fermented dairy products.
M o x a lacta m
Results from antibiotic susceptibility tests [ 1 121 led Lee and McClain [87] to add moxalac-
tam (a broad-spectrum antibiotic which is inhibitory to many gram-positive and gram-
negative bacteria, including Stuphylococcus, Proteus, and Pseudomonas) to MLA con-
taining 0.25% phenylethanol and O S % lithium chloride. The result was a highly selective
medium for recovery of L. monocytogenes from raw beef and many other foods. This
medium, Lithium chloride-Phenylethanol-Moxalactam(LPM) Agar, is recommended by
the USDA-FSIS for isolating L. monocytogenes from raw meat and poultry [ 1031 and also
has been incorporated into the current FDA procedure as a second selective plating me-
dium [74].
Ceftazidi m e
Bannerman and Bille [7] used Columbia Agar Base in combination with acriflavine and
ceftazidime (AC Agar), a broad-spectrum cephalosporin antibiotic, to isolate L. monocyto-
genes from cheese samples. AC Agar was found to be superior to FDA-Modified McBride
Listeria Agar (MMLA) [93,96], recovering approximately 50% more L. monocytogenes
isolates frorn soft cheese and cheese manufacturing environments, than did FDA-MMLA.
Except for ii few enterococci, the combination of acriflavine and ceftazidime inhibited all
other non-Listeria organisms, including yeasts and molds. However, van Netten et al.
[109] reported that PALCAM Agar, which contains polymyxin B and lithium chloride
along with half or less the concentration of acriflavine and ceftazidime found in AC Agar,
was superior to the latter medium. After comparing 13 different plating media, these au-
thors also concluded that media containing both ceftazidime and 1.5% lithium chloride
afforded more selectivity than did phenylethanol alone. However, increased selectivity
results in decreased recovery of stressed or sublethally injured cells that are frequently
present in foods.
lethanol Agar to which lithium chloride, glycine, and sheep blood are added. At least
seven subsequent changes in the original formulation of MLA have led to considerable
confusion as to the exact composition of this medium. Ironically, the first reported modifi-
cation of MLA by Bearns and Girard [9] dates back to 1959, nearly I year before the
original formulation appeared in the literature [ 1001. This medium, named Modified
McBride Medium (MLA2) by the authors and known today as one of several Modified
MLAs, is similar to the original formulation except that sheep blood is omitted and glycine
anhydride is substituted for glycine, with the anhydride form reportedly being less inhibi-
tory to L. rnonocytogenes than glycine [87]. In most instances, MLA2 was more Listeria-
selective than Nalidixic Acid Agar [49,112], Acriflavine Nalidixic Acid Agar [ 1441, or
Acridine Nalidixic Acid Agar [49]. The selectivity of MLA2 can be further improved,
without affecting recovery of Listeria, by increasing the lithium chloride content to 0.5%.
With the addition of sheep blood, this medium became partially differential and inhibitory
to background microflora, and hence it was better suited than MLA2 for recovering L.
monocytogenes from brick [ 1351, feta [ 1 151, and blue cheese [ 1 161, as well as cold-pack
cheese food [ 1341.
An earlier report in which glycine was found partially to inhibit L. monocytogenes
[87] prompted many individuals to prepare the aforementioned forms of MLA with glycine
anhydride, which is far less inhibitory to Listeria. Nevertheless, two widely used formula-
tions of the original MLA containing glycine have been commercially available since
1985 from Difco Laboratories, Detroit, MI and Bethesda Biological Laboratories (BBL)
Cockeysville, MD. Although addition of blood provides one means of identifying possible
L. monocytogenes colonies (virtually all are at least somewhat P-hemolytic) and enhances
growth of the pathogen in certain B vitamin- and/or amino acid-deficient media, many
individuals prefer to omit blood from the various formulations of MLA and examine the
plates under oblique illumination for blue to bluish green Listeria-like colonies. In 1987,
Lovett et al. [96] added cycloheximide to blood-free MLA2 and named this particularly
useful medium FDA-Modified McBride Listeria Agar (FDA-MMLA). Although one ear-
lier study claimed that TNSA was superior to MLA2, subsequent data indicated that FDA-
MMLA [93,94,96] and MLA2 [58,66,68,96,126,144], which contain glycine anhydride,
were the MLA formulations of choice for isolating Listeria spp. from foods, particularly
dairy, vegetable, and seafood products, with the FDA formulation serving for many years as
one of two plating media (the other being LPM agar) in the widely used FDA procedure [95].
LPM Agar
In 1986, Lee and McClain [87] added 4.5 g of lithium chloride and 20 mg of moxalactam to
MLA2 and named their new medium Lithium chloride-Phenylethanol-MoxalactamAgar.
Although this selective medium (commercially available in the United States from BBL
and Difco Laboratories) is particularly well suited for isolating Listeria from raw meat
and poultry, as evidenced by its inclusion as the medium of choice in an earlier version
of the USDA procedure, LPM Agar has since been replaced by Modified Oxford Agar
[27], which produces black L. monocytogenes colonies, each with a black halo following
24 h of incubation.
Oxford/MOX Agar
In 1989, Curtis et al. [34] developed an agar medium that eliminated the need for oblique
illumination. Their medium, Oxford Agar, was prepared from Columbia Agar base to
which a number of selective agents, including colistin sulfate (20 mg/L), fosfomycin (10
Methods to Detect and lsolate L. monocytogenes 235
mg/L), cefotetan (2 mg/L), cycloheximide (400 mg/L), lithium chloride (15 g/L), and
acriflavine (5 mg/L), were added. Esculin and ferric ammonium citrate also were added
as differential agents to produce black Listeria colonies from esculin hydrolysis. This
medium was slightly modified by McClain and Lee by incorporating moxalactam, with
this new medium being designated Modified Oxford Agar (MOX) [27]. In May of 1989,
the USDA-FSIS procedure was changed to incorporate MOX as the recommended plating
medium. Late in 1990, the FDA modified its procedure by replacing FDA-MMLA with
Oxford Agar (OXA). In the present version of the FDA method [74], two selective media
must now be used, either both PALCAM and OXA or OXA and LPM (or LPM plus
esculin and Fe3+).These changes have decreased reliance on the sometimes tedious Henry
illumination technique and have brought U.S. regulatory procedures into closer compli-
ance with international regulatory protocols.
PALCAM Agar
In 1988, van Netten et al. [ 1081 reported that RAPAMY Agar, a modification of TNSA
developed by Ralovich et al. [ 1261 that includes acriflavine, phenylethanol, esculin, manni-
tol, and egg yolk emulsion, was suitable for enumerating Listeria spp. Virtually identical
populations were observed when overnight broth cultures of L. monocytogenes, L. seeli-
geri, and L. ivanovii were surface-plated on RAPAMY and nonselective agar, with growth
of all non- Listeria organisms tested, except Enterococcus fizecalis and Enterococcus fae-
cium, being completely inhibited on the selective medium. Like OXA [34], RAPAMY
Agar also produced distinctive black Listeria colonies that were surrounded by a dense
black halo from esculin hydrolysis. Although such characteristic colonies were present
against a deep red background (inability to utilize mannitol) on RAPAMY Agar, E. fae-
calis and E. faecium generally produced colonies with blue-green halos. Although attempts
to eliminate growth of these two species of enterococci by adding cefoxitin (moxalactam)
to this medium failed, results suggested that RAPAMY Agar could be used to quantify
Listeria spp. in thermally processed and dried foods having total aerobic plate counts of
5 1O6CFU/gand enterococcus counts of 5 1 02CFU/g. However, as might be expected,
high populations of enterococci severely hampered detectioin of Listeria spp. in chicken,
minced meat, and mold-ripened cheese.
Further attempts by van Netten et al. [107] to eliminate growth of enterococci by
adding fosfomycin (20 mg/L) to RAPAMY Agar met with only limited success. Addition
of lithium chloride (1.5%) to RAPAMY Agar inhibited many Listeria spp.; however, an
improved selective and differential medium was obtained by adding lithium chloride to
RAPAMY Agar and omitting nalidixic acid. The resultant medium was named ALPAMY
Agar, because it contains acriflavine, lithium chloride, phenylethanol, esculin, mannitol,
and egg yolk emulsion agar. In a study with pure cultures, ALPAMY Agar allowed unin-
hibited growth of all 10 L. monocytogenes strains tested but completely prevented growth
of single strains of L. seeligeri and L. ivanovii. Selectivity tests showed that ALPAMY
Agar supported growth of only 2 of 41 non-Listeria organisms-one strain each of Staph-
~ZOCOCCUS aureus and Micrococcus spp., both of which were readily differentiated from
Listeria colonies. Subsequent studies indicate that ALPAMY Agar is far superior to
RAPAMY Agar for detecting Listeria in raw milk and soft cheeses manufactured from raw
milk, as well as in raw vegetables and chicken. This medium is the forerunner to PALCAM
agar [ 1091, which contains polymyxin B and lithium chloride along with half or less the
concentration of acriflavine and ceftazidime found in AC A.gar. It is recommended that
PALCAM Agar plates be incubated for 48 h at 30C under microaerobic conditions (5%
236 Donnelly
oxygen, 7.5% carbon dioxide, 7.5% hydrogen, and 80% nitrogen). This medium, along
with L-PALCAMY enrichment broth, is the basis for the Netherlands Government Food
Inspection Service (NGFIS) method for Listeria isolation.
Other Selective Plating Media
Interest in foodborne listeriosis during the 1980s led to development of many additional
Listeria-selective media for examining milk and dairy products. In 1984, Martin et al.
[98] developed Gum Base Nalidixic Acid Medium (GBNA)-a synthetic agar-free solid
medium superior to the MMLA of Bearns and Girard [9] for isolating L. monocytogenes
from raw milk [68]. Bailey et al. [5] also found that a modified version of this medium
containing lithium chloride and moxalactam was suitable for isolating L. monocytogenes
from raw chicken. A selective agar medium [66] based on the enrichment broth of Doyle
and Schoeni [38], from which acriflavine was omitted and Fe'' was added, compared
favorably with the original formulation of MLA [ 1001. Supplementation of selective [66]
and nonselective [30] media with Fe3+enhances growth of L. monocytogenes and may
be beneficial for isolating sublethally injured cells from food samples containing a mixed
microbial flora.
As indicated previously, attempts to isolate L. monocytogenes from food products
have focused on enhancing the selectivity of currently available blood-free plating media
which are normally viewed under oblique illumination, as well as development of alterna-
tive media that incorporate differential agents other than blood to aid microbiologists in
identifying Listeria colonies in mixed cultures. In 1987, Buchanan et al. [20] found the
combination of moxalactam, nalidixic acid, and bacitracin to be effective in allowing
growth of Listeria spp. while preventing growth of most other foodborne organisms, in-
cluding micrococci and streptococci. These selective agents were used to formulate MVJ
on which L. monocytogenes colonies appear entirely black (reduction of tellurite) on a
red background (inability to use mannitol). Thus suspect Listeria colonies could be readily
identified on MVJ without using oblique illumination. Adding the same three selective
agents to the MMLA of Bearns and Girard [9] resulted in Agricultural Research Service
Modified McBride Listeria Agar (ARS-MMLA) which could be used in conjunction with
oblique lighting to quantitate Listeria in a wide range of dairy and meat products. In a
subsequent study, Buchanan et al. [22] found that MVJ was slightly superior to ARS-
MMLA for recovery of L. monocytogenes from inoculated samples of milk, dairy prod-
ucts, meat, and coleslaw. Although ARS-MMLA was more selective than MVJ, the black
Listeria-like colonies that appeared on MVJ were more readily discernible. Initial compari-
sons of ARS-MMLA and MVJ with LPM Agar indicated that both new media functioned
well. In a follow-up study, Buchanan et al. [21] assessed the ability of MVJ and LPM
Agar to detect Listeria in retail samples of raw meat, fish, and shellfish. Listeria popula-
tions were generally too low to be detected by direct plating on either medium. However,
using USDA LEB I in a three-tube/24-h most probable number (MPN) method, compara-
ble isolation rates were obtained for both MVJ and LPM Agar. The differential capability
of MVJ was again extremely useful in selecting presumptive Listeria colonies.
Oblique Illumination
Except for plating media that contain esculin, xylose, mannitol, or other differential agents,
most formulations of Listeria-selective plating media can be classified into one of two
categories based on presence or absence of blood. Recognition of Listeria-like colonies
on blood-free media such as MMLA, TNSA, and GBNA is greatly facilitated when colo-
Methods to Detect and Isolate L. monocytogenes 237
Mirror
nies are observed under oblique illumination with a binocular scanning microscope. Using
the Henry technique [73] in which plates are examined under obliquely transmitted white
light at an angle of 45" (Fig. I), Listeria colonies are small, round, finely textured, bluish
green to bluish gray with an entire margin. In 1984, Martin et al. [98] compared the
appearance of L. rnonocytogenes on Nalidixic Acid Agar and Tryptone Soya Gum Base
Nalidixic Acid Medium and found that the uniformly transparent nature of the gum-base
medium greatly enhanced the bluish green color of Listeria colonies when observed under
oblique illumination, as described by Henry [73]. Noting that the angle of transmission
in the Henry method is 135O, Lachica [84] found that the bluish green hue of Listeria
colonies was more easily observed if plates were viewed from the backside at an angle
of 45" with a 5 X magnification hand lens while colonies were directly illuminated with
a high-intensity beam of light that traveled perpendicular to the bench surface (Fig. 2). This
View
W!Jso
5x Hand Lens
latter method has eliminated many of the problems (i.e., reproducibility and convenience)
associated with the classical technique developed by Henry [73] nearly 60 years ago.
Given enough experience, either of these two lighting techniques can be used easily to
differentiate probable Listeria colonies from background organisms, even on heavily con-
taminated plates. However, these procedures are time consuming and are not readily adapt-
able for routine use in large testing laboratories.
p-Hemo Iysis
Addition of blood to solid media also can be used to differentiate Listeria, including L.
rnonocytogenes, from other microorganisms. When grown on media containing blood,
such as MLA, L. rnonocytogenes colonies are typically surrounded by a narrow zone of
P-hemolysis. In some instances, P-hemolytic activity is so weak that the clearing zone
cannot be observed until the colony is gently removed from the agar surface. In 1989,
Blanco [ 141 proposed overlaying previously inoculated plates of blood-free Listeria selec-
tive agar with a thin layer of blood agar so that the P-hemolytic activity associated with
pathogenic Listeria could be directly observed after reincubation. According to these au-
thors, hemolysis was more readily observed using this procedure than when blood was
incorporated into plating media before incubation. However, further work using highly
contaminated samples such as raw milk showed that the success of this procedure primarily
depended on selectivity of the initial plating medium, with highly selective media yielding
the best results.
Comparative Evaluation of Direct Plating Media for
Recovery of Listeria from Foods
The need for reliable media in routine food analysis precipitated several studies to identify
the most suitable direct plating media. Golden et al. [57], Hao et al. [66], and Cassiday
et al. [28] collectively compared 20 selective plating media for their ability to recover
uninjured cells of L. rnonocytogenes from samples of pasteurized milk, Brie cheese, ice
cream mix, raw cabbage, dry cured/country-cured ham, and/or raw oysters inoculated to
contain approximately 102,104,and 106L. monocytogenes CFU/g or mL. Gum Base Nali-
dixic Acid Tryptose Soya Medium (GBNTSM), MLA2, FDA-MMLA, and Modified Des-
pierres Agar (MDA) were consistently superior to nine other media used by Golden et
al. [57] for enumerating all three inoculum levels of Listeria in samples of pasteurized
milk and ice cream mix. Ability to recover low levels of Listeria from both products was
facilitated by the lack of significant levels of non-Listeria contaminants. Five of 14 plating
media used in this study failed to recover L. rnonocytogenes from inoculated samples of
pasteurized milk as well as Brie cheese and were therefore omitted for analysis of ice
cream and raw cabbage. Examination of Brie cheese containing approximately 102and
104 L. rnonocytogenes CFU/g indicated that none of the nine remaining direct plating
media was sufficiently selective to prevent overgrowth of Listeria by molds, yeasts, and
gram-positive cocci. Despite these inherent difficulties in detecting small numbers of Liste-
ria, Modified Rodriguez Isolation Medium 111 (MRIM III), MLA2, FDA-MMLA, and
MDA were judged to be satisfactory when Brie cheese contained 1 1 0 6Listeria CFU/g.
However, subsequent results from the same laboratory [29] indicate that LPM Agar was
superior to these four media for isolating Listeria rnonocytogenes from Brie cheese. With
raw cabbage, enumeration of Listeria was a problem only at the lowest inoculum level
where large populations of microbial contaminants (i.e., gram-positive and gram-negative
rods as well as gram-positive cocci) typically interfered with recovery. At the two higher
Methods to Detect and Isolate L. monocytogenes 239
MMLA. Bailey et al. [5] found that LPM Agar and GBNA fortified with lithium chloride
and moxalactam were both superior to unfortified GBNA and MLA for recovering L.
monocytogenes as well as other Listeria spp. from naturally contaminated raw poultry.
Incubation Conditions
Most plating media used to isolate Listeria are normally incubated aerobically at 30-37C.
Plates containing popular selective media such as LPM Agar or MOX Agar normally are
incubated for 48 h, whereas plates containing pure or near-pure cultures of Listeria on
nonselective media can generally be examined after 24 h. Since growth of L. monocyto-
genes is reportedly enhanced under conditions of reduced oxygen [ 1391, inoculated plates
[38,107,108,131-133,1351 as well as selective enrichment broths [38] have been incubated
under microaerobic conditions (5% 02:10% CO2:85% N2). These latter conditions are
recommended when using PALCAM Agar.
Enrichment Media
Several food-related listeriosis outbreaks during the 1980s emphasized the need for more
sensitive Listeria detection methods. The logical approach was to use some of the previ-
ously described enrichment broths containing selective agents and to incubate samples at
an elevated temperature, generally 30C. In response to numerous requests from the food
industry, several enrichment schemes have been developed that include one or two selec-
tive enrichments.
An outbreak of listeriosis which was epidemiologically linked to consumption of
pasteurized milk [53] led Hayes et al. [68] to develop a two-stage enrichment procedure
for isolating L. rnonocytogenes from raw milk. Primary cold enrichment in ONB2 followed
by secondary enrichment at 35C in ONB2 containing potassium thiocyanate (KSCN) and
nalidixic acid, and plating on GBNA yielded the highest number of positive milk samples.
No statistically significant difference in recovery of Listeria was observed using either
Stuart Transport Medium or selective enrichment broth containing potassium thiocyanate
and nalidixic acid. Although 15 milk samples were positive when plated on GBNA me-
dium as compared with 11 on MLA2 without blood, the difference was not statistically
significant. The authors concluded that primary cold enrichment in ONB2 followed by
secondary selective enrichment at 35C and plating on GBNA medium were most useful
for identifying positive raw milk samples.
Slade and Collins-Thompson [ 1441 developed a somewhat shorter two-stage enrich-
ment procedure to isolate Listeria from foods. Their method was tested using raw milk
inoculated to contain approximately 100 L. monocytogenes CFU/mL. Results showed that
Tryptose Broth was superior to ONB2 as a primary cold enrichment medium. In addition,
diluting milk samples 1 : 10, rather than 1 :5 , increased the number of Listeria isolations
on selective media. The more dilute samples probably maintained a higher pH ( 1 6 ) during
cold enrichment as a result of fewer lactic acid bacteria and less lactose being present,
which in turn led to faster growth and increased detection of Listeria on solid media.
Original MLA without blood was the only medium tested that proved to be useful for
plating primary cold enrichments, since Tryptose Agar and Trypaflavine Nalidixic Acid
Agar were typically overgrown by competing microflora. Favorable results were, however,
obtained using Tryptose Agar after secondary enrichment at 37C. Addition of acriflavine
to Thiocyanate Nalidixic Acid Broth proved beneficial for recovery of L. monocytogenes.
Thus, following 7-14 days of cold enrichment in Tryptose Broth, L. monocytogenes was
Methods to Detect and Isolate L. monocytogenes 24 1
most frequently isolated after plating samples enriched in Thiocyanate Nalidixic Acid
Broth on either MLA+blood or Tryptose Agar.
A shortened enrichment procedure and a two-stage cold/selective enrichment
procedure were developed in Canada by Farber et al. [44] for isolating Listeria spp. from
raw milk. In the shortened enrichment procedure, milk samples underwent primary and
secondary enrichment at 30C as well as primary cold enrichment in two selective media
(FDA Enrichment Broth and University of Vermont Medium [UVM]). Although no single
step within the procedure was completely satisfactory for isolating Listeria from raw milk,
the two steps that were most helpful involved surface plating the primary FDA Enrichment
Broth culture on MLA2+blood after 1 day of incubation ai: 30C and surface plating the
30-day-old cold enriched FDA Enrichment Broth culture (initially incubated 7 days at
30C) on MLA2+blood. Collectively, these steps detected Listeria spp. in 31 of 51
(60.8%) positive raw milk samples. Although I I isolation:; were made after 1 but not 7
days of primary selective enrichment at 30C, 6 isolations were only possible after 7 days
of primary selective enrichment. Thus, incubating the primary selective enrichment at
30C for 7 days before plating on MLA2+blood markedly enhanced recovery of Listeria
from raw milk.
The two-stage cold/warm enrichment method, which was the second of two proce-
dures developed by Farber et al. [44], also detected Listeria spp. in raw milk samples.
Using this procedure, Listeria spp. were isolated from 12 samples that were negative using
the shortened enrichment procedure. Similarly, 10 samples that were positive for Listeria
spp. using the shortened enrichment procedure were negative with the two-stage cold/
warm enrichment method. Thus, when used alone, neither procedure detected Listeria in
all positive samples. Following cold enrichment, similar numbers of samples were positive
for Listeria spp. after enrichment in FDA Enrichment Broth and UVM. However, eight
raw milk samples were only positive after 2 weeks of cold enrichment as compared with
three samples in which Listeria was only detected after 4 weeks of cold enrichment. These
results are similar to those of Doyle and Schoeni [38], who also observed that Listeria
spp. could be more readily isolated from raw milk and soft, surface-ripened cheese [39]
during the first 2 weeks of cold enrichment.
Food-associated outbreaks of listeriosis along with the discovery of L. monocyto-
genes in many European varieties of soft- and smear-ripened cheese prompted two Swiss
investigators, Bannerman and Bille [7], to develop a two-stage selective/cold enrichment
procedure to recover Listeria spp. from cheese and dairy plant surfaces. Their isolation
method is similar to the shortened enrichment procedure just described [44] with the ex-
ception that the secondary selective enrichment step has been eliminated and AC Agar
has been included as an additional selective plating medium. Using this method, Listeria
spp. were isolated from 157 of 1099 (14.3%) cheese and environmental samples. A total
of 99 samples were positive for Listeria using both plating media. Following selective
enrichment, 56 of 99 (57%) and 35 of 99 (35%) samples were positive after surface-plating
enrichment cultures on AC Agar and FDA-MMLA, respectively. Increased selectivity of
AC Agar was presumably responsible for detection of approximately 50% more Listeria
isolates as compared with FDA-MMLA.
Important information concerning presence of Listeria spp. in food and environmen-
tal samples can be gained using the three procedures just described as well as procedures
developed by Hayes et al. [68] and Slade and Collins-Thompson [ 1441; however, the need
for cold enrichment in these procedures increased the length of analysis to 30-40 days.
Hence, although cold enrichment will likely remain an important research tool, the time
242 Donnelly
constraints of this method negate its use in any isolation procedure that is to be adopted
by the food industry as a standard method.
Rodriguez et al. [129] developed a complicated scheme to isolate Listeria from raw
milk which more importantly paved the way for subsequent development of several widely
used enrichment media, including UVM Enrichment Broth [27,37]. Their protocol in-
cluded three noninhibitory collection (primary enrichment) media, three selective (second-
ary) enrichment media, and one selective plating medium, RIM 111, all of which were
previously described by Rodriguez et al. [ 1271. The three selective enrichment media used
in this protocol contained nalidixic acid and trypan blue with or without polymyxin B,
whereas nalidixic acid and acriflavine were used as selective agents in the plating medium.
Milk was added to all three collection media, with Collection Medium B streaked onto
RIM I11 after 7 and 15 days of storage at 4C. Collection Medium A was incubated at
4C for 24 h, subcultured in all three secondary enrichment media, which were incubated
at 22C until a color change occurred, and then samples were streaked onto plates of RIM
11. A portion of Collection Medium A also was diluted in Collection Medium C, which
was streaked on to RIM I11 following 7 and 15 days at 4C. According to these authors,
11 L. monocytogenes isolates were obtained after primary cold enrichment, with Collection
Medium C accounting for 9 of 11 isolations. Although results for Collection Medium C
appear impressive, the increased number of isolations using this medium may have re-
sulted from a more dilute sample, approximately 1 :40 as compared with approximately
1:8 in Collection Media A and B. Under these conditions, Collection Medium C should
have maintained a higher pH during cold enrichment, since fewer lactic acid bacteria
and less lactose were likely present, thereby enhancing the growth environment for L.
monocytogenes. In contrast to cold enrichment, 49 L. monocytogenes isolates were ob-
tained following secondary enrichment at 22C with 16, 32, and 1 colony originating from
Rodriguez Enrichment Media 1, 2, and 3, respectively. Recovery of only one Listeria
isolate using Rodriguez Enrichment Medium 3 is not surprising considering that Collection
Medium A was diluted approximately 1 :68 in Collection Medium C after only 24 h of
enrichment at 4C. Since transfer of the culture after 24 h of cold enrichment provides
little opportunity for appreciable growth of L. monocytogenes, the organism was likely
diluted out of the sample. Overall, primary cold enrichment of milk samples diluted ap-
proximately 1 :8 followed by secondary enrichment in Rodriguez Enrichment Media 1
and 2 at 22C and plating on an isolation medium containing nalidixic acid and acriflavine
provided the best opportunity for detecting L. monocytogenes in raw milk.
UVM Broth
Selective media originally recommended by the FDA [93,96] and USDA [102,103] for
enrichment of food samples containing L. monocytogenes were modifications of media
proposed by Ralovich et al. [126] and Rodriguez et al. [127] as modified by Donnelly
and Baigent (University of Vermont Medium) [37], respectively. Donnelly and Baigent
explored the use of several selective enrichment media to inhibit growth of raw milk
contaminants and select for L. monocytogenes. The most successful medium for this appli-
cation was a modification of Rodriguez Enrichment Medium III [127]. This medium,
designated LEB, by Donnelly and Baigent [37], consisted of proteose peptone (5.0 g/L),
tryptone (5.0 g/L), Lab-Lemco powder (5.0 g/L), yeast extract (5.0 g/L), sodium chloride
(20.0 g/L), disodium phosphate-2-hydrate ( 12.0 g/L), potassium phosphate monobasic
(1.35 g/L), esculin (1.O g/L), nalidixic acid (40 mg/L), and acriflavine HCl (12 mg/L).
McClain and Lee [ 1021 modified this formula to contain 20 mg/L nalidixic acid, and this
Methods to Detect and lsolate L. monocytogenes 243
formulation was known as USDA LEB I. These authors further modified LEB I to contain
25 mg/L acriflavine and used this medium, LEB 11, for secondary enrichment of meat
and poultry samples. USDA-FSIS currently recommends use of UVM Broth (LEB I) for
primary enrichment of meat and poultry samples [27,76].
Fraser Broth
Fraser Broth [54] is a modification of USDA LEB I1 which contains lithium chloride (3.0
g/L) and ferric ammonium citrate (0.5 g/L). This medium reportedly was advantageous
for detecting Listeria spp. in enriched food samples. Since Listeria will turn Fraser Broth
black from esculin hydrolysis within 48 h of incubation [ 191, this broth has now replaced
USDA LE,B I1 in the USDA protocol as the preferred secondary enrichment medium for
meat and poultry samples [76].
In 1986, Doyle and Schoeni [38] used the microaeraphilic nature of L. monocyto-
genes in developing a shortened one-step enrichment procedure to isolate this organism
from milk as well as fecal and biological specimens. In their protocol, the sample was
placed inside an Erlenmeyer flask equipped with a side arm and then diluted 1 :5 in Doyle
and Schoeni Selective Enrichment Broth (DSSEB). Following 24 h of incubation at 37C
in an atmosphere of 5% 0,:10% CO,: 85% N,, a portion of the sample was streaked onto
plates of MLA (original formulation with blood), which were similarly incubated under
microaerobic conditions. Using DSSEB, L. monocytogenes was consistently isolated from
raw milk samples inoculated to contain 10 L. monocytogenes CFU/mL. In addition, about
two and five times as many L. monocytogenes isolates were recovered from fecal and
biological specimens using DSSEB rather than cold enrichment and direct plating, respec-
tively.
Another enrichment procedure, which is partially based on microaerobic incubation,
was developed by Skovgaard and Morgen [143] to isolate Listeria spp. from heavily con-
taminated samples, including feces, silage, minced meat, and poultry. In this two-step
enrichment procedure, microaerobic incubation (24 h/30C/95% air: 5% CO,) of the sam-
ple in USIIA LEB I is followed by aerobic secondary selective enrichment in USDA LEB
11, after which untreated and KOH-treated samples are surface plated on LPM Agar. Using
this isolation scheme, which, with the exception of microaerobic incubation, closely re-
sembles the original USDA procedure, numerous fecal, silage, minced beef, and poultry
samples were positive for Listeria spp., including L. monocytogenes. Based on these re-
sults, the authors concluded that their method was suitable for detecting Listeria in heavily
contaminated materials, including samples of raw ground beef and poultry. Although both
procedures just described decrease the Listeria detection time to approximately 3 days,
incubating enrichment cultures under microaerobic conditions is particularly awkward and
not feasible for large-scale testing programs.
A large listeriosis outbreak in which coleslaw was implicated as the vehicle of infec-
tion prompted Hao et al. [66] to compare various media and methods to detect L. monocy-
togenes in cabbage. Preliminary results clearly demonstrated a need for some type of
enrichment procedure before L. monocytogenes could be isolated from inoculated samples.
After comparing results from various plating and enrichment media, these investigators
proposed a two-step enrichment procedure for isolating L. ,monocytogenes from cabbage.
A cold enrichment period of 14 or 30 days at 5C in ONB2 or Brain Heart Infusion Broth
(BHI) led to increased recovery of Listeria from cabbage following secondary enrichment
(30C/48 h) in FDA Enrichment Broth or ONB2 containing potassium thiocyanate and
nalidixic acid. A comparison of nine selective plating media, both with and without an
244 Donnelly
FDA Method
The FDA method, originally developed by Lovett et al. 33,961, is the most frequently
used procedure in the United States for detecting L. monocytogenes in milk, milk products
(particularly ice cream and cheese), seafood, vegetables, and food processing environ-
ments. The original protocol [93] has been modified as shown in Figure 3 [74]. The enrich-
ment medium (LEB M52 [74]) consists of TSBYE supplemented with monopotassium
phosphate (anhydrous) 1.35 g/L; disodium phosphate (anhydrous) 9.6 g/L; and pyruvic
acid (sodium salt 10% w/v aqueous solution) 11.1 ml/L. A 25-mL liquid or 25-g solid
sample is added to 225 mL of LEB without selective agents, mixed, and incubated at
30C for 4 h. Following addition of selective agents (acriflavine HCl 10 mg/L; nalidixic
acid sodium salt 40 mg/L; cycloheximide 50 mg/L), the sample is incubated an additional
44 h at 30C for a total incubation period of 48 h. LEB was modified by increasing its
buffering capacity, thereby positioning this medium to be used in conjunction with DNA
probe and other rapid methods which are less sensitive than conventional cultural methods.
In a further modification for nondairy foods, the acriflavine concentration was reduced
from 15 to 10 mg/L so as to conform to that used for milk; and dairy products. After 24
and 48 h, LEB cultures are streaked onto OXA [34] and LPM [87] Agar prepared either
with or without esculin/Fe3+.PALCAM [ 1091 agar may be used in place of LPM agar. This
I 1
Add 25g or 25 ml sample
to 225 ml LEB
I 1
Stomach or blend
6 Incubate 4h at 30C
II 20h U 44h
30C 30C
Streak to Streak to
F a n d LPM I
I I
L
Examine for Listeria-like
colonies
FIGURE3 FDA procedure for isolating I_. monocytogenesfrom foods. (From Ref. 74.)
246 Donnelly
substitution brings the FDA method in closer alliance with other protocols used outside the
United States and decreases reliance on Henrys oblique illumination technique. OXA and
PALCAM plates are incubated (with optional use of a C02-air atmosphere) at 35C for
24-48 h, with LPM plates being incubated at 30C for 24-48 h. LPM plates can be viewed
using Henry illumination, or alternatively esculin and ferric iron salt may be added to
LPM to obtain Listeria colonies with black halos as also appear on OXA and PALCAM.
It is recommended that five or more typical colonies be picked from OXA and
PALCAM or LPM and transferred to TSAYE for confirmation. The selection of five colo-
nies increases the likelihood that multiple species of Listeria, if present, will be identified.
TSAYE plates are incubated at 30C for 24-48 h or 35C if colonies are not being used
for wet mount motility confirmation. Purified isolates are subjected to a series of standard
biochemical tests, with a total of 10-1 1 days being required to isolate and confirm the
presence of Listeria in food samples via the FDA procedure.
Present versions of the FDA procedure have greatly shortened and simplified the
isolation of Listeria spp. from many foods as compared with earlier methods that were
developed to detect the pathogen in clinical specimens, with revised procedures affording
many improvements over the original FDA protocol. In 1987, Doyle and Schoeni [39]
compared the original FDA classic cold enrichment and shortened enrichment procedures
for their ability to recover L. rnonocytogenes from 90 samples of commercially produced,
soft, surface-ripened cheese that was previously identified as likely to contain L. rnonocyto-
genes. Although L. rnonocytogenes was isolated from 41 of 90 (46%) cheeses, no single
procedure detected the pathogen in all positive samples. A total of 21 samples were posi-
tive after cold enrichment as compared with only 16 and 13 samples that were positive
using the FDA and shortened enrichment procedures, respectively. Thus, the latter two
protocols failed to recover L. rnonocytogenes from 5 of 21 (23.8%) and 8 of 21 (38.1%)
samples that were positive following cold enrichment. Furthermore, since Listeria was
never isolated from the same positive sample by all three protocols, it appears that the
original FDA method was inferior to cold enrichment. Similar results were obtained by
Doyle et al. [40] when these same three enrichment procedures were used to isolate L.
rnonocytogenes from raw milk samples after HTST pasteurization. Researchers in Canada
[45] and England [ 1181 found negligible differences between numbers of Listeria recov-
ered from naturally contaminated samples of raw milk and soft cheeses analyzed by the
FDA and cold enrichment procedures, although both methods again failed to detect Liste-
ria in all positive samples. These variable findings for the original FDA and cold enrich-
ment procedures have been attributed to nonuniform distribution of Listeria within sam-
ples. However, Doyle and Schoeni [39,40] found cold enrichment superior to the FDA
method for analysis of soft, surface-ripened cheese, where nonuniform distribution of
Listeria is expected, as well as in pasteurized milk. Hence, variations in the ability of the
FDA and cold enrichment procedures to detect Listeria in dairy products probably result
from inherent differences between the two methods (media, incubation conditions) and/
or the presence of microbial competitors rather than nonuniform distribution of Listeria
in the product. Although these results indicate that cold enrichment was generally superior
to the original FDA protocol, the time-consuming nature of cold enrichment makes this
procedure unacceptable as a commercial screening method for L. rnonocytogenes.
International Dairy Federation Method
Using the original FDA method as a starting point, the IDF initiated development of a
reference method in 1988 [146] to recover L. rnonocytogenes from dairy products.
Methods to Detect and lsolate L. monocytogenes 24 7
Development of the IDF method essentially followed that of the FDA protocol as previ-
ously reviewed by Ryser and Marth [ 1361 with the eventual elimination of both preenrich-
ment for detecting sublethally injured Lister-ier and the KOH treatment of the enrichment
broths before plating on Listc.r-icr-selective media. The present IDF method [4a] received
AOAC approval in I993 based on results from an AOAC collaborative study [ 1474 which
assessed the ability of this method to recover L. nzorzoc~toSeizL.sfrom inoculated samples
of raw milk. ice cream, Camembert cheese. Limburger cheese, and skim milk powder.
The AOAC-approved IDF method (Fig. 4) closely resembles the FDA protocol (see
Fig. 3) with the sample enriched i n IDF enrichment broth which contains the same concen-
trations of selective agents found in LEB. Following 48 h of incubation at 30C enrich-
ments are plated on Oxford Agar as opposed to the FDA procedure which calls for Oxford
Agar and either LPM without esculin/Fe or PALCAM. This method which requires a
minimum of 4 days to obtain presumptive results continues to be popular among Europe-
ans for detecting Lister-icr in dairy products.
USDA-FSIS Method
The USDA-FSIS devised a method for detecting L. i,zonoc~itoserie.sin meat and poultry
products (Fig. 5 ) 1761. The original USDA protocol developed in 1986 by Lee and McClain
[87,103] differs from both the original and revised FDA procedures in that both primary
and secondary enrichment steps are included for detecting Listrr-ici. The original USDA
procedure enabled Lister-iir detection within 3 days compared with 9- 1 1 or 5-6 days using
the original and revised FDA methods, respectively. The original USDA procedure was
revised in May of 1989 [27] and differs from the original method in that (a) LEB I1 has
been replaced by Fraser Broth [ 541 as the secondary enrichment medium; (b) LPM Agar
has been replaced by MOX; and ( c ) the regulatory sample size has been increased to 25
g. Fraser Broth and Modified Oxford Agar will both blacken during incubation, because
Lister-io spp. and other contaminants can hydrolyze esculin, with colonies of Listerici ex-
hibiting black halos on Modified Oxford Agar following 24-48 h of incubation. However.
FIGURE4 IDF procedure for isolating L. rnonocytogenes from milk and dairy prod-
ucts. (From Ref. 4a.)
248 Donnelly
U Incubate at 30C
for 20, 2411
0 1 ml + I0 ml Fraser Broth 1
U 26 f 2h
35C
I Streak to MOX
U 35C
24, 4811 U 48h
35C
FIGURE5 USDA procedure for isolating L. monocytogenes from meat and poultry
products. (From Ref. 76.)
MOX is more selective than LPM or Oxford Agar [34], with staphylococci and strepto-
cocci both generally unable to grow on MOX.
Reported inadequacies in the prior [27] USDA procedure were related to the use of
Fraser broth for secondary enrichment. False-negative results caused by reliance on Fraser
broth darkening and a 24-h secondary enrichment have been reported by several labora-
tories [6,82]. Kornacki et al. 1821 compared recovery of L. monocytogenes from Fraser
broth incubated for 26 versus 48 h. L. monocytogenes was isolated from 60 of 1088 meat
product and environmental swab samples from meat and dairy plants. False-negative rates
as high as 6.7% were attributed to the inability of L. monocytogenes to be detected in
Fraser broth at 26 h but not at 48 hours, and to the failure of Fraser broth to blacken.
Furthermore, investigators failed to detect L. monocytogenes in eight Fraser broth enrich-
ments that were positive by primary enrichment. These findings clearly stress the impor-
tance of incubating Fraser broth enrichments for 48 h.
The USDA-FSIS has therefore recommended several modifications. All Fraser broth
enrichment cultures should be streaked following 24-26 h of incubation regardless of
color. Once cultures have been streaked to MOX, Fraser broth cultures should be reincu-
bated at 35C for an additional 24 h. MOX plates streaked from 24- to 26-h Fraser broth
enrichment cultures should be examined for the presence of Listeria-like colonies. If pres-
ent, isolation should proceed. If absent, a second MOX plate should be streaked from the
48-hr Fraser broth enrichment culture. Ferron and Michard [48] compared the FDA and
Methods to Detect and Isolate L. monocytogenes 249
USDA enrichment procedures using 300 pastry samples supplied by 100 different suppli-
ers in western France. The USDA procedure was deemed superior, detecting 69% of all
positive samples compared with the FDA procedure which detected only 34%.
Netherlands Government Food Inspection Service
Using the Netherlands Government Food Inspection Service (NGFIS) protocol, food sam-
ples are enriched in L-PALCAMY enrichment broth for 48 h at 30C. After 24 and 48
h, 0.1 mL of L-PALCAMY enrichment broth is plated onto PALCAM Agar. Plates are
incubated at 30C for 48 h under microaerophilic conditions (5% oxygen, 7.5% carbon
dioxide, 7.5% hydrogen, and 80% nitrogen) [ 1091, after which presumptive Listeria colo-
nies are black and surrounded by a dense black hole from txulin hydrolysis.
Lund et al. [97] examined 300 raw milk samples for the presence of Listeria using
three primary enrichment media. A total of 84 positive sarnples were identified by one
or more of these media. PALCAMY was the most effective medium, identifying 50 of
84 positive samples, followed by UVM and LEB, which identified 46 and 42 Listeria-
positive samples, respectively. Given that the best of these primary enrichment broths
identified only 50 of 84 (59.5%) Listeria-positive samples, the use of two or more primary
enrichment broths identified an additional 34 samples and increased the overall incidence
of Listeria by almost 41%. These results once again highlight the inadequacy of relying
on a single primary enrichment broth for Listeria detection.
Noah et al. [ 1 101 evaluated the impact of more than one test procedure on recovery
of Listeria species from naturally contaminated seafood and seafood products. A total of
21 1 samples were evaluated using five different protocols. The FDA procedure [95] was
used as a control against which the efficacy of the other procedures was evaluated. A total
of 60 samples were identified as Listeria-positive by at least one of the procedures. Of
these samples, the FDA procedure missed seven samples which were subsequently found
to harbor Lzsteria via other procedures. The overall incidence of Listeria increased I 1.7%
using more than one testing procedure.
Hayes et al. [69] assessed the USDA-FSIS and cold enrichment procedures for re-
covery of 1,. monocytogenes from suspect food samples. Both procedures identified L.
monocytogenes in 28 of 51 positive samples. The USDA-FSIS procedure identified 21
samples missed by cold enrichment, whereas the cold enrichment procedure identified an
additional 2 samples that the USDA-FSIS procedure missed. Three enrichment methods
were also compared by Hayes et al. [70] during an examination of foods obtained from
the refrigerators of patients with active clinical cases of listeriosis. A total of 2229 food
samples were examined in this study, of which 11% were positive for L. monocytogenes.
Overall, the USDA-FSIS [27], FDA [95], and NGFIS [ 1091 methods were not statistically
different in their ability to isolate Listeria from 899 samples included in the comparative
evaluation. The FDA procedure [95] identified 65% of all L. monocytogenes-positive
foods, whereas the USDA-FSIS and NGFIS procedures detected L. monocytogenes in
74% of foods shown to be positive. Although none of these widely used Listeria detection
methods proved to be highly sensitive when used independently, use of any two methods
improved detectability from 65 to 74% (for individual protoco'ls)to 87-9 1% for combined
protocols.
selective enrichment procedures will not generally recover sublethally injured Listeria
which could exist in various heated, frozen, or acidified foods; or within heated, frozen,
and sanitized areas of food processing facilities. Sublethal injury of Listeria as a result
of heating, freezing, drying, irradiation, or exposure to chemicals (i.e., sanitizers, preserva-
tives, acids) is well documented [ 1,12,24,25,26,28,31,33,56,58,59,101,130,132,138].Un-
der ideal conditions, such injury is reversible with Listeria being capable of repairing
sublethal damage in foods. Repair of heat-injured L. monocytogenes has been reported in
whole and 2% milk stored at 4C [105].
Several investigators have attempted to improve the sensitivity of current detection
systems by focusing on recovery of injured Listeria that may be present in food products
and food processing environments. All current detection procedures, with the exception
of cold enrichment, involve selective enrichment and/or selective plating. Cold enrichment
is not feasible for routine testing, since several months of incubation may be necessary
to obtain positive results. By failing to consider recovery of injured Listeria, current meth-
odologies underestimate the true incidence of this organism. Several previous studies have
reported on the ability of commonly used plating media to recover injured Listeria. Among
the most commonly used selective agents examined, phenylethanol, acriflavine, poly-
myxin, and sodium chloride were found to inhibit recovery of both thermally stressed and
nonstressed Listeria [31,86,145,148,149,151].Furthermore, when examined for ability to
recover quantitatively thermally stressed Listeria, LEB agar, modified McBride's Agar
(MMA), LPM Agar [87], and FDA enrichment broth agar showed significantly impaired
recovery [221.
Warburton et al. [ 1501 examined the ability of the modified FDA and USDA meth-
ods to recover stressed cells and low levels of L. monocytogenes in food and environmental
samples. Although the modified FDA and USDA methods were comparable in their abili-
ties to isolate stressed and low level populations of L. monocytogenes, these authors failed
to assess the extent of injury within bacterial populations following exposure to sublethal
stress. The percentage of injury existing within a population of bacterial cells can pro-
foundly affect comparative results of media performance. Thus, it is difficult to determine
whether valid conclusions can be drawn from such studies.
Busch and Donnelly [26] developed an enrichment medium capable of resuscitating
heat-injured Listeria. This medium, Listeria Repair Broth (LRB), permits complete repair
of injured Listeria within 5 h at 37C after which various selective agents can be added
to inhibit the growth of competing microflora upon continued incubation. In studies com-
paring the efficacy of LRB in promoting repair/enrichment of heat-injured Listeria with
that of existing selective enrichment media, repair was not observed in FDA enrichment
broth [95],phosphate-buffered Listeria Enrichment Broth (PEB; Gene-Trak Systems, Fra-
mingham, MA), or UVM Enrichment Broth [ 1031. Final Listeria populations in selective
enrichment media after 24 h of incubation at 30C were 1.7 X 108to 9.1 X 10' CFU/
mL compared with populations in LRB which consistently averaged 2.5 X loll to 8.2 X
10'' CFU/mL [26].
Studies with LRB were extended to examine the potential for repair of freeze-injured
and sanitizer-injured L. monocytogenes [50,138]. Although variation in susceptibility of L.
monocytogenes to freeze injury was recorded, in general, L. monocytogenes is not severely
injured by freezing [43,58,114]. Percentage of injury ranged from only 40 to 60% after
Listeria populations were frozen at -9" to - 11"C for 24 h [50].As storage time increased,
an increase in percentage of injury increased to a maximum of only 70-80%. To examine
reversibility of freeze injury, low-level populations of freeze-injured L. monocytogenes
Methods to Detect and lsolafe L. monocytogenes 251
cells were added to UVM Enrichment Broth, FDA Enrichment Broth, and LRB. Repair
of freeze-injured populations occurred quickly, probably because of the low initial degree
of injury, with the pathogen again attaining high populations in LRB.
Sallam and Donnelly [ 1381 examined the ability of four commonly used dairy plant
sanitizers tc) induce injury in L. monocytogenes when exposed to sublethal concentrations.
UVM broth failed to support growth of sanitizer-injured cells, whereas LRB permitted
their recovery. Flanders et al. [52] examined the efficacy of using a repair step to increase
recovery of injured Listeria from environmental sponge samples obtained from dairy pro-
cessing plant environments. The USDA-FSIS Listeria isolation protocol using UVM mod-
ified Listeria Enrichment Broth was compared with a modified USDA-FSIS format which
utilized LRB as the primary enrichment medium. UVM and LRB broths also were used in
conjunction with a rapid DNA hybridization (Gene-Trak) and ELISA (Organon Teknika,
Durham, NC) assay. Of 80 sites positive by any method, UVM and LRB showed similar
recovery rates (87.5 and 88.8%, respectively). However, combining the cultural methods
with either rapid method for each broth increased detection to 97.5-98.8% [52]. Flanders
et al. [51] also evaluate the abilities of LRB, LRB containing ceftazidime (LRBC), and
UVM to enhance recovery of Listeria from dairy plant environmental samples. Although
no single broth could detect all Listeria-positive sites, LRBC identified 67 of 89 positive
sites (75.3%), and LRB and UVM each detected 60 of 90 positive sites (66.7%). Combin-
ing results from any two broths increased recovery from 66.7 to 75.3% to 82.2-94.4%.
The combination of LRBC and UVM detected 94.4% of positive samples, whereas LRBS
and LRBC identified 91.1% of positive samples. Pritchard et al. [121] also compared the
ability of UVM, LRB, and LRBC to isolate Listeria from dairy plant environments. Of
80 positive samples identified, 54 samples came from UVM medium, 56 were from LRB,
and 57 came from LRBC. A total of 26 samples (32.5% of positive samples) were identi-
fied by either LRB or LRBC but not by UVM media. Combining UVM with either LRB
or LRBC again substantially increased the number of positive samples identified. When
results from UVM and LRB are combined, 65 to 80 (8 1.3%) positive samples were identi-
fied. Using both UVM and LRBC, 74 of 80 (92.5%) positive samples were identified.
Despite the improved recoveries obtained by combining medja, these results illustrate the
severe limitations associated with the current regulatory procedures used to assure absence
of Listeria in foods and food processing environments.
Ryser et al. [ 1371 evaluated the ability of UVM and LRB to recover different strain-
specific ribotypes of L. monocytugenes from meat and poultry products. Forty-five paired
25-g retail samples of ground beef, pork sausage, ground turkey, and chicken underwent
primary enrichment in UVM and LRB (30C/24 h) followed by secondary enrichment in
Fraser Broth (35"C/24 h) and plating on modified Oxford Agar. A 3-h nonselective enrich-
ment period at 30C was used with LRB to allow repair of injured Listeria before adding
selective agents. Listeria spp. were detected in 73.8% and 69.4% of the 180 meat and
poultry samples tested using LRB and UVM, respectively. Although these differences
were not statistically significant, combining UVM and LRB results increased overall Liste-
ria recovery rates to 83.3%. Thus, enrichment in LRB for repair of injured cells in conjunc-
tion with the USDA-FSIS method has potential to improve recovery of Listeria from meat
and poultry products.
In the above study, following 24 h of incubation at 35"C, Listeria colonies were
biochemically confirmed and selected isolates were ribotyped using the automated Ribo-
printer Microbial Characterization System, (E.I. du Pont de Nemours and Co., Inc., Wil-
mington, DE). A total of 36 different Listeria strains comprising 16 L. monocytogenes
252 Donnelly
TABLE
2 Ribotypes of Listeria spp. Recovered from 10 Samples of Raw Chicken
Following Primary Enrichment in UVM or LRB and Secondary Enrichment
in Fraser Broth
No. of isolates
alone. These findings, combined with reports of L. innocm being able to outgrow L.
monocytogenes in UVM (and Fraser Broth) [32,119] suggest that different ribotypes of
L. monocytogenes may vary somewhat in nutritional requirements or their ability to com-
pete with other ribotypes of L. monocytogenes and/or other Listeria spp. Refinement of
existing Listeria recovery methods should consider the nutritional needs associated with
those specific genetic types widely distributed in foods.
Roth and Donnelly [ 1301 assessed survival of acid-injured L. rnonocytogenes in four
different acidic foods and also examined the efficacy of LRB and UVM to recover acid-
injured Lisreria from such foods. L. monocytogenes was injured in lactic (pH 3.0) and
acetic (pH 3.5) acids. Two levels of injury were produced anld monitored; one population
with 99.999b injury and the second with approximately 95% injury. The four acidic food
systems studied at 4 and 30C included fresh apple cider (pH 3.3), plain non-fat yogurt
(pH 4.2), fresh coleslaw (pH 4.4), and fresh salsa (pH 3.9). Acid-injured Listeria was
added to each acidic food and monitored by selective and nonselective plating. Simulta-
neously, sainples were enriched in both LRB and UVM followed by standard isolation/
identification procedures with survival of healthy L. monncytogenes also monitored. Al-
though acid-injured cells failed to repair in the acidic foods tested, the pathogen did survive
for more than a week. Storage temperatures did affect the survival rate of acid-injured cells
in that 4C storage was bacteriostatic and 30C was bacteriocidal. Parameters involved in
survival of acid-injured Listeria include the degree to which the bacterial population is
injured (percentage of injury), storage temperature, and the pH of the food. At time points
where differences were detected, LRB proved to be superior (22 of 54) in its ability to
detect injured Listeriu compared with UVM (3/54). Hence, use of LRB is recommended
when examining acidic foods for L. monocytogenes.
REFERENCES
1. Ahamad, N., and E.H. Marth. 1990. Acid injury in Listeria monocytogenes. J. Food Prot.
53126-29.
2. Albritlon, W.L., G.L. Williams, W.E. DeWitt, and J.C. Feeley. 1980. Listeria monocyto-
genes. In: E.H. Lennette, A. Balows, W.J. Hausler, Jr., and J.1. Truant, eds. Manual of Clini-
cal Microbiology. 3d ed. American Society for Microbiology, Washington, DC, pp. 139-
142.
3. Al-Ghazali, M.R., and S.K. Al-Azawi. 1988. Effects of sewage treatment on the removal of
Listeriu monocytogenes. J. Appl. Bacteriol. 65:203-208.
4. Annous, B.A., L.A. Becker, D.O. Bayles, D.P. Labed, and B.J. Wilkinson. 1997. Critical
role of anteiso-C,, fatty acid in the growth of Listeria monocytogenes at low temperatures.
Appl. Environ. Microbiol. 63:3887-3894.
4a. Association of Official Analytical Chemists. 1996. 17.10.0 1 .AOAC official method 993.12.
Listeriu monocytogenes in milk and dairy products. In: Official Methods of Analysis of the
Association of Official Analytical Chemists, AOAC International, Gaithersburg, MD.
5. Bailey. J.S., D.L. Fletcher, and N.A. Cox. 1989. Recovery and serotype distribution of Liste-
riu monocytogenes from broiler chickens in the southeastern lJnited States. J. Food Prot. 52:
148- 150.
6. Bailey. J.S., and N.A. Cox. 1992. Universal preenrichment broth for the simultaneous detec-
tion of Salmonellu and Listeria in foods. J. Food Prot. 55:256-259.
7. Bannerman, E.S., and J. Bille. 1988. A selective medium for isolating Listeria spp. from
heavily contaminated material. Appl. Environ. Microbiol. 54: 165- 167.
8. Bayles, D.O., B.A. Annous, and B.J. Wilkinson. 1996. Cold stress proteins induced in
254 Donnelly
Food Safety and Inspection Service, Laboratory Communication No. 57, Revised May 24.
USDA, Washington, DC.
28. Cassiday, P.K., R.E. Brackett, and L.R. Beuchat. 1989. Evaluation of ten selective direct
plating media for enumeration of Listeria monocytogenes in ham and oysters. Food Micro-
biol. 55: 113- 125.
29. Cassiday, P.K., R.E. Brackett, and L.R. Beuchat. 1989. Evaluation of three newly developed
direct plating media to enumerate Listeria monocytogenes in foods. Appl. Environ. Micro-
biol. 55: 1645-1648.
30. Cowart, R.E., and B.G. Foster. 1985. Differential effects of iron on the growth of Listeria
monocytogenes: minimum requirements and mechanism of acquisition. J. Infect. Dis. 151:
721 -730.
31. Crawford, R.G., C.M. Beliveau, J.T. Peeler, C.W. Donnelly. and V.K. Bunning. 1989. Com-
parative recovery of uninjured and heat-injured Listeria monocytogenes cells from bovine
milk. Appl. Environ. Microbiol. 55: 1490- 1494.
32. Curiale, M.S., and C. Lewus. 1994. Detection of Listeria rnonocytogenes in samples con-
taining Listeria innocua. J. Food Prot. 57: 1048- 1051.
33. Curtis, G.D.W., W.W. Nichols, and T.J. Falla. 1989. Selective agents for Listeria can inhibit
their growth. Lett. Appl. Microbiol. 8: 169- 172.
34. Curtis, G.D.W., R.G. Mitchell, A.F. King, and E.J. Griffen. 1989. A selective differential
medium for the isolation of Listeria rnonocytogenes. Lett. Appl. Microbiol, 8:95-98.
35. Despierres, M. 1971. Isolement de Listeria monocytogenes dans un milieu d6favorable a
Streptococcus faecalis. Ann. Inst. Pasteur 121:493-501.
36. Dijkstra, R.G. 1976. Listeria-encephalitis in cows through litter from a broiler-farm. Zen-
tralbl. Bakteriol. Hyg. I Abt. Orig. B 161:383-385.
37. Donnelly, C.W., and G.J. Baigent. 1986. Method for flow cytometric detection of Listeria
monocytogenes in milk. Appl. Environ. Microbiol. 52:689--695.
38. Doyle, M.P., and J.L. Schoeni. 1986. Selective-enrichment procedure for isolation of Listeria
monocytogenes from fecal and biologic specimens. Appl. Environ. Microbiol. 5 1 :1 127- 1129.
39. Doyle, M.P., and J.L. Schoeni. 1987. Comparison of procedures for isolating Listeria mono-
cytogenes in soft, surface-ripened cheese. J. Food Prot. 50:4-6.
40. Doyle, M.P., K.A. Glass, J.T. Beery, G.A. Garcia, D.J. Pollard, and R.D. Schultz. 1987.
Survival of Listeria monocytogenes in milk during high-temperature, short-time pasteuriza-
tion. Appl. Environ. Microbiol. 53: 1433-1438.
41. Durst, J., and G. Berencsi. 1976. Contributions to further serovariants of L. monocytogenes.
Zentralbl. Bakteriol. Hyg. I Abt. Orig. A 236531-532.
42. Elischerova, K., and S. Stupalova. 1972. Listeriosis in professionally exposed persons. Acta
Microbiol. Hung. 19:379-384.
43. El-Kest, S.E., and E.H. Marth. 1992. Freezing of Listeria monocytogenes and other micro-
organisms: a review. J. Food Prot. 55:639-648.
44. Farber, J.M., G.W. Sanders, and S.A. Malcolm. 1988. The presence of Listeria spp. in raw
milk in Ontario. Can. J. Microbiol. 34:95-100.
45. Farber, J.M., G.W. Sanders, and J.I. Speirs. 1988. Methodology for isolation of Listeria from
foods-a Canadian perspective. J. Assoc. Off. Anal. Chem. 7 1:675-678.
46. Fenlon, D.R. 1985. Wild birds and silage as reservoirs of Listeria in the agricultural environ-
ment. J. Appl. Bacteriol. 59537-543.
47. Fenlon, D.R. 1986. Rapid quantitative assessment of the distribution of Listeria in silage
implicated in a suspected outbreak of listeriosis in calves. Vet. Rec. 118:240-242.
48. Ferron, P., and J. Michard. 1993. Distribution of Listeria spp. in confectioners pastries from
Western France: comparison of enrichment methods. Int. J. Food Microbiol. 18:289-303.
49. Filice, G.A., H.F. Cantrell, A.B. Smith, P.S. Hayes, J.C. Feeley, and D.W. Fraser. 1978.
Listeria monocytogenes infection in neonates: Investigation of an epidemic. J. Infect. Dis.
138:17-23.
256 Donnelly
50. Flanders, K.J. 1991. Injury, resuscitation and detection of Listeria spp. from frozen environ-
ments. M.S. thesis, University of Vermont, Burlington, VT.
51. Flanders, K.J., C.M. Beliveau, T.J. Pritchard, and C.W. Donnelly. 1994. Enhanced recovery
of Listeria from dairy plant environments using modified selective enrichment media. IFT
Annual Meeting Technical Program: Book of Abstr. 59C, p. 166.
52. Flanders, K.J., T.J. Pritchard, and C.W. Donnelly. 1995. Enhanced recovery of Listeria from
dairy plant processing environments through combined use of repair, enrichment and selec-
tive enrichment/detection procedures. J. Food Prot. 58:404-409.
53. Fleming, D.W., S.L. Cochi, K.L. MacDonald, J. Brondum, P.S. Hayes, B.D. Plikaytis, M.B.
Holmes, A. Audurier, C.V. Broome, and A.L. Reingold. 1985. Pasteurized milk as a vehicle
of infection in an outbreak of listeriosis. N. Engl. J. Med. 312:404-407.
54. Fraser, J.A., and W.H. Sperber. 1988. Rapid detection of Listeria spp. in food and environ-
mental samples by esculin hydrolysis. J. Food Prot. 5 1 :762-765.
55. Fuzi, M., and 1. Pillis. 1961. Selektive Ziichtung von L. monocytogenes. Vortrag 3, Kongress
Ung. Mikrobiol. Gesellsch., Budapest.
56. Garayzabal, J.F., and C. Genigeorgis. 1990. Quantitative evaluation of three selective enrich-
ment broths and agars used in recovering Listeria microorganisms. J. Food Prot. 53: 105-
110.
57. Golden, D.A., L.R. Beuchat, and R.E. Brackett. 1988. Direct plating technique for enumera-
tion of Listeria monocytogenes in foods. J. Assoc. Off. Anal. Chem. 71 :647-650.
58. Golden, D.A., L.R. Beuchat, and R.E. Brackett. 1988. Evaluation of selective direct plating
media for their suitability to recover uninjured, heat-injured, and freeze-injured Listeria
monocytogenes from foods. Appl. Environ. Microbiol. 54: 1451- 1456.
59. Golden, D.A., L.R. Beuchat, and R.E. Brackett. 1988. Inactivation and injury of Listeria
monocytogenes as affected by heating and freezing. Food Microbiol. 5: 17-23.
60. Goldstein, E.J.C. 1988. Structure of the fluoroquinolone group of antibacterials-introduction.
Suppl. Urol. 32:4-8.
61. Gray, M.L. 1960. Isolation of Listeria monocytogenes from oat silage. Science 132:1767-
1768.
62. Gray, M.L., and A.H. Killinger. 1966. Listeria monocytogenes and listeric infections. Bacte-
riol. Rev. 30:309-382.
63. Gray, M.L., H.J. Stafseth, and F. Thorp, Jr. 1950. The use of potassium tellurite, sodium
azide, and acetic acid in a selective medium for the isolation of Listeria monocytogenes. J.
Bacteriol. 59:443-444.
64. Gray, M.L., H.J. Stafseth, F. Thorp, Jr., L.B. Sholl, and W.F. Riley, Jr. 1948. A new technique
for isolating listerellae from the bovine brain. J. Bacteriol. 55:47 1-476.
65. Gregorio, S.B., W.C. Eveland, and H.F. Maassab. 1986. Efficiency of various solid media
for the isolation of Listeria monocytogenes. Proc. Annual Meeting for American Society of
Microbiology, New Orleans, LA, Abstr. p. 27.
66. Hao, D.Y.-Y., L.R. Beuchat, and R.E. Brackett. 1989. Comparison of media and methods for
detecting and enumerating Listeria monocytogenes in refrigerated cabbage. Appl. Environ.
Microbiol. 53:955-957.
67. Hartmann, V., K. Friedrich, F. Beyer, and G. Terplan. 1988. Verbesserung des Listeriennach-
weises durch einen Moxalactam-enthaltenden Nahrboden. Deutsche Molkerei-Zeitung 38:
1164-1 166.
68. Hayes, P.S., J.C. Feeley, L.M. Graves, G.W. Ajello, and D.W. Fleming. 1986. Isolation of
Listeria monocytogenes from raw milk. Appl. Environ. Microbiol. 5 1 :438-440.
69. Hayes, P.S., L.M. Graves, G.W. Ajello, B. Swaminathan, R.E. Weaver, J.D. Wenger, A.
Schuchat, C.V. Broome, and the Listeria study group. 1991. Comparison of cold enrichment
and the U.S. Department of Agriculture methods for isolating Listeria monocytogenes from
naturally contaminated foods. Appl. Environ. Microbiol. 57:2 109-2 I 13.
70. Hayes, P.S., L.M. Graves, B. Swaminathan, G.W. Ajello, G.B. Malcolm, R.E. Weaver, R.
Methods to Detect and lsolate L. monocytogenes 257
Ransom, K. Deaver, B.D. Plikaytis, A. Schuchat, J.D. Wenger, R.W. Pinner, C.V. Broome,
and the Listeria Study Group. 1992. Comparison of three selective enrichment methods for
the isolation of Listeria monocytogenes from naturally contaminated foods. J. Food Prot. 55:
952-959.
71. Heisick, J.E., D.E. Wagner, M.L. Nierman, and J.T. Peeler. 1989. Listeria spp. found on
fresh market produce. Appl. Environ. Microbiol. 55:1925- 1927.
72. Heisick, J.E., F.M. Harrell, E.H. Peterson, S. McLaughlin, D.E. Wagner, I.V. Wesley, and
J. Bryner. 1989. Comparison of four procedures to detect Listeria spp. in foods. J. Food Prot.
52: 154- 157.
73. Henry, B.S. 1933. Dissociation of the genus Brucella. J . Infect. Dis. 52:374-402.
74. Hitchins, A.D. 1995. Listeria monocytogenes. In: Food and Drug Administration Bacterio-
logical Analytical Manual. ed. AOAC International, Gaithersburg, MD, pp. 10.01-
10.13.
75. Hofer, E. 1974. Study of the occurrence of L. monocytogmes in human feces. Rev. Soc.
Bras. Med. Trop. 8: 109- I 16.
76. Johnson, J.L. 1998. Isolation and identification of Listeria monocytogenes from meat, poultry
and egg products. In: USDA-FSIS Microbiology Laboratory Guidebook. 3rded. Vol. 1.
77. Kampelmacher, E.H., and L.M. van Noorle Jansen. 1969. Isolation of Listeria monocytogenes
from feces of clinically healthy humans and animals. Zentralbl. Bakteriol. I Abt. Orig. A
21 1 :153-359.
78. Kampelmacher, E.H., and L.M. van Noorle Jansen. 1972. Further studies on the isolation of
Lister-ia monocytogenes in clinically healthy individuals. 2,entralbl. Bakteriol. I Abt. Orig.
A 221 :70-77.
79. Kampelmacher, E.H., D.E. Maas, and L.M. van Noorle Jansen. 1972. Isolierung von L. mono-
cytogrnes mittels Nalidixinsauretrypaflavin. Zentralbl. Bakteriol. Parasit. Abt. 1 Orig. A 22 1 :
139- 140.
80. Khan, M.A., A. Seaman, and M. Woodbine. 1972. Differential media for the isolation of
Listeria rnonocytogenes. Acta Microbiol. Acad. Sci. Hung. 19:37 1-372.
81. KO, R., L.T. Smith, and G.M. Smith. 1994. Glycine betaine confers enhanced osmotolerance
and cryotolerance on Listeriu monocytogenes. J. Bacteriol. 176:426-43 1.
82. Kornacki, J.L., D.J. Evanson, W. Reid, K. Rowe, and R.S. Flowers. 1993. Evaluation of the
USDA Protocol for detection of Listeria monocytogenes. J Food Prot. 56:44 1-443.
83. Kramer, P.A., and D. Jones. 1969. Media selective for Listeria monocytogenes. J. Appl.
Bacteriol. 32:38 1-394.
84. Lachica, R.V. 1989. Modified Henry technique for the initial recognition of Listeria colonies.
Annual Meeting, Society of Industrial Microbiologists, Seattle, WA, August 13- 18, Abstr.
P-44.
85. Lammerding, A.M., and M.P. Doyle. 1989. Evaluation of enrichment procedures for recovery
of Listeria monocytogenes from dairy products. Proc. Annual Meeting of Institute of Food
Technology, Chicago, June 25-29, Abstr. 460.
86. Leasor, S.B., C.A. Abbas, and R. Firstenberg-Eden. 1990. Evaluation of UVM as a growth
medium for Listeria monocytogenes. Proc. Annual Meeting of American Society of Microbi-
ologists, Anaheim, CA, May 13- 19, Abstr. P-39.
87. Lee, W.H., and D. McClain. 1986. Improved Listeria monocytogenes selective agar. Appl.
Environ. Microbiol. 52: 12 15- 1217.
88. Lehnert, C. 1964. Bakteriologische, serologische und tierexperimentelle Untersuchungen zur
Pathogenese, Epizootologie und Prophylaxe der Listeriose. Arch. Exp. Vet. Med. 18:98 I -
1027,1247- 1301.
89. Leighton, I. 1979. Use of selective agents for the isolation of Listeria rnonocytogenes. Med.
Lab. Sci. 36:283-288.
90. Loessner, M.J., R.H. Bell, J.M. Jay, and L.A. Shelef. 1988. Comparison of seven plating
media for enumeration of Listeria spp. Appl. Environ. Microbiol. 54:3003-3007.
258 Donnelly
91. Loessner, M.J., M. Rudolf, and S. Scherer. 1997. Evaluation of a luciferase reporter bacterio-
phage A5 11::luxAB for detection of Listeria monocytogenes in contaminated foods. Appl.
Environ. Microbiol. 63:2961-2965.
92. Lovett, J. 1988. Isolation and enumeration of Listeria monocytogenes. Food Technol. 42(4):
172- 175.
93. Lovett, J. 1988. Isolation and identification of Listeria monocytogenes in dairy products. J.
Assoc. Off. Anal. Chem. 7 1:658-660.
94. Lovett, J., and A.D. Hitchins. 1988. Listeria isolation; Revised method of analysis. Fed.
Register 53:44 148-44 153.
95. Lovett, J., and A.D. Hitchins. 1989. Listeria isolation. Chapter 29. In: FDA Bacteriological
Analytical Manual. 6th ed. Supplement, September 1987, Association of Official Analytical
Chemists, Arlington, VA, p. 29.01.
96. Lovett, J., D.W. Francis, and J.M. Hunt. 1987. Listeria monocytogenes in raw milk: detection,
incidence, and pathogenicity. J. Food Prot. 50: 188- 192.
97. Lund, A.M., E.A. Zottola, and D.J. Pusch. 1991. Comparison of methods for the isolation
of Listeria from raw milk. J. Food Prot. 54:602-606.
98. Martin, R.S., R.K. Sumarah, and M.A. MacDonald. 1984. A synthetic based medium for the
isolation of Listeria monocytogenes. Clin. Invest. Med. 7:233-237.
99. Mavrothalassitis, P. 1977. A method for rapid isolation of Listeria monocytogenes from in-
fected material. J. Appl. Bacteriol. 43:47-52.
100. McBride, M.E., and K.F. Girard. 1960. A selective method for the isolation of Listeria mono-
cytogenes from mixed bacterial populations. J. Lab. Clin. Med. 55: 153-157.
101. McCarthy, S.A., M.L. Motes, and R.M. McPhearson. 1990. Recovery of heat-stressed Liste-
ria monocytogenes from experimentally and naturally contaminated shrimp. J. Food Prot.
53~22-25.
102. McClain, D., and W.H. Lee. 1987. A method to recover Listeria monocytogenes from meats.
Proc. Annual Meeting, American Society of Microbiology, Atlanta, May 1-6, Abstr. P-23.
103. McClain, D., and W.H. Lee. 1988. Development of a USDA-FSIS method for isolation of
Listeria monocytogenes from raw meat and poultry. J. Assoc. Off. Anal. Chem. 71:660-
664.
104. McLauchlin, J., A. Audurier, and A.G. Taylor. 1986. Aspects of epidemiology of human
Listeria rnonocytogenes infections in Britain: 1967- 1984; the use of serotyping and phage
typing. J. Med. Microbiol. 22:367-377.
105. Meyer, D.H., and C.W. Donnelly. 1992. Effect of incubation temperature on repair of heat-
injured Listeria in milk. J. Food Prot. 55579-582.
106. Murray, E.G.D., R.A. Webb, and M.B.R. Swann. 1926. A disease of rabbits characterized
by a large mononuclear leucocytosis, caused by a hitherto undescribed bacillus, Bacterium
monocytogenes (n.sp.). J. Pathol. Bacteriol. 29:407 -439.
107. Netten, P. Van, I. Perales, and D.A.A. Mossel. 1988. An improved selective and diagnostic
medium for isolation and counting of Listeria spp. in heavily contaminated foods. Lett. Appl.
Microbiol. 7:17-21.
108. Netten, P. Van, A. Van de Ven, I. Perales, and D.A.A. Mossel. 1988. A selective and diagnos-
tic medium for use in the enumeration of Listeria spp. in foods. Int. J. Food Microbiol. 6:
187-198.
109. Netten, P. Van, I. Perales, A. Van de Moosdijk, G.D.W. Curtis, and D.A.A. Mossel. 1989.
Liquid and solid selective differential media for the detection and enumeration of L. monocy-
togenes and other Listeria spp. Int. J. Food Microbiol. 8:299-316.
110. Noah, C.W., J.C. Perez, N.C. Ramos, C.R. McKee, and M.V. Gibson. 1991. Detection of
Listeria species in naturally contaminated seafood using four enrichment procedures. J. Food
Prot. 54:174-177.
111. Olson, C., Jr., L.A. Dunn, and C.L. Rollins. 1953. Methods for isolation of Listeria monocyto-
genes from sheep. Amer. J. Vet. Res. 14:82-85.
Methods to Detect and lsolate L. monocytogenes 259
112. Ortel, S. 197I . Ausscheidung von Listeria rnonocytogenes irn Stuhl gesunder Personen. Zen-
tralbl. Bakteriol. 1 Abt. Orig. 217:41-46.
113. Ortel, S. 1972. Experience with nalidixic acid-trypaflavine agar. Acta Microbiol. Acad. Sci.
Hung. 19:363-365.
114. Palumbo, S.A., and A.C. Williams. 1991. Resistance of Listeria rnonocytogenes to freezing
in foods. Food Microbiol. 8:63-68.
115. Papageorgiou, D.K., and E.H. Marth. 1989. Fate of Listeria monocytogenes during the manu-
facture, ripening and storage of Feta cheese. J. Food Prot. 52:82-87.
116. Papageorgiou, D.K., and E.H. Marth. 1989. Fate of Listeria rnonocytogenes during the manu-
facture and ripening of blue cheese. J. Food Prot. 52:459-465.
117. Patterson, M. 1989. Sensitivity of Listeria rnonocytogenes to irradiation on poultry meat and
in phosphate-buffered saline. Lett. Appl. Microbiol. 8: 181-- 184.
118. Pini, P.N., and R.J. Gilbert. 1988. A comparison of two procedures for the isolation of Listeria
monocytogenes from raw chickens and soft cheeses. Int. J. Food Microbiol. 7:33 1-337.
119. Petran, R.I., and K.M.J. Swanson. 1993. Simultaneous growth of Listeria rnonocytogenes
and Listeria innocua. J. Food Prot. 56:616-618.
120. Pritchard, T.J., and C.W. Donnelly. 1995. Combined secondary enrichment of UVM and
LRB primary enrichment broths increases the sensitivity of Listeria detection. IFT Annual
Meeting: Book of Abstracts. Abstr. 34-2, p. 96.
121. Pritchard, T.J., K.J. Flanders, and C.W. Donnelly. 1995. Comparison of the incidence of
Listeria on equipment versus environmental sites within daisy processing plants. Int. J. Food
Microbiol. 26:375-384.
122. Ralovich, B.S. 1975. Selective and enrichment media to isolate Listeria. In: M. Woodbine,
ed. Problems of Listeriosis. Proceedings of the Sixth International Symposium. Leicester
University Press, Leicester, UK, pp. 286-294.
123. Ralovich, B. 1984. Listeriosis Research-Present Situation and Perspective. Budapest: Aka-
demiai Kiado.
124. Ralovich, B., A. Forray, E. Mero, and H. Malovics. 1970. Additional data on diagnosis and
epidemiology of Listeria infections. Zentralbl. Bakteriol. 1 Abt. Orig. 214:23 1-235.
125. Ralovich, B., L. Emody, I. Malovics, E. Mero, and A. Forray. 1972. Methods to isolate
Listeria rnonocytogenes from different materials. Acta Microbiol. Acad. Sci. Hung. 19:367-
369.
126. Ralovich, B., A. Forray, E. Mero, H. Malovics, and I. Szazados. 1971. New selective medium
for isolation of L. rnonocytogenes. Zentralbl. Bakteriol. 1 Abt. Orig. 216:88-91.
127. Rodriguez, D.L., G.S. Fernandez, J.F.F. Garayzabal, and E.F.. Ferri. 1984. New methodology
for the isolation of Listeria microorganisms from heavily contaminated environments. Appl.
Environ. Microbiol. 47: 1 188- 1 190.
128. Rodriguez, D.L., J.F. Fernandez, V. Briones, J.L. Blanco, and G. Suarez. 1988. Assessment of
different selective agar media for enumeration and isolation of Listeria from dairy products. J.
Dairy Res. 55579-583.
129. Rodriguez, D.L., J.F.F. Garayzabel, J.A.V. Boland, E.R. Ferri, and G.S. Fernandez. 1985.
Isolation de microorganisms de listeria a partir de lait cru destine a le consommation humaine.
Can. J. Microbiol. 3 1 :938-941.
130. Roth. T.T., and C.W. Donnelly. 1995. Injury of Listeria rnonocytogenes by acetic and lactic
acids: mechanisms of repair and sites of sublethal damage. IFT Annual Meeting, Book of
Abstracts. Abstr. 81D-I, p. 246.
131. Ryser, ET., E.H. Marth, and M.P. Doyle. 1985. Survival of Listeria rnonocytogenes during
manufacture and storage of cottage cheese. J. Food Prot. 50:7-13.
132. Ryser, E.T., and E.H. Marth. 1987. Behavior of Listeria rnonocytogenes during the manufac-
ture and ripening of Cheddar cheese. J. Food Prot. 50:7-13.
133. Ryser, E.T., and E.H. Marth. 1987. Fate of Listeria monocytogenes during manufacturing
and ripening of Camembert cheese. J. Food Prot. 50:372-378.
260 Donnelly
134. Ryser, E.T., and E.H. Marth. 1988. Survival of Listeria monocytogenes in cold-pack cheese
food during refrigerated storage. J. Food Prot. 5 1 :6 15-62 1,625.
135. Ryser, E.T., and E.H. Marth. 1989. Behavior of Listeria monocytogenes during manufacture
and ripening of brick cheese. J. Dairy Sci. 72:838-853.
136. Ryser, E.T., and E.H. Marth. 1991. Listeria, Listeriosis and Food Safety. New York: Marcel
Dekker.
137. Ryser, E.T., S.M. Arimi, M. M.-C. Bunduki, and C.W. Donnelly. 1996. Recovery of different
Listeria ribotypes from naturally contaminated, raw refrigerated meat and poultry products
with two primary enrichment media. Appl. Environ. Microbiol. 62: 1781- 1787.
138. Sallam, S. and C.W. Donnelly. 1992. Destruction, injury and repair of Listeria species ex-
posed to sanitizing compounds. J. Food Prot. 55:77 1-776.
139. Seeliger, H.P.R. 1961. Listeriosis. New York: Hafner.
140. Seeliger, H.P.R. 1972. Reviews-A new outlook on the epidemiology and epizoology of
listeriosis. Acta Microbiol. Hung. 19:273-286.
141. Seeliger, H.P.R., F. Sander, and J. Bockemuhl. 1970. Zum kulturellen Nachweis von Listeria
monocytogenes. Z. Med. Mikrobiol. Immunol. 155:352-368.
142. Siragusa, G.R., and M.G. Johnson. 1989. Persistence of Listeria monocytogenes in yogurt
as determined by direct plating and cold enrichment methods. Int. J. Food Microbiol. 7: 147-
160.
143. Skovgaard, N., and C.-A. Morgen. 1988. Detection of Listeria spp. in faeces from animals,
in feeds, and in raw foods of animal origin. Int. J. Food Microbiol. 6:229-242.
144. Slade, P.J., and D.L. Collins-Thompson. 1987. Two-stage enrichment procedures for isolat-
ing Listeria monocytogenes from raw milk. J. Food Prot. 50:904-908.
145. Smith, J.L. and D.L. Archer. 1988. Heat-induced injury in L. monocytogenes. J. Indust. Mi-
crobiol. 3:lOS-110.
146. Terplan, G. 1988. Provisional IDF-Recommended Method: Milk and Milk Products-Detec-
tion of Listeria monocytogenes. Brussels. International Dairy Federation.
147. Truscott, R.B., and W.B. McNab. 1988. Comparison of media and procedures for the isola-
tion of Listeria monocytogenes from ground beef. J. Food Prot. 5 1 :626-628,638.
147a. Twedt, R.M., and A.D. Hitchins. 1994. Determination of the presence of Listeria monocyto-
genes in milk and dairy products: IDF collaborative study. J. AOAC Int. 77:395-402.
148. Warburton, D.W., J.M. Farber, A. Armstrong, R. Caldeira, T. Hunt, S. Messier, R. Plante,
N.P. Tiwari, and J. Vinet. 1991. A comparative study of the FDA and USDA methods
for the detection of Listeria monocytogenes in foods. Int. J. Food Microbiol. 13:lOS-118.
149. Warburton, D.W., J.M. Farber, A. Armstrong, R. Caldeira, N.P. Tiwari, T. Babiuk, P. Lacasse
and S. Read. 1991. A Canadian comparative study of modified versions of the FDA and
USDA methods for the detection of Listeria monoc-ytogenes. J. Food Prot. 54:669-676.
150. Warburton, D.W., J.M. Farber, C. Powell, N.P. Tiwari, S. Read, R. Plante, T. Babiuk, P.
Laffey, T. Kauri, P. Mayers, M.-J. Champagne, T. Hunt, P. LaCasse, K. Viet, R. Smando,
and F. Coates. 1992. Comparison of methods for optimum detection of stressed and low
levels of Listeria monocytogenes. Food Microbiol. 9: 127- 145.
151. Werner, B.S., and D.V. Lim. 1990. Growth of Listeria monocytogenes in different media.
Abstr. Ann. Mtg, Amer. Soc. Microbiol., Anaheim, CA. May 13-19, Abstr. P-41.
152. Yousef, A.E., and E.H. Marth. 1988. Behavior of Listeria monocytogenes during manufacture
and storage of Colby cheese. J. Food Prot. 5 1 :12- 15.
Rapid Methods for Detection of
Listeria
CARLA.BATT
Cornell University, Ithaca, N e w York
INTRODUCTION
Presence of Listeria monocytogenes in food products is a safety problem that warrants
attention and improvements in detection and tracking. Although normal, healthy adults
are primarily unaffected by this pathogen, infants and immunocompromised persons are
at far greater risk [38]. The traditional techniques developed :sincethe 1980s for detecting
and enumerating L. monocytogenes are not sufficiently rapid to assure the safety of perish-
able food products before consumption. Regulations limiting contamination of ready-to-
eat foods to a zero-tolerance have been the driving force behind development of rapid
tests, prompting an intense effort in both commercial and academic laboratories. These
techniques are only useful as a survey tool and as a method to track an alleged foodborne
outbreak. The need to develop quicker and more precise methods for detecting Listeria
is also a function of the similarity between L. monocytogenes and other members of the
Listeria genus. Distribution of a ready-to-eat food containing L. monocytogenes typically
leads to a class I recall. This chapter is an attempt to review objectively most of the
literature on the subject published to date with emphasis on experiences from my labora-
tory. My group has explored many different formats to detect L. monocytogenes, and over
the years, several different rapid methods have been assessed. We have focused on L.
monocytogenes because of its significance to humans (1 400 cases occurred per year during
the late 1980s [38]) and its usefulness in models for development of rapid methods.
261
262 Batt
Microbiology-Based Methods
Classic microbiology-based methods for detecting and enumerating Listeria involve en-
richment in selective media, which may include incubation at refrigeration temperatures
[31,49]. Selective enrichment media which allow only Listeria to grow have also been
developed. The wide range of Listeria-selective plating media currently available is daunt-
ing. Even though several comparative studies have been reported, no single detection
scheme appears to be so vastly superior as to be adopted universally [ 141. Recovery of
injured Listeria cells has emerged as another important issue. Sublethal thermal processing
in addition to other intrinsic and/or extrinsic factors can injure Listeria. Although injury
is not a new phenomenon, the potential significance of injured Listeria in foods deserves
greater consideration in the formulation of enrichment/recovery media.
Step 1. Annealing
I), Primer
Template
-
Step 2. Extension
Step 3. Cycle
0-
__t_
7 -0
has been developed by Gene Trak Inc. (Framingham, MA). Although the exact sequence
is proprietary, it is clearly derived from one of the variable regions of the 16s rRNA. A
novel solution hybridization assay has been formatted where final quantification is accom-
plished using an enzymatic marker [48]. Briefly, 16s rRNA is released by alkaline lysis
from cells grown in an enrichment broth. Then a capture tag consisting of the complemen-
tary sequence to a unique region of 16s rRNA and a poly-A (polyadenylic acid) tail is
allowed to hybridize to the target 16s rRNA. This hybrid is then removed from solution
through the poly A tail using a poly-T (polythymidylic acid) s,equencethat has been immo-
bilized on a polystyrene solid support (Fig. 2). Detection is accomplished using an anti-
body coupled to horseradish peroxidase and directed against a fluorescein marker cova-
lently linked to the detector probe. The detector probe recognizes sequences in 16s rRNA
as spatially distinct from the region recognized by the capturleprobe. Therefore, oxidation
of a substrate (tetramethyl benzidine) in the presence of hydrogen-peroxide by horseradish
peroxidase indicates the presence of Listeria. A more recent refinement of this approach
uses a 16s rRNA probe that is specific for L. monocytogems [58a]. Unique 16s rRNA
sequences that define L. monocytogenes have been reported [2 11, but achieving specificity
in the assay requires precise temperature control.
Virulence genes are frequent targets for nucleic acid--based probe methods, since
these genes are essential for pathogenicity and are typically conserved among a given
species. A probe derived from a putative delayed-type hypersensitivity (DTH) factor iso-
lated from L. monocytogenes 1/2a hybridized to all L. monocytogenes serogroups and L.
ivanovii but not to any other Listeria spp. tested [64]. The exact nature of the DTH gene
-
264 Batt
AAAAAAAAAA
i
FIGURE2 Sandwich hybridization capture assay.
has not yet been reported, and therefore its role in L. rnonocytogenes pathogenicity cannot
be determined. It does, however, appear to be an effective tool for detecting Listeria,
although its species specificity is not absolute for L. rnonocytogenes. For example, the
DTH gene appears to be absent from L. rnonocytogenes serogroup 4a yet present in L.
ivanovii. Thus far, it has only been used as a nucleic acid probe in colony hybridization
assays and the entire 1.1-kb DTH, which contains a fragment labeled with 32P,served as
the probe.
A sequence from what was first believed to be a putative L. rnonocytogenes
a-hemolysin gene [33] was reportedly specific for L. rnonocytogenes [22,24]. However,
subsequent analysis showed that this gene encoded for a major secreted protein (msp)
rather than a hemolysin [34]. Despite its nebulous quality, this sequence has proven to
be useful in developing nucleic acid-based detection systems for Listeria. Initially, a
colony hybridization protocol was used where suspect colonies were transferred to nitro-
cellulose filters and probed with this 32P-labeledfragment. Good specificity was shown
toward Listeria spp. which were P-hemolytic (CAMP-positive). Subsequent refinements
of this approach have included the evaluation of four synthetic 20-bp oligonucleotide
probes in lieu of the entire 500-bp fragment. Two probes which were tested against a
range of Listeria spp. hybridized to all L. monocytogenes isolates and one weakly hemo-
lytic isolate of L. seeligeri [24]. The origin of this probe has been clarified by the reported
cloning and sequencing of an invasion-associated protein (iap) [52].
Pathogenicity of L. rnonocytogenes depends on a number of factors, including the
production of one or more hemolysins. Transposon mutagenesis (Tn916) disrupts the cod-
ing sequence for listeriolysin 0 and renders L. monocytogenes avirulent to mice. The gene
coding for listeriolysin 0 has been cloned [23,54,61] and sequenced [61]. Interestingly,
Rapid Methods for Detection of Listeria 265
when the listeriolysin 0 gene is introduced into Bacillus subtilis, the organism gains the
ability to grow in macrophage-like cells in culture [8].
The listeriolysin 0 gene is presumably unique to L. monocytogenes and therefore
is an obvious target for developing a detection system. It does, however, share some amino
acid homology with other hemolysins, including streptolysin 0 and pneumolysin. The
listeriolysin 0 gene has been used as a probe in Southern hybridization analysis of DNA
purified from several Listeria spp. [ 191. A 610-bp fragment internal to the region coding
for listeriolysin 0 appears to hybridize only with hemolytic strains of L. monocytogenes.
However, under nonstringent conditions, a probe derived from sequences on the 3 of the
listeriolysin 0 gene hybridized with hemolytic strains of L,. ivanovii and L. seeligeri. Al-
though some nucleotide sequence conservation between the hemolysin genes in Listeria
apparently exists, a detailed sequence analysis will be required to determine the exact
extent of homology. Datta et al. [23] used two synthetic oligonucleotide listeriolysin 0
probes in a colony hybridization assay to detect L. monocytogenes and obtained good
specificity [24]. Such listeriolysin probes can likely be adopted to several assay formats
for analyzing food samples.
intergenic regions are likely to be highly conserved among all L. monocytogenes but PCR-
based assays have been employing 16s rRNA 14 I ,44,75,77], the intergenic spacer region
that lies between the I6 and 23 rRNA. (In some instances, the assay was diagnostic only in
the size and restriction pattern of the PCR products providing not definitive identification
[ 26,401.)
Finally, cryptic sequences have been discovered which are unique to L. morzocyto-
genes and use repetitive element sequence-based PCR (rep-PCR) [4S] and subtractive
hybridization [ S I ] . Distribution and conservation of these cryptic sequences within all L.
rnono~yt0geize.sstrains cannot, however, be intuitively deduced and must be proven by
large-scale screening studies.
Formats for PCR-based assays are varied and differ in their complexity as well
as utility. The most comtnon read-out for PCR-based assays is gel electrophoresis
accompanied by ethidium bromide staining, with the presence of a particular PCR product
being diagnostic. The disadvantages of gel electrophoresis are the lack of quantification
and the difficulty in automating post-PCR processing. Alternative means of detecting PCR
products posthybridization include (a) reverse dot-blots. (A labeled PCR product is cap-
tured by an oligonucleotide primer immobilized on a membrane [ IS].), (b) microtiter plate
capture (the labeled PCR product is captured specifically or nonspecifically in the well
of a microtiter plate [ 1 S]), (c) macroporous hydrophobic cloth [ 1 I 1, (d) immunodetection
of RNA:DNA hybrid [ 101, (e) fiberoptic biosensors [73].
A 5 nuclease PCR detection assay was first developed and perfected using L. mono-
cytogenes a\ the target organism (Fig. 4). As a nucleic acid target, listerolysin 0 (hlyA)
was chosen iis the nucleic acid target, because this sequence is unique to L. nzorzocytogenes.
We have previously used this gene as a target for a reverse dot-blot PCR assay [IS].
Although this assay was extremely sensitive, the post-PCR handling steps, including prod-
uct capture and secondary enzyme-conjugate addition, introduced potential problems in
assay throughput and contamination. The latter is of particular concern, since PCR product
contamination through aerosols frequently leads to false-positive results.
Initial work in my laboratory has reliably demonstrated the ability of the 5 nuclease
PCR detection assay to quantify L. monocytogenes in pure culture [3]. The specificity of
the PCR primers and reactions and the parameters that were used in this assay have been
documented and were supported by our data [ IS). Among all Listerici spp., significant
ARQs and amplification products, the latter observed on ethidium bromide-stained agar-
ose gels, were only obtained for L. monocytogerzes. Furthermore, addition of competing
organisms did not affect the assay until the ratio of competing to target organisms exceeded
10.
The 5 nuclease PCR detection assay using the hlyA fluorogenic probe was linear
over a range of 5 X 10 to S X 10 L. monocyfogerzes CFU with SO CFU [3] easily
detected. The yes or no assignment is an accurate scoring method which can be
used for positive and negatijre samples. Non-L. monocytogrnes strains can give a weak
positive signal only when >S X 105copies of the template are present. Even then, the
signal generated is >30 times weaker than the signal obtained from an equivalent number
templates. This assay is now being used as a format to develop
of L. r?zorzoc:~togene.s
methods for detecting L. r,zonocytogsnL.s in dairy, feed, and clinical samples.
Nucleic Acid Sequence-Based Amplification
Nucleic acid sequence-based amplification (NASBA or 3SR [29]) is a system where nu-
cleic acid targets are amplified using a series of enzymes, including a RNA polymerase
268 Batt
I Polymerization I n
foyard
5 prlmer probeU3,
v -
3 5
3
5
+ 5
Q
reyerse
primer
-
Strand displacement
5
(QI
T 3
c
t
3 5
5 3
5
~~
5 3
3 5
5 3
5
3
- w El b
5
5
5 3
5
FIGURE
4 Schematic of 5 nuclease PCR detection. Polymerization is initiated by Ta9
DNA polymerasefrom both the forward and reverse primer extending along the target
strand. Strand displacement occurs when Taq DNA polymerase encounters the
fluorogenic probe and begins to displace it from the target. The probe is labeled with
a reporter (R) and quencher (Q) dye. Cleavage of the fluorogenic probe by Ta9 DNA
polymerase releases the reporter dye. Polymerization is completed when each exten-
sion strand reaches the end of the target.
and a reverse transcriptase (Fig. 5). A target RNA molecule is first reverse transcribed to
cDNA using reverse transcriptase. RNAase H is added to digest the template RNA which
occurs only after hybridization with cDNA. The newly formed cDNA is then used as a
template for a second round of synthesis again using reverse transcriptase. The primer for
this second round carries a T7 promoter as a tail on its 5 end and therefore introduces
this promoter sequence into the second round of synthesized cDNA. At this stage, the T7
promoter containing cDNA is a substrate for RNA synthesis by T7 RNA polymerase.
Copious amounts of T7 RNA polymerase-synthesized RNA are then produced. This in-
crease in RNA can be detected easily by gel electrophoresis or sandwich hybridization,
since amplification is typically on the order of 106fold,
Since NASBA uses RNA templates, it is amenable to detection of L. monocytogenes
with 16s rRNA. Probes specific for L. monocytogenes have been developed [74]. NASBA
assays have used hZyA mRNA as the target with sensitivities as low as 10 CFU/g being
reported [12]. In this latter study, enrichment was used to induce hZyA, a problem in
mRNA based methods where the initial level per cell cannot be predicted.
Problems in Amplification Methods
Two major problems with PCR-based assays (and in general all methods that employ
enzymatic amplification) are false negatives caused by PCR inhibition and false positives
resulting from detection of nonviable cells. The former has been addressed by development
of several template purification methods which range in complexity and utility.
-
Rapid Methods for Detection of Listeria 269
ss RNA target
Primer Annealing
F
RNA-DNA
1I
Reverse Transcription (RT)
intermediate
Primer Annealing
ds DNA
ss RNA
+I
ds DNA synthesis (RT)
RNA synthesis
(T7 RNA poiymerase)
Sample preparation is a subject of intense interest but few determined efforts. Sev-
eral different approaches to sample preparation have been proposed including:
Target cell capture. In general, the most noted example of cell capture involves use
of immunomagnetic beads to which target-specific antibodies are attached [70].
The beads are used to capture cells from solution and then the recovered cells
are subjected to DNA extraction or culture enrichrnent [35]. L. rnonocytogenes
also has been recovered after centrifugation and washing to remove inhibitory
compounds in milk [20] and other foods [63].
Detergent or solvent extraction. Phenol, chloroform, and ether are examples of sol-
vents that can remove compounds that inhibit PCR [43]. Sodium iodide will gen-
erally solubilize food components and make the isolation of amplifiable DNA
possible [%I. Detergents including Tween 20 also can enhance the sensitivity
of PCR by solubilizing inhibitory compounds [68]. A two-phase solvent extrac-
tion using polyethylene glycol and dextran is reportedly effective for soft cheeses
[%I.
Filtrution. For liquid foods, most notably milk, filtration is a simple means of con-
centrating cells [20,72]. Certain filters are amenable to solvent solubilization
which aids in DNA release.
DNA capture. In addition to cell capture, target DN,4 can be captured after cell
lysis. DNA can be absorbed onto several matrices in a nonspecific manner; that
is, silica [43].
PCR cocktail. Few of the PCR inhibitors are known in specific terms. For L. monocy-
togenes and its detection in milk, calcium is thought to be a PCR inhibitor. Conse-
270 Batt
quently, increasing the amount magnesium in PCR is useful [7]. Addition of bo-
vine serum albumin or proteinase inhibitors might help spare the DNA polymerase
during amplification [65].
Viability has been a frequently cited but still unresolved problem. Amplification of archi-
val DNA from various sources documents the ability of DNA to survive well beyond
the life of the organism [57].Therefore, false positives often arise from samples whose
processing history ensures that all L. monocytogenes are nonviable. Two approaches ad-
dressing viability of target cells in PCR-based assays have been proposed. The first is to
have a mandatory culture enrichment period, where a positive result would require growth
of the target organism. The second approach involves targeting of mRNA rather than
DNA, since mRNA is less stable than DNA and should degrade in a manner that parallels
cell death. Efforts to use mRNA as a template for detecting viable L. monocytogenes have
been reported [42]. The utility of this approach may be limited because of strain differences
in target gene expression which will alter the number of mRNA molecules per cell. Second
is the difference in the history of the contaminating L. monocytogenes strain in the food
before and after processing. Since mRNA destruction is a kinetic process, thermal pro-
cessing and the time between processing and assay will be critical. Our efforts to pursue
mRNA as a target in a single-step PCR 5 nuclease assay have used the hlyA gene as a
target [ 3 2 ] . The thermostable DNA polymerase Tth has both reverse transcriptase and
DNA polymerase activity. It can be used in a single buffer reaction that contains a tempera-
ture-sensitive chelator which controls the availability of manganese. Manganese is critical
for Tth switching from reverse transcriptase to DNA polymerase activity [62]. A correla-
tion between viability (as determined by plate counts or staining with a fluorogenic esterase
substrate) and the ARQ of the assay was observed. Selection of PCR primers that hybrid-
ized to the most distal portions of the hlyA gene gave a more accurate result in monitoring
viability as compared with PCR primers that amplified an internal region.
A second means to ensure that only viable L. monocytogenes cells will be amplified
is to have a requisite enrichment period before the PCR assay. Although this might seem
to be the antithesis of rapid methodologies, the enrichment need only be a few hours
and total assay times of less than 8 h are still reasonable. We have used membrane filtration
to concentrate cells from liquid foods, including raw milk [30]. Hot detergent facilitates
filtration after which the collected cells, still on the membrane filter, are placed onto a
nutrient-soaked absorbent pad. The cells are enriched for less than 4 h, processed using
a chelating reagent, and then boiled. The total assay time is less than 8 h and sensitivities
of <I0 viable L. monocytogenes CFU are routinely obtained.
produced by fusing spleen cells isolated from BALB/c mice immunized with live L. mono-
cytogenes to NS-1 plasmacytoma cells. The immunogen consisted of live L. monocyto-
genes Scott A cells that were injected directly into the spleen of the mice [71]. Live cells
(as opposed to heat- or formalin-killed cells) and direct injection were chosen to provide
the most direct presentation of an unaltered (or minimally processed) antigen to the spleen.
Hybridomas were screened by direct ELISA assay, and of the 150 hybridomas tested,
three reacted most strongly with L. monocytogenes Scott A [27]. Although monoclonal
antibodies Mab 20-10-2, Mab 36-6-12, and Mab 59-9-16 reacted to some extent with L.
innocua and L. ivanovii in the direct binding assay, greater specificity for L. monocyto-
genes was seen in an indirect ELISA assay (Fig. 6). These antibodies were used to trap
L. monocytogenes, which was then detected by a rabbit anti-L. monocytogenes polyclonal
antiserum.
Siragusa and Johnson [69] also attempted to isolate a monoclonal antibody specific
for L. monocytogenes. These antibodies resulted from immunizations with heat-treated L.
monocytogenes as previously reported [ 171. Both immuno-dot-blot of heat-treated whole
cells and Western analysis of sodium dodecylsulphate-polyacrylamide gel electrophoresis
(SDS-PAGE) separated cell extracts were used to demonstrate reactivity. Unfortunately,
their monoclonal reacted not only with L. monocytogenes but also with L. welshimeri, L.
innocua, and possibly others. Later efforts by this group targeted another antigen that
produced monoclonal antibodies which preferentially reacted with L. monocytogenes [6].
Further specificity in terms of serotype-specific monoclonal antibodies which detect L.
monocytogenes 4b has also been reported [47].
0.9
0.8
0.7
0.6
U)
g
0
0.5
0
0.4
0.3
0.2
0.I
0
L rnonocytogenes L. ivanovii L innouca L seeligeri S. faecalis
Organism
FIGURE6 Indirect ELISA using MAB20-10-2 to trap antigen and rabbit polyclonal
anti-L. rnonocytogenes serum for detection. Bound antibodies were detected using
goat antirabbit alkaline phosphatase conjugate and p-nitrophenol phosphate. 2 x
106cells/mL; 5 x 105cells/mL. (From Ref. 37.)
Rapid Methods for Detection of Listeria 273
FUTURE DEVELOPMENTS
The long-term goals in development of any rapid method dictate that the test be fast,
simple, sensitive, accurate, and, for commercial purposes, inexpensive. However, at least
some of these desired performance attributes are mutually exclusive: for example, as an
assay is made more sensitive, the accuracy, as it is defined by the number of false positives,
increases. Most attention concerning monoclonal antibodies or nucleic acid probes for
Listeria identification (or in fact other microorganisms) is focused on the reporter mole-
cules and associated detection instrumentation. Advances in chemiluminescent-based
reporters which have sensitivities in excess of 100-fold greater than existing enzymatic-
based systems will be applicable to Listeria detection.
All of the rapid assays developed to date (July 1997) require prior enrichment, with
this step taking up to 48 h. Therefore, any claims that an assay can be completed within,
for example, 4 h, are not entirely truthful. Continued efforts to further improve media
formulations for recovery of Listeria from foods should prove beneficial as a prelude to
any rapid detection method. Another area of concern is the significance of injured Listeria
cells in a given food product and their potential for recovery either during enrichment or
in the food during long-term storage. As mentioned previously, antibodies or nucleic acid
probes can, in theory, detect both injured and dead cells. If future rapid assays are devel-
oped to detect microorganisms in food without any prior enrichment, the significance of
injured populations will need to be addressed.
At issue is whether detection systems specific for L. monocytogenes are advanta-
geous over genus detection of all Listeria. The most obvious argument for an L. monocyto-
genes-specific test is based on the fact that virtually all cases of human listeriosis are
caused by L. monocytogenes. In an ideal world, the goal would be to create a rapid test
which detects Listeria spp., which are pathogenic in humans. Until we have elucidated
the factors mediating pathogenicity of L. monocytogenes, such a goal is not feasible.
ACKNOWLEDGMENTS
The support of the Northeast Dairy Foods Research Center is greatly appreciated. The
author thanks Mary Lou Tortorello and Jerrie Gavalchin for their assistance. The author
also thanks Liz Borod for her help in the preparation of this manuscript.
REFERENCES
1. Bansal, N.S. 1996. Development of a polymerase chain reaction assay for the detection of
Listeria monocytogenes in foods. Lett. Appl. Microbiol. 22:353-356.
2. Barany, F. 199 1 . Genetic disease detection and DNA amplification using cloned thermostable
ligase. Proc. Natl. Acad. Sci. USA 88:189-193.
2a. Barricro and C.A. Batt. 1997. Unpublished data.
3. Bassler, H.A., S.J.A. Flood, K.J. Livak, J. Marmaro, R. Knorr, and C.A. Batt. 1995. Use of
Rapid Methods for Detection of Listeria 275
a fluorogenic probe in a PCR-based assay for the detection of Listeria rnonocytogenes. Appl.
Environ. Microbiol. 61 :3724-3728.
3a Batt, C.A. 1996. Unpublished data.
4. Bessenen, M.T., Q. Luo, H.A. Rotbart, M.J. Blaser, and R.T.I. Ellison. 1990. Detection of
Listeria rnonocytogenes by using the polymerase chain reaction. Appl. Environ. Microbiol.
56:2930-2932.
5. Beunier, R.R., and E. Brinkman. 1989. Detection of Listeria spp. with a monoclonal antibody-
based enzyme-linked immunosorbent assay (ELISA). Food Microbiol. 6: 17 1 - 177.
6. Bhunia, A.K., and M.G. Johnson. 1992. Monoclonal antibody-specific for Listeria rnonocyto-
genes associated with a 66-kilodalton cell surface antigen. Appl. Environ. Microbiol. 58: 1924-
1929
7. Bickley, J., J.K. Short, D.G. McDowell, and H.C. Parkes. 1996. Polymerase chain reaction
(PCR) detection of Listeria monocytogenes in diluted milk and reversal of PCR inhibition
caused by calcium ions. Lett. Appl. Microbiol. 22: 153- 158.
8. Bielecki, J., P. Youngman, P. Connelly, and D.A. Portnoy. 1990. Bacillus subtilis expressing
a haemolysin gene from Listeria rnonocytogenes can grow in mammalian cells. Nature 345:
175-176.
9. Blais, B.W. 1994. Transcriptional enhancement of the Listeria rnonocytogenes PCR and simple
immunoenzymatic assay of the product using anti-RNA:DNA antibodies. Appl. Environ. Mi-
crobiol. 60:348-352.
10. Blais. B.W., and L.M. Phillippe. 1993. A simple RNA probe system for analysis of Listeria
rnonocytogenes polymerase chain reaction products. Appl. Environ. Microbiol. 59:2795-2800.
11. Blais. B.W., and L.M. Phillippe. 1995. Macroporous hydrophobic cloth (polymacron) as a
solid phase for nucleic acid probe hybridizations. Biotechnol. Tech. 9:377-382.
12. Blais. B.W., G. Turner, R. Sooknanan, and L.T. Malek. 1997. A nucleic acid sequence-based
ampli tication system for detection of Listeria rnonocytogenes hlyA sequences. Appl. Environ.
Microbiol. 63:3 10-3 13.
13. Bohnert, M., F. Dilasser, C. Dalet, J. Mengaud, and P. Cossart. 1992. Use of specific oligonu-
cleotides for direct enumeration of Listeria rnonocytogenes in food samples by colony hybrid-
ization and rapid detection by PCR. Res. Microbiol. 143:271-280.
14. Brackett, R.E., L.R. Beuchat, D.A. Golden, and P.K. Cassiday. 1990. Assessment of the ability
of plating methods to accurately detect Listeria in foods. In: A.L. Miller, J.L. Smith, and G.A.
Somkuti, eds. Foodborne Listeriosis. New York: Elsevier, pp. 97- 103.
15. Bsat, N., and C.A. Batt. 1993. A combined modified reverse dot-blot and nested PCR assay
for the specific non-radioactive detection of Listeria rnonocytogenes. Mol. Cell. Probes 7: 199-
207.
16. Bubert, A., S . Koehler, and W. Goebel. 1992. The homologous and heterologous regions
within the Zap gene allow genus and species-specific identification of Listeria spp. by polymer-
ase chain reaction. Appl. Environ. Microbiol. 58:2625-2632.
17. Butman, B., M. Plank, R. Durham, and J. Mattingly. 1988. Monoclonal antibodies which
identify a genus-specific Listeria antigen. Appl. Environ. Microbiol. 54: 1564- 1569.
18. Cano, R.J., D.M. Norton, A.E. Inzunza, J. Gil Sanchez, and C. Oste. 1995. Polymerase chain
reaction assay coupled with fluorescence detection on microwell plates for Listeria monocyto-
genes in foods. J. Food. Prot. 58:614-620.
19. Chenevert, J., J. Mengaud, E. Gormley, and P. Cossart. 1989. A DNA probe specific for
Listeria rnonocytogenes in the genus Listeria. Int. J. Food Microbiol. 8:3 17-3 19.
20. Cooray, K.J., T. Nishibori, H. Xiong, T. Matsuyama, M. Fujita, and M. Mitsuyama. 1994.
Detection of multiple virulence-associated genes of Listeria monocytogenes by PCR in artifi-
cially contaminated milk samples. Appl. Environ. Microbiol. 60:3023-3026.
21. Czajka, J., N. Bsat, M. Piani, W. Russ, K. Sultana, M. Wiedmann, R. Whitaker, and C. Batt.
1993. Differentiation of Listeria rnonocytogenes and Listeria innocua by 16s rRNA genes
and intraspecies discrimination of Listeria monocytogenes srrains by random amplified poly-
morphic DNA polymorphisms. Appl. Environ. Microbiol. 59:304-308.
276 Batt
22. Datta, A.R., B.A. Wentz, and W.E. Hill. 1987. Detection of hemolytic Listeria rnonocytogenes
by using DNA colony hybridization. Appl. Environ. Microbiol. 53:2256-2259.
23. Datta, A.R., B.A. Wentz, and J. Russell. 1990. Cloning of the listeriolysin 0 gene and develop-
ment of specific gene probes for Listeria rnonocytogenes. Appl. Environ. Microbiol. 56:3874-
3877.
24. Datta, A.R., B.A. Wentz, D. Shook, and M.W. Trucksess. 1988. Synthetic oligodeoxyribo-
nucleotide probes for detection of Listeria rnonocytogenes. Applied Environ. Microbiol. 54:
2933-2937.
25. Donnelly, C.W., and G.J. Baigent. 1986. Method for flow cytometric detection of Listeria
rnonocytogenes in milk. Appl. Environ. Microbiol. 52:689-695.
26. Drebot, M., S. Neal, W. Schlech, and K. Rozee. 1996. Differentiation of Listeria isolates by
PCR amplicon profiling and sequence analysis of 16s-23s rRNA internal transcribed spacer
loci. J. Appl. Bacteriol. 80:174-178.
27. Epstein, S.L., and J.K. Lunney. 1985. A cell surface ELISA in the mouse using only poly-
L-lysine as cell fixative. J. Immunol. Methods 76:63-7 1.
28. Eveland, W.C. 1963. Demonstration of Listeria rnonocytogenes in direct examination of spinal
fluid by fluorescent-antibody technique. J. Bacteriol. 85: 1448- 1450.
29. Fahy, E., D.Y. Kwoh, and T.R. Gingeras. 1991. Self-sustained sequence replication (3SR):
an isothermal transcription-based amplification system alternative to PCR. PCR Methods Appl.
1125-33.
30. Farber, J.M., and J.I. Speirs. 1987. Monoclonal antibodies directed against the flagellar anti-
gens of Listeria species and their potential in EIA-based methods. J. Food Prot. 50:479-484.
31. Farber, J.M., and Peterkin, P.I. 1991. Listeria rnonocytogenes, a food-borne pathogen. Micro-
biol. Rev. 55:476-5 11.
32. Fitter, S., M. Heuzenroeder, and C.J. Thomas. 1992. A combined PCR and selective enrich-
ment method for rapid detection of Listeria rnonocytogenes. J. Appl. Bacteriol. 7353-59.
33. Flamm, R.K. 1986. Molecular genetics of Listeria rnonocytogenes: cloning of a hemoIysin
gene, demonstration of conjugation and detection of native plasmids. PhD dissertation, Wash-
ington State University, Pullman, WA.
34. Flamm, R.K., D.J. Hinrichs, and M.F. Thomashow. 1989. Cloning of a gene encoding a major
secreted polypeptide of Listeria rnonocytogenes and its potential use as a species-specific
probe. Appl. Environ. Microbiol. 55:225 1-2256.
35. Fluit, A.C., R. Torensma, M.J.C. Visser, C.J.M. Aarsman, Poppelier, M.J.J.G., B.H.I. Keller,
P. Klapwijk, and J. Verhoef. 1993. Detection of Listeria rnonocytogenes in cheese with the
magnetic immuno-polymerase chain reaction assay. Appl. Environ. Microbiol. 59: 1289- 1293.
36. Furrer, B., U. Candrian, C. Hoefelein, and J. Luethy. 1991. Detection and identification of
Listeria rnonocytogenes in cooked sausage products and in milk by in-vitro amplification of
hemolysis gene fragments. J. Appl. Bacteriol. 70:372-379.
37. Gavalchin, J., M.L. Tortorello, M. Landers, and C.A. Batt. 1991. Isolation of monoclonal
antibodies that react preferentially with Listeria rnonocytogenes. Food Microbiol. 8:325-330.
38. Gellin, B.G., and C.V. Broome. 1989. Listeriosis. J.A.M.A. 261:1313-1320.
39. Golsteyn-Thomas, E.J., R.K. King, J. Burchak, and V.P.J. Gannon. 1991. Sensitive and specific
detection of Listeria rnonocytogenes in milk and ground beef with the polymerase chain reac-
tion. Appl. Environ. Microbiol. 57:2576-2580.
40. Graham, T., E.J. Golsteyn-Thomas, V.P.J. Gannon, and J.E. Thomas. 1996. Genus- and spe-
cies-specific detection of Listeria rnonocytogenes using polymerase chain reaction assays tar-
geting the 16s-23s intergenic spacer region of the rRNA operon. Can. J. Microbiol. 42: 1155-
1162.
41. Greisen, K., M. Loeffelholz, A. Purohit, and D. Leong. 1994. PCR primers and probes for
the 16s rRNA gene of most species of pathogenic bacteria, including bacteria found in cerebro-
spinal fluid. J. Clin. Microbiol. 32:335-35 1.
42. Herman, L. 1997. Detection of viable and dead Listeria rnonocytogenes by PCR. Food Micro-
biol. 14:103- 110.
Rapid Methods for Detection of Listeria 277
43. Herman, L., and H. De Ridder. 1993. Cheese components reduced the sensitivity of detection
of Listeria monocytogenes by the polymerase chain reaction Neth. Milk Dairy J. 47:23-29.
44. Herman, L.M.F., H.F.M. De Ridder, and G.M.M. Vlaemynck. 1995. A multiplex PCR method
for the identification of Listeria spp. and Listeria monocytogenes in dairy samples. J. Food.
Prot. 58:867-872.
45. Jersek, B., E. Tcherneva, N. Rijpens, and L. Herman. 1996. Repetitive element sequence-
based. Lett. Appl. Microbiol. 23:55-60.
46. Johnson, W.M., S.D. Tyler, E.P. Ewan, F.E. Ashton, G. Wang, and K.R. Rozee. 1992. Detec-
tion of genes coding for listeriolysin and Listeria monocytogenes antigen a imaA in Listeria
spp. by the polymerase chain reaction. Microb. Pathog. 12:79-86.
47. Kathariou, S., C. Mizumoto, R.D. Allen, A.K. Fok, and A A . Benedict. 1994. Monoclonal
antibodies with a high degree of specificity for Listeria monocytogenes serotype 4b. Appl.
Environ. Microbiol. 60:3548-3552.
48. King, W., S.M. Raposa, J.E. Warshaw, A.R. Johnson, D. Lane, J.D. Klinger, and D.N. Halbert.
1989. A colorimetric assay for the detection of Listeria using nucleic acid probes. Int. J. Food
Microbiol. 8:225-232.
49. Klinger, J.D. 1988. Isolation of Listeria: a review of procedures and future prospects. Infection
16:SO8-S 105.
50. Klinger, J.D., A. Johnson, D. Croan, P. Flynn, K. Whippie, M. Kimball, J. Lawrie, and M.
Curiale. 1988. Comparative studies of nucleic acid hybridization assay for Listeria in foods.
J. Assoc. Off. Anal. Chem. 71:669-673.
51. Kohler, G., S.S. Howe, and C. Milstein. 1976. Fusion between immunoglobulin-secreting and
nonsecreting myeloma cell lines. Eur. J. Immunol. 6:292-295.
52. Kohler, S., W.M. Leimeister, T. Chakraborty, and F.A.G.W. Lottspeich. 1990. The gene cod-
ing for protein p60 of Listeria monocytogenes and its use as a specific probe for Listeria
monocytogenes. Infect. Immun. 58: 1943- 1950.
53. Lantz, P.G., F. Tjerneld, E. Borch, B. Hahn Hagerdal, and P. Radstrom. 1994. Enhanced sensi-
tivity in PCR detection of Listeria monocytogenes in soft cheese through use of an aqueous
two-phase system as a sample preparation method. Appl. Environ. Microbiol. 60:3416-
3418.
54. Leimester-Watcher, M., and T. Chakroborty. 1989. Detection of listeriolysin, the thiol-depen-
dent hemolysin in Listeria monocytogenes, Listeria ivanovii, and Listeria seeligeri. Infect.
Immun. 57:2350-2357.
55. Makino, S.I., Y. Okada, and T. Maruyama. 1995. A new method for direct detection of Listeria
monocytogenes from foods by PCR. Appl. Environ. Microbiol. 61 :3745-3747.
56. Manzano, M., L. Cocolin, P. Ferroni, V. Gasparini, D. Narduzzi, C. Cantoni, and G. Comi.
1996. Identification of Listeria species by a semi-nested polymerase chain reaction. Res. Mi-
crobiol . 147 :637-640.
57. Masters, C.I., J.A. Shallcross, and B.M. Mackey. 1994. Effect of stress treatments on the
detection of Listeria monocytogenes and enterotoxigenic Escherichia coli by the polymerase
chain reaction. J. Appl. Bacteriol. 77:73-79.
58. Mattingly, J.A., B.T. Butman, M.C. Plank, and R.J. Durham. 1988. Rapid monoclonal anti-
body-based enzyme-linked immunosorbent assay for detection of Listeria in food products.
J. Assoc. Off. Anal. Chem. 71:679-681.
58a. Mazola, M. Personal communication.
59. McLauchlin, J., A. Black, H.T. Green, J.Q. Nash, and A.G. Taylor. 1988. Monoclonal antibod-
ies show Listeria monocytogenes in necropsy tissue samples. J. Clin. Pathol. 41 :983-988.
60. McLauchlin, J., and P.N. Pini. 1989. The rapid demonstration and presumptive identification
of Listeria monocytogenes in food using monoclonal antibodies in a direct immunofluores-
cence test (DIFT). Lett. Appl. Microbiol. 8:25-27.
61. Mengaud, J., M. Vicente, J., Chenevert, J.M. Pereira, C. Geoffrey, S.B. Gicquel, F. Baquero,
D.J. Perez, and P. Cossart. 1988. Expression in Escherichia coli and sequence analysis of the
listeriolvsin 0 determinant of Listeria monocytogenes. Infect. Immun. 56:766-772.
278 Batt
62. Myers, T.W., and D.H. Gelfand. 1991. Reverse transcription and DNA amplification by a
Thermus thermophilus DNA polymerase. Biochemistry 30:766 1-7666.
63. Niederhauser, C., U. Candrian, C. Hofelein, M. Jermini, H.P. Buhler, and J. Luthy. 1992. Use
of polymerase chain reaction for detection of Listeria monocytogenes in food. Appl. Environ.
Microbiol. 58: 1564- 1568.
64. Notermans, S., T. Chakraborty, W.M. Leimeister, J. Dufrenne, K.J. Heuvelman, H. Maas, W.
Jansen, and K.A.G.P. Wernars. 1989. Specific gene probe for detection of biotyped and sero-
typed Listeria stains. Appl. Environ. Microbiol. 55:902-906.
65. Powell, H.A., C.M. Gooding, S.D. Garrett, B.M. Lund, and R.A. McKee. 1994. Proteinase
inhibition of the detection of Listeriu monocytogenes in milk using the polymerase chain reac-
tion. Lett. Appl. Microbiol. 18:59-61.
66. Rossen, L., K. Holmstrom, J.E. Olsen, and O.F. Rasmussen. 1991. A rapid polymerase chain
reaction (PCR)-assay for the identification of Listeria monocytogenes in food samples. Int. J.
Food Microbiol. 14: 145-152.
67. Sallen, B., A. Rajoharison, S. Desvarenne, F. Quinn, and C. Mabilat. 1996. Comparative analy-
sis of 16s and 23s rRNA sequences of Listeria species. Int. J. Syst. Bacteriol. 46:669-674.
68. Simon, M.C., D.I. Gray and N. Cook. 1996. DNA extraction and PCR methods for the detec-
tion of Listeria monocytogenes in cold-smoked salmon. Appl. Environ. Microbiol. 62:822-
824.
69. Siragusa, G.R., and M.G. Johnson. 1990. Monoclonal antibody specific for Listeria monocyto-
genes, Listeria innocua, and Listeria welshimeri. Appl. Environ. Microbiol. 56: 1897- 1904.
70. Skjerve, E., L.M. Rgrvik, and 0. Olsvik. 1990. Detection of Listeria monocytogenes in foods
by immunomagnetic separation. Appl. Environ. Microbiol. 56:3478-348 1.
71. Spitz, M., L. Spitz, R. Thorpe, and E. Egui. 1984. Intrasplenic primary immunization for the
production of monoclonal antibodies. J. Immunol. Methods 70:39-43.
72. Starbuck, M.A.B., P.J. Hill, and G.S.A.B. Stewart. 1992. Ultra sensitive detection of Listeria
monocytogenes in milk by the polymerase chain reaction PCR. Lett. Appl. Microbiol. 15:248-
252.
73. Strachan, N.J.C., and D.I. Gray. 1995. A rapid general method for the identification of PCR
products using a fibre-optic biosensor and its application to the detection of Listeria. Lett.
Appl. Microbiol. 21 :5-9.
74. Uyttendaele, M., R. Schukkink, B. Van Gemen, and J. Debevere. 1995. Development of
NASBA, a nucleic acid amplification system, for identification of Listeria monocytogenes and
comparison to ELISA and a modified FDA method. Int. J. Food Microbiol. 27:77-89.
75. Wang, R.F., W.W. Cao, H. Wang, and M.G. Johnson. 1993. A 16s rRNA-based DNA probe
and PCR method specific for Listeria ivanovii. FEMS 106:85-92.
76. Wernars, K., C.J. Heuvelman, T. Chakraborty, and S.H.W. Notermans. 1991. Use of the poly-
merase chain reaction for direct detection of Listeria monocytogenes in soft cheese. J. Appl.
Bacteriol. 70: 121-126.
77. Widjojoatmodjo, M.N., A.C. Fluit, and J. Verhoef. 1994. Rapid identification of bacteria by
PCR-single-strand conformation polymorphism. J. Clin. Microbiol. 32:3002-3007.
78. Wiedmann, M., F. Barany, and C.A. Batt. 1993. Detection of Listeria monocytogenes using
a nonisotopic polymerase chain reaction (PCR)-coupled ligase chain reaction (LCR) assay.
Appl. Environ. Microbiol. 59:2743-2745.
79. Wiedmann, M., J. Czajka, F. Barany, and C. Batt. 1992. Discrimination of Listeria monocyto-
genes from other Listeria species by ligase chain reaction. Appl. Environ. Microbiol. 58:3443-
3447.
80. Woese, C.R. 1987. Bacterial evolution. Microbiol. Rev. 5 1:221-27 1.
81. Wu, F.M., and P.M. Muriana. 1995. Genomic subtraction in combination with PCR for enrich-
ment of Listeria monocytogenes-specific sequences. Int. J. Food Microbiol. 27: 161- 174.
82. Ziegler, H.K., and C.A. Orlin. 1984. Analysis of Listeria monocytogenes antigens with mono-
clonal antibodies. Clin. Invest. Med. 7:239-242.
Subtyping Listeria
monocytogenes
LEWISM. GRAVES,
BALASWAMINATHAN,
AND SUSANB. HUNTER
Centers for Disease Control and Prevention, Atlanta, Georgia
Most bacterial species have sufficient phenotypic and genotypic diversity to allow for
identification of different subtypes. Therefore, phenotyping and genotyping systems used
singly or in combination often provide useful subtyping schemes for pathogenic bacteria.
The various subtyping systems reviewed in this chapter provide different degrees of dis-
crimination among Listeria monocytogenes isolates. By using these systems in epidemio-
logical studies to distinguish individual strains or groups of strains, it has been possible
to obtain information on relationships between isolates, identify disease outbreaks, identify
the source of infections in outbreaks and sporadic disease settings, and determine modes
of transmission for the organism.
We present a broad overview of the typing methods that have been applied to L.
monocytogenes and, where appropriate, discuss briefly the strengths and weaknesses of
each. In this review of the usefulness of the most commonly used subtyping methods for
L. monocytogenes, we have separated them into two major categories: conventional meth-
ods (i.e., serotyping, phage typing) and molecular methods (i.e., multilocus enzyme elec-
Use of trade names is for identification only and does not imply endorsement by the Public Health Service or
by the U.S. Department of Health and Human Services.
279
280 Graves et al.
trophoresis, DNA restriction analysis). At present, each of these methods has some utility;
however, with the extraordinary developments in nucleic acid technology and the wide
availability of these technologies, some conventional methods may cease to be used in
the future.
L. monocytogenes is among the first pathogenic bacteria for which a concerted and
internationally coordinated attempt has been made to evaluate critically various available
subtyping methods and to standardize the more useful methods. In 1996, Bille and Rocourt
organized the World Health Organization (WHO) Multicentre Listeria monocytogenes
Subtyping Study. The result from Phase I of this study were published in a special issue
of the International Journal of Food Microbiology [25] and will be referenced throughout
this chapter.
CONVENTIONAL METHODS
Serotyping
Serotyping has been a classic tool for epidemiological and sporadic case studies of L.
monocytogenes [27,38]. Strains of L. monocytogenes differ in the antigenic determinants
expressed on the cell surface. Such antigenic variations are produced by many different
surface structures, including lipoteichoic acids, membrane proteins, and extracellular or-
ganelles (e.g., flagella and fimbriae). These differences can be identified by serological
typing (serotyping). Strains of Listeria species are divided into serotypes based on somatic
(0)and flagellar (H) antigens [71]. Flagellar antigens as well as 0 antigens must be identi-
fied to type strains of serotypes 1/2a, 1/2b, 1/2c, 3a, 3b, and 3c. The remaining serotypes
all have the same flagellar antigens (A, B, and C). Serotypes 1/2a, 1/2b, 1/2c, 3a, 3b,
and 3c can be identified with two 0 antisera (one with antibodies to factor I and the other
with antibodies to both factors I and 11) and three H antisera (one with antibodies to factors
A and B, one reacting with C, and one reacting with D). With antisera for 0 factors V
and VI, VII and IX, VIII, X, XI, and XV, strains of serotypes 4a, 4b, 4c, 4d, 5 , 6a, and
6b can be typed [80]. Serotype 4bX is a variant of serotype 4b and was implicated in an
outbreak in the United Kingdom that was traced to contaminated piit6 [47].
Most (>95%) human infections are caused by strains of L. monocytogenes belong-
ing to serotypes 1/2a, 1/2b, and 4b. Therefore, serotyping alone is of limited value in
epidemiological investigations. In the WHO Multicentre L. monocytogenes Subtyping
Study, Schonberg et al. [69] found that all 80 strains tested by serotyping were typeable.
However, for only 49 (61.3%) strains was there complete agreement between the six par-
ticipating laboratories on the serotype (21 of serotype 1/2a and 28 of serotype 4b). Intra-
laboratory reproducibility, assessed on 11 duplicate strains, ranged from 82 to loo%, with
a median value of 91%. Interlaboratory reproducibility varied from 64 to 95%; no labora-
tory correctly identified the two serotype 4bX strains in the set. Schonberg et al. [69]
concluded that a critical need exists for good-quality antisera prepared from standardized
strains. Also, they emphasized the need to absorb these antisera completely and efficiently
to produce good-quality factor sera.
Serotyping has poor discriminating power when compared with other subtyping
methods. Isolates from foods and the environment are frequently nontypeable with stan-
dard typing antisera. Nevertheless, serotyping provides valuable information for rapid
Subtyping L. monocytogenes 281
Bacteriocin Typing
Bacteriocins (monocins) were first isolated from L. rnonocytogenes in 1961 and character-
ized by Sword and Pickett [78] and Hamon and P6ron [35]. Monocins are resistant to
trypsin, sensitive to heating at 56C for 30 min, and stable a.t 4C. In monocin typing, an
isolate is assessed for susceptibility to a set of bactericidal peptides produced by selected
strains [54,85]. Curtis and Mitchell [18] studied monocin interactions of 97 strains of L.
monocytogenes using an improved production method involving standardization of the
monocins against the type strain of Listeria ivanovii. Only serotype 4 strains acted as
282 Graves et al.
indicators. A typing system using 8 producer and I 1 indicator strains showed poor discrim-
ination. Bacteriocin typing has limitations similar to those described for phage typing.
Bannerman et al. [2] typed 100 strains of L. monocytogenes from sporadic cases
and epidemic outbreaks by a combination of monocin typing and phage receptorheverse
phage receptor methods. The combination monocin-phage receptor subtyping method had
a discrimination index of 0.99 for 87 epidemiologically unrelated strains, which was the
highest of seven subtyping methods evaluated. The authors suggested that the monocin-
phage reversal method was simple enough to be done in a nonspecialized laboratory and
was highly discriminatory and reproducible. However, they cautioned that the method and
the indicator test strains must be rigorously standardized.
MOLECULAR METHODS
Multilocus Enzyme Electrophoresis
Characterization of prokaryotes and eukaryotes by multilocus enzyme electrophoresis
(MEE) is based on differences in electrophoretic mobility of their metabolic enzymes.
These differences in electrophoretic mobility are a result of charge differences resulting
from amino acid substitutions in the polypeptide sequence; these charge differences, in
turn, reflect changes in the nucleotide sequence of the DNA encoding the polypeptide
[72]. In MEE, cell extracts containing the soluble metabolic enzymes are electrophoresed
in nondenaturing starch gels. After electrophoresis is completed, the gel is sliced, and
each slice is treated with a specific chromogenic substrate for a specific enzyme (e.g.,
aldolase) to render the enzyme band visible. Mobility variants of each enzyme are consid-
ered to be different electromorphs and are subjectively designated by different numbers.
Combinations of a set of electromorphs (usually 10-20) constitute an electrophoretic type
(ET), with each ET representing a multilocus genotype. Some isolates may present null
results (absence of activity for specific enzymes); this complicates analysis of MEE data.
In the early 1990s, MEE was used in the United States and Europe for epidemiologi-
cal investigations of listeriosis outbreaks [3,31,521 and to determine the extent to which
contaminated foods are involved in sporadic listeriosis [57]. Also, MEE has been useful
for taxonomic and genetic characterization studies of L. monocytogenes [7,8,56]. Boerlin
et al. [8] used MEE to estimate the genetic relatedness between various Listeria species.
The MEE data not only allowed identification of different genotypes within a population,
but also provided an estimation of the genetic relatedness between strains.
Although MEE is a very powerful tool for population genetic, taxonomic, and evolu-
tionary studies, it is only moderately discriminatory for use as a subtyping tool in epidemi-
ological investigations.
Caugant et al. [ 171 coordinated evaluation of MEE for the WHO Multicentre L.
monocytogenes Subtyping Study. Seven laboratories participated in the study, assaying a
Subtyping L. (ionocytogenes
I 283
total of 24 enzymes. Reproducibility and the discriminating power of the method varied
greatly between the laboratories. Null alleles were reported by five laboratories; in some
instances, these could be attributed to less than optimal activity of the enzyme in the
cytoplasmic extracts applied to gels, whereas in others, it was clearly related to characteris-
tics of the strains. Caugant et al. [ 171 concluded that to asceirtain immediate epidemiologi-
cal relationships of L. monocytogenes strains, one will need, in some instances, to supple-
ment MEE with other methods providing further discrimination. Similar conclusions were
reached by Norrung and Gerner-Smidt [5 11, who reported an overall discrimination index
(DI) of 0.83 for MEE. When results of MEE were combined with those of restriction
endonuclease analysis, the DI increased to 0.92. Further, MEE is a labor-intensive method
that requires techniques and equipment available in relatively few laboratories. For these
reasons, this method presently has relatively limited application in epidemiological studies.
evaluated the same probe to type 862 isolates representing serogroups 1/2, 3, and 4. Al-
though useful for subtyping serogroup 1/2 isolates, the method did not adequately dis-
criminate between serogroup 4 isolates. The cloned probe evaluated by Ridley [62] and
another probe derived from repeat sequences of L. monocytogenes DNA were assessed
in the WHO Multicentre L. monocytogenes Subtyping Study [77]. Like ribotyping, the
two cloned probes did not adequately discriminate between epidemiologically unrelated
serotype 4b isolates. Also, these probes did not discriminate between a serotype 4d
isolate and other serotype 4b isolates associated with a listeriosis outbreak. However, the
repeat sequence probe discriminated between serotype 4b isolates and serotype 4bX iso-
lates [77].
The RiboPrinter (Qualicon, Wilmington, DE) is an automated ribotyping system
that generates, analyzes, and stores riboprint patterns of bacteria. The first version of the
286 Graves et al.
RiboPrintcr was contigured t o generate ribotype patterns using only EcoRI and was used
to generate a database of patterns for 1346 isolates of L. r i i o r i o c ~ ! ~ t o g c r I c . v[ 13,371. The
RiboPrintcr was used by Ryser et al. 1651 to demonstrate that different ribotypcs of L.
were fiivorcd b j, d i ffe re n t selec t i ve c nri c h me n t pro tocol s. W i edma n n t:t
r i i o i I o( ~j~togc~iic.s
al. I841 characterized 133 isolates of L. rilorloc:\.togcrlc..v using the RiboPrintcr and tested
for pol y morph i s nis i n vi rii 1e nce-;is soc i ii t ed ge lies. They conc 1uded that the i sol ;it e s coii 1d
be separated into three distinct phylogenetic lineages: hiiman isolates were found i n lin-
eages 1 and 2. but lineage 3 nfiis coniposeed exclusi\,ely of animal isolates.
Pol y merase chain react io ti - ri bot y ping ( PC R-ri boty pi ng ) is ;i met hod that cx ploi ts
iisc of oligonucleotide primers designed to be complementary t o conser\.ed regions of the
5s. 16s. and 23s regions of the rRNA genes. These primers are amplified with purified
or criidc preparations of template DNA bj, PCR. The resulting PCR products may bc
digested with ;i restriction cndonuclease of choice or added t o an agarose gel, elcctropho-
rcsed. and \,i sii;I I i zed by et h i d i ii m bromide staining . The potential of PC R- ri bo t y p i ng for
discriminating between and within \.arious species of Listc~rier.;is well ;is strains of L.
r i ~ o r ~ o c ~ ~ ~ thas i ~ ~ ~explored
o g ebeen .~. by Sontakke and Farber 1741. who analyzed 49 strains
of L. riioiioc!togeiie.s and 12 isolates of other Listc~r-iuspecies. Gcnomic DNA isolated
from bacteria wiis sub.jcctcd to PCR amplification using the region of DNA encoding 16s
and 5s rRNA. They found that PCR-I-ibotyping distinguished beti\wn L. riiorioc:\toScric,s
serotypes 1 /2a and 1 /2b w;ith no o\-erlap i n composite ~vfiles.The sensitii~itj~ of this
metliod for differentiating serotypc 1 /2a and 1 /2b isolates appears to be ;is good ;is that
Subtyping L. monocytogenes 287
of other molecular methods. However, the PCR-ribotyping method was less discriminatory
for serotype 4b strains [26]. Sontakke and Farber [74] concluded that PCR-ribotyping
could be considered as an alternate molecular subtyping technique. However, they recom-
mended combining PCR-ribotyping with another highly discriminatory molecular subtyp-
ing method for confirming associations between isolates of serotype 4b.
FIGURE3 PFGE separation of Apal (lanes 2-7) and Ascl (lanes 9-14) macrorestriction
fragments of Listeria rnonocytogenes genomic DNA f r o m sporadic case isolates.
Lanes 1, 8, and 15, Xbal-digest o f Escherichia col; 0 1 5 7 : H7 strain.
288 Graves et al.
Brosch et al. [ 101 first demonstrated the usefulness of PFGE for subtyping L. mono-
cytogenes by applying the method to type serotype 4b strains. Using ApaI, SmaI, and
NotI, they showed PFGE can distinguish between closely related strains indistinguishable
by other typing methods. The applicability of PFGE to subtype L. monocytogenes sero-
types 1/2 and 3 was subsequently demonstrated [ 151. The applicability of PFGE for out-
break investigations was shown by Buchrieser et al. [ 141, who typed 75 L. monocytogenes
strains isolated during six major and eight smaller listeriosis outbreaks. PFGE divided
these strains into 20 subtypes. Strains within each major epidemic (Switzerland [ 1983-
19871, California [ 19851, and Denmark [ 1985-871) demonstrated indistinguishable pat-
terns, whereas strains responsible for other outbreaks were characterized by specific com-
binations of patterns. Variations within PFGE patterns occurred more frequently within
epidemiologically unrelated isolates. PFGE was used to demonstrate the link between
contaminated chocolate milk and febrile gastroenteritis among a group of people who
attended a cow show in Illinois [20,59]. Destro et al. [21] also found that RAPD and
PFGE were the most useful methods for tracing dissemination of L. monocytogenes in a
shrimp processing plant.
Brosch et al. [9] evaluated PFGE in the WHO Multicentre L. monocytogenes Subtyp-
ing Study. Four participating laboratories evaluated PFGE by analyzing 80 coded strains
of L. monocytogenes. Two restriction endonucleases (ApaI and SmaI) were used by all
laboratories; one laboratory used an additional restriction endonuclease (AscI).Agreement
among the four laboratories ranged from 79 to 90%. Sixty-nine percent of the strains
were placed in exactly the same genomic group by all four laboratories, with most of the
epidemiologically related strains being correctly identified. This study validated previous
claims that PFGE is a highly discriminating and reproducible method for subtyping L.
monocytogenes and is particularly useful for subtyping serotype 4b isolates, which are
not subtyped satisfactorily by most other subtyping methods [9].
The major disadvantages of PFGE are the time required to complete the procedure
(2-3 days), the requirement for large quantities of expensive restriction endonucleases,
and the need for relatively expensive, specialized equipment for electrophoresis.
mer primer to subtype 57 L. monocytogenes isolates and reported that the method allowed
the tracing of L. monocytogenes contamination in several food outlets to be traced back
to a food processing plant. Boerlin et al. [6] did an extensive evaluation of RAPD by
typing 100 L. monocytogenes isolates which had been characterized by serotyping, phage
typing, MEE analysis, REA, and ribotyping. They found R.APD to be highly discriminat-
ing for subtyping. ODonoghue et al. [53] found RAPD to be useful for subtyping
serogroup I /2; also, they found that the method distinguished serotype 4bX strains in-
volved in a pit&-associatedoutbreak from other serotype 4b isolates.
Wernars et al. [82] evaluated RAPD in the WHO Multicentre L. monocytogenes
Subtyping Study. Using three different 10-mer primers, the median reproducibility of
RAPD results obtained by the six participating laboratories was 86.5% (range 0-100%).
Failure in reproducibility was caused primarily by results obtained with one particular
primer. The authors concluded that RAPD is a rapid and relatively simple technique for
epidemiological subtyping of L. monocytogenes isolates that can produce reproducible
and useful results.
Despite the simplicity and high discriminating ability of RAPD, much more work
is still needed before RAPD typing becomes a widely used standard technique. Its primary
drawback is the inconsistent reproducibility of patterns. E%ecauseRAPD conditions are
less stringent to facilitate initiation of the polymerization reaction at sites having one or
more sequence mismatches, polymerization is initiated withi various efficiencies. The final
quantities of DNA produced may vary widely among the different fragments amplified
from a given isolate. Such variation is inherent in RAPD analysis and introduces two
specific problems. First, comparison and interpretation of patterns with differences in in-
tensity become quite difficult. Second, because some of the products may represent rela-
tively inefficient reactions, the actual fragments obtained from a single isolate may vary
in different amplification reactions. Consequently, a well-standardized RAPD protocol
must be followed and used consistently to obtain reliable results.
TABLE
1 Characteristics of Phenotypic and Molecular Subtyping Methods Used in the WHO Multicentre L. rnonocytogenes
Subtyping Study
methods are still too complex and appropriate targets on the genome of L. monocytogenes
have not been identified. Furthermore, a database of DNA sequences of suitable targets
of epidemiologically related and unrelated strains to facilitate interpretation of DNA se-
quencing dala is also not yet available. However, L. monocytogenes is an attractive candi-
date for implementing DNA sequence-based subtyping for the following reasons. There
is an excellent set of well-characterized, epidemiologically related and unrelated strains
of L. monocytogenes that have been subtyped by all available phenotypic, protein-based,
and DNA-RFLP-based subtyping methods. These strains will be invaluable for devel-
oping a database of DNA sequences for L. rnonocytogenes. Recent characterization of
numerous virulence-associated genes of L. monocytogenes also has provided critical infor-
mation concerning sequence heterogeneities in these genes [ 22,60,6 1,67,73,87].
The virulence-associated genes of L. monocytogenes that have been sequenced as
of November, 1997, include iup (encodes an invasion-associated protein), inlA (a family
of genes involved in internalization of the organism into the host cell), hlyA (encodes a
P-hemolysin), plcA (encodes a phosphatidyl inositol-specific phospholipase C which is
involved in lysis of the membrane during cell-to-cell spread of L. monocytogenes), mpl
(encodes a metalloprotease), actA (encodes factors involved in actin polymerization), and
the lmu operon (induces delayed-type hypersensitivity reactions in L. monocytogenes-
immune mice). In addition, theJEaA gene that encodes flagellin protein, thejuR gene that
appears to modulate DNA topology, and genes encoding 16s and 23s ribosomal RNA
in L. monocytogenes have been sequenced. Some virulence-associated genes such as
hlyA are highly conserved and may not be suitable targets for strain identification. Others
such as the inlA operon and genes encoding cell surface structures such as the cell mem-
brane and flagella, may be more polymorphic and hence more useful for discrimination
of strains.
Rasmussen et al. [61] sequenced internal fragments of theJuA, iup, hly, and 23s
rDNA genes from different L. monocytogenes serotypes of clinical, food, and environmen-
tal origin. A 150-bp region of the hly gene was sequenced in 75 strains, with 27 strains
sequenced for the other genes. Although the DNA sequence data for hly, iupJEaA were
useful for identifying three lineages, the genetic diversity within the sequencing targets
was insufficient for adequate subtyping. As methods for DNA sequencing are simplified
and methods for direct sequencing of 1- to 2-kb fragments are developed, DNA sequencing
may become viable for subtyping L. monocytogenes.
(96 of serotype 4b) from an outbreak using ribotyping and four other typing methods
(serotyping, phage typing, MEE, and REA). For serotype 4b isolates, phage typing gave
the highest DI, followed by REA, MEE, and ribotyping, with various combinations of
these methods yielding higher DIs. Graves et al. [3 11 compared ribotyping and MEE using
305 isolates of L. monocytogenes. Although MEE was more discriminating than ribotyping
for this set of isolates, neither of these methods provided adequate discrimination for
serotypes 1/2b and 4b.
At the beginning of this chapter, we referred to the World Health Organization
(WHO) Multicentre Listeria monocytogenes Subtyping Study. Phase I of this study used
a set of 80 coded L. monocytogenes strains that included 11 sets of duplicates. An overview
of the study was reported by Bille and Rocourt [ 5 ] . Because several different methods
were compared using a well-defined set of isolates, these studies are very useful in compar-
ing the utility of the various subtyping methods. Table 1 shows a comparison of the meth-
ods used in the study. On the basis of these results, serotyping, phage typing, REA, PFGE,
and RAPD were selected for standardization in Phase 11. This effort should provide a
selection of standardized subtyping methods that can be employed in a variety of epidemi-
ological investigations to make multicenter comparisons of data possible.
ACKNOWLEDGMENTS
We thank Thomas Donkar and Eric Renner for their assistance with the literature review
for this chapter.
REFERENCES
1. Baloga, A.O., and S.K. Harlander. 1991. Comparison of methods for discrimination between
strains of Listeria monocytogenes from epidemiological surveys. Appl. Environ. Microbiol.
5712324-233 1.
2. Bannerman, E., P. Boerlin, and J. Bille. 1996. Typing of Listeria monocytogenes by monocin
and phage receptors. Int. J. Food Microbiol. 3 1:245-262.
3. Bibb, W.F., B.G. Gellin, R. Weaver, B. Schwartz, B.D. Plikaytis, M.W. Reeves, R.W. Pinner,
and C.V. Broome. 1990. Analysis of clinical and food-borne isolates of Listeria monocyto-
genes in the United States by multilocus enzyme electrophoresis and application of the
method to epidemiologic investigations. Appl. Environ. Microbiol. 56:2133-2141.
4. Bille, J. 1989. Anatomy of a listeriosis outbreak. In: Foodborne Listeriosis. Proceedings of
a Symposium. Hamburg: B. Behrs GmbH and Company, pp. 29-36.
5. Bille, J., and J. Rocourt. 1996. WHO International Multicenter Listeria monocytogenes Sub-
typing Study- rationale and set-up of the study. Int. J. Food Microbiol. 32:25 1-262.
6. Boerlin, P., E. Bannerman, F. Ischer, J. Rocourt, and J. Bille. 1995. Typing Listeria monocy-
togenes: a comparison of random amplification of polymorphic DNA with 5 other methods.
Res. Microbiol. 146:35-49.
7. Boerlin, P., J. Rocourt, F. Grimont, P.A.D. Grimont, C. Jacquet, and J.C. Piffaretti. 1992.
Listeria ivanovii subsp. Zondoniensis subsp. nov. Int. J. Syst. Bacteriol. 42:69-73.
8. Boerlin, P., J. Rocourt, and J.C. Piffaretti. 1991. Taxonomy of the genus Listeria by using
multilocus enzyme electrophoresis. Int. J. Syst. Bacteriol. 41 :59-64.
9. Brosch, R., M. Brett, B. Catimel, J.B. Luchansky, B. Ojeniyi, and J. Rocourt. 1996. Genomic
fingerprinting of 80 strains from the WHO multicentre international typing study of Listeria
monocytogenes via pulsed-field gel electrophoresis (PFGE). Int. J. Food Microbiol. 32:343-
355.
10. Brosch, R., C. Buchrieser, and J. Rocourt. 1991. Subtyping of Listeria monocytogenes serovar
Subtyping L. monocytogenes 293
(REA) typing: results from the WHO collaborative study group on subtyping of Listeria rnono-
cytogenes. Int. J. Food Microbiol. 32:3 13-324.
30. Graham, T., J. Golsteyn-Thomas, V.P.J. Gannon, and J.E. Thomas. 1996. Genus- and species-
specific detection of Listeria rnonocytogenes using polymerase chain reaction assays targeting
the 16S/23S intergenic spacer region of the rRNA operon. Can. J. Microbiol. 42: 1155-1 162.
31 Graves, L.M., B. Swaminathan, M.W. Reeves, S.B. Hunter, R.E. Weaver, B.D. Plikaytis, and
A. Schuchat. 1994. Comparison of ribotyping and multilocus enzyme electrophoresis for sub-
typing of Listeria rnonocytogenes isolates. J. Clin. Microbiol. 32:2936-2943.
32. Graves, L.M., B. Swaminathan, M.W. Reeves, and J. Wenger. 1991. Ribosomal DNA finger-
printing of Listeria rnonocytogenes using a digoxigenin-labeled DNA probe. Eur. J. Epidemiol.
7:77-82.
33. Grimont, F., and P.A.D. Grimont. 1986. Ribosomal ribonucleic acid gene restriction patterns
as potential taxonomic tools. Ann. Inst. Pasteur-Microbiol. 137b: 165- 175.
34. Hadorn, K., H. Hachler, A. Schaffner, and F.H. Kayser. 1993. Genetic characterization of
plasmid-encoded multiple antibiotic resistance in a strain of Listeria rnonocytogenes causing
endocarditis. Eur. J. Clin. Microbiol. Infect. Dis. 12:928-937.
35. Hamon, Y., and Y. Piron. 1961. Etude dupouvoir bactiriocinogkne dans le genre Listeria.
C.R. Acad. Sci. 253: 1883- 1885.
36. Howard, P.J., K.D. Harsono, and J.B. Luchansky. 1992. Differentiation of Listeria rnonocyto-
genes, Listeria innocua, Listeria ivanovii, and Listeria seeligeri by pulsed-field gel electropho-
resis. Appl. Environ. Microbiol. 58:709-7 12.
37. Hubner, R.J., E.M. Cole, J.L. Bruce, C.I. McDowell, and J.A. Webster. 1995. Types of Listeria
rnonocytogenes predicted by the positions of EcoRI cleavage sites relative to ribosomal RNA
sequences. Proc. Natl. Acad. Sci. USA 925232-5238.
38. Jacobs, M.R., H. Stein, A. Buqwane, A. Dubb, F. Segal, L. Rabinowitz, U. Ellis, I. Freiman,
M. Witcomb, and V. Vallabh. 1978. Epidemic listeriosis. Report of 14 cases detected in 9
months. South African Med. J. 54:389-392.
39. Jacquet, C., J. Bille, and J. Rocourt. 1992. Typing Listeria rnonocytogenes by restriction poly-
morphism of the ribosomal ribonucleic acid gene region Zentralbl. Bakteriol. 276:356-365.
40. Lawrence, L.M., J. Harvey, and A. Gilmour. 1993. Development of random amplification of
polymorphic DNA typing method for Listeria rnonocytogenes. Appl. Environ. Microbiol. 59:
31 17-31 19.
41. Lemaitre, J.P., A. Delcourt, and A. Rousset. 1997. Optimization of the detection of bacterio-
phages induced from Listeria sp. Lett. Appl. Microbiol. 2 4 5 1-54.
42. Loessner, M.J., L.A. Estela, R. Zink, and S. Scherer. 1994. Taxonomical classification of 20
newly isolated Listeria bacteriophages by electron microscopy and protein analysis. Intervirol-
ogy. 37:3 1-35.
43. Mazurier, S.I., A. Audurier, N. Marquet-Van der Mee, S. Notermans, and K. Wernars. 1992.
A comparative study of randomly amplified polymorphic DNA analysis and conventional
phage typing for epidemiological studies of Listeria rnonocytogenes isolates. Res. Microbiol.
143507-5 12.
44. McLauchlin, J., A. Audurier, A. Frommelt, P. Gerner-Smidt, C. Jacquet, M.J. Loessner, N.
van der Mee-Marquet, J. Rocourt, S. Shah, and D. Wilhelms. 1996. WHO study on subtyping
Listeria rnonocytogenes: results of phage-typing. Int. J. Food Microbiol. 32:289-299.
45. McLauchlin, J., A. Audurier, and A.G. Taylor. 1986. Aspects of the epidemiology of human
Listeria rnonocytogenes infections in Britain 1967- 1984; the use of serotyping and phage
typing. J. Med. Microbiol. 22:367-377.
46. McLauchlin, J., A. Audurier, and A.G. Taylor. 1986. The evaluation of a phage-typing system
for Listeria rnonocytogenes for use in epidemiological studies. J. Med. Microbiol. 22:357-
365.
47. McLauchlin, J., S.M. Hill, S.K. Velani, and R.J. Gilbert. 1991. Human listeriosis and psti: a
possible association. Br. Med. J. 303:773-775.
Subtyping L. monocytogenes 295
48. Niederhauser, C., C. Hoelfelein, M. Allman, P. Burkhalter, J. Luethy, and U. Candrian. 1994.
Random amplification of polymorphic bacterial DNA: evaluation of 1 I oligonucleotides and
application to food contaminated with Listeria monocytoger,ies. J. Appl. Bacteriol. 77574-
582.
49. Nocera, D., M. Altwegg, G. Martinetti Lucchini, E. Bannerman, F. Ischer, J. Rocourt, and J.
Bille. 1993. Characterization of Listeria strains from a food borne listeriosis outbreak by rDNA
gene restriction patterns compared to four other typing methods. Eur. J. Clin. Microbiol. Infect.
Dis. 12:162- 169.
50. Nocera, D., E. Bannerman, J. Rocourt, K. Jaton-Ogay, and J. Bille. 1990. Characterization by
DNA restriction endonuclease analysis of Listeria rnonocytogenes strains related to the Swiss
epidemic of listeriosis. J. Clin. Microbiol. 28:2259-2263.
51. Norrung, B., and P. Gerner-Smidt. 1993. Comparison of multilocus enzyme electrophoresis
(MEE), ribotyping, restriction enzyme analysis (REA) and phage typing for Listeria monocyto-
genes. Epidemiol. Infect. I I 1 :7 1-79.
52. Norrung, G. 1992. Characterization of Danish isolates of Listeriu monocytogenes by multilocus
enzyme electrophoresis. Int. J. Food Microbiol. 1 5 51-59.
53. ODonoghue, K., K. Bowker, J. McLauchlin, D.S. Reeves, Ph4. Bennett, and A.P. MacGowan.
1995. Typing of Listeria monocytogenes by random amplified polymorphic DNA (RAPD)
analysis. Int. J. Food Microbiol. 27:245-252.
54. Ortel, S. 1989. Listeriocins (monocins). Int. J. Food Microbxol. 8:249-250.
55. Owens, R. 1989. Chromosomal DNA fingerprinting-a new method of species and strain
identification applicable to microbial pathogens. J. Med. Microbiol. 30:89-99.
56. Piffaretti, J.C., H. Kressebuch, M. Aeschbacher, J. Bille, E. Bannerman, J.M. Musser, R.K.
Selander, and J. Rocourt. 1989. Genetic characterization of clones of the bacterium Listeria
monocytogenes causing epidemic disease. Proc. Natl. Acad. Sci. USA 86:38 18-3822.
57. Pinner, R.W., A. Schuchat, B. Swaminathan, P.S. Hayes, K..D. Deaver, R.E. Weaver, B.D.
Plikaytis, M. Reeves, C.V. Broome, and J.D. Wenger. 1992. Role of foods in sporadic listeri-
osis 11. Microbiologic and epidemiologic investigation. J.A.M.A. 267:2046-2050.
58. Poyart-Salmeron, C., P. Trieu-Cuot, C. Carlier, A. MacGow,an, J. McLauchlin, and P. Cour-
valin. 1992. Genetic basis of tetracycline resistance in clinicsalisolates of Listeria monocyto-
genes. Antimicrob. Agents Chemother. 36:463-466.
59. Proctor, M.E., R. Brosch, J.W. Mellen, L.A. Garrett, C.W. Kaspar, and J.B. Luchansky. 1995.
Use of pulsed-field gel electrophoresis to link sporadic cases of invasive listeriosis with re-
called chocolate milk. Appl. Environ. Microbiol. 61 :3 177-3 179.
60. Rasmussen, O.F., T. Beck, J.E. Olsen, L. Dons, and L. Rossen. I99 1. Listeria monocytogenes
isolates can be classified into two major types according to the sequence of the listeriolysin
gene. Infect. Immun. 59:3945-395 I .
61. Rasmussen, O.F., P. Skouboe, L. Dons, L. Rossen, and J. E. Olsen. 1995. Listeria monocyto-
genes exists in at least three evolutionary lines: evidence from flagellin, invasive associated
protein and listeriolysin 0 genes. Microbiology 141:2053-2061.
62. Ridley, A.M. 1995. Evaluation of a restriction fragment length polymorphism typing method
for Listeria monocytogenes. Res. Microbiol. 146:21-34.
63. Rocourt, J., A. Audurier, A.L. Courtieu, J. Durst, S. Ortel, A. Schrettenbrunner, and A.G.
Taylor. 1985. A multi-centre study on the phage typing of Listeria monocytogenes. Zentralbl.
Bakteriol. Hyg. 259:489-497.
64. Rocourt, J., V. Goulet, and A. Lepoutre-Toulemon. 1993. Epidemie de listeriose en France
en 1992. Med. Mal. Infect. 23:481-484.
65. Ryser, E.T., S.M. Arimi, M.M. Bunduki, and C.W. Donnelly. 1996. Recovery of different
Listeriu ribotypes from naturally contaminated, raw refrigerated meat and poultry products
with two primary enrichment media. Appl. Environ. Microbiol. 62: 1781 - 1787.
66. Saunders, N.A., A.M. Ridley, and A.G. Taylor. 1989. Typing of Listeria monocytogenes for
epidemiological studies using DNA probes. Acta. Microbiol. Hung. 36:205-209.
296 Graves et al.
67. Schaferkordt, S., and T. Chakraborty. 1997. Identification, cloning, and characterization of
the Irna operon whose gene products are unique to Listeria rnonocytogenes. J. Bacteriol. 179:
2707-27 16.
68. Schlech, W.F., 111, P.M. Lavigne, R.A. Bortolussi, A.C. Allen, E.V. Haldane, A.J. Wort, A.W.
Hightower, S.E. Johnson, A.H. King, E.S. Nicholls, and C.V. Broome. 1983. Epidemic listeri-
osis-evidence for transmission by food. N. Engl. J. Med. 308:203-206.
69. Schonberg, A., E. Bannerman, A.L. Courtieu, R. Kiss, J. McLauchlin, S. Shah, and D. Wil-
helms. 1996. Serotyping of 80 strains from the WHO multicentre international typing study
of Listeria rnonocytogenes. Int. J. Food Microbiol. 32:279-287.
70. Schwartz, D.C., and C.R. Cantor. 1984. Separation of yeast chromosome-sized DNAs by
pulsed-field gradient gel electrophoresis. Cell 37:67-75.
71. Seeliger, H.P.R., and K. Hohne. 1979. Serotyping of Listeria rnonocytogenes and related spe-
cies. Methods Microbiol. I3:3 1-49.
72. Selander, R.K., D.A. Caugant, H. Ochman, J.M. Musser, M.N. Gilmour, and T.S. Whittam.
1986. Methods of multilocus enzyme electrophoresis for bacterial population genetics and
systematics. Appl. Environ. Microbiol. 5 1:873-884.
73. Sheehan, B., C. Kocks, S. Dramsi, E. Gouin, A.D. Klarsfeld, J. Mengaud, and P. Cossart.
1994. Molecular and genetic determinants of Listeria rnonocytogenes infectious process. Curr.
Top. Microbiol. Immunol. 192:187-21 6.
14. Sontakke, S., and J.M. Farber. 1995. The use of PCR ribotyping for typing strains of Listeria
spp. Eur. J. Epidemiol. 1 I :665-673.
75. Southern, E.M. 1975. Detection of specific sequences among DNA fragments separated by
gel electrophoresis. J. Mol. Biol. 98503-5 17.
76. Stull, T.L., J.J. LiPuma, and T.D. Edlind. 1988. A broad-spectrum probe for molecular epide-
miology of bacteria: ribosomal RNA. J. Infect. Dis. 157:280-286.
77. Swaminathan, B., S.B. Hunter, P.M. Desmarchelier, P. Gerner-Smidt, L.M. Graves, S. Har-
lander, R. Hubner, C. Jacquet, B. Pedersen, K. Reineccius, A. Ridley, N.A. Saunders, and
J.A. Webster. 1996. WHO-sponsored international collaborative study to evaluate methods for
subtyping Listeria rnonocytogenes: restriction fragment length polymorphism (RFLP) analysis
using ribotyping and Southern hybridization with two probes derived from L. rnonocytogenes
chromosome. Int. J. Food Microbiol. 32:263-278.
78. Sword, C.P., and M.J. Pickett. 1961. The isolation and characterization of bacteriophages from
Listeria rnonocytogenes. J. Gen. Microbiol. 25:24 1-248.
79. Versalovic, J., T. Koeuth, and J.R. Lupski. 1991. Distribution of repetitive DNA sequences
in eubacteria and application to fingerprinting of bacterial genomes. Nucleic Acids Res. 19:
6823-683 1.
80. Weaver, R.E. 1989. Morphological, physiological and biochemical characterization. In: Isola-
tion and Identification of Listeria rnonocytogenes. CDC Lab Manual. 1 st ed. Vol. 1. U.S. Dept.
of Health and Human Services, Centers for Disease Control, Atlanta, GA.
81. Welsh, J., and M. McClelland. 1990. Fingerprinting genomes using PCR with arbitrary prim-
ers. Nucleic Acids Res. 18:7213-72 18.
82. Wernars, K., P. Boerlin, A. Audurier, E.G. Russell, G.D.W. Curtis, L. Herman, and N. van
der Mee-Marquet. 1996. The WHO multicentre study on Listeria rnonocytogenes subtyping:
random amplification of polymorphic DNA (RAPD). Int. J. Food Microbiol. 32:325-
341.
83. Wesley, I.V., and F. Ashton. 199 I . Restriction enzyme analysis of Listeria rnonocytogenes
strains associated with food-borne epidemics. Appl. Environ. Microbiol. 57:969-975.
84. Wiedmann, M., J.L. Bruce, C. Keating, A.E. Johnson, P.L. McDonough, and C.A. Batt. 1997.
Ribotypes and virulence gene polymorphisms suggest three distinct Listeria rnonocytogenes
lineages with differences in pathogenic potential. Infect. Immun. 65:2707-27 16.
85. Wilhelms, D., and D. Sandow. 1989. Preliminary studies on monocine typing of Listeria rnono-
cytogenes strains. Acta. Microbiol. Hung. 36:235-238.
Subtyping L. monocytogenes 297
86. Williams, J.G.K., A.R. Kubelik, K.J. Livak, J.A. Rafalski, and S.V. Tingey. 1990. DNA poly-
morphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res.
18:653 1-6535.
87. Wuenscher, M.D., S. Kohler, A. Bubert, U. Gerike, and W. Goebel. 1993. The iup gene of
Listeria monocyrogenes is essential for cell viability, and its gene product, p60, has bacterio-
lytic activity. J. Bacteriol. 175:3491-3501.
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10
Foodborne Listeriosis
ELLIOTT. RYSER
Michigan State University, East Lansing, Michigan
HISTORICAL OVERVIEW
Discovery of several listeriosis outbreaks during the 1980s that were positively linked to
consumption of cheese and raw vegetables has led to inclusion of Listeria rnonocytogenes
in the current list of bonafide foodborne pathogens. However, in retrospect, the concept
of listeriosis as a foodborne illness actually can be traced back to when L. rnonocytogenes
was first isolated. Nine years before Murray et al. [167] described L. rnonocytogenes in
1926, Atkinson [42] reported an outbreak of meningitis among five 2- to 9-year-old Austra-
lian children, caused by a small gram-positive, diphtheroid-type bacillus that was probably
L. rnonocytogenes. Similarly, two additional listeriosis outbreaks [67,21I ] accounted for
7 of 36 listeriosis cases recorded in the literature between 19 17 and 1943 [ 1361. Hence,
to explain these three small outbreaks, along with at least 35 other listeriosis outbreaks
that have included 2195 documented cases since 1949 (Table I), one might reasonably
postulate that food-to-human transmission of L. rnonocytogenes occurred in at least some
instances.
Early animal feeding studies also support the notion that listeriosis can be acquired
through consumption of contaminated food. The first accurate description of L. rnonocyto-
genes in 1926 by Murray et al. [ 1671 included trials in which three of six 32-day-old
rabbits were successfully infected via the oral route. Results from subsequent postmortem
examinations agreed with the previously observed pathological findings for naturally oc-
curring listeriosis in rabbits. Fourteen years later, Julianelle [ 1321 reported that white mice
died of generalized listerial infections after consuming drinking water inoculated with
299
300 Ryser
TABLE
1 Apparent and Confirmed Common-Source Outbreaks of Listeriosis
Involving 10 or More Cases
Number Possible vehicle of
Location Year of cases infection Reference
Halle, East Germany 1949- 1957 - 100 Raw milk, sour milk, 44,l 13,185,186,
cream, cottage cheese 192,215
Jena, East Germany 1954 26 Unknown 227
Soviet Union 1956 19 Pork, mouse 116
Bremen, West Germany 1960- 1961 81 Unknown 98
Halle, East Germany 1966 279 Unknown 177
Auckland, New Zealand 1969 13 Unknown 49
Anjou, France 1975- 1976 162 Unknown 71
Johannesburg, South Africa 1977- 1978 14 Unknown I25
Western Australia 1978- 1979 12 Raw vegetables 220
Massachusetts, USA I979 20 Raw vegetables, milk 122
Auckland, New Zealand 1979- 1980 10 Unknown 190
Auckland, New Zealand 1980 22 Shellfish, raw fish 144
East Cambria, England 1981 11 Cream 104,156
Slovakia 1981 49 Unknown 190
Maritime Provinces, Canada 1981 41 Coleslaw 210
Christchurch, New Zealand 1981-1982 18 Unknown 93
Houston, Texas, USA I983 10 Unknown 69
Saxony, West Germany 1983 25 Unknown I69
Massachusetts, USA 1983 49 Pasteurized milk 99
Vaud. Switzerland 1983- 1987 122 Vacherin Mont dOr 47.66
cheese
Los Angeles, CA, USA I985 142h Mexican-style cheesea 145
Denmark 1985- 1987 35 Unknown 200,208
Linz, Austria 1986 20 Raw milk, vegetables 3,226
Los Angeles, CA, USA 1986- 1987 33 Raw eggs 214
Los Angeles, CA, USA 1987 11 Butter 154
England 1987 23 Unknown 157
England, Wales, Northern 1987- 1989 366 Pritt2 159,195
Ire 1and
New York, NY, USA 1989 10 Shrimp I94
Denmark 1989- I990 26 Blue-mold or hard 131
cheese
Western Australia 1990 11 Processed meats or p h i 139, 234
France 1992 279 Jellied pork tonguea 106,126,207
France 1993 39 Pork piit6 rilletes 126
Italy 1993 18 Rice salad 206
Illinois, Michigan, Wiscon- I994 66 Chocolate milk 75
sin, USA
France I995 33 Brie de Meaux cheesea 126
France 1997 14 Pont IEvCque cheese -c
Italy 1997 1594 Sweet corn -c
RAW MILK
Sporadic cases of bovine mastitis and abortion in which L. monocytogenes was intermit-
tently shed in milk over several lactation periods have been recorded in the literature for
more than 50 years. Dairy cows that appear healthy also can serve as reservoirs for L.
monocytogenes and secrete the organism in milk. Once obtained from the cow, milk may
be further contaminated through inadvertent contact with feces and silage, both of which
often contain Listeria and are normally present in the dairy farm environment. Considering
the present estimate that 3-4% of the milk supply contains detectable levels of L. monocy-
togenes, it is easy to understand why raw milk was suspected as one of the most likely
sources of infection in several large European outbreaks of listeriosis.
The first evidence for foodborne transmission of L. monocytogenes can be found in
a series of anecdotal reports from Germany [ 1 13,2 151. During the reconstruction period
that followed the end of World War 11, a sharp increase in the number of stillborn infants
was observed at an obstetrical clinic in Halle, with approximately 100 cases recorded up
to 1952. Working in an antiquated laboratory, Potel [ 1841 concluded that these stillbirths
resulted from infection with Corynebacterium infantiseptica. However, in 1952, Seeliger
suggested and later confirmed that this rash of stillbirths was caused by L. monocytogenes
[ 1131. Unpasteurized milk as well as sour milk, cream, and cottage cheese were suspected
by Seeliger [215] as possible vehicles of infection in several cases observed in Halle.
Foodborne Listeriosis 303
this is currently the only report linking listeriosis to consumption of human breast milk, the
medical profession should be aware of the possibility for such transmission, particularly in
apparent nosocomial cases of neonatal listeriosis.
PASTEURIZED MILK
Until 1985, the only proven foodborne listeriosis outbreaks associated with dairy products
involved consumption of raw milk. However, this changed when Fleming et al. 1991 epide-
miologically linked consumption of a specific brand of whole and 2% pasteurized milk
to 49 cases of listeriosis in Massachusetts between June and August of 1983. As seen in
Table 2,42 (86%)of the cases occurred in adults and seven (14%) in mother-infant pairs.
Fourteen of 49 individuals died giving a mortality rate of 29%. Two years later, Todd
[225] calculated the total cost of this outbreak at $1.89 million ($1.37 million-deaths,
$387,000-hospitalization, $70,00O--investigation, $6 I ,000-financial/legal costs) or
$38,6 14 per case excluding legal settlements.
Although all adults had underlying conditions that resulted in immunosuppression,
symptoms expressed during the course of illness varied depending on the persons age
and degree of immunosuppression. Forty of 49 isolates were available for serotyping, with
32 (80%) being identified as serotype 4b, which was later defined as the epidemic strain.
Two case-control studies, one matched for neighborhood of residence and the other
for the patients underlying condition, indicated that development of listeriosis was
strongly associated with drinking a specific brand of pasteurized whole or 2% milk. Further
epidemiological investigations showed (a) a correlation between increased consumption
of the specific brand of whole or 2% milk and contracting listeriosis, (b) a lower incidence
TABLE
2 Characteristics of Adult and Perinatal
Listeriosis Cases Identified in Massachusetts Between
June 30 and August 30, 1983.
Number of cases (%)
of disease among individuals who drank skim or 1% milk produced by the same dairy,
(c) an association between several listeriosis cases in Connecticut and consumption of the
same brand of whole or 2% milk, and (d) an association with a specific phage type of L.
monocytogenes (phage type 2425A), which was isolated from all 19 listeriosis victims
who reportedly drank the specific brand of whole or 2% milk.
Although these epidemiological studies strongly suggest that this outbreak resulted
from consuming whole or 2% milk, the microbiological findings were far less convincing
in that L. rnonocytogenes was never isolated from the incrirninated pasteurized milk. The
milk implicated in this listeriosis outbreak was processed at a single dairy factory and
pasteurized at 77.2C (1 7 1 O F ) / 18 s [ 1881, which is well in excess of the minimum require-
ment (7 1.7C [ 161F]/15 s) specified in the Pasteurized Milk Ordinance. Furthermore, no
defect was identified that could have led to improper pasteurization and no source of
postpasteurization contamination was ever found within the dairy factory. Shortly after
the outbreak ended, a survey conducted by the CDC [99,112,119] indicated that 15 of
124 (12%) raw milk samples collected from the factory milk supply, individual farms,
and a milk cooperative that supplied the factory contained L. monocytogenes. Several
different serotypes were identified, including 1a, 3b, 4a/b, and 4b, the epidemic serotype.
Using DNA macrorestriction analysis [ 1261, the epidemic strain was later reported to be
genetically similar to isolates responsible for two subsequent outbreaks traced to p2te in
the United Kingdom [ 1591and France [ 1261 (Table 10) but distinctly different from strains
implicated in two North American outbreaks involving coleslaw and Mexican-style cheese
[235].However, neither the phage type nor the restriction enzyme type that was epidemio-
logically linked to this presumed outbreaks of milkborne listeriosis was ever recovered
from raw milk or the incriminated pasteurized milk.
Although the epidemiological evidence gathered by Fleming et al. [99] suggests this
outbreak resulted from drinking a particular brand of pasteurized whole or 2% milk, the
means by which L. monocytogenes may have found its way into the milk remains unclear.
Postpasteurization contamination of the milk cannot be excluded; however, it seems un-
likely, since inspections failed to recover L. rnonocytogenes from the dairy factory environ-
ment. Since whole and skim milk were processed each day using the same equipment, it
is also difficult to postulate a means by which only whole milk would have been subjected
to postpasteurization contamination.
To further support the involvement of pasteurized milk in this outbreak, the authors
concluded that intrinsic contamination of the milk and siirvival of some organisms de-
spite adequate pasteurization is both consistent with the rr:sults of this investigation and
biologically plausible; the latter conclusion was based on the now faulty but at the time
frequently quoted pasteurization study by Bearns and Girard [48]. Consequently, the ap-
parent association of listeriosis with consumption of pasteurized milk raised immediate
product safety concerns, which in turn led to numerous studies examining L. monocyto-
genes heat resistance (see Chap. 6). Nearly 2 years after the outbreak was reported in the
New England Journal of Medicine, Donnelly et al. [81 I found that the open-tube
method used by Bearns and Girard [48] was flawed and concluded that freely suspended L.
monocytogenes cells were unlikely to survive normal high-temperature, short-time (HTST)
pasteurization at 71.7C (161F) for 15 s. Knowing that this pathogen can exist within
milk leukocytes, and that milk in the Massachusetts outbreak was not clarified but rather
passed through a milk sock (coarse filter) that did not remove leukocytes, many researchers
postulated that the presence of L. monocytogenes within these leukocytes enhanced the
organisms resistance to pasteurization. Subsequent studies addressing this issue have gen-
306 Ryser
erally shown insignificant differences in the degree of thermal resistance between freely
suspended and internalized L. monocytogenes cells [84,1731. Using milk containing rela-
tively large numbers of intracellular listeriae, several workers demonstrated that L. mono-
cytogenes can survive minimum requirements for pasteurization; however, commingling
of milk from many farms before pasteurization would result in much lower levels of
listeriae (i.e., 5 10 CFU/mL) [38,146,218] with many, if not most, organisms being present
only extracellularly as a result of leukocytic breakdown during the first 24-48 h of cold
storage. Hence, on this basis, both the scientific community and the WHO maintain that
L. monocytogenes will not survive minimal milk pasteurization at 16l0F/15 s [236]. In
support of this position, L. monocytogenes has not yet been demonstrated to have survived
pasteurization in a commercial dairy product that met minimum HTST pasteurization re-
quirements.
Although numerous FDA Class I recalls of pasteurized dairy products, including
2%, 1%, and skim milk as well as chocolate milk, ice milk mix, ice milk, ice cream
mix, ice cream, ice cream novelties, sherbet, butter, and various cheeses, have been well
publicized in the United States, in virtually all instances L. monocytogenes was present
in the immediate manufacturing environment, which strongly suggests postpasteurization
contamination. Given this information, the likelihood of L. rnonocytogenes having sur-
vived pasteurization at -77.2"C (17 1OF)/ 18 s in the Massachusetts outbreak appears re-
mote at best.
Since L. monocytogenes was never isolated from pasteurized milk implicated in
the Massachusetts outbreak or the dairy factory environment, some investigators have
questioned the role of milk [80]. In 1988, several design flaws were discovered in the
case-control studies [80] that included missing questions and data on questionnaires, as
well as a disproportionate number of follow-up interviews between cases and controls,
any of which might have given an unfair bias. Contrary to the authors [99], cases were
generally clustered around the Boston area in a manner that was not consistent with the
milk distribution pattern. Furthermore, inconsistencies in data were noted between expo-
sure to implicated milk and isolation of the L. monocytogenes strain supposedly responsi-
ble for the epidemic [go]. Discovery of these discrepancies in the various case-control
studies prompted a lawsuit against the CDC; however, as one might expect, a definitive
answer concerning involvement of pasteurized milk in this outbreak was never reached
through the judicial system.
Chocolate Milk
In July of 1994, Dalton et al. [75]reported that 54 of 60 (90%) previously healthy individu-
als who attended a summer picnic in Illinois developed listeriosis 9-32 h (median 20 h)
after consuming one or more 8-oz cartons of pasteurized chocolate milk with four, three,
and five related cases later reported in Illinois, Wisconsin [ 1891, and Michigan, respec-
tively. Unlike most foodborne listeriosis epidemics recorded to date, gastrointestinal symp-
toms predominated among picnickers, with victims most commonly experiencing diarrhea
(79%), fatigue (74%), fever (72%), chills (65%), headache (65%), myalgia (59%), abdomi-
nal cramps (55%), nausea (47%), and vomiting (26%) over several days. Only four indi-
viduals required short hospitalization, with one pregnant woman delivering a healthy baby
5 days after experiencing a 6-h bout of diarrhea.
In support of the aforementioned findings, the clinical strain of L. monocytogenes
identified as serotype 1 /2b was soon isolated from multiple unopened containers of choco-
Foodborne Listeriosis 307
late milk at levels of 8.8 X 108 to 1.2 X 109 CFU/mL (Le., mean infective dose of
2.9 X 10'' CFU/person), with the product's taste and quality reportedly being poor. During
a follow-up environmental survey of the implicated milk processing facility, investigators
recovered the epidemic serotype of L. monocytogenes from a floor drain beneath the choco-
late milk filler and from a valve connected to the chocolate: milk pasteurizer. Based on
multilocus enzyme electrophoresis, ribotyping and pulsed-field gel electrophoresis, the
clinical, chocolate milk, and factory environmental isolates of L. monocytogenes serotype
1/2b were indistinguishable from each other, thereby confi.rming chocolate milk as the
vehicle of infection.
Although properly pasteurized at >87"C/ 18 s after the addition of chocolate flavor-
ing and immediately cooled to <8"C, the product was held unrefrigerated for 2 h in a
malfunctioning and improperly sanitized tank before being pumped to the filling machine
over a 7-h period. In all likelihood, this outbreak occurred as a result of postpasteurization
contamination. However, given the ability for rapid growth of L. rnonocytogenes in tem-
perature-abused chocolate milk [ 1991 (see Chap. 1 l), inadequate and/or nonexistent
refrigeration during packaging (7 h), and transit (2.25 h) to the picnic are obvious contrib-
uting factors along with consumption of the product 3 days before the expiration date.
Fi 1.
2.
3.
Coordinate e n t i r e i n v e s t i g a t i o n
I n i t i a t e case-control s t u d i e s
Serve a s a c e n c r n l l a b o r a t o r y f o r L i S t e r i a - t e S t i n g
r--l
Orange County
Health Care Agency He a 1t h S e r v i c e s
J a l i s c o Mexican L3ok f o r c a u s e o f
P r o d u c t s , Inc. 4 contamination
FIGURE1 Primary roles o f local, state, and federal agencies in investigating the 1985
listeriosis outbreak in California.
310 Ryser
plex at which about 17,000 infants are delivered each year [ 128,1291.In early April, Carol
Salminen, a nurse epidemiologist who monitored infection rates at this facility, uncovered
five additional listeriosis cases among Hispanic motherhnfant pairs during the previous
2 weeks. Under normal circumstances, only three to five listerial infections would be
observed annually at this hospital. After consulting with the medical director of the labor
and delivery service, who had developed a personal interest in listeriosis and maintained
a log of listerial infections over the previous 10 years, Salminen informed health officials
at the Los Angeles County Department of Health Services on May 6 that nine listeriosis
victims had been treated at the USC Medical Center since January of 1985. Once notified,
health officials at the Los Angeles County Department of Health Services and the Orange
County Health Care Agency began surveying area hospitals for additional cases. After
the reported number of listerial infections increased from 16 to 67 in just a few days, Los
Angeles County health officials contacted the California Department of Health Services
and the CDC on May 10 for investigative assistance. At the same time, 200 area hospitals
in the counties of Los Angeles and Orange were requested to report all listeriosis cases
to the health department.
Ten days later, health officials and epidemiologists from the CDC began the first
of two case-control studies in which listeriosis victims, mostly previously healthy Hispan-
ics, were interviewed about various environmental factors, behavioral patterns, and con-
sumption of over 60 food items, including fresh fruits and vegetables, water, milk, and
cheese. On May 29, an open package of Jalisco brand Mexican-style cheese was taken
from one of the victims refrigerators and sent to the CDC in Atlanta, Georgia, for analysis.
After CDC investigators provided preliminary confirmation of L. monocytogenes in this
opened package of Mexican-style cheese on June 8, 20 packages of Mexican-style cheese
of various brands, including two packages of Queso Fresco and Cotija Jalisco brand
cheese, were purchased at area markets near the victims place of residence and sent to
the CDC for analysis [145]. On June 10, results from the case-control study described
earlier clearly demonstrated that individuals who had consumed Mexican-style cheeses
were at increased risk of contracting listeriosis. Armed with this information, investigators
immediately began a second case-control study in which individuals were questioned
about the names and brands of Mexican-style cheese consumed. On June 12, statistical
analysis of these data revealed a definite link between consumption of Jalisco brand Mexi-
can-style cheese and development of listeriosis [ 1451. FDA officials were immediately
advised of the impending problem with Jalisco cheese. Confirmation of L. monocytogenes
serotype 4b in two unopened packages each of Jalisco brand Queso Fresco and Cotija
Mexican-style cheese-the last piece of evidence needed to initiate a Class I recall of the
product-was provided by the CDC on June 13. (Subsequent studies eventually identified
the epidemic L. monocytogenes strain in 82% of all Jalisco brand products purchased at
area supermarkets.) Armed with this information, the California Department of Food and
Agriculture immediately closed the Jalisco cheese factory and announced a statewide re-
call for these two varieties of Mexican-style cheese, -80% of which was sold through
retail outlets in Los Angeles and Orange Counties. On June 14, state officials expanded
this recall to include the firms entire line of 44 products (predominantly cheese), consump-
tion of which was already blamed for at least 28 deaths. Hence, in the weeks that followed,
health officials were faced with the enormous task of checking -28,000 Los Angeles-
area supermarkets, family-owned grocery stores, and restaurants to ascertain that all Jali-
sco brand products were removed from shelves. FDA officials also ordered a Class I recall
of all Jalisco brand products distributed in California and in 12 other primarily western
Foodborne Listeriosis 31 1
states [6]. Three days later, this recall was expanded to include all 26 states in which these
products were sold as well as the United States Protectorates of Guam, American Samoa,
and the Marshal1 Islands [8]. When this recall was completed on June 22, nearly 250 tons
of Jalisco brand Mexican-style cheese and other dairy products were ready for burial in
a landfill site overlooking the San Gabriel Valley.
Even though the number of individuals who actually contracted listeriosis after
eating the tainted cheese has been a debatable issue for some time, the exact figure will
never be known, since mild listerial infections in individuals who did not seek medical
attention obviously went unreported. Newspaper accounts have placed the total number
of listeriosis cases occurring in California between January 1 and August 15 at nearly
300, including 85 fatalities. Although about half of these cases were concentrated within
the Hispanic communities of Los Angeles and Orange County, a substantial number of
listeriosis victims also reportedly resided in the San Diego area, which made the collection
of reliable data more difficult. In addition, at least 16 cheese-related listeriosis cases were
uncovered outside California (Arizona, Colorado, Oregon, Texas, and Connecticut) with
three fatalities being reported in Texas.
Although the total number of listeriosis cases reported in Los Angeles County during
the 12-month period immediately following the outbreak decreased to 94, the calculated
-
annual crude incidence rate of 12 cases/million population is still approximately twice
the national average [ 1521. Numbers of reported listerial infections have continued to
decrease in Los Angeles County, with 1990 and 1994 rates of nonperinatal and perinatal
listeriosis decreasing from 6 to 3 cased1 million population and 17 to 6 cases/100,000
live births, respectively [72,223]. The fact that these rates were previously well above the
national average is not too surprising when one considers that this outbreak certainly made
area physicians, hospital personnel, and public health authorities keenly aware of this
disease. In all likelihood, these factors in combination with mandatory reporting of this
formerly obscure illness were largely responsible for the abnormally high incidence of
listeriosis in Los Angeles County.
In 1988, Linnan and 14 other members of the investigative team [145] published
their findings concerning 142 listeriosis cases that were linked to consumption of Jalisco
brand cheese in Los Angeles County between January 1 and August 15, 1985. Although
nearly 160 additional cases occurred elsewhere in California (Orange, San Diego, and
Fresno Counties) and in other states, logistical concerns limited their studies to Los
Angeles County. During the 7.5-month epidemic period, 93 reported listeriosis cases
(65.5%) involved pregnant women or their offspring with 49 (34.5%) affecting nonpreg-
nant adults (Fig. 2, Table 3). Forty-eight of the 142 listeriosis victims died, giving an
overall mortality rate of 33.8%. Thirty deaths occurred among the 87 early fetalheonatal
cases; however, no late fetal/neonatal or maternal deaths were reported. All but 1 of the
49 nonpregnant adults had a predisposing condition such as cancer (3 patients), steroid
dependency (12 patients), chronic illness (23 patients), age :>65 ( 5 patients), or AIDS (3
patients), which placed these individuals at greater risk (than the normal population) of
developing listerial infections [91,2121.
After identifying the epidemic L. monocytogenes strain as belonging to serotype 4b,
all 105 clinical isolates available for study were phage typed and compared with the strain
isolated from Jalisco brand Mexican-style cheese. Results showed that 86 of 105 (82%)
clinical isolates were serotype 4b, with the remaining 19 non--serotype 4b isolates originat-
ing from listeriosis victims whose illnesses were presumably not related to consumption
of contaminated cheese. Of the 86 isolates identified as L. monocytogenes serotype 4b,
372 Ryser
N onpre g n an I I n
...
...
Mate
~~~
4
2
0
&
...
...
...
...
..
.
....
..
...
...
...
...
.... ..
....
.*:.
...
...
FIGURE2 Listeriosis cases classified according t o risk group in Los Angeles Country,
January 1 t o August 15, 1985. Arrow designates the time of recall 11451.
TABLE 3 Clinical and Demographic Data o n 142 Listeriosis Cases Occurring in Los
Angeles County, California, Between January 1 and August 15, 1985
Fetal or neonatal
Non pregnant
Variable Early Late Maternal adults
No. of patients 87 6 93 49
Mean age 32 weeks 38 weeks 26 yr 58 yr
gestation gestation
Race or ethnic group:
number (%)
Hispanic - - 81 (87) 14 (29)
White - - 10 (1 ) 26 (53)
Black - - 0 7 (14)
Asian - - 2 (2 2 (4)
Fatalities (%) 30 (34) 0 0 18 (37)
Epidemic phage type (%) - - 75 27
Mean birth weight (kg) 2.54 3.15 - -
Septicemia (%) 88 17 52 71
Meningitis (%) 2 67 0 14
Septicemia -t meningitis (5%) 6 17 0 14
Other positive culture (9%) 4 0 48 2
Source: Adapted from Ref. 145.
Foodborne Listeriosis 3 13
63 (73%) sfrains were of the same phage type as strains isolated from the contaminated
cheese. In several follow-up subtyping studies, clinical and cheese isolates were of the
same multilocus enzyme electrophoresis type [55,110], ribotype [ 1 101, and pulsed-field
gel electrophoresis type [65,163], and they also gave identical restriction enzyme [235]and
random amplified polymorphic DNA patterns [74], thereby confirming the involvement of
Jalisco brand cheese in this outbreak (Fig. 3). The 23 remaining clinical isolates belonging
to nonepidemic phage types presumably represented non-cheese-related background cases
of listeriosis that occurred throughout the year. Sporadic sale of tainted cheese in a few
family-owned grocery stores and restaurants, along with a likely listeriosis incubation
period of 3 days to 2 weeks, are both, at least partly responsible for those cases which
occurred beyond the middle of July with secondary infections being spread by fecal shed-
ding also a probable contributing factor [ 1531.
Although these statistics are fairly typical for human listeriosis cases, the most strik-
ing feature in Table 3 is that 81 of the 93 motherhfant pairs that contracted listeriosis
were of Hispanic origin. Furthermore, many of these economically disadvantaged Hispan-
ics sought treatment at the Los Angeles County-USC Medical Center. Clustering of cases
at a single medical facility was instrumental in uncovering, this outbreak of foodborne
listeriosis, since this epidemic would have likely gone unnoticed if the cases had been
distributed evenly among the nearly 200 major hospitals in metropolitan Los Angeles.
As previously mentioned, epidemiologists and health officials used data collected
from two case-control studies to trace this outbreak first to1 Mexican-style fresh cheese
and then to Jalisco brand Queso Fresco and Cotija cheese. The fact that these strong-
flavored cheeses are a common part of the Hispanic diet and not widely consumed by
other individuals was another key in determining the exact source of this epidemic.
Thus recognition of this outbreak was largely possible because of the use of the
Los Angeles County-USC Medical Center by many economically disadvantaged listeri-
osis victims and the predominance of cases among Hispanics, which in turn precipitated
the involvement of Mexican-style cheeses (i.e., products which few other groups of indi-
viduals consume on a regular basis). Hence one can easily speculate that this clustering
of cases would not have been observed if a nonethnic food such as Cheddar cheese, milk,
and ordinary fruits or vegetables-all of which are consumed by most individuals-had
been contaminated with L. monocytogenes. Under such circumstances, an epidemic would
probably have gone undetected. Without the increased awareness that this outbreak
brought to the scientific community, additional cases of foodborne listeriosis, including
the 1987 outbreak linked to consumption of Vacherin Mont dOr soft-ripened cheese,
would likely have gone unnoticed, as the Swiss outbreak previously had for almost 10
years. Hence although Murray et al. [ 1671 can be credited with the first accurate description
of L. monocytogenes, those individuals who investigated the I985 listeriosis outbreak in
California (and to a lesser extent, outbreaks of listeriosis associated with coleslaw and
possibly pasteurized milk in Canada and Massachusetts, respectively) can be credited with
fostering the emergence of L. monocytogenes as a serious foodborne pathogen of world-
wide concern.
Once Jalisco brand cheese was positively identified as the vehicle of infection, local,
state, and federal investigators were confronted with the task of determining how the
cheese became contaminated (see Fig. 1). Additional testing of Jalisco brand cheese indi-
cated that L. monocytogenes was present in cheese manufactured from January to mid
June, thus indicating an ongoing problem at the cheese factory. Investigators then focused
their attention on three areas: (a) raw milk supply, (b) adequacy of pasteurization, and (c)
possible contamination of the cheese during manufacture, packaging, and/or ripening.
However, before interpreting the results from these investigations, it would be prudent to
deal with methods used to manufacture those varieties of Mexican-style cheese that were
directly linked to cases of listeriosis and also with behavior of L. monocytogenes in the
finished product.
Queso Fresco, or fresh cheese (also known as Ranchero, Estilo Casero, or Quesito),
is among the most popular and widely distributed Mexican-style cheeses. Unlike most
cheese varieties, Queso Fresco is traditionally prepared without a lactic acid bacteria starter
culture. Curd is formed by coagulating warm skim milk with rennet or a similar coagulant.
The resulting curd is drained in cheesecloth, salted, packed into hoops, and pressed under
weights for several days. The final product, which is consumed without additional aging,
has a slightly grainy texture and can be sliced or shredded for cooking.
Cotija, also known as Queso Sec0 (dry cheese) or Queso Anejo (aged cheese), is
another white cheese. After cutting, the curd is pressed in large round hoops and cured
at least 3 months to produce a dry, sharp-flavored, odorous cheese, which in some respects
resembles Italian Parmesan.
It is important to realize that these cheese-making procedures produce favorable
conditions for multiplication of L. monocytogenes in the final product. The relatively high
moisture content of these Mexican-style cheeses, and absence of a starter culture which
leads to pH values 1 5 . 6 in the finished product, both played crucial roles in allowing L.
monocytogenes to grow in the cheese during refrigerated storage [ 1211. According to Lee
Foodborne Listeriosis 3 15
[ 1421, surface and interior samples of frozen Jalisco brand cheese examined some months
after the recall contained 1.4 X 104and 5.0 X 104L. rnonocytogenes CFU/g, respectively,
which supports the hypothesis that the pathogen grew in cheese during refrigerated storage.
Numbers of listeriae increase approximately 10-fold during cheese making as a result of
entrapment within curd particles. If one assumes that the pathogen did not grow in cheese
during storage, then the milk from which the cheese was prepared would have had to
contain unreasonably high levels of listeriae (- 1000-5000 CFU/mL) to produce the popu-
lations observed by Lee in the finished product. It also is noteworthy that the epidemic
strain of L. rnonocytogenes that was designated as strain California (CA) by Ryser and
Marth [202] has since proven to be less hardy in Cheddar [202], Camembert [203], brick
[204], Colby (2401, feta [ 1791, and blue cheese [ 1781 than strains Scott A (clinical isolate
from the 1983 milkborne listeriosis outbreak in Massachusetts), V7 (raw milk isolate
from Massachusetts), and/or Ohio (OH) (isolated from Liederkmnz cheese manufactured
in Ohio).
Comprehensive sanitation inspections of the cheese factory were conducted immedi-
ately after the recall to assess the possibility that the cheese was contaminated during
manufacture, packaging, and/or storage. Although the factory received a satisfactory sani-
tation rating of 85 on a scale of 100, numerous problem areas were cited which included
suspended filth on electrical wires near cheese vats, peeling paint above a pasteurizer vat,
condensate dripping on cheese in a walk-in refrigerator, and a major ant infestation. Sev-
eral L. monocytogenes isolates from environmental samples (i.e., cooler condensate,
cheese curd, insects, pasteurizer) also yielded the same restriction enzyme profile as the
epidemic strain [235], thereby confirming several possible modes of transmission. Al-
though these environmental sources could have contributed to sporadic contamination of
the finished product, the fact that the epidemic strain was isolated from 22 of 85 lots of
cheese produced between January and mid June as well as from Cotija Fresco cheese
[235] indicates that an ongoing problem existed in the factory. Hence, contact between
cheese and the factory environment was likely not to be the major route of contamination
in this outbreak.
At the time of the recall, government officials considered faulty pasteurization as
one of the most likely means by which the cheese became contaminated. Initial factory
inspections uncovered various pasteurization problems related to record keeping and re-
cording charts; however, the time and temperature at which milk was pasteurized exceeded
minimum requirements (71.7C [16l0F]/15 s). Dye testing later revealed a number of
pin-sized holes in the pasteurization units heat-transfer plates which separate raw and
pasteurized milk. However, since further inspection demonstrated that the booster pumps
of the pasteurizer had maintained a higher pressure on the pasteurized rather than raw
milk side of the heat exchanger, raw milk would not have passed through the pinholes
found in the pasteurizer plates. Hence pasteurization failure was no longer suspected as
the source of contamination [7].
Final reports indicate that L. rnonocytogenes most likely entered the cheese during
manufacture through direct addition of raw milk. Toward the end of June 1985, investiga-
tors documented that the firm received nearly 700,000 pounds (- 10%) more raw milk
between April 1 and June 12, 1985, than could have been pasteurized given the capacity
of their pasteurizer. Additionally, on several days only 150,000 of 200,000 pounds of milk
received was pasteurized. These enormous discrepancies between raw milk received and
the quantity pasteurized suggest that unpasteurized milk was deliberately mixed with pas-
316 Ryser
teurized milk for cheese making [ 1451. This conclusion also is supported by the fact that
cheese supposedly prepared from pasteurized milk contained excessive levels of alkaline
phosphatase-a native, heat-labile enzyme normally destroyed during proper pasteuriza-
tion. However, some caution must be used in interpreting these results, since Pratt-Lowe
et al. [ 1871demonstrated that California Queso Fresco cheese occasionally contains micro-
organisms which produce a heat-labile alkaline phosphatase similar to that found in raw
milk. Under these conditions, cheese prepared from properly pasteurized milk may falsely
appear as having been manufactured from raw milk. Continued preparation of Mexican-
style cheese from a mixture of raw and pasteurized milk also is compatible with the pattern
of listeriosis cases that occurred over a period of 7 months. Toward the end of July 1985,
investigators visited 27 dairy farms that supplied raw milk to the cheese factory [ 145,1501.
Although no Listeria spp. were detected in raw milk or milk filters from dairy farms
supplying the Jalisco cheese factory, the same epidemic phage and restriction enzyme
type of L. monocytogenes was isolated from jocoque (a sour cream-like product), sodium
caseinate (a jocoque ingredient), and cottage cheese by-products produced by another
company that shared the same raw milk source with Jalisco cheese [235]. However, no
cases of listeriosis were epidemiologically linked to consumption of either of these prod-
ucts. According to Hird [121], the L. monocytogenes epidemic strain was uncommon in
California during the 1 1-year period preceding the outbreak, with only three to five L.
monocytogenes serotype 4b isolates belonging to the same epidemic phage type. Even
though the epidemic strain was never isolated from raw milk, evidence described in the
preceding paragraphs strongly suggests that raw milk was the probable source of L. mono-
cytogenes.
Major outbreaks of foodborne disease not only cause great human hardship but also
major financial difficulties from lost product and employee wages as well as medical bills
and lawsuits. Jalisco Mexican Products, Inc., was forced to close its doors and declare
bankruptcy shortly after its products were recalled, because the company could no longer
meet expenses. On March 27, 1986, Los Angeles County prosecutors filed 60 misde-
meanor charges against the president and vice-president of the company for alleged short-
cuts and inadequate safety precautions that routinely occurred in the factory during cheese
making [ 151. On May 20, 1986, the vice-president of the firm was sentenced to 60 days
in jail, 2 years of probation, and was fined $9300 in connection with manufacturing and
selling Listeria-contaminated cheese [ 141. In addition to investigative costs, which report-
edly totaled $617,204, the company also is believed to be facing up to $700 million in
lawsuits filed by some of the victims [225], making this one of the costliest and deadliest
outbreaks of foodborne illness in U.S. history.
After criticism for not recalling the contaminated Jalisco brand cheese sooner, state
and federal officials issued a Class I recall for Mexican-style cheese produced by a second
Los Angeles-area firm. Although this Listeria-contaminated cheese was supposedly pre-
pared from raw milk as shown by the presence of alkaline phosphatase [ 101, the recall
was subsequently downgraded to Class I1 (i.e., a situation in which use of the product
may cause temporary or medically reversible adverse health consequences) after labora-
tory results confirmed that these cheeses contained L. innacua-a nonpathogenic Listeria
species-rather than L. monocytogenes. Later investigators also showed that this cheese
was prepared from properly pasteurized milk. Hence, one must conclude that false-
positive results were obtained with the phosphatase test, as described earlier in this
chapter .
Foodborne Listeriosis 317
that included interviews with patients and a search for L. monocytogenes in several hun-
dred food items, neither the source nor the mode of Listeria transmission could be found.
Working under the assumption that a similar listeriosis outbreak was likely the fol-
lowing winter, public health officials initiated a case-control study using listeriosis cases
that were diagnosed in French-speaking Switzerland between November 1, 1984, and
April 30, 1985 [13,52]. Overall, 16 cases (7 adults and 9 motherhfant pairs) were identi-
fied and compared with 49 controls matched for age, sex, and underlying conditions.
Fifteen of 16 (94%) patients were infected with L. monocytogenes serotype 4b, with 5 of
16 (3 1%) isolates belonging to the same phage type. Although these five cases suggest a
possible epidemic focus, data obtained from questionnaires dealing with professional and
home exposure as well as types of food consumed (e.g., milk products and raw vegetables)
were inconclusive.
In response to the 1985 listeriosis outbreak in California linked to consumption of
Mexican-style cheese, Swiss officials initiated a series of surveys to determine the inci-
dence of Listeria spp. in different dairy products, the results of which are summarized in
Chapter 12. During one such survey of soft, semihard, and hard cheeses, Breer [62] iso-
lated L. monocytogenes from 5 of 25 surface samples of Vacherin Mont dOr, a soft,
smear-ripened cheese that is only manufactured from October to March and consumed
primarily in and around the Canton of Vaud. Subsequent test results indicated that all L.
monocytogenes isolates from Vacherin Mont dOr cheese belonged to serotype 4b and
also demonstrated that two L. monocytogenes phage types isolated from this cheese were
identical to most clinical strains isolated during the 1983- 1986 epidemic period. Investiga-
tors in Switzerland [52] then examined over 200 types of domestic and imported soft
cheeses, 8- 10% of which contained L. monocytogenes. However, based on serotyping
and phage typing, these strains as well as other food and dairy product isolates were
distinctly different from those found on the surface of Vacherin Mont dOr cheese.
A subsequent review of hospital records indicated that 122 listeriosis cases involving
57 adults (Table 4) and 65 motherhnfant pairs were diagnosed in the Canton of Vaud
between 1983 and 1987 [66] (epidemic rate of -50 cases/ 1O6 population/yr) as compared
with only 28 cases between 1974 and 1982 (endemic rate of -5 cases/106 population/
yr) [37]. Interestingly, 84% of the cases that occurred during the epidemic period were
identified between October and April. Thirty-four of 122 patients died, giving a mortality
rate of 28%, with 18 of 57 (32%) adult cases proving to be fatal.
Although the two previous case-control studies failed to uncover the source of this
epidemic, a third case-control study conducted in 1987 demonstrated that 31 of 37 (84%)
cases had consumed Vacherin Mont dOr cheese as compared with only 20 of 51 (39%)
controls [54]. In addition, investigators were able to isolate the epidemic strain of L. mono-
cytogenes from a piece of Vacherin Mont dOr cheese that had been partially consumed
by one of the victims. Armed with this information, Swiss authorities halted production
of Vacherin Mont dOr cheese on November 20, 1987, and recalled the product throughout
Switzerland [20,27,54].
Overall, 111 of 120 (93%) clinical isolates available from the epidemic period be-
longed to serotype 4b, with 98 of l l 1 (85%) serotype 4b strains matching the two epidemic
phage types that were isolated from Vacherin Mont dOr cheese. Several years later, clini-
cal and cheese isolates were found to be identical based on multilocus enzyme electropho-
resis [ 1711, pulsed-field gel electrophoresis [60,64], ribotyping [%I, restriction enzyme
analysis [64,235], randomly amplified polymorphic DNA patterns [74], and pyrolysis mass
Foodborne Listeriosis 319
TABLE
4 Description of 57 Adult Listeriosis Cases Diagnosed i n Western
Switzerland from 1983 to 1987
Manifestation
~ ~~~
spectroscopy [ 1011, thus confirming Vacherin Mont dOr cheese as the infectious vehicle.
Interestingly, this epidemic strain also is of the same phage type [60,64,1811, enzyme type
[56], ribotype [56], and pulsed-field gel electrophoretic type [60,64] as strains isolated
during the 1985 listeriosis outbreak in California [181]. However, two distinct DNA re-
striction endonuclease profiles [ 1721 were eventually identified among the Swiss epidemic
strains corresponding to epidemic phage types I and 11, with Eioerlin et al. [60] also divid-
ing the epidemic electrophoretic enzyme type into two major subtypes using pulsed-field
gel electrophoresis.
The fact that the Swiss and California epidemic strains are closely related to each
other and are also phenotypically and genotypically similar to strains involved in major
outbreaks traced to coleslaw, cheese, and p2t6 in Canada, Denmark, and France, respec-
tively [65,110,126,163], raises some interesting questions as to why many of the most
serious outbreaks in North America and Europe have been confined to this one particular
strain. When Boerlin and Piffaretti [59] examined 181 Swiss L. rnonocytogenes isolates
from clinical, veterinary, food, and environmental sources, the Swiss epidemic enzyme
type comprised 26.5 and 24.2% of all strains collected during (1983-1987) and after
(1988- 1989) the Swiss outbreak, respectively (Table 5). Furthermore, the epidemic en-
zyme type was widely distributed, with this strain being recovered from humans (clinical
and fecal samples), animals (clinical and/or fecal samples from cows, sheep, and goats),
meat/meat products, cheese/milk, and the environment (soil, silage). Since this particu-
320 Ryser
TABLE
5 Swiss Epidemic and Nonepidemic L. monocytogenes Enzyme Types
Recovered from Various Sources within Switzerland During 1983-1989
Source of L. monocytogenes strain
Humans Animals Meat/Meat products Cheese/Milk Environment
Enzyme type (n = 43) (n = 49) (n = 40) (n = 19) (n = 30)
Epidemic (n = 1 ) 13 24 1 4 6
Nonepidemic (n = 49) 3O/2Oa 25/16 39/14 15/13 241 I3
aNumber of nonepidemic straindnumber of nonepidemic enzyme types.
Source: Adapted from Ref. 59.
larly virulent strain is dominant in ruminants and since a similar strain was responsible
for cheese-related outbreaks of listeriosis in California and Denmark, recovery of this L.
rnonocytogenes subtype from foods has taken on added public health significance.
However, the exact importance of this particular electrophoretic enzyme type as
compared with others in foodborne listeriosis cannot yet be adequately assessed until more
information is available regarding the distribution of different L. monocytogenes subtypes
in nature along with the ability of these various strains to grow and/or survive in dairy
products and initiate disease in both humans and laboratory animals.
Most of the tainted cheese was marketed in Switzerland; however, small quantities
were exported to other countries, including England and the United States. Hence, on
November 25, 1987, health officials in England warned the general public against consum-
ing Vacherin Mont dOr cheese, which was available at a few delicatessens and specialty
cheese shops in and around London [1,21]. Similarly, FDA officials in the United States
became concerned after a major newspaper reported that five specialty shops in New York
City and a chain of 37 stores in Connecticut had been distributing the cheese since Novem-
ber 1987 [20]. These recall efforts were largely successful, since all known listeriosis
cases linked to consumption of this cheese were confined to Switzerland.
Immediately after the recall, Swiss authorities began investigating possible routes
by which Vacherin Mont dOr cheese could have become contaminated. According to
Bille [53,54], the cheese implicated in this outbreak was produced at 40 different factories
located in western Switzerland. All contaminated cheese was reportedly prepared from
Listeria-free cows milk. Following coagulation of milk, the resulting curd was dipped
into wooden hoops and allowed to drain for 1-2 days. When thoroughly drained, the
hooped cheeses were transported to 1 of 12 cellars (i.e., caves) located throughout western
Switzerland and ripened for -3 weeks on wooden shelves during which time the cheeses
were turned daily and brushed with salt water. Once ripened, the cheeses were packaged
for sale and the wooden hoops were returned to the cheese factory.
From this description, it is apparent that ample opportunity existed for contamination
of Vacherin Mont dOr cheese, particularly during ripening. In fact, the epidemic strain
[5] was detected in 18.5% of surface (rind) samples from Vacherin Mont dOr cheese at
levels of 104-106 L. rnonocytogenes CFU/g and also on 6.8% of wooden shelves and
19.8% of brushes used in the ripening cellars.
In all likelihood, this outbreak began several years earlier when L. monocytogenes
entered one of the 40 cheese factories in raw milk from an infected dairy herd [59]. Al-
though this outbreak was first detected in 1983, the epidemic strain was initially isolated
from a listeriosis victim in 1977, which suggests that this outbreak may have been devel-
Foodborne Listeriosis 321
oping for at least 7 years. Investigations showed that nearly hall' of the 12 ripening cellars
were contaminated with one or both epidemic strains of L. monocytogenes, thus suggesting
cellar-to-cellar spread of the pathogen through production and distribution practices. This
theory is strongly supported by the fact that cheeses produced at all 40 factories were
normally transferred between different cellars for ripening and/or distribution. The prac-
tices of brushing cheeses with salt water, ripening cheese in wooden hoops, and returning
these hoops to the cheese factory also were important factors in disseminating L. manocy-
togenes to different ripening cellars.
Following the recall, all 40 factories in which Vacherin Mont d'Or cheese was manu-
factured were thoroughly cleaned and sanitized. More important, all wooden material (e.g.,
shelves, boxes, hoops) was removed from ripening cellars anti burned, The cellars were
then thoroughly cleaned, sanitized, and refitted with metal shelves and easily sanitized
equipment. Once this work was completed, experimental batches of Vacherin Mont d'Or
cheese were produced during a 2-month period and examined for the epidemic strain of
L. monocytogenes to assure government officials that the pathogen was eliminated from
all ripening cellars. These clean-up efforts proved to be highly successful, with only two
cases of listeriosis being reported in western Switzerland between January and September
of 1988 [53]. Although both of these cases resulted from nonepidemic strains, multilocus
enzyme electrophoresis later demonstrated that 45 of 145 (28%) human clinical and 44
of 116 (37%:) animal strains of L. monocytogenes isolated in Switzerland between 1988
and 1993 belonged to the previously identified epidemic enzyme type (601. After further
analysis by pulsed-field gel electrophoresis, 34 of these 26 I ( 1 3%) humadanimal isolates
matched the epidemic strain recovered from Vacherin Mont d"Or cheese, thus suggesting
a continued presence of this strain in the natural environment.
As a rr:sult of this outbreak which by one account cost an estimated $1.4 million
[232], several steps have been taken to control and limit the extent of listeriosis in Switzer-
land [52,53]. First, health authorities are systematically screening high-risk foods for L.
monocytogenes and have adopted a zero tolerance for the pathogen in 10-g samples. Sec-
ond, physicians and laboratories are now required to notify health officials of every new
case (i.e., clinical isolate) of listeriosis occurring throughout Switzerland. Finally, since
1990 the Swiss National Center for Listeriosis in Lausanne has been actively collecting
human, animal, food, and environmental isolates and further characterizing these Listeria
strains according to serotype, phage type, ribotype, enzyme type, DNA restriction pattern,
and pulsed-field gel electrophoresis profile. These efforts will serve to identify the exact
endemic rate of human listeriosis in the general population and lead to faster recognition
of possible future listeriosis outbreaks as well as the vehicleis involved.
meningitis ( IS cases) and septicemia ( 8 cases). Six of 23 adults died while hospitalized,
giving a mortality rate of 26%.
Evaluation of 90 food history questionnaires given to epideriiic/nonepideinic cases
and matched controls showed a clear epidemiological link between consumption of Danish
blue-mold cheese and cases of listeriosis, with one brand of cheese being cited in particu-
lar. Unfortunately. investigators were unable microbiologically to confirm blue-mold
cheese as the vehicle of infection. However, one year earlier. the FDA issued a Class I
recall for L. rizorzoc.?.togc.rzr.s-contaminated Danish blue cheese that had been shipped to
the United States [ 23,251. Routine dairy inspections also indicated that the epidemic strain
was present in the dairy environment as evidenced by recovery of this strain from eight
different Danish hard cheeses. The fact that these extremely popular hard cheeses were
consumed by more than 90% of both patients and controls makes these cheeses another
plausible vehicle of infection. Inability to recover the epidemic strain from other packaged
foods lends additional support for involvement of Danish blue and hard cheeses. Follow-
up studies showed this L. rizorzoc:\~to,~crzes strain to be of international importance, since
the identical phage type also was responsible for two major foodborne outbreaks in Swit-
zerland (Vacherin Mont dOr cheese, 1983- 1987) and France (jellied pork tongue. 1992).
ing. with molecular typing methods such as pulsed-field gel electrophoresis and ribotyping
being used only in epidemiological investigations. Using this surveillance system, three
minor outbreaks involving less than 16 cases were detected from 1987 to 1992, including
one in the Strasbourg area [ 143,196.198j. More important, three major foodborne listeri-
osis outbreaks were identified in 1992, 1993. and 1995. the most recent of which was
traced to Brie de Meaux cheese.
Between April 2 and 19 of 1995. the NRC received six L. rriorioc\tos:eiies human
isolatcs from hospitals in different regions of France which were soon identified as belong-
ing to an unusual phage type that had been previously responsible for only 33 cases of
listeriosis ( 1 [ < 1 % I to 8 [ 3 % ] cases annually) since 1987 [ 109,126). These findings
prompted the NRC to inform the Ministry of Health on April 28 about a possible outbreak.
A search of NRC records indicated that the same phage type had been recovered from 5
of 2200 food samples ( 1 delicatessen product, 4 Brie de Meaux cheeses) tested from
January to April. Further characterization of these strains by pulsed-field gel electrophore-
sis indicated that the six patient and four cheese isolates were identical.
Based on follow-up epidemiological investigations, all victims identified as of May
10 consumed Brie de Meaux cheese (a raw milk soft cheese) that had been cut and pur-
chased at either supermarkets or small local markets [ 1091. Additional investigative work
by the Ministry of Agriculture coupled with a case-control study soon implicated one
particular brand of Brie de Meaux cheese. with this product being recalled from the market
on May 18. Additional cheeses exported to Belgium were also recalled without incident
shortly thereafter [ 2241. Isolation of the epidemic phage type and pulsed-field gel electro-
phoresis type from eight Brie de Meaux cheeses and several other cheeses ripened at the
same facility confirmed the role of Brie de Meaux cheese in this outbreak [ 126,235). At
the time of the May 18 recall. 20 epidemic cases were documented (Fig. 5 ) , with the
victims residing in 8 of 22 French regions. Of these 20 cases, 1 I among pregnant women
led to two spontaneous abortions, four premature deliveries, and two stillbirths, with the
remaining nine cases involving immunocompromised adults (7 cases) and the elderly
(2 cases).
By the time this outbreak finally ended in July of 1995, 13 additional cases were
confirmed by phage typing and pulsed-field gel electrophoresis, bringing the total number
of cases to 33. Interestingly, this outbreak strain belonged to a new epidemic clone which
was clearly distinct from the two L. monocytogenes strains responsible for the other major
outbreaks in Canada (coleslaw 1981 ), California (Mexican-style cheese I985), Switzer-
land (Vacherin Mont dOr cheese 1983- 1987), United Kingdom (pGt6 1987- 1989), Den-
mark (blue-mold/hard cheese 1989- 1990), France (jellied pork tongue I992), and France
(pit6 1993) which either have been or will be discussed shortly.
In 1997, the NRC also identified 14 listeriosis cases over a 4-month period that were
directly traced to Pont IEvEque cheese produced in Normandy [241]. The implicated
cheese was manufactured from raw milk and contained L. monocytogenes serotype 4b at
a level of > 1000 CFU/g. Although some of this cheese was exported to Sweden, no
additional cases were reported.
serotype 1/2a recovered from the patients cerebrospinal fluid and cheese were subse-
quently found to exhibit different DNA restriction enzyme patterns, thus negating cheese
as the vehicle of infection.
MEAT PRODUCTS
Foods of animal origin have long been recognized as potential vehicles of infection, with
meat-associated cases of salmonellosis and botulism being recorded in the scientific litera-
ture since the 1890s. Following confirmation of L. monocytogenes as a human and animal
pathogen during the 1920s, listeriosis was subsequently identified as a zoonosis, a disease
transmissible from animals to humans. Hence, when listerial infections in domestic live-
stock began to emerge with some regularity during the 1930s and 1940s, some individuals,
including Wramby [238), who in 1944 first identified Listeria in raw meat, began to specu-
late that consumption of meat products could play a role in the spread of human lister-
iosis.
Listeria-laden fecal material from asymptomatically infected livestock can readily
enter the slaughterhouse environment and contaminate retail raw meats. Meat processors,
veterinarians, and others who work closely with animal carcasses will also inevitably come
in contact with L. monocytogenes as evidenced by recovery of this pathogen from the
hands and gowns of Czechoslovakian line workers during the mid 1970s [86,87]. Although
evidence is somewhat conflicting, most of the earlier studies previously reviewed by Ryser
and Marth [205] also indicated that increased exposure to L. monocytogenes in the meat
industry can lead to higher fecal carriage rates among workers. According to Elischerova
and Stupalova [86,88], six clinically healthy Czechoslovakian meat workers who had an
opportunity to consume crude and semicrude product during work were asymptomatic
fecal shedders of L. monocytogenes. These same individuals also exhibited elevated 0
and H serum agglutination titers against L. monocytogenes serotype 1, which is compatible
with oral transmission of Listeria via meat products.
Since two of the three meatborne listeriosis outbreaks identified thus far have in-
volved consumption of pit&-a ready-to-eat meat, fish, or vegetable product that is com-
monly marketed in Belgium, France, Germany, the Netherlands, and the United Kingdom
and is consumed without reheating or further cooking, it is appropriate briefly to review
the manufacture and safety-related issues surrounding this product. Preparation of meat
pit& most often involves chopping pork liver with water, seasoning, salt, and sodium
nitrite. This raw product is then either (a) cooked in a mold, decorated, sliced, and sold
as loose or vacuum-packaged pit&;or (b) cooked in small hermetically sealed containers.
The high water activity and pH of typical pi& provide an ideal growth environment for
most bacteria, including L. monocytogenes, which has an estimated doubling time of 19
h in pit& stored at 7C [76]. Hence, thorough cooking of the raw product, addition of
preservatives, and proper packaging are all essential to preventing growth of listeriae to
dangerously high levels in this food during 3 or more weeks of refrigerated storage. With
this background information in mind, the role of pit6 in one of the largest listeriosis
outbreak thus far reported will now be assessed.
Numerous reports suggesting possible involvement of meat products in human lister-
iosis can be found in the scientific literature with over 60 primarily sporadic cases docu-
mented since 1955. However, the ability of meat products to serve as vehicles of listerial
infection was not fully realized until the late 1980s when consumption of pit&was deemed
responsible for over 350 cases of listeriosis in the United Kingdom. As a result of an
Foodborne Listeriosis 327
300
250
I
$ 1
U)
200
%
0
b
-.I f \
a
E 150
\\\
3
z
O L_ _ ~ ~ - ~ _ _ _ _ J
198384 85 86 87 88 89 90 91 92 93 94
Year
FIGURE6 Reported cases of human listeriosis in England, Wales and Northern Ire-
land, 1983-1994. (Adapted from Ref. 155.)
328 Ryser
L. monocytogenes strains classified as serotype 4b phage type 6,7 and serotype 4b(x) were
responsible for 366 of 823 cases reported during this period [ 1951, with these two strains
being far less common before 1987 and after July 1989 (Fig. 7). Thereafter, the number
of human listeriosis cases decreased sharply to pre- 1987 levels.
During a routine food poisoning investigation in May of 1989, the Cardiff Public
Health Laboratory identified high levels of L. monocytogenes in pit6 taken from one vic-
tim's refrigerator. This chance finding prompted an immediate survey of primarily im-
ported pit6 sold from delicatessen display counters throughout southeast Wales between
May and August of 1989 [164,165]. Overall, L. monocytogenes was recovered from 75
of 216 (35%) pit& tested at levels ranging from <20 (42 samples) to >104 (10 samples)
CFU/g. More important, however, 32% of all positive samples harbored L. rnonocytogenes
serotype 4b(x), which was responsible for the aforementioned cluster of 23 cases identified
2 years earlier.
Results from this Welch survey prompted two actions in July of 1989: (a) a govern-
ment health warning to vulnerable individuals about eating pit6 [78] and (b) a far more
extensive survey [76] in which 1698 samples of pSt6 marketed in England and Wales
were examined for listeriae. Overall, I86 ( 10%) samples contained L. monocytogenes at
levels ranging from <200 (12 1 samples) to > 106(3 samples) CFU/g, with 37 of the I86
positive samples harboring > 103CFU/g. Investigators also noted that L. monocytogenes
levels were generally higher in (a) pSt6 prepared from fish rather than meat, (b) loose
slices rather than prepackaged pate, (c) samples marketed at >7"C, (d) pit6 tested at or
beyond the sell-by date, and (e) samples having standard bacterial plate counts of >106
CFU/g, with these findings supporting the reported ability of this pathogen to grow in
p2t6 at near-refrigeration temperatures. More important, however, 5 1 of 107 (58%) piit6s
produced in Belgium by manufacturer Y contained L. monocytogenes, with 12 of these
samples yielding >103CFU/g. Follow-up investigations [ 1591 showed that 96% (48 of 50)
of all L. monocytogenes isolates from manufacturer Ys pGt6 belonged to either serotype 4b
phage type 6,7 or serotype 4b(x), with these two strains being responsible for 30-54%
of all human listeriosis cases reported during the epidemic period. (Fig. 7). In contrast,
only 19% (6 of 31) of pitis from other producers contained these two strains, with cross
contamination among pit& handled at delicatessen counters likely contributing to appear-
ance of these otherwise rare strains. A subsequent epidemiological investigation revealed
that 13 of 15 patients infected by these epidemic strains had consumed pit6 within 3
weeks of oriset compared with 6 of 17 patients infected with nonepidemic strains.
Illness was strongly associated with piit6 consumption; however, pit6 samples were
no longer available from the victims refrigerators to microbiologically confirm this food
as the vehicle of infection. Nevertheless, all available evidence, along with the fact that
the reported decline in listeriosis cases after mid 1989 coincided with both a government
warning concerning pit6 consumption and removal of manufacturer Ys pit6 from sale,
clearly points to this particular brand of imported pit6 as being responsible for the outbreak
observed from 1987 to mid 1989. A much lower incidence of L. monocytogenes in retail
p2t6 samples tested in 1990 as compared with 1989, along with the virtual absence of
both epidemic strains in pit6 and other foods examined after 1989 [ 1051, further support
involvement of manufacturer Ys pit6 in this outbreak.
nieat products. 3 1 milk s~implcs.and 10 cnvironiiicntal samples. Among the 279 cliniciil
isolates. 249 strains exhibited the same pulsed-field gel clectrophoretic profile. with this
epidemic strain also subsequently being confirmed in I 12 saniples of jellied pork tongue
;is \veil ;is i n 19. 13, and 1 1 samples of other meat products (i.e.. ham. pit&. sausage).
cheeses. and miscellaneous foods. respectively. Furthermore. the epidemic strain was most
closely associated Lvith brand A jellied pork tongue. with high numbers being recovered
from st\re 11prc \io i i s 1y 11no pc ned con t ai ners and si x sam p 1c s s I iced at de 1i cate sse 11coii n tcrs .
Seve r;i 1 e n v i ron me 11t ;I 1 sam p 1es from brand A * s m ;in 11fac t u ri ng frici 1i ty e \re n tu a11y y i e I dcd
the epidemic strain [ 1271. with raw brine being identified iis the niost probable soiirce of
contamination during iii:iii~ifiictiire [ 207 I. These findings and results from the earlier c;ise-
control studies contirni brand A jellied pork tongue ;is the primary \.ehicle of infection.
w i t h ot he r cross con t am i 11;it ed foods at de 1i cat c sse 11 coil 11t er5 pre s i i 111;i b 1y ser\.i ng iis sec-
ondary \vehicles in approsiniately 19% of cases [ 1081. Support for the latter also comes
fro111 ii s 11b s c q 11e 11t c ;i se - c on t ro 1 s t LIct y i n which ;i st at i st i c a1 assoc i at i o11 LV ;is demonst r;i t c d
b e t ~ ~ eillnessii in patients who did not consume .jellied pork tongue and contact between
brand A jellied pork tongue and other foods at the delicatessen counter [ I08 I . Furthermore.
the epidemic strain was isolated from iitensils iised in slicing both brand A jellied pork
tongue and other delicatessen meats [ 107.197 1.
Based 011 phage typing. pulsed-tield gel electrophoresis. ribotyping. and multilocus
e11z y me e I ec t ro phore s i s. t h i s c pi de 111i c s trai 11 is phc 11ot y pi cal I y and ge 110tJ.1~ i c all y s i 111i 1;ir
to s t rai 11s re s po 11s i bl e for t he a f ore men t i oned chee se - re 1at ed 011 t b rcaks i 11 Cal i fo rn i ;i. Den-
111ark. id S\v i t ze 1.1 a11d. Th i s o bscr\.at i o n agai 17 con ti riii s that most 11i:i.j or 1is teri o s i s out-
breaks appear to be caused b!, ;i small group ot closely related strains. Gi\.cn the low
;it t ;ic k r:i tc and w i de ?cograph i c al d i s t ri bu t i 011 of 1i s te ri osi s cascs. con t i 11ued o ngo i rig sur-
i.eillance at the national Ic\.el is necessary for detection of future to,odbornc listeriosis
011t breaks.
Foodborne List eriosis 33 1
food isolates received at the NLRC since October of 1993 have matched the epidemic
phage type and pulsed-field gel electrophoresis type, respectively, thus signaling the end
of this most recent outbreak of meatborne listeriosis.
Other than the three major outbreaks just discussed, the scientific literature primarily
contains only circumstantial evidence linking or, in some instances, only suggesting
involvement of meat products in cases of human listeriosis (Table 6). Beginning in 1955,
consumption of contaminated pork (probably undercooked) was suggested as the possible
cause of 27 listeriosis cases in the former Soviet Union [115,140]. The following year,
Gudkova et al. [ 1161 isolated L. monocytogenes from the viscera of pigs on a Russian farm
where several individuals contracted listerial infections, presumably after ingesting pork
from an infected group of pigs. In 1960, Olding and Philipson [ 1751 investigated one adult
and three perinatal cases of listeriosis that occurred within a three-block area of Uppsala,
Sweden, during the previous 2 years. Although repeated attempts to isolate L. monocyto-
genes from water, milk, vegetables, and meat ended in failure, the fact that meat was the
only food item obtained from the same source by all four individuals suggests the possible
involvement of unspecified meat products in this apparently common-source outbreak.
In the only other early recorded incident involving meat products from domesticated
animals, ground meat from a dead calf was suspected of transmitting L. monocytogenes
to the wife of a Dutch farmer in the early 1960s [ 1351. Although involvement of meat in
this case of listeriosis appears plausible, the remainder of the suspected meat was sterilized
during canning, thus eliminating any hope of confirming the causative agent.
During a review of listerial infections in Canada over a 21-year period, Bowmer et
al. [61] uncovered one case in which a pregnant woman in Newfoundland delivered an
infant who died 1 month later from listerial meningitis. Ten days before the infant became
ill, the mother recalled skinning, cooking, and eating two previously frozen hares that
were brought from New Brunswick, thus suggesting rabbit meat as a possible vehicle of
infection. Although less commonly consumed, it appears that rabbit meat also may serve
sumption of such products poses no risk of listeriosis. Several shortcomings of this retro-
spective case-control study were echoed by the scientific community [26], including, (a)
omission of questions concerning cooking methods and consumption of foods such as
seafood that until recently have seldom been associated with listeriosis, (b) limited ability
to identify risk factors when exposure was very common or very rare, and (c) difficulty
in obtaining accurate diet histories with the possibility of cases more clearly recalling
what they consumed before their illness than controls. Nonetheless, results from numerous
microbiological surveys (see Chaps. 13 and 14), along with a report by the American
Meat Institute indicating that 5- 10% of prepackaged frankfurters produced in the United
States were contaminated with L. monocytogenes [22], support the possibility of con-
tracting listeriosis from consuming uncooked frankfurters or undercooked chicken as was
suggested in the case-control study by Schwartz et al. [214] and a similar case-control
study [212] reported by the CDC several years later. During their work, these researchers
[31,2141 also identified another processed meat product consumed without further cook-
ing, namely salami, as a possible risk factor in a 1987 listeriosis outbreak in Philadelphia
that claimed 14 lives. However, CDC officials again lacked the bacteriological data to
positively link consumption of the salami to illness. In a subsequent case-control study
[ 1821, L. monocytogenes strains of the same electrophoretic enzyme type were recovered
from two patients and two unopened packages of pork sausage and ground beef that were
epidemiologically linked to illness. However, inability to recover and test these products
from patients refrigerators prevented CDC investigators from positively confirming the
vehicle of infection.
As mentioned earlier, piX, jellied pork tongue, and pork pgt6 rilletes were re-
sponsible for three major meat-related listeriosis epidemics in England and France, includ-
ing two of the largest outbreaks of foodborne listeriosis recorded worldwide (see Table
1). However, it must be stressed that as of July 1998, no American-produced raw, cooked,
or otherwise processed meat product has been conclusively proven as the vehicle of infec-
tion in any case of human listeriosis. Although it is important to remember that such a
causal relationship can only be shown conclusively by isolating the identical L. monocyto-
genes strain from the patient, product consumed, and unopened packages of the implicated
food, numerous North American and European surveys have uncovered low to moderate
levels of L. monocytogenes in a wide range of commercially available raw, processed, and
ready-to-eat meat products (see Chap. 13). Even before Schwartz et al. [31,2141 announced
preliminary results from their study, the meat industry [29] maintained that susceptible
individuals who consume Listeria-contaminated dry sausage, frankfurters, luncheon
meats, and other packaged pasteurized products are at low to moderate risk of contracting
listeriosis.
Since 1988, eight isolated cases and one small outbreak of listeriosis have occurred
worldwide where meat products were suspected as the most likely vehicle of infection
(see Tables 1 and 5 ) . In the first such case, a previously healthy Italian man contracted
nonfatal meningitis several days after consuming cooked homemade pork sausage that
was later shown to contain -3 X 106L. monocytogenes CFU/g [70]. According to investi-
gators, the clinical and sausage isolates were both identified as belonging to serotype 4,
the most common serotype encountered in clinical cases of listeriosis. Unfortunately, the
exact source of contamination was never determined; however, antiquated sausage-making
practices and storage of sausage at ambient rather than refrigeration temperature were
cited as major contributing factors in this isolated case of listerial meningitis. Nevertheless,
although numbers of listeriae present in this sausage were probably more than sufficient
Foodborne Listeriosis 335
to induce illness, some caution still must be used in evaluating the role of sausage in this
case, since both isolates were never characterized beyond serotype.
Four of these unconfirmed cases of possible meatborne listeriosis have been recorded
in the United States and include (a) a 76-year-old man from Spokane, Washington, who
died from an L. rnonocytogenes serotype 4b infection after consuming cooked ground
beef; however, only serotype l a was recovered from the ground beef thus making it an
unlikely source of infection [34], (b) a case-control study in which identical L. monocyto-
genes electrophoretic enzyme types were recovered from two patients as well as retail
packages of pork sausage and ground beef [212], and (c) an incident in which L. monocyto-
genes serotype 4b was isolated from cooked Cajun pork sausage that was consumed by
an elderly San Francisco man who developed a nonfatal case of listeriosis [36]. Approxi-
mately 1000 pounds of this sausage were subsequently recalled from the market after
investigators recovered L. rnonocytogenes serotype 4b from similar unopened packages.
Even though the patient and sausage isolates were not further classified, isolation of the
same L. monocytogenes serotype from unopened packages of sausage and the ability of
investigators presumably to trace the source of contamination to natural sausage casings
imported from China [83] provides reasonably convincing evidence that Cajun pork sau-
sage was directly responsible for this case of foodborne listeriosis. The remaining listeri-
osis cases (Table 6) as well as an outbreak which included six stillbirths or mid term
miscarriages among 11 pregnant women (Table 1 ) were identified in Australia during
routine surveillance programs with processed meats and piit6 cited as possible vehicles of
infection [ 139,162,2341 based on incomplete laboratory andlor epidemiological findings.
Continued surveillance of listeriosis cases by CDC officials uncovered a direct link
between consumption of contaminated turkey frankfurters and listerial meningitis in an
Oklahoma breast cancer patient [47] (to be discussed shortly) and also led to a nationwide
recall of the product [35] along with radical changes in the U.S. Department of Agricul-
ture-Food Safety and Inspection Service (USDA-FSIS) policy regarding the presence of
L. rnonocytogenes in cooked, ready-to-eat, or otherwise processed meat and poultry prod-
ucts. In the light of this information, some public health officials are now advising high-
risk individuals (i.e., pregnant women, immunocompromise:d adults, and the elderly) to
thoroughly reheat previously cooked and chilled meat and poultry products before con-
sumption. Hence, the proven ability of L. rnonocytogenes to grow and/or survive in many
refrigerated raw, processed, and ready-to-eat foods, including meat and poultry products,
together with extensive food histories now being obtained from many listeriosis victims
in the United States, make it highly probable that meat products, particularly frankfurters
and ready-to-eat meats, will be positively linked to cases of human listeriosis in the future.
POULTRY PRODUCTS
Shedding of L. rnonocytogenes in fecal material from both clinically and subclinically
infected domestic fowl 1791 appears to place poultry workers at a somewhat higher than
normal risk of contracting superficial listerial infections, particularly conjunctivitis. This
probable association between handling infected poultry and contracting conjunctivitis is
partially based on a 1951 report by Felsenfeld [96], who, 7 years earlier, identified listerial
conjunctivitis in two employees who dressed poultry in Illinois. On further investigation,
L. rnonocytngenes was isolated from the spleens of five birds that were not dressed in the
same shop but came from an area in Illinois in which avian listeriosis was previously
observed, thus suggesting poultry as the probable source of infection. Although reports
336 Ryser
of listerial conjunctivitis can be found in the early scientific literature, including several
cases in which patients had contact with birds suffering from undetermined illnesses [ l 111,
the 1951 report by Felsenfeld [96] remains one of the few instances where avian listeriosis
was linked to listerial conjunctivitis in humans.
A search of the early scientific literature has uncovered only two reports indicating
that contact with infected poultry may lead to the systemic infections for which L. rnonocy-
togenes is best known. In 1958, Gray [ 1111 cited numerous instances in which Central
European women gave birth to Listeria-infected infants following contact with sick or
dead birds; however, evidence for the link between listeriosis and contact with infected
poultry was only circumstantial.
Similarly, Embil et al. [89] identified a woman in Nova Scotia, Canada, who gave
birth to an infected infant who died of listeriosis 1 h after delivery. Although the mother
reportedly prepared poultry for sale in a family-owned store during the previous 8 months,
researchers again failed positively to link this listeriosis case to contact with raw poultry
by not isolating the pathogen from raw chickens sold at the store.
Given the preceding evidence, Kampelmacher [135] suggested as early as 1962 that
consumption of contaminated poultry might lead to cases of human listeriosis. Although
this view also was voiced 10 years later by Mir6 and Ralovich [159a], transmission of
L. monocytogenes from contaminated poultry was not documented until November 1988
[137]. As was true for meat products, failure positively to link consumption of contami-
nated poultry to human listeriosis was until recently primarily related to difficulties in
isolating L. monocytogenes from poultry and other foods containing a complex microflora
and to a generalized lack of concern about foodborne listeriosis.
Following the two major cheese-associated outbreaks in 1985 and 1987, public
health officials in the United States and England implemented active/semiactive surveil-
lance programs to obtain more accurate data on the incidence of listeriosis in the general
population. Attempts also were made to trace the source of reported infections to consump-
tion of dairy products and other foods such as poultry which at the time had not yet been
linked to listeriosis. As a result of these efforts, three cases of listeriosis were positively
linked to consumption of poultry products, which, in turn, has led to inclusion of poultry
in the list of foods that may pose a potential threat of listeriosis to susceptible individuals.
These three recently recognized cases will now be reviewed in some detail.
Worlung in England, Kerr et al. [33,137] identified the first case of listeriosis clearly
linked to consumption of contaminated poultry. According to their November 1988 report,
a 3 1-year-old pregnant woman with a 24-h history of flu-like symptoms was admitted to
a hospital and subsequently delivered an aborted 23-week-old fetus. On further investiga-
tion, the woman reportedly consumed a heated chicken dish prepared from cooked-and-
chilled chicken 5 days before onset of symptoms, with the remaining chicken being refrig-
erated and consumed 3 days later in a salad. Thus the woman had a maximum incubation
time of only 4 days before onset of symptoms as compared with the more typical 7-
30 days for listeriosis. Following bacteriological analysis, an identical phage type of L.
monocytogenes serotype 4 was found in samples of chicken and fetal liver. Other foods
in question were tested, with no evidence of Listeria contamination, thus confirming
chicken as the vehicle of infection.
Considerable research and regulatory activity, prompted by reports suggesting that
12-25% of cook-chill poultry products marketed in England may be contaminated with
L. rnonocytogenes, uncovered a second case of poultry-associated listeriosis early in 1989.
According to this report [134], L. rnonocytogenes serotype 1/2a was cultured from the
Foodborne Listeriosis 337
blood of a 52-year-old immunocompromised woman who was receiving steroids for sys-
temic lupus erythematosus. Three to 5 days before onset of vomiting and diarrhea, the
hospitalized woman and her 29-year-old son shared some ready-cooked chicken nuggets
which he had purchased at a fast-food restaurant. Detailed questioning later revealed that
he experienced a short-lived illness with diarrhea and vomiting on the same night that his
mother became sick. Subsequently, the sons stool sample yielded L. innocua as well as
L. rnonocytogenes serotypes 1/2a and 1/2c, with the DNA homology pattern of the sero-
type 1/2a isolate being identical to that of the L. rnonocytogenes strain originally isolated
from the womans blood. Although L. innocua and an L. rnonocytogenes strain of unre-
ported serotype and DNA homology pattern were recovered from uncooked chicken nug-
gets, these investigators failed to detect L. rnonocytogenes in a subsequent lot of cooked
chicken nuggets obtained from the same source. Nonetheless, infection in both the woman
and her son presumably was acquired from commercially cooked chicken nuggets of the
fast-food variety, which, although served hot, were most likely undercooked, thus allowing
L. monocytogenes to survive in sufficient numbers to cause illness.
Although this is only the second case of poultryborne listeriosis recorded in England,
similar cases have likely gone undetected because of inadequacies in reporting and diffi-
culties encountered in linking these illnesses to consumption of poultry or any other food.
These two cases of poultryborne listeriosis and a recent survey of listeriosis cases in Scot-
land which included identification of possible food-related risk factors associated with the
disease [68] prompted the Public Health Laboratory Service (PHLS) to conduct a national
case-control study in England and Wales which attempted to correlate consumption of
high-risk foods (i.e., poultry, pitt5, cheese, prepared salads, delicatessen items) with human
listerial infections [ 1181. A total of 124 cases diagnosed from July 1990 to January 1992
were identified from both the national voluntary reporting laboratory system and the PHLS
Listeria reference laboratory and matched to 459 controls according to age, sex, underlying
illness, and pregnancy status. After obtaining dietary histories, undercooked and ready-
cooked chicken consumed either hot or cold were statistically related to development of
listeriosis in both pregnant and nonpregnant individuals. Additional epidemiological stud-
ies are needed in England, the United States, and elsewhere to expand the number of
reported foodborne listeriosis cases and generate a more comprehensive list of foods that
pose a significant public health threat.
Despite the controversial nature of many epidemiological studies, such efforts have
already played an important role in identifying possible risk factors associated with food-
borne listeriosis. As you will recall from our aforementioned discussion of meatborne
listeriosis, undercooked chicken was identified as a high-risk vehicle of infection by CDC
officials during several case-control studies [ 182,212,2141 conducted in conjunction with
an active listeriosis surveillance program in Oklahoma and five other states. In two spo-
radic cases traced to turkey frankfurters and sliced turkey ham [ 1821, L. rnonocytogenes
isolates from unopened packages of the same product brand belonged to the same electro-
phoretic enzyme type as the patient isolate, thereby implicating turkey frankfurters and
sliced turkey ham as the source of infection.
During the first of these surveillance programs, CDC officials learned of a breast
cancer patient in Oklahoma who had been infected with L. rnonocytogenes and hospitalized
for listerial septicemia and meningitis in December 1988 [35,47]. In an attempt to identify
the vehicle of infection, investigators went to the womans home, obtained foods from her
refrigerator, and eventually isolated Listeria from various products, including an opened
package of turkey frankfurters that contained > 1.1 X 103L. rnonocytogenes CFU/g. A
338 Ryser
swab sample from the refrigerators interior also yielded the pathogen. Although CDC
investigators initially concluded that the woman had contaminated the food herself, public
health officials from Oklahoma began examining the same brands of retail products from
the womans refrigerator that were positive for L. monocytogenes. Interest soon focused
on turkey frankfurters after officials learned that the woman consumed one turkey frank-
furter daily after 45-60 s of heating in a microwave oven. Four months later, the same
strain and isoenzyme type of L. monocytogenes serotype 1/2a recovered from this patient
and the opened package of turkey frankfurters was also identified in five of seven unopened
packages of identical product purchased from nearby stores [243a], thereby confirming
turkey frankfurters as the vehicle of infection in the first poultryborne listeriosis outbreak
recorded in the United States.
Once the USDA-FSIS was notified of this case by the CDC on April 14, 1989,
government officials prompted the Texas manufacturer to issue an immediate recall for
approximately 600,000 pounds of turkey frankfurters that were marketed by retail and
institutional establishments in 23 states [35]. Joint investigations initiated by the CDC and
USDA-FSIS 1 day later eventually showed that six of seven retail lots of product produced
over a 37-day period contained the implicated L. monocytogenes strain at a most probable
number (MPN) level of <0.3 CFU/g. Furthermore, environmental testing of the produc-
tion facility showed that only 2 of 40 samples taken before sausage peeling harbored L.
monocytogenes as compared with 12 of 14 samples taken after peeling, with the implicated
strain also being recovered from a conveyor belt attached to the peeler. These findings
suggest that the factory was experiencing an ongoing contamination problem at a single
point during the sausage peeling process. Although large quantities of product contained
relatively few listeriae, the ability of L. monocytogenes to grow to hazardous levels in
such processed poultry products during refrigerated storage indicates that special precau-
tions must be taken to eliminate this pathogen from the food processing environment.
This incident has since prompted the USDA-FSIS to toughen its regulatory policy regard-
ing Listeria-contaminated poultry and meat products (see Chap. 13).
nasal secretions from these birds suggests that externally contaminated eggs may constitute
a potential source of infection and a potential source for cross contamination of other
foods if the eggs are handled improperly.
As is true for meat and poultry workers, listerial infections among apparently healthy
individuals employed in the egg industry are extremely rare. In fact, a search of the litera-
ture uncovered only one documented case from 1965 in which a 39-year-old male egg
factory worker became infected with L. rnonocytogenes and subsequently died of meningi-
tis [ 135a1. Other than the fact that 29.1 and 10.6% of the egg factory workers carried L.
rnonocytogenes in their feces 4 and 16 months after the mans death, respectively, no
further evidence was reported to incriminate eggs in this fatal case of listerial meningitis.
Hence, at this time it appears that healthy egg factory workers need not take any special
precautions to guard against listeriosis.
Despite the inability to link listeriosis to consumption of eggs or egg products, the
recognized ability of L. monocytogenes to grow in egg products [ 1001 along with emer-
gence of this organism as a bonafide foodborne pathogen may change this picture in the
future. This prediction is supported by the fact that CDC officials [2 131 identified a possi-
ble cluster of listeriosis cases in Los Angeles County in which 6 of 33 cases and 4 of 101
matched controls consumed raw eggs (odds ratio = 6.4) over a 5-month period spanning
1986- 1987.
Although epidemiological investigations can never conclusively prove causality, the
possible associations discovered in such studies will likely prompt public health authorities
seriously to consider eggs and other foods (e.g., seafood, fruits) as potential vehicles of
infection, which, in turn, may lead to their implication in future cases of listeriosis.
SEAFOOD PRODUCTS
Despite the recent discovery of L. rnonocytogenes in a wide range of raw and processed
fish and seafoods, including finfish (i.e., smoked salmon, cod, trout), mussels, oysters,
shrimp, crabmeat, lobster tails, and surimi, most attempts directly to link consumption of
such products to cases of human listeriosis have proven unsuccessful. Nonetheless, a few
scattered reports attesting to possible involvement of seafoods in listerial infections along
with three reports of cases positively linked to consumption of fish, smoked mussels, and
artificial crabmeat have found their way into the scientific literature. Two early reports
from New Zealand include (a) a 1971 observation that two pregnant women delivered
Listeria-infected infants after presumably consuming raw fish sometime during their preg-
nancies [49], and (b) a cluster of 22 perinatal listeriosis cases between January and Novem-
ber of 1980 in which food histories suggested, at best, a weak association between con-
sumption of contaminated shellfishh-awfish and development of listeriosis [ 1441. In 1980,
Vilde et al. [228] published a report suggesting that a 48-year-old immunocompromised
French woman had contracted listeriosis after consuming contaminated oysters. More re-
cently, Arriold and Coble [39] identified two miscarriages among Australian women (one
case in Victoria and the other in New South Wales) in which smoked salmon was impli-
cated as the vehicle of infection, with Tan et al. [222] suggesting possible involvement
of smoked salmon and salmon cheese spread in two additional Australian cases of listeri-
osis. In one of the largest suspected seafood-related outbreaks thus far reported, 8 of 36
previously healthy adults attending a June 1989 party in New York City developed a
predominantly mild form of listeriosis characterized by fever, nausea, vomiting, diarrhea,
and musculoskeletal distress [ 1941. However, two cases of bacteremia caused by the epi-
340 Ryser
demic strain (an unusual enzyme type of L. monocytogenes serotype 4b) also occurred
among expectant mothers, with one pregnancy ending in a miscarriage. Although epidemi-
ological evidence most strongly implicated shrimp as the vehicle of infection, shrimp from
the party was unavailable for testing, and investigators also were unable to detect Listeria
in shrimp purchased 6 weeks later from the partys supplier. Thus, as already implied, a
causal link between consumption of seafood and listeriosis was never clearly proven in
any of these cases.
The first convincing evidence for direct involvement of fish or seafood in human
listeriosis is that of a 54-year-old Italian woman who in 1988 or 1989 contracted nonfatal
meningitis 3-4 days after consuming undercooked fish from which L. monocytogenes was
subsequently isolated [92]. The fact that L. monocytogenes isolates from the patients
cerebrospinal fluid and a leftover portion of the fish were of serotype 4 and were identical
based on phage typing and DNA restriction analysis confirmed fish as the vehicle of infec-
tion in this case of listerial meningitis. According to the investigators, survival and trans-
mission of the pathogen was most likely the result of undercooking, since the fish was
eaten and refrigerated after steaming. However, the mode by which this fish became con-
taminated could not be determined.
In August of 1991, the Tasmanian Health Department was notified about an other-
wise healthy 37-year-old woman and her 10-year-old son who developed malaise, chills,
fever, headache, vomiting, and diarrhea 3 days after eating 90 g of smoked mussels im-
ported from New Zealand [160,161]. On culturing, opened and unopened packages of
smoked mussels yielded L. monocytogenes at a level of 1.6 X 107CFU/g (i.e., oral in-
fective dose of 9 X log CFU), with contamination traced to three batches in which the
products shelf life was inadvertently overestimated by at least 3 months. Despite two
public warnings and withdrawal of the implicated samples from sale, one additional case
was reported a month later in an 83-year-old woman who developed similar symptoms
of gastroenteritis. Although results from strain-specific typing studies are not available,
the fact that all three victims became ill within 3 days of consuming smoked mussels
clearly points to this heavily contaminated product as the most probable source of
infection.
Working in New Zealand, Brett et al. [63] also identified two perinatal cases of
listeriosis in Aukland during November and December of 1992 in which the women gave
histories of consuming one particular brand of smoked mussels. During follow-up investi-
gations, an unopened package of smoked mussels obtained from one victims refrigerator
yielded a strain of L. monocytogenes serotype 112 that was indistinguishable from both
clinical isolates based on phage typing and pulsed-field gel electrophoresis. On further
searching, this unusual strain was identified in retail packages of smoked mussels marketed
in Aukland, New Zealand, and Brighton, England, as well as in environmental swab sam-
ples from the mussel processing factory, thereby confirming smoked mussels as the vehicle
of infection and the factory environment as the source of contamination. Although no
additional cases were confirmed in New Zealand, export of these mussels to England
combined with a reported association between mussel consumption and listeriosis in En-
gland [118] suggests that these mussels may have been responsible for additional cases
abroad. Given a 1995 report from Tasmania [219] in which L. monocytogenes was recov-
ered from 15.4% of premarket raw blue mussels and Pacific oysters collected in the wild
and from farms, public health concerns regarding shellfish safety need to be reexamined.
In 1997, Farber [94] also identified two cases of foodborne listeriosis among healthy
adults in Ontario, Canada. Samples of imitation crab meat, canned black olives, macaroni/
Foodborne Listeriosis 34 7
vegetable salad, spaghetti sauce with meatballs, and mayonnaise taken from the victims
refrigerator all yielded L. monocytogenes, with imitation crab meat containing 2.1 X 109
CFU/g. On further investigation, clinical strains and isolates from both opened and un-
opened packages of crab meat were identified as serotype 1/2b and were also indistinguish-
able by both randomly amplified polymorphic DNA analysis and pulsed-field gel electro-
phoresis, thereby confirming imitation crab meat as the vehicle of infection.
The aforementioned listerial infections along with other recently reported cases of
foodborne listeriosis that have been positively linked to consumption of dairy as well
as ready-to-eat meat and poultry products suggest it would be naive to assume that L.
monocytogvnes poses any less danger to public health when present in cooked and/or
ready-to-eat seafood than in other foods. Hence, FDA officials have maintained a policy
of zero tolerance for L. monocytogenes in all ready-to-eat foods and have (mid 1998)
issued numerous Class I recalls involving well over 45,000 pounds of contaminated
cooked/ready-to-eat seafood. These recalls and the fact that CDC officials now include
various shellfish and finfish in food history questionnaires given to listeriosis victims in
five states as well as Los Angeles County [24,28,212,214] make it likely that additional
listeriosis cases will be positively linked to fish and seafood in the future.
it seems unlikely that the listeriosis outbreak in Boston would have been reported if
Schlech et al. [210] had not published their results first.
In the outbreak described by Ho et al. [ 1221, 23 patients admitted to Boston-area
hospitals acquired systemic listerial infections during September and October of 1979,
with L. monocytogenes isolates from 20 of 23 (87%) patients identified as serotype 4b
(Fig. 10). In contrast, only 19 listeriosis cases were identified at the same eight hospitals
during the 26-month period immediately preceding the outbreak. Unlike the previously
described foodborne outbreaks, which included pregnant women, neonates, and adults, all
20 outbreak-related cases of listeriosis from which L. monocytogenes serotype 4b was
isolated were adults ranging in age from 46 to 89 years, with half the victims being immu-
nocompromised as a result of cancer, chemotherapy, or steroid treatment, Overall, 18 of
20 (90%) and 8 of 20 (40%) patients suffered from listerial bacteremia and meningitis,
respectively. Although 5 of 20 (25%) patients died, two of these deaths were related to
underlying illnesses rather than listeriosis.
A series of epidemiological studies revealed that cases were more likely than con-
trols (i.e., listeriosis patients treated at the same eight hospitals during the 26-month period
preceding the outbreak) to have (a) become infected with L. monocytogenes serotype 4b
rather than another serotype, (b) acquired listeriosis during hospitalization, (c) exhibited
gastrointestinal symptoms, and (d) received antacids or cimetidine before onset of illness.
More important, results from food histories revealed that cases were more likely than
controls to have consumed tuna, chicken salad, and cottage cheese as well as hard cheese.
Although it was at first difficult to develop a scenario by which this apparent common-
source outbreak could have resulted from consumption of three seemingly unrelated foods
obtained from different distributors, hospital kitchen records revealed that all three foods
...
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...
...
. . I
...
....
....<
...
...
...
...
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I . .
. . I
0
-R
2 3 4
5 6 7 8 9 10 11 I21
n
1977 1978 1979
were commonly used in salads containing lettuce, celery, and/or tomatoes. Thus, in light
of the 1981 Canadian listeriosis outbreak involving coleslaw., these researchers postulated
that the Boston outbreak was the result of victims having consumed raw lettuce, celery,
and/or tomatoes that were contaminated with listeriae. However, since no attempt was
made to isolate L. monocytogenes from these vegetables during the outbreak, the exact
source of infection will always remain in question.
12
I0
8 8
U
6
FIGURE11 Number of perinatal and adult cases of listeriosis recorded in the Mari-
time Provinces of Canada from January 1979 t o December 1981. (Adapted from Ref.
210.)
344 Ryser
history of foods consumed during the 3 months before onset of illness. Analysis of results
from the first case-control study failed to implicate a common environmental source for
this outbreak. Although initial data collected from food histories also failed to implicate
any particular food, results from a second questionnaire that included names of several
food items found in the refrigerator of a man who developed pneumonia and septicemia
indicated that cases were more likely than controls to have consumed both coleslaw and
radishes. Multivariate analysis later showed that ingestion of radishes was associated with
consumption of coleslaw rather than illness. However, repeated interviews with cases and
controls who had previously denied eating coleslaw revealed that 100% of the cases but
only 40% of the controls remembered consuming the product during the 3-month period
before the outbreak.
Armed with this information, investigators visited the home of one of the patients,
sampled foods from the refrigerator, and then isolated L. rnonocytogenes from coleslaw
but no other foods obtained from the patients refrigerator. The strain isolated from cole-
slaw was soon identified as serotype 4b, the same serotype isolated from all 34 victims.
To prove that the patient had not inadvertently contaminated the coleslaw, investigators
obtained two unopened packages from two different Halifax-area supermarkets and recov-
ered L. rnonocytogenes serotype 4b from each package. Audurier et al. [43] later reported
that 28 of 3 1 (90.3%) L. monocytogenes serotype 4b isolates obtained from blood, cerebro-
spinal fluid, and/or placental material between January and September of 1981 were of
the same phage type. Any remaining doubt concerning the role of coleslaw in this outbreak
was eliminated when clinical and coleslaw isolates were later shown to be of the same
electrophoretic enzyme type [40,411, pulsed-field gel electrophoretic type [65], ribotype
[ 1101, restriction enzyme pattern [235], randomly-amplified polymorphic DNA pattern
[74], and DNA macrorestriction pattern [ 1261 with this strain being closely related both
phenotypically and genotypically to those responsible for outbreaks in the United States
(Mexican-style cheese 1985) and Switzerland (Vacherin Mont dOr cheese 1983- 1987)
[126]. Thus Schlech et al. [209,210] can be credited with providing the first concrete
evidence that consumption of contaminated food, in this instance coleslaw, can cause
listeriosis in humans.
After coleslaw was confirmed as the infectious vehicle, Schlech et al. [210] at-
tempted to determine the route by which the coleslaw became contaminated. Investigators
soon traced the tainted coleslaw to a regional manufacturer whose product had been dis-
tributed exclusively in the Maritime Provinces of Canada. Although repeated microbiolog-
ical testing of environmental samples from the coleslaw factory failed to uncover the
contamination source, review of factory records along with recent data on animal listeriosis
revealed that the manufacturer had received at least 2250 kg of cabbage from a farmer
who also raised sheep. Furthermore, the farmer lost two sheep in his flock to listeriosis-
one in 1979 and one in 1981. A review of the farmers agronomic practices indicated that
cabbage was routinely fertilized with both raw and composted sheep manure obtained
from animals that were presumably fecal carriers of L. rnonocytogenes. Moreover, the
final October cabbage crop was held in a large cold-storage shed until early spring. Such
storage practices, which somewhat resemble cold enrichment, may have led to an increase
in numbers of L. rnonocytogenes in the cabbage. Although none of the implicated cabbage
crop (other than that which was previously manufactured into coleslaw and contaminated
with the epidemic strain of L. rnonocytogenes) was available for testing, and none of the
farm environmental samples, including raw sheep manure, yielded Listeria, the aforemen-
tioned circumstantial evidence still supports an indirect link between sporadic cases of
ovine listeriosis on a cabbage farm and cases of listeriosis in humans consuming a Listeria-
Foodborne Listeriosis 345
contaminated product. The fact that (a) crops grown in Canada, the United States, and
industrialized European nations are routinely fertilized with material other than raw ma-
nure, (b) 82% of all L. monocytogenes strains isolated during one large survey of raw
vegetables marketed in the United States were of serotype la [ 1201, and (c) most L. mono-
cytogenes strains isolated from animal sources, including feces, in North America are of
serotype 4 rather than 1 support transmission of L. monocytogenes from raw sheep manure
to cabbage. Hence, raw manure should not be used to fertilize vegetables that will be
consumed without cooking.
growth on the surface, the pathogen probably grew in these mushrooms during the 5
months of storage. Although the exact source of contamination was never determined, it
is noteworthy that the mans wife did not become ill after consuming thoroughly reheated
mushrooms from the same container. Hence, it appears that heavily contaminated vegeta-
bles also can be rendered safe by thorough cooking.
The final case of listeriosis directly linked to products of plant origin is markedly
different in that consumption of alfalfa tablets, a dry product in which L. rnonocytogenes
is presumably unable to grow, was directly responsible for a fatal case of listerial meningo-
encephalitis in a 55-year-old immunocompromised Canadian man [95]. As in the two
cases just discussed, L. rnonocytogenes strains of serotype 4b isolated from the victims
blood and cerebrospinal fluid as well as the remaining alfalfa tablets were all of the same
electrophoretic enzyme type, which in 1990 confirmed alfalfa tablets as the vehicle of
infection in the only listeriosis case thus far linked to ingestion of a nearly completely
dry product. However, since Czojka and Batt [74] recently found that these isolates yielded
slightly different randomly amplified polymorphic DNA patterns, the exact source of in-
fection now appears to be somewhat open to debate.
Although not directly linking consumption of raw vegetables to listerial infection,
considerable circumstantial evidence exists for involvement of vegetables in human listeri-
osis. In the first of these reports [43], a 74-year-old man contracted L. rnonocytogenes
serotype 1/2c septicemia and meningitis 1 1 days after repair of his perforated duodenal
ulcer in a London-area hospital. During the week before onset of illness, the patient con-
sumed hospital food supplemented with high protein puddings and sandwiches containing
various fillings, including cheese; however, hospital personnel failed to maintain an exact
record of foods eaten. In conjunction with a survey to determine the incidence of listeriae
in hospital-prepared foods, investigators isolated L. rnonocytogenes serotype 1/2a and L.
innocua from 1 of 15 and 2 of 15 samples of washed English round lettuce but failed to
detect Listeria spp. in 40 other food samples consisting primarily of dairy products and
other raw vegetables. Most evidence suggests that listeriosis has an incubation period of
1 to several weeks; however, three of four patients in England with confirmed cases of
foodborne listeriosis exhibited incubation periods of 5 1 week. Hence, although the incu-
bation period observed in this 74-year-old man is compatible with a hospital-acquired
listerial infection, isolation of different L. rnonocytogenes serotypes from the man and
washed English round lettuce appears to preclude direct involvement of lettuce in this
particular case of listeriosis. Nevertheless, since L. rnonocytogenes was recovered from
washed lettuce but from no other hospital-prepared food, consumption of lettuce still ap-
pears to be a possible risk factor in development of hospital-acquired listeriosis.
During the previously discussed 1986- 1987 case-control study in which consump-
tion of uncooked frankfurters and undercooked chicken was epidemiologically associated
with listeriosis, Schwartz et al. [214] isolated the same serotype and enzyme type of L.
rnonocytogenes from five Hispanic listeriosis patients who resided in Los Angeles County.
Of the four patients who voluntarily enrolled in the case-control study, all consumed let-
tuce as well as chicken and whole milk. Unfortunately, because matched controls con-
sumed these products at rates similar to those of cases, it was impossible to link consump-
tion of lettuce as well as chicken or whole milk to listeriosis. More recently, routine follow-
up investigations in Victoria, Australia, have provided some circumstantial evidence for
possible involvement of lettuce (one cluster of eight cases) [222], coleslaw (one case)
[222], raw vegetables/mayonnaise vegetable dip (one case) [201], and raw vegetables (one
case) [201] in sporadic cases of listeriosis. In the United States, active surveillance of a
Foodborne Listeriosis 347
REFERENCES
1. Acheson, D. 1987. Food hazard warning: Vacherin Mont dOr Swiss cheese. Department of
Health and Social Security, London. Release PL/CM0(87)11.
2. Adarns, M.R., and M.O. Moss. 1995. Food Microbiology. Cambridge, UK: Cambridge Uni-
versity Press.
3. Allerberger, F. 1988. Listeriosis in Austria-Report of an outbreak in Austria 1986. Proc.
X International Symposium on Listeriosis, Pecs, Hungary, Aug. 22-26, Abstr. 20.
4. Andre, P., H. Roose, R. van Noyen, L. Dejaegher, I. Uyttendaele, and K. De Schrijver. 1990.
Neuro-meningeal listeriosis associated with consumption of an ice cream. Med. Mal. Infect.
201570-572.
5. Anonymous, 1976. Report of a WHO expert committee with the participation of the FAO-
Microbiological aspects of food hygiene. WHO Tech. Rep. Series 598: 1- 103.
6. Anonymous. 1985. FDA is investigating deaths linked to Mexican-style cheese. Food Chem.
News 27( 15):42.
7. Anonymous. 1985. FDA still searching for source of Listeria in Mexican-style cheese. Food
Chem. News 27( 17):57.
8. Anonymous. 1985. Jalisco-brand Mexican style soft cheese recalled. FDA Enforcement Rep.,
June 26.
348 Ryser
37. Anonymous. 1991. Listeriosis-The situation 2 years after the outbreak caused by Vacherin
Mont-dOr soft cheese. Wkly. Epidemiol. Rec. 66(5):28-29.
38. Archer, D.L. 1988. Review of the latest FDA information on the presence of Listeria
in foods. WHO Working Group on Foodborne Listeriosis, Geneva, Switzerland, Feb. 15-
19.
39. Amold, G.J., and J. Coble. 1995. Incidence of Listeria spp. in foods in NSW. Food Austral.
47171-75.
40. Ashton, F.E., J.A. Ryan, and E.P. Ewan. 1988. Electrophoretrc analysis of enzymes produced
by Listeria monocytogenes serotype 4b. Proc. of Society for Industrial Microbiology-
Comprehensive Conference on Listeria monocytogenes, Rohnert Park, CA, Oct. 2-5, Abstr.
P-11.
41. Ashton, F.E., J.A. Ryan, and E.P. Ewan. 1988. Enzyme electrophoretic analysis of Listeria
monocytogenes serotype 4b associated with listeriosis in Canada. Proc. X International Sym-
posium on Listeriosis, Pecs. Hungary, Aug. 22-26, Abstr. P43.
42. Atkinson, E. 1917. Meningitis associated with gram-positive bacilli of diphtheroid type. Med.
J. Austral. 1:115-118.
43. Audurier, A., A.G. Taylor, B. Carbonnelle, and J. McLauchlin. 1984. A phage typing system
for Listeria monocytogenes and its use in epidemiological studies. Clin. Invest. Med. 7:229-
232.
44. Azadian, B.S., G.T. Finnerty, and A.D. Pearson. 1989. Cheese-borne Listeria meningitis in
immunocompetent patient. Lancet 1:232-233.
45. Bannister, B.A. 1987. Listeria monocytogenes meningitis associated with eating soft cheese.
J. Infect. 15:165-168.
46. Banwart, G.J., 1982. Basic Food Microbioogy. 2nd ed. New York: AV1 Publishing.
47. Barnes, R., P. Archer, J. Stack, and G.R. Istre. 1989. Listeriosis associated with consumption
of turkey franks. M.M.W.R. 38:267-268.
48. Beams, R.E., and K.F. Girard. 1958. The effect of pasteurization on Listeria monocytogenes.
Can. J. Microbiol. 4 3 - 6 1 .
49. Becroft, D.M.O., K. Farmer, R.J. Seddon, R. Sowden, J.H. Stewart, A. Vines, and D.A.
Wattie. 197 1. Epidemic listeriosis in the newborn. Br. Med. J. 3:747-75 1.
50. Bendig, J.W.A., and J.E.M. Strangeways. 1989. Listeria in hospital lettuce. Lancet 1:616-
617.
51. Berrang, M.E., J.F. Frank, and R.E. Brackett. 1988. Behavior of Listeria monocytogenes in
chocolate milk and ice cream mix made from post-expiration date skim milk. J. Food Prot.
5 1:823 (Abstr.).
52. Bille, J. 1988. Anatomy of a foodborne listeriosis outbreak. Foodborne Listeriosis-Proceed-
ings of a Symposium, Wiesbaden, Germany, Sep. 7, pp. 30-35.
53. Bille, J. 1988. Epidemiology of human listeriosis in Europe with special reference to the
Swiss outbreak. Proc. Society for Industrial Microbiology-Comprehensive Conference on
Listeria monocytogenes, Rohnert Park, CA, Oct. 2-5, Abstr. 1-11.
54. Bille, J., J. Rocourt, F. Mean, M.P. Glauser, and the Group Listeria-Vaud. 1988. Epidemic
food-borne listeriosis in Western Switzerland. 11. Epidemiology, Interscience Conference on
Antimicrobial Agents and Chemotherapy, Los Angeles, C.4, Oct. 23-26, Abstr. 1107.
55. Bibb, W.F., B.G. Gellin, R. Weaver, B. Schwartz, B.D. Plikaytis, M.W. Reeves, R.W. Pinner,
and C.V. Broome. 1990. Analysis of clinical and food-borne isolates of Listeria monocyto-
genes in the United States by multilocus enzyme electrophoresis and application of the
method to epidemiologic investigations. Appl. Environ. Microbiol. 56:2133-2141.
56. Bille, J., D. Nocera, E. Bannerman, and F. Ischer. 1992. Molecular typing of Listeria monocy-
togenes in relation with the Swiss outbreak of listeriosis. Proc. XI. Intern. Symp. on Problems
of Listeriosis, Copenhagen, Denmark, May 11- 14, pp. 195- 196.
57 * Bisping, W. von. 1957. Die Listeriose und ihre milchhygienische Bedeutung. Kieler Milchw-
irtschaftliche Forschungsberichte 9595-606.
350 Ryser
58. Blendon, D.C., and F.T. Szatalowicz. 1967. Ecological aspects of listeriosis. J. Am. Vet.
ASSOC.151 1761- 1766.
59. Boerlin, P., and J.-C. Piffaretti. 1991. Typing of human, animal, food, and environmental
isolates of Listeria monocytogenes by multilocus enzyme electrophoresis. Appl. Environ.
Microbiol. 57: 1624-1629.
60. Boerlin, P., E. Bannerman, T. Jemmi, and J. Bille. 1996. Subtyping Listeria monocytogenes
isolates genetically related to the Swiss epidemic clone. J. Clin. Microbiol. 34:2 148-
2153.
61. Bowmer, E.J., J.A. McKiel, W.H. Cockcroft, N. Schmitt, and D.E. Rappay. 1973. Listeria
monocytogenes infections in Canada. Can. Med. Assoc. J. 109: 125-135.
62. Breer, C. 1987. Listeria in cheese. In A. Schoenberg (ed.), Listeriosis-Joint WHO/ROI Con-
sultation on Prevention and Control, Berlin, West Germany, Dec. 10- 12, pp. 106- 109.
63. Brett, M., P. Short, and J. Mclauchlin. 1995. Listeriosis associated with smoked mussels.
Proc. XIIth International Symposium on Problems of Listeriosis, Perth, Western Australia,
October 2-6, p. 467.
64. Buchrieser, C., R. Brosch, and J. Rocourt. 1992. Analysis of L. monocytogenes strains which
caused human epidemics in different countries using pulsed-field gel electrophoresis and low
frequency cleavage restriction enzymes. Proc. XIth International Symposium on Problems
of Listeriosis, Copenhagen, Denmark, May 1 1- 14, pp. 60-6 1.
65. Buchrieser, C., R. Brosch, R.B. Catimel, and J. Rocourt. 1993. A new view of human listeri-
osis epidemiology: pulsed-field gel electrophoresis applied for comparing Listeria monocyto-
genes strains involved in outbreaks. Can. J. Microbiol. 36:395-401.
66. Bula, C.J., J. Bille, and M.P. Glauser. 1994. An epidemic of food-borne listeriosis in Western
Switzerland: description of 57 cases involving adults. Clin. Infect. Dis. 20:66-72.
67. Bum, C.G. 1936. Clinical and pathological features of an infection caused by a new pathogen
of the genus Listerella. Am. J. Pathol. 12:341-349.
68. Campbell, D.M. 1989. Listeriosis in Scotland, 1988. Lancet 1:492.
69. Canfield, M.A., J.N. Walterspiel, M.S. Edwards, C.J. Baker, R.B. Wait, and J.N. Urteaga.
1984. An epidemic of perinatal listeriosis serotype l b in Hispanics in a Houston hospital.
Pediatr. Infect. Dis. 4: 106.
70. Cantoni, C., C. Balzaretti, and M. Valenti. 1989. A case of L. monocytogenes human infection
associated with consumption of Testa in cascetta (cooked meat pork product). Arch. Vet.
Ital. 40: 141- 142.
71. Carbonnelle, B., J. Cottin, F. Parvery, G. Chambreuil, S. Kouyoumdjian, M. Lirzin, and F.
Vincent. 1978. Epidemic de listeriose dans Iouest de la France (1975-1976). Revue Epidt-
mioloque et de Sante Publique (Paris) 26:451-467. In: McLauchlin, J. 1987. A review-
Listeria monocytogenes, recent advances in the taxonomy and epidemiology of listeriosis in
humans. J. Appl. Bacteriol. 63: 1- 1 1.
71a. Casolari, C., R. Neglia, M. Malagoli, and U. Fabio. 1994. Foodborne sporadic neonatal listeri-
osis confirmed by DNA fingerprinting. Proc. Annual Meeting of American Society of Micro-
biology, Las Vegas, NV, May 23-27, p. 382.
72. Chao, S.M., L. Mascola, and K. Deaver-Robinson. 1995. Listeriosis surveillance in the
United States and Los Angeles County: facts, figures and trends. Proc. of XIIth International
Symposium on Problems of Listeriosis, Perth, Western Australia, October 2-6, pp. 183-
189.
73. Cliver, D.O. 1990. Foodborne Diseases. San Diego: Academic Press.
74. Czajka, J., and C.A. Batt. 1994. Verification of causal relationships between Listeria monocy-
togenes isolates implicated in food-borne outbreaks of listeriosis by randomly amplified poly-
morphic DNA patterns. J. Clin. Microbiol. 32: 1280- 1287.
75. Dalton, C.B., C.C. Austin, J. Sobel, P.S. Hayes, W.F. Bibb, L.M. Graves, B. Swarninathan,
M.E. Proctor, and P.M. Griffin. 1997. An outbreak of gastroenteritis and fever due to Listeria
monocytogenes in milk. N. Engl. J. Med. 336: 100-105.
Foodborne Listeriosis 351
76. De Boer, E., and P. Van Netten. 1990. De aanwezigheid en groei van Listeria monocytogenes
in pit& Voed. Middelen Technol. 13:15- 17.
77. Dedii, K. 1955. Beitrag zur Epizootologie der Listeriose. Arch. Exp. Vet.-Med. 9:25 1-264.
78. Department of Health. 1989. Listeria found in pitb. London: DOH press release 89/299, July
12.
79. Dijkstra, R.G. 1976. Listeria-encephalitis in cows through litter from a broiler-farm. Zbl.
Bakteriol. Hyg. I Abt. Orig. B 161:383-385.
80. Donnelly, C.W. 1989. Personal communication.
81. Donnelly, C.W., E.H. Briggs, and L.S. Donnelly. 1987. Comparison of heat resistance of
Listeria monocytogenes in milk as determined by two methods. J. Food Prot. 50:14-17, 20.
82. Doyle, M.P. (ed.). 1989. Foodborne Bacterial Pathogens. New York: Marcel Dekker,
83. Doyle, M.P. 1990. Personal communication.
83a. Doyle, M.P., L.R. Beuchat, and T.J. Montville (eds.). 1997. Food Microbiology: Fundamen-
tals and Frontiers. Washington, DC: American Society of Microbiology Press.
84. Doyle, M.P., K.A. Glass, J.T. Beery, G.A. Garcia, D.J. Pollard, and R.D. Schultz. 1987.
Survival of Listeria monocytogenes in milk during high-temperature, short-time pasteuriza-
tion. Appl. Environ. Microbiol. 53:1433-1438.
85. Eck, H. von. 1957. Encephalomyelitis listeriaca apostematosa. Schweiz. Med. Wochenschr.
912 10-2 14.
86. Elischerovi, K., and S. Stupalovi. 1972. Listeriosis in professionally exposed persons. Acta
Microbiol. Acad. Sci. Hung. 19:379-384.
87. Elischerovi, K., G. Havilkovi, and S. Stupalovi. 1976. Listeria monocytogenes isolation at
work places in the meat industry. Cs. Epidemiol. Mikrobiol. Imunol. 25:326-332.
88. Elischerovi, K., S. Stupalovi, R. Helblchovi, and J. Stepinek. 1979. Incidence of Listeria
monocytogenes in faeces of employees of meat processing plants and meat shops. Cs. Epide-
miol. Mikrobiol. Imunol. 28:97- 102.
89. Embil, J.A., E.P. Ewan, and S.W. MacDonald. 1984. Surveillance of Listeriu monocytogenes
in human and environmental specimens in Nova Scotia, 1974 to 1981. Clin. Invest. Med. 7:
325-327.
90. Evans, R.J., A.C. Allen, D.A. Stinson, R. Bortolussi, and L.J. Peddle. 1985. Perinatal listeri-
osis: report of an outbreak. Pediatr. Infect. Dis. 4:237-241.
91. Ewert, D.P., L. Lieb, P.S. Hayes, M.W. Reeves, and L. Mascola. 1995. Listeria monocyto-
genes infection and serotype distribution among HIV-infected persons in Los Angeles
County, 1985- 1992. J. Acquired Immune Defic. Synd. and Human Retrovirol. 8:46 1-465.
92. Facinelli, B., P.E. Varaldo, M. Toni, C. Casolari, and U. Fabio. 1989. Ignorance about Liste-
ria. Br. Med. J. 299:738.
93. Faoagali, J.L., and M. Schousboe. 1985. Listeriosis in Christchurch 1967-1984. N. Z1. Med.
J. 98:64-66.
94. Farber, J. 1997. A small outbreak of listeriosis linked to consumption of imitation crab meat.
Abstracts of Annual Meeting of the International Association of Milk, Food and Environmen-
tal Sanitation, Orlando, FL, July 7.
95. Farber, J.M., A.O. Carter, P.V. Varughese, F.E. Ashton, and E.P. Ewan. 1990. Listeriosis
traced to the consumption of alfalfa tablets and soft cheese. N. Engl. J. Med. 322:338.
96. Felsenfeld, 0. 1951. Disease of poultry transmissible to men. Iowa State College Vet. 13:
89-92.
97. Finle, G.G., and J.R. Long. 1977. An epizootic of listeriosis in chinchillas. Can. Vet. J. 18:
164- 167.
98. Fischer, M. 1962. Listeriose-Haufung in Raume Bremen in den Jahren 1960 und 1961. Dtsch.
Med. Wochenschr. 87:2682-2684.
99. Fleming, D.W., S.L. Cochi, K.L. MacDonald, J. Brondum, P.S. Hayes, B.D. Plikaytis, M.B.
Homes, A. Audurier, C.V. Broome, and A.L. Reingold. 1985. Pasteurized milk as a vehicle
of infection in an outbreak of listeriosis. N. Engl. J. Med. 312:404-407.
352 Ryser
100. Foegeding, P.M., and S.B. Leasor. 1990. Heat resistance and growth of Listeria monocyto-
genes in liquid whole egg. J. Food Prot, 53:9-14.
101. Freeman, R., P.R. Sisson, N.F. Lightfoot, and J. McLauchlin. 1991. Analysis of epidemic
and sporadic strains of Listeria monocytogenes by pyrolysis mass spectrometry. Lett. Appl.
Microbiol. 12:133- 136.
102. Genigeorgis, C., J.H. Toledo, and F.J. Garayzabal. 1991. Selected microbiological and chemi-
cal characteristics of illegally produced and marketed soft Hispanic-style cheeses in Califor-
nia. J. Food Prot. 54598-601.
103. Geurden, L.M.G., and A. Devos. 1952. Listerellose bij pluimvee. Vlaams. Diergeneesk.
Tijdschr. 2 1: 165- 175.
104. Gilbert, R.J., S.M. Hall, and A.G. Taylor. 1989. Listeriosis update. Public Health Lab. Serv.
Dig. 6:33-37.
105. Gilbert, R.J., J. McLauchlin, and S.K. Velani. 1993. The contamination of p$t6 by Listeria
monocytogenes in England and Wales in 1989 and 1990. Epidemiol. Infect. 110:543-551.
106. Goulet, V., A. Lepoutre, J. Rocourt, A.-L. Courtieu, P. Dehaumont, and P. Veit. 1993. Epide-
mie de listeriose en France-Bilan final et resultants de lenqeute epidemiologique. Arch.
Vet. Ital. 44: 19-24.
107. Goulet, V., I. Rebiere, Ph. Marchetti, A. Lepoutre, P. Veit, P. Dehaumont, and J. Rocourt.
1995. Listeriosis in France: lessons from the investigations of two major national outbreaks.
Proc. XIIth International Symposium on Problems of Listeriosis, Perth, Western Australia,
October 2-6, p. 159.
108. Goulet, V., A. Lepoutre, P. Marchetti, I. Rebiere, C. Moyse, J. Rocourt, 0. Pierre, and P.
Veit. 1993. Epidemiologic implication of food cross-contamination in a nation-wide outbreak
of listeriosis in France in 1992. Annual Meeting of Interscience Conference Antimicrobiol
Agents and Chemotherapy, Abst. 1458.
109. Goulet, V., C. Jacquet, V. Vaillant, I. Rebiere, E. Mouret, C. Lorente, E. Maillot, F. Stainer,
and J. Rocourt. 1995. Listeriosis from consumption of raw milk cheese. Lancet 345:1581-
1582.
110. Graves, L.M., B. Swaminathan, M.W. Reeves, S.B. Hunter, R.E. Weaver, B.D. Plikaytis,
and A. Schuchat. 1994. Comparison of ribotyping and multilocus enzyme electrophoresis
for subtyping of Listeria monocytogenes isolates. Appl. Environ. Microbiol. 32:2936-2943.
111. Gray, M.L. 1958. Listeriosis in fowls-a review. Avian Dis. 2:296-314.
112. Gray, M.L. 1960. A possible link in the relationship between silage feeding and listeriosis.
J. Am. Vet. Med. Assoc. 136:205-208.
113. Gray, M.L., and A.H. Killinger. 1966. Listeria monocytogenes and listeric infections. Bacter-
iol. Rev. 30:309-382.
114. Gray, M.L., and F. Thorp, Jr. 1957. Perinatal infection in rabbits induced by Listeria monocy-
togenes. IV. Apparent transmission through dams milk. Zbl. V e te rinhe d. 4:405-414.
115. Gudkova, E.I., and T.P. Voronina. 1956. Diagnosis of listeric angina. Zh. Mikrobiol. Epide-
miol. Immunobiol. 33:3 1-39.
116. Gudkova, E.I., K.A. Mironova, A.S. Kuzminskii, and G.O. Geine. 1958. A second outbreak
of listeriotic angina in a single populated locality. Zh. Mikrobiol. Epidemiol. Immunobiol.
35 ~24-28.
117. Hall, S.M. 1988. The epidemiology of listeriosis in England and Wales. Foodborne Listeri-
osis-Proceedings of a Symposium, Wiesbaden, West Germany, Sept. 7, pp. 38-50.
118. Hall, S.M., M. Pelerin, N. Soltanpoor, and R.J. Gilbert. 1995. A case control study of sporadic
listeriosis in England and Wales. Proc. XIIth International Symposium on Problems of Liste-
riosis, Perth, Western Australia, October 2-6, p. 157.
119. Hayes, P.S., J.C. Feeley, L.M. Graves, G.W. Ajello, and D.W. Fleming. 1986. Isolation of
Listeria monocytogenes from raw milk. Appl. Environ. Microbiol. 5 1:438-440.
120. Heisick, J.E., D.E. Wagner, M.L. Nierman, and J.T. Peeler. 1989. Listeria spp. found on
fresh market produce. Appl. Environ. Microbiol. 55: 1925- 1927.
Foodborne Listeriosis 353
121. Hird, D.W. 1987. Review of evidence for zoonotic listeriosis. J. Food Prot. 50:429-433.
122. Ho, J.L., K.N. Shands, G. Friedland, P. Eckind, and D.W. Fraser. 1986. An outbreak of type
4b Listeriu monocytogenes infection involving patients from eight Boston hospitals. Arch.
Intern. Med. 146520-524.
123. Hyslop, N. St. G. 1975. Epidemiologic and immunologic factors in listeriosis. In: M. Wood-
bine, ed. Problems of Listeriosis. Leicester, UK: Leicester University Press, pp. 94- 105.
124. Hyslop, N. St. G., and A.D. Osbome. 1959. Listeriosis: a potential danger to public health.
Vet. Rec. 71:1082-1091.
125. Jacobs, M.R., H. Stein, A. Buqwane, A. Dubb, F. Segal, L. Rabinowitz, U. Ellis, I. Freman,
M. Witcomb, and V. Vallabh. 1978. Epidemic listeriosis--report of 14 cases detected in 9
months. S. Afr. Med. J. 54:389-392.
126. Jacquet, Ch., B. Catimel, V. Goulet, A. Lepoutre, P. Veit, P. Dehaumont, and J. Rocourt.
1995. Typing of Listeria monocytogenes during epidemiological investigations of the French
listeriosis outbreaks in 1992, 1993 and 1995. Proceedings of XIIth International Symposium
on Problems of Listeriosis, Perth, Western Australia, October 2-6 p. 161-176.
127. Jacquet, C., B. Catimel, R. Brosch, C. Buchrieser, P. Dehaumont, V. Goulet, A. Lepoutre,
P. Veit, and J. Rocourt. 1995. Investigations related to the epidemic strain involved in the
French listeriosis outbreak in 1992. Appl. Environ. Microbiol. 61 :2242-2246.
128. James, S.M., S.L. Fannin, B.A. Agree, B. Hall, E. Parker, J. Vogt, G. Run, J. Williams, L.
Lieb, C. Salminen, T. Prendergast, S.B. Werner, and J. Chin. 1985. Listeriosis outbreak asso-
ciated with Mexican-style cheese-California. J.A.M.A. 254:474.
129. James, S.M., S.L. Fanning, B.A. Agree, B. Hall, E. Parker, J. Vogt, G. Run, J. Williams, L.
Lieb, C. Salminen, T. Prendergast, S.B. Werner, and J. Chin. 1985. Listeriosis outbreak asso-
ciated with Mexican-style cheese-California. M.M.W.R. 34(24):357-359.
130. Jay, J.M. 1996. Modem Food Microbiology, 5th ed.: New York, Van Nostrand Reinhold Co.
131. Jensen, A., W. Frederiksen, and P. Gerner-Smidt. 1994. Risk factors for listeriosis in Den-
mark, 1989-1990. Scand. J. Infect. Dis 26:171-178.
132. Julianelle, L.A. 1940. The function of Listerellu in infection. Ann. Intern. Med. 14:608-620.
133. Junttila, J., and M. Brander. 1989. Listeriu monocytogenes septicemia associated with con-
sumption of salted mushrooms. Scand. J. Infect. Dis. 21 :339-342.
134. Kaczmarski, E.B., and D.M. Jones. 1989. Listeriosis and ready-cooked chicken. Lancet 1:
549.
135. Kampelmacher, E.H. 1962. Animal products as a source of listeric infection in man. In M.L.
Gray, ed. Second Symposium on Listeric Infection, Montana State College, Bozeman, MT,
Aug. 29-31, pp. 146-151.
135a. Kanipelmacher, E.H., and L.M. van Noorle Jansen. 1969. Isolation of Listeriu monocytogenes
from faeces of clinically healthy humans and animals. Zbl. Bakteriol. I Abt. Orig. 21 1:353-
359.
136. Kaplan, M.M. 1945. Listerellosis. N. Engl. J. Med. 232:755-759.
137. Kerr, K.G., S.F. Dealler, and R.W. Lacey. 1988. Materno-fetal listeriosis from cook-chill
and refrigerated food. Lancet 2: 1 133.
138. Khomenko, G.I., V.A. Matsievskii, and O.P. Lebedeva. 1953. Data to clinical picture and
diagnosis of listerellosis. Vrach. Delo. No. 12:1099- 1 104. In: Ralovich, B. 1984. Listeriosis
Research-Present Situation and Perspective, Akademiai Kiado, Budapest.
139. Kittson, E. 1992. A case cluster of listeriosis in Western Australia with links to p%e consump-
tion. Proc. XIth International Symposium on Problems of Listeriosis, Copenhagen, May 1 1 -
14, p. 39-40.
140. Kuzminskii, A S . 1956. Epidemiology of listerellosis outbreak. Zh. Mikrobiol. Epidemiol.
Immunobiol. 33:25 -30.
141. Kvenberg, J.E. 1988. Outbreaks of listeriosis/Listeriu-contaminated foods. Microbiol. Sci.
5:355-358.
142. Lee. W.H. 1989. Personal communication.
354 Ryser
143. Lemagny, F., I. Rebiere, J. Rocourt, and B. Hubert. 1989. Listeriose humaine: enquete epide-
miologique de deux episodes epidemiques en France, en 1988 et 1989. Bull. Epidemiol.
Hebdom. 39: 162-163.
144. Lennon, D., B. Lewis, C. Mantell, D. Becroft, B. Dove, K. Farmer, S. Tonkin, N. Yeates,
R. Stamp, and K. Mickleson. 1984. Epidemic perinatal listeriosis. Pediatr. Infect. Dis. 3:30-34.
145. Linnan, M.J., L. Mascola, X.D. Lou, V. Goulet, S. May, C. Salminen, D.W. Hird, M.L.
Yonkura, P. Hayes, R. Weaver, A. Audurier, B.D. Plikaytis, S.L. Fannin, A. Kleks, and C.V.
Broome. 1988. Epidemic listeriosis associated with Mexican-style cheese. N.Engl. J. Med.
3 19:823-828.
146. Lovett, J., D.W. Francis, and J.M. Hunt. 1987. Listeria monocytogenes in raw milk: Detec-
tion, incidence, and pathogenicity. J. Food Prot. 50: 188- 192.
147. Malinverni, R., M.P. Glauser, J. Bille, and J. Rocourt. 1986. Unusual clinical features of an
epidemic of listeriosis associated with a particular phage type. Eur. J. Clin. Microbiol. 5:
169- 171.
148. Malinverni, R., J. Bille, Cl. Perret, F. Regli, F. Tanner, and M.P. Glauser. 1985. Listkriose
kpidkmizue-Observation de 25 cas en I5 mois au centre hospitalier universitaire vaudois.
Schweiz. Med. Wochenschr. I 15:2- 10.
149. Marchetti, P., A. Lepoutre, A.F. Miegeville, J. Rocourt, and V. Goulet. 1995. Listeriosis in
non-pregnant adults in 1992 in France: clinical presentation of 225 cases. Proc. XIIth Interna-
tional Symposium on Problems of Listeriosis, Perth, Western Australia, October 2-6,
pp. 145-146.
150. Mascola, L., S.L. Fannin, and M. Linnan. 1989. Epidemic listeriosis-response to letter. N.
Engl. J. Med. 320:538.
151. Mascola, L., L. Chun, and J. Thomas. 1988. A case-control study of a cluster of perinatal
listeriosis identified by an active surveillance system in Los Angeles County. Proceedings
of Society for Industrial Microbiology-Comprehensive Conference on Listeria rnonocyto-
genes, Rohnert Park, CA, Oct. 2-5, Abstr. P-10.
152. Mascola, L., F. Sorvillo, J. Neal, K. Iwakoshi, and R. Weaver. 1989. Surveillance of listeri-
osis in Los Angeles County, 1985- 1986. Arch. Intern. Med. 149:1569- 1572.
153. Mascola, L., F. Sorvillo, V. Goulet, B. Hall, R. Weaver, and M. Linnan. 1992. Fecal carriage
of Listeria monocytogenes-Observations during a community-wide, common-source out-
break. Clin. Infect. Dis. 15:557-558.
154. Mascola, L., L. Chun, J. Thomas, W.F. Bibe, B. Schwartz, C. Salminen, and P. Heseltine.
1988. A case-control study of a cluster of perinatal listeriosis identified by an active surveil-
lance system in Los Angeles County. Proceedings of Society for Industrial Microbiology-
Comprehensive Conference on Listeria rnonocytogenes, Rohnert Park, CA, Oct. 2-5, Abstr.
P- 10.
155. McLauchlin, J., and L. Newton. 1995. Human listeriosis in England, Wales and Northern
Ireland: A changing pattern of infection. Proc. XIIth International Symposium on Problems
of Listeriosis, Perth, Western Australia, October 2-6, pp. 177- 181.
156. McLauchlin, J., A. Audurier, and A.G. Taylor. 1986. Aspects of the epidemiology of human
Listeria monocytogenes infections in Britain 1967- 1984; the use of serotyping and phage
typing. J. Med. Microbiol. 22:367-377.
157. McLauchlin, J., N. Crofts, and D.M. Campbell. 1989. A possible outbreak of listeriosis
caused by an unusual strain of Listeria monocytogenes. J. Infect. 18:179- 187.
158. McLauchlin, J., M.H. Greenwood, and P.N. Pini. 1990. The occurrence of Listeria monocyto-
genes in cheese from a manufacturer associated with a case of listeriosis. Int. J. Food Micro-
biol. 10:255-262.
159. McLauchlin, J., S.M. Hall, S.K. Velani, and R.J. Gilbert. 1991. Human listeriosis and pit&
a possible association. Br. Med. J. 303:773-775.
159a. Mkr6, E., and B. Ralovich. 1972. Present situation of human listeriosis in Hungary. Acta
Microbiol. Acad. Sci. Hung. 19:30 1-3 10.
Foodborne Listeriosis 355
160. Misrachi, A., A.J. Watson, and D. Coleman. 1991. Listeria in smoked mussels in Tasmania.
Commun. Dis. Intell. 15:427.
161. Mitchell, D.L. 1991. A case cluster of listeriosis in Tasmania. Commun. Dis. Intell. 15:427.
162. Mogyorosy, R.L., J.I. Wells, and T.V. Riley. 1995. Epidemiology of Listeria monocytogenes
infections in Western Australia, 1978 to 1993. Proceedings of XIIth International Symposium
on Problems of Listeriosis, Perth, Western Australia, October 2-6, p. 480.
163. Moore. M.A., and A.R. Datta. 1994. DNA fingerprinting of Listeria monocytogenes strains
by pulsed-field gel electrophoresis. Food Microbiol. 1 1 :3 1-38.
164. Morris, I.J., and C.D. Ribeiro. 1989. Listeria monocytogenes and pit&.Lancet 2: 1285- 1286.
165. Morris. I.J., and C.D. Ribeiro. 199 1. The occurrence of Listcria species in pit&: the Cardiff
experience 1989. Epidemiol. Infect. 107:11I-I 17.
166. Mossel, D.A.A., J.E.L. Corry, C.B. Struijk, and R.M. Baird. 1995. Essentials of the Microbi-
ology of Foods. New York: Wiley.
167. Murray, E.G.D., R.A. Webb, and M.B.R. Swann. 1926. A disease of rabbits characterized
by a large mononuclear leucocytosis, caused by a hitherto undescribed bacillus Bacterium
monocytogenes (n. sp.). J. Pathol. Bacteriol. 29:407-439.
168. Negri, F., M.L. Massone, M. Cingolani, A. Morando, M. Somenzi, M.A. Barretta, and C.
Savioli. 1994. Fatal neonatal listeriosis after maternal infection acquired with ingestion of
home-made cheese. Minerva Pediatr. 46:395-399.
169. Nicolai-Scholten, M.-E.. J. Potel, J. Natzschka, and St. Pekker. 1985. High incidence of
listeriosis in Lower Saxony, 1983. Immun. Infekt. 13:76-77.
170. Nicholas, J.-A., and N. Vidaud. 1987. Contribution a Iitude des Listeria presentes dans
les denries dorigine animale destinies a la consommation humaine. Recueil de Medecine
Veterinaire I63:283-285.
171. Nocera, D.A., M.H. Fonjallaz, E. Bannerman, J.C. Piffaretti, J. Rocourt, and J. Bille. 1989.
Restriction endonuclease analysis of genomic DNA associated with multilocus enzyme elec-
trophoresis for Listeria monocytogenes strains. Annual Meeting of American Society Micro-
biology, New Orleans, LA, May 14-18, Abstr. D-233.
172. Nocera, D., E. Bannerman, J. Rocourt, K. Jaton-Ogay, and J. Bille. 1990. Characterization
by DNA restriction endonuclease analysis of Listeria monocytogenes strains related to the
Swiss epidemic of listeriosis. J. Clin. Microbiol. 28:2259-2263.
173. Northolt, M.D., H.J. Beckers, U. Vecht, L. Toepoel, P.S.S. Soentoro, and H.J. Wissenlink.
1988. Listeria monocytogenes: Heat resistance and behavior during storage of milk and whey
and making of Dutch types of cheese. Neth. Milk Dairy J. 42:207-219.
174. Oh, M.H.L., H.S. Howe, and M.L. Boey. 1992. Co-existing Listeria and pneumococcal infec-
tion in a chronic alcoholic. J. R. Soc. Med. 85:362.
175. Olding, L., and L. Philipson. 1960. Two cases of listeriosis in the newborn, associated with
placental infection. Acta Pathol. Microbiol. Scand. 48:24-30.
176. Olsen, J.A., A.E. Yousef, and E.H. Marth. 1988. Growth and survival of Listeria monocyto-
genes during making and storage of butter. Milchwissenschaft 43:487-489.
177. Ortel, S. 1968. Bakteriologische, serologische und epidemiologische Untersuchungen wah-
rend einer Listeriose-Epidemie. Dtsch. Gesundheitswesen 2.3:753-759.
178. Papageorgiou, D.K., and E.H. Marth. 1989. Fate of Listeria nronocytogenes during the manu-
facture and ripening of blue cheese. J. Food Prot. 52:459-4.65.
179. Papageorgiou, D.K., and E.H. Marth. 1989. Fate of Listeriu nionocytogenes during the manu-
facture, ripening and storage of Feta cheese. J. Food Prot. 52:82-87.
180. Pierson, M.D., and N.J. Stern. 1986. Foodborne Microorganisms and Their Toxins: Devel-
oping Methodology. New York: Marcel Dekker.
181. Piffaretti, J.-C., H. Kressebach, M. Aeschbacher, J. Bille, E. Bannerman, J.M. Musser, R.K.
Selander, and J. Rocourt. 1989. Genetic characterization of clones of the bacterium Listeria
monocytogenes causing epidemic listeriosis. Proc. Natl. Acad. Sci. USA 86:38 18-3822.
182. Pinner, R.W., A. Schuchat, B. Swaminathan, P.S. Hayes, K.A. Deaver, R.E. Weaver, B.D.
356 Ryser
Plikaytis, M. Reeves, C.V. Broome, J.D. Wenger, and the Listeria study group. 1992. Role
of foods in sporadic listeriosis-11. Microbiologic and epidemiologic investigation. JAMA
267 :2046-2050.
183. Piolino, M., and J.-P. de Kalbermatten. 1968. La list6riose du systkme nerveux central.
Schweiz. Med. Wochenschr. 98:822-828.
184. Potel, J. 1952. Zur Granulomatosis-Infantiseptica. Zbl. Bakteriol. Parasitenk. Abt. I Orig.
l58:329-33 1.
185. Potel, J. 1952/ 1953. Uber die diaplazentare Ubertragung von Listeria Infantiseptica. Wiss.
Z. Martin-Luther Univ. Halle-Wittenberg 2: 15-47.
186. Potel, J. l953/ 1954. Aetiologic der Granulomatosis Infantiseptica. Wiss. Z. Martin Luther
Univ. 3:341-354.
187. Pratt-Lowe, E.L., R.M. Geiger, T. Richardson, and E.L. Barrett. 1988. Heat resistance of
alkaline phosphatase produced by microorganisms isolated from California Mexican-style
cheese. J. Dairy Sci. 7 1 : 17-23.
188. Prentice, G.A., and P. Neaves. 1988. Listeria rnonocytogenes in food: Its significance and
methods for its detection. Bull. Int. Dairy Fed. 223: 1-6.
189. Proctor, M.E., R. Brosch, J.W. Mellen, L.A. Garrett, C.W. Kasper, and J.B. Luchansky.
1995. Use of pulsed-field gel electrophoresis to link sporadic cases of invasive listeriosis
with recalled chocolate milk. Appl. Environ. Microbiol. 6 I :3177-3 179.
190. Ralovich, B. 1984. Listeriosis Research-Present Situation and Perspective. Budapest: Aka-
demiai Kiado.
191. Ray, B. 1996. Fundamental Food Microbiology. Boca Raton, FL: CRC Press.
192. Reiss, H.J., J. Potel, and A. Krebs. 195 1. Granulomatosis Infantiseptica eine durch eine spezi-
fischen Erreger hervorgerufene fetale Sepsis. Klin. Wochenschr. 29:29.
193. Riemann, H., and F.L. Bryan. 1979. Foodborne Infections and Intoxications. 2nd ed., New
York: Academic Press.
194. Riedo, F.X., R.W. Pinner, M. de Lourdes Tosca, M.L. Cartter, L.M. Graves, M.W. Reeves,
R.E. Weaver, B.D. Plikaytis, and C.V. Broome. 1994. A point-source foodborne listeriosis
outbreak: Documented incubation period and possible mild illness. J. Infect. Dis. I70:693-
696.
195. Roberts, D. 1994. Listeria rnonocytogenes and food: the U.K. approach. Dairy Food Environ.
Sanitat. 14:198, 200, 202-204.
196. Rocourt, J., E.P. Espaze, A.F. Miegeville, B. Catimel, and A.L. Courtieu. 1992. La listeriose
en France en 1990-Etude a partir des souches adressees au Centre National de Reference.
Bull. Epidemiol. Hebdom. 16:69-70.
197. Rocourt, J., V. Goulet, A. Lepoutre, P. Dehaumont, and P. Veit. 1994. Epidemiologie de la
listeriose. Cah. Nutr. Diet. 29:98- 101.
198. Rocourt, J., E.P. Espaze, R. Minck, B. Catimel, B. Hubert, and A.L. Courtieu. 1989. Cluster
of listeriosis isolates with different serovar and phagovar characteristics. Lancet 2: 1217- 1218.
199. Rosenow, E.M., and E.H. Marth. 1987. Growth of Listeria rnoncytogenes in skim, whole
and chocolate milk, and in whipping cream during incubation at 4, 8, 13, 21, and 35C. J.
Food Prot. 50:452-459.
200. Rothgardt, N.P., S. Samuelsson, A. Carvajal, and W. Fredericksen. 1988. Human listeriosis
in Denmark I98 1- 1987. Proc. X International Symposium on Listeriosis, Pecs, Hungary,
Aug. 22-26, Abstr. P27.
201. Russell, E.G. I99 1. Clinical cases of listeriosis. Food Austral. 43: 105- 107.
202. Ryser, E.T., and E.H. Marth. 1987. Behavior of Listeria rnonocytogenes during the manufac-
ture and ripening of Cheddar cheese. J. Food Prot. 50:7-13.
203. Ryser, E.T., and E.H. Marth. 1987. Fate of Listeria rnonocytogenes during manufacture and
ripening of Camembert cheese. J. Food Prot. 50:372-378.
204. Ryser, E.T., and E.H. Marth. 1989. Behavior of Listeria rnonocytogenes during manufacture
and ripening of brick cheese. J. Dairy Sci. 72:838-853.
Foodborne Listeriosis 357
205. Ryser, E.T., and E.H. Marth. 1991. Listeria, Listeriosis and Food Safety. 1st ed. New York:
Marcel Dekker.
206. Salamina, G., E. Dalle Donne, A. Niccolini, G. Poda, D. Cesaroni, M. Bucci, R. Fini, M.
Maldini, A. Schuchat, B. Swaminathan, W. Bibb, J. Rocourt, N. Binkin, and S. Salmaso.
1996. A foodborne outbreak of gastroenteritis involving Listeria monocytogenes. Epidemiol.
Infect. 1 17:429-436.
207. Salvat, G., M.T. Toquin, Y. Michel, and P. Colin. 1995. Control of Listeria monocytogenes
in the delicatessen industries: the lessons of a listeriosis outbreak in France. Intern. J. Food
Microbiol. 25:75-8 1.
208. Samuelsson, S., N.P. Rothgardt, A.C. Christensen, and W. Fredericksen. 1990. An epidemio-
logical study of human listeriosis in Denmark 1981 - 1987 including an outbreak November
1985- March 1987. J. Infect. 20:25 1-259.
209. Schlech, W.F. 1984. New perspectives on the gastrointestinal mode of transmission in inva-
sive Listeria monocytogenes infection. Clin. Invest. Med. 7:32 1-324.
210. Schlech, W.F., P.M. Lavigne, R.A. Bortolussi, A.C. Allen, E.V. Haldane, A.J. Wort, A.W.
Hightower, S.E. Johnson, S.H. King, E.S. Nicholls, and C.V. Broome. 1983. Epidemic listeri-
osis: Evidence for transmission by food. N. Engl. J. Med. 308:203-206.
21 1. Schmidt, V., and A. Nyfeldt. 1938. Ueber Mononucleosis Infectiosa und Meningoencephali-
tis. Acta Oto-Laryngol. 26:680-688.
212. Schuchat, A., K.A. Deaver, J.D. Wenger, B.D. Plikaytis, I,. Mascola, R.W. Pinner, A.L.
Reingold, C.V. Broome, and the Listeria study group. 1992. Role of foods in sporadic listeri-
osis---I. Case-control study of dietary risk factors. JAMA 267:204 1-2045.
213. Schwartz, B., D. Hexter, C.V. Broome, A.W. Hightower, R.B. Hirschhom, J.D. Porter, P.S.
Hayes, W.F. Bibb, B. Lorber, and D.G. Faris. 1989. Investigation of an outbreak of listeriosis:
New hypothesis for the etiology of epidemic Listeria monocytogenes infections. J. Infect.
Dis. 159:680-685.
214. Schw;irtz, B., C.V. Broome, G.R. Brown, A.W. Hightower, C A . Ciesielski, S. Gaventa, B.G.
Gellin, L. Mascola, and the Listeriosis Study Group. 1988. Association of sporadic listeriosis
with consumption of uncooked hot dogs and undercooked chicken. Lancet 2:779-782.
215. Seeliper, H.P.R. 1961. Listeriosis. New York: Hafner.
216. Simpson, D.M. 1996. Microbiology and epidemiology in foodborne disease outbreaks: the
whys and why nots. J. Food Prot. 59:93-95.
217. Sipka, M., B. Stajner, and S. Zakula. 1973. Detection of Listeria in milk. Wien tierarztl.
Monatsschr. 60(2/3):50-52.
218. Slade, P.J., and D.L. Collins-Thompson. 1988. Enumeration of Listeria monocytogenes in
raw milk. Lett. Appl. Microbiol. 6: 12 1 - 123.
219. Soontharanont, S., and C.D. Garland. 1995. The occurrence of Listeria in temperate aquatic
habitats. Proceedings of XIIth International Symposium on Problems of Listeriosis, Perth,
Western Australia, October 2-6, pp. 145- 146.
220. Souef, P., N. Le, and B.N.J. Walters. 1981. Neonatal listeriosis-a summer outbreak. Med.
J. Austral. 2: 188- 191.
221. Swaminathan, B., C. Dalton, P. Mead, P.S. Hayes, W.F. Bibb, and A. Schuchat. 1995. Update
on listeriosis in the United States. Proceedings of XIIth International Symposium on Prob-
lems of Listeriosis, Perth, Western Australia, October 2-6, p. 489.
222. Tan, A., H. Li, S. Heaton, and J.R.L. Forsyth. 1995. Probing epidemiological associations
of Listeria monocytogenes with PFGE techniques. Proceedings of XIIth International Sympo-
sium on Problems of Listeriosis, Perth, Western Australia, October 2-6, pp. 191-194.
223. Tappero, J.W., A. Schuchat, K.A. Deaver, L. Mascola, and J.D. Wenger. 1995. Reduction
in the incidence of human listeriosis in the United States--effectiveness of prevention ef-
forts? JAMA 273: I 1 18- 1 122.
224. Tjomb, P. 1995. La "Listeria" a frappe le Brie de Meaux. Rev. Econ. Technol. Indust.
Aliment. 539: 13.
358 Ryser
225. Todd, E. 1988. Cost of foodborne listeriosis. Proc. X International Symposium on Listeriosis,
Pecs, Hungary, Aug. 22-26, Abstr. P4 I .
226. Tulzer, G., R. Bauer, W.D. Daubek-Puza, F. Eitelberger, C. Grabner, E. Heinrich, L. Hohen-
auer, M. Stojakovic, and F. Wilk. 1987. A local epidemic of neonatal listeriosis in Austria-
report of 20 cases. Klin. Padiar. 199:325-328.
227. Urbach, H., and G.1. Schabinski. 1955. Zur Listeriose des Menschen. 2. Hyg. 141:239-248.
228. Vilde, J.L., A. Huchon, M. Mignon, H. Scherrer, E. Bergogne-Berezin, and J. Pierre. 1980.
Infection dallure typique due i Listeria monocytogenes apris absorption dhuhres. Nouvelle
Presse Med. 9:3281.
229. Vizcaino, L.L., M.-J. Cubero, and A. Contreras. 1988. Listeric abortions in ewes and cows
associated to orange peel and artichoke silage feeding. Proc. X International Symposium on
Listeriosis, Pecs, Hungary, Aug. 22-26, Abstr. P29.
230. Vlahovic, S.M.,D. Pantic, M. Pavicic, and J.H. Bryner. 1988. Transmission of Listeria mono-
cytogenes from mothers milk to her baby and to puppies. Lancet 2:1201.
23 1 Vries, J., and R. Strikwerda. 1957. Ein Fall klinishcher Euter-Listeriose beim Rind. Zbl.
Bakteriol. Abt. I. Orig. 167:229-232.
232. Waites, W.M., C.E.R. Dodd, and K.J. Bolton. 199 I . Microbial food poisoning: problems
and solutions. Br. Food J. 93:4-9.
233. Watson, G.L., and M.G. Evans. 1985. Listeriosis in a rabbit. Vet. Pathol. 22:191-193.
234. Watson, C., and K. Ott. 1990. Listeria outbreak in Western Australia. Commun. Dis. Intell.
24:9- 12.
234a. Wenger, J.D., B. Swaminathan, P.S. Hayes, S.S. Green, M. Pratt, R.W. Pinner, A. Schuchat,
and C.V. Broome. 1990. Listeria monocytogenes contamination in turkey franks: evaluation
of a production facility. J. Food Prot. 53:1015-1019.
235. Wesley, I.V., and F. Ashton. 199I . Restriction enzyme analysis of Listeria monocytogenes
strains associated with food-borne epidemics. Appl. Environ. Microbiol. 57:969-975.
236. WHO Working Group. 1988. Foodborne listeriosis. Bull. WHO 66:421-428.
237. Wramby, G.O. 1944. Ovn Listerella monocytogenes bokteriologi ach our forekomst av lister-
ellainfection has djur. Skandinavisk Veterinar Tidskrift 34:278-290.
238. Wramby, G.O. 1944. Unpublished data.
239. Yersin, B.R., M.P. Glauser, and F. Regli. 198I . Infections i Listeria monocytogenes chez
Iadulte-Etude de 10 cas et revue de la littkrature. Schweiz. Med. Wochenschr. 1 1 1 :1596-
1602.
240. Yousef, A.E., and E.H. Marth. 1988. Behavior of Listeria monocytogenes during the manu-
facture and storage of Colby cheese. J. Food Prot. 5 1 : 12- 15.
241. Goulet, V. 1998. Personal communication.
Incidence and Behavior of Listeria
monocytogenes in Unfermented
Dairy Products
ELLIOTT. RYSER
Michigan State University, East Lansing, Michigan
INTRODUCTION
Recognition of raw milk as a potential source of Listeria monocytogenes led to speculation
that consumption of such milk was at least partly responsible for the previously described
listeriosis outbreak in post-World War I1 Germany. After this listeriosis epidemic, only
scattered reports of individuals drinking raw milk, along with assurances that raw milk
was being properly pasteurized, virtually eliminated the threat of any further outbreaks
of milkborne listeriosis. Consequently, research in this area also subsided. However, in
1983, concerns about the possibility of milkborne listeriosis were rekindled when con-
sumption of pasteurized milk was epidemiologically linked to an outbreak of listeriosis
in Massachusetts. Two events, namely, publication of an article in the New England Jour-
nal of Medicine detailing this outbreak in Massachusetts and a report in June of 1985 that
as many as 300 people in California had acquired listeriosis after eating Mexican-style
cheese contaminated with L. monocytogenes, caused considerable concern in the United
States about the presence of Listeria in dairy products. This problem subsequently took
on international proportions with the 1987 report of another cheese-related outbreak in
which consumption of tainted Vacherin Mont dOr soft-ripened cheese was directly linked
to numerous cases of listeriosis in Switzerland. Despite considerable progress, such epi-
demics continue to plague the dairy industry, with pasteurized chocolate milk and Pont
359
360 Ryser
1EvSque cheese being responsible for the most recent dairy-related outbreaks of listeriosis
in the United States and France, respectively.
In response to questions raised by milk producers, dairy processors, health officials,
and the general public, a plethora of work has been conducted worldwide since 1983 to
determine the incidence and behavior of L. rnonocytogenes in unfermented (raw milk,
pasteurized milk, chocolate milk, cream, butter, ice cream, other frozen dairy desserts) as
well as fermented (cheese, yogurt, cultured milk) dairy products. The incidence and behav-
ior of L. rnonocytogenes in unfermented dairy products will be dealt with in this chapter;
similar information about fermented dairy products appears in Chapter 12.
TABLE
1 Incidence of Listeria spp. in Raw Milk Produced in the United States and Canada
Number of positive samples (%)
Number of
Location samples L. inonocyogenes L. innocuu L. ri.eist'iirneri Others Ref.
USA
California 200 14 (7.0) 19 (9.5) 0 0 141
100 0 4 (4.0) 0 1 (1.0y 141
Massachusetts 121 15 (12.4) ND ND ND 129
Massachusetts, 939 15 (1.6) ND ND ND 87
Vermont
Minnesota 300 9 (3.0) 77 (25.7) 5 (1.7) 0 144
84 0 6 (7.1) 1 (1.2) 0 161
Nebraska 200 8 (4.0) 10 (5.0) 0 0 139
Ohio, Kentucky, 350 13 (3.7) 27 (7.7) 6 (1.5) 3 (0.9)a 141
and Indiana
Pennsylvania 251 1 79 (3.1) ND ND ND 88
Tennessee 292 12 (4.1) ND ND ND 177
Wisconsin 50 0 ND ND ND 89
55 0 0 0 0 20 1
Total 5 197 165 (3.2) 143 (11.1) 12 (0.9) 4 (0.3)
Canada
Alberta 426 8 (1.9) ND ND ND 106
252 4 (1.6) ND ND ND 200
Manitoba 256 4 (1.6) ND ND ND 80
Ontario 445 6 (1.3) 43 (9.7) 6 (1.3) 0 97
3 15 17 (5.4) 26 (8.2) 1 (0.3) 0 190
Total 1694 39 (2.3) 69 (9.1) 7 (0.9) 0 (0)
ND, not determined (omitted from total).
Two L. ivnnovii and one L. seeligeri
362 Ryser
TABLE
2 Incidence of Listeria spp. in Raw Milk Collected Outside of North America
Number of positive samples (%)
Number of
Location samples L. monocytogenes L. innocua L. welshimeri Others Ref.
Europe
Czechoslovakia 177 6 (3.4) ND ND ND 151
123 4 (3.3) ND ND ND 152
Denmark 1,227,053 278 (0.02) ND ND ND 167
Finland 256 13 (5.1) ND ND ND 167
59 1 (1.7) ND ND ND 167
France 1409 85 (6.0) ND ND ND 43
561 21 (3.8) ND ND ND 49
337 14 (4.2) 5 (1.5) 0 0 120,121
51 10 (19.6) 10 (19.6) 18 (35.3) 9 (17.6) 120,121
635 2 (0.3) ND ND ND 115, 197
80 3 (3.8) ND ND ND 174
50 2 (4.0) ND ND ND 174
Ireland 589 29 (4.9) 20 (3.4) ND ND 173
50 4 (8.0) ND ND ND 133
Italy 290 8 (2.8) 16 (5.6) 0 2 (0.7) 83
142 0 1 (0.6) 0 0 192
98 2 (2.0) ND ND ND 138
85 0 0 0 0 117
50 0 1 (2.0) 0 0 199
40 0 0 0 0 148
32 0 2 (6.3) ND ND 112
Netherlands 137 6 (4.4) ND ND ND 68
Poland 134 2 (1.5) ND ND ND 136
81 6 (7.4) ND ND ND 178
L. monocytogenes in Unfermented Dairy Products 363
that contamination of raw milk on the farm is an ongoing process. Whereas these authors
did not quantitate L. monocytogenes in any of the samples examined, Slade and Collins-
Thompson [ 1891 reported that positive raw milks from bulk tanks in southwestern Ontario
always contained <5 L. monocytogenes CFU/mL when samples were analyzed by direct
plating and a most probable number (MPN) enrichment procedure. Thus the positive raw
milk samples encountered in three Canadian studies likely contained only low levels of
L. monocytogenes. In two subsequent surveys, L. monocylogenes was identified in 1.6
[200] and 1.9% [106] of all raw milk bulk tank samples examined in the province of
Alberta. However, both surveys also indicated substantially higher contamination rates
for commingled raw milk before processing with milk in 5 of 72 (6.9%) tank trucks [ 1061
and in 4 of 15 (26.6%) milk silos [200] testing positive for L. monocytogenes. As was
true for the three United States surveys [ 1411just discussed, L. innocua also was the most
common Listeria sp. isolated from Canadian raw milk, with 8.2 and 9.7% of the samples
being reported as positive. Overall, 2.3 and 9.1% of all Canadian raw milk samples con-
tained L. rnonocytogenes and L. innocua, respectively, as compared with 3.2 and 1 1.1 %
of raw milk samples examined in the United States. Thus the incidence of listeriae in raw
milk from both countries appears to be similar.
Public concern and economic hardships brought about by several recalls of French
Brie cheese imported into the United States prompted French scientists to begin surveying
raw milk for L. monocytogenes. Results from three such surveys [43,49,120] (see Table
2) conducted between 1986 and 1988 indicated that 85 of 1409 (6.0%), -21 of 561 (3.8%),
and 14 of 337 (4.2%) raw milk samples from French bulk tanks were positive for L.
monocytogrnes, with L. innocua being detected in about one third as many samples in
the latter study [120]. During January of 1986, 51 raw milk samples were submitted to
the French Central Laboratory of Food Hygiene. Both L. monocytogenes and L. innocua
were found in 19.6% of these samples. The unusually high incidence of listeriae ob-
served in this survey, as compared with other studies described thus far, may be the re-
sult of nonrandom sampling or seasonal variations, the latter of which will be discussed
shortly.
Frequent isolation of L. monocytogenes from dairy products prompted additional
surveys of the raw milk supply throughout Europe and later elsewhere, with primary focus
being given to countries with sizeable dairy industries (see Table 2). Results from two
comprehensive year-long surveys in the United Kingdom [ 1 181 and Scotland [ 1071 re-
vealed an incidence of L. monocytogenes in raw milk similar to that observed in the United
States and Canada, with actual Listeria populations in positive samples also being esti-
mated to be extremely low. As in North America, L. monocytogenes strains belonging to
serotype 1/2 appear to predominate in European raw milk [ 107,108,126,1781. Although
four subsequent surveys from the United Kingdom yielded similar findings [ 107,119,
126,1571, L. monocytogenes contamination rates of 14 to 15% have been reported from
both Scotland [ 1081 and Northern Ireland [ 1271, thus suggesting considerable local vari-
ability. Harvey and Gilmour [127] also found that 33% of raw milk samples collected
from processing centers harbored L. monocytogenes, which again emphasizes the impact
of commingling milk. However, levels of listeriae in such milk again appear to be quite
low, with actual numbers of L. monocytogenes typically being < 10 CFU/mL in positive
samples from Scotland [ 1081.
In 1987, Beckers et al. [68] reported culturing L. monocytogenes from 6 of 137
(4.4%) raw milk samples obtained from farms in the Utrecht region of The Netherlands.
As was true for raw milk tested in the United States and Canada, milk samples from The
366 Ryser
Netherlands again contained < 100 L. monocytogenes CFU/mL. Similar findings have
been reported from most other European surveys, with 3-5% of raw milk samples harbor-
ing low levels of L. monocytogenes. However, two nationwide surveys conducted in Swit-
zerland during and after the outbreak involving Vacherin Mont dOr cheese indicated a
far lower incidence, with only 0.4 and 0.6% of the raw milk samples being positive for
L. monocytogenes [66]. Several years earlier, Terplan [ 1971 detected L. monocytogenes
in only 2 of 635 (0.3%) raw milk samples obtained from farm bulk tanks in Wurtemburg,
Germany. Subsequent attempts to isolate this pathogen from 448 quarter-milk and 30
separator sludge samples failed, along with attempts to culture this organism from raw
milk samples obtained from tank trucks and storage tanks. More recently, findings from
an exhaustive survey in Denmark of over 1 million raw milk samples demonstrated an
L. monocytogenes contamination rate of only 0.2%, with several additional small-scale
Italian surveys yielding negative results. Thus, when the Danish results are excluded, 3.6%
of all European raw milk would be expected to contain low levels of L. monocytogenes
as compared with 3.0% of the raw milk produced in North America.
In response to the aforementioned North American and European surveys, investiga-
tors in Australia, New Zealand, the Middle East, South America, Africa, and the Orient
also have begun assessing the incidence of L. monocytogenes contamination in their own
raw milk supplies. In the first of these surveys, workers in New Zealand [ 1941 collected
and analyzed 71 raw milk bulk tank samples between August 1986 and March 1987 for
listeriae using both warm and cold enrichment. Although L. monocytogenes was appar-
ently absent from this milk as well as from milk examined in a later Australian survey,
isolation of other Listeria spp. from these samples strongly suggests that such milk is
unlikely to be completely free of L. monocytogenes. Although similar negative findings
have been obtained from surveys conducted in Brazil [73] and Costa Rica [63], Arias
et al. [63] also cited another survey in which 20% of hand-milked samples harbored L.
monocytogenes. Working in Japan, Takai et al. [196] reported the absence of Listeria spp.
from 120 raw milk samples, whereas L. monocytogenes was recovered from 6 of 150
(4.0%) samples in a subsequent Japanese survey [186]. Given these findings along
with additional reports of L. monocytogenes in raw milk from Egypt [93,1], Iran
[172], Turkey [124], Morocco [94], and South Africa [204], it is now clear that
milkborne listeriosis constitutes a worldwide threat. However, confirmation of dairy-
related listeriosis cases in such developing countries will likely remain very difficult
given their lack of resources and understandable preoccupation with far more immediate
health concerns.
Examination of data summarized in Tables 1 and 2 indicates that 3.2, 2.3, and 3.6%
of the raw milk produced in the United States, Canada, and Europe, respectively, can be
expected to contain low levels of L. monocytogenes, with similar contamination rates also
likely occurring in other parts of the world. Except for the European samples, L. innocua
was isolated from raw milk more frequently than L. monocytogenes, with the former found
in 11.1, 9.1, and 3.4% of the samples analyzed for all Listeria spp. in the United States,
Canada, and Europe, respectively. This observation also is supported by additional survey
findings from Australia, Costa Rica, Egypt, and New Zealand.
Although L. innocua is nonpathogenic, isolation of this organism and other listeriae
from dairy products and dairy processing facilities is taken very seriously in the United
States, since nonpathogenic listeriae and L. monocytogenes are assumed to occur in similar
environmental niches. Use in the United States of L. innocua as a potential indicator of
L. monocytogenes is supported by data from the raw milk surveys just described (see
L. monocytogenes in Unferrnented Dairy Products 367
Table 1) in that isolation of listeriae other than L. morzocytogenes from dairy products
and processing facilities suggests that the factory environment may be contaminated with
raw milk; that is, a product that can be expected to contain L. monocytogenes 3-4% of
the time. Although long theorized, the spread of identical L. nzonocytogenes strains from
the farm environment into the raw milk supply and ultimately to dairy processing facilities
now has been confirmed using various strain-specific typing methods, including restriction
fragment length polymorphism [ 1271 and automated ribotyping [64].
In addition to assessing the general incidence of listeriae in raw milk, several of the
surveys just described also dealt with seasonal variations in the incidence of L. monocyto-
genes and L. innocuci in raw milk produced in the United States [88,139,141] and Canada
(971. However, since all of the aforementioned studies differ in numbers and sizes of
samples analyzed as well as the Listericl isolation procedures employed, it is difficult to
make any definitive statement concerning the seasonal occurrence of Listerin in raw milk
[ 107,190]. Nonetheless, several distinct trends can be observed from selected data in Fig-
ure 1. First. the overall incidence of L. rnonocytogenes in raw milk was highest in spring
(5.8%) followed by winter (4.5%), fall (2.8%), and summer (2.5%). Second, a somewhat
similar seasonal variation also can be observed for the incidence of L. innocun in raw
milk, with the highest overall percentage of positive samples again occurring in winter
(1 1.7%) followed by spring (10.2%). summer (6.0%), and fall (4.2%). These findings
again suggest that L. innocirn can be used as a potential indicator for the presence of L.
inonocytogenes.
Although not fully understood, current herd management and feeding practices may
be at least partly responsible for seasonal differences observed in isolation rates for L.
inonocytogenes and possibly L. iiinocun. During cold winter months, silage comprises a
major component of the diet. While investigating a listeriosis outbreak, Donnelly [85]
observed that 8 of 44 Holstein cows fed Listeria-contaminated silage shed the organism
in their milk. Furthermore, milk from these animals was free of L. monocytogenes 1 month
after feeding of contaminated silage ceased. Ruminants that ingest contaminated silage
may either succumb to infection or carry L. monocytogenes asymptomatically ; however,
if the animal lives, the organism can be shed for many months in feces and in milk from
lactating animals. Extended survival of L. monocytogenes in fecal material, soil, and grass
can perpetuate the infectious cycle shown in Figure 2, particularly when animals are win-
tered in cramped quarters. Once dairy cattle resume grazing on pastures during late spring,
summer, and early fall, L. monocytogenes becomes dispersed over a wide area, which, in
turn, weakens the infectious cycle by decreasing the likelihood that animals will come in
contact with contaminated material.
Seasonal differences in the incidence of Listeria spp. in raw milk also may be related
to breeding practices. Dairy cattle typically bear their young in late winter or early spring.
During winter gestation, dairy cattle develop a weakened immune system as a direct result
of pregnancy, which, in turn, makes these animals more susceptible to listerial infections
and abortions. These events can then culminate in the shedding of L. monocytogenes in
milk and fecal material. Increased environmental stress and changes in habitat that occur
during winter, along with increased difficulties in providing proper herd hygiene, all can
serve to decrease the natural defense system in dairy cattle, which again increases the
likelihood for listerial infections. Once an asymptomatic animal begins shedding L. mono-
cytogenes in feces, the organism is likely to spread quickly to other animals that are housed
in close proximity to the shedder. In this way, confinement of dairy cattle may play an
important role in increasing the number of animals that shed L. monocytogenes in their
milk during late winter and early spring.
Survival in
s o i l and grass / Ingestion
by ruminants
confinement
4
-
L. monocytogenes
excreted in feces
-
L. monocytogenes
secreted in milk
TABLE
3 Incidence of L. rnonocytogenes in Raw Ewes and Goats Milk
Number of Number of samples
Type of milk Location samples positive (%) Ref.
Ewe England/ Wales 56 1 (1.8) 126
Italy 40 0 71
Italy 34 0 76
Spain 1052 23 (2.2)a 175
Turkey 302 0 78
Total 1484 24 (1.6)
Goat United States 450 17 (3.8) la
England/Wales 480 4 (0.8) 126
Italy 24 0 71
Portugal 25 1 (4.0) 79
Spain 1445 37 (2.6) 116
Australia 69 1 (1.4) 125
Total 2493 60 (2.4)
earned a reputation for being both safe and nutritious, with dairy products accounting for
<1.5% of all foodborne illnesses [67]. Pasteurized milk is responsible for 5 5 % of all
reported dairy-related illnesses [ 1501. Despite these impressive findings, public confidence
in the safety of pasteurized milk began to erode in July of 1982 following an outbreak
of yersiniosis in Tennessee, Arkansas, and Mississippi [ 1951 and again in 1983 when the
CDC claimed that consumption of pasteurized milk was responsible for 49 cases of listeri-
osis in Massachusetts. In 1985, the dairy industry was dealt another blow when at least
16,000 culture-confirmed cases of salmonellosis were associated with drinking a particular
brand of pasteurized milk produced in the Chicago area [183].
These three outbreaks, along with the previously discussed listeriosis outbreak in
California linked to consumption of contaminated Mexican-style cheese, prompted the
FDA to take corrective action in the form of a large-scale testing program commonly
referred to as the FDA Dairy Initiative Program [134] (Fig. 3). This program, begun in
April of 1986 in cooperation with individual state agencies and members of the National
Conference on Interstate Milk Shipments, was designed to examine every interstate milk
shipment (IMS) pasteurization facility in the United States for potential safety problems
related to pasteurization, postpasteurization contamination, cleaning and sanitizing regi-
mens, equipment maintenance, and educationaUtraining programs for dairy factory per-
sonnel. As part of the FDA Dairy Initiative Program, the agency also established the
Microbiological Surveillance Program, which was designed to detect L. monocytogenes,
Salmonella spp., Yersinia enterocolitica, Campylobacter jejuni, and C. coli (Campylo-
bacter omitted in 1987) as well as Staphylococcus aureus, which was added in 1987 for
dry and fluid milk [16]. Dairy products tested under this program included fluid milk,
nonfat dry milk, cream, butter, ice cream, ice milk, and other dairy commodities over
1 ! I
I I
April 1986: Begin dairy initiatives. Portion of IMS and non-PIS
inventory sampled f o r Listeria, Salmonella, Yersinia and
Campylobacter. Plant inspections in cooperation with
state agencies. 1174 samples analyzed during fiscal 1986.
1
~
-I
Staphylococcus aureus and Yersinia; Dry milk: Listeria,
Salmonella, and Scaphylococcus aureus.
FIGURE3 FDA Dairy Initiatives Program. IMS, interstate milk shipment. (Adapted
from Ref. 134)
L. monocytogenes in Unfermented Dairy Products 371
which the FDA has jurisdiction. Analysis of cheeses (except cottage cheese) was covered
under a series of separate programs, which will be discussed in Chapter 12 concerning
fermented dairy products. Under provisions of this program, FDA inspectors collected 30
retail-sized containers of as many as five different products available from dairy factories
at the time of inspection. Duplicate 25-g or 25-mL samples obtained after combining 30
retail-sized samples per product were then analyzed for L. monocytogenes and other lister-
iae using the original and later versions of the FDA procedure.
Since raw milk containing L. monocytogenes will enter every dairy factory in the
United States from time-to-time, it is logical to assume that finished products also may
become contaminated with this pathogen. During the first 2 years of the FDA Dairy Initia-
tive Microbiological Surveillance Program (Table 4), L. monocytogenes was isolated from
2 of 350 samples of pasteurized whole milk and 5 of 415 samples of chocolate milk,
which suggests that approximately 0.67% of the pasteurized milk available in the United
States could contain this pathogen unless factories take corrective action to reduce this
value. In contrast, L. monocytogenes was isolated from 3 of 99, 23 of 659, and 30 of 351
samples of ice milk, ice cream, and novelty ice cream, respectively. Only two of the
positive ice cream samples were analyzed quantitatively for L. monocytogenes. One con-
tained an average of 15 L. monocytogenes CFU/g, whereas the other sample contained
between 1 and 5 CFU/g. Thus during the time of this survey, approximately 5% of frozen
dairy products manufactured in the United States presumably contained low levels of L.
monocytogenes.
Furthermore, L. innocua was isolated from three of four product categories (see
Table 4) which also contained samples positive for L. monocytogenes. Since both organ-
TABLE
4 Incidence of Listeria spp. in Unfermented Dairy Products Manufactured
in the United States during 1986 and 1987
Number of positive samples (%)
Number of -
Product samples tested L. rnonocytogenes L. innocua
Whole milk 350 2 (0.57) 0
Lowfat milk 182 0 2 (1.10)
Skim milk 98 0 0
Chocolate milk 415 5 (1.20) 1 (0.24)
Cream 52 0 0
Half and half 42 0 0
Ice milk 99 3 (3.03) 0
Ice cream 659 23 (3.03) 12a(1.82)
Novelty ice cream 35 1 30 (8.55) lob (2.85)
Butter 30 0 0
Nonfat dry milk 44 0 0
Casein/Milk protein hydrolysate 15 0 0
Other products 171 0 0
Total 2518 63 (2.50) 25 (0.99)
L. seeligeri also detected in 7 of 659 (1.06%) samples.
L. gruyi also detected in I of 351 (0.28%) samples.
Dairy blend whey, eggnog.
Source: Refs. 62 and 135.
372 Ryser
isms likely occupy similar niches in the natural environment and dairy processing facili-
ties, isolation of L. innocua from a dairy product should raise immediate concerns about
the possible presence of L. monocytogenes.
Greater success in isolating L. monocytogenes from chocolate rather than whole
milk is likely related to the organisms ability for enhanced growth in this product as
compared with other fluid dairy products. Reasons for increased growth of Listeria in
chocolate milk will be discussed shortly in conjunction with the behavior of listeriae in
autoclaved fluid dairy products.
The higher incidence of L. monocytogenes in frozen rather than fluid dairy products
coincides with the relatively complex handling of ice milk, ice cream, and particularly
ice cream novelties during manufacture and packaging. This, in turn, suggests that these
products are most likely contaminated after pasteurization through either direct or indirect
contact with listeriae within the dairy factory environment. This hypothesis is supported
by frequent isolations of L. monocytogenes from many areas within dairy factories, includ-
ing floors, ceilings, drains, and coolers. In addition, this organism also has been found in air
and condensate and on various pieces of equipment, including conveyor belts. A detailed
discussion of the incidence of L. monocytogenes in food processing facilities, including
dairy factories, can be found in Chapter 17.
Inability of the FDA to detect L. monocytogenes in skim and low-fat milk as well
as half and half, cream, and butter may have resulted from the separation processes used
for adjusting milk-fat content that these products undergo. Such centrifugal separation
processes tend to decrease levels of listeriae, particularly if leukocytes containing the
organism are still present after initial clarification of the milk.
Failure to isolate L. monocytogenes from nonfat dry milk and casein/ protein hydro-
lysates may be partly related to the heat treatments necessary to manufacture these prod-
ucts. This theory is supported by the work of Doyle et al. [90], who demonstrated that
populations of L. monocytogenes decreased 1 9 0 % during conversion of skim milk into
nonfat dry milk via spray drying. However, failure to isolate L. monocytogenes from dried
dairy products also may result from the generally recognized inability of the FDA method
to detect cells of L. monocytogenes that have been sublethally injured during thermal
processing. Thus the methodology employed to detect L. monocytogenes will predetermine
whether or not the organism can be isolated from a particular food.
According to the Food, Drug and Cosmetic Act of 1938, a food may be considered
adulterated and therefore unfit for human consumption if the product contains poisons or
other harmful substances (e.g., pathogenic microorganisms) at detrimental concentrations.
Although the oral infective dose for L. monocytogenes is presently unknown, evidence
from the California listeriosis outbreak involving Mexican-style cheese suggests that the
number of L. monocytogenes cells needed to induce this life-threatening illness may be
quite low-perhaps as few as several hundred to a few thousand total cells for certain
segments of the population. Although not directly applicable to the human population,
several independent studies involving immunocompromised mice have demonstrated LDSo
values (the dose of cells which is lethal to 50% of a given population) in the range of
approximately 10 [ 1231 and 4 to 480 [77,193] L. monocytogenes cells when the pathogen
was administered orally and intraperitoneally, respectively. Consequently, because of a
moral obligation to the public, the FDA has adopted and is continuing to uphold a policy
of zero tolerance regarding presence of L. monocytogenes in ready-to-eat foods.
In accordance with Title 21 of the United States Code of Federal Regulations, Sec-
tion 7.40 [ 1 1 I], the FDA can request that firms voluntarily recall any product that contains
L. monocytogenes in Unfermented Dairy Products 373
5 Chronological List of Voluntary Class I Recalls Issued in the United States During 1986 and 1987 for Milk and Unfermented
TABLE
Dairy Products Contaminated with L. monocytogenes
Month/ year State of
Type of dairy product of recall manufacture Distribution Quantity Ref.
~~~~~~~~~~ ~ ~
Ice cream 4/87 New York New York, Pennsylvania -316 gal 27,30
Ice cream (48 flavors), ice 7/87 California California, Oregon -60,000 gal 20,40
milk (6 flavors), sher-
bert (5 flavors)
Ice cream nuggets 7/87 Maryland Connecticut, Florida, 20,400 boxes 21,28
Maryland, New Jersey,
New York, North Caro-
lina, Ohio, Pennsylva-
nia, Virginia
Ice cream, ice milk 7/87 Nebraska Colorado, Iowa, Kansas, -30,000 gal 17
Nebraska, North Da-
kota, South Dakota, Wy-
oming
Ice cream sundae cones 8/87 Florida Alabama, Arizona, British Unknown 22
West Indies, Florida,
Louisiana, Mississippi,
North Carolina, Ohio,
Puerto Rico, South Caro-
lina, Tennessee, Vir-
ginia, West Virginia
Chocolate ice cream 8/87 Kentucky Florida, Puerto Rico -956 gal 14
Ice cream nuggets 9/87 Maryland Nationwide Unknown 23
Ice cream novelties-sand- 9/87 Ohio Nationwide Unknown 24
wiches, bars, pieces,
slices, sundae cones
Ice cream bars 10187 Ohio Michigan, Ohio, Pennsyl- 5 1,780 bars 18
vania, West Virginia
Chocolate ice cream 2/88 Georgia Georgia, North Carolina Unknown 32,35
Ice cream, sherbet, ice 7/88 Ohio Ohio >1083 gal 31
milk
Ice cream 8/88 Connecticut Connecticut, New York, 5.6-8.4 gal 37
Massachusetts
Ice cream 8/88 Connecticut Connecticut 30 gal 36
Ice cream pies 9/88 Connecticut Connecticut, New York, 1700 pies 33
Massachusetts
376 Ryser
TABLE
5 Continued
Monthlyear State of
Type of dairy product of recall manufacture Distribution Quantity Ref.
Despite millions of gallons of frozen dairy products that have been recalled both
formally and internally, it must be stressed that only one case of listeriosis has been posi-
tively linked to consumption of a contaminated frozen dairy product in Belgium [4] (see
Chapter 10). On this basis, the International Ice Cream Association and the Milk Industry
Foundation have contended that a Class I recall may be too harsh a response for a frozen
dairy product containing presumably very low levels of L. monocytogenes. However, until
the oral infectious dose and relative risk for susceptible individuals can be firmly estab-
lished, the FDA is likely to maintain its zero tolerance policy for L. monocytogenes and
continue requesting recalls of products containing this pathogen at any detectable level.
The U.S. government has developed one of the most stringent policies regarding
presence of L. monocytogenes in ready-to-eat foods, whereas most other countries have
adopted more relaxed policies (i.e. not >100 CFU/g or ml), particularly where consump-
tion of contaminated products that have not yet been firmly linked to cases of listeriosis
is concerned. As an example of the latter attitude, the Canadian government has decided
to confine all formal recalls to only those foods that have been linked to major outbreaks
of listeriosis, namely, coleslaw, soft cheese, and pasteurized milk, with the role of pasteur-
ized milk in foodborne listeriosis still being highly debated [ 1331. Hence, no recalls were
issued when researchers at the Health Protection Branch of Health and Welfare Canada
(analogous to the U.S. FDA) identified L. monocytogenes in 1 of 394 (0.25%) and 1 of
51 (2.0%) samples of ice cream and ice cream novelties, respectively [96], during their
own federal inspection program. Although subsequent investigations were presumably
conducted to identify (a) the source of contamination, (b) proper corrective measures, and
(c) possible links to human illness, Canadian officials maintained that recalling the two
contaminated lots would be inappropriate without proof that consumption of Listeria-
contaminated ice cream can lead to listeriosis. Many individuals and most manufacturers
will undoubtedly argue in favor of the more relaxed Canadian position. When one consid-
ers the numerous recalls of Listeria-contaminated ice cream in the United States, that
worldwide only one case of listeriosis has been positively linked to ice cream containing
unusually high numbers of listeriae, the inability of L. monocytogenes to grow in this
product during frozen storage and the normal exposure rate of the human population to
listeriae, it appears that the risk of contracting listeriosis from contaminated ice cream is
extremely low. Although current scientific data mandate the immediate removal of fluid
dairy products and cheeses that support growth of L. monocytogenes, it appears that a
scientifically valid argument can be made against recalling certain dairy products in which
listeriae will not proliferate such as ice cream and dried goods which, if contaminated,
typically contain very low numbers of listeriae as postpasteurization contaminants.
As a result of several large recalls of French Brie cheese and a listeriosis outbreak
in Switzerland that was traced to consumption of Vacherin Mont dOr soft-ripened cheese,
European scientists have logically focused their attention on the incidence of listeriae in
cheese. However, numerous recalls of unfermented dairy products in the United States
also have heightened public health concerns about the presence of listeriae in pasteurized
dairy products manufactured outside of North America.
In one of the first European surveys of finished products reported in 1988, research-
ers in Germany [ 1971 failed to isolate Listeria spp. from pasteurized milk (39 samples),
nonfat dry milk (1 1 samples), caseidcaseinate (30 samples) and various dried products,
including baby food (Table 6). During the same year, investigators in Hungary [ 1001 and
The Netherlands [69] also failed to recover L. monocytogenes from samples of pasteurized
milk, with similar negative findings being obtained from most other subsequent surveys
L. monocytogenes in Unfermented Dairy Products 379
of properly pasteurized milk and cream produced in Europe, Australia, the Middle East,
and North Africa (Table 6). However, L. monocytogenes was eventually demonstrated in
11 of 1039 (1.1%), 4 of 115 (3.5%), and 1 of 95 (1.1%) pasteurized milk samples examined
in the United Kingdom [ 119,126,1821 for a combined contarnination rate of 1.3%, with
these findings generally being similar to those observed in the United States. According
to Garayzabal et al. [113], 21.4, 89.2, 10.7, and 3.6% of pasteurized milk samples from
one particular milk processing facility in Madrid contained I,. monocytogenes, L. grayi,
L. innocua, and L. welshimeri, respectively. These same authors [ 114,1761 previously
reported similar Listeria contamination rates for raw milk entering the same processing
facility. Furthermore, after pasteurization these same samples had a total mesophilic aero-
bic plate count of 2.5 X 107CFU/mL, which is well above the maximum allowable limit
of 1 X 104CFU/mL for properly pasteurized milk in the United States. Hence, improper
pasteurization caused by leaking pasteurizer plates, as suggested by Northolt et al. [ 1561,
and/or postpasteurization contamination from the factory environment appear to be most
likely responsible for the unusually high incidence of listeriae in pasteurized milk sam-
ples from this particular dairy factory. Although results from these aforementioned surveys
of pasteurized milk, cream, and dried products are very encouraging, the isolation methods
used in these studies were generally unable to detect sublethally injured listeriae. Hence,
the true incidence of listeriae in pasteurized milk, cream, and dried products may well be
somewhat higher. To enhance recovery of injured cells, the International Dairy Federation
has recommended that such dairy products undergo preenrichment in a nonselective me-
dium (i.e., buffered peptone water) before primary enrichment in various selective broths
and plating on Listeria-selective media [37,198]. Further details concerning recovery of
sublethally injured listeriae can be found in Chapter 7.
Results from a 1989 International Dairy Federation survey [ 1331 indicated that pub-
lic health issues regarding the presence of listeriae in pasteurized milk were clearly spread-
ing beyond the continental boundaries of Europe and North America, with the many afore-
mentioned surveys from Table 6 attesting to these concerns. More recently, the safety of
several additional dairy products, including flavored milks, chocolate milk, ice cream, and
butter has attracted international attention with the FDA Initiatives Program, the many
Class I recalls of Listeria-contaminated dairy products, and fears of international trade
embargoes fueling these concerns. Following the 1987 discovery of L. monocytogenes in
Australian ricotta cheese, New Zealand and Australian officials instituted Listeria-moni-
toring programs for caseidcaseinate products as well as high-moisture cheese, pasteurized
milk, ice cream, and milk powders. Results from one 10-month survey begun in April
1988 [202] revealed the presence of L. monocytogenes in 1 of 206 (0.48%) samples of
pasteurized flavored/unflavored milk processed in and around Melbourne. Subsequent
identification of heat-labile alkaline phosphatase in the contaminated product (pasteurized
milk to which a pasteurized flavored syrup was added) suggested that improper pasteuriza-
tion was most likely responsible for the presence of L. monocytogenes in the final product.
However, unsatisfactory storage of the flavored syrup also may have contributed to con-
tamination. In keeping with Listeria policies developed in the United States and Canada,
Australian officials withdrew the affected product from the marketplace and prohibited
the sale of all subsequently produced product until 12 consecutive lots of Listeria-free
pasteurized flavored milk could be produced from the same product line.
As in the United States, recent foreign surveys also have shown a higher incidence
of L. monocytogenes in chocolate milk (1 1.6%), ice cream (2.0-13.9%), and butter (3.8-
6.7%) as compared with pasteurized milk and dried products which are seldom contami-
380 Ryser
TABLE
6 Incidence of Listeria spp. in Pasteurized Dairy Products Produced Outside the United States and Canada
Number of positive samples (%)
Country of Number of
Product origin samples L. monocytogenes L. innocua L. welshimeri Other Ref.
Milk Australia 77 0 0 0 0 125
33 0 ND ND ND 65
Brazil 220 0 2 (0.9) 0 0 155
20 0 0 0 0 73
Czechoslovakia 30 0 ND ND ND 151
15 0 ND ND ND 152
Germany 39 0 0 0 0 197
Hungary 100 0 ND ND ND 131
50 0 0 0 0 100
Italy 348 0 0 0 0 117
50 0 0 0 0 199
Morocco 20 0 ND ND ND 94
Netherlands 41 0 0 0 0 69
Poland 73 0 0 0 7a 178
Turkey 22 0 ND ND ND 187
United Arab Emirate 182 0 0 0 0 122
L. monocytogenes in Unfermented Dairy Products 381
United Kingdom
England/Wales 1039 11 (1.1) ND ND ND 126
Scotland 115 4 (3.5) ND ND ND 182
Northern Ireland 95 1 (1.1) ND ND ND 119
Chocolate milk Hungary 60 7 (11.6) 0 0 0 171
Flavored milk Australia 206 1 (0.5) ND ND ND 202
Ice cream Australia 166 23 (13.9) ND ND ND 65
Costa Rica 50 1 (2.0) ND ND ND 154
England/Wales 40 0 ND ND ND 118
Turkey 50 5 (10.0) 6 (12.0) 0 0 75
Cream Australia 12 0 0 0 0 125
England/Wales 40 0 ND ND ND 126
Hungary 15 0 ND ND ND 131
Morocco 20 0 ND ND ND 94
Butter Hungary 15 1 (6.7) 0 0 Ib 131
Italy 130 5 (3.8) ND ND ND 169
Nonfat dry milk Germany 11 0 0 0 0 197
Caseidcaseinate Germany 30 0 0 0 0 I97
Dry infant formula Germany 120 0 0 0 0 197
ND, not determined.
a Seven non-L. monocytogenes isolates.
nated (Table 6). The increased incidence of listeriae in ice cream and butter is clearly the
result of postpasteurization contamination during handling and packaging as evidenced
by the highest contamination rates in ice cream bars and novelties. The fact that Listeria
spp. are more commonly found in chocolate milk, as opposed to unflavored milk, is also
not surprising given that the added ingredients can serve as an additional source for
listeriae.
Raw Milk
Despite longtime recognition of L. monocytogenes as a raw milk contaminant, relatively
few studies assessing the behavior of this organism in raw milk can be found in the litera-
ture. In 1958, Dedie [82]found that L. monocytogenes survived 210 days in naturally
contaminated raw milk stored in an ice chest. Thirteen years later, Dijkstra [84]reported
results from a much longer storage study in which 36 samples of naturally contaminated
raw milk (obtained from cows that experienced Listeria-related abortions) were held at
5C and examined for viable L. monocytogenes over a period of 9 years. Although 4 of
36 (1 1%) samples were free of L. monocytogenes within 6 months, the pathogen was still
detected in 16 of 36 (44%) samples following 2 years of refrigerated storage. The number
of samples from which listeriae could be isolated continued to decrease, with 9 of 36
(25%) samples being positive after 4 years of storage. However, the pathogen was still
present in 4 of 36 (1 1%) raw milk samples after 8-9 years of storage. These early findings
emphasize the importance of establishing proper cleaning and sanitizing programs for all
phases of milk production. If routinely used, such programs will likely prevent this organ-
ism from finding an appropriate niche within the farm or dairy factory environment and
greatly reduce the threat of this pathogen surviving long term.
The studies just described adequately demonstrate that L. monocytogenes can persist
in raw milk for long periods; however, until several outbreaks of milkborne and cheese-
borne listeriosis were reported in the 198Os, little attention had been given to the potential
for growth of L. monocytogenes in raw milk.
In 1988, Northolt et al. [ 1561 examined the behavior of listeriae in samples of freshly
drawn raw milk that were inoculated to contain approximately 500 L. monocytogenes
CFU/mL and incubated at 4 and 7C. As shown in Figure 4, Listeria populations decreased
approximately 4- and 8.5-fold in raw milk during the first 2 days of incubation at 4 and
7C, respectively. These authors suggested that naturally occurring bacterial substances
L. monocytogenes in Unfermented Dairy Products 383
lo4 I
10 L
0 2
Raw Milk
L l L L
4 6
Days
in raw milk (i.e., lactoperoxidase and lysozyme) may have partially inhibited growth of
listeriae during the first 2 days of incubation. However, in a Canadian study which will
be discussed shortly [98], no such decrease was observed when incubated samples of
naturally contaminated raw milk were surface plated on FDA Modified McBride Listeria
Agar. Hence, a more likely explanation is that the plating medium Trypaflavine Nalidixic
Acid Serum Agar used by Northolt et al. [ 1561 was less than ideal for recovering listeriae,
as also was observed during concurrent work with pasteurized milk. Although L. monocy-
togenes failed to grow in raw milk samples incubated at 4C for up to 7 days, Listeria
populations increased approximately 10-fold during this period when the incubation tem-
perature was raised to 7C. Following 3 days of incubation at 4 and 7OC, Listeria popula-
tions began doubling every 3.5 and 1.0 day, respectively. Two years later, Wenzel and
Marth [203] reported that populations of L. monocytogenes strain V7 remained constant
in inoculated raw milk during 5 days of storage at 4 and 7"C, with numbers of listeriae
also being unaffected by the presence of a commercial raw milk lactic acid bacteria inocu-
lant designed to suppress the growth of primarily gram-negative psychrotrophic bacteria.
Since L. monocytogenes failed to grow during 3-5 days of incubation at 7"C, it
appears that the 3-day period during which raw milk is sometimes held in farm bulk tanks
is insufficient to allow growth of the organism. However, the temperature of raw milk in
farm bulk tanks will fluctuate every time freshly drawn raw milk at 37C is commingled
with bulk tank milk at -4C from previous milkings. In 1985, Oz and Farnsworth [I591
found that raw milk in farm bulk tanks attained temperatures of 30-3 1OC, 10- 14"C, I2"C,
and 9C when freshly drawn raw milk was added after the first, second, third, and fourth
milking periods, respectively. Moreover, 6 h were generally needed for the milk to cool
to 4C after each milking period. In view of these findings, it appears that temperatures
obtained after adding warm milk to farm bulk tanks may be sufficient to allow at least
limited growth of L. monocytogenes, particularly when raw milk from early millungs
384 Ryser
enters the bulk tank. Although the temperature of bulk tank milk will eventually decrease
to -4"C, exposure to temperatures as high as 9C when raw milk is trucked to processing
facilities during summer [99] also may lead to some multiplication of the pathogen.
Discovery of a naturally infected cow in Canada that shed freely suspended and
phagocytized cells of L. monocytogenes in milk (maximum of 104CFU/mL in milk from
one of four quarters of the mammary gland) continuously for nearly 3 years provided
Farber et al. [98] with a unique opportunity to study growth of L. monocytogenes in natu-
rally rather than artificially contaminated raw milk during extended storage. When raw
milk from this cow was analyzed for numbers of L. monocytogenes,no appreciable growth
of the pathogen was observed during the first 3 days and 1 day of incubation at 4 and
IO'C, respectively (Fig. 5). The delay in onset of growth was less than 1 day at 15C.
Immunological staining of milk smears indicated that some multiplication of L. monocyto-
genes had occurred within macrophages after I and 2 days of incubation at 15 and 10C,
respectively, with 1 0 4 0 % of the macrophages containing 1-20 intracellular listeriae.
Nonetheless, as previously noted by Doyle et al. [91], rapid deterioration of macrophages
shortly thereafter was followed by appearance of freely suspended listeriae in milk with
few intact macrophages remaining after 5 days regardless of incubation temperature. Fol-
lowing the lag phase, L. monocytogenes entered a period of logarithmic growth, with
generation or doubling times of 25.3, 10.8, and 7.4 h being calculated for raw milk samples
held at 4, 10, and 15OC, respectively. Although maximum L. monocytogenes populations
were approximately 2 X 107CFU/mL after 10, 7, and 3 days of incubation at 4, 10, and
15"C, respectively, the highest achievable population in raw milk was independent of
7.0 -
m- 4C
t- 10C
.- 15OC
0 2 4 6 8 10 12 14
incubation temperature (Fig. 6). As in the previous study by Northolt et al. [156], these
findings again stress the importance of maintaining raw milk at 1 4 C during storage and
transport to milk processing facilities.
Investigations dealing with behavior of listeriae in raw milk have not been limited
to cow's milk. Reports of ovine listeriosis in Europe prompted Ikonomov and Todorov
[132] to examine the behavior of L. monocytogenes in raw ewe's milk inoculated with
the pathogen. Their results show that L. monocytogenes remained viable for long periods
and persisted in the milk even after coagulation at 10 and 20C. In 1987, a pregnant
woman in the United States reportedly aborted after consuming feta cheese contaminated
with L. monocytogenes. Since feta and other cheeses such as Roquefort, Manchego, Gjeost,
and Chachcaval are traditionally manufactured from ewe's or goat's milk, interest in the
behavior of listeriae in these milks as well as in ethnic-type cheeses manufactured from
these milks has increased over the last several years.
&ITS?'-Pasteurized Milk
I/ 4 "C
0 2 4 6 0 2 4 6
Days Days
i-
91-
7 - -+- - - - - - - - -
6 -
-
-
a SkimMilk
5 -
---- Whole Milk
-A Chocolate Milk
Days
The organism also grew markedly faster in pasteurized than in raw milk when both prod-
ucts were incubated at 7C. In contrast to their data for raw and pasteurized milk, lag
times for L. rnonocytogenes were reduced considerably when the organism was grown in
intensively pasteurized milk incubated at 4 and 7C. Furthermore, numbers of listeriae in
intensively pasteurized milk increased approximately 100-fold following 3 and 6 days of
incubation at 7 and 4OC, respectively. When L. rnonocytogenes was later grown in ultra-
high temperature (UHT) sterilized milk, Rajkowski et al. [ 1711 reported generation times
of 4.7, 1.7, 1.0, and 0.9 h for samples incubated at 12, 19, 28, and 37"C, respectively.
Hence, these findings suggest that the growth rate for L. rnonocytogenes in milk is directly
related to the degree of heat applied to milk. Further work is needed to define more clearly
the effect of competing microorganisms on growth of listeriae in raw and pasteurized
milk as compared with intensively pasteurized and UHT-sterilized milk with biochemical
changes that occur in milk during thermal processing (i.e., protein denaturation, enzyme
inactivation, carmelization) also likely influencing listeriae growth in these products.
microbial competitors, readers should keep in mind that growth rates for L. monocytogenes
are likely to be somewhat faster in autoclaved than in pasteurized or especially in raw milk
products. Nevertheless, L. monocytogenes clearly can grow to dangerously high levels in
all three types of milk during extended refrigeration.
In 1987, Rosenow and Marth [180] published results from a definitive study in
which autoclaved (12 1"Cl15 min) samples of whole, skim, and chocolate milk as well as
whipping cream were each inoculated separately with four strains of L. monocytogenes
(Scott A, V7, V37CE, or California), incubated at 4, 8, 13, 21, or 35"C, and examined
for numbers of listeriae at suitable intervals by surface plating appropriate dilutions on
Tryptose Agar. Growth rates of L. monocytogenes were generally similar in all four prod-
ucts at a given temperature and increased with an increase in incubation temperature. At
4"C, listeriae began growing after an initial delay of approximately 5- 10 days depending
on the bacterial strain and type of product (see Fig. 7). All four strains generally attained
maximum populations of 2 1O7 CFU/mL after 30-40 days of incubation, with little change
in numbers occurring after 30-40 days of additional storage. Overall, chocolate milk sup-
ported development of the highest Listeria populations followed by skim milk, whole
milk, and whipping cream. Generation times for growth at 4C ranged between 28.16 and
45.55 h. Average generation times for L. monocytogenes in all four products are shown
in Table 7. Although these results clearly demonstrate the ability of L. monocytogenes to
reach potentially hazardous levels in fluid dairy products held at 4"C, more recent data
suggest that slow growth of this organism can even occur in milk held at 0C. Thus
the only way to avoid a public health problem with fluid dairy products is to prevent L.
monocytogenes from entering such products before, during, and after manufacture.
Increasing the incubation temperature from 4 to 8C decreased the lag period to
1.5-2 days (Fig. 8) and nearly tripled the growth rate for L. monocytogenes in all four
products (Table 7) [ 180,1811. After 10- 14 days of incubation, the growth curves at 4 and
8OC were similar, with highest Listeria populations again being found in chocolate milk.
Theoretical calculations based on these data indicate that Listeria populations could in-
crease from 10 to 4.2 X 106organismdqt (947 mL) of milk during 10 days of storage at
8C (46"F), a temperature that commonly occurs in some home and commercial refrigera-
tors. These findings, which since have been confirmed by Siswanto and Richard [188]
using skim milk, raise additional safety concerns about reclaiming and reprocessing re-
turned products that have likely undergone some degree of temperature abuse.
As is true for 8"C, 13C (55F) also represents a temperature that dairy products
occasionally encounter during transportation and storage. Following a 12-h lag period, all
9. -
8. -
7. -
-
6. -
-
5. -
-
- A-. Chocolare milk
4.
3. -
four Listeria strains grew nearly twice as fast at 13C as at 8C (see Table 7) and generally
attained levels of 2 106CFU/mL in all four products by the third day [ 1801. These genera-
tion times are somewhat longer than those observed by Farber et al. [98] when naturally
contaminated raw milk was incubated at 4 (25.3 h), 10 (10.8 h), and 15C (7.4 h). L.
monocytogenes also attained maximum populations that were approximately 1 0-fold lower
in raw than in sterile milk, which in turn suggests possible depletion of essential nutrients
by raw milk contaminants or production of substances inhibitory to growth of the patho-
-
gen. Maximum Listeria populations of 1 O9 CFU/mL were again observed in chocolate
milk, with numbers generally being 10-fold lower in skim milk, whole milk, and whipping
cream [ 1801. Increasing the incubation temperature to 2 1"C doubled the growth rate (see
Table 7) and led to maximum Listeria populations of I Ox- 10' CFU/mL within 48 hs. As
expected, L. monocytogenes grew most rapidly at 35"C, with populations of 1Ox- 10' CFU/
mL being observed after only 24 h of incubation.
In another study examining the influence of temperature and milk composition on
growth of listeriae, Donnelly and Briggs 1861 found that five L. monocytogenes strains
began growing in inoculated samples of autoclaved (121 "C/ 10 min) whole, skim, and
reconstituted nonfat dry milk (1 1 % total solids) after approximately 24-48, 2-24, 4-12,
and 0.5-4.0 h of incubation at 4, 10, 22, and 37OC, respectively. Although growth rates for
all Listeria strains were primarily determined by the incubation temperature, two strains
of L. monocytogenes serotype 4b grew considerably faster in whole rather than skim or
L. monocytogenes in Unfermented Dairy Products 389
reconstituted nonfat dry milk during incubation at 4 and 10C. These observations led
Donnelly and Briggs [86] to suggest a possible relationship between levels of milkfat and
the growth rate of L. monocytogenes in milk during refrigerated storage. Furthermore,
these authors suggested that enhanced psychrotrophic growth in whole milk may be related
to a listerial lipase produced by both P-hemolytic strains of L. monocytogenes serotype
4b. Unlike both of these strains, the three remaining L. monocytogenes strains of serotypes
1 and 3 failed to exhibit enhanced growth in whole milk at 10C and had little if any
hemolytic activity on McBride Listeria Agar containing sheep blood.
In contrast to what might be expected from the study just described, Rosenow and
Marth [ 1801 failed to observe any significant difference in growth rates among four strains
of L. monocytogenes (two serotype 4b, two serotype 1) when they were incubated in
autoclaved samples of whole and skim milk at 4, 8, 13, 21, and 35C. The pathogen also
attained lower maximum populations in whipping cream than in whole, skim, or chocolate
milk at all incubation temperatures. In support of these findings, Marshall and Schmidt
[ 1451 failed to observe enhanced growth of L. monocytogenes strain Scott A (serotype
4b) in whole rather than skim milk during 8 days of incubation at 10C. Finally, in a
study to be discussed in greater detail in Chapter I2 [ 1851, four strains of L. monocytogenes
(three serotype 4b and one serotype 1) frequently attained higher maximum populations
in whey samples that were defatted by centrifugation, filter sterilized, and incubated at
6C than would be expected to occur in autoclaved skim milk, whole milk, or whipping
cream after prolonged incubation at 8C. Thus, although some L. monocytogenes strains
are lipolytic as reported by Marshall and Schmidt [ 1461, one must presently conclude that
psychrotrophic growth of L. monocytogenes is not generally enhanced by the normal level
of milk fat found in fluid milk.
Recognizing the vital importance of carbohydrates in microbial metabolism, re-
searchers at the CDC [ 1661 attempted to define growth of Listeria spp. in terms of sugar
utilization. An initial experiment using aerobically incubated broth media indicated that
five strains of L. monocytogenes and one strain each of L. Jnnocua, L. seeligeri, and L.
ivanovii utilized only the glucose moiety of lactose, whereas single strains of L. grayi and
L. murruyi utilized both the glucose and galactose of lactose. Overall, maximum cell
populations, as determined by optical density, were directly proportional to the concentra-
tion of glucose ( S O . 125%) in the growth medium. However, marked differences were
observed in the ability of L. monocytogenes and L. innocua to utilize lactose, with three
strains of L. monocytogenes (isolated from Mexican-style cheese in connection with the
1985 listeriosis outbreak in California) being unable to grow in a medium containing
lactose as the only carbohydrate. Although these observations agree with several reports
[102,145,146] indicating that the pH of fluid milk is unaffected by L. monocytogenes
growth, Quinto et al. [I701 did report a sharp pH decrease in such milk after 16 and 24
days of incubation at 14 and 7"C, respectively, with these differences most likely being
related to strain variation.
Growth of L. monocytogenes in autoclaved samples of whole and skim milk was
generally similar to that previously observed by Rosenow and Marth [ 1801, with maximum
populations of 5 5 X IO* CFU/mL developing after extended incubation at 5 and 25C.
Except for L. seeligeri, the behavior of L. innocua and L. ivanovii did not differ markedly
from that of L. monocytogenes in these samples (Fig. 9). However, as noted by Northolt
et al. [ 1561, higher maximum populations and increased survival rates were again observed
when these organisms were grown in autoclaved rather than pasteurized whole milk. Ex-
amination of milk by gas-liquid chromatography indicated that lactic, acetic, isobutyric,
390 Ryser
L. rnonocvloaeneg + 0
Lseeliaeri m o
C ivanovil A A
\ innocua e o
9.0
8.0
7.0
6.0
0 4 8 12 16 20 24
Days
isovaleric, and 2-hydroxy isocaproic acids were formed during incubation. Since this milk
initially contained -81 -85 mg of glucose/L, the aforementioned acids likely resulted, at
least in part, from fermentation of glucose. Considerably lower populations of L. monocy-
togenes as well as L. innocua, L. grayi, and L. murrayi also developed in glucose oxidase-
treated (an enzyme that degrades glucose) rather than untreated milk during both aerobic
and anaerobic incubation, and so it is evident that glucose is one of the major substrates
for growth of listeriae in milk. However, when incubated anaerobically in glucose oxi-
dase-treated milk, two lactose-negative L. monocytogenes isolates from Mexican-style
-
cheese still attained final populations of 10' CFU/mL; thus suggesting the involvement
of other as yet unidentified growth factors.
In the aforementioned study by Rosenow and Marth [ 1801, maximum populations
of L. monocytogenes were typically about 10-fold higher in chocolate milk than in other
fluid dairy products. To explain the enhanced growth .of L. monocytogenes in chocolate
milk, several investigators at the University of Wisconsin examined the effect of major
chocolate milk constituents (i.e., cocoa, sugar, and carrageenan) on growth of this organ-
ism in autoclaved skim milk and laboratory media. Rosenow and Marth [ 1791 found that
L. monocytogenes in Unfermented Dairy Products 391
growth of L. monocytogenes at 13C was only slightly enhanced in skim milk containing
5% cane sugar, and that the organism attained higher final populations when commercial
cocoa power (1.3%) and carrageenan stabilizer (0.5%) were used in place of cane sugar
(Fig. 10). Carrageenan also enhanced the growth rate of L. monocytogenes in the presence
of cocoa; however, the organism attained similar maximum populations regardless of the
presence or absence of carrageenan. These findings suggest that carrageenan may be more
important in increasing contact between cocoa particles and Listeria than as a source of
nutrients. Highest final populations and shortest generation times were observed when L.
monocytogerzes was grown in skim milk containing cocoa, sugar, and carrageenan. In
addition, maximum Listeria populations obtained in skim milk containing all three ingredi-
ents (see Fig. 10) were similar to populations observed in initial work with commercially
produced chocolate milk (see Figs. 7 and 8).
Subsequently, Pearson and Marth [ I621 examined growth of L. monocytogenes strain
V7 at 13C in skim milk containing various concentrations of cocoa, sugar, and carra-
geenan. Since some Listeria strains can utilize sucrose, it is not surprising that L. monocy-
togenes developed significantly higher final populations (see Fig. 1 1 ) and had shorter
generation times (5.05 vs 5.17 h) as the concentration of cane sugar (sucrose) in skim
milk was increased from 0 to 12%. (Peters and Liewen [ 1651 also reported that addition of
7% sucrose to ultrafiltered (concentrated) skim milk caused rnaximum L. monocytogenes
populations to increase rather than decrease.) A near-linear relationship between increas-
ing sugar concentration and maximum attainable populations of L. monocytogenes also
was observed for all but one combination of sugar, cocoa, and carrageenan tested; that is,
10
-- 2%milk(m)
rl
- ------ 2%m + sugar (s)
r n20 ~ 40 ' 60 l 80
' l 100 ' 120 ' 140
' 1160 ' 180 I 200
' l
2 O
8.90
8.80
8.70
8.60
8.50
8.40
0 3 6 9 12
12% sugar and 0.03% carrageenan (see Fig. 11). Although addition of 0.03% carrageenan
significantly lengthened generation times and decreased maximum populations compared
with those observed in skim milk without carrageenan, L. monocytogenes achieved highest
populations in skim milk containing 0.75% cocoa with or without carrageenan, which in
turn indicates that the apparent ability of cocoa to stimulate growth of this organism in
skim milk containing 0- 12% sugar is independent of carrageenan. Since cocoa contains
only trace amounts of fermentable carbohydrates, these authors theorized that cocoa en-
hanced growth of L. monocytogenes in skim milk by providing increased levels of peptides
and amino acids, particularly valine, leucine, and cysteine, which are reportedly essential
for growth. Additional work showed that agitation, combined with the presence of cocoa,
sugar, and/or carrageenan in skim milk, enhanced growth of the pathogen at 30C when
compared with growth in the same medium that was incubated quiescently. However,
growth of Listeria in skim milk alone was better without rather than with agitation. Thus
agitation most likely increased the availability of extractable nutrients from cocoa, which
in turn led to enhanced growth of the pathogen.
In 1968, anthocyanins in cocoa were reported to inhibit growth of salmonellae in
laboratory media; however, the inhibitory effect of cocoa could be neutralized with casein
[72]. These early findings prompted Pearson and Marth [164] to investigate the effect of
cocoa with and without casein on growth of L. monocytogenes strain V7. Using Modified
Tryptose Phosphate Broth containing 0.2% tryptose, addition of 0.75- 10% cocoa in-
creased the generation time for L. monocytogenes at 30C (1.02-1.12 h) as compared
with samples without cocoa (0.94 h). However, the pathogen generally attained higher
populations when grown in media with (1.1 - 1.5 X 109CFU/mL) rather than without (6.4
X 108CFU/mL) cocoa. Interestingly, when the same medium was inoculated to contain
L. monocytogenes in Unfermented Dairy Products 393
during manufacture reduces its water activity (a,) to -0.83, which is well below the
minimum a, value of 0.90 reported for growth of L. monocytogenes. Unlike sweetened
condensed milk, the relatively high a, value for evaporated milk (-0.986) allowed profuse
growth of listeriae, with lowest inoculum levels increasing approximately 4 orders of
magnitude after 7 and 14 days of incubation at 21 and 7OC, respectively. In addition, no
decrease in numbers of listeriae was noted during continued incubation at either tempera-
ture. Thus, since L. monocytogenes can survive >42 days in sweetened condensed milk
and grow rapidly in evaporated milk, special precautions should be taken to prevent lister-
iae from entering these products during packaging, storage, and subsequent use.
Ultrafiltered Milk
Ultrafiltration, a mechanical process by which milk is filtered under pressure and concen-
trated, results in major compositional changes in the finished product when compared with
the starting material. During ultrafiltration, 94- 100% of the milk proteins and protein-
bound vitamins (i.e., vitamin BI2and folic acid) remain in the retentate along with milk
fat, whereas lactose is equally divided between the retentate and permeate. Increased use
of ultrafiltered milk in cheesemaking prompted El-Gazzar et al. [92] to investigate the
growth characteristics of L. monocytogenes in 2X and 4X retentate as well as the corre-
sponding permeate obtained from ultrafiltered pasteurized milk. When samples were inoc-
ulated to contain about 104CFU/mL of L. monocytogenes strain V7 or CA and incubated
at 4"C, growth of both organisms was enhanced 10- to 100-fold in retentate as compared
with unfiltered skim milk. Increasing the concentration of ultrafiltered (UF) skim milk
retentate from 2X to 5X also resulted in faster growth, with the pathogen attaining a
population of 106CFU/mL in 5X and 2X retentate after approximately 7 and 10-12 days
of refrigerated storage, respectively. L. monocytogenes also grew to dangerous levels in
permeate with maximum levels of 10' CFU/mL as compared with 10' CFU/mL in reten-
tate following 30 days of incubation. When identical tyndalized samples were incubated
at 32 and 4OoC, both Listeria strains grew similarly in skim milk and retentate, with
populations of 10' CFU/mL generally being reported after 24 h of incubation. However,
as was true for samples incubated at 4OC, maximum numbers of listeriae were again 10-
to 100-fold lower in permeate than in retentate and unfiltered skim milk. Hence, the same
care should be given to production of unfiltered milk to prevent contamination and subse-
quent growth of Listeria in the product during cold storage.
L. nionocyrogenes
--- +-
L. niotiocylogenes
+ P.fragi
------Q.---
L. nronocyrogenes
+ P . flicorescens
Days
generally did not differ significantly. As was true for whole and skim milk, L. monocyto-
genes attained populations of 1 X 107to 5 X 107CFU/mL in reconstituted nonfat dry
milk, with highest numbers occurring in milk preincubated with P. jluorescens rather than
P. fragi.
Flavobacterium is another genus of gram-negative psychrotrophic bacteria that is
frequently recovered from raw milk, pasteurized milk, and butter. Hence, Farrag and Marth
[104] also examined behavior of L. monocytogenes in the presence of flavobacteria in
skim milk at 7 and 13C. Growth of L. monocytogenes strains Scott A, CA, and V7 in
autoclaved skim milk was enhanced by the presence of F. lutescens during 14-42 days
of a 56-day incubation period at both 7 and 13"C, with these higher populations again
being attributed to proteolysis of milk proteins by F. lutescens. However, Flavobacterium
sp. ATCC 21429 failed to impact the growth of L. monocytogenes at 7C and proved to
be slightly inhibitory to the same three Listeria strains when samples were held at 13C.
One strain of Bacillus spp. [ 1431 also prevented Listeria growth in raw milk.
These results dispel the previous theory and indicate that L. monocytogenes can
readily compete with P. fragi, P. jluorescens, and certain Flavobacterium spp. for nutrients
in milk and at the same time can outgrow these organisms at refrigeration temperatures
L. monocytogenes in Unfermented Dairy Products 397
17 -
16 - L. monocytogenes
- L. monocytogenes
15 - P . fluorescens
L. monocytogenes
14 - P.fragi
13 -
12 -
11 -
10 -
6
Whole Skim Nonfat Milk Solids
Product
Ice Cream
Frequently, pasteurized milk that has not been sold in retail stores is returned to dairy
factories and reprocessed into chocolate ice cream. Since large commercial refrigeration
units often fail to maintain a constant temperature of 4"C, virtually all reclaimed milk has
undergone some degree of temperature abuse during the period in which the product was
on sale. In addition to possible growth of L. monocytogenes during this 2-week period of
"cold enrichment,'' pseudomonads also can grow in milk and produce an environment
that is more favorable for growth of Listeria even after pasteurization.
The numerous Class I recalls issued since the late 1980s for Listeria-contaminated
ice cream prompted Berang et al. [70] to investigate the behavior of L. monocytogenes
in inoculated samples of chocolate ice cream (and chocolate milk as discussed earlier)
prepared from fresh skim milk and commercial skim milk that was held beyond the expira-
tion date. Although growth of listeriae was certainly not expected in ice cream held at
- 18 to -24"C, the pathogen survived equally well in both types of chocolate ice cream.
Hence, use of returned milk in chocolate ice cream did not appear to enhance Listeria
survival. Long-term survival of L. monocytogenes was also confirmed in a later study
[160] in which the pathogen persisted for 14 weeks in ice cream stored at - 18C with
no apparent cell death or injury. In 1996, Dean and Zottola [81] assessed the fate of L.
monocytogenes V7 in full-fat ( 10%) and reduced-fat (3%) soft-serve ice cream prepared
with and without 14 ppm nisin. Regardless of fat content, L. monocytogenes populations
remained constant in ice cream during freezing and 3 months of storage at - 18C. How-
ever, nisin effectively reduced Listeria survival in both full- and reduced-fat ice cream
during manufacture, with Listeria populations generally decreasing 2 and 3 orders of mag-
nitude in full- and low-fat ice cream, respectively, following 1 month of frozen storage
at - 18C. Although not currently approved in the United States as an ice cream ingredient,
incorporation of nisin into ice cream formulations appears to be an effective, albeit costly,
means of inactivating listeriae and reducing the number of Class I Listeria-related recalls
that continue to plague the dairy industry.
In 1989, Amelang and Doores [2,3] determined the generation times for L. monocy-
togenes in nine formulations of commercially produced ice cream mix that varied in type
and level of fat (cream, butter), sugar (cane sugar, corn sweetener), and milk solids (con-
densed milk, skim milk, whey powder). To simulate postprocessing contamination, all
-
samples were inoculated to contain 103L. monocytogenes strain Scott A or V7 CFU/
mL and incubated at 4, 2 1, and 35C. Overall, L. monocytogenes had average generation
times of 21.6, 1.08, and 0.79 h in ice cream mixes incubated at 4, 21, and 35"C, respec-
tively, with similar growth rates occurring in mixes containing 10, 14, and 15% fat and
held at the same temperature. It is noteworthy that these generation times are markedly
shorter than those calculated by Rosenow and Marth [ 1801 for growth of the same strains
in whole milk, skim milk, chocolate milk, and whipping cream (see Table 5). Although
L. monocytogenes generally behaved similarly in all ice cream mixes incubated at 4 and
2 1"C, differences in generation times were noted at 35C when the pathogen was cultured
in ice cream mixes made with alternative fat and milk solids. At 35"C, growth of listeriae
was somewhat enhanced in mixes containing butter rather than cream, skim milk powder,
or whey powder rather than condensed skim milk and egg yolk as additional sources of
solids. Although the pathogen grew most rapidly in ice cream mix containing a 50 :50
ratio of cream to butter, partial replacement of cane sugar (sucrose: glucose + fructose)
L. monocytogenes in Unfermented Dairy Products 399
with corn sweetener (glucose and maltose) or high fructose corn syrup failed to signifi-
cantly shorten generation times.
Butter
In 1988, Olsen et al. [158] examined the fate of L. monocytogenes during manufacture
and storage of butter in the event that the product is prepared from contaminated cream.
According to their report, pasteurized cream was inoculated to contain 104- 10' L. monocy-
togenes CFU/g and churned into butter. After removing the buttermilk, washed butter
grains were salted to a level of 1.2% and resultant butter was analyzed weekly for listeriae
during 10 weeks of storage at - 18, 4-6, and 13C. During manufacture -95% of the L.
monocytogenes population was lost in buttermilk, with the remaining 5% of the population
appearing in butter. The pathogen was present at levels of 1.7 X 104to 1.8 X 10' CFU/
g in cream as compared with 1.5 X 103to 1.6 X 104CFU/g in butter, indicating that like
Staphylococ-cus aureus [ 1531, L. monocytogenes also favors the water rather than lipid
phase during butter making. As shown in Figure 14, Listeria populations increased 1.9
and 2.7 orders of magnitude in butter stored at 4-6 and 13OC, with maximum numbers
being observed after 49 and 42 days of storage, respectively. These findings along with
similar results by Lanciotti et al. [ 1371 for commercially prepared light butter stored at 4
and 20C indicate that enough milk solids were trapped in the water phase (containing
-6% salt) to support growth of listeriae during storage. Numbers of listeriae then began
to decrease; however, the organism was still present at levels >104 CFU/g following 70
days of refrigerated storage. Although freezing the contaminated butter prevented growth
6.00
1
I-/ / \ / 4 to 6 O C
t
2.001'
0
'
20
* A '
40
' . a '
60
" ' J
80
Days
of L. monocytogenes, the organism was still present at levels of -103 CFU/g after 70
days of storage at -18"C, as was also reported by Slavchev et al. [191].
Thus far L. monocytogenes has not been isolated from pasteurized cream manufac-
tured in the United States; however, given the massive Listeria recall of Texas-produced
fluid dairy products, including half-and-half and whipping cream, in May of 1986 (see
Table 5 ) , one cannot assume that all pasteurized cream and butter manufactured in the
United States and elsewhere will be universally free of listeriae. As you will recall from
Chapter 10, one cluster of listeriosis cases in southern California was attributed to con-
sumption of contaminated butter [ 1471. Hence, since at least four Class I recalls have been
issued for L. monocytogenes-contaminated butter, and since growth of L. monocytogenes
has been demonstrated experimentally in both cream and butter during refrigerated storage,
it is necessary to ensure that cream is pasteurized and that recontamination of pasteurized
cream is prevented before and during its churning into butter.
presumably at very low levels, it may be possible to eliminate this pathogen by holding
the product at room temperature for several months.
1. Abou-Donia, S.A., and A.K. Al-Medhagi. 1992. Detection and survival of Listeria monocyto-
genes in Egyptian dairy products. J. Dairy Sci. 75 (suppl. 1): 138.
1a. Abou-Eleinin, A.M., E.T. Ryser, and C.W. Donnelly. 1998. Unpublished data.
2. Amelang, J., and S. Doores. 1989. The effect of ingredients in ice cream formulations on the
growth of Listeria monocytogenes. Annual Meeting of the Institute of Food Technologists,
Chicago, June 25-29, Abstr. 468.
3. Amelang, J., and S. Doores. 1989. The effect of medium, growth phase and temperature on
the growth of Listeria monocytogenes in ice cream mix. Annual Meeting of the Institute for
Food Technologists, Chicago, June 25-29, Abstr. 469.
4. Andre, P., H. Roose, R. Van Noyen, L. Dejaegher, I. Vyttendaele, and K. De Schrijver. 1990.
Neuro-meningeal listeriosis associated with consumption of an ice cream. Med. Mal. Infect.
20:570-572.
5. Anonymous. 1986. Ice cream bars recalled. FDA Enforcement Report, July 16.
6. Anonymous. 1986. Ice cream recalled. FDA Enforcement Report, Oct. 22.
7. Anonymous. 1986. Ice cream recalled. FDA Enforcement Report, Oct. 29.
8. Anonymous. 1986. Ice cream, sherbet and glacee recalled. FDA Enforcement Report, Sept.
3.
9. Anonymous. 1986. Ice milk mix recalled. FDA Enforcement Report, June 25.
10. Anonymous. 1986. Large class I recall made of ice cream because of Listeria. Food Chem.
News 28(24):11-12.
11. Anonymous. 1986. Listeria causes class I recalls of ice milk mix, milk. Food Chem. News
28( 16):22.
12. Anonymous. 1986. Milk, chocolate milk, half and half, cultured buttermilk, whipping cream,
ice milk, ice milk mix and ice milk shake mix recalled. FDA Enforcement Report, June 25.
13. Anonymous. 1986. Sherbets, non-dairy products, ice milk products, gelati-da products and
ice cream recalled. FDA Enforcement Report, Aug. 27.
14. Anonymous. 1987. Chocolate ice cream recalled. FDA Enforcement Report, Sept. 16.
15. Anonymous. 1987. Class I recall made of cheese because of Listeria. Food Chem. News
28(50):52.
16. Anonymous. 1987. FDA launching two-year pathogen surveillance program. Food Chem.
News 29(31):10-12.
17. Anonymous. 1987. Ice cream and ice milk recalled. FDA Enforcement Report, Aug. 26.
18. Anonymous. 1987. Ice cream bars recalled. FDA Enforcement Report, Nov. 4.
19. Anonymous. 1987. Ice cream, ice milk and sherbet recalled. FDA Enforcement Report, Feb.
11.
20. Anonymous. 1987. Ice cream, ice milk and sherbet recalled. FDA Enforcement Report, Aug.
19.
21. Anonymous. 1987. Ice cream nuggets recalled. FDA Enforcement Report, Aug. 5.
22. Anonymous. 1987. Ice cream products recalled. FDA Enforcement Report, Sept. 2.
23. Anonymous. 1987. Ice cream products recalled. FDA Enforcement Report, Sept. 16.
24. Anonymous. 1987. Ice cream products recalled. FDA Enforcement Report, Sept. 23.
25. Anonymous. 1987. Ice cream recalled. FDA Enforcement Report, Jan. 28.
26. Anonymous. 1987. Ice cream recalled. FDA Enforcement Report, Feb. 11.
27. Anonymous. 1987. Ice cream recalled. FDA Enforcement Report, May 27.
28. Anonymous. 1987. Ice cream recalled because of Listeria, pottery because of lead. Food
Cheni. News 29(21):16-17.
402 Ryser
29. Anonymous. 1987. Milk industry has spent $66 million on recalls and related expenses, Witte
says. Food Chem. News 29( 17):29-30.
30. Anonymous. 1987. More ice cream recalled because of Listeria. Food Chem. News 28(48):
33.
31. Anonymous. 1988. Frozen dessert products recalled. FDA Enforcement Report, July 27.
32. Anonymous. 1988. Ice cream, cheese recalled because of Listeria. Food Chem. News 30(6):
27.
33. Anonymous. 1988. Ice cream pies recalled. FDA Enforcement Report, Dec. 28.
34. Anonymous. 1988. Ice cream products, cheese recalled because of Listeria. Food Chem.
News 30(9):47.
35. Anonymous. 1988. Ice cream recalled. FDA Enforcement Report, April 6.
36. Anonymous. 1988. Ice cream recalled. FDA Enforcement Report, Sept. 7.
37. Anonymous. 1988. Ice cream recalled. FDA Enforcement Report, Sept. 14.
38. Anonymous. 1988. Ice cream recalled. FDA Enforcement Report, Nov. 2 .
39. Anonymous. 1988. International Dairy Federation: Group E64-Detection of Listeria rnono-
cytogenes-sampling plans for Listeria rnonocytogenes in foods, Feb. 9. Brussels.
40. Anonymous. 1988. More cheese, ice cream linked to possible Listeria. Food Chem. News
29( 11):37-38.
41. Anonymous. 1989. Ice cream bars recalled. FDA Enforcement Report, Feb. 15.
42. Anonymous. 1989. Ice cream recalled. FDA Enforcement Report, April 19.
43. Anonymous. 1989. Le contr6le des rbsidus dans les produits laitiers. Bull. 1nf.-Minist. Agric.,
France I273:22-24.
44. Anonymous. 1990. Frozen yogurt recalled. FDA Enforcement Report, Feb. 7.
45. Anonymous. 1990. Ice cream and frozen yogurt novelties recalled. FDA Enforcement Report,
July 10.
46. Anonymous. 1990. Ice cream bars recalled. FDA Enforcement Report, April 25.
47. Anonymous. 1990. Ice cream recalled. FDA Enforcement Report, Nov. 7.
48. Anonymous. 1990. Sherbet, ice milk and ice cream recalled. FDA Enforcement Report, Dec.
5.
49. Anonymous. 1990. USDA, FDA officials report apparent decrease in Listeria isolations. Food
Chem. News 32( 1): 12- 15.
50. Anonymous. 1991. Butter recalled. FDA Enforcement Report, Aug. 7.
51. Anonymous. 1991. Ice cream and ice milk recalled. FDA Enforcement Report, June
19.
52. Anonymous. 1992. Butter and butterine recalled. FDA Enforcement Report, July 22.
53. Anonymous. 1992. Ice milk and ice cream recalled. FDA Enforcement Report, Dec. 30.
54. Anonymous. 1993. Ice cream bars recalled. FDA Enforcement Report, Sept. 29.
55. Anonymous. 1994. Butter products recalled. FDA Enforcement Report, Oct. 12.
56. Anonymous. 1994. Ice cream recalled. FDA Enforcement Report., Oct. 5.
57. Anonymous. 1995. Ice cream novelties recalled. FDA Enforcement Report, Dec. 13.
58. Anonymous. 1996. Frozen yogurt recalled. FDA Enforcement Report, Mar.6.
59. Anonymous. 1996. Ice cream and sherbet recalled. FDA Enforcement Report, April 10.
60. Anonymous. 1996. Ice cream, frozen yogurt, sherbet, sorbet and ice cream mix recalled.
FDA Enforcement Report, Jan. 3 1.
61. Anonymous. 1996. Ice cream recalled. FDA Enforcement Report, Jan. 3 I .
62. Archer, D.L. 1988. Review of the latest FDA information on the presence of Listeria in
foods. WHO Working Group on Foodborne Listeriosis, Geneva, Feb. 15-19.
63. Arias, L., R. Monge, F. Antillon, and E. Glenn. 1994. Occurrence of the bacteria Listeria
spp. in raw milk in Costa Rica. Rev. Biol. Trop. 42:711-713.
64. Arimi, S.M., E.T. Ryser, T.J. Pritchard, and C.W. Donnelly. 1997. Diversity of Listeria ribo-
types recovered from dairy cattle, silage and dairy processing environments. J. Food Prot.
60:8 1 1-8 16.
L. monocytogenes in Unfermented Dairy Products 403
65. Arnold, G.J., and J. Coble. 1995. Incidence of Listeria species in foods in NSW. Food Austra-
lia 47:7 1-75.
66. Bachman, H.P., and U. Spahr. 1995. The fate of potentially pathogenic bacteria in Swiss
hard cheese and semihard cheeses made from raw milk. J. Dairy Sci. 78:476-483.
67. Bean, N.H., J.S. Goulding, C. Lao, and J.F. Angulo. 1996. Surveillance of foodborne disease
outbreaks-United States, 1988- 1992. M.M.W.R. 45: 1-66.
68. Beckers, H.J., P.S.S. Soentoro, and E.H.M. Delfgou-van Asch. 1987. The occurrence of Liste-
ria monocytogenes in soft cheeses and raw milk and its resistance to heat. Intern. J. Food
Microbiol. 4:249-256.
69. Beckers, H.J., P.H. int Veld, P.S.S. Soentoro, and E.H.M. Delfgou-van Asch. 1988. The
occurrence of Listeria in food. Foodborne Listeriosis-Proceedings of a Symposium, Wies-
baden, Germany, Sept. 7, pp. 84-97.
70. Berrang, M.E., J.F. Frank, and R.E. Brackett. 1988. Behavior of Listeria monocytogenes in
chocolate milk and ice cream mix made from post-expiration date skim milk. J. Food Prot.
5 1 :823 (Abstr.).
71. Brindani, F., and E. Freschi. 1988/1989. Ricerca di Listerirz monocytogenes nel latte di ovi-
caprini ed in alcuni tipi di formaggio. Annal. Fac. Med. Vet. 8-9:205-219.
72. Busta, F.F., and M.L. Speck. 1968. Antimicrobial effect of cocoa on salmonellae. Appl.
Microbiol. 16:424-425.
73. Casarotti V.T., R.G. Claudio, and R. Camargo. 1994. Occurrence of Listeria monocytogenes
in raw milk, pasteurized C type milk and minas frescal cheese commercialized in Piracicaba-
S.P. Arch. Latinoamer. Nutr. 44: 158-163.
74. Cheng, C.C., S.B. Shiau, and H.S. Lin. 1993. Incidence and characterization of Listeria
monocytogenes in raw milk and feeds. Taiwan J. Vet. Med. Anim. Husb. 6159-
65.
75. Ciftcioglu, G., M.T. Ulgen, and K. Bostan. 1992. An investigation on the presence of Listeria
monocytogenes in ice cream. J. Fac. Vet. Istanbul 18:1-8.
76. Colonna, V., A.M. DiNoto, E.M. Russo Alesi, C. Emanuele, and S. Caracappa. 1994. Obser-
vations on the presence of Listeriu monocytogenes in milk products of ovine origin. In Prog-
ressi scientifici e technolgici in tema di patologia e di allevamento degli ovini e dei caprini.
Societa Italiana di Patologia e di Allevamento degli Ovini e dei Caprini. Atti XI Congress0
Nazionale. Perugia, Italy, June 1-4, p. 439-442.
77. Conner, D.E., V.N. Scott, S.S. Sumner, and D.T. Bernard. 1989. Pathogenicity of foodborne,
environmental and clinical isolates of Listeria monocytogenes in mice. J. Food Sci. 54: 1553-
1556.
78. Coskun, S., 0. Onal, M. Keskin, T. Okyay, A. Yuce, and B. Erel. 1993. Investigation of
Listeria in raw milk and comparison of culture and ELIS.4 methods. Turkish J. Infect. 7:
329- 332.
79. Da Cruz, I.M.V., M.I. Fernandes, and M.M. Sol. 1990. Incidence of Listeria monocytogenes
in Portuguese raw goats milk. In: Posters and Brief Communications of the XXIII Interna-
tional Dairy Congress, Montreal, Oct. 8-12. Abst. 77.
80. Davidson, R.J., D.W. Sprung, C.E. Park, and M.K. Rayman. 1989. Occurrence of Listeria
monocytogenes, Campylobacter spp., and Yersinia enterocolitica in Manitoba raw milk. Can.
Inst. Food Sci. Technol. J. 22:70-74.
81. Dean, J.P., and E.A. Zottola. 1996. Use of nisin in ice cream and effect on the survival of
Listeria monocytogenes. J. Food Prot. 59:476-480.
82. Dedie, K. 1958. Weitere experimentelle Untersuchungsbefunde zur Listeriose bei Tieren. In
R. Roots and D. Strauch (eds.), Listeriosen, Zbl. Veterinarmed. Beiheft, pp. 99-109.
83. DErrico, M.M., P. Villari, G.M. Grasso, F. Romano, and I.F. Angelillo. 1990. Isolamento
di Listeria spp. da latte e formaggi. Riv. Soc. Ital. Sci. Aliment. 19:47-52.
84. Dijkstra, R.G. 1971. Investigations on the survival times 0 1 Listeria bacteria in suspensions
of brain tissue, silage and faeces and in milk. Zbl. Bakteriol. I Abt. Orig. 216:92-95.
404 Ryser
85. Donnelly, C.W. 1986. Listeriosis and dairy products: Why now and why milk? Hoards Dairy-
man 131:663, 687.
86. Donnelly, C.W., and E.H. Briggs. 1986. Psychrotrophic growth and thermal inactivation of
Listeria rnonocytogenes as a function of milk composition. J. Food Prot. 49:994-998.
87. Donnelly, C.W., G.J. Baignet, and E.H. Briggs. 1988. Flow cytometry for automated analysis
of milk containing Listeria rnonocytogenes. J. Assoc. Off. Anal. Chem. 71:655-658.
88. Doores, S., and J. Amelang. 1990. Personal communication.
89. Doyle, M.P., and J.L. Schoeni. 1986. Selective-enrichment procedure for isolation of Listeria
rnonocytogenes from fecal and biologic specimens. Appl. Environ. Microbiol. 5 1:1127-1 129.
90. Doyle, M.P., L.M. Meske, and E.H. Marth. 1985. Survival of Listeria monocytogenes during
the manufacture and storage of nonfat dry milk. J. Food Prot. 48:740-742.
91. Doyle, M.P., K.A. Glass, J.T. Beery, G.A. Garcia, D.J. Pollard, and R.D. Schultz. 1987.
Survival of Listeria monocytogenes in milk during high-temperature, short-time pasteuriza-
tion. Appl. Environ. Microbiol. 53: 1433- 1438.
92. El-Gazzar, F.E., H.F. Bohner, and E.H. Marth, 1991. Growth of Listeria monocytogenes at
4, 32 and 40C in skim milk and in retentate and permeate from ultrafiltered skim milk. J.
Food Prot. 54:338-342, 348.
93. El-Leboudy, A.A., and M.A. Fayed. 1992. Incidence of Listeria in raw milk. Assuit Vet.
Med. J. 27: 134-146.
94. El Marrakchi, A., A. Hamama, and F. El Othmani. 1993. Occurrence of Listeria rnonocyto-
genes in milk and dairy products produced or imported into Morocco. J. Food Prot. 56:256-
259.
95. Eyles, M. 1992. Raw milk cheese: the issues. Austral. J. Dairy Technol. 47:102-105.
96. Farber, J.M., G.W. Sanders, and M.A. Johnston. 1989. A survey of various foods for the
presence of Listeria species. J. Food Prot. 52:456-458.
97. Farber, J.M., G.W. Sanders, and S.A. Malcom. 1988. The presence of Listeria spp. in raw
milk in Ontario. Can. J. Microbiol. 34:95-100.
98. Farber, J.M., G.W. Sanders, and J.I. Speirs. 1990. Growth of Listeria monocytogenes in
naturally-contaminated raw milk. Lebensm. Wiss. Technol. 23:252-254.
99. Farber, J.M., G.W. Sanders, J.I. Speirs, J.-Y. DAoust, D.B. Emmons, and R. McKellar.
1988. Thermal resistance of Listeria rnonocytogenes in inoculated and naturally contaminated
raw milk. Intern. J. Food Microbiol. 7:277-286.
100. Farkas, G.Y., S. Szakaly, and B. Ralovich. 1988. Occurrence of Listeria strains in a Hungar-
ian dairy plant-Pecs. Proc. X International Symposium on Listeriosis, Pecs, Hungary, Aug.
22-26, Abstr. P59.
101. Farrag, S.A., and E.H. Marth. 1989. Behavior of Listeria rnonocytogenes when incubated
together with Pseudornonas species in tryptose broth at 7 and 13C. J. Food Prot. 52536-
539.
102. Farrag, S.A., and E.H. Marth. 1989. Growth of Listeria monocytogenes in the presence of
Pseudornonasfluorescens at 7 or 13C in skim milk. J. Food Prot. 52:852-855.
103. Farrag, S.A., F.E. El-Gazzar, and E.H. Marth. 1990. Fate of Listeria monocytogenes in sweet-
ened condensed and evaporated milk during storage at 7 or 21C. J. Food Prot. 53:747-750.
104. Farrag, S.A., and E.H. Marth. 1991. Behavior of Listeria rnonocytogenes in the presence of
flavobacteria in skim milk at 7 and 13C. J. Food Prot. 54:677-680.
105. Farrag, S.A., and E.H. Marth. 1991. Variation in initial populations of Pseudornonas Juo-
rescens affects behavior of Listeria rnonocytogenes in skim milk at 7 and 13C. Milchwis-
senschaft 46:7 18-72 1.
106. Fedio, W.M., and H. Jackson. 1990. Incidence of Listeria rnonocytogenes in raw bulk milk
in Alberta. Can. Inst. Food Sci. Technol. J. 23:236-238.
107. Fenlon, D.R., and J. Wilson. 1989. The incidence of Listeria monocytogenes in raw milk
from farm bulk tanks in North-East Scotland. J. Appl. Bacteriol. 66:191-196.
108. Fenlon, D.R., T. Stewart, and W. Donachie. 1995. The incidence, numbers and types of
L. monocytogenes in Unfermented Dairy Products 405
Listeria rnonocytogenes isolated from farm bulk tank milks. Lett. Appl. Microbiol. 2057-
60.
109. Fistrovici, E., and D.L. Collins-Thompson. 1990. Use of plasmid profiles and restriction
endonuclease digest in environmental studies of Listeria spp. from raw milk. Intern. J. Food
Microbiol. 10:43-50.
110. Fleming, D.W., S.L. Cochi, K.L. MacDonald, J. Brondum, P.S. Hayes, B.D. Plikaytis, M.B.
Holmes, A. Audurier, C.V. Broome, and A.L. Reingold. 1985. Pasteurized milk as a vehicle
of infection in an outbreak of listeriosis. N. Engl. J. Med. 312:404-407.
111. Food and Drug Administration. 1989. Code of Federal Regulations, Title 21, Code Fed. Reg.,
U.S. Dept. Health Human Services, Washington, DC.
112. Franzin, L. 1992. Comparison of five isolation media for the recovery of Listeria from river
water and raw milk. In: XI International Symposium on Problems of Listeriosis, Copenhagen,
May 11-14, p. 160-161.
113. Garayzabal, J.F.F., L.D. Rodriguez, J.A.V. Boland, J.L.B . Cancelo, and G.S. Fernandez.
1986. Listeria rnonocytogenes dans le lait pasteurisi. Can. J. Microbiol. 32: 149-150.
114. Garayzabal, J.F.F., L.D. Rodriguez, J.A.V. Boland, E. Gomez-Lucia, E.R. Ferri, and G.S.
Fernandez. 1987. Occurrence of Listeria rnonocytogenes in raw milk. Vet. Rec. 120:258-
259.
115. Gasparovic, E. von, M. Sabolic, W. Unglaub, and G. Terplan. 1989. Untersuchungen uber
das Vorkommen von Listeria rnonocytogenes in Rohmilch in Sudwurttemberg. Tieriirztl.
Umschau 44:783-790.
116. Gaya, P., C. Saralegui, M. Medina, and M. Nunez. 1996. Occurrence of Listeria rnonocyto-
genes and other Listeria spp. in raw caprine milk. J. Dairy Sci. 79:1936-1941.
117. Gelosa, L. 1990. La Listeria rnonocytogenes quale contarninante di prodotti lattiero-caseari.
Indust. Aliment. 29: 137-139.
118. Gilbert, R.J. 1990. Personal communication.
119. Gilmour, A., and J. Harvey. 1990. The incidence of Listeria spp. in Northern Ireland dairy
products. In: Posters and Brief Communications of the XX.111 International Dairy Congress,
Montreal, Oct. 8-12, Abst. 230.
120. Gledel, J. 1986. Epidemiology and significance of listeriosis in France. In A. Schonberg ed.
Listeriosis-Joint WHO/ROI Consultation on Prevention and Control, West Berlin, December
10- 12, 1986, Institut fur Veterinkedizin des Bundesgesundheitsamtes, Berlin, pp. 9-20.
121. Gleclel, J. 1988. Listeria and the dairy industry in France. Foodborne Listeriosis-Proceed-
ings of a Symposium, Wiesbaden, Germany, Sept. 7, pp. 72-82.
122. Gohil, V.S., M.A. Ahmed, R. Davies, and R.K. Robinson. 1995. Incidence of Listeria spp.
in retail foods in the United Arab Emirates. J. Food Prot. 58:102-104.
123. Golnazarian, C.A., C.W. Donnelly, S.J. Pintauro, and D.B. Howard. 1989. Comparison of
infectious dose of Listeria rnonocytogenes F58 17 as determined for normal versus compro-
mised C57B 1/6J mice. J. Food Prot. 52:696-701.
124. Goz, M. 1992. Distribution of Listeria strains which are isolated in Turkey. In: XI Interna-
tional Symposium on Problems of Listeriosis, Copenhagen, May l l - 14, p. 188- 189.
125. Greenaway, C., and P.G. Drew. 1990. Survey of dairy products in Victoria, Australia for
Listeria species. In: Posters and Brief Communications of the XXIII International Dairy
Congress, Montreal, Oct. 8-12, Abst. 234.
126. Greenwood, M.H., D. Roberts, and P. Burden. 1991. The occurrence of Listeria species in
milk and dairy products: a national survey in England and Wales. Int. J. Food Microbiol.
12:197-206.
127. Harvey, J., and A. Gilmour. 1992. Occurrence of Listeria species in raw milk and dairy
products produced in Northern Ireland. J. Appl. Bacteriol. 72: 119- 125.
128. Hayes, P.S. 1988. Personal communication.
129. Hayes, P. S., J.C. Feeley, L.M. Graves, G.W. Ajello, and D.W. Fleming. 1986. Isolation of
Listeria monocytogenes from raw milk. Appl. Environ. Microbiol. 5 1:438-440.
406 Ryser
130. Ibrahim, A., and I.C. MacRae. 1991. Incidence of Aeromonas and Listeria spp. in red meat
and milk samples in Brisbane, Australia. Int. J. Food Microbiol. 12:263-270.
131. Ibrahim, G.A.M., H. Domjan-Kovacs, A. Fabian, and B. Ralovich. 1992. Listeria in milk
and dairy products in Hungary. In: XI International Symposium on Problems of Listeriosis,
Copenhagen, May 1 1 - 14, p. 297-298.
132. Ikonomov, L., and D. Todorov. 1964. Studies of the viability of Listeria monocytogenes in
ewes milk and dairy products. Vet. Med. Nauki, Sofiya 7:23-29.
133. International Dairy Federation. 1989. Pathogenic Listeria- Abstracts of replies from 24
countries to questionnaire 1288/B on pathogenic Listeria. Circular 89/5, March 3 1, Intern.
Dairy Fed., Brussels.
134. Kozak, J.J. 1986. FDAs dairy program initiatives. Dairy Food Sanit. 6: 184- 185.
135. Kozak, J., T. Balmer, R. Byrne, and K. Fisher. 1996. Prevalence of Listeria monocytogenes
in foods-incidence in dairy products. Food Control 7:215-221.
136. Kwiatek, K., B. Wojton, J. Rola, and H. Rozanska. 1992. The incidence of Listeria rnonocyto-
genes and other Listeria spp. in meat, poultry and raw milk. Bull. Vet. Inst. Pulawy. 35:7-11.
137. Lanciotti, R., S. Massa, M.E. Guerzoni, and G. DiFabio. 1992. Light butter: natural microbial
population and potential growth of Listeria monocytogenes and Yersinia enterocolitica. Lett.
Appl. Microbiol. 15:256-258.
138. Legnani, P., E. Leoni, F. Soppelsa, and P. Bisbini. 1995. Prevalence of Listeria spp. in food
products in the province of Belluno (Italy). L Igiene Moderna 103:143- 155.
139. Liewen, M.B., and M.W. Plautz. 1988. Occurrence of Listeria monocytogenes in raw milk
in Nebraska. J. Food Prot. 5 1 :840-841.
140. Liewen, M.B., D.L. Peters, and M.W. Plautz. 1987. Incidence of L. monocytogenes in raw
milk in Nebraska. Annual Meeting of the Institute of Food Technologists, Las Vegas, NV,
June 16-19, Abstr. 118.
141. Lovett, J., D.W. Francis, and J.M. Hunt. 1987. Listeria monocytogenes in raw milk: detection,
incidence, and pathogenicity. J. Food Prot. 50: 188- 192.
142. Luisjuan-Morales, A., R. Alaniz-de la 0, M.E. Vazquez-Sandoval, and B.T. Rosas-Barbosa.
1995. Prevalence of Listeria monocytogenes in raw milk in Guadalajara, Mexico. J. Food
Prot. 58:1139-1141.
143. Lund, A.M., and E.A. Zottola. 1990. Inhibition of Listeria species by Bacillus in raw milk.
J. Food Prot. 59:903 (abstr.).
144. Lund, A.M., E.A. Zottola, and D.J. Pusch. 1991. Comparison of methods for isolation of
Listeria from raw milk. J. Food Prot. 54:602-606.
145. Marshall, D.L., and R.H. Schmidt. 1988. Growth of Listeria monocytogenes at 10C in milk
preincubated with selected pseudomonads. J. Food Prot. 5 1 :277-282.
146. Marshall, D.L., and R.H. Schmidt. 1991. Physiological evaluation of stimulated growth of
Listeria monocytogenes by Pseudomonas species in milk. Can. J. Microbiol. 37594-599.
147. Mascola, L., L. Chun, J. Thomas, W.F. Bibe, B. Schwartz, C. Salminen, and P. Heseltine.
1988. A case-control study of a cluster of perinatal listeriosis identified by an active surveil-
lance system in Los Angeles County. Society for Industrial Microbiology-Comprehensive
Conference on Listeria monocytogenes, Rohnert Park, CA, Oct. 2-5, Abstr. P-10.
148. Massa, S., D. Cesaroni, G. Poda, and L.D. Trovatelli. 1990. The incidence of Listeria spp.
in soft cheeses, butter, and raw milk in the province of Bologna. J. Appl. Bacteriol. 68:153-
156.
149. Mattingly, J.A., B.T. Butman, M.C. Plank, R.J. Durham, and B.J. Robison. 1988. Rapid
monoclonal antibody-based enzyme-linked immunosorbant assay for detection of Listeria in
food products. J. Assoc. Off. Anal. Chem. 7 1 :679-68 1.
150. McBean, L.D. 1988. A perspective on food safety concerns. Dairy Food Sanit. 8: 112-1 18.
151. Mickova, V. I99 1 . Listeria monocytogenes in foods. Vet. Med. (Praha) 36:745-750.
152. Mickova, V., and S. Konecny. 1990. Listeria monocytogenes in foods. Veterinarstyi 40:327-
328.
L. monocytogenes in Unfermented Dairy Products 407
153. Minor, T.E., and E.H. Marth. 1972. Staphylococcus aureus and enterotoxin A in cream and
butter. J. Dairy Sci. 55:1410-1414.
154. Monge, R., D. Utzinger, and L. Arias. 1994. Incidence of Listeria in pasteurized ice cream
and soft cheese in Costa Rica, 1992. Rev. Biol. Trop. 43:327-328.
155. Moura, S.M., M.T. Destro, and B.D. Franco. 1993. Incidence of Listeria species in raw and
pasteurized milk produced in Sao Paulo, Brazil. Int. J. Food Microbiol. 19229-237.
156. Northolt, M.D., H.J. Beckers, U. Vecht, L. Toepoel, P.S.S. Soentoro, and H.J. Wisselink.
1988. Listeria monocytogenes: heat resistance and behavior during storage of milk and whey
and making of Dutch types of cheese. Neth. Milk Dairy J. 42:207-219.
157. ODonnell, E.T. 1995. The incidence of Salmonella and Listeria in raw milk from farm bulk
tanks in England and Wales. J. Soc. Dairy Technol. 48:25-29.
158. Olsen, J.A., A.E. Yousef, and E.H. Marth. 1988. Growth arid survival of Listeria monocyto-
genes during making and storage of butter. Milchwissenschaft 43:487-489.
159. Oz, H.H., and R.J. Farnsworth. 1985. Laboratory simulation of fluctuating temperature of
farm bulk tank milk. J . Food Prot. 48:303-305.
160. Palumbo, S.A., and A.C. Williams. 1991. Resistance of Listeria monocytogenes to freezing
in foods. Food Microbiol. 8:63-68.
161. Patterson, R.L., D.J. Pusch, and E.A. Zottola. 1989. The isolation and identification of Liste-
ria spp. from raw milk. J. Food Prot. 52:745.
162. Pearson, L.J., and E.H. Marth. 1990. Behavior of Listeria monocytogenes in the presence of
cocoa, carrageenan, and sugar in a milk medium incubated with and without agitation. J.
Food Prot. 53:30-37.
163. Pearson, L.J., and E.H. Marth. 1990. Behavior of Listeria monocytogenes in the presence of
methylxanthines-caffeine and theobromine. J. Food Prot. 53:47-50, 55.
164. Pearson, L.J., and E.H. Marth. 1990. Inhibition of Listeria monocytogenes by cocoa in a
broth medium and neutralization of this effect by casein. J . Food Prot. 53:38-46.
165. Peters, D.L., and M.B. Liewen. 1988. Growth and survival of Listeria monocytogenes in
unfiltered milk. Annual Meeting of the Institute of Food Technologists, New Orleans, June
19-22, Abstr. 326.
166. Pine. L., G. B. Malcolm, J.B. Brooks, and M.I. Daneshvar. 1989. Physiological studies on
the growth and utilization of sugars by Listeria species. Can. J. Microbiol. 35:245-254.
167. Prentice, G.A. 1994. Listeria monocytogenes. In: The Significance of Pathogenic Microor-
ganisms in Raw Milk. Brussels, International Dairy Federation Ref. S.I. 9405, p. 101-
115.
168. Proctor, M.E., R. Brosch, J.W. Mellen, L.A. Garrett, C.W. Kasper, and J.B. Luchansky.
1995. Use of pulsed-field gel electrophoresis to link sporadic cases of invasive listeriosis
with recalled chocolate milk. Appl. Environ. Microbiol. 6 1 :3177-3 179.
169. Quagilo, G., C. Casolari, G. Menziani, and A. Fabio. 1992. The incidence of Listeria monocy-
togenes in milk and milk products. LIgiene Moderna 97565-579.
170. Quinto, E.J., C.M. Franco, C.A. Fente, B.I. Vazquez, and A. Cepeda. 1996. Effects of Pseu-
domonas Jluorescens on the growth of Listeria monocytogenes and Listeria innocua in
skimmed milk. Arch. Lebensmittelhygiene 47: 107- 1 10.
171. Rajikowski, K.T., S.M. Calderone, and E. Jones. 1994. Effect of polyphosphate and sodium
chloride on the growth of Listeria monocytogenes and Staphylococcus aureus in ultra-high
temperature milk. J. Dairy Sci. 77: 1503- 1508.
172. Razavi-Rohani, M., and Y. Hedaiatinia. 1990. A study of the contamination of milk to Liste-
ria in Urmia, Iran. In: Posters and Brief Communications of the XXIII International Dairy
Congress, Montreal, Oct. 8- 12. Abst. 364.
173. Rea, M.C., T.M. Cogan, and S. Tobin. 1992. Incidence of pathogenic bacteria in raw milk
in Ireland. J. Appl. Bacteriol. 73:331-336.
174. Rodler, M., and W. Korbler. 1988. Examination of Listeria monocytogenes in milk products.
X International Symposium on Listeriosis, Pecs, Hungary, Aug. 22-26, Abstr. 47.
408 Ryser
174a. Rodler, M., and W. Korbler. 1989. Examination of Listeria monocytogenes in dairy products.
Acta Microbiol. Hung. 36:259-261.
175. Rodriguez, J.L., P. Gaya, M. Medina, and M. Nunez. 1994. Incidence of Listeria monocyto-
genes and other Listeria spp. in ewes raw milk. J. Food Prot. 57571-575.
176. Rodriguez, L.D., J.F.F. Garayzabal, J.A.V. Boland, E.R. Ferri, and G.S. Fernandez. 1985.
Isolation de micro-organismes du genre listeria h partir de lait cru destin6 h la consommation
humaine. Can. J. Microbiol. 3 1:938-941.
177. Rohrbach, B.W., F.A. Draughon, P.M. Davidson, and S.P. Oliver. 1992. Prevalence of Liste-
ria monocytogenes, Campylobacter jejuni, Yersinia enterocolitica and Salmonella in bulk
tank milk: risk factors and risk of human exposure. J. Food Prot. 55:93-97.
178. Rola, J., K. Kwiatek, B. Wojton, and M.M. Michalski. 1994. Incidence of Listeria monocyto-
genes in raw milk and dairy products. Medycyna Wet. 50:323-325.
179. Rosenow, E.M., and E.H. Marth. 1987. Addition of cocoa powder, cane sugar, and carra-
geenan to milk enhances growth of Listeria monocytogenes. J. Food Prot. 50:726-729, 732.
180. Rosenow, E.M., and E.H. Marth. 1987. Growth of Listeria monocytogenes in skim, whole
and chocolate milk, and in whipping cream during incubation at 4, 8, 13, 21, and 35C. J.
Food Prot. 50:452-459.
181. Rosso, L., S. Bajard, J.P. Flandrois, C. Lahellec, J. Fournaud, and P. Veit. 1996. Differential
growth of Listeria monocytogenes at 4 and 8C: Consequences for the shelf life of chilled
products. J. Food Prot. 59:944-949.
182. Roy, R.N. 1992. Listeria monocytogenes in dairy products and water. In: XI International
Symposium on Problems of Listeriosis, Copenhagen, May 11-14, p. 327-328.
183. Ryan, C.A., M.K. Nickels, N.T. Hargrett-Bean, M.E. Potter, T. Endo, L. Mayer, C.W. Lang-
kop, C. Gibson, R.C. McDonald, R.T. Kenney, N.D. Puhr, P.J. McDonnell, R.J. Martin,
M.L. Cohen, and P.A. Blake. 1987. Massive outbreak of antimicrobial resistant salmonellosis
traced to pasteurized milk. J.A.M.A. 258:3269-3274.
184. Ryser, E.T. 1998. Public health concerns. In: Applied Dairy Microbiology. Marth, E.H, and
J.L. Steele (eds.). Marcel Dekker, New York.
185. Ryser, E.T., and E.H. Marth. 1988. Growth of Listeria monocytogenes at different pH values
in uncultured whey or in whey cultured with Penicillium camemberti. Can. J. Microbiol. 34:
730-734.
186. Satio, A., Y. Tokumaru, H. Masaki, T. Itaya, and A. Aoki. 1991. Evaluation of enrichment
and plating media for the isolation of Listeria monocytogenes from raw milk and the state
of contamination of raw milk by Listeria. J. Japan Vet. Med. Assoc. 44:378-383.
187. Sharif, A., and N. Tunail. 1991. Listeria monocytogenes contamination of raw milk from
different regions of Anatolia and pasteurized milk sold in Ankara. Mikrobiyol. Bul. 25: 15-
20.
188. Siswanto, H.P., and J. Richard. 1992. Growth rate of Listeria monocytogenes and other spe-
cies of Listeria in milk at sub-optimum temperatures. Le Lait 72:265-275.
189. Slade, P.J., and D.L. Collins-Thompson. 1988. Enumeration of Listeria monocytogenes in
raw milk. Lett. Appl. Microbiol. 6: 121-123.
190. Slade, P.J., D.L. Collins-Thompson, and F. Fletcher. 1988. Incidence of Listeria species in
Ontario raw milk. Can. Inst. Food Sci. Technol. J. 21:425-429.
191. Slavchev, G., S . Milaski, I. Stefanov, and S. Stoyanova. 1994. Survival of Listeria monocyto-
genes in milk and dairy products. Khranif. Promish. 43:28-30.
192. Soncini, G., and L. Piantoni. 1993. The occurrence of Listeria monocytogenes in raw milk.
Latte 18:846-848.
193. Stelma, G.N. Jr., A.L. Reyes, J.T. Peeler, D.W. Francis, J.M. Hunt, P.L. Spaulding, C.H.
Johnson, and J. Lovett. 1987. Pathogenicity test for Listeria monocytogenes using immuno-
compromised mice. J. Clin. Microbiol. 25:2085-2089.
194. Stone, D.L. 1987. A survey of raw whole milk for Campylobacter jejuni, Listeria monocyto-
genes and Yersinia enterocolitica. New Zealand J. Dairy Sci. Technol. 22:257-264.
L. monocytogenes in Unfermented Dairy Products 409
195. Tacket, C.O., J.P. Narain, R. Sattin, J.P. Lofgren, C. Konigsberg, R.C. Rendtorff, A. Rausa,
B.R. Ilavis, and M.L. Cohen. 1984. A multistate outbreak of infections caused by Yersinia
enterocolitica transmitted by pasteurized milk. J.A.M.A. 25 1:483-486.
196. Takai, S., F. Orii, K. Yasuda, S. Inoue, and S. Tusbaki. 1990. Isolation of Listeria rnonocyto-
genes from raw milk and its environment at dairy farms in Japan. Microbiol. Immunol. 34:
63 1-634.
197. Terplan, G. 1988. Factors responsible for the contamination of food with Listeria rnonocyto-
genes. WHO Working Group on Foodborne Listeriosis. Geneva, Feb. 15-19.
198. Terplan, G. 1988. Provisional IDF-recommended method: Milk and milk products-detec-
tion of Listeria rnonocytogenes. International Dairy Federation, Brussels, Belgium.
199. Tiscione, E., A. Lo Nostro, R. Donato, L. Galassi, R. Pancinj, and B. Ademollo. 1994. Prob-
lemi microbiologi ei relativi ai latticini in riferimento alla loro possible funizone di veicoli
ricera di Listeria spp. e Pseudornonas spp. nel ciclo di produzione delle mozzarelle. Igiene
e Sanitab. Pubblica. 50:27-36.
200. Tiwari, N.P., and S.G. Aldenrath. 1990. Occurrence of Listeria species in food and environ-
mental samples in Alberta. Can. Inst. Food Sci. Technol. J. 23: 109-1 13.
201. Vassau, N. 1988. Personal communication.
202. Venables, L.J. 1989. Listeria rnonocytogenes in dairy products-the Victorian experience.
Food Australia 41:942-943.
203. Wenzel, J.M., and E.H. Marth. 1990. Behavior of Listeria rnonocytogenes at 4 and 7C in
raw milk inoculated with a commercial culture of lactic acid bacteria. Milchwissenschaft 45:
772-774.
204. Wnorowski, T. 1990. The prevalence of Listeria species in raw milk from the Transvaal
region. Sud.-Afrik. Tydsk. Suiwelk. 22: 15-21.
205. Anonymous. 1997. Chocolate ice cream recalled. FDA Enforcement Report, August 13,
206. Anonymous. 1998. Ice cream bars recalled. FDA Enforcement Report, Jan. 14.
207. Anonymous. 1998. Ice cream recalled. FDA Enforcement Report, Mar. 4.
208. Anonymous. 1998. Ice cream sandwiches recalled. FDA Enforcement Report, Jan. 7.
209. Anonymous. 1997. Frozen strawberry yogurt recalled. FDA Enforcement Report, Sept. 17.
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12
Incidence and Behavior of Listeria
monocytogenes in Cheese and
Other Fermented Dairy Products
ELLIOTT. RYSER
Michigan State University, East Lansing, Michigan
INTRODUCTION
On June 14, 1985, L. monocytogenes emerged from relative obscurity to the front page
of many American newspapers because of a large listeriosis outbreak in California that
was directly linked to consumption of Mexican-style cheese manufactured in metropolitan
Los Angeles. By the time this outbreak subsided in August 1985, as many as 300 cases
of listeriosis were reported, including 85 deaths-at least 40 of which were traced to the
tainted cheese. In response to this foodborne outbreak of listeriosis, U.S. Food and Drug
Administration (FDA) officials added L. monocytogenes to their list of pathogenic organ-
isms that should be of concern to cheesemakers and began surveying various soft domestic
cheeses for listeriae.
Approximately 6 months later, isolation of L. monocytogenes from several imported
Brie cheeses purchased at a supermarket led to the eventual recall of approximately
300,000 tons of Brie cheese imported from France and to a real concern about the inci-
dence of this pathogen in other European cheeses. Recall of this cheese prompted two
corrective measures: (a) adoption of a cheese certification program by the United States
and France to prevent importation of Listeria-contaminated cheese and (b) initiation of
numerous large-scale surveys to determine the extent of Listeria contamination in virtually
all types of cheese manufactured in the United States, Canada, and Western Europe.
411
4 12 Ryser
Throughout 1986 and most of 1987, the impact of Listeria on European cheesemak-
ers was primarily in the form of economic losses from destruction of contaminated prod-
uct. However, L. rnonocytogenes struck again late in 1987 with the report of a large listeri-
osis outbreak in Switzerland (see Chap. 10) in which Vacherin Mont d'Or soft-ripened
cheese was incriminated as the vehicle of infection. Most recently, tainted Brie cheese
prepared from raw milk was responsible for a major listeriosis outbreak in France.
These cheeseborne listeriosis outbreaks have prompted worldwide efforts to deter-
mine the incidence of Listeria spp. in various cheeses and examine the behavior of L.
rnonocytogenes during manufacture and storage of numerous fermented dairy products.
The first portion of this chapter summarizes Listeria-related recalls of cheese in the United
States and results from surveys dealing with the incidence of listeriae in domestic and
imported fermented dairy products. The second half of this chapter addresses the fate of L.
rnonocytogenes during manufacture and storage of buttermilk, yogurt, and various cheeses
(including whey) and the potential for cheese ingredients, such as rennet, salt brine, and
coloring agents, to serve as vehicles of contamination during cheesemaking.
cedure. Cheese samples also were tested for the presence of enteropathogenic strains of
E. coli and for phosphatase activity, which if present generally indicates improper pasteur-
ization of cheesemilk and/or subsequent contamination with raw milk. However, suitabil-
ity of the phosphatase test for cheese has since been questioned.
Less than 2 months into this program, FDA officials isolated a pathogenic strain of
L. monocytogenes from one sample of domestically produced Liederkranz cheese (see
Table 1). The manufacturer subsequently recalled the product nationwide. Following pre-
liminary FDA reports of further Listeria contamination, this recall was extended to include
all lots of Brie and Camembert cheese manufactured at the same facility [8,12]. However,
final laboratory reports indicated that both Brie and Camembert cheese were contaminated
with L. inrzocua, which is nonpathogenic, rather than L. monocytogenes. Although the
Domestic Soft Cheese Surveillance Program also was responsible for temporarily closing
two soft cheese factories in California that produced phosphatase-positive cheese [9], it
must be stressed that L. monocytogenes was never isolated from cheeses produced at either
facility.
In general, FDA inspections of other soft cheese factories uncovered problems simi-
lar to those encountered during inspections of Grade A fluid milk factories: (a) potential
bypasses of the pasteurizer, (b) postpasteurization blending of product, and (c) a general
lack of education and/or training of plant personnel [223]. Items of particular concern to
cheesemakers and that were not generally found during visits to Grade A milk factories
included defects in the pasteurization process, discrepancies in pasteurization/production
records, and a higher incidence (than in Grade A milk factories) of pathogenic microorgan-
isms (including L. monocytogenes) on environmental surfaces in production and storage
areas.
Inspections of domestic cheese factories continued throughout 1986, 1987, and 1988
under four separate programs (see Fig. l), with FDA officials reaching nearly half of the
400 soft cheese factories in the United States by April of 1986 and the remaining factories
(including follow-up inspections of problem factories) by late 1987 [46]. According to
FDA records [loll, L. monocytogenes was confirmed in 12 of 658 (1.82%) domestic
cheese samples analyzed during 1986. During these inspection programs, six Class I recalls
were issued for various ethnic-type soft and semisoft cheeses containing L. monocyto-
genes. In response to (a) a 1987 report of a woman who developed listeriosis in San
Bernadino, California, after consuming illegally produced Mexican-style cheese and (b)
the widespread availability of uninspected, unbranded Mexican-style cheese illegally pro-
duced from raw milk in metropolitan Los Angeles [71a], Genigeorgis et al. [175], in
conjunction with the California Department of Food and Agriculture, U.S. Department of
Agricultures Food Safety and Inspection Service (USDA-FSIS), the Immigration and
Naturalization Service, and the Los Angeles District Attorneys Office, surveyed 100 Cali-
fornia-produced soft Hispanic-style cheeses that were either seized or purchased under-
cover between June and November of 1988, Overall, two samples each were positive for
L. monocytogenes and L. innocua. These four Listeria-contaminated cheeses had a pH of
6.2-6.5 and were presumably prepared from raw milk as evidenced by a positive alkaline
phosphatase test. Given the ability of L. monocytogenes to grow in such cheeses during
refrigerated storage and marketing, Hispanic-style cheeses continue to constitute a signifi-
cant public health threat, with these varieties accounting for 7 of 21 recalls issued through
1996, including one large recall in June 1990 involving approximately 500,000 Ib of prod-
uct. As previously mentioned, although all products containing L. monocytogenes must
be retrieved from the marketplace, formal Class I recalls do not have to be issued for
4 14 Ryser
TABLE
1 Chronological List of Class I Recalls in the United States for Domestic Cheese Contaminated with L. monocytogenes
Date recall
Type of cheese initiated Origin Distribution Quantity (lb) Ref.
Jalisco brand soft Mexican-style: Cotija, 6/13/85 California Arizona, Arkansas, California, Colorado, -500,000 10, 11
Queso Fresco, and 20 other varieties Georgia, Guam, Hawaii, Idaho, Illinois,
Kansas, Louisiana, Marshal1 Islands, Mas-
sachusetts, Nevada, New Jersey, New Mex-
ico, New York, Oklahoma, Oregon, Rhode
Island, Samoa, Texas, Utah, Washington
state
Liederkranz (Brie,a Camembert) 8/ 14/85 Ohio Nationwide, Puerto Rico -10,000 8, 9, 12
Soft Mexican-style: Queso Fresco and 5 3/5/86 California Arizona, California, Oregon, Texas 127,607 20, 38
other varieties
Semisoft Salvador-style white 911 1/86 Virginia Virginia, Washington, DC 10,850 42, 54
Soft-ripened: Old Heidelberg 4117/87 Illinois Illinois, North Carolina, Ohio, Pennsylvania 1150 52
Soft-ripened: Bonbel and Gouda 5/6/87 Kentucky Nationwide -13,800 51
Raw milk sharp Cheddar 8/21187 Wisconsin California, Washington state -1400 53, 280
Soft Mexican-style: Cotija, Queso Fresco, 1/29/88 California Arizona, California, Florida, Texas, Washing- Unknown 64
+
and 8 other varieties Baby Jack and ton state
Monterey Jack
Mexican-style soft cheese 1 1/6/90 California Arizona, California, Idaho, Nevada, Oregon, 500,000 78
Washington state
Cheese spread 2/1/91 Florida Southeastern United States -1362 81
Mozzarella 2/14/91 Wisconsin Connecticut, Georgia, Illinois, Michigan, >89.000 80
New York, Ohio, Pennsylvania, Texas,
West Virginia, Wisconsin
Ricotta 711 1/91 New York Florida, New York 1109 82
Jack 10/28/91 Wisconsin Iowa, Minnesota, Wisconsin 12,500 79
Cold-pack cheese food 3110192 Wisconsin Arizona, California, Colorado, Florida, Geor- Unknown 83
gia, Illinois, Indiana, Maryland, Michigan,
Minnesota, New York, Ohio, Pennsylva-
nia, Tennessee, Texas, Vermont, Virginia,
Wisconsin
Queso fresco 10/14/92 Washington Oregon, Washington state Unknown 85
Limburger 1211 8/92 Wisconsin Wisconsin 1500 86
L. monocytogenes in Fermented Dairy Products 4 15
Cheese spread 3/4/93 Tennessee Alabama, Illinois, Indiana, Kentucky, Missis- 11,789 84
sippi, Tennessee
Cream cheese 10119/93 Wisconsin California, Florida, Georgia, Illinois, Indiana, 3075 88
Iowa, Minnesota, Nebraska, North Caro-
lina, Ohio, South Carolina, South Dakota,
Tennessee, Wisconsin
Queso prensado 4/15/94 Wisconsin Florida, New Jersey, Wisconsin 1429 94
Cream cheese and lox 511 1/94 Massachusetts Connecticut, Georgia, Massachusetts 20 89
Mexican-style soft white 5120194 Texas Texas Unknown 92
Mexican-style soft white 512 I 194 Texas Texas Unknown 91
Mexican-style soft white 5/23/94 Texas Texas Unknown 91
Queso blanco 5/24/94 Wisconsin New Jersey 1220 93
Goat milk cheese 6/15/94 California California, Colorado, Georgia, Illinois, Mas- -5,682 90
sachusetts, Michigan, New York, Oregon,
Texas
Torte loaf cheese 8111194 Missouri Illinois, Indiana, Kansas, Louisiana, Mis- 301 96
souri, Texas
Swiss cold-pack cheese food 811 1 194 Wisconsin Missouri, Ohio 510 95
Swiss I0/28/94 Ohio Pennsylvania 2270 97
Gorgonzola 2/2/96 Wisconsin California, Colorado, Florida, Georgia, Illi- 4500 98
nois, Minnesota, New Jersey, New York,
North Carolina, Pennsylvania, Tennessee,
Washington state, Wisconsin
Cream cheese with vegetables 10/30/97 Massachusetts Connecticut, Maine, Massachusetts, New 7,340 lOOa
Hampshire, New Jersey, New York,
Rhode Island, Pennsylvania, Vermont
Cream cheese 1 1114197 Massachusetts Connecticut, Maine, Massachusetts, New Unknown lOOb
Hampshire, New Jersey, New York,
Rhode Island, Vermont
Queso fresco 2/4/98 Wisconsin Nationwide 248,938 IOOe, lOOf
Queso fresco 3/23/98 Domestic Alabama, Florida, Georgia, North Carolina, Unknown lOOg
South Carolina, Tennessee, Virginia
Blue cheese 411 1/98 Wisconsin Nationwide Unknown lOOc
Blue cheese salad dressing 511198 Louisiana Nationwide Unknown lOOd
1 l-
* 2
r
Aged and ripened cheese survey -
begun January 1986
r
Continuation of s o f t cheese survey -
March 1987 cheese testing program - begun April 1986
1
Survey of cheese manufactured
I-
1
L. monocytogenes isolated from Italian
Romano cheese - June 1987
I 1
Italian cheese surveillance program
begun July 1987
I
-
I
-I
surveillance program -
Cheese under general pathogen
1988
I
I
status -
Survey of import cheese in domestic
begun December 1987 I
L
FIGURE1 Surveillance programs for Listeria spp. in domestic and imported cheese. (Adapted from Ref. 101.)
L. monocytogenes in Fermented Dairy Products 417
contaminated products that have not yet reached retail stores. Since such situations typi-
cally lead to nonpublished ''internal recalls" issued by the manufacturer, far more cheese
was likely destroyed during this 11-year period than has actually been reported. Several
such informal recalls involved a part-skim milk cheese manufactured in California [57]
as well as ricotta, Parmesan, and mozzarella cheese of uncertain origin [233].
Following a report by Ryser and Marth [259] that L. rnonocytogenes can survive
more than 1 year in Cheddar cheese (i.e., well beyond the mandatory 60-day aging period
for Cheddar cheese manufactured from raw milk), the FDA modified its Domestic Cheese
Program in August of 1987 to include cheese prepared from unpasteurized milk [46].
Between April and October of 1987, 181 samples of domestic aged (held a minimum of
60 days at 1:1.7"C [35"F]) natural cheese manufactured from raw milk, as well as similar
imported cheeses in domestic status, were collected from retail stores by FDA field person-
nel and analyzed for L. rnonocytogenes (Table 2). These efforts uncovered one positive
sample-a sharp Cheddar cheese manufactured in Wisconsin, which was subsequently
recalled from the market in July of 1987 (see Table 1).
Late in 1987, the FDA announced plans for a 2-year pathogen surveillance program
[43] which was designed to examine domestic and imported cheese as well as other high-
risk foods (i.e., milk, vegetables, and seafood) for the presence of L. rnonocytogenes and
other selected pathogens, including Vibrio cholerae, V. parahaernolyticus, Escherichia
coli, enteropathogenic E. coli, Staphylococcus aureus, Salmonella spp., Yersinia entero-
colitica, Clarnpylobacterjejuni, and C. coli. Under this program, samples of soft-ripened
and raw milk cheese as well as imported hard and artificial blended cheese were examined
for all of the aforementioned organisms except Vibrio spp. Domestic cheeses were col-
lected at the wholesale level, whereas samples of imported cheese were obtained from
retail stores. Although this program prompted only one Listeria -related recall of domestic
cheese during 1989 and 1990, five separate recalls of Anari and Halloumi cheese imported
TABLE
2 Incidence of L. monocytogenes in
"Domestic" Cheese Manufactured from Raw
Milk-FDA 1987a
Number of Number of
samples positive samples
Type of cheese analy zed (%>
Blue 18 0
Brick 5 0
Cheddar 71 l b (1.4)
Colby 8 0
Edam 4 0
Goat 6 0
Gouda 1 0
Monterey Jack 9 0
Swiss 42 0
Other 17 0
Total 181 1 (0.55)
~
from Cyprus were reported during this same 2-year period along with one additional recall
of Italian soft-ripened/semisoft cheese. However, since additional cheese-related recalls
after 1990 have been limited to three imported cheeses, the present FDA inspection pro-
gram in combination with increased vigilance on the part of cheesemakers appears to be
highly effective in limiting consumer exposure to both domestic and imported Listeria-
contaminated cheese.
Several other fermented dairy products also were examined for L. monocytogenes
in conjunction with the FDA Dairy Initiative Program [ l o l l (see Chap. 11). In 1986, 10
samples of cottage cheese were found to be free of listeriae. Other than cheese and cheese
food, 1% fat cultured buttermilk [33,35] and frozen yogurt [7 1 ] are the only other domesti-
cally produced, fermented dairy products known to have been contaminated with L. mono-
cytogenes. The first of these products was included in a 1986 Class I recall involving
approximately 1 million gallons of dairy products (fluid milk, chocolate milk, half-and-
half, whipping cream, ice milk, ice milk mix, ice milk shake mix, ice cream, and ice cream
mix), all of which presumably contained L. monocytogenes. Three years later, officials
from the Wisconsin Department of Agriculture, Trade and Consumer Protection issued a
statewide recall for one particular brand of frozen yogurt after routine testing revealed
the presence of L. monocytogenes in one sample of mandarin orange frozen yogurt [73].
Both of these products were retrieved from the marketplace without incident.
Imported Cheese
France
International concern over the potential health hazard of consuming Listeria-contaminated
cheese also is rooted in the California listeriosis outbreak of 1985. This outbreak and an
earlier link between consumption of French Brie and/or Camembert cheese and several
outbreaks of foodborne illness in the United States and Europe caused by enterotoxigenic
and/or enteropathogenic E. coli [2 17,234,2961prompted a meeting in September of 1985
between FDA officials and representatives of the French Embassy/French Delegation on
Food Safety and Food Distribution to discuss the FDAs plans for inspecting imported
soft cheese [ 131. Late in September, representatives from the Codex Committee on Food
Hygiene and the International Dairy Federation agreed with FDA officials that a Code
of Hygienic Practices should be developed for manufacturing fresh and soft cheese [7].
Use of raw milk (a known source of L. monocytogenes) to improve organoleptic properties
of certain cheeses was cited as a particular area of concern. Although a basic certification
program for soft cheese produced in France was in operation for some time, agreement
on a general Code of Hygienic Practices for manufacture of soft cheese was not reached
during the remainder of 1985.
In January of 1986, as part of a research effort to enhance recovery of listeriae from
cheese, FDA officials inadvertently isolated a pathogenic strain of L. monocytogenes from
two uninoculated control samples of French Brie cheese purchased at a local supermarket
[ 19,1011. Ironically, both cheeses were prepared from pasteurized milk in a cheese factory
certified by the French government under the existing soft-ripened cheese agreement. In
response to these findings, the first in a series of six nationwide recalls was issued in
February of 1986 for Listeria-contaminated French Brie cheese (Table 3). The following
week, the French firm that manufactured the tainted cheese agreed to cease all production
[19]. Shipment of additional cheese that was previously certified as Listeria free by an
L. monocytogenes in Fermented Dairy Products 4 19
In this proposal, FDA officials stressed that other methods used to detect Listeria
in cheese should conform to the 7-day FDA method described in Chapter 7 and also
suggested that 1 / 16-in-thick slices from the cheese surface (sample with highest pH) be
analyzed for listeriae rather than cross-sectional plugs of cheese.
Following FDA threats to halt importation of soft-ripened cheese, the French Minis-
try of Agriculture agreed to begin lot-by-lot testing in April of 1986, as outlined in the
March FDA proposal [25]. However, French authorities stressed that such a program
would not be practical on a long-term basis and hoped that the FDA would accept an
expansion ofthe existing factory/product certification program to include Listeria testing
in the near future. Beginning in May 1986, FDA officials announced that all shipments
of French soft-ripened cheese lacking certification of analysis for Listeria would be de-
tained [23]. Inspections during the next 2 months uncovered L. rnonncytogenes in two
French cheeses-a noncertified Brie and a 6-lb certified lot of Muenster [23]-both of
which were presumably recalled internally.
Continued problems with Listeriu-contaminated French soft-ripened cheeses
prompted FDA officials to revise the imported cheese surveillance program in August of
1986 [IS]. These changes allowed immediate detention of French cheeses that were: (a)
420 R yser
TABLE
3 Chronological List of Class I Recalls in the United States for Imported Cheese Contaminated with L. rnonocytogenes
Date recall Country of
Type of cheese initiated manufacture Distribution Quantity Ref.
Brie 2112/86 France Bermuda, Nationwide 57,000 2-6-lb wheels 17, 19, 26, 29
Brie 2114/86 France Georgia, New Jersey 40 cases 16
Brie 2/14/86 France Colorado, Connecticut, Florida, Louisi- 100 cases 16
ana, Maryland, Massachusetts, New
Jersey, New York, Ohio, Washing-
ton, DC
Brie 2/14/86 France Florida, New York, Washington, DC 10 cases 16
Brie 2/14/86 France Nationwide Unknown 16, 37
Brie 212 1186 France Oregon, Washington state Unknown 15, 37
Brie 2/24/86 France Illinois, Minnesota, New Jersey 909 cases 17, 39
Brie 3/14/86 France Nationwide -660 million lb 36, 40
Brie 411/86 France Colorado, Connecticut, Georgia, New -230 Ib 27, 36
Jersey, New York, North Carolina,
Texas, Washington, DC
Soft-ripened: Tourre de 1Aubier and 6/23/86 France New York, Ohio, Pennsylvania Unknown 28,31
Fromage des Burons
Soft-ripened: Tourre de 1Aubier 8/13/86 France California, Illinois, Maine, Massachu- 1056 Ib 32
setts, New Jersey, New York, Oregon
Semisoft: Morbier Rippoz 8118/86 France Illinois, Massachusetts, Michigan -1600 lb 28, 30, 34
Soft-unripened, full fat 4/16/87 France New Jersey, New York, Texas 15 wheels 47, 48
Semisoft 1/27/88 Italy Nationwide 410cartons 59
L. monocytogenes in Fermented Dairy Products 421
Semiseft: LP,mu!ette Dmish Esrem 2/ 11/88 EefiF-Ek CdifGrni2 Unknown 56, 57, 60
Semisoft: LAmulette Danish Esrom 21 12/88 Denmark East, Midwest, North, South -11,500 lb 56, 57, 61
Semisoft: LAmulette Danish Esrom 21 18/88 Denmark Florida, New Jersey, New York, Massa- -1,150 lb 56-58, 62
chusetts
Blue 4/6/88 Denmark California, Florida, Illinois, Massachu- -5,000 Ib 55, 56
setts, Michigan, Minnesota, New Jer-
sey, New York, North Carolina, Ore-
gon, Pennsylvania, Texas
Anari 5/26/89 Cyprus New York 50 cases 68
Anari 7/27/89 Cyprus Illinois, Texas 79 cases 69
Anari 8/9/89 Cyprus New York 80 cases 70
Halloumi 9/15/89 Cyprus Florida, New Jersey, New York 14,400 Ib 75
Italian soft ripened and semisoft 7/27/90 Italy California, Connecticut, New Jersey, Unknown 76
New York, Pennsylvania
Fontina 4/6/93 Sweden California, Connecticut, Maryland, Mas- 85,080 lb 87
sachusetts, Minnesota, New Hamp-
shire, New York, North Carolina,
Pennsylvania, Rhode Island, Washing-
ton state
Limburger 2/29/96 Germany Florida, Indiana, Maine, Massachusetts, 813 lb 100
New Jersey, New York, Ohio, Utah,
Virginia
jarisberg 6i7i96 Norway Alaska, Cdifoniia, Guam, Hawaii, 30,727 lb 99
Idaho, Montana, Nevada, Oregon,
Utah, Washington state
422 R yser
The language used in such press releases also has received considerable attention.
These messages to the public must be firm enough to accomplish the goals of the recall
but not so alarming as to create an undue panic.
After considerable consultation, the governments of France and the United States
reached agreement on a French certification program for soft cheese [49,101]. Under this
program, which began February 15, 1987, cheeses were tested before shipping using meth-
ods that were mutually acceptable by both governments. French cheeses manufactured at
certified factories would be sampled at the 5% level, whereas other French cheeses (and
cheeses manufactured in other countries without certification programs) would be analyzed
at the 20% level. In the event of a Listeria-positive shipment, personnel at the French
cheese factory would be required to investigate the potential source of contamination and
analyze every lot of cheese for listeriae in at least the next 20 consecutive shipments
destined for the United States. Although a positive finding would not automatically result
in suspension of the certified status for a cheese factory under this program, FDA officials
reserved the right to initiate detentions and/or recalls if a product was found to contain
L. monocytogenes. After this certification program was accepted, only one additional recall
involving a French soft-ripened full-fat cheese has been reported (see Table 3).
Other Western European Countries
Despite the adverse publicity that the French cheese industry received throughout 1986
and 1987, it must be recognized that the problem of Listeria-contaminated cheese was
not limited to France. Between October and December of 1986, FDA inspectors isolated
Listeria spp. from 4 of 74 (5.4%) cheeses imported from Italy, two cheeses of which also
contained high levels of phosphatase [45]. After finding similar percentages of positive
samples during January, February, and March of 1987, FDA officials told representatives
of the Italian government either to submit a draft for a certification program (or recommend
an alternate solution) or face a ban on importation of potentially hazardous cheeses into
the United States. As of April 30, 1987, only 13 of all Italian cheese samples analyzed
complied with current FDA safety standards: free of Listeria, phosphatase and enteropath-
ogenic strains of E. coli. Additionally, 144 cheese samples examined as part of an import
alert were suspected of containing L. monocytogenes [44].
After isolating listeriae from Italian Pecorino Romano cheese prepared from goats
milk (see Fig. 1) in June of 1987, [280], the previous import alert was extended to include
L. monocytogenes in Fermented Dairy Products 423
both soft and hard varieties of Italian cheese [50]. (This was the first instance in which L.
monocytogenes was isolated from hard cheese.) Subsequently, the FDA ordered intensified
sampling of soft and hard cheese for the next 2 months [44].
Late in 1987, FDA officials also increased the number of cheeses sampled from
Austria, Denmark, Germany, Italy, and Switzerland as part of the agencys ongoing im-
ported cheese surveillance program (see Fig. 1) [57]. Although this action prompted the
recall of several Danish cheeses in early 1988 (see Table 3), no additional Class I recalls
were issued during the remainder of 1988 for imported cheese contaminated with L. mono-
cytogenes. Heightened concern over the presence of this pathogen in European cheeses
(which sterns from the 1987 cheeseborne outbreak of listeriosis in Switzerland) and subse-
quent initiation of corrective action are probably both responsible for the lack of Class I
recalls issued during the remainder of 1988 and early 1989. However, during the latter
half of 1989, FDA officials issued (a) four separate Class I recalls for Listeria-contami-
nated soft cheeses manufactured in Cyprus (see Table 3) and (b) an import alert for con-
taminated soft and hard cheeses produced by two Italian firm [ 1651. The overall situation
regarding presence of L. monocytogenes in imported cheese has greatly improved since
1986 [77] with only four additional recalls of imported cheese issued since 1990. However,
sporadic detection of listeriae in imported cheeses suggests that limited surveillance of
such products is still necessary to safeguard public health.
Canada
Reports from federal monitoring programs in both Canada and the United States [57,196]
indicate that the incidence of listeriae in Canadian cheese (and nonfermented dairy prod-
ucts as described in Chap. 11) is relatively low. In the only Canadian survey thus far
reported, Farber et al. [ 1661 examined 182 samples of soft and semisoft cheese for listeriae
using the original FDA method. The cheeses analyzed in this survey were produced at
61 different cheese factories, most of which were located in the provinces of Ontario and
Quebec. Although all cheeses examined were Listeria-free, 19 of 79 samples (24%) were
positive for phosphatase, which suggests that these cheeses may have been prepared, at
least in part, from raw milk. The only additional information concerning Canadian cheese
is the unconfirmed isolation of L. monocytogenes from Cheddar and Colby cheeses [207],
424 Ryser
both of which were manufactured from raw milk and held a minimum of 60 days at
2 1.7"C (235F) as required by the Canadian government.
Although the incidence of L. monocytogenes in Canadian-produced cheese appears
to be low, the pathogen has been detected in cheese exported to Canada from several
Western European countries, including Denmark, France, Switzerland, and Germany
[66,196]. In conjunction with the Canadian survey just discussed, Farber et al. [ 1661 also
examined 187 samples of Western European soft and semisoft cheese (98 different brands
from 12 different countries) that were regularly exported to Canada. Three soft and semi-
soft cheeses produced by the same manufacturer in France were positive for Listeria spp.
(Table 4). Two cheeses contained L. monocytogenes alone, whereas the third contained
both L. monocytogenes and L. innocua. In keeping with the previously described 1988
policy regarding foods that have been directly linked to major listeriosis outbreaks, Cana-
dian officials immediately recalled the contaminated cheese. Although some of the tainted
cheese was likely consumed before the recall, no cases of listeriosis linked to consumption
of this cheese were reported.
Despite being labeled as manufactured from pasteurized milk, the three French
cheeses from which listeriae were isolated yielded positive results with the phosphatase
test as did other cheeses imported from Denmark, Finland, and Switzerland. Such findings,
along with unpublished reports of phosphatase in pasteurized dairy products, have raised
serious questions as to the validity of the phosphatase test. Results from one study [244]
demonstrated that certain heat-labile, microbially produced alkaline phosphatases can
mimic the natural phosphatase found in milk and produce false-positive results in the
Scharer test. Hence, the ability of the phosphatase test to determine whether or not a
dairy product such as cheese was made from pasteurized milk (or from pasteurized milk
contaminated with raw milk) needs to be reexamined.
TABLE
4 Incidence of Listeria spp. in Soft/Semisoft European Cheeses Exported
to Canada Between October 1985 and March 1987
France
Beginning in early 1986, sporadic Listeria-contamination problems have been associated
with French soft cheeses exported to the United States and Canada as well as England
[ 1961, Germany [74], the Netherlands [ 1 141, Norway [ 196,3081, Sweden [ 1961, and Aus-
tralia [ 1911. Hence, in an effort to bolster public confidence in the safety of cheeses pro-
duced in France, the French government, in cooperation with the Veterinary Service for
Food Hygience in France, conducted a series of systematic surveys to determine the inci-
dence of Listeria spp. in French cheeses destined for domestic and foreign markets (Table
5) [ 1 19,181 1. Overall, I .34% of predominantly soft, 30-day-old French cheeses examined
during 1986 and 1987 contained detectable levels of both L. monocytogenes and other
Listeria spp., with few differences observed between cheeses destined for domestic or
foreign consumption. It also is noteworthy that comparable levels of contamination were
seen in soft cheeses prepared from raw and pasteurized milk. These findings agree with
those of most other surveys which suggest that soft cheeses are most likely to become
contaminated with L. monocytogenes during the latter stages of manufacture and ripening.
Although L. monocytogenes also was recovered from 10.3% of soft/semisoft French
cheeses marketed in Sweden from 1989 to 1993 [210], most surveys have suggested con-
tamination rates of < 10% with I03 of 2275 (4.5%) French cheeses surveyed (Table 6)
reportedly harboring L. monocytogenes. However, contamination rates of 46.9 and 87.0%
have been reported for soft surface-ripened cheeses prepared from raw milk. In the latter
survey [279], L. monocytogenes populations of 106 CFU/g were detected on the cheese
surface, with serotype 1/2 predominating.
In another French survey [ 1SO], workers at the Veterinary Service for Food Hygiene
recovered I,. monocytogenes as well as L. innocua and other Listeria spp. from 0.3 to
3.5% of cottage, soft-ripened, and semihard cheeses examined (Table 6). However, unlike
the aforementioned survey, comparable contamination rates were observed for soft-rip-
ened cheese prepared from raw and pasteurized milk.
Additional efforts in France have focused on characterizing listeriae isolates from
cheese and other milk products. Listeria spp. recovered from French dairy products during
1986 included L. monocytogenes (370 strains), L. innocua (134 strains), L. seeligeri (17
TABLE
5 Incidence of Listeria spp. in French Cheese Destined for Domestic
(France) and Foreign Markets during 1986 and/or 1987
Number of positive samples (%)
Number of ~
samples Other
Market Type of cheese analyzed L. monocytogenes Listeria spp.
DomesticJ Soft 192 2 (1.0) 3 (1.6)
Other I35 0 I (0.7)
Foreignb Soft (pasteurized milk) 736 1 1 (1.5) 1 0 (1.4)
Soft (raw milk) 355 6 (1.7) 5 (1.4)
Total 1418 19 (1.34) 19 (1.34)
TABLE
6 Incidence of Listeria spp. in Cheeses Manufactured Outside the United States
Number of Other
samples L. monocy- Listeria
Country Type of cheese analyzed togenes L. innocua SPP. Ref.
Europe
Belgium Soft 886 62 (6.9) ND ND 210
Unspecified 929 214 (23) ND ND 151
Unspecified 262 35 (13.4) ND 68 (25.9) 300
Unspecified 37 0 0 0 119
Czechoslovakia Soft ripened 77 6 (7.8) ND ND 229
Sheep's milk 10 0 ND ND 229
Hard 33 0 ND ND 229
Unspecified 24 2 (8.3) ND ND 229
Denmark Soft/semisoft 46 0 ND ND 210
Unspecified 25 8 (32.0) ND 8 (32.0) 119
France Soft ripened (raw milk) 330 3 (0.9) 6 (1.8) 1 (0.3) 180
Soft, surface-ripened (raw milk 23 20 (87.0) ND ND 228
Soft (raw milk) 32 15 (46.9) 13 (40.6) 1 (3.1)" 164
Soft (heat-treated milk) 5 0 2 (40.0) 0 164
Soft ripened (pasteurized milk) 873 12 (1.4) 5 (0.6) 3 (0.3) 180
Soft (pasteurized milk) 32 3 (9.4) 5 (15.6) 0 164
Soft semisoft 174 18 (10.3) ND ND 210
Semihard 289 10 (3.5) 1 (0.3) 1 (0.3) 180
Blue 126 0 0 0 180
Cottage 149 2 (1.3) 0 0 180
Unspecified 242 20 (8.3) ND 61 (25.2) 257
Germany Soft 712 33 (4.6) 58 (S.1) 4 (0.6)" 287
Soft 248 3 (1.2) 16 (6.4) 0 272
Soft 166 7 (4.2) ND ND 290
Soft (raw milk) 22 2 (9.1) 2 (9.1) 0 164
Soft (unripened) 8 0 0 0 302
Soft (mold-ripened) 117 4 (3.4) 5 (4.3) 0 302
L. monocytogenes in Fermented Dairy Products 427
TABLE
6 Continued
Number of Other
samples L. monocy- Listeria
Country Type of cheese analyzed togenes L. innocua SPP- Ref.
TABLE
6 Continued
Number of Other
samples L. monocy- Listeria
Country Type of cheese analyzed togenes L. innocua SPP. Ref.
L. welshimeri.
432 Ryser
strains), and L. ivanovii (1 strain), with 299 of 370 (80%) and 48 of 370 (13%) L. monocy-
togenes strains belonging to serovars I /2 and 4b, respectively. Additional surveys con-
ducted in France [ 1 19,1811 and Belgium [ 1031 from 1985 to 1990 indicated that a dispro-
portionately large number of L. monocytogenes strains isolated from cheese and other
dairy products were serovar 1/2. This situation appears to be reversed in the United States,
with isolates of serovar 4b typically outnumbering those of serovar 1/2.
Phage typing has become a useful means of characterizing particular L. monocyto-
genes strains isolated from dairy products and of trackmg the probable source of foodborne
listeriosis outbreaks. Although only 33 3 % of all L. monocytogenes strains isolated from
French dairy products during 1986 and 1987 were typeable using the available set of
phages, some phage types were unique to particular regions within France [ 1191. In some
instances, excellent correlations were observed between specific phage types and certain
cheese varieties, with some phage types even being specific to a particular dairy. Such
findings have lead to better control of the listeriosis problem within the dairy industry.
The inadvertent isolation of L. monocytogenes from French soft-ripened cheese by
FDA officials in January of 1986 prompted several additional surveys of French cheese
exported to other Western European countries. Working in The Netherlands, Beckers et
al. [ 114,1151 examined 69 samples of French soft cheese (i.e., Brie and Camembert) for
L. monocytogenes using both direct plating and cold enrichment. The pathogen was recov-
ered from 7 of 69 (10.1%) cheeses at levels ranging between 103and 106 CFU/g. Cold
enrichment uncovered three additional cheeses with L. monocytogenes for a total of 10
positive samples. Although all 10 Listeria-positive cheeses were prepared from raw milk,
comparable rates of contamination have frequently been reported for cheese manufactured
from raw and pasteurized milk [ 119,1801.
Germany
As mentioned in Chapter 10, Germany experienced a major outbreak of listeriosis shortly
after World War 11. This outbreak, which may have resulted from consuming contaminated
raw milk, led to an increased interest in listeriosis research, and this in turn prompted
Prof. H. P. R. Seeliger to publish his time-honored monograph Listeriosis in 1961. Dur-
ing the last 40 years, the late Prof. Seeliger emerged as one of the worlds leading au-
thorities on listeriosis. In addition, he operated a listeriosis research center at the In-
stitut fur Hygiene und Mikrobiologie der Universitat Wurzburg to which Listeria isolates
could be sent for biochemical and serological confirmation. Hence, it is not surprising
to learn that the incidence of listeriae in cheese has received considerable attention in
Germany.
Although spared from the heavy economic losses experienced by the United States
and France, Germany and most other European countries have not escaped the Listeria
problem completely unscathed. Despite rigorous testing, Listeria-laden German blue-
veined cheese was recalled from France [196], with a similar recall being issued for sour
milk cheese exported to Canada [66,196] and The Netherlands [196]. Consequently, a
series of Listeria-monitoring programs were introduced for German soft, semisoft, semi-
hard, and hard cheeses as well as cultures, cheese byproducts, and the general environment
within cheese factories. Since 1990, German officials have been enforcing a policy similar
to that adopted in Canada in which only contaminated foods previously associated with
foodborne listeriosis outbreaks are recalled from the marketplace.
Results from various surveys made since 1986 (see Table 6) indicate that 0-9.1 %
L. monocytogenes in Fermented Dairy Products 433
(average of 3.9%) and 0-28.6% (average of 3.6%) of the soft and semisoft cheeses mar-
keted in Germany contained L. monocytogenes, respectively, with the highest incidence
of listeriae generally occurring in smear-ripened varieties. With few exceptions, L. innocua
was isolated more frequently from soft and semisoft cheese than was L. monocytogenes.
Although somewhat similar average percentages were reported for the incidence of L.
monocytogenes in semihard (3.8%) and hard cheese (4.8%), the two hard cheeses that
contained L. monocytogenes were reportedly manufactured from ewes rather than cows
milk. Overall, it appears that the Listeria contamination rate for hard cheeses prepared
from cows milk may still be relatively low, as also was observed in Switzerland (see
Table 6). Hence, these results from Germany generally agree with those from other surveys
in that L. monocytogenes was found more frequently in high-rather than low-moisture
cheese.
Of the three remaining categories of German cheese shown in Table 6, only acid
curd cheese was positive for listeriae. The apparent absence of Listeria spp. from samples
of fresh (i.e., cottage) and processed cheese is not entirely unexpected, since procedures
used to manufacture these cheeses include relatively severe heat treatments. Even if a few
listeriae survived the manufacturing process, most, if not all, of the survivors would have
been sublethally injured during exposure to heat and/or acid and would therefore be unable
to grow in most selective enrichment broths that are commonly used for examining cheese.
In the only other study thus far reported, Weber et al. [302] examined various Ger-
man cheeses, including 11 types manufactured from ewes and goats milk, for listeriae
(Table 7). Although all cheeses prepared from ewes or goats milk were free of L. monocy-
togenes, L. innocua was detected in one sample of fresh goats milk cheese.
Despite the ability of lactating sheep and goats to shed L. monocytogenes in their
milk, as further evidenced by isolation of L. monocytogene:?from approximately 4 of 480
(0.8%) samples of raw goats milk in England [188], relatively few additional studies
have dealt with the incidence of listeriae in ewes and goats milk cheese. Nevertheless,
in addition to the aforementioned survey of German hard cheese produced from ewes
milk [287l, Tham [291] also reported isolating L. monocytogenes from one sample of 8-
week-old goat cheese marketed in Sweden.
TABLE
7 Incidence of Listeria spp. in Domestic and Imported Cheese
Analyzed in Germany Between October 1987 and June 1988
~
Italy
Public health concerns raised in the United States following the isolation of L. monocyto-
genes from imported cheese prompted over 15 surveys of cheeses manufactured in Italy
(see Table 6). Overall, L. monocytogenes was recovered from 196 of 6382 (3.1%) Italian
cheeses surveyed, with this pathogen being most prevalent in Gorgonzola (7.3%), followed
by mozzarella (6.3%) and various soft cheeses (4.2%). In another study, Cantoni et al.
[128] reportedly isolated L. monocytogenes from 14 of 375 (3.7%), 14 of 216 (6.5%), and
5 of 95 (5.3%) samples of Gorgonzola (blue-veined), Tallegio (soft, surface-ripened), and
other Italian cheeses, respectively. Although a follow-up study demonstrated L. monucytu-
genes at levels of <100 to 12,000 CFU/g in Gorgonzola and Taleggio cheese, respectively,
the pathogen was never recovered from 1150 samples of soft, semisoft, semihard, or hard
cheese or from 72 samples of pasta filata-type cheese such as provolone and mozzarella.
When present, however, L. monocytogenes serovar 1 typically predominated [242] as has
been reported for other European cheeses.
Between January 1987 and September 1988, Massa et al. [222] also examined 54
soft rindless (i.e., Mascarpone, mozzarella, Crescenza) and 67 soft thin-rind (i.e., Italico,
Caciotta) cheeses produced by both large and small northern Italian factories for listeriae
and E. coli. Listeria rnonocytogenes was detected in only 2 of 47 (4.2%) thin-rind cheeses
manufactured by one small factory, with core samples from these two positive cheeses
being negative for the pathogen. Although all other thin-rind and rindless soft
cheeses were free of L. monocytogenes, two mozzarella cheeses contained detectable lev-
els of L. innucua. According to these investigators, E. coli populations in these cheeses
ranged from <10 to 8 X 105 CFU/g, with the two L. rnonocytogenes-positive cheeses
containing 2 104E. coli CFU/g. However, since 14 similar Listeria-free cheeses also con-
tained >103 E. coli CFU/g, E. coli is clearly a poor indicator organism for possible pres-
ence of listeriae.
Switzerland
Although several major Western European countries have experienced various degrees of
economic loss from Listeria-contaminated cheese, thus far only Switzerland, France, and
the United States have been forced to deal with major outbreaks of cheeseborne listeriosis.
Well before the 1987 listeriosis outbreak in Switzerland linked to consumption of Vacherin
Mont dOr soft-ripened cheese, Swiss officials began examining various cheeses for lister-
L. monocytogenes in Fermented Dairy Products 435
iae. Although these surveys apparently were prompted by the 1985 listeriosis outbreak in
California, an unusually high incidence of listeriosis in certain areas of Switzerland which
could not yet be explained may have provided added incentive to initiate these surveys.
A two-stage Listeria-monitoring program was later established for cheese and other dairy
products with random testing of 10-g samples obtained at both the factory and retail level
[ 1961. According to the Federal Bureau of Health, such samples must be completely free
of L. monocytogenes before the product is deemed acceptable.
Working in Switzerland, Breer [ 1231 examined 799 domestic and imported cheese
samples for listeriae during the winter of 1985/1986. Various Listeria spp. were detected
in 19.2% of the soft surface-ripened cheeses, all of which were traced to 10 Swiss and a
few foreign manufacturers. During follow-up investigations of these 10 cheese factories
in Switzerland, Listeria spp. were isolated from surfaces of various cheeses and also from
curing and smearing brines, waste-water sinks, and surfaces of wooden boards used in
cheese ripening. In addition, identical serovars of L. monocytogenes (1 /2b and/or 4b) and/
or L. innocua were isolated repeatedly from the same cheese factories. These findings
demonstrate that ample opportunity existed for cheese to become contaminated with lister-
iae during the later stages of manufacture and ripening.
Subsequently, Breer [ 1231 reported that 4.9 and 4.7% of all cheeses sold in Switzer-
land contained L. monocytogenes or L. innocua, respectively (see Table 6). Of equal im-
portance is the fact that both Listeria spp. were isolated more frequently from soft (6.6,
6.3%) than semisoft cheese (1.9,0%) and that neither organism was detected in 88 samples
of hard cheese.
During 1986, Breer [122] also found that 12.9 and 10.0% of soft surface-ripened
cheeses manufactured in Switzerland were contaminated with L. monocytogenes and L.
innocua, respectively (Table 8). The incidence of both Listeria spp. was generally twice
as high in smear- rather than mold-ripened cheese. As in previous studies, the rate of
Listeria contamination was typically independent of the type of milk (raw or pasteurized)
from which smear-ripened cheeses were manufactured.
These Swiss studies, along with several of the aforementioned German surveys,
indicate a greater likelihood of isolating L. monocytogenes and L. innocua from high rather
than low-moisture cheese, with special emphasis on mold- and smear-ripened varieties.
In support of this observation, Bannerman and Bille [ 11I] found that 110 of 449 (24.5%)
rinds from soft cheese produced in Switzerland were contaminated with Listeria spp.,
including a high percentage of samples with L. monocytogenes. During an additional sur-
vey made between October 1986 and September 1987 [41], L. monocytogenes was de-
tected in 4 of 18 (22.2%) and 6 of 67 (8.7%) smear-ripened soft (i.e., Limburger, Romadur,
Muenster, Reblochon) and semisoft (i.e., St. Paulin, Tilsiter, Mutschli, Raclette) cheeses,
respectively, with many cheeses also containing L. innocua. From the apparent widespread
distribution of Listeria within some cheese factory environments, it follows that cheeses
prepared from raw and pasteurized milk are equally likely to contain listeriae. Additional
information concerning the incidence and control of listeriae in dairy factories and other
food processing facilities is given in Chapter 17.
TABLE
9 Incidence of L. monocytogenes in Cheeses Marketed in Sweden from 1989 t o 1993
Type of cheese Type of cheese milk
Country of origin White mold Green/Blue mold Smear-ripened Other Heat-treated Raw
Austria __ 0/1" o/ 1 -
France 15/119 (12.6) 0123 3/18 (16.7) 0/14 5/144 (3.5) 13/30 (43.3)
Germany 0/8 1/19 (5.3) o/ 1 0/3 1/31 (3.2) -
Greece - __ - o/ 1 o/ 1 -
Italy 0/5 0/25 1/2 (50.0) 0/4 1/36 (2.8) -
Netherlands - 0/2 - 0/2 -
Norway o/ 1 - - o/ 1 -
Romania - - o/ 1 o/ 1 -
Spain - o/ 1 - - o/ 1 -
Sweden 0/3 0/13 0/3 0/8 0126 o/ 1
Total 15/154 (9.7) 1/ 122 (0.8) 4/26 (15.4) 0/3 1 7/302 13/31 (41.9)
~~~ ~~
goats milk cheese that contained L. monocytogenes at levels >107CFU/g (see Chap. 10).
During follow-up investigations at the factory [224], the same L. monocytogenes strain
also was isolated from 8 of 11 and 4 of 8 factory and/or retail samples of Halloumi and
Cheddar cheese, respectively, as well as single samples of Gjestost and soft chive cheese.
In addition, L. innocua also was recovered from several samples of Halloumi and Cheddar
cheese. As in the previously described studies by Pini and Gilbert [241] and Massa et al.
[222], no clear relationship was observed between the presence of L. monocytogenes/L.
innocua and coliformslE. coli. According to several additional surveys, some Costa Rican
[2311 and Turkish cheeses [ 1481 contained 104L. monocytogenes and 2 102coliform CFUI
g. Hence coliforms appear to be relatively poor indicators of Listeria contamination.
The Public Health Laboratory Service in London coordinated a large-scale survey in
which various dairy products marketed in England and Wales were sent to 46 laboratories
throughout the country for Listeria testing. Results from this comprehensive survey (see
Table 6) indicated that 8.2, 1.1, 1.5 and 4.1% of the soft-ripened, soft-unripened, hard,
and goats milk cheese manufactured in England and Wales contained L. monocytogenes;
75, 42 and 7 isolates classified as serovar 1/2, 4b and 4, respectively. Among the soft
ripened varieties, 13 cheeses harbored > 103 L. monocytogenes CFU/g with 3 samples
exceeding 105 CFU/g. Of these 13 cheeses, 7 were prepared from raw milk, with only
one of the cheeses being manufactured in the United Kingdom. In contrast, only 2 of 33
cheeses prepared from ewes or goats milk contained >500 L. monocytogenes CFU/g.
Overall, the incidence of this pathogen was similar in imported (7.4%) and UK-produced
cheese. These incidence rates and serovar distribution patterns for L. monocytogenes in
soft-ripened and unripened cheese are generally similar to those observed in most other
European studies [209,2101.
As just suggested, numerous surveys for incidence of listeriae in cheese also have
been completed in many of these aforementioned countries which, with the exception of
Denmark and France, have not experienced major economic problems associated with
Listeria-contaminated cheese. Following the 1986 report of an English woman who con-
tracted listeriosis after consuming French soft cheese [ 1121, two English researchers [241]
examined 45 domestic soft cheeses as well as 177 soft cheeses imported from France,
Italy, Cyprus, Germany, Denmark, and Lebanon for Listeria spp. and E. coli. (Table 10).
Overall, L. monocytogenes was isolated from 2 of 45 (4.4%) English cheeses and 21 of
177 (11.9%) soft cheeses imported from France, Italy, and Cyprus. Populations of L.
monocytogenes in contaminated cheese ranged from <102 to 105 CFU/g, with 9 of 12
French cheeses containing 2 104CFU/g. Despite differences in media and methods used
in various surveys, the contamination rate of 14.1% for soft French cheeses calculated in
this study was close to the 14.5% previously observed for French soft cheese exported to
The Netherlands. As was true for previous surveys of French dairy products, all strains
of L. monocytogenes (except one nontypable strain) were of serovar 1/2 or 4b, with the
former predominating. Listeria innocua, the only other Listeria sp. detected during this
survey, was isolated from 9 of 85 (10.6%), 7 of 44 (15.9%), 2 of 45 (4.4%), and 1 of 6
(16.7%) soft cheeses produced in France, Italy, England, and Denmark, respectively, with
6 of 222 (2.7%) cheeses containing both Listeria spp. Although E. coli populations ex-
ceeded 10 CFU/g in 73 of 222 (32.9%) cheeses examined, no correlation was again ob-
served between the presence of L. monocytogenes or L. innocua and contamination with
E. coli. In fact, E. coli was detected at >10 CFU/g in only 10 of 23 (43.5%) cheeses that
contained the pathogen. In this study, 10 of 23 (43.5%) cheeses contaminated with L.
monocytogenes were prepared from pasteurized milk, whereas 2 and 11 of the remaining
positive cheeses were manufactured from raw milk and milk of undetermined processing,
L. monocytogenes in Fermented Dairy Products 439
TABLE
10 incidence of L. rnonocytogenes and E. coli in Soft Cheese Sampled in
England During 1987
L. monocytogenes
Number of Number of Number of samples
samples positive samples with >I0 E. coli
Country of origin analyzed Level/g CFU/g (%)
England 85 12 (14.1) <102-105 32 (37.6)
France 45 2 (4.4) <:102 14 (31.1)
Italy 44 7 (15.9) < 102-1 0 4 12 (27.3)
Cyprus 20 2 (10.0) <:102 3 (15.0)
West Germany 17 0 ND 9 (52.9)
Denmark 6 0 ND 2 (33.3)
Lebanon 5 0 ND 1 (20.0)
Total 222 23 (10.4) ND- 105 73 (32.9)
ND, not detected.
Source: Adapted from Ref. 241.
respectively. Thus, as in previous studies, the type of milk (i.e., raw or pasteurized) from
which cheese is made appears to be a poor indicator of possible Listeria contamination.
Problems regarding the occasional presence of listeriae in soft cheese also have
surfaced in the Scandinavian countries, with L. monocytogenes being recovered from 0.3%
of Norwegian cheeses [ 1511 and also identified in Danish Esrom and Blue Costello cheese
that was exported to Norway [196,308], Sweden [196], and the United States. Although
four Class I recalls were issued for Danish Esrom and Blue cheese in the United States
(see Table 3), both of these cheeses (-20% of which were contaminated) were on sale
for up to 2 months in Norway before being removed from the market, apparently without
incident [3O5]. Danish officials also took steps to prevent unsold cheese from reaching
consumers and have since developed a Listeria surveillance program [67] similar to that
instituted in the United States with routine testing of various cheeses as well as cheesemak-
ing facilities.
Additional concern over the microbiological safety of various cheeses also led to
isolation of L. monocytogenes serovar 1/2b from two presumably French soft-ripened
cheeses (one prepared from raw milk and one from pasteurized milk) that were exported
to Norway and Sweden [292]. Surface and interior samples from the raw milk cheese
contained 7.5 X 105and 1.0 X 102L. monocytogenes CFU/g, respectively, whereas corre-
sponding samples from the pasteurized milk cheese contained 4.0 X 106 and 1.O X 106
L. monocytogenes CFU/g. The reasons for nonuniform distribution of listeriae in soft-
ripened cheese will be explored in the second half of this chapter. Although cheese pre-
pared from raw milk contained 3 X 106to 7 X 106colifomr CFU/g, coliform tests indi-
cated that the remaining cheese manufactured from pasteurized milk was fit for consump-
tion. These findings reinforce the fact that coliform-free cheese may not necessarily be
free of L. monocytogenes.
Other Countries
Reports of Listeria-contaminated cheese in countries beyond North America and Europe
also are beginning to surface (see Table 6). In 1987, L. monocytogenes was recovered
440 Ryser
from ricotta cheese manufactured in Melbourne, Australia [298]. This event, along with
identification of L. monocytogenes in the same imported brands of Danish blue and French
brie cheese that were recalled in the United States [ 1911 prompted Venables [298] to
determine the incidence of listeriae in Camembert, blue vein, ricotta, cottage, pasta filata,
high-moisture, low-acid, and other cheese varieties manufactured in and around Mel-
bourne. Overall, L. monocytogenes was recovered from 6 of 338 (13%)cheeses produced
by five different manufacturers, with the pathogen identified as being present in pasta
filata (three samples), ricotta (two samples), and shredded (one sample) cheese. One cheese
also contained L. seeligeri. Simultaneous identification of L. monocytogenes in environ-
mental samples from all factories producing Listeria-positive cheese strongly suggests
that these cheeses were contaminated during manufacture and/or ripening. In keeping
with U.S. policies, attempts were made to remove tainted cheese from the marketplace.
Furthermore, after thoroughly cleaning and sanitizing the factory, government officials
required that Listeria-free cheese be produced for 12 consecutive days before being re-
leased to the public. Recent discovery of nonpathogenic listeriae in raw milk from neigh-
boring New Zealand also has prompted authorities in that country to institute a similar
environmental monitoring program for all cheesemakers who export their products.
Information regarding the presence of listeriae in dairy products produced elsewhere
is still reasonably scant, with results from a March 1989 IDF Survey [ 1961 indicating that
L. monocytogenes had not yet been isolated from any dairy products manufactured in
South Africa, Israel or the former Soviet Union. Most cheese-related surveys from other
countries have yielded negative results, with detection of L. monocytogenes being limited
to certain high-moisture domestic cheeses produced in Costa Rica [23 I], Egypt [ 1671, and
Venezuela [ 1471 along with imported (presumably European) soft cheeses marketed in
Japan [232] and the United Arab Emirates 11841.
Given the enormous volume of dairy products exported to other countries and the
fact that L. monocytogenes has been isolated from the natural environment of all seven
continents except Antarctica, it appears that developing countries are unlikely to remain
completely untouched by the problems associated with Listeria-contaminated foods. Con-
sequently, interest in the incidence of listeriae in dairy products and other ready-to-eat
foods will likely continue in the years ahead.
begins as well as afterward as a contaminant of the finished product. Thus far most studies
have dealt with behavior of L. monocytogenes in fermented dairy products inoculated with
the pathogen either before or after fermentation, with relatively few studies addressing
the fate of listeriae in fermented dairy products manufactured from naturally contaminated
raw milk.
Although the extent to which L. monocytogenes survives in cultured dairy products
is partly dictated by whether or not the pathogen enters the product before or after fermen-
tation, viability of Listeria in fermented dairy products, particularly cheese, depends on
the type of product in which the pathogen is found as well as the degree of acid tolerance
possessed by the contaminating strain [ 1721. Hence, to better understand the complex
interactions between the various factors that affect viability of listeriae in cheese (i.e.,
amount, activity and type of starter culture, a,, pH, salt content, temperature during manu-
facture and storage), it is appropriate to begin this section by first discussing the behavior
of L. monocytogenes in milk fermented with niesophilic arid thermophilic lactic starter
cultures. Commingled with this information will be data concerning the fate of this food-
borne pathogen during manufacture and storage of cultured buttermilk, cream, and yogurt.
The viability of L. monocytogenes in coagulants (e.g., calf rennet, microbial rennet, and
bovine-pepsin rennet extract), coloring agents (e.g., annatto), and starter distillates (e.g.,
natural flavor compounds derived from cultured milk) used in cheesemaking also will be
considered before our discussion of natural cheeses and cold-pack cheese food. Two addi-
tional areas of concern to cheesemakers, namely, the fate of L. monocytogenes in whey
and salt brine solutions, will be examined at the end of this chapter.
-1
mm] =0.10/0 = 1.0%
-E
2 1.50
c1
M
tl, 1.25
0
-g,
Y
.d
1 .o
0
a
'c 0.75
;
I
.- 0.50
M
c
cd
v 0.25
crerrioris
S. _
- __ S. lactis
--
-
the starter culture medium was inoculated to contain 103 L. monocytogenes CFU/mL
together with either a 0.25 or 1.0% inoculum of S. lactis or S. cremoris and inoculated
at 21 or 30C for 30 h. Growth of the pathogen was only partially inhibited by S. lactis
and S. cremoris when compared with starter-free controls, with the greatest inhibition
occurring at the higher inoculum level and higher temperature. However, Listeria popula-
tions of 10'- 106and 104-10' CFU/mL developed in samples fermented with S. lactis and S.
cremoris, respectively, when these cultures were ready for use (pH 5.5) after 15 to 18 h of
incubation. Since neither conventional bulk starter technology nor internal pH-controlled
media will completely inhibit this pathogen, manufacturers of cheese and other fermented
dairy products should not discount the starter culture as a possible source for Listeria but
rather should adopt rigorous sanitation standards and Hazard Analysis Critical Control
Point (HACCP) programs to minimize Listeria contamination and potential product loss.
Ultrafiltered milk, a type of concentrated milk sometimes used for commercial man-
ufacture of certain cheeses including mozzarella, ricotta, cottage, and Cheddar, also pos-
sesses a higher buffering capacity than that of unfiltered milk because of higher concentra-
tions of proteins and insoluble salts. Consequently, listeriae may have greater opportunity
to grow in ultrafiltered as opposed to unfiltered milk during fermentation. El-Gazzar et
al. I1581 examined this question by inoculating samples of unfiltered skim milk as well
as retentate (concentrated twofold and fivefold by volume) and permeate from unfiltered
skim milk to contain 103-10' L. monocytogenes CFU/mL together with 107-108 L. lactis
subsp. cremoris CFU/mL. In contrast to the aforementioned studies involving skim milk
and internal pH-controlled bulk starter media, Listeria failed to grow in ultrafiltered milk
containing starter culture, with populations remaining constant in unfiltered slum milk and
decreasing up to 10- and 100-fold in 2-fold retentate and permeate, respectively, after 36
h of incubation at 30C. Increased inactivation of L. monoc-ytogenes in the permeate as
compared with retentate and unfiltered skim milk is again related to the lower buffering
capacity of the permeate which results from ultrafiltration. When these samples were re-
frigerated at 4"C, L. monocytogenes persisted 4-6 weeks in slum milk (pH 4.2), 3-5
weeks in retentate (pH 4.6), and 1 week in permeate (pH 4.1). Thus, fermentation of
ultrafiltered milk at 30C will not guarantee complete inactivation of Listeria even after
the finished product is moved to refrigerated storage.
Cultured Buttermilk
Cultured buttermilk is essentially pasteurized skim milk that has undergone a 12- to 15-
hour fermentation at 20C with S. cremoris or S. lactis (0.5% initial inoculum) and certain
flavor-enhancing lactic acid bacteria such as Leuconostoc cremoris or L. dextranicum.
After fermentation, the final product is packaged and refrigerated until consumed.
As part of a follow-up study [270], all 15-h-old fermented milk samples from the
aforementioned study by Schaack and Marth [269] were stored at 4C and examined for
L. monocytogenes. Survival of the pathogen ranged from an average of 5 weeks in skim
milk fermented at 30C with a 5.0% inoculum of S. cremoris to 12.5 weeks in skim milk
fermented at 21C with a 0.1% inoculum of S. cremoris (Table 11). Similarly, Listeria
viability averaged 2.5- 13.0 weeks in skim milk previously fermented at 30 and 2 1"C with
5.0 and 0.1% S. lactis, respectively. Slower inactivation of the organism in skim milks
fermented at 21 rather than 30C may be related to the rate of acid production during
fermentation, since pH values for fermented milks ranged between 4.3-6.0 and 4.2-4.6
immediately after 15 h of incubation at 2 1 and 3OoC,respectively. The fact that L. monocy-
togenes can survive 10.5 weeks in this refrigerated product (fermented 15 h at 21C with
444 Ryser
are inactivated faster than L. monocytogenes in cultured buttermilk imply that coliform-
free buttermilk may not necessarily be free of listeriae. These findings again emphasize
the importance of good sanitation in producing Listeria-free buttermilk.
Cultured Cream
Unlike cultured buttermilk, far less is known about the viability of L. monocytogenes in
cultured cream. In the only study reported thus far, Stajner et al. [281] manufactured
cultured cream from naturally contaminated raw cows milk containing approximately 5
X 1O5 L. monocytogenes CFU/mL. According to these Yugoslavian authors, viable lister-
iae were detected in the finished product throughout 7 days of storage at 3-5C.
Thermophilic Starter Cu Itures
Practically speaking, thermophilic fermentations used to produce yogurt and certain
cheeses (e.g., Swiss, Parmesan, mozzarella, and Romano) are not normally continued be-
yond 4-6 h. The only two exceptions are in production of Bulgarian buttermilk and acido-
philus milk, which require thermophilic fermentations of 10- 1 2 and 18-24 h, respectively.
Therefore, primary emphasis will be placed on behavior of Listeria during the first 6 h
of fermentation.
In addition to determining the fate of L. monocytogenes in the presence of mesophilic
starter cultures [269], Schaack and Marth [268] also investigated the ability of this organ-
ism to grow during fermentation of skim milk with thermophilic lactic acid bacteria. As
in the previous study, samples of autoclaved skim milk were inoculated to contain -103
L. monocytogenes CFU/mL. After adding 0.1, 1 .O, or 5.0% (Streptococcus thermophilus,
Lactobacillus bulgaricus, or a mixture of the two species, all samples were examined for
numbers of listeriae during 15 h of incubation at 37 and 42C.
Limited growth of L. monocytogenes occurred in all samples, with the organism
generally attaining maximum populations after 6 h of incubation at either temperature
(Fig. 3). At this point, Listeria populations were generally I .O-1.5 orders of magnitude
lower in fermented than in unfermented control samples, as was also reported by Luka-
sova, [212], indicating that growth of the pathogen was markedly suppressed by the ther-
mophilic starter culture, particularly when used at inoculuni levels of 5%. In addition,
greater inhibition of listeriae was consistently observed in rnilks fermented at 42 rather
than 37C.
Listeria monocytogenes behaved similarly in milks fermented with S. thermophilus,
L. bulgaricus, and a mixture of both starter cultures during the initial 6 h of incubation;
however, viability of the pathogen in milks fermented beyond 6 h varied with the species of
lactic acid bacterium used in the fermentation. Although populations of listeriae remained
relatively unchanged in all milks fermented 6-15 h with S. thermophilus (final pH of
4.55-4.90), the pathogen frequently survived only 9-15 h in milks fermented with L.
bulgaricus alone. Rapid inactivation of the pathogen coincided with pH values 1 4 . 0 which
developed in milks fermented 9-15 h with L. bulgaricus. The combination of L. bulgaricus
and S. thermophilus was more inhibitory to Listeria than was S. thermophilus alone but
less inhibitory than was L. bulgaricus alone. Although populations of listeriae failed to
decrease in milks fermented at 37C with the mixed starter culture, some inactivation was
noted in all corresponding samples incubated at 42C. As was true when L. bulgaricus
was used alone, inactivation of listeriae by the mixed starter culture again was most pro-
nounced in samples having pH values 54.0.
Following 15 h of incubation, all fermented milks in this study were stored at 4C
446 Ryser
1.75
-E
h
5 1.50
LL
U
2
1.25
c!
c
.g
- 1.0
*I
1
a
0
a 0.75
3 0.50
B;:[
.
to
0)
2 0.25
...
6 ...
...
...
0
37oc 42C 37C 42C 37C 42OC
S, thermophilug -
L. bulrraricu$ -
L. bulearicus arid
S. IhermoohiluS
and monitored for listeriae by Schaack and Marth [270]. Using S. thermophilus alone,
L. monocytogenes survived 21-32 and 5-15 weeks in milks fermented at 37 and 42OC,
respectively (Table 13). Failure of these milks to attain pH values 14.0 after fermentation
with S. thermophilus helps explain the unusually long survival of listeriae. As expected,
L. bulgaricus was most detrimental to listeriae with the pathogen surviving beyond 15 h
only in samples fermented with the lowest inoculum. Using a 0.1 % L. bulgaricus inoculum
the pathogen was eliminated from milks fermented at 37 and 42C following 7 and 3 days
TABLE
13 Weeks of Survivalaof L. monocytogenes
in Skim Milks Fermented with S. thermophilus or S.
thermophilus + L. bulgaricus (LEST) at 37 and 42C
for 15 h and then Stored at 4C
S. thermophilus LBST
Inoculum (%) 37C 42C 37C 42C
0.1 28.5 15.0 7.5 1.5
1.o 32.0 8.5 1.5 12 h
5 .O 21.0 5 .O 1.o 15 h
aAverage of two trials.
Source: Adapted from Ref. 268.
L. monocytogenes in Fermented Dairy Products 447
to occur after rather than before fermentation. Choi et al. [ 1331 simulated postfermentation
contamination of yogurt by inoculating two commercial brands of plain and vanilla-fla-
-
vored custard-and fluid-style yogurt to contain 104- 1O5 L. monocytogenes CFU/mL.
The pathogen survived an average of 2 1.2-24.7 days in yogurt held at 4C (Table 12),
with most listeriae being inactivated during the first 8-12 days of refrigerated storage.
Khattab et al. [203] reported similar findings with listeriae persisting 18-24 days in experi-
-
mentally produced Egyptian yogurt (pH 3.8-3.9) inoculated to contain 106L. monocyto-
genes CFU/mL. After inoculating various formulations of experimentally produced yogurt
-
to contain 106L. monocytogenes CFU/mL, Griffith and Deibel [ I891 also detected the
pathogen in yogurt samples having a pH value of 4.3 following 28 days of storage at 4C.
Although the type of yogurt had no apparent effect on Listeria survival in these studies,
the point at which yogurt became contaminated greatly influenced survival of listeriae,
with Schaack and Marth [270] showing that the pathogen survived only 1- 12 days when
added to yogurt mix before fermentation.
Siragusa and Johnson [279] conducted a similar study in which three different brands
of commercial, unflavored, low-fat yogurt were inoculated to contain approximately 1O2
and 107L. monocytogenes CFU/g and then stored at 5C. Listeriae survived <3 days in
yogurt inoculated with low levels of the pathogen even though pH values of yogurt were
similar to those in the study by Choi et al. [133] (Table 12). Using the high inoculum,
viable listeriae were found for only 9 days, with populations decreasing approximately
100-fold each after 3 and 6 days of refrigerated storage. Ayre et al. [ 1061 reported similar
results using an L. monocytogenes inoculum level of 107CFU/g. In contrast, Ribeiro and
Carminati [251] examined the effect of yogurt pH on Listeria survival by inoculating
experimentally produced (pH 4.5), commercial fruit (pH 4.05) and plain yogurt (pH 3.76)
to contain -104 L. monocytogenes CFU/mL. Overall, the pathogen survived 2-3 days,
3-4 days, and 12-14 days in refrigerated yogurts having pH values of 3.76, 4.05 and 4.5,
respectively, thereby verifying the impact of pH on Listeria survival. However, Griffith
and Deibel [ 1891 reported that L. monocytogenes populations decreased approximately
four orders of magnitude in artificially acidified (pH 4.2) rather than fermented yogurt
during the first 6 days of storage at 4C. Hence, decreased tenacity of L. monocytogenes
in inoculated yogurt samples in these studies as compared with the work by Choi et al.
[ 1331 again demonstrates that factors other than pH also are contributing to the demise
of Listeria.
Many yogurts and cultured buttermilks marketed today have pH values of approxi-
mately 4.0 and 4.3, respectively. Since large populations of L. monocytogenes were inacti-
vated faster in yogurt than in buttermilk during refrigerated storage, one would expect a
lower incidence of listeriae in commercial yogurt. Evidence from FDA surveys discussed
earlier in this chapter supports this view, since thus far the pathogen has been detected
in commercial buttermilk but not yogurt, with 100 retail and farm-produced samples also
negative for L. monocytogenes in the United Kingdom [202]. However, as was true for
buttermilk, E. coli and Enterobacter aerogenes are also inactivated faster than L. monocy-
togenes in yogurt during refrigerated storage [ 1821. Hence, coliform-free yogurt may not
necessarily be free of Listeria. This is important to remember when results of coliform
tests on these products are interpreted. It is evident from this discussion that good sanita-
tion practices are of utmost importance in producing Listeria-free yogurt, buttermilk, and
other fermented milk products.
Since yogurt is occasionally used as an ingredient in other foods, Sikes [277] investi-
gated the fate of L. monocytogenes in low-moisture (1.9% water), medium-acid (pH 4.9)
L. monocytogenes in Fermented Dairy Products 449
yogurt-based dairy bars supplied to the U.S. military. These bars, which contained -34%
heavy cream, 27.5% yogurt, 27.5% cream cheese, and I I % of other ingredients (i.e., sugar,
sunflower oil, whey), were inoculated to contain approximately 1 X 10' L. monocytogenes
strain Scott A CFU/g and then periodically examined for numbers of listeriae during
extended storage at 25C. Results indicated that Listeria populations decreased only ap-
proximately 100-fold after 40 days of incubation, thus demonstrating the ability of this
organism to persist in low-moisture, medium-acid foods. As will soon be discussed, similar
behavior has been reported for L. monocytogenes in semihard cheeses, such as Cheddar,
Colby, and Gouda, which also have pH values near 5.0.
lowing approximately 5 h of incubation at 42"C, the product is salted (1% NaCl), poured
into muslin bags, and hung in a cooler for 48 h. Gentle mixing to obtain a smooth consis-
tency follows, after which the finished product (pH 3.8) is packaged for sale. These manu-
facturing steps which afford many opportunities for contamination prompted Gohil et al.
[ 1851 to assess the fate of L. rnonocytogenes in labneh as a postfermentation contaminant.
When commercially produced labneh was inoculated to contain l O4 L. rnonocytogenes
CFU/g and stored at 4"C, the pathogen survived 2 and 7 days in product of pH 3.8 and
4.5, respectively, with the addition of 1% NaCl not appreciably altering Listeria survival.
Raising the holding temperature to 10, 20, or 30C led to more rapid demise of listeriae,
with samples generally being free of L. rnonocytogenes after 2-3 days of storage.
Coagulants
To produce cheese curd, milk must first be coagulated or clotted, which can be done either
by acidification or addition of a coagulating enzyme. In the first method, an active lactic
starter culture is used to lower the pH of the milk to 4.6-4.7 (isoelectric point of casein), at
which point the casein micelles in the milk precipitate and form a coagulum. Alternatively,
coagulation is occasionally accomplished by adding food-grade acids directly to milk.
Coagulation of milk by either means of acidification is primarily confined to the manufac-
ture of cottage cheese and a few ethnic varieties of fresh cheese.
The second method, in which a coagulating enzyme is added to destabilize the casein
micelles and clot milk at a near-neutral pH, is used to manufacture virtually all other
types of cheese. Coagulants presently used in cheesemaking include calf rennet extract,
chymosin, bovine pepsin-rennet extract, and microbial rennet. Traditionally, calf rennet
is extracted from the lining of the abomasum (fourth stomach) of suckling calves and
contains two enzymes-pepsin and chymosin (the latter is most important for coagulation
of milk). Shortage of calf rennet following World War I1 led to the use of bovine pepsin-
rennet, an extract obtained from the abomasum of somewhat older calves, that can be
substituted for calf rennet. Increased production costs of both calf rennet and bovine pep-
sin-rennet have in turn prompted development of several rennets of microbial origin. Thus
far enzyme preparations obtained from molds belonging to the genus Mucor (particularly
M. rniehei) have proven to be the most satisfactory substitutes for animal rennet.
Since two of four coagulants used in cheesemaking are of animal origin, and since
these animals sometimes carry L. rnonocytogenes, this pathogen might occasionally appear
L. monocytogenes in Fermented Dairy Products 451
in both crude enzyme preparations and finished coagulant at the time of shipping. Although
microbial rennet should be free of Listeria spp. when manufactured, the presence of lister-
iae within the rennet manufacturing facility or the cheese factory environment could con-
taminate any of these products if mishandled. Given the recovery of an apparent sodium
benzoate-resistant strain of L. monocytogenes from commercially produced calf rennet
extract [ 1571, the possible presence of L. monocytogenes in coagulants should be of con-
cern to cheesemakers, with the International Dairy Federation also contemplating the addi-
tion of rennet to its list of cheesemaking ingredients to be examined for Listeria
spp. [288].
During 1988 and 1989, El-Gazzar and Marth published results from three studies
examining the viability of listeriae in calf [ 1531, bovine pepsin [ 1541, and microbial rennet
[ 1561. In each of these studies, commercially produced, Listeria-free rennet was inoculated
to contain approximately l 03,l 04,l 05,or 1O6 L. monocytogenes CFU/mL and analyzed for
listeriae during 56-70 days of storage at 7C using both direct plating and cold enrichment.
All samples of calf and bovine pepsin-rennet inoculated with the two lowest levels
of Listeria were free of the pathogen after 14-28 days of storage at 7C (Table 14).
Even though 42-56 days of storage were required to eliminate the pathogen from samples
containing initial inocula of approximately 1O5 and 1O6 L. rnonocytogenes CFU/mL, the
reader is reminded that all four inoculum levels used in these studies were many times
greater than levels that might occur naturally in commercially produced coagulants. Hence,
barring contamination in the cheese factory, these findings suggest that calf and bovine
pepsin-rennet are normally held long enough in distribution channels to ensure cheesemak-
ers that both coagulants are Listeria-free. Inactivation of L. monocytogenes in calf rennet
and pepsin-rennet probably results from the combined effects of 5% propylene glycol,
2% sodium propionate, 0.1% (or more) sodium benzoate, 14-21 % salt, and a relatively
low pH of 5.6. Results from several studies assessing the viability of L. monocytogenes
in the presence of benzoic acid and sodium propionate are discussed in Chapter 6.
Unlike calf and bovine pepsin-rennet, more than 70 days of storage were required
to eliminate L. monocytogenes at even the lowest inoculuni level from microbial rennet
(see Table 14). Enhanced survival of listeriae in microbial rennet may be related to the
nature of the coagulant itself as well as the presence of fewer preservatives. Although L.
monocytogenes is unlikely to enter microbial rennet during manufacture, the relatively
high incidence of listeriae in cheese factories may lead to inadvertent contamination of
the coagulant during cheesemaking. Considering the tenacity of L. monocytogenes in mi-
crobial rennet and the long shelf life of this product, it may be prudent for cheesemakers
periodically to verify that the microbial rennet they are using is indeed Listeria-free.
TABLE
14 Survival of L. monocytogenes Strain CA in Three Milk Coagulants Stored a t 7C
~~ ~ ~
Bovine pepsin-rennet extract 9.5 X 103 10 <10 (-) <10 (-) <10 (-) <10 (-) -
2.0 x 10' 30 <10 (-) <10 (-) <10 (-) <10 (-) -
7.5 x 105 1.0 x 103 1.0 x 10' <10 (+) <10 (+) <10 (-> -
1.0 x 106 2.0 x 104 1.0 x to2 <10 (+) <10 (+) <10 (-) -
Microbial rennet 6.0 x 103 1.5 x 103 7.6 X 10' 2.8 X 10' 3.3 x 102 1.2 x 10' 40
7.0 X 103 1.7 X 103 1.0 x 103 2.2 x 102 4.4 x 10' 1.4 X 102 90
2.0 X 10' 1.7 X 10' 2.3 X 10' 2.5 X 10' 2.3 X 10' 1.6 X 10' 8.5 X 103
1.0 x 106 1.5 x 105 4.0 X 10' 3.6 X 10' 9.3 x 10' 7.0 X 10' 9.0 X 10'
mately 103--1O7 L. monocytogenes strain CA/mL and stored at 22C. Regardless of the
initial inoculum level, populations of listeriae immediately decreased 2 4 orders of magni-
tude in all colorants, with the pathogen being completely inactivated immediately after
addition to three of five extracts. The almost instantaneous death of Listeria in these three
colorants was attributed to the presence of propylene glycol and a pH of 13.3 (one extract).
Although Listeria populations of 5800 CFU/mL were observed in the two remaining
colorants immediately after inoculation, with the highest level of listeriae, these samples
were free of the pathogen following 7 days of ambient storage. Overall, these findings
indicate that the length of time that these colorants spend at ambient temperatures during
distribution and before use at the cheese factory is more than adequate to inactivate small
numbers of listeriae that might enter as chance contaminants.
Small levels of starter distillates, that is, mixtures of natural flavor compounds such
as diacetyl obtained by distilling specially cultured milks, are frequently used to enhance
the flavor of cottage and processed cheese as well as ice cream, margarine, butter, yogurt,
snack foods, and certain types of candy. Hence, in connection with the study just described,
El-Gazzar and Marth [ 1551 also examined the fate of listeriae in a commercially available
starter distillate that was inoculated to contain 102- I O6 L. monocytogenes strain CA, Scott
A, or V7 CFU/mL and held at 7C. Overall, strain CA decreased to nondetectable levels
in all samples after 2-7 days of storage depending on the initial inoculuni, whereas 7-
28 days of incubation were required to eliminate strains Scott A and V7 from similar
samples. Therefore, barring inadvertent contamination in the cheese factory, the time in-
volved in shipping and distributing these starter distillates should be more than sufficient
to eliminate any inadvertent Listeria contaminants.
Mold-Ripened Cheeses
Mold-ripened cheeses can be divided into two categories: (a) white mold cheeses, which
are surface-ripened by either Penicillium camemberti, Penicillium caseicolum, or Penicil-
lium candidum (i.e., Brie and Camembert), and (b) blue-mold or blue-veined cheeses in
which ripening results from growth of Penicillium roqueforti or P. glaucum throughout
the cheese (i.e., Roquefort, blue, Gorgonzola). The relatively high moisture content of
these surface-ripened cheeses, along with a nearly neutral pH in fully ripened cheese,
allows rapid growth of L. monocytogenes as well as other foodborne pathogens that would
normally be inhibited in more acidic cheeses. Since mold-ripened cheeses also are highly
susceptible to surface contamination during ripening, it is not surprising that Brie and
Camembert were among the first varieties of cheese in which L. monocytogenes was de-
tected and the behavior of the organism studied.
Camembert Cheese
Pasteurized milk was inoculated to contain approximately 500 L. monocytogenes strains
Scott A, V7, CA, or OH CFU/mL and manufactured into Camembert cheese by Ryser
-
and Marth [ 2601. Following 10 days of storage at I5"C/95% relative humidity (RH) to
permit proper growth of P. camemberti on the cheese surface, all cheeses were wrapped
in foil and ripened at 6C. Wedge (pie-shaped), surface, and interior samples of cheese
were diluted in Tryptose Broth and analyzed for listeriae at appropriate intervals using
both direct plating and cold enrichment.
Populations of L. monocytogenes increased 5- to 10-fold during the first 24 h after
manufacture; however, this increase probably did not result from growth of the organism
454 Ryser
during cheesemaking. Numerous studies have shown that bacterial populations typically
increase 5- to 10-fold during curd formation as a direct result of entrapment of organisms
in curd particles, with the exact level of increase dependent on the moisture content of the
cheese. In all likelihood, L. monocytogenes was similarly concentrated during formation of
Camembert cheese curd. Entrapment of L. monocytogenes in the curd is further supported
by the fact that, in this study, only 1.3% of the original Listeria inoculum in the milk was
lost in the whey. Yousef et al. [308] later demonstrated that the failure to observe L.
monocytogenes population increases of approximately 5- to 10-fold after formation of
Camembert as well as Cheddar and cottage cheese curd was probably related to the method
of sample preparation. Their improved procedure in which curd samples were homoge-
nized in warm (45C) Tryptose Broth containing 2% trisodium citrate was subsequently
used to examine behavior of L. monocytogenes during manufacture and storage of brick
[264], Colby [306], feta [238], blue [237], and Parmesan cheese [307].
During the initial 17 days of cheese ripening, the first 10 days of which occurred
-
at 15"C, populations of three of four L. monocytogenes strains decreased 10- to > 1000-
fold, with lowest numbers generally being observed in surface samples (Fig. 4). On further
ripening at 6"C, all four Listeria strains grew (particularly between 25 and 30 days of
storage) and attained maximum populations of 106- 108CFU/g in wedge and surface sam-
ples from fully ripened cheese; however, maximum listeriae populations were generally
8.0 8.0
s!
3
/
.-c
E
;3
35.0
ULLld 4 . 5
" 0 "5 10 15 20 75 30 35 40 45 50 55 60 65
Days
10- to 100-fold lower in interior samples from the same cheeses. Although growth of L.
monocytogenes clearly paralleled the increase in pH of the cheese during ripening, with
growth usually commencing after the cheese attained a pH value of 5.75-6.25, decreased
viability of three of four Listeria strains in surface samples having pH values of 6.25-
6.50 suggests that factors other than pH, including the presence of potentially inhibitory
surface bacteria and yeasts, may also be involved in controlling growth of this pathogen
in Camembert cheese.
Results from a subsequent study by Ryser and Marth [ 2621 showed greater growth
of L. monoc-ytogenesin filter-sterilized Camembert cheese whey previously cultured with
P. camemberti than in uncultured whey adjusted to pH values of 5.60-6.80 and thus
suggest that P. camemberti is not involved in reducing Listeria populations on the surface
of Camembert cheese. In support of these findings, Geisen et al. [ 1731 also failed to ob-
serve any antilisterial activity among several strains of P. camemberti that were tested
against L. monocytogenes in vitro. However, in the work by Ryser and Marth [260], possi-
ble antilisterial activity from yeasts and non-lactic acid bacteria (i.e., micrococci, coryne-
forms) that are naturally present on the cheese surface during initial stages of ripening
was not precluded.
To simulate contamination of cheese in the ripening room, Ryser and Marth [260]
also inoculated surfaces of 10-day-old wheels of Listeria-free Camembert cheese to con-
tain 2-40 L. monocytogenes (four strains tested separately) CFU/20 cm2.All cheeses were
then ripened at 6C for 60 days, during which time 10-g surface samples were analyzed
for listeriae. Three of four L. monocytogenes strains grew on the surface of the cheese
-
and attained maximum populations of 10'- 105CFU/g (Fig. 5 ) . Although the remaining
Listeria strain failed to grow on the cheese surface after 60 days of storage, the pathogen
was routinely detected throughout the ripening period using cold enrichment. These find-
ings, along with the unfortunate recall of over 300,000 tons of French Brie cheese, stress
the importance of manufacturing surface-ripened soft cheeses from high-quality, Listeria-
free milk and observing good sanitary practices in the ripening room. Nonetheless, even
under ideal manufacturing and ripening conditions, such cheeses may still inadvertently
become contaminated with listeriae.
Since Vacherin Mont d'Or and Brie de Meaux (a raw milk cheese) were directly
involved in two major outbreaks of listeriosis in Europe (see Chap. lO), scientists in both
Europe and North America have been exploring various means of eliminating this patho-
gen from such cheeses that are surface ripened with mold and/or bacteria. Not surprisingly,
Banks [ 1 101 reported that L. monocytogenes grew rapidly in Camembert cheese prepared
from raw milk, reaching a population of 106 CFU/g in fully ripened cheeses. Although
heat treating this Listeria-contaminated milk at subpasteurization temperatures (i.e., 62.8
or 65.6"C) led to markedly lower Listeria populations in the cheese immediately after
manufacture, the pathogen was not completely inactivated, with some growth being re-
ported during 7 weeks of cheese ripening. In another report [ 1451, addition of lactoperoxi-
dase system components to the surface of soft bacterial smear-ripened French cheese con-
taining 102--1 O6 L. monocytogenes CFU/g led to complete inactivation of the pathogen
following 4 days of storage at 15C. In 1989, Hughey et al. [ 1931 showed that lysozyme
was only bacteriostatic to L. monocytogenes in Camembert cheese. Incorporating 2- 10%
carrot juice into homogenized Brie cheese was also effective in minimizing growth of L.
monocytogenes in refrigerated samples [ 1 171. Although several additional reports also
attest to the usefulness of x-ray [ 12I] and gamma irradiation [ 1621 in eliminating high
456 Ryser
./
/
Days
Nisin was most effective when the milk for cheesemaking contained 10' L. monocytogenes
CFU/mL, with the pathogen being absent in 25-g samples even after 6 weeks of ripening.
These findings agree with those of Sulzer and Busse [285], who used a different nisin-
producing strain of L. lactis subsp. lactis. Although the nisin system has limits to pre-
venting regrowth of Listeria during cheese ripening, use of nisin-producing starter cultures
appears to be a relatively simple and effective means of minimizing Listeria growth during
Camembert cheese manufacture provided that the cheese milk is of good hygienic quality
and contains < 103L. monocytogenes CFU/mL.
At least five additional studies have addressed the fate of L. rnonocytogenes during
manufacture and ripening of Camembert cheese [ 108,225,282,284,2891.Regardless of the
method of inoculation (i.e., milk, surface, brine), the basic conclusions from these reports
were similar to those reached by Ryser and Marth [260] years earlier in that (a) L. monocy-
togenes failed to grow during cheesemaking, (b) low numbers of listeriae were recovered
during the period of rapid growth of Penicillium candidum on the cheese surface, (c)
populations of listeriae in cheese increased rapidly after 21 to 28 days of ripening, and
(d) maximum Listeria populations of >106 CFU/g were detected in surface slices from
fully ripened cheese. However, Terplan et al. [289] found that interior samples from 4-
to 56-day-old Camembert cheeses ripened at 5C consistently contained < 100 L. monocy-
togenes CFU/g and never attained pH values >5.6 even after 56 days of ripening. Hence,
under these conditions, growth of the pathogen was probably suppressed or severely re-
tarded. However, some cells may have been sublethally injured during continuous expo-
sure to this acidic environment, which in turn would have probably decreased the number
of Listeria colonies observed on selective plating media. Two of these studies also ad-
dressed the influence of cheese ripening temperature on Listeria growth [ 108,2841. As
expected, growth of L. monocytogenes was enhanced as the cheese storage temperature
was increased from 3 to 15C in response to more rapid ripening of the cheese and a
concomitant increase in pH.
Current evidence suggests that this pathogen behaves similarly in naturally contami-
nated, conimercially produced soft and semisoft mold-ripened cheese. While conducting
a survey of soft/semisoft cheese sold in Canada, Farber et al. [ 1661 discovered eight 4-
month-old French cheeses, presumably of the BrieKamembert variety, that contained
- 104-105L. monocytogenes CFU/g. Following 1 year of continuous storage at 4"C, Liste-
ria populations remained constant in one cheese and decreased only 10- to 100-fold in
the seven remaining cheeses. In view of these results, it is easy to understand why this
pathogen has been most frequently detected in soft/sernisoft cheeses that have been surface
ripened by molds.
Blue Cheese
Blue-veined cheeses such as blue, Roquefort, and Gorgonzola that are ripened internally
and sometimes externally with P. roqueforti or P. glaucum also have been examined for
their ability to support growth and survival of listeriae. Papageorgiou and Marth [237] used
the modified Iowa method to manufacture blue cheese from pasteurized milk inoculated to
contain approximately 1000 L. rnonocytogenes (strains Scott A or CA) CFU/mL. All
cheeses were ripened 84 days at 9-12C (90-98% RH) and then held an additional 36
days at 4C.
Numbers of listeriae increased by an average of 1.50 log,, CFU/g during the first
24 h of manufacture, with increases of 0.62 and 0.71 log,, CFU/g being attributed to
entrapment of the organism within curd particles and growth, respectively. Growth of L.
458 Ryser
monocytogenes occurred primarily during the first 9 h of manufacture and ceased when
the pH of the cheese dropped below 5.0. As expected, somewhat less growth occurred in
two lots of cheese with particularly rapid acid production.
Unlike the behavior of L. monocytogenes in Camembert cheese, the pathogen not
only failed to grow during ripening of blue cheese, but decreased in numbers by two to
nearly three orders of magnitude during the first 56 days of storage at 5C (Fig. 6). These
decreases, which occurred despite favorable pH values that developed during ripening
from growth of P. roqueforti, were most likely caused by formation of free fatty acids
[27 11, with listeriostatic/listeriocidal levels of caproic, caprylic [204], lauric, and other
medium-chain fatty acids [205] produced as by-products of P. roqueforti growth during
ripening of blue-veined cheeses. However, at least eight different P. roqueforti strains can
also produce listeriocins in laboratory media [ 1731. Nonetheless, combined effects of a
relatively high pH and low storage temperature were probably responsible for both Listeria
strains surviving at least 120 days in all lots of blue cheese. Although additional tests
f +T r i a l
__t_ Trial 2
I
._f_ l r i a l 3
?i
5
-* Trial 1
Trial 2
Trial 3
4
0 20 40 60 80 100 120 140
Days
showed that strain Scott A was evenly distributed throughout blocks of 120-day-old blue
cheese, strain CA was far less tolerant to environmental conditions on the cheese surface
and was detected in such samples only after cold enrichment. The lengthy survival of L.
rnonocytogeizes in blue cheese, coupled with the recall of Danish blue cheese and isolation
of L. rnonocytogenes from Italian Gorgonzola cheese, all stress the importance of preparing
blue-mold cheeses from properly pasteurized milk under good hygienic conditions to pre-
vent a possible public health problem involving Listeria.
week-old slice (pH 6.0-6.5), surface (pH 6.5-6.9), and interior (pH 5.6-6.2) samples,
respectively. During the remaining 20 weeks of ripening at 1O"C, numbers of strains OH
and Scott A generally decreased only 1- to 7-fold in mild brick cheese. Both strains also
behaved similarly in aged and Limburger-like cheese during smear development and ex-
tended storage at 10C.
In contrast, strains CA and V7 failed to grow appreciably during or after smear
development, despite favorable pH values of 6.8-7.4 in fully ripened cheese. Although
strains CA and V7 were detected only sporadically in 4- to 26-week-old samples of mild,
aged, and Limburger-like cheese at levels ranging between 2.7 and 4.6 log,, CFU/g, both
strains were routinely recovered from 24- to 26-week-old slice, surface, and interior sam-
ples after cold enrichment. Hence, all four L. monocytogenes strains survived beyond the
normal shelf life of brick cheese. Subsequent experiments [26 11 dealing with possible
antilisterial effects of several sulfur compounds (i.e., methyl sulfide, dimethyl disulfide,
and methyl trisulfide) produced during ripening of brick cheese failed to explain the inabil-
ity of strains CA and V7 to grow in mild, aged, and Limburger-like brick cheese. Addi-
tional possibilities include (a) inhibition of strains CA and V7 by smear-ripening organ-
isms such as Geotrichum candidum [2 151, Lactobacillus plantarum [ 1631, Brevibacterium
linens [2 181, enterococci [ 178,2661, coryneform bacteria [266], and/or certain staphylo-
cocci [266], all of which can reportedly produce bacteriocin-like substances active against
listeriae or (b) heightened sensitivity of these L. monocytogenes strains to the inhibitory
effects of certain listeriocidal fatty acids (i.e., linoleic) and monocglycerides [301] pro-
duced during cheese ripening.
In conjunction with the previously mentioned European study involving Camembert
cheese, Terplan et al. [289] also assessed the behavior of Listeria during manufacture and
ripening of red smear-ripened ("brick-like") cheese. When this cheese was produced from
pasteurized milk inoculated to contain 95 L. monocytogenes CFU/mL, numbers of listeriae
increased 10-fold after the coagulum was cut as a result of entrapment within the curd;
however, no growth of the pathogen was detected during the remainder of cheese manufac-
ture. In fact, unlike the study by Ryser and Marth [264], Listeria populations decreased
10-fold by the time the cheese (pH 4.9) was ready for brining, with the cheese containing
only 9 L. rnonocytogenes CFU/g after brining. Following 8 days of smear development
at 16SC/93% RH, all cheeses were ripened at 5C for an additional 62 days. Listeria
populations close to the cheese surface increased from 2.5 X 10' CFU/g immediately
after smear development to 1.5 X 104 CFU/g in 14-day-old cheese, during which time
the pH increased from 4.9 to 5.1. Continued ripening of brick-like cheese at 5C led to
development of stable L. monocytogenes populations of 2.5 X 105CFU/g in 1-cm thick
surface slices of 42-day-old cheese. However, unlike the study by Ryser and Marth [264],
the pathogen was never detected in interior cheese samples that were more than 4 days
old despite pH values of 5.7 in interior samples of 56-day-old cheese. Although results
of Ryser and Marth [264] suggest that L. monocytogenes should at least have been isolated
occasionally from interior samples of brick-like cheese, the FDA procedure used in this
study was unable to detect listeriae in these samples, possibly because of acid injury which
may have occurred during exposure to pH values 5 5.2 for as long as 6 months.
Ti lsiter Cheese
In 1995, Bachmann and Spahr [ 1071 manufactured Tilsiter cheese (a semifirm, slightly
yellow, smear-ripened variety similar to brick cheese) from milk inoculated to contain
104 L. monocytogenes CFU/mL. Overall, their findings were similar to those observed
L. monocytogenes in Fermented Dairy Products 461
for brick cheese containing strains L. rnonocytogenes CA and V7 [264], with Listeria
populations varying between 103 and 104 CFU/g in Tilsiter cheese during 90 days of
ripening at 10- 13C.
Trappist Cheese
The ability of L. monocytogenes to survive the Trappist cheesemaking process and persist
during 90 days of ripening was investigated by Kovincic et al. [206]. Cheeses were pre-
pared from pasteurized milk inoculated to contain I 02- 1O5 L. rnonocytogenes CFU/mL
and a 1% starter culture inoculum of L. lactis subsp. Zactis and L. Zactis subsp. crernoris.
After rennet coagulation, the curd was cooked at 39C for 45 min, hooped, drained, and
pressed for 10-12 h. Thereafter, the cheese was brine salted (18% NaC1,4 h), dried (5 days
at 16-18Q waxed, and aged at 10C for up to 90 days. Populations of L. rnonocytogenes
increased -10-fold in the finished cheese during the first 30 days of ripening, stabilized
over the next 30 days, and then gradually decreased to levels approaching the original
inoculum after 90 days of storage. Similar results were also obtained when L. rnonocyto-
genes was added to the curd/whey mixture rather than the pasteurized milk during cheese-
making. The limited growth and extended survival of L. rnonocytogenes in Trappist cheese
(pH 4.9, 30% moisture, 1.4% NaCI) is generally similar to what has been observed for
several common varieties of semisoft/hard cheeses to be discussed shortly.
Mozza re1la
The severe heat treatment that the cheese curd receives and the reported thermal tolerance
of listeriae led Buazzi et al. [125] to assess the fate of L. rnonocytogenes during manufac-
ture of mozzarella cheese. When this cheese was prepared from pasteurized milk inocu-
lated to contain 104- 105L. rnonocytogenes strain OH, CA, or V7 CFU/mL, Listeria popu-
lations of 104-105 CFU/mL were reported in the curd after cutting, cooking, and
cheddaring. However, immersing and stretching the curd 3 to 4 min in hot water (77C)
led to the complete demise of the pathogen. Given that the temperature of the curd was
maintained at 65C for at least 2 min and that L. monocytogenes has a reported D-value
of 28.1 s at 65C [214], mozzarella cheese should be Listeria-free even if small numbers
of the pathogen are present in the curd before stretching. These findings are generally
similar to those reported during manufacture of traditional mozzarella cheese from buffalo
milk [299], with L. rnonocytogenes populations decreasing at least 100-fold during brief
stretching of the curd at 90-95C. Although a few survivors remained after curd stretching
and molding, all cheeses prepared from milk inoculated to contain 1O3 and I O5 L. rnonocy-
462 Ryser
togenes CFU/mL were free of this pathogen after 24 and 48 h of refrigerated storage,
respectively.
The heat treatment given to mozzarella cheese curd is clearly sufficient to inactivate
small numbers of listeriae that might be present. However, ample opportunity exists for
postprocessing contamination as evidenced by recent surveys and a Class I recall that was
issued in early 1991 for over 89,000 lb of mozzarella cheese harboring L. monocytogenes.
Stecchini et al. [283] addressed the issue of postprocessing contamination by inoculating
the surface and packaging fluid of mozzarella cheese with L. monocytogenes and then
storing the product at 5C for up to 21 days. Under these conditions, numbers of listeriae
increased about 10,000-fold during 21 days of storage, with inclusion of a crude heat-
treated bacteriocin preparation from L. lactis subsp. lactis yielding final populations only
10-fold lower as compared with untreated controls. Thus, manufacturers of mozzarella
cheese must adhere to good manufacturing and sanitary practices to prevent contamination
and growth of L. monocytogenes to potentially hazardous levels in this product during
storage.
original level in pasteurized milk. However, some Listeria growth was noted during manu-
facture, with populations increasing an additional fourfold in normal Gouda and Maasdam
cheese before brining. Six hours after manufacture, slightly higher Listeria populations
were detected in Gouda cheese of high rather than normal moisture. Although numbers
of listeriae in interior samples from both Gouda and Maasdam cheese remained relatively
constant at 104CFU/g during the first 2 weeks of ripening, the pathogen was not detected
I-
in cheese samples taken at or near the surface. After 6 weeks of ripening, L. monocytogenes
reappeared in surface samples from all cheeses at levels between 102and 104CFU/g. In
contrast, numbers of listeriae in interior samples from 6-week-old cheese were only four-
to eightfold lower than populations in the same cheeses immediately after brining. Al-
though L. monocytogenes survived best in high-moisture Gouda cheese which had a pH
of 6.0, the selective plating medium used in this study, TrypaBavine Nalidixic Acid Serum
Agar, proved to be less than optimal for recovery of stressed or acid-injured listeriae that
probably were present in fully ripened Gouda and Maasdam cheese having pH values of
5.48 and 5.44, respectively.
Colby Cheese
Yousef and Marth [306] prepared Colby cheese from pasteurized milk inoculated to con-
tain 102-103L. monocytogenes (strain V7 or CA) CFU/mL. Following manufacture, all
blocks of cheese were held at 4C for 140 days.
During cheesemaking, most Listeria cells were trapped in curd, with an average of
only 2.4% of the original inoculum escaping in whey. Populations of L. monocytogenes
in cheese increased an average of 1.27 orders of magnitude after pressing-about 29 h
after the start of manufacture (Fig. 7). Since an increase of no more than one order of
magnitude can be attributed to entrapment of listeriae within curd particles after cutting,
these findings suggest that slight growth of the organism did occur, particularly during
"1
Time
the later stages of cheesemaking and pressing. Numbers of both Listeria strains remained
relatively constant in cheese during the first 40 days of ripening, after which populations
decreased almost linearly (Fig. 7). Viability of L. rnonocytogenes was strongly influenced
by moisture content with strain V7 decreasing more than twice as fast in cheese containing
38.5% (D-value of 54 days) rather than 42.3% (D-value of 124 days) moisture, which is
well above the maximum allowable moisture content of 40.0% for Colby cheese.
Behavior of L. rnonocytogenes in cheese of normal moisture content also was strain
dependent, with strain CA being less stable than strain V7. However, strains V7 and CA
were still detected in 140-day-old Colby cheese by direct plating, with cold enrichment
results from a follow-up study [308] indicating that both strains were still viable in 5- to
8-month-old Colby cheese stored at 4C. According to the FDA, Colby and other selected
cheeses can be manufactured from raw or heat-treated (subpasteurization) milk provided
that the finished cheese is held a minimum of 60 days at or above 1.7"C (35F) before
sale in an attempt to eliminate pathogenic microorganisms. These results (and those for
Cheddar cheese to follow) have recently prompted the FDA to reconsider the adequacy
of this aging requirement for cheeses prepared from raw milk.
Cheddar Cheese
Normal stirred-curd Cheddar cheese, which has a moisture content only slightly less than
Colby cheese (i.e., 36-38%), was manufactured by Ryser and Marth [259] from pasteur-
ized whole milk inoculated to contain approximately 5 X 102L. rnonocytogenes (strain
Scott A, V7, or CA) CFU/mL. The resulting 10-lb blocks of cheese were ripened at 6
and 13C and assayed for numbers of listeriae at appropriate intervals.
All curd samples examined during manufacture contained approximately 5 X 102
L. rnonocytugenes CFU/g, which suggests that the organism was only minimally concen-
trated in the curd and failed to grow during cheesemaking. However, since only 6.4% of
the initial Listeria inoculum was recovered in the whey, the expected 10-fold increase
from entrapment of the organism in curd particles probably went unnoticed because of
inadequate sample preparation methods which have since been improved in our laboratory
[308]. Numbers of listeriae increased slightly in cheese during pressing, with all three
-
strains attaining maximum populations of 3.50-3.75 log,, CFU/g after 14-35 days of
ripening at 13 (Fig. 8) and 6C (Fig. 9). This population increase, which was approximately
10-fold higher than that of the original inoculum in milk, probably resulted because of
enhanced recovery of the pathogen from older cheese which was easier to homogenize
rather than from actual growth, as shown by Yousef et al. [308]. After 35 days of storage
at either temperature, Listeria populations in cheese began to decrease, with all cheeses
maintaining pH values of 5.04-5.09 throughout ripening. Strains Scott A, V7, and CA
survived 70-224, 126-196, and 70-126 days in cheese ripened at 13"C, respectively,
whereas the same strains remained viable for 70-154, 126-434, and 70-154 days in
cheese aged at 6C. Thus, except for strain V7, which was still present in one block of
434-day-old cheese at a level of 30 CFU/g, the remaining two strains survived equally
well in cheeses ripened at either temperature. These findings suggest different acid toler-
ances among the L. rnonocytogenes strains tested, as has also been reported by Gahan et
al. [172]. Additional experiments with strains V7 and CA demonstrated that L. rnonocyto-
genes was uniformly distributed in Cheddar cheese during at least the first 98 days of
ripening at 6C. Working in England, Banks [ 1101 also reported that L. rnonocytogenes
persisted 8-9 months and up to 7 months in Cheddar cheese prepared from Listeria-
contaminated raw and subpasteurized (i.e., 62.8 or 65.6"C) milk, respectively. Hence,
L. monocytogenes in Fermented Dairy Products 465
4.0 r
2.01
-- * Trial 4
-A Trial 5
\
--4 Trial 6
b
\
\
\
\
1 .o L
t
0 0
A A2 A A
0 0
these data provide some of the strongest evidence for the inadequacy of the 60 day/? 1.7"C
minimum holding period for cheeses manufactured from raw milk.
Several subsequent studies examined the effect of Cheddar cheese compositional
changes on Listeria survival during cheese ripening. Mehta and Tatini [226] assessed the
behavior of L. monocytogenes strains Scott A and V7 in stirred-curd Cheddar cheese
containing 1.3 or 2.5% NaCl or an equal molar mixture of NaCl and KCl. Lowering the
level of NaCl enhanced destruction of Listeria, with 1.3% NaC1, 2.5% Na/KCl, and 2.5%
NaCl decreasing populations 4.3-, 2.3- and 0.4-orders of magnitude in cheese, respec-
tively, after 10 weeks of aging at 7C. Thus, in addition to being healthier, low-sodium
Cheddar cheese appears to be safer in regard to listeriae. When these same investigators
[227] prepared stirred-curd Cheddar cheese from whole milk and reduced fat milk (1.55
or 2.0% milkfat), L. monocytogenes strains Scott A and V7 persisted in the finished cheese
during 20 months of storage at 7OC, with no survival differences being observed between
full fat (28.9% fat) and reduced fat (20.7%) cheese. However, Listeria survival in Cheddar
cheese is strongly influenced by milk fat composition and release of free fatty acids during
cheese ripening. Schaffer et al. [271] increased the levels of both long-chain (c18, c18.2)
and unsaturated fatty acids in milk by feeding cows a diet of extruded soybeans or sun-
flower seeds and then using this milk to manufacture stirred-curd Cheddar cheese con-
taining L. monocytogenes strains Scott A and V7 as previously described. During manufac-
ture, numbers of listeriae increased 1.O- 1.5 orders of magnitude as previously reported
466 Ryser
-- Trial 4
+Trial 5
-4 Trial 6
'w-- <
\
\
\
\
I
L
by Ryser and Marth [259]. More important, after 120 days of ripening at 7C L. monocyto-
genes populations were five- to eight orders of magnitude lower in cheeses prepared from
modified fat milk as compared with unmodified milk. Although some inconsistences were
noted in performance of sunflower-and soybean-modified milk, inactivation of L. monocy-
togenes always occurred most rapidly in cheeses containing the highest levels of free fatty
acids, with oleic, linoleic, lauric, and myristic acids shown to be major contributors to
Listeria destruction during cheese ripening.
Swiss Cheese
Unlike the aforementioned cheeses, manufacture and ripening of Swiss cheese involves
several decidedly different steps, including cooking of the curd at 50-53C and ripening
the finished cheese at an elevated temperature for "eye" development. These observations
prompted Buazzi et al.[ 1261 to examine the fate of L. monocytogenes during manufacture
and ripening of Swiss cheese. When rindless Swiss cheese was prepared from pasteurized
milk inoculated to contain 104-105 L. monocytogenes strain V7, CA, or OH CFU/mL,
the pathogen was generally unable to grow during cheesemaking, with populations increas-
ing 43% during the early stages of cooking owing to physical concentration and curd
shrinkage. Thereafter, about 57% of the population in the curd was inactivated after 30-
40 min of cooking at 50C. After pressing, the curd contained 50% fewer listeriae, with
this population decreasing most sharply after 30 h of brining at 7C. Storing the finished
cheese (pH 5.2-5.4) 10 days at 7C reduced the Listeria population to very low numbers.
Complete inactivation of the pathogen occurred after 66-80 days of ripening at 24"C,
L. monocytogenes in Fermented Dairy Products 467
E
107
Manufacture
106
lofi
104
-
-
Batch # 1
103 --.I Batch # 3
Batch H6
-
102 -
--
-
-
-
10 J L
0 1 2 3 0 40 80 120
Days
Hours
ples from 35-day-old cheeses. Compositional analysis of these cheeses suggested that the
amount of moisture lost after 35 days of ripening (a, 0.95-0.96) may have offset the
benefit for Listeria growth caused by the increase in pH.
Hispanic Cheeses
Traditional Hispanic-type cheeses comprise a wide range of white cheeses produced in
Mexico and in Central and South America. Some of the most popular varieties, including
Queso Blanco, Queso Fresco, and Queso de Puna, are high-moisture fresh cheeses con-
sumed shortly after manufacture, whereas others, such as Queso Anejo, Queso de Bola,
Queso de Crema, Queso de 10s Ibores, and Queso de Prensa, are lower in moisture and
undergo various degrees of aging. Although 30 min of heating at 80-85C is more than
adequate to inactivate L. monocytogenes during manufacture of Queso Blanco cheese
[ 1791, typical production practices for Hispanic-type cheeses involve extensive curd mani-
pulations, including hand stirring, salting, and molding, any of which can easily lead to
L. monocytogenes in Fermented Dairy Products 469
product contamination. Queso Blanco and Queso Fresco cheese pose a particular threat
to the industry given the involvement of these cheeses in the 1985 listeriosis outbreak in
California.
Queso Blanco Cheese
In 1995, Glass et al. [179] reported on the behavior of L. monocytogenes in starter culture-
free Queso Blanco cheese containing citric, malic, or acetic acid as acidulants and ALTA
(a commercial bacteriocin preparation resembling pediocin AcH) as an antilisterial agent.
After the finished product (pH 5.2) was inoculated to contain 106L. monocytogenes CFU/
g, populations increased about 10-fold in cheeses containing citric or malic acid during
42 days of storage at 4C, whereas numbers of listeriae decreased slightly in cheeses
prepared with acetic acid. These findings are consistent with those of other investigators
who used various laboratory media acidified with malic, citric, or acetic acid (see Chap. 6).
Addition of 0.6% ALTA to these cheeses yielded slightly lower Listeria counts as com-
pared with cheeses without ALTA. Using an L. monocytogenes inoculum level of 102
CFU/g, these workers concluded that acetic acid was significantly more effective than
malic or citric acid in reducing numbers of L. monocytogenes in Queso Blanco cheese
and that addition of ALTA provided added protection against this pathogen. Similar bene-
fits also were reported when Queso Blanco cheese was prepared using a nisin-producing
starter culture, with L. monocytogenes populations being about 1000-fold lower in such
cheeses (pH 5.3) after 21 days of storage at 4 or 12C than in nisin-free controls [144].
Although direct addition of Nisaplin (1000 AU/mL) to the cheese milk yielded Listeria
populations 100-fold lower in 1-day-old cheeses than in Nisaplin-free controls, the patho-
gen recovered to control levels within 21 days at 12C. Hence, incorporating a nisin-
producing starter culture was superior to direct addition of Nisaplin for minimizing sur-
vival of L. monocytogenes in Queso Blanco cheese during storage.
Queso de 10s lbores Cheese
The fate of L. monocytogenes in Queso de 10s Ibores cheese (a hard, ripened cheese of
pH -5) also was indirectly determined by Mas and Gonzalez-Crespo [219] using commer-
cially available cheeses of various ages. Overall, detecting Listeria spp. in 5 of 10, 2 of
10, and 1 of 10 cheeses that had been aged for 7, 30, and 60 days, respectively, suggests
that this hard, low-moisture cheese will not support long-term survival of listeriae.
Pickled Cheeses
The terms pickled and white-brined are often used to describe a group of soft/semisoft,
white curd cheeses to which large quantities of salt are added as a preservative. Cheeses
belonging to this group are principally manufactured in countries bordering the Mediterra-
nean Sea and include such varieties as feta (Greece), Turkish white-brined cheese (Tur-
key), Teleme (Bulgaria), Domiati (Egypt), and Kareish (Egypt). Some of these cheeses
are frequently prepared from ewes, goats, or buffalos milk. Depending on the cheese
variety, salt either can be added directly to the milk or curd, or the finished cheese can
be stored in salt brine, salted whey, salted skim milk, or dry salt. The extreme tolerance
of L. monocytogenes to high concentrations of salt, along with the organisms ability to
grow at refrigeration temperature, has made these cheeses of particular interest to food
microbiologists working with Listeria.
470 Ryser
- 7
Average pIl
%I
. . 6
PH
- s
Cp- Trial 1
- 4
&-- Trial 2
Trial 3
' I . , ' I t , , , , , I
- 3
-20 0 20 40 60 80 100 120 I40
Hours
6% brine solution. Both salt brines in which feta cheese was ripened and/or stored were
positive for listeriae (these details appear in our discussion of brine solutions at the end
of this chapter). Although all feta cheeses older than 5 days maintained a pH of 4.3, both
L. monocytogenes strains survived >90 days in finished cheese stored at 4C (Fig. 12).
However, differences between the two Listeria strains were noted, with populations of
strains Scott A and CA decreasing 1.28 and 3.07 log,, CFU/g in 90-day-old as compared
with 2-day-old feta cheese, respectively. Sarumehmetoglu and Kaymaz [267] reported
that L. monocytogenes behaved similarly in Turkish white-brined cheese prepared from
artificially contaminated raw milk, with numbers of listeriae generally decreasing < 100-
fold in the finished cheese during 90 days of refrigerated storage. Although feta and Turk-
ish white-brined cheese can be prepared from raw milk, ripening such cheese at or above
1.7"C for 60 days will not in any way guarantee that the final product is Listeria-free with
long-term survival of this pathogen highly probable. Hence, it would appear prudent to
manufacture these cheeses only from pasteurized milk under good hygienic conditions to
decrease the chance of a public health problem involving Listeria.
Domiati Cheese
Domiati cheese, a popular fresh white-brined cheese most commonly consumed in Egypt
and other parts of the Middle East, was prepared by Tawfik [286] using a 1 : 1 mixture
of pasteurized cow: buffalo milk to which 7.5% NaCl, 0.5% Lactobacillus casei, and 106
L. monocytogenes were added. Listeria populations increased approximately 10-fold in
the finished cheese as a result of entrapment within the curd and then decreased over time,
with the pathogen surviving 4-8 weeks when the cheese was stored in salted whey at
20-25C. Similar findings were reported by Ahmed et al. [ 5 ] , with L. monocytogenes
strain V7 being inactivated in Domiati cheeses (pH 4.5-5.5) containing 5 and 10% NaCl
Trial 1
&- Trial 2
Trial 3
2 1 8 I I I I I I I I I I 1
0 20 40 60 80 100
Days
Twenty-one years later, Katic [200] prepared a similar white-brined cheese from
artificially contaminated milk containing 0.8% starter culture and found that after 40 days
of storage at 4C, L. monocytogenes populations had increased 100- and 43-fold in cheeses
stored in 10 and 16% brine, respectively. In contrast, numbers of listeriae increased only
4- and 12-fold in identical cheeses that were stored in 10 and 16% brine solutions at 8C,
respectively. Thus, given the same brining conditions, higher storage temperatures were,
as expected, more detrimental to Listeria survival.
-0 L . m o n o c y t o g e n e s in cheese VI
-4 L . m o n o c y f o g e n e s in cheese V
-A Total aerobic count in cheese V
;- ;
9.0 - 4 Total aerobic count in cheese VI
I
Added I, m o n o c y t o g e n e s per ml milk
-
8.0 *
-L-
7.0 .
6.0 .
5.0 -
L
I I I 1 I I 1 I I I
0 2 4 6 8 10 12 14 16 18
Weeks
rnonocytogenes populations increasing 10- and 10,000-fold in cheeses prepared with and
without starter culture, respectively, after 4 weeks of ripening at 4C.
As you will recall from Chapter 10, a middle-aged English woman developed liste-
rial meningitis in February of 1988 after consuming 2-3 oz of commercially produced
Anari-type goats milk cheese containing 107L. rnonocytogenes CFU/g [63]. During a
series of follow-up investigations [224], L. monocytogenes populations of < 10 CFU/g
were discovered in one 2- to 3-day-old Anari cheese and in three 2- to 3-day-old Halloumi
cheeses produced by the same manufacturer. After these naturally contaminated cheeses
were stored at 4C for 4-5 weeks Listeria populations as high as 8 X 104 and 1 X 106
CFU/g developed in Anari (pH 5-6) and Halloumi cheese (pH 6), respectively. Assuming
a lag time of zero and an original L. rnonocytogenes population of 1-9 CFU/g, these
authors calculated generation times of 47-56 and 32-37 h for this pathogen in Anari and
Halloumi cheese, respectively. Both of these generation times are similar to those previ-
ously reported for L. rnonocytogenes in refrigerated fluid milks (see Chapter 1 1, Table 7).
Assuming that these cheeses were on sale for up to 3 months after distribution, potentially
hazardous levels of listeriae easily could have developed in such products during retail
storage. Hence, as with cheeses prepared from cows milk, it is imperative that goats
milk and ewes milk cheeses also be manufactured from high-quality pasteurized milk
under the best possible hygienic conditions.
L. monocytogenes was not detected in samples of curd or whey that were directly plated
on McBride Listeria Agar. However, following cold enrichment in Tryptose Broth, L.
monocytogenes was detected in four of eight, two of eight, one of eight, and two of eight
samples of cooked curd, whey, wash water, and washed curd, respectively, which suggests
that some Listeria cells were only sublethally injured during cooking of the curd at pH
4.6-4.7. As indicated in Chapter 7, such injury may preclude growth on McBride Listeria
Agar which contains both lithium chloride and phenylethanol as selective agents.
Examination of the finished product indicated that L. monocytogenes survived in
both creamed and uncreamed cottage cheese at levels generally <100 CFU/g during 28
days of refrigerated storage. Although there was no evidence for growth of listeriae in
either cheese during storage, probably because of pH values generally <5.5, the pathogen
was recovered more frequently and at higher numbers in creamed rather than uncreamed
cottage cheese. Higher pH values in 3-day-old creamed (pH 5.32-5.45) rather than un-
creamed (pH 5.12-5.22) cottage cheese may have been responsible for increasing the
repair rate of injured cells, thereby increasing recovery of listeriae.
Although behavior of L. monocytogenes in cheese failed to gain widespread attention
until 1985, a search of the scientific literature has uncovered an earlier study by Stajner
et al. [281] which examined the viability of Listeria in unsalted small-curd skim milk
cheese (similar to uncreamed cottage cheese) manufactured from naturally infected milk
containing approximately 5 X 105L. monocytogenes CFU/mL. Results from these Yugo-
slavian investigators support the findings of Ryser and Marth [265] in that the pathogen
survived at least 7 days in finished cheese (pH 4.55-4.75) stored at 3-5C.
More recently, El-Shenawy and Marth [160] studied the behavior of listeriae in
cottage cheese prepared from pasteurized skim milk that was inoculated to contain 106
L. monocytogenes CFU/mL and then coagulated over a period of 3 h using hydrochloric
acid, gluconic acid, or bovine rennet rather than a lactic acid bacteria starter culture during
which time the temperature of the milk was gradually increased from 2 to 32C. The
resulting coagulum was then cut and cooked using the aforementioned procedure of Ryser
et al. [265].
As might be expected, acidification of the milk to a pH of 4.7-4.8 followed by
heating was again detrimental to survival of listeriae during manufacture of cottage cheese.
Overall, L. monocytogenes populations decreased -4.5 and >6.0 orders of magnitude in
fully cooked (57.2"C/30 min) curd obtained by adding hydrochloric and gluconic acid,
respectively (Fig. 14). However, using cold enrichment, Listeria was recovered from sam-
ples of fully cooked gluconic acid curd. Numbers of listeriae decreased faster in acidified
whey than curd, with cold enrichment results indicating that the pathogen was eliminated
from gluconic but not hydrochloric acid whey after 30 min of cooking at 57.2"C. Nonethe-
less, direct acidification of milk (pH 4.7-4.8) for cottage cheesemaking followed by cook-
ing the resultant curd at 572C for 30 min should be more than sufficient to eliminate
expected numbers of listeriae (<10 CFU/mL) that might inadvertently enter pasteurized
milk as postprocessing contaminants.
In contrast to acid curd/whey, populations of listeriae in freshly cut rennet curd and
whey were virtually identical to those initially observed in milk (Fig. 14). Furthermore,
slight increases in numbers of listeriae were noted midway through manufacture with fully
cooked rennet curd (pH 6.6) and whey still containing 104 and 103 L. monocytogenes
CFU/g or CFU/mL, respectively. Thus, listericidal effects associated with cooking were
greatly enhanced under acidic conditions. Subsequent experiments with selective plating
media confirmed that substantial numbers of L. monocytogenes cells were sublethally in-
L. monocytogenes in Fermented Dairy Products 477
3,
0 - Rennet Curd
0 - -- Rennet Whey
2, A HC1 Curd
A - - HC1 Whey
I- GACurd
1,
0 - - GA Whey
0 1 1 I I
A B C D E F
jured during manufacture of cottage cheese, as was suggested by Ryser and Marth [265]
five years earlier. Sublethal injury was far more evident in whey rather than curd samples,
probably because curd afforded some thermal protection to listeriae. Not surprisingly, the
degree of sublethal injury was also closely related to coagulant type (i.e., acidity) and
cooking temperature, with less injury being observed in rennet rather than acid curd/whey
and partially rather than fully cooked samples of curd and whey. Heat alone was primarily
responsible for rennet-associated injury, whereas the combined effects of heat and acid
led to injury of listeriae in acid curd and whey.
With the exception of cottage cheese prepared from milk acidified with gluconic
acid, both of these studies demonstrated limited survival of L. monocytogenes in cottage
cheese. However, the fact that the pathogen failed to grow in the product and decreased
drastically in numbers during manufacture suggests that cottage cheese poses far less of
a public health threat than do those varieties that are surface ripened with molds or bacteria.
Lower health risks associated with consumption of cottage cheese are also supported by the
extremely low incidence of L. monocytogenes in commercially produced cottage cheese
examined in American and European surveys.
The likelihood of L. monocytogenes entering cottage cheese during creaming and/
or packaging of the product is far greater than having listeriae present in pasteurized milk
at sufficiently high levels to survive in the cooked curd. Although most recently published
work has addressed the fate of L. monocytogenes in cottage cheese as a postmanufacturing
contaminant, results from these efforts have been somewhat conflicting. Based on the
findings of three studies conducted in the United States [240] and England [169,192],L.
478 Ryser
Whey Cheeses
A few cheeses such as ricotta, Broccio, and Ricotone are prepared from sweet whey de-
rived from the manufacture of mozzarella, Cheddar, Swiss, Tilsiter, and feta cheese. Manu-
facture of these whey cheeses is based on the direct acidification of whey, whey/milk,
and whey/cream mixtures to pH 5.9-6.0 using food-grade acids (i.e., citric, acetic), lactic
starter cultures, or acid whey powder followed by cooking at 180-190F to precipitate
the whey protein. The fine precipitate which eventually rises to the vat surface is then
removed, drained, matted, and either marketed as whey cheese or a dairy ingredient. Sev-
eral additional extremely low-moisture ( I 3 to 18%) whey cheeses, including Gjetost, My-
sost, and Gudbrandsdalsost, are unique to Norway and are prepared by thermally concen-
trating and then boiling a blend of goat's and cow's milk whey until the mixture carmelizes
and becomes viscous. This plastic mass is then cooled, extruded, and cut into extremely
dense blocks for marketing.
As was true for mozzarella cheese, L. monocytogenes will be completely inactivated
during manufacture of these whey cheeses. However, the potential still exists for contami-
nation during packaging as is evidenced by at least one Class I recall of ricotta cheese in
July of 1991 and the report of a small cluster of listeriosis cases traced to Anari whey
cheese produced in Cyprus (see Chap. 10).Consequently, Papageorgiou et al. [239] exam-
ined the fate of L. monocytogenes as a postprocessing contaminant in Greek Myzithra
(identical to Anari cheese), Anthotyros, and Manouri cheese, all of which are starter cul-
ture-free, soft (50-70% moisture), low-acid (pH 6.0-6.5) whey cheeses. Immediately after
commercial manufacture, these cheeses were inoculated to contain -500 L. monocyto-
480 Ryser
genes strain Scott A or CA CFU/g. Regardless of cheese type or Listeria strain, the patho-
gen grew rapidly and attained maximum populations of 107-108CFU/g after 24-30 days,
5-12 days, and 56-72 h of storage at 5, 12, and 22OC, respectively. Consequently, strict
hygienic practices must be followed to prevent Listeria contamination during packaging,
with postpackaging pasteurization of the product also being recommended as an added
safeguard.
Cold-Pack C h e e s e Food
Unlike the natural cheeses discussed thus far, cold-pack cheese food is typically prepared
by comminuting and blending aged Cheddar cheese (or another variety) with nonfat dry
milk, dried whey, water, cream, plastic cream (composition similar to butter), salt, acidu-
lants (i.e., lactic and/or acetic acid), preservatives (i.e., potassium sorbate and/or sodium
propionate), and other optional ingredients into a homogeneous mass without heating.
Since all of the dairy ingredients and some of the optional ingredients used in manufactur-
ing cold-pack cheese food can potentially harbor L. monocytogenes, Ryser and Marth
[263] investigated the behavior of this pathogen in nine different formulations of cold-
pack cheese food inoculated to contain approximately 500 L. monocytogenes strains Scott
A, V7, CA, or OH CFU/g.
During 182 days of storage at 4OC, populations of all four Listeria strains decreased
less than 10-fold in nonacidified, (pH 5.20) preservative-free cheese food, with the patho-
gen surviving throughout the product's entire 6-month shelf life (Fig. 15).In sharp contrast
to these findings, addition of preservatives with or without acidifying agents led to the
--a Scott A
1.0 - --0 v7
- * CA
-5 -
--+ OH
10
eventual demise of listeriae in cheese food stored at 4C (Fig. 16). In nonacidified cheese
food (pH 5.20) preserved with 0.3% sodium propionate, L. monocytogenes survived an
average of 142 days as compared with 118, 103, and 98 days in the same product adjusted
to pH 5.0-5.1 with lactic, acetic, and lactic plus acetic acid, respectively. Using 0.3%
sorbic acid in place of sodium propionate, the pathogen survived an average of 130 days
in nonacidified cheese food (pH 5.45) as compared with 112, 93, and 74 days in cheese
food acidified to pH 5.0-5.1 with lactic, lactic plus acetic, and acetic acids, respectively.
Thus, sorbic acid was consistently more antagonistic to listeriae than sodium propionate.
In addition, antilisterial effects of both preservatives were more pronounced in cheese food
acidified with acetic rather than lactic acid. Since organic acids are far more bactericidal in
the undissociated than dissociated state, increased inactivation of listeriae in the presence
of acetic acid probably resulted from the higher proportion of undissociated acetic (-36%)
rather than lactic acid (-5.9%) in cheese food acidified to pH 5.0-5.1. These findings
indicate that it would be prudent to consider (a) adding preservatives, particularly sorbic
acid, to cold-pack cheese food and (b) reducing the pH of the product to 5.0 by adding
small amounts of lactic and/or acetic acid to minimize L. rnonocytogenes survival in the
finished product. Additional information on conditions leading to inhibition and/or inacti-
vation of this pathogen by sorbic, propionic, lactic, and acetic acids can be found in
Chapter 6.
150 -
140 -
S SorbicAcid
130 - p NaPropionata
A Acetic Acid
120 - L LacticAcid
I I
01
110 -
6
h
100-
? -
'$
90
vI 80 -
70 -
60 -
50 I
P S P+L S+L P+A P+L+A S+L+A S4A
Additive
TABLE
15 Behavior of L. monocytogenes During Cheese Ripening as Affected by Cheese Composition
Estimated
salt in Ripening Log,, of L. monocytogenes CFU/g
PH temp. Survival
Moisture Estimated water phase
Cheese @) a, (%) Initial" Final ("C) Initiala Maximum Final (days) Ref.
Camembert 54.4 0.975 4.72 4.6 7.5 15/6 3.1-3.6 6.7-7.5 6.7-7.5 65 260
Blue 38.9 0.950 11.52 4.6 6.3 9-12/4 4.0-5.0 4.0-5.0 1.0-2.3 120 237
Brick 43.0 0.990 1.89 5.3 7.3 15/10 3.0-4.7 4.6-6.7 2.7-6.1 168 264
Feta 54.7 0.975 4.57 4.7 4.4 22/4 5.2-6.2 5.7-6.2 2.8-4.6 90 238
Cheddar 37.2 0.975 4.61 5.1 5.1 6 2.5-3.2 2.6-3.8 0-1.5 70-434 259
Cheddar 37.2 0.975 4.61 5.1 5.1 13 2.6-3.4 3.0-3.7 0 70-224 259
Colby 40.0 0.975 3.91 5.1 5.1 4 3.5-4.5 3.6-4.6 2.3-4.1 112-140 306
Parmesan 32.0 0.935 4.96 5.1 5.1 13 3.3-4.3 3.3-4.3 1.0-1.3 21-1 12 307
Hard Italian NR' 0.950 2.12d 5.3 5.7 4 4.5-5.1 4.5-5.6 2.0 35 I35
Cold-pack 41.4 0.975 4.90 5.3 5.1 4 2.4-2.8 2.4-2.8 1.1-2.0 180 263
cheese foodb
-
useful in predicting whether or not this pathogen will grow in other cheeses having similar
microbiological, biochemical, and physical characteristics.
In addition to being present in milk at the time of cheesemaking, Listeria also can
easily contaminate the finished cheese during packaging, ripening, and storage. Conse-
quently, Genegeorgis et al. [ 1761 evaluated the fate of Listeria as a postprocessing contam-
inant by inoculating 49 retail cheeses (24 typed28 brands) with L. monocytogenes and
then storing these cheeses at 4-30C for up to 36 days. As expected, Listeria growth was
primarily confined to high-moisture varieties, including Brie, Camembert, ricotta, and the
soft Hispanic cheeses, all of which had a pH 2 6 and low to moderate levels of salt in
the moisture phase (Table 16). Back et al. [ 1081 also reported temperature-dependent
surface growth of L. monocytogenes on several additional retail European soft cheeses
including Cambazola, English Brie, Blue Lymeswold, and White Lymeswold, with Liste-
ria populations remaining relatively stable on Blue Stilton, White Stilton, Mycella, and
Chaume cheese during short-term storage. In addition, commercial [ 1311 and experimen-
tally produced [170] Arzua cheese (a soft, low-acid Spanish cheese prepared from raw
cow's milk) supported growth of Listeria as evidenced by populations >loo0 CFU/g in
the finished product. All of these findings again point to the high-moisture, low-acid
cheeses as being of primary public health importance.
TABLE
16 Growth and Inactivation of L. monocytogenes in Surface-
Inoculated Retail Cheeses During Storage at 4-30C
% NaCl in
Cheese category and type PH moisture phase Growth
Soft mold ripened
Brie 6.0-7.7 2.5-3.6 +
Camembert 7.3 2.5 +
Blue 5.1 6.1
Bacterial surface ripened
Limburger 7.2 4.8
Muenster 5.5 3.8
Soft Italian
Provolone 5.6 4.6
String cheese 5.5 4.4
Semisoft and hard ripened
Monterey Jack 5.0-5.2 1.O-3.0
Colby 5.5 4.9
Cheddar 4.9-5.6 2.6-5.4
Swiss 5.5 2.7
Hispanic
Queso Fresco 6.5-6.6 4.5-6.6 +/-
Queso Rancher0 6.2 4.1 +
Queso Panella 6.2-6.7 2.5-3.9 +
Cotija 5.5-5.6 9.6- 12.5 -
Pickled cheese
Feta 4.2-4.3 2.2-7.5
Ewes milk cheese
Kasseri 4.8-5.3 5.5-5.8
Soft unripened
Cottage Cheese 4.9-5.1 1.0-1.2 +/-
Cream Cheese 4.8 <0.9 -
Whey cheeses
Ricotta 5.9-6.1 <0.7 +
Processed cheese
American 5.7 2.1
Monterey Jack 5.7 4.4
Piedmont 6.4 5.1
Source: Adapted from Ref. 176.
demonstrated that ripening Parmesan cheese for 10 months, as legally required, is suffi-
cient to produce a high-quality, Listeria-free product, desirable flavor and texture charac-
teristics are not easily attainable in sharp Cheddar and Swiss cheese prepared from pasteur-
ized milk. Hence, alternative means should be developed to enhance the safety of these
products. Such methods might include cold sterilization of the milk via microfiltration or
addition of various flavor- and texture-enhancing enzymes (or microorganisms) to pasteur-
ized milk, which would allow the cheesemaker to obtain a higher quality product [199].
However, as important as it is to manufacture cheese from Listeria-free milk, it is equally
important to prevent contamination of the product during manufacture, ripening, and
storage by using good manufacturing practices. Information concerning problem areas
486 Ryser
and safeguards during manufacture of dairy products and other, foods can be found in
Chapter 17.
Whey
Listeria monocytogenes has not yet been isolated from commercially produced cheese
whey. However, studies have shown that when various cheeses were experimentally pro-
duced from pasteurized milk inoculated with L. monocytogenes, between 1-5% of the
original inoculum was lost in the whey during cheesemaking (Table 17). These findings
again demonstrate that the pathogen is concentrated 8- to 10-fold in curd during milk
coagulation. Unlike other wheys, populations of L. monocytogenes in acid whey (pH 4.6)
obtained from the manufacture of cottage cheese were reduced more than 10,000- fold
after cooking the curdlwhey mixture at 57.2"C (1 35F) for 30 min. However, as previously
noted, Listeria was detected in several whey samples after 6 weeks of cold enrichment
[265], which, in turn, suggests that some cells were only sublethally injured during cooking
of the curd/whey mixture.
These observations prompted Ryser and Marth [262] to examine the behavior of L.
monocytogenes in wheys from Camembert cheese that were filter-sterilized and adjusted
to pH values of 5.0-6.8. All whey samples were then inoculated to contain -100-500
L. monocytogenes strains Scott A, V7, CA, or OH CFU/mL and incubated at 6C.
Although the four L. monocytogenes strains failed to grow in wheys having pH
values 15.4, the pathogen survived in all samples with populations decreasing 110-fold
during 35 days of refrigerated storage. In contrast, L. monocytogenes grew in all remaining
samples after a 3-day lag period and attained average maximum populations of 7.48, 7.87,
and 7.84 log,, CFU/mL in wheys adjusted to pH 5.6, 6.2, and 6.8, respectively, following
35 days of incubation. As previously noted, these Listeria populations in whey were
slightly higher than those that would be expected to develop in skim or whole milk during
refrigerated storage. Generation times for L. monocytogenes in wheys adjusted to pH 5.6,
6.2, and 6.8 ranged between 25.2-31.6 h, 14.8 and 21.1 h, and 14.0 and 19.4 h, respec-
tively, depending on the individual strain. Although doubling times were similar for all
strains at the same pH value, generation times were significantly longer at pH 5.6 than
% of original
L. monocytogenes inoculum in
Cheese CFU/mL of whey whey Ref.
Camembert 8 1.3 26 1
Blue 43 3.6 237
Brick 12 2.5 264
Feta 15 3.2 238
Gouda 5 1 .o 235
Colby 21 2.4 306
Cheddar 22 5.0 259
Average 18 21.7
L. monocytogenes in fermented Dairy Products 487
at pH 6.2 and 6.8. Interestingly, Hughey et al. [193] observed that L. monocytogenes could
be inactivated in similar samples of demineralized whey by adding lysozyme. However,
this enzyme did not decrease viability of the pathogen in normal whey, which in turn
suggests that lysozyme activity is neutralized by whey minerals and/or proteins.
Using a different approach to examine the behavior of Listeria in whey, Northolt
et al. [235] inoculated heat-treated (68"C/lO s) wheys (pH 6.5) to contain 500-1000 L.
monocytogenes CFU/mL and incubated the samples at temperatures between 7-30C.
Following a 6- to 24-h lag period, the pathogen grew in all samples with doubling times
of 12 h, 6 h, 4 h, and 40 min in wheys incubated at 7, 12, 20, and 30"C, respectively.
Although incubation of all whey samples was terminated before L. monocytogenes reached
the stationary growth phase, the pathogen did attain populations of 104-106 CFU/mL at
the conclusion of the experiment.
In the only study thus far reported dealing with nonsterile whey, researchers in
France [ 1131 produced whey containing 2-7 L. monacytogenes CFU/mL from previously
inoculated milk and examined samples for listeriae after 101, 156, and 25 1 days of storage
at 4C. Moderate growth and extended survival of the pathogen were observed in whey
collected immediately after coagulation of the milk (pH 5.4), with 156- and 251-day-old
whey samples at pH 4.8 containing 2.5 X 104 and 7.0 X 102 L. monocytogenes CFUI
mL, respectively. As predicted by Ryser and Marth [262], the pathogen also failed to
grow in more acidic wheys (pH 5.2-5.3) collected after hooping with listeriae no longer
observed in 101- and 156-day-old wheys having pH values of 3.28 and 4.26, respectively.
Not surprisingly, increasing the incubation temperature to 6, 14, and 20C led to faster
demise of listeriae in 21 similar wheys (pH 3.75-5.72) initially containing 60-96 L. mono-
cytogenes CFU/mL, with the pathogen being eliminated from all but one sample (pH
5.72) examined after 114 days of incubation at 6C. These findings demonstrate that L.
monocytogenes can grow to high numbers in fluid wheys having pH values of 25.4 and
remain viable in more acidic wheys during many months of refrigerated storage.
Given results of a study described in Chapter 11 in which numbers of L. monocyto-
genes decreased only 1-5 orders of magnitude during manufacture of nonfat dry milk
[ 1501, it appears that this organism also is likely to survive the spray-drying process used
in converting fluid whey into whey powder. However, to our knowledge, no Listeria spp.
have yet been isolated from dried whey manufactured commercially in Europe or the
United States. Although Gabis et al. [171] failed to find any Listeria spp. in 23 environ-
mental samples from whey processing factories, these authors did isolate L. monocyto-
genes from a floor drain that was located within a raw milk receiving room of a dry milk
processing factory. Additionally, Listeria spp. other than L. monocytogenes were isolated
from several drains and trenches in the powder production area of a second factory that
manufactured dry milk products. Considering the widespread use of dried whey (and non-
fat dried milk) as an ingredient in numerous products including cheese food, ice cream,
sherbet, candy, beverages, and baked goods, strict enforcement of good manufacturing
practices for dried whey and nonfat dry milk should be continued to prevent a possible
public health problem involving listeriae.
Brine Solutions
Since L. monocytogenes is quite halotolerant, it is not surprising to learn that brine solu-
tions in which cheeses are salted and/or ripened also can serve as potential reservoirs for
this organism. Current evidence suggests that these brine solutions may become contami-
488 Ryser
nated with L. monocytogenes through directhdirect contact with the cheese factory envi-
ronment (i.e., equipment, condensate on walls, floors, and ceilings) as well as actual shed-
ding of L. monocytogenes into the brine solution from Listeria-contaminated cheese. Breer
[ 1231 and Terplan [28] reported isolating L. monocytogenes from commercial brine solu-
tions in Europe. In one instance, the pathogen was detected in brine tanks 4 days after
soft/semisoft cheeses were removed from the salt brine. Since one 1991 recall of mozza-
rella cheese in the United States [80] was presumably traced to a contaminated brine tank,
interest in the incidence of listeriae in brine solutions will likely increase.
Migration of L. monocytogenes into brine solutions and salted whey during salting
of artificially contaminated cheese has been well documented. Ryser and Marth [26] brine
-
salted brick cheese containing 103-104 L. monocytogenes CFU/g at 10C. Cold enrich-
ment of membrane filters through which 50-mL portions of 22% brine solution were fil-
tered indicated that the pathogen leached from the cheese into the salt solution during 24
h of brining. Furthermore, viable listeriae were detected in samples of 22% brine stored
at 10C at least 5 days after blocks of cheese were removed from the brine. When Abdalla
et al. [ l ] brine salted Sudanese white pickled cheese in whey containing 8% NaCI, L.
monocytogenes once again leached from the cheese into the salted whey, with populations
increasing 2.5-3.5% orders of magnitude during 65 days of storage at 4C.
In conjunction with their study on the fate of L. monocytogenes during manufacture,
ripening, and storage of feta cheese, Papageorgiou and Marth [238] also examined Listeria
viability in the brine solution in which cheese was salted and stored. After 1 day of salting,
a 12% brine solution contained an average Listeria population of 2.63 log,, CFU/g, which
again indicates that the pathogen leached from cheese into the brine (Fig. 17). However,
no growth of L. monocytogenes was observed in the 12% brine solution despite ample
migration of cheese nutrients into salt brine as well as favorable values for temperature
and pH. After transferring feta cheese to a 6% brine solution, the pathogen leached from
cheese into salt brine and grew rapidly, with similar populations being observed in both
cheese and 6% brine after 6 days of incubation at 22C. Although numbers of listeriae
I I I I I I I I , I I ,<' 1 I I I
0 1 2 3 4 5 20 34 48 62 76
Days
decreased in both cheese and salt brine during 90 days of refrigerated storage, the pathogen
was inactivated slower in 6% salt brine than in feta cheese.
Larsen et al. [208] reported that L. monocytogenes began growing in salt-free whey
(pH 5.6) and whey containing 3.6% NaCl and/or KC1 after 7-14 days of storage at 4"C,
with Listeria growth generally being unrelated to the type of salt used. However, L. mono-
cytogenes populations remained unchanged in identical samples containing 4.8% NaCl
and/or KCI. When similar whey samples were incubated at 25"C, L. monocytogenes popu-
lations increased 3-4 orders of magnitude, with more rapid growth being observed in
wheys containing 3.6% rather than 4.8% NaCl and/or KCI. However, Listeria growth at
25C could be prevented by adding a L. l a d s subsp. lactis or L. lactis subsp. cremoris
starter culture before incubation. In 1994, Rajkowski et al. [249] also reported that addition
of 4.5% NaCl to ultrahigh temperature (UHT)-sterilized milk decreased the growth rate
of L. monocytogenes in samples incubated at 12-37C. However, fortification of these
same samples with 0.5 or 1 .O% polyphosphate did not alter Listeria growth characteristics.
Since feta and other white-pickled cheeses such as Teleme, Halloumi, and Domiati
are frequently cured in whey or skim milk containing 6 or 12% salt, Papageorgiou and
Marth [2361 examined behavior of Listeria in salted whey and skim milk. Autoclaved
samples of skim milk (pH 6.0-6.2) and deproteinated whey (pH 5.5-5.7) containing 6
-
and 12% NaCl were inoculated to contain 103 L. monocytogenes (strains Scott A or
CA) CFU/niL and incubated at 4 and 22C.
After a lag period of 5- 10 days, L. monocytogenes grew rapidly in 6% salted whey
and 6% salted skim milk, with the pathogen attaining maximum populations of 107-10R
CFU/mL following 50-55 days of refrigerated storage (Table 18). In the study discussed
earlier, Ryser and Marth [262] reported that these same Listeria strains had shorter lag
periods and generation times but achieved similar maximum populations in unsalted, filter-
sterilized whey (pH 5.6) after 24 days of incubation at 6C. Hence these findings suggest
that addition of NaCl or KCI to whey and milk plays a major role in decreasing the growth
rate of Listeria.
Increasing the incubation temperature to 22C resulted in lag periods of 6-12 h for
both Listeritr strains in whey and skim milk containing 6% salt. Generation times were
TABLE
18 Generation Times (GT) and Maximum Populations
(MP) of L. monocytogenes Strains Scott A and CA in 6% Salted
Whey and Skim Milk Incubated at 4 and 22C
MP
Strain Product GT (h) (logl,,CFU/mL)
Incubation at 4C
Scott A Whey 46.8 1 7.97
Scott A Skim milk 45.23 7.58
CA Whey 37.49 8.04
CA Skim milk 49.43 7.69
Incubation at 22C
Scott A Whey 3.67 8.02
Scott A Skim milk 4.3 1 7.70
CA Whey 3.56 8.10
CA Skim milk 4.42 7.89
5 -
ScottA Whey
f
0
3 -
2 -
1 - CAWhey
@- CA Skim milk
0 1 I I I I I I I I I I I
-25 0 25 50 75 100 125 150
Days
similarly reduced with both strains exhibiting faster growth rates and higher maximum
populations in salted whey rather than salted skim milk (see Table 18). These findings
agree with those of other researchers who also observed that L. monocytogenes grew faster
and attained higher maximum populations in unsalted whey [262] than in skim milk [255].
Unlike the previous findings obtained with 6% salt, growth of L. rnonocytogenes
was completely inhibited in 12% salted whey and skim milk, with populations of both
strains decreasing < 10-fold during 130 days of storage at 4C. Although strain Scott A
also persisted >130 days in 12% salted whey and skim milk incubated at 22"C, strain
CA proved to be less salt tolerant, surviving only 80 and 105 days in 12% salted skim
milk and whey, respectively (Fig. 18). Increased destruction of L. monocytogenes in salt
solutions held at ambient rather than refrigeration temperatures has been well documented.
Results from several of these studies dealing with viability of listeriae in salted Tryptose
Broth 112761 and cabbage juice [ 1391 are discussed in Chapter 6. Based on these observa-
tions, acidification of brine solutions used in cheesemaking to pH values <5.0 has been
recommended to prevent growth of L. monocytogenes, particularly if such solutions con-
tain 510% salt. In 1989, Hughey et al. [193] also noted that addition of 0.35% H 2 0 2to
a 23% brine solution caused numbers of L. monocytogenes to decrease by six orders of
magnitude within 24 h. However, unlike organic acids, the bactericidal activity of H 2 0 2
dissipates fairly rapidly. Hence addition of H202to salt brine provides only temporary
protection against listeriae that might be present within the cheesemaking environment.
REFERENCES
1. Abdalla, O.M., G.L. Christen, and P.M. Davidson. 1993. Chemical composition of and Liste-
ria rnonocytogenes survival in white pickled cheese. J. Food Prot. 56:841-846.
2. Abdalla, O.M., P.M. Davidson, and G.L. Christen. 1993, Survival of selected pathogenic
bacteria in white pickled cheese made with lactic acid bacteria or antimicrobials. J. Food
Prot. 56:972-976.
L. monocytogenes in Fermented Dairy Products 491
3. Abou-Donia, S.A., and A.K. Al-Medhagi. 1992. Detection and survival of Listeria monocyto-
genes in Egyptian dairy products. J. Dairy Sci. (suppl. 1) 75:138
4. Ahmed, A.A.-H. 1989. Behaviour of Listeria rnonocytogenes during preparation and storage
of yoghurt. Assuit Vet. Med. J. 22:76-80.
5. Ahmed, A.A.-H., S.H. Ahmed, M.K. Moustafa, and N.M. Saad. 1989. Growth and survival
of Listeria rnonocytogenes during manufacture and storage of Damietta cheese. Assiut Vet.
Med. J. 22:88-94.
6. Anonymous. 1949. Cheeses, processed cheeses, cheese foods, cheese spreads, and related
foods, definitions and standards of identity. Fed. Reg. April 22: 1960- 1992.
7. Anonymous. 1985. Code of hygienic practices for soft cheeses to be elaborated. Food Chem.
News 27(3 1): 12.
8. Anonymous. 1985. FDA finds Listeria in 3 brands of General Foods soft cheeses. Food
Chem. News 27(25):33.
9. Anonymous. 1985. FDA is checking facilities of cheese plant after finding Listeria. Food
Chem. News 27(24):25.
10. Anonymous. 1985. FDA is investigating deaths linked to Mexican-style cheese. Food Chem.
News 27(15):42.
11. Anonymous. 1985. Jalisco-brand Mexican style soft cheese recalled. FDA Enforcement Re-
port, June 26.
12. Anonymous. 1985. Liederkranz, Camembert and Brie cheese recalled. FDA Enforcement
Report, Sept. 4.
13. Anonymous. 1985. Planning program for inspection of imported soft cheese. Food Chem.
News 27(30):35.
14. Anonymous. 1985. Soft cheese manufacturers to be inspected under FDA priority program.
Food Chem. News 27(18):22.
15. Anonymous. 1986. Brie cheese recalled. FDA Enforcement Report, April 2.
16. Anonymous. 1986. Brie cheese recalled. FDA Enforcement Report, April 9.
17. Anonymous. 1986. Cheese recalls extended by General Foods, U.S. importer. Food Chem.
News 27(52):25-26.
18. Anonymous. 1986. FDA advises import alert on imported soft cheese. Food Chem. News
28(2:3):22-23.
19. Anonymous. 1986. FDA finds Listeria in Brie from French certified plant. Food Chem. News
27(50):35.
20. Anonymous. 1986. FDA finds Listeria in Mexican-style soft cheese. Food Chem. News
28( 1):54.
21. Anonymous. 1986. FDA may block list all soft-ripened cheese from France. Food Chem.
News 28(2):64-65.
22. Anonymous. 1986. FDA proposes soft-ripened cheese testing program to France. Food
Cheni. News 28(4):3-4.
23. Anonymous. 1986. FDA to detain all soft-ripened French cheese lacking certification. Food
Chem. News 28(10):9-10.
24. Anonymous. 1986. FDA to sample 20% of soft cheeses from all foreign countries. Food
Chem. News 28(6):27-28.
25. Anonymous. 1986. France agrees to temporary FDA soft-ripened cheese testing program.
Food Chem. News 28(7):24-26.
26. Anonymous. 1986. French Brie cheese recalled. FDA Enforcement Report, March 12.
27. Anonymous. 1986. French Brie cheese recalled. FDA Enforcement Report, May 14.
28. Anonymous. 1986. French cheeses recalled because of Listeria. Food Chem. News 28(24):
12.
29. Anonymous. 1986. French firm agrees to stop shipments of Brie cheese. Food Chem. News
27(51):12-14.
30. Anonymous. 1986. French semi-soft cheese recalled. FDA Enforcement Report, August 20.
492 Ryser
31. Anonymous. 1986. French soft-ripened cheese recalled. FDA Enforcement Report, August
13.
32. Anonymous. 1986. French soft-ripened cheese recalled. FDA Enforcement Report, Sept. 24.
33. Anonymous. 1986. Listeria causes class I recalls of ice milk mix, milk. Food Chem. News
28( 16):22.
34. Anonymous. 1986. Listeriosis hazard still being evaluated, FDA tells France. Food Chem.
News 18(26):29-32.
35. Anonymous. 1986. Milk, chocolate milk, half and half, cultured buttermilk, whipping cream,
ice milk, ice milk mix and ice milk shake mix recalled. FDA Enforcement Report, June 25.
36. Anonymous. 1986. More recalls made of French cheese, animal feeds. Food Chem. News
28(7):52- 53.
37. Anonymous. 1986. Recall of Brie cheese is extended by FDA. Food Chem. News 28( 1):54.
38. Anonymous. 1986. Soft Mexican-style cheeses recalled. FDA Enforcement Report, April 9.
39. Anonymous. 1986. Soft, ripened Brie cheese recalled. FDA Enforcement Report, April 9.
40. Anonymous. 1986. Soft-ripened French Brie cheese recalled. FDA Enforcement Report,
April 23.
41. Anonymous. 1987. Annual Report, Swiss Institute for Dairy Research, Liebefeld-Berne,
pp. 344-353.
42. Anonymous. 1987. Class I recall made of cheese because of Listeria. Food Chem. News
28(50):52.
43. Anonymous. 1987. FDA launching two-year pathogen surveillance program. Food Chem.
News 29(3 1): 10- 12.
44. Anonymous. 1987. FDA orders sampling of Italian hard cheese for microorganisms. Food
Chem. News 29(22):36.
45. Anonymous. 1987. FDA renews threat of automatic detention for Italian soft cheese. Food
Chem. News 29(6):3-4.
46. Anonymous. 1987. FDA sets separate program for cheese from unpasteurized milk. Food
Chem. News 29(8): 17- 18.
47. Anonymous. 1987. French full fat soft cheese recalled. FDA Enforcement Report, May 13.
48. Anonymous. 1987. French full fat cheese recalled because of Listeria. Food Chem. News
29(9): 16.
49. Anonymous. 1987. French soft cheese certification to be in place Feb. 15. Food Chem. News
28 (50):36.
50. Anonymous. 1987. Hard cheese from Italian firm blocklisted. Food Chem. News 29(23): 19.
51. Anonymous. 1987. Mini Bonbel and Gouda semi-soft cheese recalled. FDA Enforcement
Report, June 3.
52. Anonymous. 1987. Old Heidelberg soft-ripened cheese recalled. FDA Enforcement Report,
June 3.
53. Anonymous. 1987. Raw milk sharp Cheddar cheese recalled. FDA Enforcement Report, Aug.
19.
54. Anonymous. 1987. Salvador-style white semi-soft cheese recalled. FDA Enforcement Re-
port, Feb. 1 I .
55. Anonymous. 1988. Blue Castello Danish blue cheese recalled. FDA Enforcement Report,
May 4.
56. Anonymous. 1987. Danish cheese recalled because of Listeria. Food Chem. News 30(4):42.
57. Anonymous. 1988. FDA finds Listeria in foreign soft cheeses. Food Chem. News 29(49):
36-37.
58. Anonymous. 1988. Ice cream, cheese recalled because of Listeria. Food Chem. News 30(6):
27.
59. Anonymous. 1988. Italian semi-soft cheese recalled. FDA Enforcement Report, March 30.
60. Anonymous. 1988. LAmulette Danish Esrom cheese recalled. FDA Enforcement Report,
March 23.
L. monocytogenes in Fermented Dairy Products 493
61. Anonymous. 1988. LAmulette Danish Esrom cheese recalled. FDA Enforcement Report,
March 30.
62. Anonymous. 1988. LAmulette Danish Esrom cheese recalled. FDA Enforcement Report,
April 6.
63. Anonymous. 1988. Listeriosis: Goats milk cheese. Communicable Disease Report, Feb. 12.
64. Anonymous. 1988. Mexican-style, baby Jack and Monterey Jack cheese recalled. FDA En-
forcement Report, March 9.
65. Anonymous. 1988. Nisin preparation affirmed as GRAS for cheese spreads. Food Chem.
News 30(6):37-38.
66. Anonymous. 1988. Soft cheese warning because of Listeria monocytogenes. Food Chem.
News 29(49):2.
67. Anonymous. 1989. Controlling Listeria-the Danish solution. Dairy Indust. Intern. 54(5):3 1-
32.
68. Anonymous. 1989. Cyprus Anari cheese recalled. FDA Enforcement Report, July 12.
69. Anonymous. 1989. Cyprus Anari cheese recalled. FDA Enforcement Report, Sept. 27.
70. Anonymous. 1989. Cyprus Anari cheese recalled. FDA Enforcement Report, Nov. 1.
71. Anonymous. 1989. Frozen yogurt recalled. FDA Enforcement Report, Nov. 7.
71a. Anonymous. 1989. Home-made chorizo bust in L.A. year-long investigation. Lean trim-
mings. Western States Meat Association, April 7.
72. Anonymous. 1989. Pasteurized process cheese spread: Amendment of standard of identity.
Fed. Reg. 54:6 120-6 121.
73. Anonymous. 1989. Salmon recalled because of Listeria; blocklist covers hard cheeses. Food
Cheni. News 3 1(37):27-28.
74. Anonymous. 1989. Umweltministerium warnt vor zwei Weichkasesorten. Deutsche Molkerei
Zeitung 110:898.
75. Anonymous. 1990. Cyprus Halloumi cheese recalled. FDA Enforcement Report, Jan.
17.
76. Anonymous. 1990. Robiola Interbiola soft ripened and semi soft cheese recalled. FDA En-
forcement Report, Nov 7.
77. Anonymous. 1990. USDA, FDA officials report apparent decrease in Listeria isolations. Food
Cheni. News 32(1):12-15.
78. Anonymous. 199 1. Cheese and sour cream products recalled. FDA Enforcement Report, Mar.
13.
79. Anonymous. 199 1. Jack semi-soft cheese recalled. FDA Enforcement Report, Dec. 18.
80. Anonymous. 1991. Mozzarella cheese recalled. FDA Enforcement Report, May 15.
81. Anonymous. 1991. Pimento cheese spread recalled. FDA Enforcement Report, Mar. 27.
82. Anonymous. 1991. Ricotta cheese recalled. FDA Enforcement Report, Oct. 2.
83. Anonymous. 1992. Cold pack cheese food recalled. FDA Enforcement Report, Apr. 29.
84. Anonymous. 1993. Cheese spread recalled. FDA Enforcement Report, May 19.
85. Anonymous. 1993. El Ranchito queso fresco soft Mexican cheese recalled. FDA Enforcement
Report, Jan. 6.
86. Anonymous. 1993. Limburger cheese recalled. FDA Enforcement Report, Feb. 17.
87. Anonymous. 1993. Swedish fontina cheese recalled. FDA Enforcement Report, May 19.
88. Anonymous. 1994. Butter products and cream cheese recalled. FDA Enforcement Report,
Jan. 19.
89. Anonymous. 1994. Cream cheese and lox spread recalled. FDA Enforcement Report, July
13.
90. Anonymous. 1994. Goat milk cheese recalled. FDA Enforcement Report, Aug. 24.
91. Anonymous. 1994. Mexican style soft white cheese recalled. FDA Enforcement Report, Sept.
28.
92. Anonymous. 1994. Ochoa Mexican style soft white cheese recalled. FDA Enforcement Re-
port, Sept. 14.
494 Ryser
93. Anonymous. 1994. Queso blanco semi-soft cheese recalled. FDA Enforcement Report, Aug. 10.
94. Anonymous. 1994. Queso Prensado semi soft cheese recalled. FDA Enforcement Report,
Aug. 10.
95. Anonymous. 1994. Swiss cold pack cheese food recalled. FDA Enforcement Report, Oct. 5.
96. Anonymous. 1994. Torte loaf cheeses recalled. FDA Enforcement Report, Oct. 5.
97. Anonymous. 1995. Swiss cheese recalled. FDA Enforcement Report, Jan. 25.
98. Anonymous. 1996. Creamy Gorgonzola cheese recalled. FDA Enforcement Report, Apr. 17.
99. Anonymous. 1996. Jarlsberg cheese recalled. FDA Enforcement Report, July 17.
100. Anonymous. 1996. Limburger cheese recalled. FDA Enforcement Report, Apr. 3.
100a. Anonymous. 1997. Cream cheese with vegetables recalled. FDA Enforcement Report, Dec.
10.
100b. Anonymous. 1997. Cream cheese and tuna salad recalled. FDA Enforcement Report, Dec. 24.
1ooc. Anonymous. 1998. Blue cheese recalled. United Press International Release, April 11.
1OOd. Anonymous. 1998. Blue cheese salad dressing recalled. Associated Press Release, May 1.
100e. Anonymous. 1998. Queso fresco cheese recalled. FDA Enforcement Report, April 29.
100f. Anonymous. 1998. Queso fresco cheese recalled. FDA Enforcement Report, May 6.
1oog. Anonymous. 1998. Queso fresco Mexican-style cheese recalled. Food Chem. News 40(5):
39.
101. Archer, D. L. 1988. Review of the latest FDA information on the presence of Listeria in
foods. WHO Working Group on Foodborne Listeriosis. Geneva, Switzerland, Feb. 15- 19.
102. Arnold, G.J., and J. Coble. 1995. Incidence of Listeria species in foods in NSW. Food Austra-
lia 47:7 1-75.
103. Art, D., and P. Andre. 1991. Clinical and epidemiological aspects of listeriosis in Belgium.
Zbl. Bakt. 275~549-556.
104. Ashenafi, M. 1994. Fate of Listeria monocytogenes during the souring of Ergo, a traditional
Ethiopian fermented milk. J. Dairy Sci. 77:696-702.
105. Asperger, H., B. Url, and E. Brand]. 1989. Interactions between Listeria and the ripening
flora of cheese. Neth. Milk Dairy J. 43:287-298.
106. Ayre, H.A., H. Williams, K. Hood, I.R. McFadyen, and C.A. Hurt. 1992. Survival of Listeria
monocytogenes in food. In: Proceedings of XIth International Symposium on Problems of
Listeriosis, Copenhagen, Denmark, May 1 1 - 14, p. 123- 124.
107 Bachmann, H.P., and U. Spahr. 1995. The fate of potentially pathogenic bacteria in Swiss
hard and semihard cheeses made from raw milk. J. Dairy Sci. 78:476-483.
108. Back, J.P., S.A. Langford, and R.G. Kroll. 1993. Growth of Listeria monocytogenes in Cam-
embert and other soft cheeses at refrigeration temperatures. J. Dairy Res. 60:42 1-429.
109. Ballare, G., A. Bertona, C. Airoldi, and A. Moiraghi Ruggenini. 1989. In tema di contaminaz-
ione di alimenti da parte di Listeria monocytogenes indaginerelativa a campioni di latte e
formaggi nellarco di un biennio (1987-1989). Microbiol. Medica 4: 110- 114.
110. Banks, W. 1994. Microbiology of milk. Milk Industry (Tech. Process. Suppl.) 96: 18-20.
111. Bannerman, E.S., and J. Billie. 1988. A selective medium for isolating Listeria spp. from
heavily contaminated material. Appl. Environ. Microbiol. 54: 165- 167.
112. Bannister, B.A. 1987. Listeria monocytogenes meningitis associated with eating soft cheese.
J. Infect. 15:165-168.
113. Barnier, E., J.P. Vincent, and M. Catteau. 1988. Survie de Listeria monocytogenes dans les
saumures et sirurn de fromagerii. Sci. Aliments 8: 175-178.
114. Beckers, H.J., P.S.S. Soentoro, and E.H.M. Delfgou-van Asch. 1987.The occurrence of Liste-
ria monocytogenes in soft cheeses and raw milk and its resistance to heat. Int. J. Food Micro-
biol. 4:249-256.
115. Beckers, H.J., P.H. int Veld, P.S.S. Soentoro, and E.H.M. Delfgou-van Asch. 1988. The
occurrence of Listeria in food. Foodborne Listeriosis-Proceedings of a Symposium, Wies-
baden, Germany, Sept. 7, pp. 84-97.
L. monocytogenes in Fermented Dairy Products 495
116. Benkerroum, N., and W.E. Sandine. 1988. Inhibitory action of nisin against Listeria monocy-
togenes. J. Dairy Sci. 71:3237-3245.
117. Beuchat, L.R., and M.P. Doyle. 1995. Survival and growth of Listeria monocytogenes in
foods treated or supplemented with carrot juice. Food Microbiol. 12:73-80.
118. Bilney, F., R. Armstrong, and A. Vickerman. 1991. Listeria in food: Report of the West and
North Yorkshire Joint Working Group on a two year survey on the presence of Listeria in
food. Environ. Health 99: 132-1 37.
119. Bind, J.-L. 1988. Review of latest information concerning data about repartition of Listeria
in France. WHO Working Group on Foodborne Listeriosis, Geneva, Switzerland, February
15- 19.
120. Bockemuhl, J., G. Schulze, G. Marcy, and H.P.R. Seeliger. 1992. Number and distribution of
Listeria monocytogenes in soft cheese: how relevant is the natural contamination for causing
disease? In: Proceedings of the 3rd World Congress on Foodborne Infections and Intoxica-
tions, Berlin, Germany, June 16- 19, pp. 496-500.
121. Bougle, D.L., and V. Stahl. 1994. Survival of Listeria monocytogenes after irradiation treat-
ment of Camembert cheeses made from raw milk. J. Food Prot. 572311-813.
122. Breer, C. 1986. The occurrence of Listeria spp. in cheese. In: Proceedings of 2nd World
Congress on Foodborne Infections and Intoxications, Institute of Veterinary Medicine, Robert
von Ostertag Institute, Berlin, pp. 230-233.
123. Breer, C. 1988. Occurrence of Listeria spp. in different foods. WHO Working Group on
Foodborne Listeriosis, Geneva, Switzerland, Feb. 15- 19.
124. Brindani, F., and E. Freschi. 1988/1989 Ricerca di Listeriu monocytogenes nel latte di ovi-
caprini ed in alcuni tipi di formaggio. Ann. Fac. Med. Vet. 8-9:205-219.
125. Buazzi, M.M., M.E. Johnson, and E.H. Marth. 1992. Fate of Listeria monocytogenes during
the manufacture of mozzarella cheese. J. Food Prot. %:SO-83.
126. Buazzi, M.M., M.E. Johnson, and E.H. Marth. 1992. Survival of Listeria monocytogenes
during the manufacture and ripening of Swiss cheese. J. Dairy Sci. 75:380-386.
127. Buldrini, A., C. Maini, and G. Sanavio. 1990. Isolamento di Listeria spp. Da prodotti lattiero-
caseari e da alimenti carnei. Indust. Aliment. 2930-552.
128. Cantoni, C., M. Valenti, and G. Comi. 1988. Listeriu in formaggi e in salumi. Ind. Alimentari
27:859-861.
129. Casarotti Vania, T., R. Gallo Claudio, and Rodolpho Camargo. 1994. Occurrence of Listeria
monocytogenes in raw milk, pasteurized C type milk and minas frescal cheese commercial-
ized in Piracicaba-S.P. Arch. Latinoamer. Nutr. 44: 158- 163.
130. Centeno, J.A., A. Cepeda, J.L. Rodriguez-Otero, and F. Docampo. 1995. Hygenic study of
Arzua cheese. Alimentaria 33:9 1-96.
131. Centeno, J.A., J.L. Rodriguez-Otero, and A. Cepeda. 1994. Microbiological study of Arzua
cheese (NW Spain) throughout cheesemaking and ripening. J. Food Safety 14:229-241.
132. Chen, J.H., and J.H. Hotchkiss. 1992. Growth of Listeria monocytogenes and CZostridium
sporogenes in cottage cheese in modified atmosphere packaging. J. Dairy Sci. 76:972-
977.
133. Choi, H.K., M.M. Schaack, and E.H. Marth. 1988. Survival of Listeria monocytogenes in
cultured buttermilk and yogurt. Milchwissenschaft 43:790-792.
134. Comi, C., and C. Cantoni. 1988. Alcuni aspetti della presenza di L. monocytogenes nei
formaggi. Ind. Alimentari 27: 104-106.
135. Comi, G., and M. Valenti. 1988. Survival and growth of Listeria monocytogenes in Italian
type cheese. Latte 13:956-958.
136. Comi, G., C. Cantoni, and S. dAubert. 1987. Indagine sulla presenza di Listeria monocyto-
genes nei formaggi. Ind. Alimentari 26:216-218.
137. Comi, G., C. Cantoni, M. Valenti, and M. Civilini. 1990. Listeria species in Italian cheese.
Microbiol. Aliments Nutr. 8:377-382.
496 Ryser
138. Comi, G., M. Maifreni, C. Cantoni, F. DAurelio, and M. Valenti. 1990. Direct and
M.P.N. methods for quantitative analysis of Listeria spp. in foods. Indust. Aliment. 29:985-
990.
139. Conner, D.E., R.E. Brackett, and L.R. Beuchat. 1986. Effect of temperature, sodium chloride,
and pH on growth of Listeria monocytogenes in cabbage juice. Appl. Environ. Microbiol.
52159-63.
140. Cottin, J., F. Picard-Bonnaud, and B. Carbonnelle. 1990. Study of Listeria monocytogenes
survival during the preparation and the conservation of two kinds of dairy products. Acta
Microbiol. Hung. 37: 119-122.
141. Coveney, H.M., G.F. Fitzgerald, and C. Daly. 1994. A study of the microbiological status
of Irish farmhouse cheeses with emphasis on selected pathogenic and spoilage microorgan-
isms. J. Appl. Bacteriol. 77:621-630.
142. Dalu, J.M., and S.B. Feresu. 1996. Survival of Listeria monocytogenes in three Zimbabwean
fermented milk products. J. Food Prot. 59:379-383.
143. Darwish, S.M., E.A. El-Difrawy, H.M. Osman, and J.M. Debever. 1995. Effect of water
activity, pH, and lysozyme on the growth of some pathogenic bacteria in cottage and Karish
cheeses. Alex. J. Agric. Res. 40: 149-157.
144. Degnan, A.J., N.Y. Farkye, M.E. Johnson, and J.B. Luchansky. 1996. Use of nisin to control
Listeria monocytogenes in Queso Fresco cheese. J. Food Prot. 59 (Suppl.):44.
145. Denis, F., and J.-P. Ramet. 1989. Antibacterial activity of the lactoperoxidase system on
Listeria monocytogenes in trypticase soy broth, UHT milk and French soft cheese. J. Food
Prot. 52:706-7 11.
146. DErrico, M.M., P. Villari, G.M. Grasso, F. Romano, and I.F. Angelillo. 1990. Isolamento
di Listeria spp. da latte e formaggi. Rev. Soc. Ital. Sci. Aliment. 19:47-52.
147. De Urbina, E.O., M.T. de Daza, and L.D. Miranda. 1993. Incidencia de Listeria monocyto-
genes en productos lacteos de alto consumo en cojedes, Venezuela. Rev. Unellez Ciencia
Tecnol. 11:41-49.
148. Digrak, M., 0. Yilmaz, and S. Ozcelik. 1994. Microbiological and some physicochemical
characteristics of Erzincan Tulum (Savak) cheese samples sold in shops in Elazig. Gida 19:
38 1-387.
149. Dominguez, L., J.F.F. Garayzabal, J.A. Vazquez, J.L. Blanco, and G. Suarez. 1987. Fate of
Listeria monocytogenes during manufacture and ripening of semi-hard cheese. Lett. Appl.
Microbiol. 4:125-127.
150. Doyle, M.P., L.M. Meske, and E.H. Marth. 1985. Survival of Listeria monocytogenes during
the manufacture and storage of nonfat dry milk. J. Food Prot. 48:740-742.
151. Durand, M.P. 1992. Listeria-Listeriosis et produits laitiers. Bull. Soc. Vet. Prat. France 76:
5 17-539.
152. Eilertz, I., M.L. Danielsson-Tham, K.-E. Hammarberg, M.W. Reeves, J . Rocourt, H.P.R.
Seeliger, B. Swaminathan, and W. Tham. 1993. Isolation of Listeria monocytogenes from
goat cheese associated with a case of listeriosis in goat. Acta Vet. Scand. 34:145-149.
153. El-Gazzar, F.E., and E.H. Marth. 1988. Loss of viability by Listeria monocytogenes in com-
mercial calf rennet extract. J. Food Prot. 5 I :16- 18.
154. El-Gazzar, F.E., and E.H. Marth. 1988. Loss of viability of Listeria monocytogenes in com-
mercial bovine pepsin-rennet extract. J. Dairy Sci. 72: 1098- 1102.
155. El-Gazzar, F.E., and E.H. Marth. 1989. Fate of Listeria monocytogenes in some food colours
and starter distillate, Lebensm. Wiss. Technol. 22:406-4 10.
156. El-Gazzar, F.E., and E.H. Marth. 1989. Loss of viability by Listeria monocytogenes in com-
mercial microbial rennet. Milchwissenschaft 44:83-86.
157. El-Gazzar, F.E., and E.H. Marth. 199 1 . An apparent benzoate-resistant strain of Listeria
monocytogenes recovered from a milk clotting agent of animal origin. Milchwissenschaft
461350-354.
158. El-Gazzar, F.E., H.F. Bohner, and E.H. Marth. 1992. Antagonism between Listeria monocy-
L. monocytogenes in Fermented Dairy Products 497
togenes and lactococci during fermentation of products from ultrafiltered skim milk. J. Dairy
Sci. 75:43-50.
159. El-Marrakchi, A., A. Hamama, and F. El-Othmani. 1993. Occurrence of Listeria monocyto-
genes in milk and dairy products produced or imported into Morocco. J. Food Prot. 56:256-
259.
160. El-Shenawy, M.A., and E.H. Marth. 1990. Behavior of Listeria monocytogenes in the pres-
ence of gluconic acid and during preparation of cottage cheese curd using gluconic acid. J.
Dairy Sci. 73: 1429-1438.
161. El-Sukhon, S.N. 1993. Bacteriological analysis of white brined (Nablusian) cheese with refer-
ence to Listeria monocytogenes in Jordan. J. Egypt. Vet. Med. Assoc. 53: 139-144.
162. Ennahar, S., F. Kuntz, A. Strasser, M. Bergaentzle, C. Hasselmann, and V. Stahl. 1994.
Elimination of Listeria monocytogenes in soft and red smear cheeses by irradiation with low
energy electrons. Intern. J. Food Sci. Technol. 29:395-403.
163. Ennahar, S., D. Aoude-Werner, 0. Sorokine, A. Van Dorsselaer, F. Bringel, J.-C. Hubert,
and C. Hasselmann. 1996. Production of pediocin AcH by Lactobacillus plantarum WHE
92 isolated from cheese. Appl. Environ. Microbiol. 62:438 1-4387.
164. Eppert, I., E. Lechner, R. Mayr, and S. Scherer. 1995. Lkterien und coliforme Keime in
echten und fehldeklarierten Rohmilchweichkasen. Archiv. Lebensmittelhyg. 46:85-87.
165. Farber, J.M., G.W. Sanders, and S.A. Malcom. 1988. The presence of Listeria spp. in raw
milk in Ontario. Can. J. Microbiol. 34:95- 100.
166. Farber, J.M., M.A. Johnston, U. Purvis, and A. Loit. 1987. Surveillance of soft and semi-
soft cheeses for the presence of Listeria spp. Int. J. Food Microbiol. 5:157-163.
167. Fathi, Sh.M., and N. Saad. 1992. A survey of some selected food items for the presence of
Listeria monocytogenes and other Listeria species. Assuit Vet. Med. J. 27: 1 14- 120.
168. Fedio, W.M., A. Macleod, and L. Ozimek. 1994. The effect of modified atmosphere
packaging on the growth of microorganisms in cottage cheese. Milchwissenschaft 49:622-
629.
169. Ferreira, M.A.S.S., and B.M. Lund. 1996. The effect of nisin on Listeria monocytogenes in
culture medium and long-life cottage cheese. Lett. Appl. Microbiol. 22:433-438.
170. Franco, C.M., S. Menendez, E.J. Quinto, C.A. Fente, B. Vazquez, L. Dominguez, and C.
Cepeda. 1996. Behaviour of L. rnonocytogenes and L. innocua in Arzua type Galician
cheese. Effect of wrapping in self-adhesive plastic film. Alimentaria 34:s 1-85.
171. Gabis, D.A., R.S. Flowers, D. Evanson, and R.E. Faust. 1989. A survey of 18 dry dairy
product processing plant environments for Salmonella, Listeria and Yersinia. J. Food Prot.
52: 122- 124.
172. Gahan, C.G.M., B. ODriscoll, and C. Hill. 1996. Acid adaptation of Listeria monocytogenes
can enhance survival in acidic foods during milk fermentation. Appl. Environ. Microbiol.
62:3128-3132.
173. Geisen, R., E. Glenn, and L. Leistner. 1988. Effects of Penicilfium roquefortii, P. camemberti
and P. nalgiovense on growth of Listeria monocytogenes in vitro. Mitteilungsblatt der Bunde-
sanstalt fuer Fleischforschung, Kulmbach 101:8088-8092.
174. Gelosa, L. 1990. La Listeria monocytogenes quale contaminante di prodotti lattierocaseari.
Indust. Aliment. 29: 137- 139.
175. Genigeorgis, C., J.H. Toledo, and F.J. Garayzabal. 1991. Selected microbiological and chemi-
cal characteristics of illegally produced and marketed soft hispanic-style cheeses in Califor-
nia. J. Food Prot. 54:598-601.
176. Genigeorgis, C., M. Carniciu, D. Dutulescu, and T.B. Farver. 1991. Growth and survival of
Listeria monocytogenes in market cheeses stored at 4 to 30C. J. Food Prot. 54:662-668.
177. Gilbert, R.J. 1995. Zero tolerance for Listeria monocytogenes in foods-Is it necessary or
realistic? In: Proceedings of the XIIth International. Symposium on Problems of Listeriosis,
Perth, Australia, October 2-6, p. 35 1-356.
178. Giraffa, G., E. Neviani, and G.T. Tarelli. 1994. Antilisterial activity by enterococci in a model
498 Ryser
predicting the temperature evolution of Taleggio, an Italian soft cheese. J. Dairy Sci. 77:
1176-1 182.
179. Glass, K.A., B.B. Prasad, J.H. Schlyter, H.E. Uljas, N.Y. Farkye, and J.B. Luchansky. 1995.
Effects of acid type and AltaTM2341 on Listeria monocytogenes in a Queso Blanco type of
cheese. J. Food Prot. 58:737-741.
180. Gledel, J. 1986. Epidemiology and significance of listeriosis in France. In: A. Schonberg,
ed. Listeriosis-Joint WHO/ROI Consultation on Prevention and Control, Berlin, December
10- 12, Institut fur Veterinkedizin des Bundesgesundheitsamtes, Berlin, pp. 9-20.
181. Gledel, J. 1988. Listeria and the dairy industry in France. Foodborne Listeriosis-Proceed-
ings of a Symposium. Wiesbaden, Germany, Sept. 7, pp. 72-82.
182. Goel, M.C., D.C. Kulshrestha, E.H. Marth, D.W. Francis, J.G. Bradshaw, and R.B. Read,
Jr. 1971. Fate of coliforms in yogurt, buttermilk, sour cream, and cottage cheese during
refrigerated storage. J. Milk Food Technol. 3454-58.
183. Goepfert, J.M., N.F. Olson, and E.H. Marth. 1968. Behavior of Salmonella typhimuriurn
during manufacture and curing of Cheddar cheese. Appl. Microbiol. 16:862-866.
184. Gohil, V.S., M.A. Ahmed, R. Davies, and R.K. Robinson. 1995. Incidence of Listeria spp.
in retail foods in the United Arab Emirates. J. Food Prot. 58:102-104.
185. Gohil, V.S., M.A. Ahmed, R. Davies, and R.K. Robinson. 1996. Growth and survival of
Listeria monocytogenes in two traditional foods from the United Arab Emirates. Food Micro-
biol. 13:159-164.
186. Goz, M., and A.T. Cengiz. 1992. Incidence of Listeria monocytogenes in various cheese
species. In: Proceedings of the XIth International Symposium on Problems of Listeriosis,
Copenhagen, Denmark, May 11- 14, p. 160- 161.
187. Greenaway, C., and P.G. Drew. 1990. Survey of dairy products in Victoria, Australia for
Listeria species. In: Posters and Brief Communications of the XXIII International Dairy
Congress, Montreal, Canada, Oct. 8-12. Abst. 234.
188. Greenwood, M.H., D. Roberts, and P. Burden. 1991. The occurrence of Listeria species in
milk and dairy products: a national survey in England and Wales. Int. J. Food Microbiol.
12:197-206.
189. Griffith, M., and K. Deibel. 1988. Survival of Listeria monocytogenes in yogurt and acidified
milk. Annual Meeting of American Society for Microbiology, Miami Beach, FL, May 8-
13, Abstr. P-2 1.
190. Gul, K., A. Suay, M.N. Dag, M. Mete, and 0. Mete. 1995. Isolation of Listeria species from
cheese samples collected in the province of Diyarbakir. Turkish J. Infect. 9:45-46.
191. Hapke, B. 1989. Personal communication.
192. Hicks, S.J., and B.M. Lund. 1991. The survival of Listeria rnonocytogenes in cottage cheese.
J. Appl. Bacteriol. 70:308-314.
193. Hughey, J.L., P.A. Wilger, and E.A. Johnson. 1989. Antibacterial activity of hen egg white
lysozyme against Listeria monocytogenes Scott A in foods. Appl. Environ. Microbiol. 63 1-638.
194. Ibrahim, G.A.M., H. Domjan-Kovacs, A. Fabian, and B. Ralovich. 1992. Listeria in milk
and dairy products in Hungary. In: Proceedings of the XIth International Symposium on
Problems of Listeriosis, Copenhagen, Denmark, May 1 1-14, p. 297-298.
195. Ikonomov, L., and D. Todorov. 1964. Studies of the viability of Listeria rnonocytogenes in
ewes milk and dairy products. Vet. Med. Nauki, Sofiya 7:23-29.
196. International Dairy Federation. 1989. Pathogenic Listeria-Abstracts of replies from 24
countries to questionnaire 1288/B on pathogenic Listeria. Circular 89/5, March 3 1, Interna-
tional Dairy Federation, Brussels.
197. Johnson, E.A., J.H. Nelson, and M. Johnson. 1990. Microbiological safety of cheese made
from heat-treated milk, Part I. Executive summary, introduction and history. J. Food Prot.
531441-452.
198. Johnson, E.A., J.H. Nelson, and M. Johnson. 1990. Microbiological safety of cheese made
from heat-treated milk, Part 11. Microbiology. J. Food Prot. 5 3 5 19-540.
L. monocytogenes in Fermented Dairy Products 499
199. Johnson, E.A., J.H. Nelson, and M. Johnson. 1990. Microbiological safety of cheese made
froni heat-treated milk, Part 111. Technology, discussion, recommendations, bibliography. J.
Food Prot. 53:610-623.
200. Katic, V. 1995. The survival of Listeria monocytogenes in white brined cheese. Acta Vet.
(Beograd) 45:3 1-36.
201. Kaufmann, U. 1990. Behavior of Listeria monocytogenes in raw milk hard cheeses. Revue
Suisse Agric. 225-9.
202. Ken-, K.G., N.A. Rotowa, and P.M. Hawkey. 1992. Listeria in yoghurt? J. Nutr. Med. 3:
27-29.
203. Khattab, A.A., A.M. El-Leboudy, and H.M. Ahmad. 1993. Survival of Listeria monocyto-
genes in yoghurt and acidified milk during storage at 4-5C. Egypt. J. Food Sci. 21:41-48.
204. Kinderlerer, J.L., and B.M. Lund. 1992. Inhibition of Listeria monocytogenes and Listeria
innocua by hexanoic and octanoic acids. Lett. Appl. Microbiol. 14:271-274.
205. Kinderlerer, J.L., H.E. Matthias, and P. Finner. 1996. Effect of medium-chain fatty acids in
mould ripened cheese on the growth of Listeria monocytogenes. J. Dairy Res. 63593-606.
206. Kovincic, I., I.F. Vujicic, M. Svabic-Valhovic, M. Vulic, M. Gagic, and I.V. Wesley. 1991.
Survival of Listeria monocytogenes during the manufacture and ripening of Trappist cheese.
J. Food Prot. 54:418-420.
207. Lafaivre, J. 1988. Personal communication.
208. Larson, A., E.A. Johnson, and J.H. Nelson. 1993. Behavior of Listeria monocytogenes and
Salmonella heidelberg in rennet whey containing added sodium and/or potassium chloride.
J. Food Prot. 56:385-389.
209. Lewis, S.J., and J.E.L. Corry. 1991. Comparison of a cold enrichment and the FDA method
for isolating Listeria monocytogenes and other Listeria spp. from ready-to-eat food on retail
sale in the U.K. Int. J. Food Microbiol. 12:281-286.
210. Loncarevic, S., M.-L. Danielsson-Tham, and W. Tham. 1995. Occurrence of Listeria mono-
cytogenes in soft and semi-soft cheeses in retail outlets in Sweden. Intern. J. Food Microbiol.
26:245-250.
211. Lopez-Diaz, T.M., J.A. Santos, C.J. Gonzalez, B. Moreno, and M.L. Garcia. 1995. Bacterio-
logical quality of a traditional Spanish blue cheese. Milchwissenschaft. 50503-505.
212. Lukasova, J. 1993. Influence of milk cultures on the survival of Listeria monocytogenes in
milk. Prumsyl Potravin 44: 158- 160.
213. MacGowan, A.P., K. Bowker, J. McLauchlin, P.M. Bennett, and D.S. Reeves. 1994. The
occurrence and seasonal changes in the isolation of Listeria spp. in shop bought food stuffs,
human faeces, sewage and soil from urban sources. Int. J. Food Microbiol. 21:325-334.
214. Mackey, B.M., and N. Bratchell. 1989. The heat resistance of Listeria monocytogenes: A
review. Lett. Appl. Microbiol. 9:89-94.
215. Maheswari, R.R.A., and M. Gueguen. 1990. Geotrichurn candidurn inhibiteur de Listeria
monocytogenes? Posters and Brief Communications of the XXIIIrd International Dairy Con-
gress, Montreal, Canada, Oct. 8- 12, Abst. 701.
216. Maisner-Patin, S., N. Deschamps, S.R. Tatini, and J. Richard. 1992. Inhibition of Listeria
monocytogenes in Camembert cheese made with a nisin-producing starter. Lait 72:249-
263.
217. Marier, R., J.G. Wells, R.C. Swanson, W. Callahan, and I.J. Mehlman. 1973. An outbreak
of enteropathogenic Escherichia coli foodborne disease traced to imported French cheese.
Lancet 2: 1376- 1378.
218. Martin, von F., K. Friedrich, F. Beyer, und G. Terplan. 1995. Antagonistische Wirkungen
von Brevibacterium linens-Stammen gegen Listerien. Arch. Lebensmittelhygiene 46:7- 1 1.
219. Mas, M., and J. Gonzalez-Crespo. 1993. Control de microorganismos pathogenos en Queso
de los Ibores. Alimentaria 3 1:41-44.
220. Massa, C.C. 1996. Microbiological quality of cheese: importance of good processing. Ali-
mentaria 34:69-72.
500 Ryser
221. Massa, S., L.D. Trovatelli, and F. Canganella. 1991. Survival of Listeria monocytogenes in
yogurt during storage at 4C. Lett. Appl. Microbiol. 13:I 12- 1 14.
222. Massa, S., D. Cesaroni, G. Poda, and L.D. Trovatelli. 1990. The incidence of Listeria spp.
in soft cheeses, butter and raw milk in the province of Bologna. J. Appl. Bacteriol. 68: 153-
156.
223. McBean, L.D. 1988. A perspective on food safety concerns. Dairy Food Sanit. 8: 1 12- 1 18.
224. McLauchlin, J., M.H. Greenwood, and P.N. Pini. 1990. The occurrence of Listeria rnonocyto-
genes in cheese from a manufacturer associated with a case of listeriosis. Int. J. Food Micro-
biol.
225. Matsusaki, S., A. Katayama, M. Okada, R. Endo, K. Tanaka, K. Sekiya, and K. Shibata.
1991. Behavior of Listeria monocytogenes during the manufacture, ripening and storage of
Camembert cheese after contamination of pasteurized milk and brine with this organism. J.
Food Hyg. Soc. Japan 32:498-503.
226. Mehta, A., and S.R. Tatini. 1992. Behavior of Listeria monocytogenes in Cheddar cheese
made with NaCl or equimolar mixture of NaCl and KCI. J. Dairy Sci. 75 (suppl. 1):93.
227. Mehta, A., and S.R. Tatini. 1994. An evaluation of the microbiological safety of reduced-
fat Cheddar-like cheese. J. Food Prot. 57:776-779.
228. Michard, J., N. Jardy, and J.L. Gey. 1989. Enumeration and localization of Listeria rnonocyto-
genes in soft surface-ripened cheese made from raw milk. Microbiol. Aliments Nutr. 7: 131-
137.
229. Mickova, V., and S. Konecny. 1990. Listeria monocytogenes in foods. Veterinarstyi 40:327-
328.
230. Moir, C.J., M.J. Eyles, and J.A. Davey. 1993. Inhibition of pseudomonads in cottage cheese
by packaging in atmospheres containing carbon dioxide. Food Microbiol. 10:345-35 1.
231. Monge, R., D. Utzinger, and L. Arias. 1994. Incidence of Listeria in pasteurized ice cream
and soft cheese in Costa Rica, 1992. Rev. Biol. Trop. 42:327-328.
232. Nakama, A., T. Maruyama, Y. Kokubo, T. Iida, and F. Umeki. 1992. Incidence of Listeria
monocytogenes in foods in Japan. In: Proceedings of the XIth International. Symposium on
Problems of Listeriosis, Copenhagen, Denmark, May 1 1- 14, p. 162- 163.
233. Nichols, J.G. 1987. Personal communication.
234. Nooitgedagt, A.J., and B.J. Hartog. 1988. A survey of the microbiological quality of Brie
and Camembert cheese. Neth. Milk Dairy J. 4257-72.
235. Northolt, M.D., H.J. Beckers, U. Vecht, L. Toepoel, P.S.S. Soentoro, and H.J. Wisselink.
1988. Listeria monocytogenes: Heat resistance and behavior during storage of milk and whey
and making of Dutch types of cheese. Neth. Milk Dairy J. 42:207-219.
236. Pacini, R., L. Panizzi, E. Quagli, R. Galassi, L. Malloggi, and R. Morganti. 1993. Listeria
rnonocytogenes presence in food products. Indust. Aliment. 32: 1086- 1089.
236a. Papageorgiou, D.K., and E.H. Marth. 1989. Behavior of Listeria monocytogenes at 4 and 22C
in whey and skim milk containing 6 or 12% sodium chloride. J. Food Prot. 52:625-630.
237. Papageorgiou, D.K., and E.H. Marth. 1989. Fate of Listeria monocytogenes during the manu-
facture and ripening of blue cheese. J. Food Prot. 52:459-465.
238. Papageorgiou, D.K., and E.H. Marth. 1989. Fate of Listeria monocytogenes during the manu-
facture, ripening and storage of feta cheese. J. Food Prot. 52:82-87.
239. Papageorgiou, D.K., M. Bori, and A. Mantis. 1996. Growth of Listeria monocytogenes in
the whey cheeses Myzithra, Anthotyros, and Manouri during storage at 5, 12, and 22C. J.
Food Prot. 59: 1 193- 1 199.
240. Piccinin, D.M., and L.A. Shelef. 1995. Survival of Listeria monocytogenes in cottage cheese.
J. Food Prot. 58: 128- 131.
241. Pini, P.N., and R.J. Gilbert. 1988. The occurrence in the U.K. of Listeria species in raw
chickens and soft cheese. Int. J. Food Microbiol. 6:3 17-326.
242. Pinto, B., and D. Reali. 1996. Prevalence of Listeria monocytogenes and other listerias in
Italian-made soft cheeses. Zbl. Hyg. 199:60-68.
L. monocytogenes in Fermented Dairy Products 50 1
243. Pinto, B., S. Rosati, and D. Reali. 1992. Listeria monocytogenes in Italian soft cheese: detec-
tion and incidence. In Proceedings of the 3rd World Congress on Foodborne Infections and
Intoxications, 1:358-360, Berlin, Germany, June 16- 19.
244. Pralt-Lowe, E.L., R.M. Geiger, T. Richardson, and E.L. Barrett. 1988. Heat resistance of
alkaline phosphatase produced by microorganisms isolated from California Mexican-style
cheeses. J. Dairy Sci. 7 1: 17-23.
245. Pucci, M.J., E.R. Vedamuthu, B.S. Kunka, and P.A. Vandebergh. 1988. Inhibition of Listeria
moriocytogenes by using bacteriocin PA- 1 produced by Pediococcus acidilactici PAC 1 .O.
Appl. Environ. Microbiol. 54:2349-2353.
246. Quagilo, G., C. Casolari, G. Menziani, and A. Fabio. 1992. The incidence of Listeria monocy-
togcwes in milk and milk products. LIgiene Moderna 97565-579.
247. Quinto, E., C. Franco, J.L. Rodriguez-Otero, C. Fente, and A. Cepeda. 1994. Microbiological
quality of Cebrero cheese from Northwest Spain. J. Food Safety 14:l-8.
248. Raccach, M., R. McGrath, and H. Daftarian. 1989. Antibiosis of some lactic acid bacteria
including Lactobacillus acidophilus toward Listeria monocytogenes. Int. J. Food Microbiol.
912-5-32.
249. Rajkowski, K.T., S.M. Calderone, and E. Jones. 1994. Effect of polyphosphate and sodium
chloride on the growth of Listeria monocytogenes and Staphylococcus aureus in ultra high
temperature milk. J. Dairy Sci. 77: 1503- 1508.
250. Raris, M., L. Bedin, L. Carraro, M. Pincin, and M. Scagnelli. 1994. Bacteriological survey of
soft cheeses on enforcement of Regione Veneto guidelines (N. 26 August 27, 1990). LIgiene
Moderna 101:217-228.
251. Ribeiro, S.H.S., and D. Carminati. 1996. Survival of Listeria monocytogenes in fermented
milk and yogurt: effect ofpH, lysozyme content and storage at 4C. Sci. Aliments 16:175- 185.
252. Richard, J. 1993. Inhibition of Listeria monocytogenes during cheese manufacture by adding
nisiii to milk and/or using a nisin-producing starter. 1n:Food Ingredients Europe-Confer-
ence Proceedings, Porte de Versailles, Paris, France, Oct. 4-6, p. 58-64.
253. Rodler, M., and W. Korbler. 1989. Examination of Listeria monocytogenes in dairy products.
Acta Microbiol. Hung. 36:259-261.
254. Rorvik, L.M., and M. Yndestad. 1991. Listeria monocytogenes in foods in Norway. Intern.
J. Food Microbiol. 13:97- 104.
255. Rosenow, E.M., and E.H. Marth. 1987. Growth of Listeria monocytogenes in skim, whole
and chocolate milk, and in whipping cream during incubation at 4, 8, 13, 21 and 35C. J.
Food Prot. 50:452-459.
256. Rota, C., J. Yanguela, D. Blanco, J.J. Carraminana, and A. Herrera. 1992. Isolation and
identification of microorganisms of the genus Listeria in soft cheese samples, ripened cheese
samples and processed cheese samples. Alimentaria 3059-62.
257. Roucset, A., and M. Rousset. 1989. Listeria monocytogenes isolkes de fromages prilev6s au
niveau diffirents points de vente. Sci. Aliments 9: 129- 131.
258. Roy, R.N. 1992. Listeria monocytogenes in dairy products and water. In: Proceedings of the
XIth International Symposium on Problems of Listeriosis, Copenhagen, Denmark, May 1 1-
14, 13. 327-328.
259. Ryser, E.T., and E.H. Marth. 1987. Behavior of Listeria monocytogenes during the manufac-
ture and ripening of Cheddar cheese. J. Food Prot. 50:7- 13.
260. Ryser, E.T., and E.H. Marth. 1987. Fate of Listeria monocytogenes during manufacture and
ripening of Camembert cheese. J. Food Prot. 50:372-378.
261. Ryser, E.T., and E.H. Marth. 1987. Unpublished data.
262. Ryser, E.T., and E.H. Marth. 1988. Growth of Listeria monocytogenes at different pH values
in uncultured whey or whey cultured with Penicillium camemberti. Can. J. Microbiol. 34:
730--734.
263. Ryser, E.T., and E.H. Marth. 1988. Survival of Listeria monocytogenes in cold-pack cheese
food during refrigerated storage. J. Food. Prot. 5 1 :615-621, 625.
502 Ryser
264. Ryser, E.T., and E.H. Marth. 1989. Behavior of Listeria monocytogenes during manufacture
and ripening of brick cheese. J. Dairy Sci. 72:838-853.
265. Ryser, E.T., E.H. Marth, and M.P. Doyle. 1985. Survival of Listeria monocytogenes during
manufacture and storage of cottage cheese. J. Food Prot. 48:746-750, 753.
266. Ryser, E.T., S. Maisner-Patin, J.J. Gratadoux, and J. Richard. 1994. Isolation and identifica-
tion of cheese-smear bacteria inhibitory to Listeria spp. Int. J. Food Microbiol. 2 1:237-246.
267. Sarumehmetoglu, R., and S. Kaymaz. 1994. Turk salamura bey az peynirinde yapim ve 01-
gunlasma asamalarinin Listeria monocytogenes uzerine etkisi. Vet. Fak. Derg. 41 :234-242.
268. Schaack, M.M., and E.H. Marth. 1988. Behavior of Listeria monocytogenes in skim milk
and in yogurt mix during fermentation by thermophilic lactic acid bacteria. J. Food Prot. 5 1:
607-614.
269. Schaack, M.M., and E.H. Marth. 1988. Behavior of Listeria monocytogenes in skim milk
during fermentation with mesophilic lactic starter cultures. J. Food Prot. 5 1:600-606.
270. Schaack, M.M., and E.H. Marth. 1988. Survival of Listeria monocytogenes in refrigerated
cultured milks and yogurt. J. Food Prot. 5 12348-852.
271. Schaffer, S.W., S.R. Tatini, and R.J. Baer. 1995. Microbiological safety of blue and Cheddar
cheeses containing naturally modified milk fat. J. Food Prot. 58:132-138.
272. Schoen, R., and G. Terplan. Personal communication.
273. Schonberg, A., P. Teufel, and E. Weise. 1989. Serovars of Listeria monocytogenes and Liste-
ria innocua from food. Acta Microbiol. Hung. 36:249-253.
274. Seeliger, H.P.R. 1961. Listeriosis, Hafner Publishing Co., New York.
275. Seeliger, H.P.R., and D. Jones. 1987. Listeria. In: Bergys Manual of Systematic Bacteriol-
ogy. Williams & Wilkins, Baltimore, pp. 1235- 1245.
276. Shahamat, M., A. Seaman, and M. Woodbine. 1980. Survival of Listeria monocytogenes in
high salt concentrations. Zbl. Bakteriol. Hyg. I Abt. Orig. A 246506-51 1.
277. Sikes, A. 1989. Fate of Staphylococcus aureus and Listeria monocytogenes in certain low
moisture military rations during processing and storage. Annual Meeting of Institute of Food
Technologists, Chicago, June 25-29, Abstr. 466.
278. Sipka, M., S. Zakula, I. Kovincic, and B. Stajner. 1974. Secretion of Listeria monocytogenes
in cows milk and its survival in white brined cheese. 19th International Dairy Congress IE
157.
279. Siragusa, G.R., and M.G. Johnson. 1989. Persistence of Listeria monocytogenes in yogurt
as determined by direct plating and cold enrichment methods. Int. J. Food Microbiol. 7:147-
160.
280. Skinner, K.J. 1989. Listeria-Battling back against one tough bug. Dairy Food Environ.
Sanit. 9:23-24.
281. Stajner, B., S. Zakula, I. Kovincic, and M. Galic. 1979. Heat resistance of Listeria monocyto-
genes and its survival in raw milk products. Vet. Glasnik 33: 109-1 12.
282. Stauber, N.V., R. Braatz, H. Gotz, G. Sulzer, and M. Busse. 1990. Influence of microorgan-
isms on the growth of Listeria in cheese. Deutsche Milchwirtschaft (Hildesheim) 41 :1 126-
1130.
283. Stecchini, M.L., V. Aquili, and I. Sarais. 1995. Behavior of Listeria monocytogenes in mozza-
rella cheese in the presence of Lactococcus Zactis. Int. J. Food Microbiol. 25:301-310.
284. Sulzer, G., and M. Busse. 1993. Behaviour of Listeria spp. during the production of Camem-
bert cheese under various conditions of inoculation and ripening. Milchwissenschaft 48: 196-
199.
285. Sulzer, G., and M. Busse. 1991. Growth inhibition of Listeria spp. on Camembert cheese
by bacteria producing inhibitory substances. Int. J. Food Microbiol. 14:287-296.
286. Tawfik, N.F. 1993. Growth and inactivation of Listeria monocytogenes in Domiati cheese.
Egyptian J. Dairy Sci. 21:l-9.
287. Terplan, G. 1988. Factors responsible for the contamination of food with Listeria monocyto-
genes. WHO Working Group on Foodborne Listeriosis, Geneva, Switzerland, Feb. 15- 19.
L. monocytogenes in Fermented Dairy Products 503
JEFFREY
M. FARBER
AND PEARL
1. PETERKIN
Health Canada, Ottawa, Ontario, Canada
INTRODUCTION
Only within the past 10 years have there been cases of human listeriosis traced to
meat, with evidence for transmission of listeriosis through consumption of contaminated
meats and meat products (see Chap. 10). Products such as piit&, turkey frankfurters,
and sausages have been implicated [ 11 1,1151. In a valuable review, Jay [ 1 111 has
summarized reports on the prevalence of listeriae in meat products from 1971 to 1994
worldwide.
Recently, leading researchers in the United States [13] and the United Kingdom
[ 1441 have stated that all Listeria monocytogenes strains should be considered as poten-
tially pathogenic. With this in mind, and given the ubiquity of this pathogen within slaugh-
terhouse and meat-packing environments, it is not surprising that the incidence and behav-
ior of L. monocytogenes in meat products are receiving increased attention worldwide. In
the beginning of this chapter, results are given for both the IJ.S. Department of Agricul-
ture-Food Safety Inspection Service (USDA-FSIS) Listeria-testing program for cooked
and ready-to-eat meat products and the Agriculture Canada Listeria monocytogenes-moni-
toring program for ready-to-eat meat products. Thereafter results from nonregulatory, in-
dependent surveys of raw meat and of various meat products marketed in North America,
505
506 Farber and Peterkin
I
Cooked beef, roast beef.
and cooked c o r m beef -
September 1987 to present sausage - September
MONITORING: MONITORING:
Intact retail packages - e.g., Non-intact samples from a large
frankfurters, sausage, luncheon unpackaged product - e.g., roast beef,
1
corned beef
meat
Homogenize 25-g sample in 225 ml
1
Homogenize 25-8 sample in 225m1
+
enrichment broth
L. monocytogenesdetected
enrichment broth
4
L. monocytogenesdetected using
using USDA Method USDA Method
+
1
1. FSIS rgquests immediate Class I
recall of the sample production
VERTFICATTON:
Intact sample fiom subsequent
lots 1
Homogenize 25-g sample in
lot 225 ml enrichment broth
2. FSIS initiates
+
L. monocytogenes detected using
(a) Hold-test program for USDA Method
contaminated product
tions for conditions that may allow growth of listeriae, and (d) take any additional appro-
priate in-factory action to prevent production of contaminated product.
The current policy regarding large products that are not normally available in retail-
sized packages (e.g., roast beef, corned beef) (see Fig. 2) more closely resembles the
September 1 987 version of the monitoring/verification program in that USDA-FSIS offi-
cials will not request an immediate Class I recall if L. rnonocytogenes is detected in one
or more monitoring samples. In such instances, verification samples (intact samples from
subsequent lots) will be collected and analyzed for L. rnonocytogenes using current USDA
methodology. Any verification sample that is positive for L. monocytogenes will automati-
cally trigger action similar to that just described for products in intact retail-sized packages
weighing 1 3 lb; included is the issuance of an immediate Class I recall in the unlikely
event that the firm failed to hold all sampled lots until results of Listeria testing became
known.
Raw Meat
In January 1987, the USDA-FSIS began a monitoring and sampling program to determine
the incidence of L. rnonocytogenes in domestic raw beef. This program was not undertaken
to initiate regulatory action against particular firms but rather to provide the agency with
critical background information. During the 26-month period from January 1987 to Febru-
ary 1990, L. rnonocytogenes was isolated from 122 of 1726 (7.1%) 25-g samples of domes-
508 Farber and Peterkin
tically produced raw beef [64]. In 1995, the FSIS reported on the levels of bacteria, includ-
ing L. monocytogenes, in ground beef [21]. The survey, based on 600 1-lb samples of
ground beef from 661 plants, reported an 18% incidence for the organism.
TABLE
1 Incidence of Listeria rnonocytogenes in USDA-FSIS Monitoring Samples
of Cooked and Ready-to-Eat Meat Products, 1993-1996
Number of lots sampleda (% positive)
products examined (see Table 1). In June 1988, monitoring of meat/poultry salads and
spreads was begun, with incidences of 2.2-4.7% being reported from 1993 to 1996.
After a request from the food industry that the FDA reconsider its policy of zero
tolerance for the presence of L. rnonocytogenes in cooked and ready-to-eat foods, the
FDA stated that it would need the support of scientific data on infectious dose to justify
any change in its regulation that the pathogen should not be present in such foods [ 111.
Later, the FDA announced that its pathogen-monitoring program would concentrate on
selected high-risk foods, including prepared sandwiches [22]. The agency stated that if a
prepared sandwich is found to be positive for L. monocytogenes, then a comprehensive
follow-up inspection of the firm is warranted, including sampling of raw materials, food
processing surfaces, and finished product.
TABLE
2 Chronological List of Recalls (Class I or Voluntary) Issued in the United States for Cooked and Ready-to-Eat Meat Products
Contaminated with Listeria rnonocytogenes, 1990-1997
Date recall
Product initiated Origin Distribution Quantity Ref.
Sandwiches-BBQ beef, ham and cheese, etc. 1991 LA LA, FL,AL, MS -4860 units 16
Ham salad 1991 MN MN 460 lb 16
Sandwiches-roast beef, etc. 07/03/9 1 LA LA, FL, AL, MS -8057 units 16
Skinless hot dogs 1991 MI MI 3700 lb 14
Ham salad 1991 wv wv 600 lb 15
Frankfurters-beef, pork 03/20/92 CT CT 3578 lb 17
Sandwiches-roast beef, turkey, subs, ham and cheese, BBQ 06118/92 LA LA, FL,AL, MS NA 18
beef, etc.
Sandwiches-beef, ground beef, pork, ham and cheese, ham 09/25/92 TN TN, NC, SC, GA, AL, NA 19
salad, hot dog, BBQ beef, etc. MS, KY, VA, AS
Sandwiches-ham and cheese, ham salad, etc. 09/08/95 MI MT, IN, OH, IL, WI 20, 563 units 16
Sandwiches-roast beef, hot dog, burgers, sausage, meat ball, 02/16/96 LA LA, FL, AL, MS 20-25,000 units 16
ham and cheese, etc.
Sandwiches-hot dog, ham and cheese, sausage, beef, salami, 0 1122197 MS MS NA 23
etc.
Smoked ham 07131/98 NH NH, VT NA 25a
NA. not available.
Listeria monocytogenes in Meat Products 51 1
TABLE
3 Incidence of Listeria monocytogenes in Agriculture Canada Testing
Samples of Domestic and Imported Ready-to-Eat Meat Products and in Processing
Plants, 1989- 1994
Domestic Imported products
products
no. of samples
no. of samples (% positive) Plant environment
Year (% positive) USA Others no. of samples (% positive)
1989 396 (12) 50 (4) 5 (0) 677 (17)
1990 66 (35) 1279 (2) 3 (0) 2267 (8)
1991 19 (0) 984 (2) 14 (0) 3314 (13)
1992 35 (3) 469 (3) 9 (0) Phase Ia Phase I1 Phase I11
~- ~ ~
positive products showed an incidence of 46% for meat products (data not shown) and
an average of 12% for environmental samples. The incidence dropped sharply during
1991-1992 to levels of 0-3% for meat products, but the contamination in the factory
environment remained about the same. After 1992, only factory environmental samples
were taken for testing domestic products, but a more rigorous testing procedure involving
three phases was instituted. Phase I of the program consisted of taking 10 postprocess
environmental samples of a product contact surface. These 10 samples were composited
into one sample for analysis. Any positive Phase I analysis triggered entrance into Phase
11, consisting of 10 repeat environmental samples, analyzed individually, and a review of
good manufacturing practices (GMP) throughout the plant. Phase 111 was entered if one
or more analyses in Phase I1 were positive, in which case a thorough review of all manufac-
turing procedures was undertaken before collecting further environmental samples for
individual analysis. Between 1992- 1994, the incidence of L. monocytogenes in food pro-
cessing environments dropped sharply, indicating the value of this approach.
The presence of L. monocytogenes in imported ready-to-eat meat products during
this period remained low, with incidences ranging from 0 to 5% (see Table 3). The impor-
tance to Canadian consumers of imports from the United States compared with other
countries is evident from the the relatively large number of American samples tested.
TABLE
4 Class II Recalls issued in Canada for Processed and Ready-to-Eat Meat
Products Contaminated with Listeria monocytogenes, 1989-August 1997
Number of recalls
dry
fermented sandwiches1 sliced cooked
Year wieners sausages subs roast beef ham miscellaneousa
1989 4 2
1990 1 1 2
1991 1 8 3
1992 1 1
I993 1
1994 1
1995 1 2 4 3
1996 15
1997 2 1 1 2
Totals 7 20 19 12 9 8
aPizza, burrito, cretons, pork ribs, ham salad
Source: From Refs. 9 and 24.
1989-1997, 20 recalls were ordered for dry, fermented sausages, some of which were
smoked (Table 4). An almost equal number of recalls were initiated for sandwiches, in-
cluding submarine sandwiches, containing meat. Sliced cold meats of various types also
were found to contain L. monocytogenes during this period, possibly because of contami-
nation from retail slicers. Wieners and miscellaneous products comprised the balance of
the recalls.
TABLE
5 Incidence of Listeria monocytogenes in R a w M e a t a n d Ready-to-Eat M e a t
Products in t h e USA a n d Canada, 1990-1997
Percentage of
Number of positive samples Levels of
samples - Listeria spp.
Product analyzed Listeria spp. LM (CFU/g) Ref.
Raw meats-USA
beef roast 50 116
pork roast 50 LM- 10 1 I6
lamb roast 10 116
ground beef 39 4-560 192
ground pork 20 4-240 192
pork sausage 17 240-2 1000 192
lamb patty 2 4-56 192
beef 658 111
pork loin 135 159
Raw meats-Canada
ground beef 22 73
ground pork 19 73
ground veal 3 73
ground meat 11 135
meat cuts 18 135
wild animal meat- 10 135
moose, deer, bear
RTE products -USA
wieners 93 181
wieners 24 181
wieners 30 20
RTE products -Canada
fermented sausages 30 73
cooked meat 16 170
wieners 38 I70
luncheon meats 67 170
sausages 9 170
LM, L. monocytogenes; ND, not determined; RTE, ready-to-eat.
TABLE
6 Incidence of Listeria monocytogenes in Raw Meat in European and Other
Countries, 1990-1997
% of positive
Number of samples
samples
Country Product analyzed Listeria spp. LM Ref.
United Kingdom Pork sausage 59 79
Beef 26 136
Lamb 20 136
Pork 32 136
Sausage 23 136
Meat 15 96
Beef 1295 184
Lamb 37 184
Pork 794 184
Ireland Beef 20 163
Ground beef 85 163
Pork 20 163
Lamb 20 163
Sausage 20 163
Frozen beef burgers 94 163
Norway Ground meat 40 157
Germany Ground meat 21 111
Rinderhack 59 111
Italy Calf 19 138
Horse 19 138
Pork 19 138
Mutton 18 138
Sausage 156 43
casing surface 116 43
Pork 67 179
Beef 99 179
Ground beef 148 52
Pork 153 52
Ground meat 308 53
Beef 174 53
Raw meat 82 131
Sausage 30 131
Switzerland Ground meat 85 38
Beef 18 38
Pork 31 38
Sausage meat 102 38
Dried meat 44 38
Ham-uncooked 19 38
Spain Ground meat 168 61
Ground pork 42 139
Ground beef 41 139
Poland Pork 245 126
Beef 114 126
Bosnia/Hercegovina Beef 20 133
Pork 50 133
Listeria monocytogenes in Meat Products 515
TABLE
6 Continued
~-
% of positive
Number of samples
samples .
Country Product analyzed Listeria spp. LM Ref.
Bulgaria Beef and pork 234 151
Trinidad Beef 76 1
Ground beef 35 1
Mutton 66 1
Goat meat 70 1
Pork 71 1
United Arab Emirates Beef 15 86
Goat 17 86
Sheep 24 86
Camel 14 86
Australia Beef 50 110
Lamb 50 110
Pork 50 110
Malaysia Beef 12 26
India Cattle 54 37
Buffalo 54 37
Sheep 54 37
Goat 54 37
Japan Beef 15 158
Ground beef 5 158
Pork 18 158
Ground pork 6 158
China Pork 25 182
Beef 10 182
Lamb 14 182
Taiwan Beef steak 25 187
Pork 34 187
LM, L. rnonocytogenes; ND, not determined.
a 5-500 Listerra CFU/g.
According to Gilbert 1791, 49% of raw pork sausage harbored L. monocytogenes, with
MacGowan et al. [136] reporting a similar incidence of 35%.
Working in Ireland, Sheridan et al. [ 1631 observed high incidences of listeriae rang-
ing from 45 to 85% in 20 samples each of beef, pork, and lamb, with L. monocytogenes
being present in 1 5 5 0 % of the samples. Interestingly, less contamination was observed
in comminuted meats (94 samples of frozen beef burgers, 85 samples of ground beef, and
20 samples of sausage), with L. monocytogenes being present in only 5- 18% of the sam-
ples. An incidence of 5% for L. monocytogenes in ground meat was found in Norway
[157], with the pathogen belonging to serotype 1 .
In Germany, about 50% of ground beef and raw beef products reportedly contain
L. rnonocytogenes 11113. Since raw minced meat is a popular dish in Germany, the German
5 16 Farber and Peterkin
Ministry of Health decided to restrict the sale of contaminated product [7]. For samples
containing <100 CFU/g, the factory or retail outlet undergoes further surveillance, and
a public warning is issued if numbers of listeriae reach 2 1000 CFU/g.
Workers in Italy have examined many types of raw meat for listeriae. Maini et al.
[138] reported that in 19 samples each of calf, horse, pork, and mutton, the incidences of
listeriae were 63, 26,53, and 56%, respectively. Cantoni et al. [43] showed that the preva-
lences of the organism on sausages and their casing surfaces were roughly equal; that is,
60 and 47% for listeriae and 13 and 12% for L. monocytogenes, respectively. Other surveys
of fresh pork and beef showed incidences ranging from 21 to 47% for listeriae and 13 to
22% for L. monocytogenes, with serotype 4 being found in 7% of these samples [43].
Comminuted meats and sausages showed higher incidences for Listeria spp. (44-54%)
than for L. monocytogenes (9- 19%) [52,53,13I], with serotype 1/2c predominating [52].
Listeria-positive samples generally contained < 100 CFU/g.
Breer and Schopfer [38] reported that listeriae were recovered from 11-45% of 209
samples of beef, pork, and pork products collected in Switzerland, with an incidence of
0- 15% for L. monocytogenes. In comminuted meat products, the corresponding incidences
were 40-65% and 8- 15%, respectively. Reports from Spain showed that of 25 1 samples
of ground meat, 63-80% contained listeriae, with L. monocytogenes being recovered from
17-29% of the samples [61,139].
In Eastern European countries, workers have observed similar levels (8-20%) of
listeriae contamination in 613 samples of fresh beef and pork to those found in Western
Europe [ 126,133,1511. The incidence of L. monocytogenes in these samples ranged from
7 to 10%, with a prevalence (94%) of serotypes 1 to 3 and the remaining being serotype
4 [126].
Other Countries
Reports from elsewhere indicate that the problem of Listeria-contaminated meat is world-
wide and deserves the serious attention of the industry. In general, the prevalence of Liste-
ria spp., and more particularly, L. monocytogenes, is in the same range as that already
discussed for North America and Europe.
In Trinidad, Adesiyun [ I ] identified listeriae in 0- 10% of samples of fresh beef, mut-
ton, and goat meat, with L. monocytogenes being present in 0-4% of the samples. The organ-
isms were prevalent in ground beef samples at similar levels: 1 1 and 6%, respectively. L.
monocytogenes serotypes 1/2c and 4 were found in both local and imported meat.
Similar incidences of listeriae have been reported in the Asian countries. In the
United Arab Emirates, Gohil et al. [86] examined fresh beef ( I 5 samples), goat (17 sam-
ples), sheep (24 samples), and camel (I4 samples) and found that listeriae were present
in 0-21% of the samples. No samples contained L. monocytogenes. Among 50 samples
each of fresh beef, lamb, and pork examined in Australia, listeriae were found in 34, 40,
and 30% and L. monocytogenes in 24, 16, and 10%, respectively [ 1 101. In Malaysia, 50%
of 12 beef samples contained L. monocytogenes [26]. Examination of 54 samples each of
cattle, buffalo, sheep, and goat in India by Erahmbhatt and Anjaria [37] yielded a low
incidence of L. monocytogenes (4-6%). Ryu et al. [ 1581 reported high incidences for
listeriae in Japanese meats, with 40 and 13% of fresh beef and and 61 and 39% of fresh
pork yielding Listeria spp. and L. monocytogenes, respectively. In ground beef and pork
samples, the prevalence was higher, with 80 and 60% and 100 and 67% of the samples
being positive, respectively.
A study done by Wang et al. [182] in China showed a high incidence (from 43 to
Listeria monocytogenes in Meat Products 517
70%) of listeriae in pork, beef, and lamb. However, the incidence of L. monocytogenes
was much lower, ranging from 0 to 28%, with serotypes 1/2a, 1/2b, and 1/2c being identi-
fied. In contrast, meats from Taiwan showed a high incidence, with 24 and 59% of all
beef steak and pork samples respectively being positive for L. monocytogenes [ 1871.
TABLE
7 Incidence of Listeria monocytogenes in Sausages and Ready-to-Eat Meat
Products in European and Other Countries, 1990-1997
% of positive
No. of samples Levels of
samples Listeria spp.
Country /Product analyzed Listeria spp. LM (CFUlg) Ref.
United Kingdom
pit6 696 145
RTE meat and poultry 3939 145
sliced meat
ham 303 177
salami 128 177
tongue 28 177
corned beef 27 177
mixed meats 119 177
prepared sandwiches 91 108
cooked meats 1551 <20- 1000 12
pit6 239 <20- 100 12
salad with meat 15 <20-100 12
sandwiches with meat 237 20- 100 12
cook-chill-meat and 736 79
poultry
salami 67 79
pit6 1834 LM-<200-106 80
pit6 626 LM-<200- 105 80
cook-chill food-meat 992 LM-< 10- 103 101
cook-chill food-meat 854 LM-< 10-103 101
pit6 216 79
cured or smoked meat 29 10
cooked tripe 44 109
Denmark
sliced ham 80 156
sliced rolled sausage 80 156
sliced smoked pork loin 78 156
frankfurters 67 156
Norway
VP processed meat 35 157
France
sausages, pitis, ham 990 128
Italy
pork sausage 55 132
sausage-mixed meat 20 132
ground beef rissoles 45 132
sausage 82 47
wiirstel 118 LM- 10-224 47
pit6 48 47
Switzerland
sliced cured dried beef 26 LM-20 172
salami 59 LM-20 172
mettwurst 14 172
Listeria monocytogenes in Meat Products 5 19
TABLE
7 Continued
% of positive
No. of samples Levels of
samples - Listeria spp.
Country/Product analyzed Listeria spp. LM (CFU/g) Ref.
Spain
cured sausilge 17 30
cooked ham or sausage 15 30
luncheon meat 60 1-100 127
pit6 36 1-100 127
Hungary
dry cured sausage 136 124
fermented sausage 21 124
smoked sausage 23 124
Yugoslavia
fermented sausage 21 40
hot smoked sausage 15 40
VP hot smoked sausage 14 40
South Africa
Vienna sausage 47 180
ham 43 180
cervelat 44 180
Australia
piit6 7 162
luncheon meat 28 162
pit6 25 98
processed meat 25 98
luncheon meat 20 171
VP salami 19 91
VP corned beef 72 91
VP ham 71 91
VP luncheon meat 13 91
salami 132 176
ham 90 176
corned beef 39 176
pit6 7 176
luncheon meat 16 176
New Zealand
RTE pork 34 103
RTE beef 18 103
RTE lamb 3 103
LM, L. monocytogenes; RTE, ready-to-eat; ND, not determined; VP, vacuum-packed.
respectively. Levels of the organism in this study ranged from <20 to 1000 CFU/g. An-
other report on 91 samples of preprepared sandwiches showed a 17% incidence for L.
rnonocytogenes [ 1OS]. Of 13 strains examined, 10 were serotype 1 /2 and 3 were serotype
4. A survey of 1834 and 626 pi% samples in 1989 and 1990 showed incidences of 10
and 4%, respectively, for L. rnonocytogenes with levels of the organism up to 106CFU/
g [80]. Both serotypes 1/2 and 4 were present. In another study, L. rnonocytogenes was
520 Farber and Peterkin
present in 16% of 67 salami samples [79]. Cooked tripe, which is often eaten without
further heating, showed a 9% incidence of L. monocytogenes [ 1091. In cook-chill catering,
often used by hospitals, food is prepared and cooked in a traditional manner, cooled very
rapidly, and maintained chilled (0-3C) for up to 5 days until reheated for use. In three
surveys of cook-chill foods containing meat (736, 992, and 854 samples), the incidence
of L. monocytogenes was 2, 3, and 9%, respectively [79,101]. Of 28 strains typed, 7 were
serotype 1/2, 17 were serotype 3, and 4 were serotype 4 [ l o l l . These results must give
rise for concern in light of the many highly vulnerable patients in hospitals.
In Denmark, 305 samples of wieners and sliced meats showed similar incidences,
10% of which contained L. monocytogenes, at a level of < 100 CFU/g [ 1561. A Norwegian
study of vacuum packaged processed meat showed that 11% of 35 samples contained L.
monocytogenes [ 1571.
In France, 22% of 18 dry sausages harbored L. monocytogenes, whereas in Germany,
9% of 11 mettwurst samples contained the organism [ 1111.
Several surveys of sausages in Italy showed similar incidences to the above. Levrk
et al. [132], reporting on 120 samples of pork sausage/mixed chicken, turkey, pork
sausage/beef rissoles, found that overall as many as 90% of these samples contained lister-
iae, with 33% being positive for L. monocytogenes. In another study carried out over a
2-year period (1990- 1991), Casolari et al. [47], demonstrated similar incidences in sau-
sage, with 88% and 28% of 82 samples showing the presence of Listeria and L. monocyto-
genes, respectively. Additionally, 36% of 166 meat product samples (wurstel, p2t6) con-
tained listeriae, with L. monocytogenes being present in 4% of these samples at a level
of 10-224 CFU/g.
A survey in Switzerland taken during the production of cured and air-dried meat
products [ 1721 uncovered a substantial number of Bundnerfleisch (cured, air-dried beef),
salami, and mettwurst samples that contained listeriae. Overall, of samples taken during
production, Bundnerfleisch (19 samples), salami (30 samples), and mettwurst (3 samples)
contained 26, 50, and 100% of listeriae and 11, 10, and 0% of L. monocytogenes, respec-
tively (data not shown). The endproducts showed listeriae at incidences ranging from 15
to 93%, in contrast to incidences ranging from 0 to 5% for L. monocytogenes. The organ-
ism was present at levels of <20 CFU/g. Listeriae were isolated only from the surface
of Bunderfleisch. L. monocytogenes isolates were of serotype 1/2 (86%), and serotype 4
(14%). Since most L. monocytogenes isolates from human listeriosis patients in Switzer-
land belong to serotype 4b, these data suggest that transmission of the pathogen via meat
is relatively uncommon.
Surveys of ready-to-eat meat products in Spain showed a 13% incidence for Listeria
spp. in cured or cooked sausages and ham (a total of 32 samples) [30]. Twelve percent of
the cured sausages contained L. rnonocytogenes,with the pathogen being absent in ham and
cooked sausage [30]. Among sliced meat (60 samples) and pi%&(36 samples), 42 and 14%
of samples contained Listeria spp., with 22 and 3% of samples being positive for L. monocyto-
genes, respectively [ 1271. Both organisms were found at levels of 1- 100 CFU/g.
In 1991, a Hungarian researcher, KovAcsn6 Domjan [ 1241, recovered listeriae from
37 of 136 (27%), 7 of 21 (33%), and 15 of 23 (65%) samples of dry-cured, fermented,
and smoked pork sausage, respectively. L. monocytogenes was present in 10, 10, and 13%
of these samples, respectively. Working in Yugoslavia, Buntid [42] found that 28 and
19% of 2 1 fermented sausage samples contained listeriae and L. monocytogenes, respec-
tively. In contrast, listeriae were not recovered from hot, smoked sausage. Of 14 surface
samples of vacuum-packed hot smoked sausage, 35 and 2 1% yielded listeriae and L. mono-
cytogenes, respectively, indicating recontamination before and during vacuum packaging.
Listeria monocytogenes in Meat Products 52 1
Overall, the results from Table 7 indicate that a substantial portion of European
processed meat is contaminated with listeriae, including L. monocytogenes. The presence
of listeriae other than L. monocytogenes in processed as well as raw meat may be indicative
of possible contamination with L. rnonocytogenes.
Other Countries
A recent report by Vorster et al. [ 1801 in South Africa showed a low prevalence for listeriae
in 134 retail samples of processed, ready-to-eat meats, with an overall incidence of 8%
in Vienna sausage, ham, and cervelat (see Table 7). None of the samples yielded L. mono-
cytogenes.
Several surveys from Australia show incidences in processed meats which are simi-
lar to those reported from North America and Europe. According to Robertson and co-
workers [162,171], l of 20 (5%) and 3 of 28 (1 l%) sliced meat samples yielded Listeria
spp., with L. monocytogenes being recovered at rates of 0 and 7%, respectively. No Listeria
spp. were recovered from seven samples of pgt6. Hobson et al. [98] reported that in 25
samples each of pgt6 and processed meat, L. monocytogenes was present in 8 and 4%,
respectively. When 175 samples of vacuum-packaged processed meat were examined,
Grau and Vanderlinde [91] reported that 60 of 72 (88%) siimples of corned beef, 29 of
71 (41%) samples of ham, 3 of 13 (23%) samples of luncheon meats, and 1 of 19 ( 5 % )
samples of salami yielded listeriae, with 72,34, 15, and 0% of these samples being contam-
inated with L. monocytogenes, respectively. Sixteen corned beef samples had listeriae
counts >50 CFU/g, and, of these, six counts were >I000 CFU/g. Listeria counts for
salami were <50 CFU/g, and no L. rnonocytogenes was found. In another survey involving
284 samples of ready-to-eat meat products, Varabioff [ 1761 found that except for pit&,
which was free of listeriae, the incidence of listeriae ranged from 5 to 41% (see Table
7); L. rnonocytogenes was present in 4-19% of the samples. Of the isolates recovered,
62% were serotype 1 and 36% were serotype 4. Additional testing of cutting utensils and
cutting boards showed that most of the contamination occurred at the retail level.
A New Zealand survey of 55 samples of ready-to-eat meat products showed listeriae
incidences ranging from 23 to 67% [ 1031. Interestingly, even though mixed-source prod-
ucts had a lower incidence (23%) of listeriae than products made from a single meat (50-
67%), at least half of the isolates from mixed meats were L. monocytogenes, whereas the
pathogen was seldom recovered from single meat products. The authors also reported a
significantly higher incidence for L. monocytogenes in smoked products than in meats
preserved by other methods.
Additional information concerning the various habitats, niches, and relative inci-
dence of listeriae in all facets of the meat industry is needed, as it is now evident that the
presence of L. rnonocytogenes in processed meat products may pose a serious health hazard
to certain individuals. Therefore, it is necessary to control all listeriae in meat-processing
facilities and to design procedures and treatments that will eliminate L. rnonocytogenes
from ready-to-eat processed meat products.
including organ as well as muscle tissue. Thus, the first definitive studies on the behavior
of L. monocytogenes in raw meat were not begun until the 1970s. Even then, to quantitate
this organism in meat products during extended storage accurately, it was generally
deemed necessary to use specially treated sterile meat rather than raw meat products
containing a normal microbial background flora.
Outbreaks of foodborne listeriosis linked to consumption of contaminated cheese
prompted concerns about the microbiological safety of raw and, particularly, ready-to-eat
meat products marketed in North America and Europe. These outbreaks also demonstrated
an urgent need for methods to detect listeriae rapidly and accurately in a wide range of
foods. Subsequent development of the USDA procedure to detect listeriae in meat and
poultry products provided researchers with a method by which to determine the growth
and survival of L. monocytogenes in raw and ready-to-eat products. Recent meat-oriented
epidemiological studies along with several large listeriosis outbreaks linked to meat prod-
ucts suggest that the behavior and control of L. monocytogenes in meat products is likely
to remain an active area of research for some time to come. As in the previous section
of this chapter, information concerning the behavior of L. monocytogenes in raw meat
will be presented first, followed by a discussion of the fate of this pathogen in processed
meat products.
Localization In Tissues
In 1988, Johnson et al. [ 1 141 reported results from a study in which samples of muscle,
organ, and lymphoid tissue as well as feces and blood from several Holstein cows were
Listeria monocytogenes in Meat Products 523
ing an adsorption time of 30 min, approximately 30 and 50% of the original Listeria
inoculum migrated from lean and fat tissue, respectively, to lean tissue after 5 min of
direct contact at ambient temperature. When contamination between fat and lean tissue
was simulated using shorter contact times of 15-60 s, a greater percentage of listeriae
migrated from inoculated fat to uninoculated lean tissue, which in turn likely reflects the
transfer of cells in unadsorbed water from hydrophobic fat to hydrophilic lean tissue. More
important, the fact that bacterial transfer also occurred at 5C with 0.6-9.5% of the original
Listeria population migrating from inoculated to uninoculated lean and/or fat tissue after
an 18-h adsorption period provides a reasonable explanation for the spread of this pathogen
to Listeria-free meat during storage in walk-in coolers.
These findings attest to the hardy nature of L. monocytogenes on the surface of raw
meats and to the need for effective means of reducing surface contamination on carcasses.
Regarding the latter, Chung et al. [50] reported that wash solutions containing nisin effec-
tively delayed growth of L. monocytogenes on surfaces of raw meats, particularly when
such products were incubated at refrigeration, rather than ambient temperatures. Although
nisin-producing bacterial starter cultures have been used in the dairy industry for many
years, with the exception of certain types of cheese spread, present laws in North America
still forbid direct addition of nisin to most foods, including raw meat.
Populations of enteric pathogens (i.e., Salmonella spp., enteropathogenic Esche-
richia coli, and Yersinia spp.) on raw meats can be sharply reduced by exposing the
water phase of meat surfaces to 0.2 M lactic acid (pH 2.5) at 21C [ 1141. Although L.
monocytogenes is generally recognized as being more acid tolerant than the previously
mentioned enteric pathogens, this organism is nevertheless inactivated at pH values <4.
Hence, provided that L. monocytogenes is exposed to lactic acid for sufficient time, acid
washes may be somewhat helpful in decreasing Listeria populations on the surface of
animal carcasses.
As noted by Johnson et al. [ 1141, L. monocytogenes was routinely detected in muscle
tissue from cows that were killed 2 days after being inoculated intravenously with the
pathogen. Although some contamination of muscle tissue might have occurred during
sampling, results from this study suggest that L. monocytogenes can enter muscle tissue
via the blood stream.
To further investigate this hypothesis, Johnson et al. [ 1121 examined muscle, liver,
and spleen tissue from two lambs and one calf that had been inoculated intravenously with
L. monocytogenes. Microscopic examination of tissues stained with immunoperoxidase or
Azure A revealed L. monocytogenes cells at levels of 103-104CFU/g in muscle tissue
and 103-106 CFU/g in both liver and spleen tissue. Although L. monocytogenes appeared
to be associated with phagocytes in liver and spleen tissue, the pathogen was observed
in loose connective tissue between muscle fibers and also within the muscle fibers
themselves.
Using the USDA enrichment method, Johnson et al. [ 1 161 subsequently detected L.
monocytogenes serotype 1/2a at 5 10 CFU/g in aseptically removed interior samples from
2 of 50 (4%) and 3 of 50 (6%) retail whole muscle beef and pork roasts, respectively.
One beef sample also was positive for L. innocua and L. welshimeri. Although the presence
of these two nonpathogenic (i.e., noninvasive) Listeria spp. within a whole muscle roast
suggests possible contamination during sampling, other results from Johnson et al. [ 114-
1161 strongly support at least limited transmission of L. monocytogenes from the blood
stream into muscle tissue.
Listeria monocytogenes in Meat Products 525
Raw Beef
Growth and Survival
Interest in behavior of Listeria in raw meat products dates back to at least 1966 when
Sielaff [ 165al inoculated beef, pork, and rabbit with L. rnonocytogenes immediately after
slaughter and examined these samples for listeriae during extended storage at 3-4C.
Under these conditions, the pathogen survived at least 15 days in all three products.
Although this study was among the first to examine the ability of L. rnonocytogenes
to survive in raw beef, information concerning actual growth of this pathogen in raw beef
was not available until 1978. In that year, Gouet et al. [87] reported results from a study
which examined multiplication of L. monocytogenes in sterile minced beef alone and
in combination with a defined microflora. L. monocytogenes failed to grow alone in minced
beef (pH 5.8) stored at 8C, and populations decreased < 10-fold during 17 days of incuba-
tion. In contrast, numbers of listeriae decreased approximately 100-fold in samples of
minced beef (pH 5.8) that were simultaneously inoculated with Lactobacillus plantarurn
and held at 8C for 17 days. These researchers also found that higher concentrations of
L. rnonocytogenes (106 CFU/g) enhanced growth of L. plantarurn. When samples of
minced beef were simultaneously inoculated to contain equal numbers of L. rnonocyto-
genes, Pseudornonasjluorescens, and Escherichia coli, Listrria populations decreased ap-
proximately 10-fold after 24 h of incubation at 8OC, with numbers rapidly increasing after
day 7 (Fig. 3). Rapid growth of listeriae during the latter half of incubation was likely
caused by proteolysis of meat proteins by P. jluorescens, which in turn gradually increased
-
the pH of the meat from 5.8 to 6.8. With a complex microflora consisting of 103CFUI
g each of L. plantarurn, P. jluorescens, E. coli, Micrococcus sp., Clostridiurn perjhkgens,
and Enterococcus (Streptococcus)faecalis, behavior of L. rnonocytogenes was similar to
that previously observed for the pathogen in the presence of P.jluorescens and E. coli, with
listeriae populations reaching approximately 6 X 105CFU/g in minced beef following 17
days at 8C (Fig. 4). A rapid increase in numbers of P. jluorescens before growth of L.
plantarurn again appeared instrumental in raising the pH from 5.8 to 7.2 which in turn
stimulated growth of listeriae. Hence, results from this early study suggested that L. rnono-
cytogenes can grow in temperature-abused retail ground beef, since the microbial composi-
tion of this product was fairly similar to that found in the ground beef inoculated with
the seven different organisms.
In a similar investigation completed 11 years later, Kaya and Schmidt [ 1201 also
found that growth of L. rnonocytogenes in artificially contaminated sterile minced meat
during extended incubation at 8-20 C was suppressed by adding 106Lactobacillus CFUI
g but was unhindered in the presence of 1O6 Pseudornonas sp. CFU/g. However, in contrast
to the previous study by Gouet et al. [87], this L. monocytogenes strain readily grew in
the absence of other microorganisms, with populations increasing approximately 2 and
2 4 orders of magnitude in sterile minced meat after 10 and 1-5 days of storage at 4
and 8-2OoC, respectively. In this study, proteolysis of meat proteins by pseudomonads
apparently was not a prerequisite for growth of L. monocytogenes in sterile minced meat
having an initial pH value of 5.8-6.0.
During 1988 and 1989, three additional studies were done to determine the behavior
of listeriae in ground beef; however, unlike previous investigations, the meat was not
pretreated to eliminate the normal background flora. When ground beef at pH 5.6-5.9
was inoculated to contain 105and 106L. rnonocytogenes Scott A or V7 CFU/g, packaged
526 Farber and Peterkin
- 7.0
l90 1 4
7iI /
/
/
- 6.5
'd
=e
- 6.0
'1
0 ' I I I I - 5.5
1 3 7 11 17
Days
10
1
1 I I I I
0 1 1' 3 7 11 17
Days
on ground beef of pH 6.14 than of pH 5.47. For example, after 28 days of storage at 4"C,
serotypes 3b, 3a, and 1/2a had increased by 2.87, 2.64, and 2.24 logs, respectively, in
high-pH ground beef, whereas strain Scott A did not change significantly in numbers [29].
The authors felt that the antimicrobial effect of low pH may also impact Listeria indirectly,
since altering the natural microbial flora could possibly promote growth of bacteriocin-
producing lactic acid bacteria.
L. monocytogenes appears to behave similarly on the surface of fresh intact beef
muscle. In two studies conducted shortly before L. monocytogenes emerged as a serious
foodborne pathogen, Lee et al. [129,130] dealt indirectly with the incidence and subse-
quent behavior of Listeria spp. along with many other psychrotrophic and mesophilic
organisms present on the surface of hot-boned and conventionally boned beef. In this
study, hot-boned beef was obtained from five steers 2 h after slaughter, vacuum packaged,
and cooled from 32 to 21C. In contrast, conventionally processed beef was obtained from
carcasses that were hung in a cold room at 2C for 2 days after slaughter. Both types of
beef were then examined for mesophilic and psychrotrophic organisms at day 0 when the
surface temperature had decreased to 2 1"C (<1 h for conventionally processed beef) and
again following 14 days of storage at 2C. Nearly 1250 bacterial isolates were subse-
quently identified by computer analysis of 116 miniaturized testdisolate. Although Liste-
ria spp. were never isolated from slow or moderately chilled, hot-boned beef at day 0,
9.1-13.5% of all microorganisms present on the surface of such beef after 14 days were
identified as Listeria spp., some isolates of which were likely L. monocytogenes (Table 8).
Overall, only one isolate from conventionally processed beef was identified as belonging to
the genus Listeria. From these data one can infer that Listeria can grow on the surface
of vacuum-packaged hot-boned beef but not on the surface of unpackaged conventionally
processed beef during 2 weeks of storage at 2C. Since the high water-binding properties
TABLE
8 Generation (GT) and Lag Times (LT) of L.
monocytogenes in Meats
of hot-boned beef make this product particularly well suited for sausage making, wide-
spread use of hot-boned beef by the processed meat industry may be partially related to
the relatively high incidence of listeriae in ready-to-eat meat products.
In a more definitive Australian study reported in 1988, Grau and Vanderlinde [90]
examined the ability of L. monocytogenes to grow on the surface of artificially contami-
nated (102--l O3 CFU/cm2) vacuum-packaged, nonsterile beef striploin during extended
incubation at 0 and 5.3"C. Although L. monocytogenes populations increased in all sam-
ples (and in product exudates that developed in packages) during storage, the extent of
Listeria growth was markedly influenced by incubation temperature, pH of the sample
(5.6 vs 6.01, and type of tissue (lean vs fat). Overall, higher Listeria populations consis-
tently developed on fat tissue, with growth also being more rapid at the higher of the two
incubation temperatures and pH values. Numbers of listeriae on fat tissue of pH 5.6 in-
creased from 5 X 103to 3 X 107 CFU/cm2 during 16 days of incubation, whereas the
pathogen was just beginning to grow on corresponding samples after 7- 14 days of storage
-
at 0C. These researchers also noted that Listeria populations increased < 10- and 1000-
fold on vacuum-packaged meats of pH 5.6 and 6.0, respectively, after 10-1 1 weeks of
storage at 0C. Thus, it appears that two conditions, (a) a storage temperature of 0C and
(b) a product pH value of 55.6, must be met simultaneously to prevent significant growth
of L. monocytogenes in vacuum-packaged raw meats destined for export. Grau and Vand-
erlinde [92] extended their studies by using two models: the modified Arrhenius and
square-root model to examine aerobic growth of L. monocyiogenes on lean and fatty raw
beef tissue. For both lean and fatty tissue, the modified Arrhenius model gave better fits
and estimates of the growth rates. The effect of temperatures between 0 and 30C on the
growth rate could be described by a modified Arrhenius equation: Ln (gen/h) = -205.73
+ 1.2939 X 105/K - 2.0298 X 107/K2,where K = OK. The combined effect of tempera-
ture and pH on the growth rate of the organism on lean beef was best described by the
following equation: Ln (gen/h) = -232.64 + 1.4041 X 105/K - 2.1908 X 107/K2 +
1.1586 X 102/pH - 4.0952 X 102/pH2.For lean meat at pH values of about 5.5-5.6 and
6.0-7.0, the latter equation applied at 2.5 to 35C and 0 to 35"C, respectively. There was
considerable scatter in the measured lag periods for both types of meat, and therefore a
poor fit was observed for both models. In two trials, both models predicted growth on
lean tissue where no growth was observed experimentally. In the first, L. monocytogenes
failed to grow on lean tissue with a mean pH of 5.61 during 13 weeks of storage at OOC,
whereas in the other no growth was observed after 48 h at 43.2"C and pH 5.46. However,
growth of the organism was observed on lean tissue having a mean pH of 5.61 at 2.5"C,
on lean meat of pH values 16.0, and all fatty tissue (pH 5.5-5.7) regardless of storage
temperature. One should be aware, however, that these investigators used only one strain
of L. monocytogenes (which was not a meat strain), and that meats where the contaminat-
ing flora exceeded 10% of the Listeria count were discarded, thus partially eliminating
the effects of background microflora on growth of the organism.
Growth rates predicted by the best equations for lean tissue were compared with
literature data for aerobic growth of L. monocytogenes on several foods. Predicted growth
rates for lean meat were higher than those for corn, clarified cabbage juice, and milk,
whereas food supporting growth rates similar to lean beef included UHT milk, raw and
cooked chicken, and cooked ground meat [92].
Other researchers have found that some models derived from the growth of L. mono-
cytogenes in broth cannot reliably predict growth of the organism in raw pork [83]. For
530 Farber and Peterkin
example, L. monocytogenes grew on pork fat tissue without lag at -0.3"C. At higher
temperature, growth rates were greater than those in unacidified Tryptic Soy Broth (TSB)
held at lower temperatures. Faster growth of the organism on fat tissue as compared with
TSB suggests that fat tissue may contain micronutrients which are not present in TSB
[83]. In addition, the organism grew slower on pork muscle tissue at temperatures 2 15.4"C
than in acidified (pH 5.5) TSB, implying that pork muscle tissue may contain growth
inhibitors which are either absent or present in very low levels both in TSB and fat tissue.
All models published thus far predict that the organism will grow in broth at pH 5.5 and
5C in contrast to the failure of L. monocytogenes to grow on normal pH raw pork muscle
even at temperatures as high as 15C. Only one published study has shown that the organ-
ism can grow on raw meat (beef muscle) of normal pH at low temperatures [92].
sine, serine, and valine), which are reportedly essential for growth of L. monocytogenes,
as first suggested by Khan et al. [ 12 1 ] in 1973.
Luncheon Meats
Since previous studies found that luncheon meat, cooked ham, and cooked breast meat
were the most frequently contaminated cooked meats in The Netherlands, these products
were used in a study to determine the survival and growth of L. monocytogenes [34].
Products were inoculated with low levels (10 CFU/g) of the organism and then stored
under vacuum or an atmosphere of 30% c o 2 / 7 0 % N 2 at 7C for 4-6 weeks. Growth on
vacuum-packed product was similar to that of modified-atmosphere packaged (MAP)
stored meats, with counts increasing up to 108CFU/g after 35 days (Fig. 5). High numbers
of lactic acid bacteria were present but did not affect growth of the organism. However,
the pathogen decreased in number on saveloy (fermented sausage; pH 5.5-5.7) and raw
532 Farber and Peterkin
chicken breast
8 A luncheon meat
0 cooked ham
6
--.
bD
z
$ 4
c(
I I I I I I I
0 5 10 15 20 25 30 35
Time (days)
Coburger ham (pH 4.3-4.5), most likely as a result of the acidity of these products. Grau
and Vandelinde [91] examined growth of L. monocytogenes on both naturally contami-
nated and artificially inoculated processed corned beef and ham. For corned beef stored
at O"C, L. monocytogenes grew at about half the rate of the other microflora, whereas at
9"C, both groups of organisms grew at similar rates. On ham stored at 5OC, the organism
grew at only one third the rate of the other flora. Meat composition, that is, pH, salt, and
residual nitrite, played a role in determining the growth potential for this pathogen. For
example, at OOC, the organism failed to grow on ham containing 170 ppm residual nitrite,
but it did grow on ham with 11 ppm nitrite. Although L. monocytogenes grew at similar
rates on ham stored at 15"C, as the storage temperature decreased, the organism again
grew slower on ham containing the higher level of residual nitrite. Fastest growth was
observed on corned beef of pH 6.2, a, 0.97, and <5 ppm nitrite, with slowest growth
being seen on ham of pH 6.6, a, 0.97, and 170 ppm nitrite. From these inoculated pack
studies, equations were developed to describe the growth of both L. monocytogenes and
the other flora; that is, lactic acid bacteria and Brochothrix thermospacta on ham and
corned beef (Fig. 6). For naturally contaminated corned beef, good agreement was ob-
tained between predicted and actual growth of the organism. As predicted, L. monocyto-
genes could not grow on naturally contaminated ham stored at 0.loC, although slight
growth (i.e., from -0.3 to 6.2 CFU/g) of the organism was observed on product stored
at 4.8"C. Juneja et al. [ 1171 examined the potential for outgrowth of various foodborne
pathogens on cooked ground beef during cooling from 54.4 to 7.2"C within 6, 9, 12, 15,
18 or 21 h. L. monocytogenes was inoculated into ground beef at a level of 103 CFU/g,
the meat was then heated linearly to 60C within 1 h, and then cooled as described above.
The organism was not detected in any beef sample examined. Presumably, the slight heat-
ing and cooling regime was sufficient to reduce levels of the organism below the detectable
level; levels at which the organism remained during the duration of the cooling period
[117]. The fate of L. monocytogenes on unirradiated and irradiated cook-chill roast beef
Listeria monocytogenes in Meat Products 533
I I
7.5
p
4
6
3 2 5.5
3
I
J
bD
.-cd
0 3.5 sB
0
-2 1.5
0 10 20 30 20 40 60
Time (Days)
FIGURE6 Changes in the number of L. monocytogenes (II) and of other (lactic acid
bacteria and B. thermosphacta) flora ( 0 )o n the lean tissue of commercial vacuum
packs of corned beef stored at (a) 4.8"C or (b) 0.1"C. Note the different vertical scales
for Listeria and other flora. Off odors first detected (?I. (Adapted from Ref. 91.)
and gravy was examined at 5 and 10C [89]. The organism grew well on both products,
increasing 5 logs in number on unirradiated beef and gravy over 15 days at 5C and by
6 logs in irradiated products stored at 10C for 23 days. Although the observed lag phase
for L. rnonocytogenes was longer on irradiated as compared with unirradiated product,
the specific growth rates were similar at each storage temperature, suggesting that the
background microflora of beef and gravy did not interfere with growth of L. rnonocyto-
genes. The authors concluded that there would be no increased risk of listeriosis if cook-
chill roast beef and gravy were to be irradiated with 2 kGy [89]. L. rnonocytogenes also
grows well on cooked beef [102] (Table 8). Interestingly, the organism grew at similar
rates both aerobically and anaerobically, although the authors claimed that samples stored
under aerobic conditions probably became anaerobic during the course of the experiment
[102]. L. rnonocytogenes also survived pasteurization (e.g., 91 and 96C for 3 or 5 min)
of precooked beef roasts and then grew when samples were stored at 4 and 10C for up
to 56 and 12 days, respectively [94]. Products stored at 10C supported far better growth
of the surviving listeriae than those stored at 4C; that is, at IOOC, L. rnonocytogenes
reached levels in 12 days that took up to 56 days to attain at the lower storage temperature.
It is well known that the organism can repair itself much better at higher temperatures.
Van Laack et al. [ 1751 examined the effect of three packaging treatments, that is, vacuum
packed directly after hot boning (hot packaged), vacuum packed after chilling for 1 day
(cold packaged), or unpackaged, on survival of L. rnonocytogenes on pork loins stored at
1C for up to 9 days. Populations increased about 1 log (Table 9), demonstrating that the
organism can grow on raw refrigerated pork in the presence of numerous competitors.
Although the data were not analyzed statistically, the type of packaging did not appear
to greatly influence growth.
534 Farber and Peterkin
TABLE
9 Influence of Packaging Treatment on
Numbers of L. rnonocytogenes on Pork Loins During
9 days storage at 1 1C *
Average log,,, CFU/cm2a
Sampling Packaging
day treatment Trial 1 Trial 2
HP 2.68 (5/8)h 2.42 (8/8)
CP 2.48 ( 5 / 8 ) 2.86 (8/8)
cu 2.65 (618) 2.86 (8/8)
HP 2.98 (6/8) 2.86 (818)
CP 2.48 (5/8) 2.86 (8/8)
cu 2.91 (7/8) 2.73 (8/8)
HP 3.36 (8/8) 3.1 1 (8.8)
CP 2.68 ( 5 / 8 ) 2.77 (7/8)
cu 2.64 (6/8) 2.91 (7/8)
HP 3.77 (7/8) 3.1 1 (8/8)
CP 2.92 (7/8) 3.36 (8/8)
cu 2.73 (8/8) 3.19 (7/8)
HP 3.73 (8/8) 3.23 (8/8)
CP 3.19 (7/8) 3.61 (7/8)
cu 3.48 (5/8) 3.23 (5/8)
The issue of whether L. rnonocytogenes can grow on raw and cooked meats is com-
plex. As seen in this section, factors such as pH, a,, background microflora, sodium nitrite
and NaC1, length and temperature of storage, strain type, and history all play a role in
determining the fate of Listeria on a meat surface. Another seemingly important factor
which is often overlooked is the initial inoculum on the product. Although early work
from modeling experiments suggested that initial numbers of organisms present on a food
surface had little or no influence on subsequent outgrowth and growth rate, more recent
work does not substantiate this theory. For example, Farber and Daley [70] found that
when L. monocytogenes was present in very low numbers on meats such as sliced ham,
turkey breasts, wieners, and pSt6 stored at 4OC, its numbers did not increase. Similar results
have been observed for foods other than meats and poultry (J.M. Farber, unpublished
results).
Unfermented Sausage
Even though potentially contaminated raw meats find their way into enormous quantities
of sausage products, with over 200 varieties manufactured in the United States alone, no
information pertaining to the behavior of L. monocytogenes during manufacture and stor-
age of these popular meat products appeared in the scientific literature before 1988. Al-
though the California listeriosis outbreak of 1985 eventually led to the aforementioned
surveys in which Listeria was detected in raw and processed meats, including sausage,
Listeria monocytogenes in Meat Products 535
the early consensus was that consumption of such products did not pose a serious threat
to public health, as shown by a lack of any confirmed cases of meatborne listeriosis.
However, this situation changed in December, 1988, following the report of a breast cancer
patient who developed listerial meningitis and eventually died after consuming turkey
frankfurters that were contaminated with L. monocytogenes.
Unfermented sausages are best classified according to the following five categories,
which are based on the method of manufacture: (a) fresh sausage (e.g., fresh pork sausage,
bratwurst), (b) cooked sausage (e.g., liver sausage, Braunschweiger), (c) cooked smoked
sausage (e.g., frankfurters, bologna), (d) uncooked smoked sausage (e.g., Mettwurst,
smoked country-style pork sausage, kielbasa), and (e) cooked meat specialty items (e.g.,
head cheese). Research efforts have dealt primarily with the behavior of Listeria in sau-
sages belonging to the first three categories.
Fresh Sausage
Although fresh pork sausage is by far the most widely manufactured type of fresh sausage,
this category also includes other well-known varieties such as fresh Italian, breakfast, and
beef sausage as well as fresh bratwurst, Thuringer, and bockwurst. The last two are most
popular in Germany. All varieties of fresh sausage are normally prepared from coarse or
finely comminuted pork, beef, or veal to which water is added along with an array of
spices which varies with the type of sausage. In the United States, certain varieties of
fresh sausage also may contain binders and/or extenders (e.g., cereal, vegetable starch,
nonfat dry milk, dried whey) at levels not exceeding 3.5% by weight. After being
stuffed into natural or artificial casings, the product is twisted and cut to form individual
sausage links which are cooled rapidly to preserve freshness and flavor. Unlike cooked
and fermented sausages, fresh sausages have a short shelf life and must be kept
refrigerated to prevent growth of spoilage organisms, including lactic acid bacteria
and micrococci.
When commercially prepared fresh bratwursts were surface-inoculated to contain
approximately 0.1 or 600 L. monocytogenes CFU/g, vacuum packaged, and stored at
4.4OC, Glass and Doyle [84] found that the pathogen attained populations of 106CFU/g
on organoleptically acceptable 4-week-old bratwursts regardless of the initial inoculum.
As with ham, profuse growth of listeriae on fresh bratwurst was attributed to a pH value
>6 which was maintained by the product throughout the first 4 weeks of refrigerated
storage.
In another study involving fresh sausage, Hughey et al. [ 1061 investigated the ability
of lysozyrne to prevent growth of L. monocytogenes in bratwurst prepared from coarsely
ground pork. After addition of commercial bratwurst spice, distilled water was added
with or without 100 ppm lysozyme and 5 mM ethylenediaminetetraacetic acid (EDTA),
a generally recognized as safe chelating agent that enhances the antibacterial activity of
lysozyme. This meat mixture was then inoculated to contain -4 X 103 CFU/g of L.
monocytogenes and stuffed into natural hog casings which were subsequently linked, sepa-
rated, vacuum packaged, and stored at 5C for 45 days.
As expected from the previous study, Listeria populations increased rapidly in fresh
bratwurst (pH z 6) without added lysozyme or EDTA, reaching levels of > 106CFU/g
following 10 days of refrigerated storage (Fig. 7). L. monocytogenes also behaved similarly
in bratwurst containing lysozyme alone; however, the presence of EDTA alone resulted
in a 15-day lag period, thus preventing the pathogen from reaching populations of 10'
536 Farber and Peterkin
t t
/
/
/
/
/ CI
/
/
/
/
4.......+
/
1 1 I I I
CFU/g until nearly 30 days of storage. In contrast, lysozyme and EDTA acted synergisti-
cally to retard growth of L. monocytogenes in fresh bratwurst. Under these conditions,
the pathogen exhibited a lag period of nearly 2 1 days, that is, approximately 7 days beyond
the normal shelf life of the product, and reached populations of <105 CFU/g following
44 days of refrigerated storage. Although only listeriostatic, the combined use of lysozyme
and EDTA appears to be an effective means of controlling Listeria growth during the
normal shelf life of fresh bratwurst. Furthermore, once growth is prevented, low levels
of L. monocytogenes that occasionally appear in fresh bratwurst (<103CFU/g) should be
readily eliminated by proper cooking.
necessary, frankfurters and other similar sausages are frequently hung in smoking rooms
either before or after cooking. Alternatively, commercially available liquid smoke products
can be added to the sausage emulsion or applied directly to the frankfurter surface before
or during heating. In either event, beside imparting a pleasant smoked flavor to the finished
product, some smoke components (i.e., formaldehyde, acetic acid, creosote, and phenols
with high boiling points) possess bacteriostatic and/or bactericidal properties. After cook-
ing, frankfurters are carefully cooled, packaged, and refrigerated during shipment to
wholesale and retail markets. Skinless frankfurters, which are very popular, are produced
in a similar manner except that the artificial casings are mechanically peeled from the
sausage after cooking or smoking.
Epidemiological data from the Centers for Disease Control and Prevention (CDC)
showing an apparent association between listeriosis and undercooked frankfurters
prompted several thermal resistance studies. Zaika et al. [ 1931 prepared frankfurters from
-
a sausage emulsion inoculated to contain 1O8 CFU/g of L. monocytogenes. After stuffing,
all frankfurters were thermally processed (without smoke) according to a standard com-
mercial heating schedule. These USDA officials found that L. monocytogenes populations
decreased approximately 1000-fold in frankfurters that were heated to an internal tempera-
ture of 7 1.1"C (160F). Based on these data, cooking frankfurters to an internal tempera-
ture of 7 1,I "C would probably eliminate maximum levels of L. monocytogenes (<103
CFU/g) that could conceivably occur in raw frankfurter eniulsions.
Data gathered by the American Meat Institute in 1988 pointed to frankfurters as
being likely carriers of L. monocytogenes and also suggested that poor environmental
conditions before packaging could play a major role in contaminating the finished product
[4]. Moreover, Glass and Doyle [84] reported that this pathogen can proliferate on vacuum-
packaged, artificially contaminated (-0.0 1 L. monocytogenes CFU/g) retail frankfurters
at 4.4"C with populations two to five orders of magnitude higher on organoleptically
acceptable samples after 4 weeks of refrigerated storage. Similarly, L. monocytogenes
increased in number from 5 X 10' to 2.1 X 105MPN/g on vacuum-packed frankfurters
stored at 4C for 20 days. Interestingly, uninoculated control samples which were initially
negative for Listeria contained 1.2 X 102MPN/g after the 20-day storage period [40]. In
a more detailed study examining growth of L. monocytogenes on vacuum-packaged all-
beef, poultry or beef/pork wieners at 5C for up to 28 days, McKellar et al. [ 1431 found
that of 61 wieners analyzed, 40 (65.6%) supported growth of L. monocytogenes. For those
samples supporting growth, an average increase of 1.26 logs was observed within a 14-
day period. Unlike NaCl levels, concentrations of phenol, nitrite and lactic acid bacteria
varied considerably during storage. In addition, average pH levels decreased significantly
by 0.19 pH units during storage. Several statistical models were derived in an attempt to
describe growth and death of the organism in all wiener samples. Although no single
model was completely adequate, the best model implicated initial and final lactic acid
bacteria counts and initial pH as factors influencing growth of L. monocytogenes. Pre-
venting Listeria contamination and subsequent growth is further complicated by present
consumer demands for reduced levels of salt and preservatives, along with longer shelf
life, smaller packages, and greater convenience, all of which will require the processed
meat industry to develop even stricter requirements for processing, cooking, handling,
packaging, and refrigeration of products.
Several studies were initiated by the food industry to examine the feasibility of using
heat to eliminate L. monocytogenes from the surface of finished frankfurters. In one such
study [6], frankfurters were dipped in a broth culture of L. monocytogenes ( 106-108 CFU/
538 Farber and Peterkin
TABLE
10 Fate of L. monocytogenes o n the Surface o f Beef
Frankfurters Sprayed with CharSol Poly-I0 or CharSol
Supreme Liquid Smoke and Stored at 4C for 72 h
L. monocytogenes
Level Initial
(oz/ 100 lb inoculum Inactivation
Treatment frankfurters) (CFU/g) after 72 h (%)
Control 0 5.28 0
CharSol Poly - 10 1.7a 5.30 Growth
3.4a 5.28 0
5.1a 5.16 23.6
8.5 4.84 63.1
12.0 4.67 75.2
CharSol supreme 1 5.02 44.7
3.6a 5.04 42.1
5.4 4.73 71.5
9.0 4.4 1 86.3
12.6 4.30 89.5
aOrganoleptically acceptable concentration of liquid smoke.
Source: Adapted from Ref. 183.
three to four orders of magnitude above initial levels on organoleptically acceptable sam-
ples after 4 weeks at 4.4"C. These findings stressed the importance of following good
manufacturing practices, which will in turn greatly reduce the possibility of listeriae con-
taminating ready-to-eat meats during slicing and packaging.
ent temperature. However, some health concerns have arisen with beef jerky following
linkage to several outbreaks of salmonellosis [48].
This situation prompted Harrison and Harrison [95] to examine the fate of E. coli
0 157 :H7, Salmonella typhimurium, and L. monocytogenes in beef jerky during prepara-
tion and storage. Half of the inoculated beef loin strips were marinated at 4C overnight
and then dried at 60C for 10 h. The remaining samples were heated in marinade to 7 1.7"C
and then dried. L. monocytogenes populations decreased by 1.8 and 6.0 logs after 3 and
10 h of drying, respectively. Cooking to 7 1.7"C before drying led to a 4.5-log decrease
in numbers of the organism, with a further 2-log reduction in numbers occurring during
the 10-day drying period (Fig. 8). After 8 weeks of storage at 25"C, none of the beef
jerky samples yielded pathogens.
Much attention has been given to p2t6 following its incrimination in at least two
foodborne listeriosis outbreaks. However, the growth potential of L. monocytogenes in
p2t6 still remains controversial. De Boer and van Netten [60] reported that inoculated
retail pit6 was a good growth menstruum for L. monocytogenes, with the pathogen reach-
ing levels as high as >8.3 log,, CFU/cm2 after 7 days at 12.5"C when the background
microflora was low (<2.3 CFU/cm2). An inverse correlation was observed between Liste-
ria growth and the presence of lactic acid bacteria. When high numbers of lactic acid
bacteria were present, the pH was low (average of 4.9), and L. monocytogenes increased
slightly or decreased in numbers after 1 week of storage at 7 or 12.5"C. Morris and Ribeiro
[ 1481 also found that L. monocytogenes grew on some naturally contaminated pgtis, reach-
ing levels as high as 2 X 108CFU/g after 21 days of refrigerated storage. However,
other samples failed to support growth. Several other investigators also reported that L.
monocytogenes failed to multiply on retail inoculated p2t6 stored at 4C for up to 3 weeks
[70]. To more fully assess the potential health hazards posed by L. monocytogenes in liver
pit6 products, multifactorial design experiments were conducted to examine the influence
of temperature (4 and lO"C), NaCl (1 and 3%), sodium nitrite (0 and 200 ppm), sodium
erythrobate (0 and 550 ppm), and spice (0 and 0.4%) on growth of L. monocytogenes on
experimental pit6 [75]. A total of 16 different liver p2t6 formulations were prepared and
7
0 Lm (unheated)
6 1
5- Lm (heated)
Y
.3
Y 4-
a
E
3-
5 2-
1-
3
- -
0 "
6 10
Drying time (h) in dehydrator
stored at 4 and 10C. When analysis of variance was usecl to assess the impact of the
various factors on maximum growth rate, temperature was the only factor that affected
the growth rate, with L. monocytogenes growing well in all experimental piit6s.
The generation and lag times for the organism in piit6 can be seen in Table 8. Overall,
potential growth of L. rnonocytogenes in pgt6 appears to be related to pSt6 composition
and pH, initial numbers of lactic acid bacteria, and storage temperature and time.
Fermented Sausage
Sausages classified as fermented undergo a controlled lactic acid-type fermentation, usu-
ally through the action of a commercially produced starter culture added to the meat.
Although all fermented sausages can be further classified according to moisture content
as either semidry or dry, manufacturing procedures for both types are generally similar
until the point of drying. Fermented sausages are normally prepared from comminuted
beef and/or pork to which sugar and various spices are added along with sodium or potas-
sium nitrate and/or nitrite. This meat preparation, known as a mix rather than an emulsion,
is inoculated with a commercial lactic acid bacteria starter culture, which frequently in-
cludes species of Pediococcus (particularly P. cerevisiae and P. acidilactici), Lactobacil-
lus, and Leuconostoc. After stuffing the inoculated sausage mix into natural or artificial
casings, the strings of sausage links are hung in ripening or "green-rooms" at 27-40" C/
80-90% RH. Within 2-3 days, sugar added to the mix is fermented to lactic acid by the
starter culture, which in turn decreases the pH to -5.1 and produces the characteristic
tangy flavor found in fermented sausages. As in cheese making, controlled lowering of
the sausage pH to levels near the isoelectric point of meat protein is crucial for proper
removal of water during later stages of sausage manufacture. Following fermentation,
sausages destined to become semidry varieties containing -50% moisture (e.g., Cervelat-
type sausages and Lebanon bologna) zre normally placed in smokehouses where they are
smoked and cooked to internal temperatures of 60-68C. In contrast, dry sausages which
will ultimately contain -35% moisture (e.g., pepperoni, Genoa, and Milano salamis) are
moved to drying rooms (10-17"C/65-80% RH) where they remain for various times,
depending on the type and size of sausage. Some varieties also may be exposed to cool
smoke before drying; however, unlike semidry varieties, dry sausages are never cooked.
Although fermented sausages keep well because of their relatively high salt content, low
pH, and low moisture (a,) content, both varieties, particularly semi-dry types, should be
refrigerated.
Semidry Fermented Sausage
The relatively severe heat treatment that semidry sausages receive during manufacture is
generally sufficient to eliminate most commonly encountered non-spore-forming food-
borne pathogens. Hence, despite concerns regarding the presence of listeriae in meat prod-
ucts, behavior of L. monocytogenes during manufacture and storage of semidry fermented
sausage has received relatively little attention.
Although L. monocytogenes is unlikely to survive during manufacture of semidry
sausage, ample opportunity exists for this pathogen to contaminate the finished product
during slicing and packaging. Hence, to simulate postprocessing contamination, Glass and
Doyle [84] inoculated slices of commercially produced, fermented semidry sausage to
contain approximately 0.01 or 100 L. monocytogenes CFU/g, after which all samples were
vacuum-packaged and quantitatively examined for listeriae during prolonged incubation
542 Farber and Peterkin
at 4.4"C. Unlike ham, bologna, and frankfurters, the pathogen failed to grow on fermented
semidry sausage of pH 4.8-5.2, with populations generally decreasing 5 10-fold on organ-
oleptically acceptable samples after 6- 12 weeks of refrigerated storage.
Recognizing the likelihood of listeriae being introduced into semidry sausage during
slicing/packaging and surviving throughout the normal shelf life of the product, Cirigliano
et al. [5 11 investigated the possibility of eliminating L. monocytogenes from inoculated
slices of German-type and Polish-type beef sausage ( 104- 105 L. monocytogenes strain
Scott A or V7 CFU/g) by exposing vacuum-packaged product to temperatures of 32.2-
5 1.7"C (90- 125F) for up to 72 h. According to the authors, L. monocytogenes populations
failed to change in product stored at 32.2"C (90F); however, numbers of both Listeria
strains decreased approximately 100-fold after product was held at 373C (100F) for 72
h, with a slightly faster rate of inactivation being observed in Polish-type than German-
type sausage. Although increasing the temperature to 43.3"C (1 10F) eliminated strain
V7 from Polish-type and German-type sausage after 8 and 48 h, respectively, strain Scott
A was not eliminated from either product until completion of a 72-h heat treatment. When
exposed to 48.9 and 51.7"C (120 and 125"F), strain V7 was inactivated in Polish-type
and German-type sausages within 4 and 24 h, respectively. Although somewhat more heat
resistant, strain Scott A was eliminated from both products after 24 h at 51.7"C (125F).
In some instances, objectionable fat losses were observed for both product types; however,
the authors concluded that mild heat treatments can be used to salvage Listeria-contami-
nated German-type or Polish-type sausage without seriously affecting product quality. In
fermented "tea" sausages inoculated with 8 X 106MPN/g of L. monocytogenes, the organ-
ism decreased about 1.5 logs during the initial 4-day ripening period where the pH dropped
from 5.47 to 4.80, decreased another 1.5 logs during the 9-day drying period, and then
remained constant in number during the 20 days of storage at 18-22C [41]. Similar
results were obtained when sausages were inoculated with lower levels of the organism.
The initial a, value as well as those after drying and after storage were 0.974, 0.933, and
0.861, respectively.
Dry Fermented Sausage
Unlike semidry varieties, dry fermented sausages are never exposed to Listeria inactivation
temperatures. Consequently, dry sausages have attracted more attention, as shown by sev-
eral studies that examined the fate of L. monocytogenes during the fermentation, drying,
and storage of hard salami, pepperoni, and several other sausages.
In the first such study, Johnson et al. [ 1141 prepared hard salami from naturally
contaminated (i.e., meat from cows inoculated intravenously with L. monocytogenes) as
well as artificially inoculated ground beef, both of which contained -104 CFU/g of L.
monocytogenes. Glucose, spices, sodium nitrite, and salt were added to the ground beef
along with a glucose-fermenting strain of Pediococcus acidilactici. After stuffing the mix
into casings, all sausages were fermented at 40C for 24 h, dried at 13C for 9 days,
vacuum packaged in gas-impermeable film, and stored at 4C for 12 weeks. As shown in
Fig. 9, listeriae populations decreased approximately 10- and 100-fold during fermentation
(40C/24 h) of hard salami prepared from naturally contaminated and artificially inocu-
lated ground beef, respectively. Inactivation of listeriae during fermentation was primarily
attributed to production of lactic acid (or other metabolites) by the starter culture, with
pH values decreasing from approximately 5.7 to 4.4 by the end of fermentation. Although
numbers of listeriae remained relatively constant in naturally contaminated hard salami
following 9 days of drying (13"C/65% RH), populations in product prepared from artifi-
Listeria monocytogenes in Meat Products 543
5.0
4.0
t-- Naturally Contaminated
\ \
2.0
a
1.0 e----.
a 0
A A A A A A A
t -0- , ' 1 1 I I I-
0 1 2 4 6 8 1 0 14 28 42 56 70 84
Days
cially inoculated ground beef decreased nearly 100-fold during drying. L. monocytogenes
was detected in both products during 8 weeks of refrigerated storage; however, higher
levels of listeriae were recovered from naturally contaminated rather than artificially inoc-
ulated hard salami, thus suggesting that behavior of this pathogen is best studied using
sausage prepared from naturally contaminated rather than artificially inoculated ground
beef. Compositionally, both products were very dry, having a, values of 0.79-0.81 as
compared with -0.9 1 for commercially produced hard salami. Although L. monocyto-
genes might be expected to survive more readily in higher moisture commercial products,
growth of the pathogen in retail hard salami appears unlikely given the presence of 5-
7% NaCl and 100- 150 ppm sodium nitrite combined with a pH of 4.3-4.5 and a relatively
low storage temperature.
In contrast to the study just described, Triissel and Jemmi [I721 reported that L.
monocytogenes populations in salami prepared from a mix inoculated to contain approxi-
mately 103or 107CFU/g of L. monocytogenes decreased 5 10-fold in product of pH 5 5 . 6
during 7 days of ripening at 12-22"C/82-95% RH. After 8 weeks of drying at 10- 17"Cl
78-82% RH, numbers of listeriae in salami of pH 5.4-5.7 decreased to <10 CFU/g
regardless of the initial inoculum. However, using an enrichment procedure, the pathogen
was detected in these sausages after an additional 6-1 1 weeks of drying at 10-17"C/35-
50% RH to a, values of 0.68-0.69. Thus, as was true for certain fermented dairy products
discussed in Chapter 12, small numbers of L. monocytogenes cells also can persist in
fermented dry sausages for at least 14-19 weeks. Farber et al. [74] examined the fate of
544 Farber and Peterkin
effect on 1,. monocytogenes, with viable populations decreasing only about 10-fold. Al-
though holding pepperoni for 4 h at 5 l .7"C reduced Listeria populations to undetectable
levels (as determined by direct plating and enrichment), the pathogen was sporadically
recovered from 5- to 22-day-old sausage using the USDA enrichment procedure. Subse-
quent holding of the same pepperoni (pH 4.6) at an internal temperature of 51.7"C for 4
h immediately after 26 days of drying at 123C completely inactivated the pathogen as
determined by direct plating and enrichment procedures. Additional experiments con-
ducted on pepperoni containing 5.3 X 103CFU/g of L. monocytogenes after 19 days of
drying verified that a minimum heat treatment of 4 h at 51.7"C was required to obtain a
Listeria-free product. Thus, although normal processes used to manufacture pepperoni
will not eliminate L. monocytogenes from heavily contaminated product, holding pep-
peroni and possibly other dry sausages at an internal temperature of 5 1.7"C for at least
4 h may prove to be a viable means of salvaging contaminated product.
Although the antibotulinal properties of nitrate, and particularly nitrite, have been
recognized for many years, much remains to be learned concerning the effect of these
preservatives on Listeria behavior in dry fermented sausage. Junttila et al. [ 1 181 examined
the ability of L. monocytogenes to survive in dry Finnish sausage containing various levels
of potassium nitrate, sodium nitrite, and salt. All sausage was prepared from a mixture
of ground beef and pork to which sugar, spices, and 3.0 or 3.5% salt were added along
with 50- 1000 pprn potassium nitrate and/or sodium nitrite. After inoculation to contain
- 105CFU/g of L. monocytogenes and a starter culture consisting of Staphylococcus car-
nosus and Lactobacillus plantarum, the sausage mix was stuffed into casings. All sausage
links were fermented 2 days at 23"C, smoked 5 days at 20--22"C, and then dried 1 week
each at 18 and 10C. L. monocytogenes populations in sausage containing commonly used
levels of salt (3.0%) and sodium nitrite ( I 20 ppm) decreased 1.14 orders of magnitude
over 21 days (Fig. 10). Similar findings also were reported when 3.5 rather than 3.0%
salt was used. Increasing the levels of sodium nitrite (200 ppm) and potassium nitrate
(330 ppm) to those commonly used 30 years ago led to somewhat faster inactivation of
listeriae in dry fermented sausage, with inactivation again being most pronounced during
the later stage of drying. Over the same 21-day period, Listeria populations decreased
approximately 3.3 orders of magnitude in sausage containing 3.5% salt and 1000 ppm
potassium nitrite; however, this concentration of potassium nitrate is no longer permitted
in dry fermented sausage. Growth of L. monocytogenes in this product was apparently
suppressed by the combination of salt, sodium nitrite, and a pH of 4.7; however, given
the pathogen's known tolerance to salt, acid, and low temperatures, addition of commonly
used levels of sodium nitrite to fermented sausage was only marginally effective in inacti-
vating listeriae. Thus, although this and other studies have provided valuable information
concerning the behavior of L. monocytogenes in sausage products, an understanding of
interactions between various factors such as starter cultures, food additives, and various
heat treatments is still needed to develop suitable methods to eliminate L. monocytogenes
from fermented sausage and other processed meat products.
Modified-Atmosphere Packaging
Modified-atmosphere packaging (MAP) can extend the shelf life of many perishable foods
such as meats and poultry. The C02-enriched atmosphere which is created within a meat
pack can inhibit normal spoilage flora and select for certain groups of organisms such as
the lactic acid bacteria [69]. Concerns have been raised about the ability of L. monocyto-
genes to outgrow the normal spoilage flora on MAP foods. In addition, MAP foods have
546 Farber and Peterkin
,
8
Dryingat 18C ,
I
Drying at 10C
I
3.0 I
1 I I I I I 1
I,
N I 1
0 1 2 3 4 5 6 7 14 21
Days
a relatively long shelf life, which in turn can give extra time for psychrotrophic foodborne
pathogens such as L. monocytogenes to grow to high levels. Although Listeria can grow
on vacuum-packed meats such as beef, lamb, and pork, as discussed earlier, the effect of
intermediate to high levels of CO2 on survival and growth of this pathogen on meat and
poultry is not clear [77].
Beef
Growth of L. monocytogenes was observed on samples of vacuum-packaged high-pH (>6)
beef stored at 0, 2, 5, and 10C but not on those vacuum-packs stored at -2C. However,
long lag periods were usually observed, with the organism growing at a slower rate than
the spoilage flora [81]. When samples were packaged under CO2, Listeria only grew at
10C and not at any of the lower storage temperatures tested. However, when normal
ultimate-pH beef (pH 5.3-5.5) was tested, L. monocytogenes was unable to grow on sam-
ples stored in CO2 packs at 5 or 10C [27]. Hence, the lower pH of normal as compared
with dark firm dry (DFD) meat, combined with the high CO2environment, was probably
sufficient to inhibit growth and partially inactivate the organism. As in the findings of
Grau and Vanderlinde [ 8 3 ] ,L. rnonocytogenes grew well on vacuum-packaged meat stored
at 5 and 10C. It is interesting that in the study by Avery et al. [27], L. monocytogenes
outgrew the spoilage flora on vacuum-packaged beef, which is in contrast to the results
obtained by Gill and Reichel [81]. Perhaps L. monocytogenes can compete better with
spoilage organisms at a lower pH. A follow-up study by Avery et al. [28] was designed
to assess the effects of previous high CO2 exposure of Listeria to its subsequent growth
Listeria monocytogenes in Meat Products 547
during abusive retail display. Beef steaks of normal pH were inoculated with L. monocyto-
genes, individually packaged in CO2 packs, and then stored at - 1.5"C for <3 h and 5
or 8 weeks. At the end of each storage period, samples were removed, overwrapped, and
placed on retail display at 12C for up to 140 h. Even after only a brief (<3 h) exposure
to COz, L. rizonocytogenes grew slightly, if at all, during retail display, with demonstrated
lag phases of >75 h. However, no comparative controls were used to confirm growth of
the organism following inoculation and storage at 12C. Experiments were also done
whereby steaks were removed from storage and then rinsed to remove some cells of L.
monocytogmes, which were reinoculated onto freshly cut beef steaks to simulate cross
contamination. Under these conditions, Listeria cells previously exposed to CO, for 5 or
8 weeks did not grow on cross-contaminated steaks. Consequently, it was concluded that
(a) prior exposure of L. monocytogenes-contaminated beef steaks to high CO, environ-
ments will not increase the likelihood for growth of the organism when the steaks are
placed on retail display and possibly temperature abused; and (b) the risk of growth is
minimal when cross contamination occurs between high-C02 stored beef and fresh raw
beef before retail display. Additional experiments have examined survival and growth of
this pathogen on sliced roast beef stored under vacuum or CO2 at - I .5 and 3.0"C. Al-
though unable to grow under CO, at - l SoC, L. monocytogrwes did grow under all other
test conditions. At 3"C, the organism grew three times faster on vacuum-packaged as
compared with CO2-stored roast beef (see Table 8). When growth occurred, maximum
populations were attained only at the end of product shelf life.
Sausages
Experiments were done to determine survival of L. monocytogenes on sliced frankfurters
incubated under 20-80% CO, at 4,7, and 10C for up to 6 weeks [ 1251. Although numbers
of L. mono~ytogenesin vacuum packs, 20% CO,, and 30% CO2 increased by 2.5, 1.0,
and 0.5 logs, respectively, during the commercial minimum shelf life of 3 weeks at 4"C,
the pathogen was inhibited in the presence of both 50 and 80% CO,. During an additional
3 weeks of storage, the organism grew in the presence of 50% but not 80% COz. However,
increasing the storage temperature to 7C permitted growth of the organism during this
3-week storage period even in the presence of 80% COz. Therefore, under commercial
conditions (4-10C, 3-week shelf life), only 80% CO2prevented growth of the organism.
However, since this level of CO2,caused undesirable organoleptic changes in the product,
CO, levels between 50 and 80% may be more appropriate [ 1251.
Lamb
Sheridan et al. [165] examined growth of L. monocytogenes on both raw minced lamb
and lamb pieces stored at 0 and 5C under various atmospheres (vacuum; 80% 02:20%
CO,; 50% 0,:50% CO,; and 100% CO,). No growth was observed on lamb stored at
0C. At 5"C, L. monocytogenes grew on aerobically packaged and vacuum-packaged lamb
pieces but not on minced lamb (Table 1 1 ) . Disregarding aerobically packaged samples,
highest Listcria populations were observed on pieces and minced lamb stored under 80%
0,:20% CO1 and 50% Oz:5O%CO2,respectively, after 42 days of storage at 5C. Again,
L. monocytogenes failed to grow on lamb stored under 100% CO2.
Pork
When the microbial ecology of fresh MAP pork was assessed at various storage tempera-
tures, listeriae were one of the predominant organisms on product stored at - I "C but not
on samples stored at 4.4 or 10C [ 1461. In fact, most bacteriocin-producing organisms
548 Farber and Peferkin
were isolated from samples stored at the two higher temperatures. No growth of L. rnonocy-
togenes was observed on fresh pork longissimus dorsi at 1C regardless of storage atmo-
sphere. At 7"C, L. rnonocytogenes grew on aerobically stored samples but not on those
stored under 100% N,, 80% 02:20% CO, or 60% 0,:40% CO, [140]. No additional
hazards were identified using modified atmosphere (MAs) for packaging fresh pork of
normal pH. According to Davies [59], under an atmosphere of 80% 02:20%CO,, growth
of L. rnonocytogenes on cooked ham was no greater than that observed in aerobically
stored control samples. Manu-Tawiah et al. [ 1411 examined the influence of 20 and 40%
CO2on growth of L. rnonocytogenes and Yersinia enterocolitica on fresh pork chops stored
at 4C for 35 days. Levels of L. rnonocytogenes on air- or vacuum-packaged pork generally
did not differ significantly from the numbers on chops packaged in 20 and 40% CO2,
with populations increasing 1.5 to 2.0 logs (from 3.7-106 CFU/cm2) after 35 days. The
growth rates for Listeria and the aerobic psychrotrophic spoilage flora were faster in air
as compared with C02-packaged samples, with the aerobic psychrotrophic spoilage flora
always outgrowing L. rnonocytogenes. In contrast to the results of Wimpfheimer et al.
[185], no differences were observed in the numbers of L. rnonocytogenes on chops pack-
aged in 60% N2:40% CO, or 50% N,:lO% 02:40% CO2. Y. enterocolitica outgrew L.
rnonocytogenes in all MA-packaged samples, increasing to nearly 108CFU/cm2 after 35
days of storage at 4C. The fact that Yersinia grew much better in MA-packaged than in
aerobically stored chops is disconcerting from a public health standpoint.
The application of additional hurdles in addition to MAP is a strategy that will be
more commonly used in the future. As a case in point, the combined effect of nisin and
MAP on growth and survival of L. rnonocytogenes on cooked pork tenderloin was exam-
ined during storage at 4 and 20C [67]. The organism grew on pork tenderloin packaged
under 100% CO, at both storage temperatures. However, when nisin ( 104 IU/mL) was
added, the combination proved to be bactericidal (Table 12). Although a lower concentra-
tion of nisin ( 103IU/mL) also was listericidal in pork stored at 4O, but not at 20"C, simulta-
neous growth of Pseudornonas fragi, a spoilage organism, was observed. Fewer pseu-
domonads were generally seen in MA-stored samples, with these levels being unaffected
Listeria monocytogenes in Meat Products 549
by nisin. The authors used the general concept of a safety index, which compares the
relative numbers of spoilage organisms with pathogens. In the MAP samples containing
nisin, numbers of P. fragi increased during storage relative to the growth of L. monocyto-
genes. However, such growth was not observed in nisin-free samples. MAP also has been
used in conjunction with irradiation to minimize growth of foodborne pathogens on raw
pork [88]. In nonirradiated pork stored under an atmosphere of 75% N2: 25% COz, L.
monocytogenes populations increased about 2 logs after 9 days of storage at 10C, with
the pathogen being undetected in irradiated (1.75 kGy) control samples. Similar results
were obtained using a higher initial inoculum (106 versus 103 CFU/g). After 9 days of
incubation at 10C, Listeria counts numbered about 4.5 X 104 and 1 X 10s CFU/g in
irradiated and nonirradiated treated samples, respectively. Potential benefits of using irra-
diation after meat packaging were also evident, since lactic acid microflora outgrew L.
monocytogenes and several other pathogens tested during storage [88]. Since Lactobacillus
sake usually predominates the microflora of irradiated MAP pork, sakacin A (a bacteriocin
produced by this organism) may have been responsible for inhibiting the growth of L.
monocytogenes. However, L. monocytogenes grew to similar levels in both irradiated and
nonirradiated samples.
Given these findings, L. monocytogenes will likely grow in the presence of up to
50% CO2, with growth at higher CO2 concentrations mainly dependent on the interplay
between the gas atmosphere, pH, temperature, and other microbial competitors.
coal-fired grill at 110-120C and cooked for 15 min to an internal temperature of 78-
85C. After grilling, L. monocytogenes was isolated from all meatballs that originally
contained 104-1OS listeriae/g. However, the pathogen was recovered from only one of
four meatballs inoculated to contain 103listeriae/g and was absent from meatballs that
originally contained 1O2 listeriae/g. Since data from the European surveys discussed earlier
indicate that retail raw beef may occasionally contain up to 103Listeria CFU/g (some of
which are likely to be L. monocytogenes), thorough cooking of raw meat is presently
advised to eliminate L. monocytogenes as well as E. coli 0157:H7, salmonellae, Campylo-
bacter spp., and other organisms that have been associated with foodborne illness. Al-
though these findings attest to the hardy nature of listeriae in fresh ground beef, L. monocy-
togenes also was equally tenacious in artificially contaminated frozen ground beef, with
populations remaining unchanged at 10' CFU/g during 6 months of storage at - 18C.
Concern about possible resistance of L. monocytogenes to pasteurization of milk,
along with recovery of this pathogen from cooked meats, prompted interest in the possibil-
ity of L. monocytogenes surviving thermal processing steps commonly used to convert
raw meat into ready-to-eat products. Boyle et al. [36] investigated the thermal destruction
of L. monocytogenes strain Scott A in ground beef (-20% fat) by submerging sealed
tubes containing ground beef with 10' listeriae/g in a water bath at 75C until the internal
temperature of samples reached 50, 60, 65, or 70C. Samples then were examined for
listeriae by direct plating as well as selective and cold enrichment. According to these
researchers, Listeria levels remained constant in samples heated to an internal temperature
of 50C over 6.2 min. Numbers of listeriae decreased 4.4-6.1 orders of magnitude in
samples of ground beef during 8.4 and 10.6 min of heating to 60 and 65"C, respectively,
with similar results also being reported in 1988 by Farber et al. [72]. Heating ground pork
to 62C over a period of 25 min was sufficient to inactivate 5.8-7.35 log CFU/g of L.
monocytogenes depending on the pork formulation. Most additives, such as kappa-carra-
geenen, sodium lacate, and algin/calcium binders used in the ground pork formulations,
did not influence thermal resistance of the organism [188]. Similarly, Yen et al. [190]
found that sodium phosphate, sodium erythorbate, and added water had little or no effect
on survival of various L. monocytogenes strains in ground pork during heating. Interest-
ingly, although an added cure decreased Listeria inactivation by 2.0-2.2 logs in ground
pork cooked to 62C as had been noticed previously [69a, 1891, this protective effect was
only seen at temperatures below 67C.
Several studies were done to determine the D- and z-values for L. monocytogenes
in various ground and whole meat products (Table 13). D,,,o,-values for most products
range from 1.8 to 8.3 min. Carlier et al. [45] examined destruction of L. monocytogenes
in whole hams cooked to an internal temperature of 583C. When stored for 2 months
at 9"C, survivors were found among hams inoculated to contain 4 X 10s CFU/g of L.
monocytogenes but not in those inoculated with <10 CFU/g. It was suggested that a
minimum core temperature of 65C be attained in these products, with an F700c-value of at
least 40 min. Another study examined survival of L. monocytogenes in vacuum-packaged,
nitrite-free beef roasts prepared with brines containing selected antimicrobial agents [ 1741.
The brines used included sodium chloride, sodium tripolyphosphate, Brifisol 4 14, acetic
acid, sodium lactate, Lauralac, and potassium sorbate. Meats were cooked in a bag once
or twice to 623C under conditions simulating product contamination from pumping
brines or from slicing/cutting after cooking. Survival of L. monocytogenes on the surface
of beef roasts was surprisingly high considering that cells had possibly been exposed to
temperatures 280C for more than 30 min. Irrespective of the number of cookings, Liste-
Listeria monocytogenes in Meat Products 551
TABLE
13 Heat Resistance of L. rnonocytogenes in Meats
Temp. D-value z-value
Product ("C) ("C; min.) ("Cl Ref.
Ground beef 60 3.12 5.3 72
Fermented sausage mix 60 16.7 4.6
Ground beef roast 60 4.47 160
Ground beef 60 1.62
Beaker sausage 60 9.13
Meat slurry 60 2.54 36
70 0.23
Meat slurry 60 7.3 6.8 155
Beef 60 3.8 7.2 137
70 0.14
Beef steak 60 8.32, 6.27 5.98, 5.98 78
70 0.20, 0.14
Liver sausage slurry 60 2.42 6.2 35
Lean ground beef 62.8 0.6 9.3 68
Fatty ground beef 1.2 11.4
Meats (predicted value) 60 3.82 6.8 I37
70 0.13
Ham 60 1.82 5.05 44
60 3.48a 6.74
Ground pork 62 6.5-7.7 123
Ground pork 60 1.14- 1.7 5.05-5.45 152
aCells heat shocked at 42C for 1 h.
ria survival rates in internally cooked inoculated roasts were similar for all brine treatments
except NaC1-sodium tripolyphosphate, raising the possibility that sublethal heating may
have induced a heat-shock response in these organisms. The highest number of Listeria-
positive samples was seen among roasts processed with standard NaC1-phosphate brines,
which most closely resembles product currently being marketed. In contrast, greatest de-
struction of the organism was observed with brines containing a phosphate blend and
sodium lactate or glycerol monolaurin in combination with one, and especially two, cook-
ings [174].
Steam pasteurization is gaining acceptance as a means of reducing surface levels
of pathogenic microorganisms on meats. Some advantages of using steam pasteurization
include its ability to uniformly heat the entire carcass surface, and uniformly cover irregu-
larly shaped surfaces. Furthermore, waste water accumulation is not an issue, and, by
virtue of automation, the process is not subject to operator misuse. In one study, frankfurt-
ers were inoculated with L. innocua and steam pasteurized in a small pasteurizer designed
in-house. The heating chamber was evacuated for 15 s after which the product was steam
treated at a set time, temperature, and pressure. Treatment times of 32 and 40 s at 136
and I15"C, respectively, led to a &log reduction in counts of L. innocua on the meat
surface while only slightly affecting color and weight [58]. Another study compared steam
pasteurization (S; 15-s steam pasteurization) to traditional methods for reducing pathogens
on meat surfaces [ 1531. The latter methods, which were tested both individually and in
combination, included knife trimming (T), water washing (W; 35"C), hot water/steam
vacuum spot cleaning (V), and spraying with 2% vol/vol lactic acid (pH 2.25 at 54C)
552 Farber and Peterkin
TABLE
14 Effectiveness of Combination and Individual Decontamination
Treatments in Reducing L. monocytogenes on Surfaces of Freshly Slaughtered
Beef
Mean Mean
Treatmenta Initialh reductionc Treatmenta Initialb reductionc
TW 5.52 f. 0.22 4.96 f. 0.34d T 5.26 f 0.33 2.54 f. 0.33'
TWS 5.57 f 0.15 4.56 t 0.34d' W 5.27 t 0.35 1.28 2 0.33g
ws 5.46 f. 0.03 4.40 -t 0.34def V 5.37 f 0.20 3.33 f. 0.33ef
vw 5.56 k 0.12 3.49 2 0.34f S 5.38 f. 0.16 3.44 k 0.33ef
vws 5.49 t 0.04 3.84 ? 0.34" VWLS*5 5.18 f. 0.35 4.51 k 0.33d
TWLS 5.51 k 0.19 5.07 f. 0.34d VWLS*lO 5.21 f. 0.33 4.23 -+ 0.33de
VWLS 5.51 & 0.13 5.01 t 0.34d
a Order of treatment within abbreviation indicates order of application: T, trim; W, 35C water wash; S, 15-s
steam pasteurization; V, hot water/steam vacuum spot cleaning; L, 2% lactic acid spray; S* 5 and S* 10, 5-
and 10-s exposure time, respectively, for steam pasteurization.
Mean initial pathogen population (log CFU/cm') from four replications * standard error of mean.
Mean reduction in pathogen population (log CFU/cm') from four replications 5 standard error of mean.
d,ef.g Means having the same superscript within columns are not significantly different ( P < .05).
.o
2*o*
1Before
treatment
After
treatment
2 7
Time (Days)
14 21
FIGURE11 Effects of moist heat interventions on the initial levels and subsequent
outgrowth of Listeria innocua (least squares means, log CFU/cm2:error bars denote
standard error of the mean) during a combination of aerobic and vacuum storage at
5C for 21 days. (Adapted from Ref. 63.)
[147,169]. Since the general use of bacteriocins to control listeriae has been discussed
earlier in this book (Chap. 6), the following discussion of bacteriocins will be confined
to meat applications.
Raw Ground Meat
In one of the first bacteriocin-related studies, Buchanan and Klawitter [39] assessed the
ability of Curnobacterium piscicola strain LK5 to inhibit growth of L. monocytogenes in
a variety of different foods. Foods were inoculated with 103CFU/g of L. monocytogenes
either with or without 104CFU/g of strain LK5. In sterile raw ground beef, strain LK5
inactivated L. monocytogenes at 5C and prevented its growth at 19C. C. piscicola had
no effect on L. monocytogenes in nonsterile ground beef (or chicken roll), with no growth
of the pathogen being observed in control samples. The bacteriocin-producing strain gener-
ally was most effective in foods containing a background microflora. Similar studies using
sterile and nonsterile ground beef were done with Lactobacillus casei and its associated
bacteriocin, lactocin 705 [ 1781. Meat was inoculated with either L. casei or the pure bacte-
riocin and then stored at 20C for 24 h. In general, inactivation of L. monocytogenes was
greatest at the highest level of lactocin 705 tested ( 1 6,80OAU/mL), with the fewest listeriae
being recovered from a meat slurry and autoclaved ground beef.
Inhibition of L. monocytogenes by starter cultures also has been assessed in mini-
mally heat-treated vacuum-packaged beef cubes either with or without gravy and/or glu-
cose. For these experiments, Lactobacillus bavaricus strain MN was inoculated into beef
at 10sor 103CFU/g, along with 102CFU/g of L. monocytogenes, then vacuum sealed,
and stored at 4 or 10C [ 1861. Strain MN grew and produced bacteriocin during the early
stages of growth, with inhibition of L. monocytogenes being most pronounced at the higher
MN inoculum level and lower incubation temperature. Bacteriocin production was inde-
pendent of the presence of glucose in the meat. Addition of sugar enhanced the antilisterial
activity even though the pH was not greatly reduced in those meats with gravy and glucose.
554 Farber and Peterkin
Greater antilisterial activity was seen in meats containing gravy with glucose, suggesting
that the gravy may have enhanced diffusion of the bacteriocin.
Bacteriocin-producing lactic acid bacteria also have been used to minimize growth
of L. rnonocytogenes on frankfurters. In these experiments, either high ( 107CFU/g) or
low ( l 03- 1O4 CFU/g) levels of a bacteriocin-producing strain of Pediococcus acidilactici,
as well as its plasmid-cured, bacteriocin-negative derivative (bac- ), were inoculated sepa-
rately onto frankfurters along with a five-strain cocktail of L. rnonocytogenes. Frankfurters
were stored both aerobically and anaerobically at 4 and 15C. The presence of P. acidilac-
tici on the frankfurters inhibited listeriae to various degrees depending on the pediococci
levels, storage temperature, and packaging atmosphere [32]. Yousef et al. [ 1913 reported
that L. rnonocytogenes grew in two of five wiener exudates tested, with the best growth
being observed in exudates containing the lowest concentration of phenols, the lowest
indigenous levels of lactic acid bacteria, and the highest pH. Those exudates which did
not support growth of the organism, including those at 4OC, proved to be listericidal. Glass
and Doyle [85] previously showed that L. monocytogenes could grow on wieners stored
at 4.4"C. These conflicting results could be explained by the loss of wiener exudate during
manipulation, uneven distribution of exudate on the wiener surface, or variations in the
intensity of smoking within and among different brands of wieners [ 19I]. Both P. acidilac-
tici H and pure pediocin AcH inactivated listeriae that had been inoculated into one brand
of wiener exudate and stored at 25C for 8 days. In control samples, L. rnonocytogenes
populations increased from about 104to nearly 107CFU/g within 4 days. In a follow-up
study, the latter authors assessed the ability of the same lactic acid bacteria to limit growth
of L. rnonocytogenes in temperature-abused vacuum-packaged wieners stored at 4 and
25C. At 25OC, the presence of P. acidilactici strain LB42 inhibited but did not completely
inactivate L. rnonocytogenes. However, P. acidilactici strain JBL 1095 was listericidal,
decreasing counts of L. rnonocytogenes by 2.7 logs. This inactivation was not solely caused
by pH, since pH values in wieners inoculated with both strains of pediococci were similar.
The only difference between the two strains was that production of pediocin AcH was
confined to JBL 1095, strongly suggesting that this bacteriocin enhanced the antilisterial
activity of lactic acid bacteria in vacuum-packaged wieners.
Fermented Sausages
Similar work also has been done to examine the effects of starter cultures and/or their
bacteriocins on survival and growth of L. rnonocytogenes in sausages. Foegeding et al.
[76] used pediocin-producing strains of P. acidilactici (along with an isogenic mutant as
a control) to minimize growth of L. rnonocytogenes on dry fermented American-style
sausages. Recovery of the organisms during fermentation was made easier by using antibi-
otic-resistant strains of pediococci and listeriae. The study was unique in showing that in
combination with other fermentation endproducts, inhibition of L. rnonocytogenes was
enhanced by bacteriocin production in situ during fermentation and drying. Berry et al.
[31] had also previously shown the benefits of using P. acidilactici as a starter culture to
minimize growth of listeriae during sausage fermentation. According to these authors, a
commercial summer sausage mix was inoculated to contain - 106CFU/g of L. rnonocyto-
genes and fermented with either a bacteriocin-producing or non-bacteriocin-producing
strain of P. acidilactici. Following a 12- to 14-h fermentation at 37.8"C, populations of
listeriae decreased approximately 100-and 10-fold in summer sausage fermented with bac-
teriocin-producing and non-bacteriocin-producing strains of P. acidilactici, respectively.
The pathogen also was inactivated in sausages with slower acid production (pH >5.5),
Listeria monocytogenes in Meat Products 555
REFERENCES
1. Adesiyun, A.A. 1993. Prevalence of Listeria spp., Curnpylobacter spp., Salmonella spp.,
Yersinia spp. and toxigenic Escherichia coli on meat and seafoods in Trinidad. Food Micro-
biol. 10:395-403.
2. Adesiyun, A.A., and C. Krishnan. 1995. Occurrence of Yersinia enterocolitica 0:3, Listeria
rnonocytogenes 0 :4 and thermophilic Curnpylobacter spp. in slaughter pigs and carcasses
in Trinidad. Food Microbiol. 12:99- 107.
3. Amtsberg, G. von, A. Elsner, H.A. Gabbar, and W. Winkenwerder. 1969. Die epidemiolog-
ische und lebensmittelhygienische Bedeutung der Listerieninfektion des Rindes. Dtsch. tier-
arztl. Wochenshr. 76:497-536.
4. Anonymous. 1988. AM1 data show frankfurters most likely Listeria carrier in meat. Food
Chem. News 30(16):37-39.
5. Anonymous. 1988. Hot dog processing borderline for Listeria destruction: ARS. Food
Chem. News 30(41):46.
6. Anonymous. 1988. Listeria destruction in cooked meat products ineffective: Hormel. Food
Chem. News 30( I5):32-34.
7. Anonymous. 1989. Listeria in raw meat restricted in Germany. Food Chem. News 31(31):
28-29.
8. Anonymous. 1989- 1994. Annual Reports. Agri-Food Safety Division, Food Production and
Inspection Branch, Agriculture Canada, Ottawa, Canada.
9. Anonymous. 1989- 1996. Food recalls. Health Protection Branch, Health and Welfare Can-
ada, Ottawa, Canada.
10. Anonymous. 1990. The microbiological safety of food. Part 1. Report of the Committee on
the Microbiological Safety of Food. Her Majestys Stationery Office, p 137.
I I . Anonymous. 1990. Data needed to change zero Listeria tolerance stance: FDA. Food Chem.
News 32(40):9- 10.
12. Anonymous. 199 1. Listeria in food. Report of the West and North Yorkshire Joint Working
Group on a two year survey of the presence of Listeria in food. Environ. Health 99:132-
137.
13. Anonymous. 1991. All L. rnonocytogenes said to be potentially pathogenic. Food Chem.
News 32(45): 18- 19.
14. Anonymous. I99 1 . Skinless hot dogs recalled for Listeria. Food Chem. News, 33( 19):39.
15. Anonymous. 1991. Ham salad recalled due to Listeria. Food Chem. News, 33(40):38.
16. Anonymous. I99 1 - 1997. Food and Drug Administration Enforcement Reports, 1991 - 1997.
Food and Drug Administration, Washington, DC.
17. Anonymous. 1992. Frankfurters recalled by Connecticut firm for Listeria. Food Chem. News
34(5):5 1-52.
18. Anonymous. 1992. Mrs. Drake, Good n Fresh sandwiches recalled due to Listeria. Food
Chem. News, 34(30):36.
19. Anonymous. 1992. Class I recalls involve botulinum, Listeria. Food Chem. News, 34(36):
42.
20. Anonymous. 1993. Listeria found in 20% of hot dogs in L.A. Times survey. Food Chem.
News 35(2 1):45-46.
21. Anonymous. 1995. FSIS issues snapshot of raw meat, poultry microbiological profile. Food
Chem. News 37(43): 19-20.
22. Anonymous. 1996. FDA targets high risk foods in pathogen monitoring program. Food
Chem. News 38(8): 15-1 6.
23. Anonymous. 1997. Possible L. rnonocytogenes contamination prompts recall of fresh sand-
wiches. Food Chem. News 38(49):41.
24. Anonymous. 1997. Food recalls. Canadian Food Inspection Agency, Ottawa, Canada.
25. Anonymous. 1997. Microbiological monitoring of ready-to-eat products, 1993- 1996. United
States Department of Agriculture, Food Safety and Inspection Service, Washington, DC.
Listeria monocytogenes in Meat Products 557
25a. Anonymous. 1998. Goulds smoked breakfast ham recalled from New Hampshire for Liste-
ria. IJSDA-FSIS press release, July 3 1.
26. Arumugaswamy, R.K., G.R.R. Ali, and S.N. Hamid. 1994. Prevalence of Listeria monocyto-
genes in foods in Malaysia. Int. J. Food Microbiol. 23: 1 17- 121.
27. Avery, S.M., J.A. Hudson, and N. Penney. 1994. Inhibition of Listeria monocytogenes on
normal ultimate pH beef (pH 5.3-5.5) at abusive storage temperatures by saturated carbon
dioxide controlled atmosphere packaging. J. Food Prot. 57:33 1-333.
28. Avery, A.M., A.R. Rogers, and R.G. Bell. 1995. Continued inhibitory effect of carbon diox-
ide packaging on Listeria monocytogenes and other microorganisms on normal pH beef dur-
ing abusive retail display. Int. J. Sci. Technol. 30:725-735.
29. Barbosa, W.B., J.N. Sofos, G.R. Schmidt, and G.C. Smith. 1995. Growth potential of individ-
ual strains of Listeria monocytogenes in fresh vacuum-packaged refrigerated ground top
rounds of beef. J. Food Prot. 58:398-403.
30. Benezet, A., J.M. De La Osa, M. Boras, N. Olmo, and F.P. Florea. 1993. Study of Listeria
monocytogenes in meat products. Alimentaria 30: 19-23.
31. Berry, E.D., M.B. Liewen, R.W. Mandigo, and R.W. Hutkins. 1990. Inhibition of Listeria
monocytogenes by bacteriocin-producing Pediococcus during manufacturing of fermented
semitlry sausage. J. Food Prot. 53: 194- 197.
32. Berry, E.D., R.W. Hutkins, and R.W. Mandigo. 1991. The use of bacteriocin-producing Pedi-
ococcus acidilactici to control post processing Listeria monocytogenes contamination of
frankfurters. J. Food Prot. 54:68 1-686.
33. Beuchat, L.R., R.E. Brackett, D.Y.-Y. Hao, and D.E. Conner. 1986. Growth and ther-
mal inactivation of Listeria rnonocytogenes in cabbage juice. Can. J. Microbiol. 32:79 1-
795.
34. Beumer, R.R., M.C. te Giffel, E. de Boer, and F.M. Rombouts. 1996. Growth of Listeria
monocytogenes on sliced cooked meat products. Food Microbiol. 13:333-340.
35. Bhaduri, S.P., W. Smith, S.A. Palumbo, C.O. Turner-Jones, J.L. Smith, B.S. Marmer, R.L.
Buchanan, L.L. Zaika, and A.C. Williams. 1991. Thermal destruction of Listeria monocyto-
genes in liver sausage slurry. Food Microbiol. 8:75-78.
36. Boyle, D.L., J.N. Sofos, and G.R. Schmidt. 1989. Thermal destruction of Listeria monocyto-
genes in a meat slurry and in ground beef. J. Food Sci. 55:327-329.
37. Brahmbhatt, M.N., and J.M. Anjaria, 1993. Analysis of market meats for possible contamina-
tion with listeria. Ind. J. Anim. Sci. 63:687.
38. Breer, C., and K Schopfer. 1988. Listeria and food. Lancet ii: 1022.
39. Buchanan, R.L., and L.A. Klawitter. 1992. Effectiveness of Carnobacterium piscicola LK5
for controlling the growth of Listeria monocytogenes Scott A in refrigerated foods. J. Food
Safety 12:219-236.
40. BunEid, S. 199 I . The incidence of Listeria monocytogenes in slaughtered animals, in meat,
and in meat products in Yugoslavia. Int. J. Food Microbiol. 12:173- 180.
41. BunEid, S., L. Paunovic, and D. Radisic. 1991. The fate of Listeria monocytogenes in fer-
mented sausages and in vacuum-packaged frankfurters. J. Food Prot. 54:413-4 17.
42. Campanini, M., I. Pedrazzoni, S. Barbuti, and P. Baldini. 1993. Behaviour of Listeria mono-
cytogenes during the maturation of naturally and artificially contaminated salami: effect of
lactic-acid bacteria starter cultures. Food Microbiol. 20: 169- 175.
43. Cantoni, C., S. dAubert, M. Valenti, and G. Comi. 1989. Listeria species in cheese and
fresh sausage products. Indust. Aliment. 28: 1068- 1070.
44. Carlier, V., J.C. Augustin, and J. Rozier. 1996. Heat resistance of Listeria monocytogenes
(phagovar 2389/2425/3274/267 1/47/ 108/340): D-and Z-vidues in ham. J. Food Prot. 59:
588-59 1 .
45. Carlier, V., C.A. Jean, and R. Jaques. 1996. Destruction of Listeria monocytogenes during
a ham cooking process. J. Food Prot. 59592-595.
46. Carlin, F., C. Nguyen-the, and A.A. Da Silva. 1995. Factors affecting the growth of Listeria
monocytogenes on minimally processed fresh endive. J. Appl. Bacteriol. 78:636-646.
558 Farber and Peterkin
47. Casolari, C., A. Fabio, G. Menziani, P. Messi, and P. Quaglio. 1994. Characterization of
Listeria monocytogenes strains detected in meat and meat products. LIgiene Moderna 101:
193-21 5.
48. Centers for Disease Control. 1995. Outbreak of salmonellosis associated with beef jerky-
New Mexico, 1995. M.M.W.R. 44:785-788.
49. Chung, K.-T., J.S. Dickson, and J.D. Crouse. 1989. Attachment and proliferation of bacteria
on meat. J. Food Prot. 52:173-177.
50. Chung, K.-T., J.S. Dickson, and J.D. Crouse. 1989. Effects of nisin on growth of Listeria
monocytogenes on meat. Annual Meeting of American Society for Microbiology, New Or-
leans, May 14- 18, Abstr. P-1 1 .
51. Cirigliano, M.C., R.M. Ehioba, and R.T. McKenna. 1989. Personal communication.
52. Comi, G., R. Frigerio, and C. Cantoni. 1992. Listeria monocytogenes serotypes in Italian
meat products. Lett. Appl. Bacteriol. 15:168- 171.
53. Contato, E., M.L. Tempieri, A. Sartea, G. Rossetti, C. Barbieri, G. Mirolo, and G. Bucci.
1994. Isolation of Listeria from food sources. LIgiene Moderna 102:13-21.
54. Cottin, J., H. Genthon, C. Bizon, and B. Carbonnelle. 1985. Recherche de Listeria monocyto-
genes dans des viandes prdevies sur 524 bovins. Sci. Aliment. 5:145-149.
55. Crawford, L.M. 1989. Food Safety and Inspection Service-revised policy for controlling Lis-
teria monocytogenes. Fed. Reg. 54:22345-22346.
56. Cutter, C.N., and G.R. Siragusa. 1994. Decontamination of beef carcass tissue with nisin
using a scale model carcass washer. Food Microbiol. 1 I :48 1-489.
57. Cutter, C.N., and G.R. Siragusa. 1996. Reductions of Listeria innocua and Brochothrix ther-
mosphacta on beef following nisin spray treatments and vacuum packaging. Food Microbiol.
13:23-33.
58. Cygnarowicz-Provost, M., R.C. Whiting, and J.C. Craig, Jr. 1994. Steam surface pasteuriza-
tion of beef frankfurters. J. Food Sci. 59:l-5.
59. Davies, A.R. 1995. Fate of food-borne pathogens on modified-atmosphere packaged meat
and fish. Int. Biodeter. Biodegrad. 407-410.
60. de Boer, E., and P. van Netten. 1990. The presence and growth of Listeria monocytogenes
in pit&.Voedings Middelen Technol. 28: 15-17.
61. De Sirnon M., C. Tarrago, and M.D. Ferrer. 1992. Incidence of Listeria monocytogenes in
fresh foods in Barcelona (Spain). Int. J. Food Microbiol. 16:153-156.
62. Dickson, J.S. 1990. Transfer of Listeria monocytogenes and Salmonella typhimurium be-
tween beef tissue surfaces. J. Food Prot. 53:5 1-55.
63. Dorsa, W.J., C.N. Cutter, and G.R. Siragusa. 1997. Effects of steam-vacuuming and hot
water spray wash on the microflora of refrigerated beef carcass surface tissue inoculated with
Escherichia coli 0 157:H7, Listeria innocua, and Clostridium sporogenes. J. Food Prot. 60:
114-1 19.
64. Dubbert, W.H. 199 1. Personal Communication.
65. El-Khateib, T.A., A.E. Yousef, and H.W. Ockerman. 1993. Inactivation and attachment of
Listeria monocytogenes on beef muscle treated with lactic acid and selected bacteriocins. J.
Food Prot. 56:29-33.
66. Fang, T.J., and L.-W. Lin. 1994. Inactivation of Listeria monocytogenes on raw pork treated
with modified atmosphere packaging and nisin. J. Food & Drug Anal. 2:189-200.
67. Fang, T.J., and L.-W. Lin. 1994. Growth of Listeria monocytogenes and Pseudomonas fragi
on cooked pork in a modified atmosphere packaginghisin combination system. J. Food Prot.
57~479-485.
68. Fain Jr., A.R., J.E. Line, A.B. Moran, L.M. Martin, R.V. Lechowich, J.M. Carosella, and
W.L. Brown. 1991. Lethality of heat to Listeria monocytogenes Scott A: D-value and z-
value determinations in ground beef and turkey. J. Food Prot. 54:756-761.
69. Farber, J.M. 199 1. Microbial aspects of modified-atmosphere packaging technology-a re-
view. J. Food Prot. 54:58-70.
Listeria monocytogenes in Meat Products 559
69a. Farber, J.M., and B.E. Brown. 1989. The effect of prior heat shock on the heat resistance
of Listeria rnonocytogenes in meat (abstr). J. Food Prot. 52:750.
70. Farber, J.M., and E. Daley. 1994. Presence and growth of Listeria rnonocytogenes in natu-
rally-contaminated meats. Food Microbiol. 22:33-42.
71. Farber, J.M., and J. Harwig. 1996. The Canadian position on Listeria rnonocytogenes in
ready-to-eat foods. Food Control 7:253-258.
72. Farber, J.M., A. Hughes, R. Holley, and B. Brown. 1989. Thermal resistance of Listeria
rnonocytogenes in sausage meat. Acta Microbiol. Hung. 36:273-275.
73. Farber, J.M., G.W. Sanders, and M.A. Johnston. 1989. A survey of various foods for the
presence of Listeria species. J. Food Prot. 52:456-458.
74. Farber, J.M., E.F. Daley, R. Holley, and W.R. Usborne. 1993. Survival of Listeria rnonocyto-
genes during the production of uncooked German, American and Italian-style fermented sau-
sages. Food Microbiol. 10:123-1 32.
75. Farber, J.M., R.C. McKellar, and W.H. Ross. 1995. Modelling and the effects of various
parameters on the growth of Listeria monocytogenes on liver p5t6. Food Microbiol. 12:447-
453.
76. Foegeding, P.M., A.B. Thomas, D.H. Pilkington, and T.R. Klaenhammer. 1992. Enhanced
control of Listeria rnonocytogenes by in situ-produced pediocin during dry fermented sausage
production [published erratum appears in Appl Environ Microbiol 1992, 58:2102]. Appl En-
viron Microbiol 58:884-890.
77. Garcia de Fernando, G.D., G.J.E. Nychas, M.W. Peck, and J.A. Ordhez. 1995. Growth/
survival of psychrotrophic pathogens on meat packaged under modified atmospheres. Int. J.
Food Microbiol. 28:221-23 1.
78. Gaze, J.E., G.D. Brown, D.E. Gaskell, and J.G. Banks. 1989. Heat resistance of Listeria
rnonocytogenes in homogenates of chicken, beef steak and carrot. Food Microbiol. 6:25 1-
259.
79. Gilbert, R.J. 1991. Occurrence of Listeria rnonocytogenes i n foods in the United Kingdom.
Proceedings International Conference on Listeria and Food Safety, Laval, France, pp. 82-88.
80. Gilbert, R.J., J. McLauchlin, and S.K. Velani. 1993. The contamination of p5t6 by Listeria
rnonocytogenes in England and Wales in 1989 and 1990. Epidemiol. Infect. 110:543-
551.
81. Gill, C.O., and M.P. Reichel. 1989. Growth of the cold-tolerant pathogens Yersinia enterocol-
itica, Aerornonas hydrophila and Listeria monocytogenes on high-pH beef packaged under
vacuum or carbon dioxide. Food Microbiol. 6:223-230.
82. Gill, C.O., and T. Jones. 1995. The presence of Aerornonas, Listeria and Yersinia in carcass
processing equipment at two pig slaughtering plants. Food Microbiol. 12:135-141.
83. Gill, C.O., G.G. Greer, and B.D. Dilts. 1997. The aerobic growth of Aerornonas hydrophila
and Listeria rnonocytogenes in broths and on pork. Int. J. Food Microbiol. 35:67-74.
84. Glass, K.A., and M.P. Doyle. 1989. Fate of Listeria rnonocytogenes in processed meat prod-
ucts during refrigerated storage. Appl. Environ. Microbiol. 55: 1565- 1569.
85. Glass, K.A., and M.P. Doyle. 1989. Fate and thermal inactivation of Listeria monocytogenes
in beaker sausage and pepperoni. J. Food. Prot. 52:226-231, 235.
86. Gohil, V.S., M.A. Ahmed, R. Davies, and R.K. Robinson. 1995. Incidence of Listeria spp.
in retail foods in the United Arab Emirates. J. Food Prot. 58:102-104.
87. Gouet, Ph., J. Labadie, and C. Serratore. 1978. Development of Listeria rnonocytogenes in
monoxenic and polyxenic beef minces. Zbl. Bakteriol. Hyg., I Abt. Orig. B 166:87-94.
88. Grant, I.R., and M.F. Patterson. 1991. Effect of irradiation and modified atmosphere packag-
ing on the microbiological safety of minced pork stored under temperature abuse conditions.
Int. J. Food Sci. Technol. 26521-33.
89. Grant, I.R., C.R. Nixon, and M.F. Patterson. 1993. Comparison of the growth of Listeria
rnonocytogenes in unirradiated and irradiated cook-chill roast beef and gravy at refrigeration
temperatures. Lett. Appl. Microbiol. 17:55-57.
560 Farber and Peterkin
90. Grau, F.H., and P.B. Vanderlinde. 1990. Growth of Listeria monocytogenes on vacuum pack-
aged beef. J. Food Prot. 53:739-741.
91. Grau, F.H., and P.B. Vanderlinde. 1992. Occurrence, numbers, and growth of Listeria mono-
cytogenes on some vacuum-packaged processed meats. J. Food Prot. 55:4-7.
92. Grau, F.H., and P.B. Vanderlinde. 1993. Aerobic growth of Listeria monocytogenes on beef
lean and fatty tissue: equations describing the effects of temperature and pH. J. Food Prot.
56:96- 101.
93. Green, S. 1997. Personal communication.
94. Hardin, M.D., S.E. Williams, and M.A. Harrison. 1993. Survival of Listeria monocytogenes
in postpasteurized precooked beef roasts. J. Food Prot. 56:655-660.
95. Harrison, J.A., and M.A. Harrison. 1996. Fate of Escherichia coli 0157:H7, Listeria monocy-
togenes and Salmonella typhimurium during preparation and storage of beef jerky. J. Food
Prot. 59: 1336- 1338.
96. Harvey, J., and A. Gilmour. 1993. Occurrence and characteristics of Listeria in foods pro-
duced in Northern Ireland. Int J. Food Microbiol. 19:193-205.
97. Herald, P.J., and E.A. Zottola. 1988. Attachment of Listeria monocytogenes to stainless steel
surfaces at various temperatures and pH values. J. Food Sci. 53:1549-1552, 1562.
98. Hobson, P., I. Baldwin, and P. Weinstein. 1991. Listeria monocytogenes as a contaminant
of food in South Australia. Commun. Dis. Intell. 15:421-426.
99. Hohne, K., 1972. Uber die Entwicklung eines neuen selecktiven Kombinations-Medium zum
Nachweis von Listeria monocytogenes mit einem Hinweis auf den Listeriabefall des
Schlachtschweines. Inaug. Diss., Univ. Wurzburg. In Hohne, K., B. Loose, and H.P.R. See-
liger. 1975. Isolation of Listeria monocytogenes in slaughter animals and bats of Togo (West
Africa). Ann. Inst. Pasteur Microbiol. 126A:501-507.
100. Hohne, K., B. Loose, and H.P.R. Seeliger. 1975. Isolation of Listeria monocytogenes in
slaughter animals and bats of Togo (West Africa). Ann. Inst. Pasteur Microbiol. 126A:501-
507.
101. Houang, E., and R. Hurley. 1991. Isolation of Listeria species from precooked chilled foods.
J. Hosp. Infect. 19:231-238.
102. Hudson, J.A., and S.J. Mott. 1993. Growth of Listeria monocytogenes, Aeromonas hydrophila
and Yersinia enterocolitica on cooked beef under refrigeration and mild temperature abuse.
Food Microbiol. 10:429-37.
103. Hudson, J.A., S.J. Mott, K.M. Delacy, and A.L. Edridge. 1992. Incidence and coincidence
of Listeria spp., motile aeromonads and Yersinia enterocolitica on ready-to-eat fleshfoods.
Int. J. Food Microbiol. 16:99-108.
104. Hudson, J.A., S.J. Mott, and N. Penney. 1994. Growth of Listeria monocytogenes, Aeromonas
hydrophila, and Yersinia enterocolitica on vacuum and saturated carbon dioxide controlled
atmosphere-packaged sliced roast beef. J. Food Prot. 57:204-208.
105. Hugas, M., M. Garriga, M.T. Aymerich, and J.M. Monfort 1995. Inhibition of Listeria in
dry fermented sausages by the bacteriocinogenic Lactobacillus sake' CTC494. J. Appl. Bacte-
riol. 79:322-330.
106. Hughey, V.L., P.A. Wilger, and E.A. Johnson. 1989. Antibacterial activity of hen egg white
lysozyme against Listeria monocytogenes Scott A in foods. Appl. Environ. Microbiol. 55:
63 1-638.
107. Humphrey, T.J., and D.M. Worthington. 1990. Listeria contamination of retail meat slicers.
PHLS Microbiol. Dig. 7:57.
108. Hunter, P.R., H. Hornby, I. Green, and Cheshire Chief Experimental Health Officers Food
Group. 1990. The microbiological quality of pre-packed sandwiches. Br. Food J. 92: 15- 18.
109. Hunter, P.R., B. Cooper-Poole, and H. Hornby. 1992. Isolation of Aeromonas hydrophila
from cooked tripe. Lett. Appl. Microbiol. 15222-223.
110. Ibrahim, A., and I.C. MacRae. 1991. Incidence of Aeromonas and Listeria spp. in red meat
and milk samples in Brisbane, Australia. Int. J. Food Microbiol. 12:263-269.
Listeria monocytogenes in Meat Products 561
111. Jay, J.M. 1996. Prevalence of Listeria spp. in meat and poultry products. Food Contr. 7:
209- 2 14.
112. Johnson, J.L., R.G. Cassens, M.P. Doyle, and J.T. Berry. 1988. Microbiological and histo-
chemical examination of muscle for Listeria monocytogenes. Annual Meeting of Institute of
Food Technologists, New Orleans, June 19-22, Abstr. 324.
113. Johnson, J.L., M.P. Doyle, and R.G. Cassens. 1988. Survival of Listeria monocytogenes in
ground beef. Int. J. Food Microbiol. 6:243-247.
114. Johncon, J.L., M.P. Doyle, R.G. Cassens, and J.L. Schoeni. 1988. Fate of Listeria monocyto-
genes in tissues of experimentally infected cattle and in hard salami. Appl. Environ. Micro-
biol. 54:497-501.
115. Johnson, J.L., M.P. Doyle, and R.G. Cassens. 1990. Listeria monocytogenes and other Liste-
ria spp. in meat and meat products: a review. J. Food Prol. 53:81-91.
116. John5on, J.L., M.P. Doyle, and R.G. Cassens. 1990. Incidence of Listeria spp. in retail meat
roasts. J. Food Sci. 55572, 574.
117. Juneja, V.K., O.P. Snyder Jr., and B.S. Marmer. 1997. Potential for growth from spores of
Bacillus cereus and Clostidium botulinum and vegetative cells of Staphylococcus aureus,
Listeria monocytogenes, and Salmonella serotypes in cooked ground beef during cooling. J.
Food Prot. 60:272-275.
118. Junttila, J., J. Hirn, P. Hill, and E. Nurmi. 1989. Effect of different levels of nitrite and nitrate
on the survival of Listeria monocytogenes during the manufacture of fermented sausage. J.
Food Prot. 52: 158- I6 1.
119. Karaioannoglou, P.G., and G.C. Xenos. 1980. Survival of Listeria monocytogenes in meat-
balls Hell. Vet. Med. 23:lll-117.
120. Kaya, M., and U. Schmidt. 1989. Verhalten von Listeria monocytogenes im Hackfleisch bei
Kuhl-und Gefrierlagerung. Fleischwirtschaft 69:6 1 7-620.
121. Khan, M.A., C.V. Palmas, A. Seaman, and M. Woodbine. 1973. Survival versus growth of
a facultative psychrotroph in meat and products of meat. Zbl. Bakteriol. Hyg., I Abt. Orig.
B 157:127-282.
122. Khan, M.A., LA. Newton, A. Seaman, and M. Woodbine. 1975. Survival of Listeria monocy-
togerzes inside and outside its host. In: Problems of Listeriosis (M. Woodbine, ed.). Leicester
University Press, Surrey, UK, pp 75-83.
123. Kim, K.-T., E.A. Murano, and D.G. Olson. 1994. Heating and storage conditions affect sur-
vival and recovery of Listeria monocytogenes in ground pork. J. Food Sci. 59:30-59.
124. KovAcsni Domjin, H. 1991. Occurrence of Listeria infection in meat-industry raw materials
and end-products. Hung. Vet. J. 46:229-233.
125. Kranier, K.H., and J. Baumgart. 1992. Bruhwurst cold cuts. Fleischwirtschaft 72:666-67.
126. Kwiatek, K. 1991. Incidence of Listeria monocytogenes in beef, pork and poultry meat. Med-
ycyna Wet. 47:69-7 1.
127. Lafarge Gil, M.A., A. Ferrindez Salva, M.P. Martinez Girneno, B. Grasa Quintin, and J.J.
Marcan Letosa. 1994. Study of Listeria monocytogenes and Listeriu spp. in pork-turkey cold
meats and pgtis. Alimentaria 3 1:25-28.
128. Lahellec, C., G. Salvat, and A. Brisebois. 1996. Incidence des Listeria dans les denries ali-
mentaires. Path. Biol. 44:808-8 15.
129. Lee, C.Y., D.Y .C. Fung, and C.L. Kastner. 1982. Computer-assisted identification of bacteria
on hot-boned and conventionally processed beef. J. Food Sci. 47:363-367, 373.
130. Lee, C.Y., D.Y.C. Fung, and C.L. Kastner. 1985. Computer-assisted identification of mi-
croflora on hot-boned and conventionally processed beef Effect of moderate and slow chill-
ing rate. J. Food Sci. 50553-557.
131. Legnani, P., E. Leoni, F. Soppelsa, and P. Bisbini. 1995. Prevalence of Listeria spp. in food
products in the Province of Belluno (Italy). LIgiene Moderna 103:143- 155.
132. Levrk, E., P. Valentini, and G. Caroli. 1993. Listeria spp. in meat products. LIgiene Moderna
100:404-4 16.
562 Farber and Peterkin
133. Loncarevic, S., A. Milanovic, F. Caklovica, W. Tham, and M.-L. Danielsson-Tham. 1994.
Occurrence of Listeria species in an abattoir for cattle and pigs in Bosnia and Hercegovina.
Acta Vet. Scand. 35:ll-15.
134. Lovett, J., D.W. Francis, and J.G. Bradshaw. 1988. Outgrowth of Listeria monocytogenes
in foods. Society for Industrial Microbiology-Comprehensive Conference on Listeria mo-
nocytogenes, Rohnert Park, CA, Oct. 2-5, Abstr. 1-26.
135. Lowry, P.D., and I. Tiong. 1988. The incidence of Listeria monocytogenes in meat and meat
products and factors affecting distribution. Proceedings 34th International Congress Meat
Science Technology Part B:528-530.
136. MacGowan, A.P., K. Bowker, J. McLauchlin, P.M. Bennett, and D.S. Reeves. 1994. The
occurrence and seasonal changes in the isolation of Listeria spp. in shop-bought food stuffs,
human faeces, sewage and soil from urban sources. Int. J. Food Microbiol. 21:325-334.
137. Mackey, B.M., C. Pritchet, A. Norris, and G.C. Mead. 1990. Heat resistance of Listeria:
strain differences and effects of meat type and curing salts. Lett. Appl. Microbiol. 10:251-
255.
138. Maini, P., R. Gaiani, I. Piva, L. Zampino, B. Biccochi, and G. Bucci. 1989. Listeria spp.
and enteric pathogens in raw meat: a survey in the Ferrara area. Boll. 1st. Sieroter. Milan.
68 142-44.
139. Maiieru, L., and I. Garcia-Jal6n. 1995. Listeria rnonocytogenes in foods from the Pamplona
market. Alimentaria 33:39-43.
140. Mano, S.B., G.D. Garcia de Fernando, D. Lbpez, M.D. Selgas, M.L. Garcia, M.I. Cambero,
and J.A. Ord6iiez. 1995. Growth/survival of Listeria monocytogenes on refrigerated pork
and turkey packaged under modified atmospheres. J. Food Safety 15:305-319.
141 Manu-Tawiah, W., D.J. Myers, D.G. Olson, and R.A. Molins. 1993. Survival and growth
of Listeria monocytogenes and Yersinia enterocolitica in pork chops packaged under modi-
fied gas atmospheres. J. Food Sci. 58:475-479.
142. Martins da Silva, R.Z., and U. Schmidt. 1992. Survival of Listeria in fermented sausage
made from poultry meat. Flair 6:79-84.
143. McKellar, R.C., R. Moir, and M. Kalab. 1994. Factors influencing the survival and growth
of Listeria monocytogenes on the surface of Canadian retail wieners. J. Food Prot. 57:387-
392.
144. McLauchlin, J. 1997. The pathogenicity of Listeria monocytogenes: a public health perspec-
tive. Rev. Med. Microbiol. 8:l-14.
145. McLauchlin, J., and R.J. Gilbert. 1990. Listeria in food. PHLS Lab. Dig. 7:54-55.
146. McMullen, L.M., and M.E. Stiles. 1994. Microbial ecology of fresh pork stored under modi-
fied atmosphere at -1, 4.4 and 10C. Int. J. Food Microbiol. 18:l-14.
147 McMullen, L.M., and M.E. Stiles. 1996. Potential for use of bacteriocin-producing lactic
acid bacteria in the preservation of meats. J. Food Prot. (suppl):64-7 1.
147a. Messina, M.C., H.A. Amad, J.A. Marchello, C.P. Gerba, and M.W. Paquette. 1988. The
effect of liquid smoke on Listeria monocytogenes. J. Food Prot. 5 1:629-63 1, 638.
148. Morris, I.J., and C.D. Ribeiro. 1991. The occurrence of Listeria species in pgt6: the Cardiff
experience 1989. Epidemiol Infect 107:111- 1 17.
149. Muriana, P.M. 1996. Bacteriocins for control of Listeria spp. in food. J. Food Prot. (suppl):
54-63.
150. Nesbakken, T., E. Nerbrink, 0.-J. Rotterud, and E. Borch. 1994. Reduction of Yersinia enter-
ocolitica and Listeria spp. on pig carcasses by enclosure of the rectum during slaughter. Int.
J. Food Microbiol. 23: 197-208.
151. Nitcheva, L., V. Yonkova, V. Popov, and C. Maney. 1990. Listeria isolation from foods of
animal origin. Acta Microbiol. Hung. 37:223-225.
152. Ollinger-Snyder, P., F. El-Gazzar, M.E. Mattews. E.H. Marth, and N. Unklesbay. 1995. Ther-
mal destruction of Listeria monocytogenes in ground pork prepared with and without soy
bulls. J. Food Prot. 58:573-576.
Listeria monocytogenes in Meat Products 563
153. Phebus, R.K., A.L. Nutsh, D.E. Schafer, R.C. Wilson, M.J. Rieman, J.D. Leising, C.L. Kas-
tner, J.R. Wolf, and R.K. Prasai. 1997. Comparison of steam pasteurization and other methods
for reduction of pathogens on surfaces of freshly slaughtered beef. J. Food Prot. 60:476-
484.
154. Pociecha, J.Z., K.R. Smith, and G.J. Manderson. 1991. Incidence of Listeria monocytogenes
in meat production environments of a South Island (New Zealand) mutton slaughterhouse.
Int. J. Food Microbiol. 13:321-328.
155. Quintavalla, S., and M. Campanini. 1991. Effect of rising temperature on the heat resistance
of Listeria monocytogenes in meat emulsion. Lett. Appl. Microbiol. 12:184- 187.
156. Qvist, S., and D. Liberski. 199 1. Listeria monocytogenes in frankfurters and ready-to-eat
sliced meat products. Dan. Veterinaertidsskr. 74:773-778.
157. Rorvik, L.M., and M. Yndestad. 1991. Listeria monocytogenes in foods in Norway. Int. J.
Food Microbiol. 13:97- 104.
158. Ryu, C.-H., S. Igimi, S. Inoue, and S. Kumagai. 1992. The incidence of Listeria species in
retail foods in Japan. Int. J. Food Microbiol. 16:157-160.
159. Saide-Albornoz, J.J., C.L. Knipe, E.M. Murano, and G.W. Beran. 1995. Contamination of
pork carcasses during slaughter, fabrication, and chilled storage. J. Food Prot. 58:993-997.
160. Schoeni, J.L., K. Brunner, and M.P. Doyle. 1991. Rates of thermal inactivation of Listeria
monocytogenes in beef and fermented beaker sausage. J. Food Prot. 54:334-337.
161. Schwartz, B., C.V. Broome, G.R. Brown, A.W. Hightower, C.A. Ciesielski, S. Gaventa,
B.G. Gellin, and L. Mascola. 1988. Association of sporadic listeriosis with consumption of
uncooked hot dog and undercooked chicken. Lancet ii:779-782.
162. Seneviratna, P., J. Robertson, I.D. Robertson, and D.J. Hampson. 1990. Listeria species in
food of animal origin. Aust. Vet. J. 67:384.
163. Shelef, L.A. 1980. Survival of Listeria monocytogenes in ground beef or liver during storage
at 4 and 25C J. Food Prot. 52:379-383.
164. Sheridan, J.J., G. Duffy, D.A. McDowell, and I.S. Blair. 1994. The occurrence and initial
numbers of Listeria in Irish meat and fish products and the recovery of injured cells from
frozen products. Int. J. Food Microbiol. 22: 105-1 13.
165. Sheridan, J.J., A. Doherty, P. Allen, D.A. McDowell, I.S. Blair, and D. Harrington. 1995.
Investigations on the growth of Listeria monocytogenes on lamb packaged under modified
atmospheres. Food Microbiol. 12:259-266.
165a. Sielaff, H. 1966. Die lebensmittelhygienische Bedeutung der Listeriose. Monatsh. Veteri-
niirmed. 21:750-758.
166. Siragusa, G.R., J.S. Dickson, and E.K. Daniels. 1993. Isolation of Listeria spp. from feces
of feedlot cattle. J. Food Prot. 56:201-205.
167. Snyder, O.P. 1996. Use of time and temperature specifications for holding and storing food
in retail food operations. Dairy Food Environ. Sanit. 16:374-388.
168. Stegeman, H., B.J. Hartog, F.K. Stekelenburg, and J.P.M. Den Hartog. 1988. The effect
of heat pasteurization on Listeria monocytogenes in canned cured ham. Tenth International
Symposium on Listeriosis, Pecs, Hungary, Aug. 22-26. Abstr. P55.
169. Stiles, M.E. 1996. Biopreservation by lactic acid bacteria. Antonie van Leeuwenhoek. 70:
33 1-345.
170. Tiwari, N.P., and S.G. Aldenrath. 1990. Occurrence of Listeria species in food and environ-
mental samples in Alberta. Can. Inst. Food Sci. Technol. J. 22: 109-1 13.
171. Trott, D., P. Seneviratna, and J. Robertson. 1991. Listeria in cooked chicken, pit6 and mixed
smallgoods. Aust. Vet. J. 68:249-250.
172. Triissel, M. 1989. The incidence of Listeria in the production of cured and air-dried beef,
salami and mettwurst. Schweiz. Arch. Tierheilk. 131:409-421.
173. Triissel, M., and T. Jemmi. 1989. The behavior of Listeria monocytogenes during the ripening
and storage of artificially contaminated salami and Mettwurst. Fleischwirtschaft 69: 1586-
1593.
564 Farber and Peterkin
174. Unda, J.R.R., A. Molins, and H.W. Walker. 199 1. Clostridium sporogenes and Listeria mono-
cytogenes: survival and inhibition in microwave-ready beef roasts containing selected antimi-
crobials. J. Food Sci. 56: 198-206.
175. Van Laack, R.L.J.M., J.L. Johnson, C.J.N.M. Van der Palen, F.J.M. Smulders, and J.M.A.
Snijders. 1993. Survival of pathogenic bacteria on pork loins as influenced by hot processing
and packaging. J. Food Prot. 56:847-85 1.
176. Varabioff, Y. 1992. Incidence of Listeria in smallgoods. Lett. Appl. Microbiol. 14:167- 169.
177. Velani, S., and R.J. Gilbert. 1990. Listeria monocytogenes in prepacked ready-to-eat sliced
meats. PHLS Microbiol. Dig. 756.
178. Vignolo, G., S. Fadda, M.N. de Kairuz, A.A.P. de Ruiz Holgado, and G. Oliver. 1996. Control
of Listeria monocytogenes in ground beef by lactocin 705, a bacteriocin produced by Lacto-
bacillus casei CRL 705. Int. J. Food Microbiol. 29:397-402.
179. Villari, P., M.M. DErrico, E. Prospero, G.M. Grasso, and F. Romano. 1991. Isolation of
Listeria spp. in fresh meats produced in Campania. LIgiene Moderna 96:274-278.
180. Vorster, S.M., R.P. Greebe, and G.L. Nortjk. 1993. The incidence of Listeria in processed
meats in South Africa. J. Food Prot. 56:169-172.
181. Wang, C., and P.M. Muriana. 1994. Incidence of Listeria monocytogenes in packages of
retail franks. J. Food Prot. 57:382-386.
182. Wang, G.-H., K.-T. Yan, X.-M. Feng, S.-M. Chen, A.-P. Lui, and Y. Kokubo. 1992. Isolation
and identification of Listeria monocytogenes from retail meats in Beijing. J. Food Prot. 55:
56-58.
183. Wendorff, W.L. 1989. Effect of smoke flavorings on Listeria monocytogenes in skinless
franks. Seminar presentation, Department of Food Science, University of Wisconsin-Madi-
son, Jan. 13.
184. Wilson, I.G. 1995. Occurrence of Listeria species in ready to eat foods. Epidemiol. Infect.
115:519-526.
185. Wimpfheimer, L., N.S. Altman, and J.H. Hotchkiss. 1990. Growth of Listeria monocytogenes
Scott A, serotype 4, and competitive spoilage organisms in raw chicken packaged under
modified atmosphere and in air. Int. J. Food Microbiol. 11:205-214.
186. Winkowski, K., A.D. Crandall, and T.J. Montville. 1993. Inhibition of Listeria monocyto-
genes by Lactobacillus bavaricus MN in beef systems at refrigeration temperatures. Appl.
Environ. Microbiol. 59:2552-2557.
187. Wong. H.-C., W.-L. Chao, and S.-J. Lee. 1990. Incidence and characterization of Listeria
monocytogenes in foods available in Taiwan. Appl. Environ. Microbiol. 56:3 101-3 104.
188. Yen, L.C., J.N. Sofos, and G.R. Schmidt. 199 1. Effect of meat curing ingredients on thermal
destruction of Listeria monocytogenes in ground pork. J. Food Prot. 54:408-4 12.
189. Yen, L.C., J.N. Sofos, and G.R. Schmidt. 1992. Destruction of Listeria monocytogenes by
heat in ground pork formulated with kappa-carrageenan, sodium lactate and the algin/calcium
meat binder. Food Microbiol. 9:223-230.
190. Yen, L.C., J.N. Sofos, and G.R. Schmidt. 1992. Thermal destruction of Listeria monocyto-
genes in ground pork with water, sodium chloride and other curing ingredients. Lebensm.
Wiss. Technol. 25:61-65.
191. Yousef, A.E., J.B. Luchansky, A.J. Degnan, and M.P. Doyle. 1991. Behavior of Listeria
monocytogenes in wiener exudates in the presence of Pediococcus acidilactici H or pediocin
AcH during storage at 4 or 25C. Appl. Environ. Microbiol. 57:1461-1467.
192. Yu, L.S.L., R.K. Prasai, and D.Y.C. Fung. 1995. Most probable number of Listeria species
in raw meats detected by selective motility enrichment. J. Food Prot. 58:943-945.
193. Zaika, L.L., S.A. Palumbo, J.L. Smith, F. Del Corral, S. Bhaduri, C.O. Jones, and A.H. Kim.
1990. Destruction of Listeria monocytogenes during frankfurter processing. J. Food Prot. 53:
18-21.
Incidence and Behavior of Listeria
monocytogenes in Poultry and
Egg Products
NELSON
A. Cox AND J. STANLEY
BAILEY
Agricultural Research Service, U.S. Department of Agriculture,
Athens, Georgia
ELLIOTT. RYSER
Michigan State University, East Lansing, Michigan
INTRODUCTION
Avian listeriosis was first recognized in 1932 when TenBroeck isolated Listeria monocyto-
genes (then Bacterium monocytogenes) from diseased chickens [ 1 1 1,1121. Chickens have
remained a common avian host for L. monocytogenes since avian listeriosis was first recog-
nized. Listeriosis also has been observed in at least 22 other avian species, including
such frequently consumed fowl as turkeys [25,59,94], ducks [55,113], geese [55,96], and
pheasants [55].Large outbreaks of listeriosis in domestic fowl are relatively rare [93];
however, sporadic cases occur much more frequently and are often accompanied by
asymptomatic shedding of Listeria in feces. According to one report, 4.7% of cecal sam-
ples from Danish broiler chickens harbored L. monocytogenes [96]. Hence, as was true
for beef, pork, and lamb, poultry meat destined for human consumption also is at risk of
becoming contaminated with L. monocytogenes, particularly when birds are slaughtered,
defeathered, and eviscerated. Several early studies suggested that poultry- and egg-pro-
cessing workers can contract Listeria infections by handling contaminated birds
565
566 Cox et al.
[40,71,721. Additionally, several reports from England during the 1970s indicated that L.
monocytogenes could be isolated with some frequency from raw chicken as well as turkey,
duck, and pheasant. Nevertheless, consumption of poultry products has been only recently
linked to listeriosis in humans. An added concern with poultry relates to eggs that might
become contaminated with this pathogen during collecting and processing.
The emergence of L. rnonocytogenes as a bonafide foodborne pathogen following
the Mexican-style cheese outbreak of 1985 prompted immediate concern about presence
of Listeria in dairy products and also generated a parallel interest in the incidence and
behavior of L. rnonocytogenes in meat and poultry products; the latter is the topic of this
chapter. Interest in this area has increased as a result of listeriosis cases that were directly
linked to consumption of turkey frankfurters and ready-to-eat/cook-chill poultry products
in the United States and England, respectively. A discussion of the incidence and behavior
of L. monocytogenes in egg products appears toward the end of this chapter.
USDA-FSIS LISTERIA-MONITORING/VERIFICATION
PROGRAM FOR COOKED/READY-TO-EAT POULTRY
PRODUCTS
Public health concerns about Listeria-contaminated raw and, particularly, processed ready-
to-eat poultry products sold in the United States also stem directly from the 1985 listeriosis
outbreak in California associated with consumption of Mexican-style cheese. Soon thereaf-
ter U.S. Department of Agriculture-Food Safety Inspection Service (USDA-FSIS) offi-
cials announced their intentions to develop Listeria-monitoring/verification programs for
cooked and ready-to-eat meat as well as poultry products. Since these monitoring/verifica-
tion programs for meat and poultry products developed in parallel and were both similar
in terms of sampling scheme, methodology, and action taken when L. rnonocytogenes is
found in a product, the following discussion focuses on the various products tested and
pertinent results rather than on an in-depth analysis of this program.
A Listeria-monitoring/verification program for cooked/ready-to-eat poultry was
first suggested in December 1985 and was to cover all such products prepared in federally
inspected establishments as well as those produced by certified foreign manufacturers [60].
However, actual testing of poultry sausage, that is, the first category of ready-to-eat poultry
products examined, did not begin until September 1988, 1 year after the Listeria-
monitoring/verification program was first instituted for cooked beef, roast beef, and
cooked corned beef.
Before April 1989, the USDAs Listeria policy, which gave firms the opportunity
to clean up their facility before additional verification samples were analyzed under hold-
test procedures, was consistent with the fact that listeriosis had not yet been linked to
consumption of poultry products. However, the official USDA-FSIS position regarding
the presence of L. monocytogenes in cooked and ready-to-eat poultry products changed
radically on April 14, 1989, when Centers for Disease Control and Prevention (CDC)
investigators directly linked consumption of contaminated turkey frankfurters to a case
of listerial meningitis in a breast cancer patient in Oklahoma [ 141. After isolating L. mono-
cytogenes serotype 1/2a of the same electrophoretic enzyme type from the woman and
opened, as well as unopened, retail packages of turkey frankfurters, USDA-FSIS officials
requested that the manufacturer issue an immediate Class I recall for approximately
Listeria monocytogenes in Poultry and Egg Products 567
600,000 pounds of Texas-produced turkey frankfurters that were marketed by retail and
institutional establishments in Alaska, Arizona, Arkansas, California, Florida, Georgia,
Idaho, Illinois, Indiana, Kentucky, Louisiana, Mississippi, Missouri, New Jersey, New
York, Ohio, Oklahoma, Pennsylvania, Tennessee, Texas, Utah, and Washington. As ex-
pected, this recall immediately prompted an intensified monitoring/verification program
to determine the extent of Listeria contamination in a far wider range of cookedheady-
to-eat poultry products marketed in the United States.
Despite pleas by the National Turkey Association to adopt tolerance levels for L.
rnonocytogenes in cooked and ready-to-eat poultry products [9], USDA-FSIS officials
maintained that since an acceptable level of L. monocytogenes in such products could
at that time not be determined, the only acceptable alternative was to adopt a policy of
zero tolerance for this pathogen in cookedheady-to-eat poultry and meat products [ 101.
Consequently, under the program [36] which is identical to that developed for cooked and
ready-to-eat red meat products, USDA-FSIS officials request firms to issue a Class I recall
for all lots of cooked and ready-to-eat poultry products in which L. rnonocytogenes is
detected in monitoring samples taken from intact packages of product. However, Listeria-
positive lots that are still under direct control of the manufacturer can be recalled internally,
thus avoiding adverse publicity. If the pathogen is initially detected in monitoring samples
from unpackaged products, USDA-FSIS officials do not request firms immediately to re-
call the sampled lot. Instead, government officials will analyze subsequent lots and, if
necessary, initiate further steps (i.e., hold-test programs) to prevent distribution of contam-
inated products.
Our knowledge concerning the incidence of Listeria in cookedh-eady-to-eat poultry
products marketed in the United States has come primarily from the USDA-FSIS Listeria-
-
monitoringherification programs with results indicating that 1.5-2.0% of all such prod-
ucts are contaminated with L. rnonocytogenes [7,18,31]. Poultry products in which L.
rnonocytogenes has been found include chicken patties [7], chicken thighs [7], chicken
salad [7], diced poultry [7], poultry salad [18], poultry spread [18], poultry frankfurters
[7], poultry bologna [7], and turkey sausage [7]. In all likelihood, the pathogen entered
these products during the later stages of manufacture and/or packaging. Since most manu-
factures now retain all sampled lots of product until results of Listeria testing become
known, formal Class I recalls for such products are quite limited and include the aforemen-
tioned recall of turkey frankfurters [14], two recalls of chicken salad [4,6], and one addi-
tional incident involving 13,000 pounds of chicken spread produced by a Virginia-based
firm [ 151. However, numerous Class I recalls have been issued for prepared delicatessen-
type sandwiches, with at least two of these recalls [ 11,171 involving items that also con-
tained processed chicken and/or turkey.
several surveys were initiated to determine the extent to which raw chicken and turkey
meat are contaminated with Listeria and Salmonella.
Chicken
The aforementioned concerns prompted USDA-FSIS officials to initiate a poultry back/
neck testing program for L. monocytogenes, Salmonella, and Escherichia coli serotype
0157 :H7 in January of 1989. L. monocytogenes and Salmonella were detected in 508 of
2686 (18.9%) and 792 of 2739 (28.9%) samples, respectively, with E. coli serotype 0157 :
H7 being absent from 2696 samples [ 181. Bailey et al. [20] determined the incidence of
L. monocytogenes and other Listeria spp. on the surface of broiler carcasses processed in
the southeastern United States. They also compared L. monocytogenes serotypes isolated
from raw chicken with those that are commonly associated with human cases of listeriosis.
Using an enrichment procedure together with three selective plating media, Listeria spp.
were detected in rinse samples from 34 of 90 (37.8%) chicken carcasses; however, recov-
ery of Listeria varied widely with three lots of 10 birds each being reported as negative.
More important, 21 of 90 (23.3%) carcasses contained L. monocytogenes, with 64, 18, 6
and 12% of the isolates being identified as serotypes I /2b, 1/2c, 3b, and nontypable strains,
respectively. Although only 7 of 1 15 (6.1%) L. monocytogenes isolates from listeriosis
victims in the United States were of serotype 1/2b or 1/2c, the fact that most L. monocyto-
genes strains isolated from chickens were pathogenic to mice, suggests chicken meat as
a possible vehicle in human cases of listeriosis.
Between June 1988 and May 1989, Genigeorgis et al. [45,46] conducted two large
surveys which examined the incidence of Listeria spp. on fresh and/or semifrozen, chicken
and turkey parts obtained from retail and slaughterhouse sources. According to their results
for chicken, 12.5% of fresh wings, 16.0% of fresh legs, and 15.0% of fresh livers purchased
at three supermarkets in northern California contained detectable levels of L. monocyto-
genes (Table 1). Furthermore, with the exception of fresh chicken liver, L. innocua was
generally two to three times more prevalent in the remaining samples than was L. monocy-
togenes. Overall, the highest incidence of Listeria spp. was observed for fresh legs (54.0%)
followed by fresh wings (42.5%) and fresh livers (32.5%). In contrast to fresh products,
only 10% of semifrozen chicken wings, legs, and livers contained Listeria spp. However,
finding L. monocytogenes alone in 1 of 10 semifrozen legs and livers points to the ability
of this pathogen to survive in semifrozen raw chicken and turkey, as also was observed
by Palumbo and Williams [98].
In addition to these efforts, Genigeorgis et al. [45] also attempted to trace the route
of Listeria contamination on fresh chicken wings, legs, and livers by examining samples
at various stages of production and storage. Although all chicken parts from the beginning
of the production line were free of L. monocytogenes, results in Table 2 indicate that most
contamination occurred during the latter stages of production when carcasses came in
direct contact with Listeria-laden fecal material, since at the time of packaging, 70, 30,
and 33% of chicken wings, legs, and livers contained L. monocytogenes, respectively. Not
surprisingly, L. innocua, which was virtually absent from chicken parts at the beginning
of production, also was routinely isolated from wings, livers, and particularly legs at the
end of production. Despite these relatively high contamination rates, both Listeria spp.
failed to grow on all three packaged products during the first 4 days of refrigerated storage.
Wimpfheimer et al. [ 1321 observed a 3- to 4-day lag phase for L. monocytogenes when
inoculated samples of raw minced chicken were held at 4C. Given this information, the
Listeria monocytogenes in Poultry and Egg Products 569
TABLE
1 Incidence o f Listeria spp. on Fresh and/or Semi-Frozen Chicken a n d Turkey Parts Purchased f r o m Three California
Supermarkets Between J u n e 1988 a n d M a y 1989
TABLE
2 Incidence of Listeria spp. on Commercially Produced Fresh Chicken and
Turkey Parts Before and After Being Packaged and/or Stored at 4C
4-day -old
packaged
Production line product
Type and part stored at
of poultry Listeria sp. beginning end 4C
Chicken
wings L. monocytogenes 0/20a 21/30 (70) 18/25 (72)
L. innocua 0/20 6/30 (20) 4/25 (16)
legs L. monocytogenes 0/20 11/30 (37) 13/25 (52)
L. innocua 0/20 19/30' (67) 17/25 (68)
livers L. monocytogenes 0/3 1 5/15 (33) 6/15 (40)
L. innocua 2/31 (6.5)b 4/15 (27) 4/15 (27)
Turkey
wings L. monocytogenes 1/30 (3.3) 0/30 NDd
L. welshimeri 1/30 (3.3) 4/30 (13.3) ND
legs L. monocytogenes 0/30 2/30 (6.7) ND
L. welshimeri 1/30 (3.3) 1/30 (3.3) ND
livers L. monocytogenes 0/30 0/30 ND
L. welshimeri 1/30 (3.3) 5/30 (16.7) ND
Percentage positive.
Strain of L. welshimeri also detected.
Source: Adapted from Refs. 48 and 49.
recovered from the intestinal tract of broiler chickens at the time of slaughter, 25% of
postprocessing and retail-level carcasses contained L. monocytogenes. In another study
involving perorally dosed chicks, Husu et al. [65] reported that L. monocytogenes was
generally eliminated within 9 days, which again suggests that intestinal carriage of L.
monocytogenes is most often transient.
It is important to remember that like other meats, poultry products also can be used
for purposes other than human consumption. Al-Sheddy and Richter [ 11 determined the
incidence of L. monocytogenes in frozen ground meat that contained raw chicken together
with chopped beef by-products. Although not conclusive, recovery of L. monocytogenes
from all five samples examined and the fact that this pathogen is more commonly found
in chicken rather than beef products leads one to conclude that raw chicken was the most
likely source of contamination. Hence, considering the high incidence of L. monocytogenes
in raw chicken, it may be prudent to eliminate raw poultry products from the diet of zoo
animals to curb the number of listeriosis cases occurring in zoological parks.
The scientific literature relating to pathogens commonly associated with processed
poultry is extensive, and a review of this literature has been published [129]. In another
review paper [69] covering the years 1971-1994, the prevalence of L. monocytogenes in
meats appears to be highly variable, with approximately 16?6 of products being positive.
In general, the highest numbers of L. monocytogenes have been found in processed meat
and poultry products, with fresh meats generally containing much lower numbers. Al-
though serotypes 1/2a, 1/2b, and 1/2c are most frequently isolated from meats, most
human outbreaks have been traced to serotype 4b, thus suggesting that poultry products
play a relatively minor role in foodborne listeriosis.
Turkey
Since chickens and other types of domesticated fowl are similarly processed, one would
expect various Listeria spp., including L. monocytogenes, superficially to contaminate
other raw poultry products, including turkeys, ducks, and pheasants. After completing the
aforementioned survey of California chicken parts for Lisleria [45], Genigeorgis et al.
[46] initiated a similar study to determine the prevalence of various Listeria spp. on fresh
turkey parts obtained from retail sources and slaughterhouses.
Listeria contamination rates were generally similar to those previously observed for
fresh chicken, with 45.0% of fresh turkey wings, 28.3% of fresh legs, and 23.3% of fresh
tails obtained from three local northern California supermarkets harboring various Listeria
spp. (see Table 1). Although their findings further demonstrate that L. monocytogenes is
equally common on fresh chicken and turkey parts, with isolation rates of 10.0-16.0%
and 11.7-20.0%, respectively, the same cannot be said for I,. innocua and L. welshimeri.
In fact, L. innocua, the Listeria sp. most commonly detected on fresh chicken, was recov-
ered from only 3 of 180 (1.7%) fresh turkey parts. Similarly, L. welshimeri, the dominant
Listeria sp. on fresh turkey, was only rarely observed on fresh chicken. Although both
surveys were confined to fresh chicken and poultry parts available from three local super-
markets, these findings still suggest the interesting possibility that chickens and turkeys
may be preferential hosts for L. innocua and L. welshimeri, respectively. However, addi-
tional data need to be gathered from other parts of the country to prove or disprove this
theory.
In a subsequent survey [33], 9 of 42 turkey skin samples harbored L. monocytogenes
with L. monocytogenes contamination rates apparently unrelated to the incidence in the
572 Cox et al.
flock before processing or after defeathering. As was true for fresh chicken, additional
testing at a local slaughterhouse once again demonstrated that fresh turkey parts are most
likely to become contaminated with Listeria during later stages of processing (i.e., eviscer-
ation, chilling) (see Table 2). This scheme, mentioned earlier as the route by which fresh
poultry becomes contaminated, was further confirmed by identifying various Listeria spp.,
including L. monocytogenes, in 4 of 15 (26.7%) samples of mechanically deboned raw
turkey meat obtained from the same slaughterhouse. Ryser et al. [ 1081 further stressed
the importance of postprocessing contamination when they reported that 33 of 45 (73%)
retail samples of ground turkey contained Listeria spp., including a diverse group of L.
monocytogenes strains belonging to nine different ribotypes.
No. (%) of
No. of positive
Origin Type of poultry carcasses examined carcasses Ref.
Denmark Fresh chicken 17 8 (47.1) 120
England/ Fresh chicken 51 27 (52.9) 75
Wales
Fresh chicken 38 19 (50.0) 75
Fresh chicken 50 33 (66.0) 102, 103
Fresh chicken 6 2 (33.3) 51
Fresh chicken 100 60 (60.0) 104
Fresh chicken 30 15 (50.0) 63
Fresh chicken 16 10 (62.5) 88
Fresh chicken 32 21 (65.6) 83
Frozen chicken 64 41 (64.0) 75
Frozen chicken 50 27 (54.0) 102, 103
Frozen chicken 56 7 (12.5) 51
Turkey 4 1 (25.0) 75
Fresh turkey 1 0 51
Frozen turkey 3 0 51
Duck 3 3 (100.0) 75
Frozen duck 2 1 (50.0) 51
Wild pheasant 2 1 (50.0) 75
Italy Fresh chicken --200 0 34
Fresh chicken 50 18 (36) 44
Sweden Fresh chicken 45 0 123
Switzerland Fresh chicken 24 5 (20.8) 29
West Germany Fresh/frozen chicken 100 85 (85.0) 110, 122
Unspecified poultry 30 6 (20.0) 97
Unspecified poultry 11 3 (27.3) 122
indicates that improperly handled poultry products other than chicken also may pose a
potential threat to consumers.
One year later, Gitter [51] published results from a similar study which examined
the incidence of L. monocytogenes on surfaces of various oven-ready poultry products
purchased at 26 different shops and supermarkets in southern England. Using a combina-
tion of direct plating and cold enrichment, L. monocytogenes was identified on 7 of 56
(12.5%) frozen and 2 of 6 (33.3%) fresh chickens as well as on 1 of 2 frozen ducks (see
Table 3). Although the incidence of L. monocytogenes on raw poultry products was mark-
edly lower than that previously found by Kwantes and Isaac [75], L. monocytogenes iso-
lates identified as serotype 4 again outnumbered those of serotype 1/2.
Following emergence of L. monocytogenes as a serious foodborne pathogen in June
1985, Pini and Gilbert [ 1031 determined the prevalence of this pathogen in uncooked fresh
and frozen chickens obtained from retail outlets throughout metropolitan London. Unlike
previous studies, which relied on swab samples from carcasses, these researchers exam-
ined two different samples from each chicken carcass whenever possible-one sample
574 Cox et al.
consisting of edible offal (trimmings and/or viscera) and the other a composite sample
of skin and carcass remnants. Using cold enrichment in conjunction with the Food and
Drug Administration (FDA) procedure, L. rnonocytogenes was recovered from 33 of 50
(66%) fresh and 27 of 50 (54%) frozen chickens. These results are similar to those reported
from other 1987- 1994 surveys [49,63,83,88,104] in which L. rnonocytogenes was detected
on 10 of 16 (62.5%), 15 of 30 (50%), 60 of 100 (60%) and 21 of 32 (66%) fresh chicken
carcasses marketed in England and Wales (see Table 3). According to a second report by
Pini and Gilbert [102], other Listeria spp., including L. innocua, L. seeligeri, and L.
welshirneri, also were detected either alone or together with L. rnonocytogenes in 26 and
30% of the fresh and frozen chicken samples tested, respectively. Overall, 74 L. rnonocyto-
genes strains representing serotypes 1/2, 3a, 3b, 3c, 4b, 4d, and two nontypable strains,
with serotype 1/2 predominating, were isolated from 160 samples consisting of 60 edible
offal and 100 composite samples. Composite samples yielded more isolates of L. rnonocy-
togenes (57%) than did edible offal samples (22%) and also a higher percentage of other
Listeria spp. (23%) than did edible offal samples (15%). Despite differences in methodolo-
gies and types of samples analyzed in the above-mentioned studies, averaging the results
in Table 3 indicates that 187 of 323 (58%) fresh chickens and 75 of 170 (44.1%) frozen
chickens marketed in England and Wales between 1971 and 1994 contained L. monocyto-
genes. That the 1986 findings of Pini and Gilbert [103,104] are similar to those obtained
in both American and European surveys as far back as the mid 1970s underscores the
continuing need for proper kitchen hygiene, cooking of raw poultry products, and continu-
ous inspection of carcasses (e.g., identification of liver and heart lesions), along with use
of good manufacturing and sanitizing practices in poultry-processing facilities. Since 152
of 214 (71%) clinical L. rnonocytogenes isolates obtained from British patients between
November 1986 and 1987 [87] were of serotype 4b, poultry products, in which L. rnonocy-
togenes serotype 1/2 predominates, may be a less common vehicle for listeriosis than
other foods. However, pit&, which are in essence poultry spreads prepared from chicken
or goose liver, may be an exception.
European concern about the incidence of L. rnonocytogenes in raw poultry products
consumed outside of England/Wales dates back to at least 1982 when two Swedish work-
ers, Ternstrom and Molin [ 1231, examined 45 chickens obtained from two local slaughter-
houses (see Table 3). Although these researchers failed to isolate L. rnonocytogenes from
any of the chickens examined, it appears that the Listeria isolation/detection methods used
in this study were primarily responsible for their lack of success, since listeriosis in Swed-
ish poultry is relatively common, with 112 cases being diagnosed in the 10-year period
between 1948 and 1957 [94]. In view of the high incidence of L. rnonocytogenes in raw
poultry marketed in the United States and England, inadequate isolation/detection methods
of the early 1980s also were likely responsible for the inability of Comi and Cantoni [34]
to recover this pathogen from approximately 200 chicken samples (i.e., carcass, skin,
entrails) obtained from slaughterhouses in northern Italy, with more recent Italian surveys
[44,84] showing L. monocytogenes contamination rates of 10.6 and 36% for raw poultry.
After the 1985 cheeseborne listeriosis outbreak in California, western European
scientists began to determine the incidence of Listeria in a wide range of foods, includ-
ing fresh poultry products. In the first of these studies, which was published in 1988,
Skovgaard and Morgen [ 1201 visited two large Danish poultry slaughterhouses and exam-
ined chilled chicken carcasses for evidence of Listeria contamination. According to these
authors, Listeria spp. were detected in neck-skin samples from 16 of 17 (94.1 %) chicken
carcasses, with L. innocua being identified in all but two Listeria-positive samples. Al-
Listeria monocytogenes in Poultry and Egg Products 575
though most of the poultry processed at these two facilities was heavily contaminated
with L. innocua, 8 of 17 (47.l%), 1 of 17 (5.9%), and 2 of 17 (1 1.8%) carcasses also
contained detectable levels of L. monocytogenes (see Table 3), L. innocua, and other
Listeria spp. (L. welshimeri, L. murrayi, and/or L. denitrificans), respectively. Thus the
L. monocytogenes contamination rate for chickens processed in Denmark was only slightly
lower than the average (56.7%) for fresh chicken carcasses marketed in England and
Wales. These Danish researchers also identified Listeria spp., including L. monocytogenes,
in chicken feces and transport cage material, further supporting the widespread belief that
poultry carcasses most likely become contaminated with Listeria during evisceration and
subsequent handling, as also was suggested by Genigeorgis et al. [45,46] and Cox [35]
based on results of surveys of poultry-processing facilities in California and Georgia.
Interest in the incidence of Listeria in European fresh poultry again intensified fol-
lowing reports that 34 individuals in Switzerland died after consuming contaminated
Vacherin Mont dOr soft-ripened cheese. Breer [29] isolated L. monocytogenes, L. inno-
cua, and 1,.seeligeri from 5 of 24 (20.8%), 6 of 24 (25.0%), and 1 of 24 (4.2%) raw
chickens purchased in Switzerland, (see Table 3), respectively. Using a modified version
of the FDA procedure, German researchers [ 110,121], found Listeria spp. and L. monocy-
togenes in 94 of 100 (94%) and 85 of 100 (85%) chicken carcasses, respectively. However,
results from two smaller surveys of German poultry [97,121] suggested far lower L. mono-
cytogenes contamination rates, with only 20.0-27.3% of unspecified fresh poultry car-
casses (presumably chicken) containing this pathogen (see Table 3). Similarly, Rijpens
et al. [ 1051 more recently recovered Listeria spp. and L. monocytogenes from 35.5% and
15.5%, respectively, of poultry samples examined in Belgium, with the incidence of Liste-
ria spp. markedly higher in unpackaged (41.7%) than in prepackaged (1 1.1%) poultry. L.
monocytogenes also has been detected in French poultry since 1988 [26]. During a subse-
quent large-scale survey of poultry carcasses in France and Belgium, Uyttendaele et al.
[ 1271 reported that the L. monocytogenes contamination rate decreased from 32.1% in
1992 to 9.2% in 1995, with 87%-98% of the carcasses tested yielding < I L. monocyto-
genes CFU/cm2. Contamination of poultry parts also decreased from 25.8% to 3% during
this same 4-year period, with L. monocytogenes-positive samples primarily being confined
to chicken legs and wings. In another survey similar to that conducted in the United States
by Genigeorgis et al. [45], Franco et al. [43] identified Listeria spp. on 96, 84, 80, and
0% of the fresh chicken legs, wings, breasts, and livers, respectively, that were processed
at a Spanish facility, with Listeria counts ranging from <2.0 to 2.8 log CFU/g on chicken
wings. Follow-up testing of the processing environment again showed that most of the
contamination occurred during the later stages of production as carcasses entered the quar-
tering room and exited on conveyor belts. In a Danish survey [96], L. monocytogenes was
not observed in cecal samples from over 2000 broilers representing 90 randomly selected
broiler flocks. However, L. monocytogenes was isolated from 0.3 to 18.7% of all poultry-
processing environmental samples examined. Using several strain-specific typing meth-
ods, L. monocytogenes contamination was shown to be primarily localized in the pro-
cessing facility, with the incidence reducible through improved hygiene.
Elsewhere, 10 of 30 domestically grown fresh chickens from the United Arab Emir-
ates [54] contained Listeria spp., with 1 of these carcasses being positive for L. monocyto-
genes. However, Listeria spp. were detected on 32 of 39 (82%) imported frozen chickens,
18 (46%) of which contained L. monocytogenes. Similarly, Rusul et al. [47] identified L.
monocytogenes in 4 of 16 (25%) poultry samples collected from six local Malaysian mar-
kets, with one of these markets subsequently yielding L. monocytogenes in 15 of 24
576 Cox et al.
Cooked/Ready-to-Eat Poultry
Increasing evidence indicates that contamination of ready-to-eat foods, including cooked
chicken, is most likely to originate from the processing environment. With this premise
in mind, Lawrence and Gilmour [76] examined the incidence of Listeria spp., including
L. monocytogenes, in one poultry-processing facility, along with raw and cooked chicken
processed at this same facility in Northern Ireland between March and August 1992.
Within the raw and cooked poultry-processing environments, 36 of 79 (46%) and 51 of
173 (29%) samples harbored Listeria spp., whereas 21 of 79 (26%) and 27 of 173 (15%)
yielded L. monocytogenes, respectively. Contamination rates were fairly uniform, with
several environmental sites yielding L. monocytogenes throughout the study. Among raw
and cooked products tested, 53 of 58 (91%) and 8 of 96 (8%) contained Listeria spp.,
whereas 34 of 58 (59%) and none of 96 cooked samples yielded L. monocytogenes, respec-
tively. Although L. monocytogenes was absent from all cooked products tested, the pres-
ence of other Listeria spp. and a subsequent report by the same authors [77] attesting to
isolation of identical L. monocytogenes strains from raw poultry, cooked poultry, and the
processing environment (some environmental strains of which persisted for up to 1 year)
confirms the importance of minimizing postprocessing contamination.
An association between human listeriosis and consumption of cooked/cooked-
chilledheady-to-eat poultry products which are cooked, rapidly chilled, and frequently
held refrigerated for at least 5 days before being consumed without further heating has
been observed in both the United Kingdom and the United States. According to one
survey conducted by the Public Health Laboratory Service in London [49,50,104], L.
monocytogenes was present in 63 of 527 (1 2.0%) precooked ready-to-eat poultry products
collected from London-area retail establishments between mid November 1988 and mid
January 1989. Little information is available concerning actual numbers of L. monocyto-
genes present in cooked poultry products; however, 14 samples that were examined quanti-
tatively contained < 100 CFU/g. In addition L. monocytogenes was isolated from 13 of
74 (18%) retail chilled meals, most of which were poultry products given to hospital
patients. The pathogen was also discovered in 6 of 24 (25%) cook-chilled poultry products
[13], 7 cook-chilled poultry dishes at levels up to 700 L. monocytogenes CFU/g, and 2
cooked chicken products labeled ready-to-eat which contained up to 400 L. monocyto-
genes CFU/g.
Working at the Cardiff Public Health Laboratory Service in Wales, Morris and
Ribeiro [90] determined the potential Listeria-related risks associated with consumption
of pit&,an appetizer-type poultry spread that is typically prepared from chicken or goose
liver. According to their report, 14 varieties of pit& (primarily imported from Belgium)
were obtained in bulk from area delicatessen counters or in unopened packages from
supermarket refrigerators and examined for L. monocytogenes using established methods.
Overall, this pathogen was isolated from 37 of 73 (50.4%) pitis, with 28 (75.7%) and 9
(24.3%) of these positive samples originating from delicatessens and supermarket refriger-
ators, respectively. These pit& were subsequently withdrawn from the market after offi-
cials discovered dangerously high L. monocytogenes populations of l 0 4 - r 1O5 CFU/g in
Listeria monocytogenes in Poultry and Egg Products 577
seven samples [8]. L. monocytogenes strains of serotype 4b, the serotype responsible for
-80% of all human listeriosis cases in England and Wales, were isolated from 36 of 37
positive samples, with one strain of L. monocytogenes serotype 4b matching clinical iso-
lates from a 1987 cluster of listeriosis cases in which the exact origin of illness could
never be determined.
These findings prompted Public Health Laboratory officials greatly to expand their
survey of pihi. By March 1990 [48,104], workers at 48 of 53 (90.6%) participating labora-
tories in England and Wales isolated L. monocytogenes from 187 of 1834 ( 10.2%) samples
of imported and domestic pit& As in the previous survey, -10% of all positive samples
contained 104-> 106L. monocytogenes CFU/g, with over half of all isolates belonging to
serotype 4. Following this pitk-related outbreak discussed in Chapter 8, the number of
listeriosis cases reported in England and Wales decreased to approximately half the level
reported in 1988. Although some individuals expected to see a further decline [48,104],
the incidence of human listeriosis in the United Kingdom has since stabilized, with about
100-125 cases, being reported annually over the last 5 years.
In one other European survey of cookedheady-to-eat poultry products, Lieval et al.
[80] isolated L. monocytogenes, L. seeligeri, and L. innocua from one of nine, two of
nine, and one of nine chicken sandwiches obtained from cafes in and around Paris. Al-
though identical efforts to recover Listeria from 20 fast-food fried chicken items ended
in failure, the ability of such foods to harbor Listeria, including L. monocytogenes, has
been well established by the previously discussed surveys in England.
Growth-Raw Chicken
Wimpfheimer et al. [ 1321 examined the behavior of L. monocytogenes in raw chicken. In
their study, raw minced chicken meat was inoculated to contain 10, L. monocytogenes
CFU/g, packaged anaerobically (75% CO2:25% N2), microaerobically (72.5% CO,:
22.5% N 2 : S % O,), or aerobically (air) and examined for numbers of L. monocytogenes
as well as aerobic spoilage organisms during storage at 4, 10, or 27C. Neither L. monocy-
togenes nor aerobic spoilage organisms grew in anaerobically packaged raw chicken dur-
578 Cox et al.
ing extended storage at any of the three temperatures, with both populations decreasing
to <10 CFU/g after 6 days of storage at 4C (Fig. 1). When packaged microaerobically
under conditions more closely simulating commercial practices, numbers of L. monocyto-
genes in raw chicken increased rapidly during extended storage at 4"C, whereas growth
of aerobic spoilage organisms was strongly inhibited (see Fig. 1). Under these conditions,
L. monocytogenes can rapidly proliferate in normal unspoiled raw chicken during refriger-
ated storage. Zeitoun and Debevere [133] later reported on the successful use of a 10%
lactic acid/sodium acetate buffer (pH 3) in conjunction with modified atmosphere packag-
ing (90% CO2, 10% 0,) to prevent growth of L. monocytogenes on uncooked chicken
legs and extend the product's shelf life at 6C to 17 days. According to Wimpheimer et
al. [132], the ability of L. monocytogenes to grow in microaerobically packaged raw
chicken was not affected by initial levels of Listeria (<10' or 102CFU/g) or aerobic
spoilage organisms ( l O4 or 1O8 CFU/g). However, the ratio of Listeria to spoilage organ-
isms was strongly temperature dependent, with both organisms reaching populations of
107-10Rand 109-10'" CFU/g in microaerobically packaged chicken following <2 and 8
days of storage at 10 and 27"C, respectively. Neither L. monocytogenes nor aerobic spoil-
age organisms were inhibited in aerobically packaged raw chicken, with Listeria and spoil-
age organisms attaining populations > l O7 and 1O9 CFU/g, respectively, in products stored
at 4, 10, and 27C. Thus with exception of the microaerobically packaged product, raw
chicken would likely become overtly spoiled before L. monocytogenes could proliferate
L. monocytogenes 0
c.v
Aerobic Spoilage
98 1 Organisms /
67l P'
/
0 2 4 6 8 10 12 14 16 18 20 22
Days
to the point where the pathogen might be detectable in minimally cooked chicken. Never-
theless, it is important to remember that consumers must take special precautions to pre-
vent cross contamination between raw chicken that may contain L. monocytogenes and/
or Salmonella spp. and ready-to-eat products including cooked chicken.
TABLE
4 Populations of L. monocytogenes (log,, CFU/g) in Artificially Contaminated Cooked/Ready-to-Eat Poultry Products of
Acceptable Organoleptic Quality During Extended Refrigerated Storage
10
I 1
9
6
m
3
U-
5
* I
r 4
cs,
0
J
.-a 3
5
*-.
0
a 2
m
1
I 1 I
1 4 6
Days
magnitude lower than those in the controls. Two additional studies on fermented summer
sausage prepared from ground chicken [ 191 and turkey [8 I] also demonstrated the impor-
tance of an active starter culture in limiting growth of L. rnonocytogenes during fermenta-
tion. When these sausages were prepared from a chicken or turkey batter (pH 6.6) con-
taining a pediococcal starter culture and L. rnonocytogenes at a level of 1O4--1O7CFU/g,
numbers of Listeria decreased 0.9- 1.8 orders of magnitude after an I 1-h fermentation
(pH 5). Replacing this pediococcal starter culture with a pediocin-producing strain of
Pediococcus acidilactici essentially doubled the inactivation rate of L. rnonocytogenes
during fermentation. However, regardless of the starter culture used, all remaining listeriae
were subsequently inactivated during 45 min of cooking to an internal temperature of
66.5"C. Thus, an active fermentation combined with normal thermal processing before
packaging should result in a Listeria-free product.
Finally, several investigations also have assessed behavior of Listeria in inoculated
samples of chicken gravy and chicken broth during cooling and/or refrigerated storage.
According to Huang et al. [62], L. monocytogenes populations in individual 1000-g sam-
ples of artificially contaminated chicken gravy (prepared from poultry stock, spices, waxy
maize wash, and chicken base) increased by 2 orders of magnitude as the product cooled
from 40 to 9C during 24 h of storage in a refrigerator at 7C (Fig. 3). Even though
generation times for L. rnonocytogenes approximately doubled after the chicken gravy
stabilized at 7"C, the pathogen still attained a maximum population of 109CFU/g in 8-
day-old gravy. Huang et al. [61] subsequently reported a reduction in L. rnonocytogenes
582 Cox et al.
+ L. monocvtoaenes
0- - Temperature
0 1
-- 2
- -0- -- -0- - -e- -a-- - 0 - - -0
3 4
I
5
1
6
I
7 8
I
Days
when similar chicken gravy was held at 65C for 1.3 min; however, such a heat treatment
is clearly inadequate to inactivate higher Listeria populations that can develop in chicken
gravy during prolonged refrigerated storage. Walker et al. [ 1301 also demonstrated the
ability of three L. monocytogenes strains to multiply in artificially contaminated sterile
chicken broth (pH E 6.4) held at heat-freezing temperatures, with Listeria populations in
this product increasing 100-fold during extended incubation at 03C (Fig. 4). In fact,
growth of this pathogen was also evident in samples of chicken broth that were held at
temperatures as low as -0.4"C, below which the broth froze and was no longer sampled.
Results from these investigations stress the importance of cooling foods as rapidly
as possible and show that hazardous situations can easily develop if refrigerated foods
are subjected to mild temperature abuse, that is, holding at temperatures above 4C. In
an effort to increase the safety of cooked, cooked-chilled, and ready-to-eat poultry, USDA
officials lowered the recommended long-term storage temperature for such products from
4.4 (40F) to 1.7"C (35F) and also have developed stricter guidelines that require faster
(than previously recommended) cooling of warm products at the end of manufacture [3].
Continued attention to rapid cooling of finished products and avoidance of postprocessing
contamination are both essential to decrease the incidence of this psychrotrophic pathogen
in cookedh-eady-to-eat poultry products.
Listeria monocytogenes in Poultry and Egg Products 583
I
I I I I 1
0 10 20 30 40 50
Days
Thermal Inactivation
Heating is the most obvious means of destroying L. monocytogenes and other foodborne
pathogens i n any raw food, including poultry. However, numerous reports attesting to
unusual thermal tolerance of L. monocytogenes in various foods, coupled with discovery
of L. monocytogenes in several cooked poultry products that were directly linked to cases
of listeriosis, have raised questions about the exact thermal processing times and tempera-
tures required to eliminate this pathogen completely from raw poultry products. In re-
sponse to these concerns, Carpenter and Harrison conducted three studies in which raw
-
chicken breasts were surface inoculated to contain 1OS- l O7 L. monocytogenes CFU/g
and cooked to internal temperatures of 65.6, 71.1, 73.9, 76.7, or 82.2C using dry heat
[32], moist heat [57], and microwave radiation [58].All cooked chicken breasts were then
vacuum-packaged or wrapped in oxygen-permeable film and analyzed for numbers of
Listeria during 4 weeks of storage at 4 and 10C.
Overall, L. monocytogenes was recovered from chicken breasts cooked to all five
internal temperatures, using dry heat, moist heat, and microwave radiation. As expected,
the magnitude of lethality was directly related to cooking temperature. Since chicken
breasts contained somewhat different levels of L. monocytogenes before heating, one can-
not directly compare the effectiveness of the three cooking methods used in these studies.
However, assuming that L. monocytogenes populations in these chicken breasts decreased
linearly during heating (admittedly, some tailing of the survivor curve likely occurred
at the three highest temperatures using dry heat, moist heat, and microwave irradiation),
then the number of survivors in chicken breasts that contained any initial inoculum can
be estimated. Thus, if Carpenter and Harrison had used an initial population of 1 .O X 106
L. monocytogenes CFU/g in all three studies, one would expect their results to have been
similar to the estimated number of survivors shown in Table 5. Considering these approxi-
mations, it appears that L. monocytogenes was generally more tolerant of microwave radia-
tion than dry or moist heat, with numbers of Listeria decreasing less than 4 orders of
magnitude on chicken cooked to an internal temperature of 822C. Of greater importance
is the fact that Listeria populations decreased 1 2 orders of magnitude on chicken breasts
cooked to an internal temperature of 7 1.1 C, the minimum internal temperature to which
poultry must be heated to designate the product as fully cooked in the United States [2].
Although numbers of Listeria decreased approximately 5 orders of magnitude when
584 Cox et al.
TABLE
5 Estimated Decrease in Numbers of L. rnonocytogenes o n the Surface of
Chicken Breasts Inoculated to Contain 6.00 log,, CFU/g and Cooked t o Various
Internal Temperatures Using Dry Heat, Moist Heat, or Microwave Radiation
NO.^ of L. monocytogenes (log,, CFU/g) decrease after cooking to
internal temp. of
~ ~
chicken was cooked to higher internal temperatures using either dry or moist heat, these
authors [56] later demonstrated that moist heating of surface-inoculated chicken breasts
to an internal temperature of 73.9"C also failed to completely inactivate more realistic L.
monocytogenes populations of 102-104CFU/g. Overall, microwave heating was less effec-
tive than either dry or moist heating, with Listeriu populations decreasing less than 4
orders of magnitude on chicken breasts cooked to an internal temperature of 82.2"C (see
Table 5). In 1989, researchers in England [82] also reported that microwave heating was
less effective than other forms of cooking for eliminating L. monocytogenes from the
surface and interior (stuffing) of whole stuffed chickens (- 1.7 kg each) inoculated to
contain 106and 107 Listeria CFU/g of skin and stuffing, respectively. Uneven heating,
which is an inherent problem in microwave cooking, accounted for the 20 min of additional
standing time that was required after 38 min of cooking (final skin temperature of 80-
99C) to completely inactivate the pathogen on the surface of whole chickens. However,
low levels of L. monocytogenes (<10 CFU/g) were still detected in stuffed samples from
one of two similarly treated whole chickens that attained temperatures of 72-85C after
20 min of standing. Thus, these findings serve as a warning to people who regularly cook
large stuffed birds (particularly turkeys) in microwave ovens, and they also stress the
importance of postcooking standing time for further inactivation of Listeriu and other
foodborne pathogens in poultry products after microwave cooking.
Not surprisingly, follow-up work by Carpenter and Harrison [32,57,58] demon-
strated that L. monocytogenes survivors (likely sublethally injured during heating) can
persist and multiply on both oxygen-permeable film-wrapped and vacuum-packaged
cooked chicken during extended storage at 4 and 10C. As shown in Figure 5, growth of
Listeriu on chicken breasts packaged in oxygen-permeable film was most evident after 2
weeks of refrigerated storage with larger populations generally developing on products
that were cooked using microwave radiation, followed by those given moist or dry heat.
Most important, L. monocytogenes was recovered via direct plating from all 4-week-old
aerobically packaged chicken breasts except those that were cooked to an internal tempera-
ture of 82.2"C using moist heat. In addition, higher numbers of Listeriu also developed
on aerobically packaged chicken breasts that were exposed to less severe heat treatments.
As expected, increasing the incubation temperature also led to much faster growth of
Listeria, with the pathogen generally attaining populations of 106-107CFU/g on aerobi-
cally packaged chicken breasts after only 7 days at 10C.
Behavior of L. monocytogenes on chicken breasts was influenced by the heating
Listeria monocytogenes in Poultry and Egg Products 585
P
3
U.
0
I I 1 I
0 1 2 3 4
Weeks
methodkreatment, temperature at which the cooked product was ultimately stored, and
type of packaging material. According to these authors, Listeria populations were gener-
ally 1 to 2 orders of magnitude lower in vacuum-packaged than in aerobically wrapped
product following 4 weeks of storage at 4C; however, the pathogen was present in all
4-week-old samples except those that were originally cooked to an internal temperature
of 82.2"C using moist or dry heat (Fig. 6). Although numbers of Listeria were again
generally 1-2 orders of magnitude lower in vacuum-packaged than in aerobically wrapped
chicken breasts following 1 week of storage at 10C, populations were as much as 5 orders
of magnitude lower in vacuum-packaged chicken breasts that were cooked to an internal
temperature of 82.2"C using moist heat.
Since raw chicken normally contains <loo0 L. monocytogenes CFU/g [88] and
Listeria populations generally decrease approximately 3-5 orders of magnitude in fully
cooked poultry heated to an internal temperature of 63.9"C, as specified in the U.S. Code
of Federal Regulations, it appears that present cooking temperatures are, at best, only
marginally adequate to eliminate this pathogen from raw poultry products. In fact, Harrison
Cox et al.
I 1 I I
1 2 3 4
Weeks
and Carpenter [56] reported that moist heating of inoculated chicken breasts to an internal
temperature of 73.9"C failed to completely inactivate L. monocytogenes surface popula-
tions of 102-104CFU/g, with the pathogen reestablishing itself at levels equal to or greater
than the original inoculum level after 4 weeks at 4C. Nonetheless, the adequacy of current
poultry-processing methods was maintained by another report [5] which indicated that a
turkey meat emulsion (containing salt, sodium tripolyphosphate, carrageenan, and water)
inoculated to contain 5.78 L. monocytogenes log,,, CFU/g was free of the pathogen after
holding the product at 7 1.1 "C for 2 min (estimated D71,10C= 0.28 min). However, in view
of at least three human listeriosis cases linked to cooked-chilled chicken meat and turkey
frankfurters (the latter was reportedly warmed 45-60 s in a microwave oven before con-
sumption), and that small numbers of L. monocytogenes survived a wide range of heat
treatments given to chicken breasts and frequently grew in these products during refrig-
eration, it is prudent for poultry processors to cook their products to somewhat higher
internal temperatures (76.7-82.2"C) than is now routinely practiced until the adequacy
of the current minimum heat treatment can be firmly established. Future studies should
address the effect of processed poultry ingredients (i.e., salt, preservatives) on thermal
resistance of Listeria during conventional as well as microwave heating, since Harrison
and Carpenter [58] found that the latter cooking method was less successful in eliminating
L. monocytogenes from the surface of chicken breasts than either dry or moist heating.
Listeria monocytogenes in Poultry and Egg Products 587
Chemical Treatments
Various food additives have been evaluated for their ability to inactivate Listeria spp. on
fresh poultry. Dipping inoculated chicken wings in 10% trisodium phosphate (TSP) at
10C for 15 s and hot water (95C) for 5 s resulted in a 79.49% reduction in numbers of
L. monocytogenes, with minimal changes in subcutaneous temperature [ 1061. Hwang and
Beuchat [66] also found that washing chicken skin in 1% TSP or 1% lactic acid signifi-
cantly reduced viable populations of L. monocytogenes compared with washing in water.
According to Shelef and Yang [ 1 161, growth of L. rnonocytogenes in sterile comminuted
chicken was slowed during refrigerated storage by adding 4% sodium or potassium lactate.
When used at levels 10.3%, sodium diacetate also was inhibitory to L. rnonocytogenes
in poultry slurries [ 1091, with the antilisterial activity of sodium diacetate being further
enhanced by supplementing the product with 0.25% ALTA, a commercially available
microbially produced shelf life extender.
monocytogenes populations in chicken breast meat from 1O6 CFU/g to undetectable levels,
with no growth of the pathogen being observed during 5 weeks of storage at 8C. Hence,
in the absence of multiple treatments, these findings suggest that small numbers of Listeria
may either escape sublethal injury during irradiation or undergo repair and grow on these
carcasses during refrigerated storage.
From this information, it appears that a gamma radiation dose of 2.5 kGy may be
only marginally sufficient to inactivate levels of Listeria that one might reasonably expect
to find on naturally contaminated raw poultry. Nevertheless, provided that irradiated poul-
try products are properly packaged to prevent recontamination (and subsequent growth)
with Listeria and other foodborne pathogens, this procedure should markedly decrease the
risk of contaminating ready-to-eat foods (e.g., salads, raw vegetables) when raw poultry is
prepared by the consumer. Unfortunately, although the scientific community generally
contends that foods exposed to such low levels of radiation are safe for human consump-
tion, irradiated foods have not yet gained full acceptance by consumers. Perhaps the con-
tinued outpouring of scientific evidence will eventually curb the remaining unfounded
fear of irradiated foods in the mind of the general public.
EGG PRODUCTS
Listeria monocytogenes is most frequently isolated from heart, liver, and spleen tissue of
poultry suffering from listeriosis; however, according to the early scientific literature, this
pathogen also has been detected in necrotic oviduct lesions of several infected hens [55]
and in follicles of one artificially infected chicken [72]. These observations prompted a
large-scale survey in 1958 [70] in which 600 intact hens eggs were examined and found
to be negative for L. monocytogenes. In keeping with these findings, consumption of eggs
and egg products also has not yet been linked to a single case of listeriosis. However, the
possible presence of L. monocytogenes on egg shells which may contain minute cracks
along with the ability of this pathogen to survive 90 and > 14 days on eggs stored at 5 [22]
and 10C [24], respectively, persist on inoculated eggs treated with sodium hypochlorite
containing 100 ppm available chlorine [24], and grow in artificially contaminated eggs
stored at refrigeration as well as ambient temperatures [22] suggests that eggs cannot be
ignored as a possible source of listerial infection. Hence, it is not surprising that recent
concerns about contaminated poultry products also have prompted efforts to define both
the incidence and behavior of L. monocytogenes in eggs and egg products, including pas-
teurized liquid and dried egg.
Incidence
As mentioned in the preceding paragraph, isolation of L. monocytogenes from intact whole
eggs has not yet been documented; however the same is not true for broken eggs. Ac-
cording to Leasor and Foegeding [78], Listeria spp. were isolated from 15 of 42 (36%)
previously frozen samples of raw commercially broken solids-adjusted liquid whole egg
(21 samples), natural-proportion liquid whole egg (20 samples), and yolk (1 sample) ob-
tained on several occasions from 6 of 11 (54%) commercial manufacturers located
throughout the United States. On closer examination of the data, L. innocua and L. monocy-
togenes were identified in 15 of 15 (100%) and 2 of 15 (13.3%) positive samples, respec-
tively. Twelve of 15 (80%) and 3 of 15 (20%) positive samples, including one sample
each with L. monocytogenes, were classified as solids-adjusted and natural-proportion
Listeria monocytogenes in Poultry and Egg Products 589
liquid whole egg, respectively. Increased handling during manufacture and, as suggested
by the authors, a higher solids content which may enhance growth and survival of Listeria
are just two of many possible reasons why a higher incidence of Listeria was observed
in solids-adjusted rather than natural-proportion liquid whole egg. Although results from
direct plating indicated that the two L. monocytogenes-positive samples contained approxi-
mately 1 and 8 L. monocytogenes CFU/g at the time of analysis, the fact that these samples
were held frozen for up to 4.5 months and subjected to two freeze-thaw cycles suggests
that numbers of Listeria likely decreased by at least 50% during storage (see Chap. 6).
Hence, both samples probably contained < 100 L. monocytogenes CFU/g before being
frozen.
Working in the United Kingdom, Moore and Madden [23] recovered Listeria from
125 of 173 (72%) in-line filters used to remove shell fragments from raw blended whole
egg; 78 and 47 samples of which contained L. innocua and L. monocytogenes, respectively,
generally at levels <400 CFU/mL. However, since 500 pasteurized egg samples yielded
negative results for listeriae, pasteurization at 64.4"C for 2.5 min, as required in the United
Kingdom, appears to afford a high degree of safety. During a subsequent survey of
three Australian egg factories in New South Wales, Desmarchelier et al. [37] identified
L. innocua in 9 of 13 (69%) samples of unpasteurized liquid whole egg, with no other
Listeria spp. being observed. Floors, drains, and mobile equipment in raw processing areas
of these factories were later confirmed as major sources of contamination through strain-
specific typing of environmental Listeria isolates. Although 7 samples of raw sugared
yolk and 26 peeled boiled eggs were Listeria free, L. innocua was recovered from 1 of
51 samples of pasteurized liquid whole egg, with this organism also being detected in 2
of 14 peeled boiled eggs following 3 weeks of modified-atmosphere storage at 4C. Hence,
under certain conditions, Listeria can multiply to detectable levels in egg products during
extended cold storage.
Growth
The ability of Listeria to grow in hen's eggs was first recognized in 1940 when Paterson
[ 1001 inoculated a laboratory culture of L. monocytogenes into the chorioallantoic mem-
brane of a chicken embryo. This procedure was traditionally used to determine virulence
of L. rnonocytogenes isolates [ 1211. Information concerning growth of this pathogen in
nonfertile eggs and egg products is limited. A search of the scientific literature has uncov-
ered only two studies pertaining to growth of L. monocytogenes in raw whole eggs or egg
components. According to results from the first such paper published in 1955, Urbach
and Schabinski [ 1251 found that populations of L. monocytogenes in intact experimentally
infected nonfertile eggs increased nearly 6 orders of magnitude during 10 days of storage
at ambient temperature. Following this study, 20 years passed until viability of L. monocy-
togenes was again examined in artificially contaminated raw, as well as cooked (1 2 1"C/
15 min) whole egg, albumen, and egg yolk during extended storage at 5 and 20C [73].
Results for raw whole and separated egg showed that growth of L. monocytogenes was
primarily confined to egg yolk (Fig. 7), with the pathogen exhibiting respective generation
times of 1.7 days and 2.4 h at 5 and 20C. Overall, Listeria populations in raw whole
egg generally varied less than 1 order of magnitude from the original inoculum during
extended storage at either temperature; however, numbers of Listeria in raw albumen (pH
8.9) decreased 3 and 5 orders of magnitude during prolonged incubation at 5 and 2OoC,
respectively. Despite the reported ability of L. monocytogenes to grow in laboratory media
590 Cox et al.
0 10 20 30
Days
having pH values as high as 10 [ 1131, loss of Listeria viability in raw albumen was pH
related, with numbers of Listeria decreasing less than 2 orders of magnitude in samples
that were preadjusted to pH 7 and held at 5C. Unlike raw whole egg, albumen, and egg
yolk, Listeria grew rapidly in corresponding cooked samples. Generation times for the
pathogen in cooked whole egg, egg yolk, and albumen were 1.9,2.3, and 2.4 days, respec-
tively, at 5"C, and 2.6, 2.6, and 3.5 h at 20C. These authors speculated that loss of the
aforementioned antilisterial properties of raw albumen resulted from inactivation of one
or more binding proteins (i.e., ovotransferrin, ovoflavoprotein, avidin) during heating.
Since L. monocytogenes can grow rapidly in cooked whole as well as separated eggs,
investigators of foodborne outbreaks should not overlook these products as potential vehi-
cles of infection.
In 1990, Sionkowski and Shelef [ I171 provided the first information concerning
growth of L. monocytogenes in pasteurized egg products. To simulate postpasteurization
contamination, pasteurized (64.4OU2.5 min) samples of liquid egg and reconstituted dried
-
egg were inoculated to contain 104- I O5 L. monocytogenes CFU/mL and examined for
numbers of Listeria during 7 days of storage at 4C. As shown in Fig. 8, the pathogen grew
-
similarly in both products, reaching populations 1O7 CFU/mL after 7 days of refrigerated
storage. Although transmission of L. monocytogenes by pasteurized egg products has not
yet been documented, these findings suggest that a possible public health problem could
develop if L. monocytogenes enters pasteurized liquid egg, particularly since the shelf life
of some of these refrigerated products now has been extended to several months.
Growth of L. monocytogenes in commercially processed, liquid whole egg was first
assessed by Foegeding and Leasor [41]. In this study, commercially broken, liquid whole
egg was ultrapasteurized (68"C/ I 18 s), homogenized, inoculated to contain one of five
L. monocytogenes strains (Scott A [clinical isolate], F5069 [milk isolate], ATCC 191I 1
[poultry isolate], NCF-U2K3, and NCF-FI KKr [raw liquid whole egg isolate]) at a level
of 5 X 102to 1 X 103 CPU/mL, overlaid with mineral oil to prevent oxygen transfer,
and examined for numbers of Listeria during extended incubation at 4, 10, 20, and/or
30C.
Generation times and maximum populations were generally similar to those previ-
Listeria monocytogenes in Poultry and Egg Products 591
107
106
Tl
A Iiquidegg
0 Reconstituted dried egg
1 0 5 1 1
I I I I I I I
0 1 2 3 4 5 6 7
Days
ously observed in fluid milk products (see Chap. 11) with the exception that strain Scott
A failed to grow in liquid whole egg during prolonged incubation at 4 and 10C (Table
6). Although growth of strain Scott A in fluid milk, cheese, and cabbage juice during
refrigeration is well documented, Buchanan et al. [30] recently reported that this strain
failed to grow in meat and poultry products incubated at 4C. As shown in Table 6,
generation times for the five L. monocytogenes strains ranged from 24.0 to 51.0, 8.0 to
31.0, 7.5 to 26.0 and 4.3 to 15.0 h at 4, 10, 20, and 30"C, respectively. Maximum popula-
tions ranged from 5.0 to 7.0, 5.48 to 8.48, 6.85 to 8.0, and 7.0 to 8.0 L. monocytogenes
log,, CFU/g in liquid whole eggs incubated at 4, 10,20, and 3OoC,respectively. However,
Sheldon and Schuman [ 1151 reported that when stored at 4"C, L. monocytogenes popula-
tions in an inoculated commercially available reduced-cholesterol liquid whole egg prod-
uct could be reduced as much as 3.9 orders of magnitude by adjusting the pH of the
product to 6.6 with citric acid and adding nisin at a level of 1000 IU/mL. Considering
current distribution and marketing practices, it is likely that perishable products such as
liquid whole egg will occasionally encounter periods of temperature abuse. Hence, from
these data, it follows that even brief exposure to temperatures 210C can lead to a dra-
matic increase in both growth rate (i.e., decreased generation time) and maximum popula-
592 Cox et al.
2 days of incubation at 4C and then slowly increased to levels near or slightly above
the original inoculum level after 5 additional days of refrigerated storage. Although L.
monocytogenes generally exhibited similar behavior patterns in nonalcoholic samples in-
cubated at 22OC, initial population decreases were far more abrupt, with the pathogen then
increasing to populations 1 to 3 orders of magnitude lower than the original inoculum after
7 days of incubation. Unlike alcohol-free samples, L. monocytogenes was slowly inactivated
in product containing 7% ethanol, with populations typically 1-2 and more than 4 orders of
magnitude lower in 7-day-old samples held at 4 and 22"C, respectively, than were present
initially. Hence, given the normal 2-week refrigerated shelf life of similar commercially
available nonalcoholic eggnog-like products, recontamination of these beverages during
packaging could lead to potential public health problems involving Listeria and other foodb-
orne pathogens, with Salmonella enteritidis and S. typhimurium reportedly also remaining
viable in artificially contaminated samples during 63 days of refrigerated storage.
Thermal Inactivation
Interest in possible heat resistance of L. monocytogenes in eggs is of recent origin; how-
ever, concerns by European scientists regarding potential transmission of Listeria through
eggs prompted a 1955 study by Urbach and Schabinski [ 1251, which examined the ability
of this pathogen to survive in artificially infected eggs that were fried. According to these
authors, L. monocytogenes was isolated from fried eggs (congealed white, soft yolk) pre-
pared from inoculated raw eggs in which the pathogen had previously grown to levels
>5 X 105CFU/g.
Whereas the aforementioned work appears to be fairly crude by current standards
and is now primarily only of historical interest, Foegeding and Leasor [41] conducted a
more sophisticated study in which D-values were determined for five strains of L. monocy-
togenes (Scott A [clinical isolate], F5069 [milk isolate], ATCC 19111 [poultry isolate],
NCF-U2K3 and NCF-FlKK4 [raw liquid whole egg isolates]) in sterile raw egg. Inocu-
lated samples of raw liquid whole egg were added to glass capillary tubes which were
heat-sealed and immersed in a water or oil bath at 51.0, 55.5, 60.0, and 66.0C. After
various times, tubes were removed and contents examined for survivors. Numbers of
Listeria decreased linearly in raw egg during all four heat treatments, with D-values for
the five L. monocytogenes strains ranging from 14.3 to 22.6, 5.3 to 8.2, 1.3 to 1.7, and
0.06 to 0.20 min at 51.0,55.5,60.0, and 66.OoC,respectively. Strain Scott A was generally
less heat resistant than were the other four strains, particularly at the two lower tempera-
tures; however, strain F5069 and the two isolates from raw egg exhibited moderate thermal
tolerance at all four temperatures. Muriana et al. [92] subsequently reported similar
D-values at 60C for L. monocytogenes strain Scott A when inoculated samples of liquid
whole egg were tested using either capillary tubes (D-value of 1.8 min) or a flow injection
system (D-value of 1.95 min). Although this pathogen appears to exhibit a similar degree
of heat resistance in both raw whole milk (see Chap. 6) and raw liquid whole egg, survival
of L. monocytogenes is enhanced by supplementing liquid whole egg or egg yolk with
-
10% NaCl [89]. At 64"C, L. monocytogenes exhibited D-values of 10 and 10.5 min in
-
salted liquid whole egg and egg yolk, respectively, as compared with 1 .O and 1.8 min
for unsalted samples, with increased thermal tolerance attributed to a decrease in water
activity. In contrast, adding 10% sucrose to unsalted samples neither increased thermal
resistance of listeriae nor altered the product's water activity.
In more practical terms, USDA officials currently require that liquid whole egg be
594 Cox et al.
pasteurized at a minimum of 60C for 3.5 min to effect a 9-order of magnitude (9-D)
kill of Salmonella spp. [ 1261. Although results from the study just discussed indicate that
minimum pasteurization of liquid whole egg would yield only a 2.1- to 2.7-D kill of
L. monocytogenes, one must remember that current estimates place L. monocytogenes
populations in liquid whole egg at < 100 CFU/g [4 11. Hence, as is true for milk pasteuriza-
tion, current minimum pasteurization requirements for raw liquid whole egg appear ade-
quate to inactivate normal levels of Listeria that might be present in the product. However,
it is important to stress that current minimum pasteurization requirements for liquid whole
egg, as specified in the USDA Egg Pasteurization Manual, indicate that the margin of
safety is approximately 6 orders of magnitude lower for L. monocytogenes than for most
Salmonella spp. Furthermore, such pasteurization treatments appear to be inadequate for
salted liquid whole egg and egg yolk.
In 1987, Ball et al. [2 11 documented that ultrapasteurization (i.e., pasteurization
at>60"C for <3.5 min) in combination with aseptic processing and packaging can be
used to produce liquid whole egg with a refrigerated shelf life of 3-6 months. After results
from this study were published, FDA officials issued a temporary permit allowing a North
Carolina firm to market ultrapasteurized liquid whole egg [ 12,1071. Although two of the
four objectives of the process were to render the product free of Salmonella and L. monocy-
togenes, the exact time/temperature requirements to completely inactivate L. monocyto-
genes in liquid whole egg were not specified in the FDA temporary permit.
Based on extrapolations from the aforementioned survivor curves which showed no
evidence of tailing, Foegeding and Leasor [4 11 predicted that the ultrapasteurization pro-
cesses used by Ball et al. [21] would effect a 1- to 34-D (average of 14-D) kill of L.
monocytogenes in liquid whole egg. Assuming that the ultrapasteurization times and tem-
peratures used in conjunction with the temporary FDA permit are those values that were
previously determined by Ball et al. [21], Foegeding and Leasor [41] went on to speculate
that four of the 10 thermal treatments used by Ball and coworkers may not conform
to the definition of ultrapasteurization in the temporary permit, depending on how one
views the necessity for a 9-D reduction in numbers of Listeria. However, it appears that
Listeria-free ultrapasteurized liquid whole egg having a refrigerated shelf life of 1 to sev-
eral months can be produced, provided that the raw product is processed using one of the
six more severe timehemperatwe treatments proposed by Ball et al. [21] and then is asep-
tically packaged to eliminate postpasteurization contamination.
Foegeding and Stanley [42] verified the previous predictions concerning heat resis-
tance of Listeria by determining the thermal death time (F-value) for L. monocytogenes
strain F5069 in liquid whole egg. Using their previously described submerged capillary
tube method [39], they found L. monocytogenes was eliminated from inoculated samples
(1.0 X 10' to 4.0 X 108CFU/mL) of sterile liquid whole egg after processing at 62, 64,
66, 69, and 72C for 16.0, 8.0, 4.5, 1.6, and 0.6 min, respectively.
Although results from this study confirm that minimum pasteurization (60C/3.5
min) will not result in a Listeria-free product if initial populations are large, the need for
a 9-D kill as currently required by the USDA may not be appropriate for L. monocytogenes,
since present estimates place the population of this pathogen at < 100 CFU/g in raw liquid
whole egg. Hence, based on maximum expected L. monocytogenes levels in raw liquid
whole egg, pasteurization by current standards should render such products free of
Listeria. The situation regarding ultrapasteurization appears to be somewhat different since
the thermal death-time data obtained by Foegeding and Stanley [42] indicate that 4 of
the 10 ultrapasteurization processes proposed by Ball et al. [21] (63.7OU26.2 s, 63.8*C/
Listeria monocytogenes in Poultry and Egg Products 595
92.0 s, 67.7OU9.2 s, and 71.5OC12.7 s) would likely fail to produce a 9-D decrease in
numbers of Listeria in raw liquid whole egg. Nonetheless, the >9-D kill effected by
the remaining six ultrapasteurization processes proposed by Ball et al. [2 I ] indicates that
ultrapasteurization processes can be designed to produce Listeria-free liquid whole egg
with an anticipated refrigerated shelf life of 3-6 months.
REFERENCES
1. Al-Sheddy, and E.R. Richter., 1989. Microbiological quality/safety of zoo food. Annual
Meeting of the Institute of Food Technologists, Chicago, June 26-29, Abstr. 476.
2. Anonymous. 1988. Code of Federal Regulations, Title 9, Section 381.150.
3. Anonymous. 1988. FSIS recommends 35F for long-term storage of meat, poultry, Food
Chem. News 30( 12):25-28.
4. Anonymous. 1989. Chicken salad recalled in New England due to Listeria. Food Chem.
News 3 1(42):65-66.
5. Anonymous. 1989. Current meat processing may not kill Listeria, study shows. Food Chem.
News 30(52):57-58.
6. Anonymous. 1989. Listeria-contaminated chicken salad recalled from 3 states. Food Chem.
News 3 1(35):5 1.
7. Anonymous. 1989. Listeria found by FSIS in small number but wide range of products. Food
Chem. News 3 1 (30):47-48.
8. Anonymous. 1989. Listeria rnonocytogenes: P&i. Common. Dis. Rep. 89(27): 1.
9. Anonymous. 1989. Listeria tolerances asked by meat, poultry group. Food Chem. News
3 1( 1 4):46-48.
10. Anonymous. 1989. Listeria zero tolerance is warranted, USDA says. Food Chem. News
3 I (19):41-48.
11. Anonymous. 1989. Refrigerated fresh and frozen sandwiches recalled. FDA Enforcement
Report, Dec. 20.
12. Anonymous. 1989. Temporary permit granted antimicrobial liquid eggs. Food Chem. News
30(47):49.
13. Anonymous. 1989. U K establishes committee to investigate food safety. Food Chem. News
30(5 1):39-40.
14. Anonymous. 1989. USDA to toughen regulatory policy on Listeria in meat, poultry. Food
Chem. News 3 1(8):52-53.
15. Anonymous. 1990. Chicken, potato salad recalled by Campbell unit due to Listeria. Food
Chem. News 32(9):61-63.
16. Anonymous. 1990. Irradiation in the production, processing and handling of food. Fed. Reg.
55: 18538.
17. Anonymous. 1990. Prepared sandwiches recalled. FDA Enforcement Report, Jan. 3 1.
18. Anonymous. 1990. USDA monitoring finds Listeria in ready-to-eat products at 78 plants.
Food Chem. News 32(7):71-73.
19. Baccus-Taylor, G., K.A. Glass, J.B. Luchansky, and A.J. Maurer. Fate of Listeria rnonocyto-
genes and pediococcal starter cultures during the manufacture of chicken summer sausage.
Poultry Sci. 72: 1772-1778.
20. Bailey, J.S., D.L. Fletcher, and N.A. Cox. 1989. Recovery and serotype distribution of Liste-
ria rnonocytogenes from broiler chickens in the southeastern United States. J. Food Prot. 52:
148--150.
21. Ball, H.R., Jr., M. Hamid-Samimi, P.M. Foegeding, and K.R. Swartzel. 1987. Functionality
and microbial stability of ultrapasteurized, aseptically packaged, refrigerated whole egg. J.
Food Sci. 52:1212-1218.
596 Cox et al.
22. Baranenkov, M.A. 1969. Survival rate of Listeria on the surface of eggs and the development
of methods for disinfecting them. Tr. Vses. Nauch.-Issled. Inst. Vet. Sanit. 32:453-458.
23. Bartlett, F.M., and A.E. Hawke. 1995. Heat resistance of Listeria monocytogenes Scott A
and HAL 957E1 in various liquid egg products. J. Food Prot. 58:1211-1214.
24. Bartlett, F.M. 1993. Listeria rnonocytogenes survival on shell eggs and resistance to sodium
hypochlorite. J. Food Safety 13:253-261.
25. Belding, R.C., and M.L. Mayer. 1957. Listeriosis in the turkey-two case reports. J. Am.
Vet. Med. Assoc. 131:296-297.
26. Bind, I.-L. 1988. Review of latest information concerning data about repartition of Listeria
in France. WHO Working Group on Foodborne Listeriosis, Geneva, Feb. 15-19.
27. Brackett, R.E. and L.R. Beuchat. 1991. Survival of Listeria monocytogenes in whole egg
and egg yolk powders and in liquid whole eggs. Food Microbiol. 8:331-337.
28. Brackett, R.E. and L.R. Beuchat. 1992. Survival of Listeria monocytogenes on the sur-
face of egg shells and during frying of whole and scrambled eggs. J. Food Prot. 55:862-
865.
29. Breer, C. 1988. Occurrence of Listeria spp. in different foods. WHO Working Group on
Foodborne Listeriosis, Geneva, Feb. 15- 19.
30. Buchanan, R.L., H.G. Stahl, and D.L. Archer. 1987. Improved plating media for simplified,
quantitative detection of Listeria monocytogenes in foods. Food Microbiol. 4:269-275.
31. Carosella, J. 1989. Personal communication.
32. Carpenter, S.L., and M.A. Harrison. 1989. Survival of Listeria monocytogenes on processed
poultry. J. Food Sci. 54556-557.
33. Clouser, C.S., S. Doores, M.G. Mast, and S.J. Knabel. 1995. The role of defeathering in the
contamination of turkey skin by Salmonella species and Listeria monocytogenes. Poultry
Sci. 74:723-73 1.
34. Comi, G., and C. Cantoni. 1985. Listeria spp. in poultry from slaughterhouses of Lombardia.
Indust. Aliment. 24521-525.
35. Cox, N.A., J.S. Bailey, and M.E. Berrang 1997. The presence of Listeria monocytogenes in
the integrated poultry industry. J. Appl. Poultry Res. 6: 116-1 19.
36. Crawford, L.M. 1989. Food Safety and Inspection Service-Revised policy for controlling
Listeria monocytogenes. Fed. Reg. 54:22345-22346.
37. Desmarchelier, P., J. Cox, and R. Esteban. 1995. Study of Listeria spp. contamination in the
egg industry. In Proceedings of XI1 International Symposium on Problems of Listeriosis,
Perth, Western Australia, Oct. 2-6, Promaco Conventions Pty. Ltd., Canning Bridge, West-
ern Australia, pp. 257-260.
38. Dykes, G.A., I. Geornaras, M.A. Papathanasopoulos, and A. von Holy. 1984. Plasmid
profiles of Listeria species associated with poultry processing. Food Microbiol. 11:
5 19-523.
39. Erickson, J.P. and P. Jenkins. 1991. Comparative Salmonella spp. and Listeria mono-
cytogenes inactivation rates in four commercial mayonnaise products. J. Food Prot. 54:
913-916.
40. Felsenfeld, 0. 1951. Diseases of poultry transmissible to man. Iowa State College Vet. 13:
89-92.
41. Foegeding, P.M., and S.B. Leasor. 1990. Heat resistance and growth of Listeria monocyto-
genes in liquid whole egg. J. Food Prot. 53:9-14.
42. Foegeding, P.M. and N.W. Stanley. 1990. Listeria monocytogenes F5069 thermal death times
in liquid whole egg. J. Food Prot. 53:6-8, 25.
43. Franco, C.M., E.J. Quinto, C. Fente, J. L. Rodriguez Otero, L. Dominguez, and A. Cepeda.
1995. Determination of the principal sources of Listeria spp. contamination in poultry meat
and a poultry processing plant. J. Food Prot. 58: 1320-1325.
44. Galli, R., C.G.T. Sannipoli, and C. Valente. 1992. Listeria monocytogenes as broiler carcasses
contaminant. Indust. Aliment. 3 1:21-23.
Listeria monocytogenes in Poultry and Egg Products 597
45. Genigeorgis, C.A., D. Dutulescu, and J. F. Garayzabal. 1989. Prevalence of Listeria spp. in
poultry meat at the supermarket and slaughterhouse level. J. Food Prot. 52:618-624.
46. Genigeorgis, C.A., P. Oanca, and D. Dutulescu. 1990. Prevalence of Listeria spp. in turkey
meat at the supermarket and slaughterhouse level. J. Food Prot. 53:282-288.
47. Ghulam-Rusul, R., Aziah-Ibrahim, and Fatimah-Abu-Bakar. 1992. Prevalence of Listeria
monocytogenes in retail beef and poultry. Pertanika 14:249-255.
48. Gilbert, R.J. 1990. Personal communication.
49. Gilbert, R.J., S.M. Hall, and A.G. Taylor. 1989. Listeriosis update. Public Health Lab. Serv.
Dig. 5:33-37.
50. Gilbert, R.J., K.L. Miller, and D. Roberts. 1989. Listeria monocytogenes and chilled foods.
Lancet 1:383-384.
51. Gitter, M. 1976. Listeria monocytogenes in oven ready poultry. Vet. Rec. 99:336.
52. Glass, K.A., and M.P. Doyle. 1989. Fate of Lisreria monocytogenes in processed meat prod-
ucts during refrigerated storage. Appl. Environ. Microbiol. 55: 1565- 1569.
53. Glass, K.A., and M.P. Doyle. 1991. Fate of Salmonella and Listeria monocytogenes in com-
mercial, reduced calorie mayonnaise. J. Food Prot. 54:69 1-695.
54. Gohil, V.S., M.A. Ahmed, R. Davies, and R.K. Robinson. 1995. Incidence of Listeria spp.
in retail foods in the United Arab Emirates. J. Food Prot. 58:102-104.
55. Gray, M.L. 1958. Listeriosis in fowls-A review. Avian Dis. 2:296-314.
56. Harrison, M.A., and S.L. Carpenter. 1989. Fate of small populations of Listeria monocyto-
genes on poultry processed using moist heat. J. Food Prot. 52:768-770.
57. Harrison, M.A., and S.L. Carpenter. 1989. Survival of large populations of Listeria monocy-
togenes on chicken breasts processed using moist heat. J. Food Prot. 52:376-378.
58. Harrison, M.A., and S.L. Carpenter. 1989. Survival of Listeria monocytogenes on microwave
cooked poultry. Food Microbiol. 6: 153-157.
59. Hatkin, J.M., and W.E. Phillips, Jr. 1986. Isolation of Listeria monocytogenes from an eastern
wild turkey. J. Wildlife Dis. 22: 111- 1 12.
60. Houston, D.L. 1987. Food Safety and inspection Service-Testing for Listeria monocyto-
genes. Fed. Reg. 52:7464-7465.
61. Huang, I.-P.D., A.E. Yousef, E.H. Marth, and M.E. Matthews. 1992. Thermal inactivation
of Listeria monocytogenes in chicken gravy. J. Food Prot. 55:492-496.
62. Huang, D., A.E. Yousef, M.E. Matthews, and E.H. Marth. 1993. Growth and survival of
Listeria monocytogenes in chicken gravy during cooling and refrigerated storage. J. Food
Sew. Syst. 7:185-192.
63. Hudson, W.R., and G.C. Mead. 1989. Listeria contamination at a poultry processing plant.
Lett. Appl. Microbiol. 9:211-214.
64. Huhtanen, C.N., R.K. Jenkins, and D.W. Thayer. 1989. Gamma radiation sensitivity of Liste-
ria monocytogenes. J. Food Prot. 52:610-613.
65. Husu, J.R., J.T. Beery, E. Nurmi, and M.P. Doyle. 1990. Fate of Listeria monocytogenes in
orally dosed chicks. Int. J. Food Microbiol. 11:259-269.
66. Hwang, C.-A., and L.R. Beuchat. 1995. Efficacy of selected chemicals for killing pathogenic
and spoilage microorganisms on chicken skin. J. Food Prot. 58:19-23.
67. Ingham, S.C., and C.L. Tautorus. 1991. Survival of Salmonella typhimurium, Listeria mono-
cytogenes and indicator bacteria on cooked uncured turkey loaf stored under vacuum at 3C.
J. Food Safety 11:285-292.
68. Ingham, S.C., J.M. Escude, and P. McCown. 1990. Comparative growth rates of Listeria
monocytogenes and Pseudomonas fragi on cooked chicken loaf stored under air and two
modified atmospheres. J. Food Prot. 53:289-29 1.
69. Jay, J.M. 1996. Prevalence of Listeria spp. in meat and poultry products (abstr). Food Control.
7 ~209-214.
70. Kampelmacher, E.H. 1958. Berichten uit het Rijksinstitut voor de Volksgezondheit, Utrecht,
The Netherlands. In H.P.R. Seeliger. Listeriosis New York: Hafner, 1961.
598 Cox et al.
71. Kampelmacher, E.H. 1962. Animal products as a source of listeric infection in man. In M.L.
Gray, ed. Second symposium on listeric infection, Montana State College, Bozeman, Mon-
tana, pp. 146- 151.
72. Kampelmacher, E.H., and L.M. van Noorle Jansen. 1969. Isolation of Listeria monocytogenes
from faeces of clinically healthy humans and animals. Zbl. Bakteriol. I Abt. Orig. 21 1:353-
359.
73. Khan, M.A., LA. Newton, A. Seaman, and M. Woodbine. 1975. Survival of Listeria monocy-
togenes inside and outside its host. In M. Woodbine, ed. Problems of Listeriosis. Surrey,
UK, Leicester University Press, pp. 75-83.
74. Kwantes, W., and M. Isaac. 1971. Listeriosis. Br. Med. J. 4:296-297.
75. Kwantes, W., and M. Isaac. 1975. Listeria infection in West Glamorgan. In M. Woodbine
ed. Problems of Listeriosis. Surrey, UK, Leicester University Press, pp. 112-1 14.
76. Lawrence, L.M., and A. Gilmour. 1994. Incidence of Listeria spp. and Listeria monocyto-
genes in a poultry processing environment and in poultry products and their rapid confirma-
tion by multiplex PCR. Appl. Environ. Microbiol. I2:4600-4604.
77. Lawrence, L.M., and A. Gilmour. 1995. Characterization of Listeria monocytogenes isolated
from poultry products and from poultry-processing environment by random amplification of
polymorphic DNA and multilocus enzyme electrophoresis. Appl. Environ. Microbiol. 6 1:
2 139-2 144.
78. Leasor, S.B., and P.M. Foegeding. 1989. Listeria species in commercially broken raw liquid
hole egg. J. Food Prot. 52:777-780.
79. Lewis, S.J., and J.E.L. C o y . 1991. Survey of the incidence of Listeria monocytogenes and
other Listeria spp. in experimentally irradiated and in naturally unirradiated raw chickens.
Int. J. Food Microbiol. 12:281-286.
80. Lieval, F., J. Tache, and M. Poumeyrol. 1989. Qualite microbiologique et Listeria sp. dans
les produits de la restauration rapide. Sci. Aliment. 9: I 11-1 15.
81. Luchansky, J.B., K.A. Glass, K.D. Harsono, A.J. Degnan, N.G. Faith, B. Cauvin, G. Baccus-
Taylor, K. Arihara, B. Bater, A. J. Maurer, and R. G. Cassens. 1992. Genomic analysis of
Pediococcus starter cultures used to control Listeria monocytogenes in turkey summer sau-
sage. Appl. Environ. Microbiol. 5 8:3053-3059.
82. Lund, B.M., M.R. Knox, and M.B. Cole. 1989. Destruction of Listeria monocytogenes during
microwave cooking. Lancet 1 :2 18.
83. MacGowan, A.P., K. Bowker, J. McLauchlin, P.M. Bennett, and D.S. Reeves. 1994. The
occurrence and seasonal changes in the isolation of Listeria spp. in shop bought food stuffs,
human faeces, sewage and soil from urban areas. Int. J. Food Microbiol. 21:325-334.
84. Marino, M., M. Maifreni, G. Comi, and G. Soncini. 1995. Microbiological quality of poultry
meat marketed in Italy. Ingegn. Aliment. Conserve Animali 1 1 :27-33.
85. Marshall, D.L., P.L. Wiese-Lehigh, J.H. Wells, and A.J. Farr. 1991. Comparative growth of
Listeria monocytogenes and Pseudomonas juorescens on precooked chicken nuggets stored
under modified atmospheres. J. Food Prot. 54:84 1-843, 85 1.
86. McKellar, R.C., R. Moir, and M. Kalab. 1994. Factors influencing the survival and growth
of Listeria monocytogenes on the surface of Canadian retail weiners. J. Food Prot. 57:387-
392.
87. McLauchlin, J., N.A. Saunders, A.M. Ridley, and A.G. Taylor. 1988. Listeriosis and food-
borne transmission. Lancet 1 :177- 178.
88. Mead, G.C., W.R. Hudson, and R. Ariffin. 1990. Survival and growth of Listeria monocyto-
genes on irradiated poultry carcasses. Lancet 1:1036.
89. Moore, J., and R.H. Madden. 1993. Detection and incidence of Listeria species in blended
whole egg. J. Food Prot. 56:652-654, 660.
90. Morris, I.J., and C.D. Ribeiro. 1989. Listeria rnonocytogenes and pit&.Lancet 2: 1285- 1286.
91. Mulder, R.W.A.W., S. Notermans, and E.H. Kampelmacher. 1977. Inactivation of salmonel-
lae on chilled and deep frozen broiler carcasses by irradiation. J. Appl. Bacteriol. 42: 179- 185.
Listeria monocytogenes in Poultry and Egg Products 599
92. Muriana, P.M., H. Hou, and R.K. Singh. 1996. A flow-injection system for studying heat
inactivation of Listeria monocytogenes and Salmonella enteritidis in liquid whole egg. J.
Food Prot. 59:121-126.
93. Nagi. M.S., and J.D. Verma. 1967. An outbreak of listeriosis in chickens. Indian J. Vet. Med.
44: 539-543.
94. Nilsson, A., and K.A. Karlsson. 1959. Listeria monocytogenes isolations from animals in
Sweden during 1948 to 1957. Nord. Vet. Med. 11:305-315.
95. Notermans, S., P.S.S. Soentoro, and E.H.M. Delfgou-van Asch. 1990. Survival of pathogenic
microorganisms in an egg-nog-like product containing 7% ethanol. Int. J. Food Microbiol.
10:2O9-2 18.
96. Ojeniyi, B., H.C. Wegener, N.E. Jensen, and M. Bisgaard. 1996. Listeria rnonocytogenes in
poultry and poultry products: epidemiological investigations in seven Danish abattoirs. J.
Appl. Bacteriol. 80:395-401.
97. Ozari, R. von, and F.A. Stolle. 1990. Zum Vorkommen von Listeria monocytogenes in
Fleisch und Fleisch-Erzeugnissen einschliesslich Geflugelfleisch des Handels. Arch. Lebens-
mittelhyg. 41 :47-50.
98. Paluinbo, S., and A.C. Williams. 1989. Freezing and freeze-injury in Listeria monocyto-
geney. Annual Meeting American Society Microbiologists, New Orleans, I, May 14- 18,
Abstr. P-1.
99. Paterson, J. St. 1937. Listerelfa infection in fowls-Preliminary note on its occurrence in
East Anglia. Vet. Rec. 49: 1533- 1534.
100. Paterson, J. St. 1940. Experimental infection of the chick embryo with organisms of the
genus Listerella. J. Pathol. Bacteriol. 5 1 :437-440.
101. Patterson, M. 1989. Sensitivity of Listeria monocytogenes to irradiation on poultry meat and
in phosphate-buffered saline. Lett. Appl. Microbiol. 8: 181 - 1 84.
102. Pini, P.N., and R.J. Gilbert. 1988. A comparison of two procedures for the isolation of Listeria
monocytogenes from raw chickens and soft cheeses. Int. J. Food Microbiol. 7:33 1-337.
103. Pini, P.N., and R.J. Gilbert. 1988. The occurrence in the U.K. of Listeria species in raw
chickens and soft cheese. Int. J. Food Microbiol. 6:3 17-326.
104. Richmond, M. 1990. Report of the Committee on the Microbiological Safety of Food,
HMSO, pp. 133-137.
105. Rijpens, N.P., G. Jannes, and L.M. Herman. 1997. Incidence of Listeria spp. and Listeria
monocytogenes in ready-to-eat chicken and turkey products determined by polymerase chain
reaction and line probe assay hybridization. J . Food Prot. 60548-550.
106. Rodriguez De Ledesma, A. M., H.P. Riemann, and T.B. Farver, 1996. Short-time treatment
with alkali and/or hot water to remove common pathogenic and spoilage bacteria from
chicken wing skin. J. Food Prot. 59:746-750.
107. Ronk, R.J. 1989. Liquid eggs deviating from the standard of identity; temporary permit for
market testing. Fed. Reg. 54: 1794-1795,
108. Ryser, E.T., S.M. Arimi, M.M.-C. Bunduki, and C.W. Donnelly. 1996. Recovery of different
Listcria ribotypes from naturally contaminated, raw refrigerated meat and poultry products
with two primary enrichment media. Appl. Environ. Microbiol. 62: 1781 - 1787.
109. Schlyter, J.H., A.J. Degnan, J. Loefelholz, K.A. Glass, and J.B. Luchansky. 1993. Evaluation
of sodium diacetate and ALTATM2341 on viability of Listeria rnonocytogenes in turkey
slurries. J. Food Prot. 56:808-8 10.
110. Schonberg, A., P. Teufel, and E. Weise. 1988. Isolates of Listeria monocytogenes and Listeria
innocuu. 10th International Symposium on Listeriosis, Pecs, Hungary, Aug. 22-26, Abstr.
45.
111. Seastone, C.V. 1935. Pathogenic organisms of the genus Listerefla. J. Exp. Med. 62:203-
212.
112. Seeliger, H.P.R. 1961. Listeriosis. New York: Hafner.
113. Seeliger, H.P.R., and D. Jones. 1987. Listeria. In Bergys Manual of Systematic Bacteriology,
600 Cox et al.
9th ed. P.H.A. Sneeth, N.S. Mair, N.E. Sharpe, and J.G. Holt, eds., Williams & Wilkins,
Baltimore, pp 1235-1245.
114. Shamsuzzaman, K., L. Lucht, and N. Chuaqui-Offermanns. 1995. Effects of combined elec-
tron-beam irradiation and sous-vide treatments on microbiological and other qualities of
chicken breast meat. J. Food Prot. 58:497-501.
115. Sheldon, B.W., and J.D. Schuman. 1996. Thermal and biological treatments to control psych-
rotrophic pathogens. Poultry Sci. 75: 1126-1 132.
116. Shelef, L.A., and Q. Yang. 1991. Growth suppression of Listeria monocytogenes by lactates
in broth, chicken, and beef. J. Food Prot. 54:283-287.
117. Sionkowski, P.J., and L.A. Shelef. 1990. Viability of Listeria monocytogenes strain Brie-1
in the avian egg. J. Food Prot. 53:15-17, 25.
118. Siragusa, G.R., and M.G. Johnson. 1988. Detection by conventional culture methods and a
commercial ELISA test of Listeria monocytogenes added to cooked chicken (abstr). Poultry
Sci 67(suppl 1):157.
119. Siragusa, G.R., K.J. Moore, and M.G. Johnson. 1988. Persistence on and recovery of Listeria
from refrigerated processed poultry. J. Food Prot. 5 1 9 31-832.
120. Skovgaard, N., and C.-A. Morgen. 1988. Detection of Listeria spp. in faeces from animals,
in feeds, and in raw foods of animal origin. Int. J. Food Microbiol. 6:229-242.
121. Steinmeyer, S. von, and G. Terplan. 1990. Listerien in Lebensmitteln-eine aktuelle Uber-
sicht zu Vorkommen, Bedeutung als Krankheitserreger, Nachweis und Bewertung. DMZ
Lebensmittelindustrie und Milchwirtschaft 11: 150- 155.
122. Steinmeyer, S. von, R. Schoen, and G. Terplan. 1987. Zum Nachweis der Pathogenitat von
aus Lebensmitteln isolierten Listerien am bebriiteren Huhnerei. Arch. Lebensmittelhyg. 38:
95-99.
123. Ternstrom, A., and G. Molin. 1987. Incidence of potential pathogens on raw pork, beef and
chicken in Sweden, with special reference to Erysipelothrix rhusiopathiae. J. Food Prot. 50:
141- 146,149.
124. Thayer, D.W. 1995. Use of irradiation to kill enteric pathogens on meat and poultry. J. Food
Safety 15:181- 192.
125. Urbach, H., and G.L. Schabinski. 1955. Zur Listeriose des Menschen. Z.Hyg. 141:239-248.
126. USDA. 1969. Egg Pasteurization Manual. ARS 74-48. Poultry Laboratory, Agriculture Re-
search Service, USDA, Albany, CA.
127. Uyttendaele, M.R., K.D. Neyts, R.M. Lips, and J.M. Debevere. 1997. Incidence of Listeria
rnonocytogenes in poultry products obtained from Belgian and French abbatoirs. Food Micro-
biol. 14:339-345.
128. Varabioff, Y., G.E. Mitchell, and S.M. Nottingham. 1992. Effects of irradiation on bacterial
load and Listeria monocytogenes in raw chicken. J. Food Prot. 55:389-391.
129. Waldroup, A.L. 1996. Contamination of raw poultry with pathogens (abstr). Worlds Poultry
Sci. 52:7-25.
130. Walker, S.J., P. Archer, and J.G. Banks. 1990. Growth of Listeria monocytogenes at refrigera-
tion temperatures. J. Appl. Bacteriol. 68: 157-162.
131. Wederquist, H.J., J.N. Sofos, and G.R. Schmidt. 1994. Listeria monocytogenes inhibition in
refrigerated vacuum packaged turkey bologna by chemical additives. J. Food Sci. 59:498-
500, 516.
132. Wimpfheimer, L., N.S. Altman, and J.H. Hotchkiss. 1990. Growth of Listeria monocytogenes
Scott A, serotype 4 and competitive spoilage organisms in raw chicken packages under modi-
fied atmospheres and in air. Int. J. Food Microbiol. 11:205-214.
133. Zeitoun, A.A.W., and J.M. Debevere. 1991. Inhibition of Listeria monocytogenes on poultry
as influenced by buffered lactic acid treatment and modified atmosphere packaging. Int. J.
Food Microbiol. 14:161- 169.
15
Incidence and Behavior of Listeria
monocytogenes in Fish and
Seafood*
MELW. EKLUND**
U.S.National Marine Fisheries Service, Northwest Fisheries
Science Center, Seattle, Washington
INTRODUCTION
Listeria monocytogenes is ubiquitous in nature. Many aquatic creatures, including fin fish,
oysters, shrimp, crabs, lobsters, squid and scallops, are harvested from natural environ-
ments; therefore, fish and seafood have been targeted as potential sources of Listeria in
the human diet. Many of these products also undergo various processing procedures, some
of which can inactivate Listeria present on the raw product. Listeria also can enter the
* The views expressed here are those of the authors and are not necessarily endorsed by the U.S. Food and
Drug Administration, National Marine Fisheries, or the Government of the United States.
** Retired.
60 1
602 Jinneman et al.
product both during and after processing by poor sanitation conditions or manufacturing
practices. The psychrotrophic nature of L. monocytogenes allows survival or even multipli-
cation of this potential pathogen during refrigerated storage or temperature abuse situa-
tions. This is a special concern for those products which receive minimal or no heat treat-
ment before consumption. Since the first time L. monocytogenes was isolated from
imported cooked crabmeat in 1987, at least 112 Class I recalls (i.e., a situation where
reasonable probability exists that the use of or exposure to a violative product will cause
serious adverse health consequences or death) have been issued by the U S . Food and
Drug Administration (FDA) for more than 250,000 pounds of ready-to-eat domestidim-
ported fish and seafood, with this pathogen routinely being found in 8.7% of all such
products marketed in the United States. The first of several cases of listeriosis positively
linked to consumption of fish or seafood was not reported until 1989 when a 54-year-old
woman in Italy contracted listerial meningitis 4 days after consuming steamed fish from
which L. monocytogenes was later isolated [36]. This case and the potential hazard associ-
ated with consumption of other Listeria-contaminated ready-to-eat food such as cooked
crabmeat, cooked shrimp, and smoked salmon has prompted studies to determine the inci-
dence and control of Listeria in various seafoods. The incidence and behavior of L. mono-
cytogenes in fish and seafood have been addressed in several review papers [ 13,26,
32,391.
In this chapter, data reviewed are from a series of FDA surveys from I987 to 1996.
These were designed to determine the incidence of L. monocytogenes in domestic and
imported shrimp, crab, and various other fish and seafood products. As in previous chap-
ters, Class I recalls that have been issued for Listeria-contaminated fish and seafoods also
will be mentioned. Surveys of fish and seafood products for Listeria conducted by many
other international groups will be reviewed. The behavior of L. monncytogenes in these
foods, data concerning growth and thermal resistance of L. monocytogenes in seafoods,
as well as measures used such as the application of lactic acid for controlling growth of
Listeria in seafood also will be covered.
Listeria and Escherichia coli in all frozen crabmeat shipped to the United States from
Mexico.
Less than 1 month after this product was recalled, the FDA in Seattle, Washington,
detected L. monocytogenes in samples of imported frozen raw shrimp [3] and lobster tails
[15]. Although no recalls were issued for these products, which are almost invariably
cooked before consumption, confirmation of Listeria in these seafoods together with the
finding in cooked crabmeat noted previously prompted the FDA to initiate two surveys
in July of 1987.
In the first of these surveys, six imported samples of frozen raw shrimp were col-
lected monthly and examined for Listeria at each district office. The samples represented
as many different countries as possible (Table 1) [3]. Additionally, each district also was
requested to collect three domestic samples of frozen raw shrimp per month at the whole-
sale or retail level. Using the original FDA method [69], Listeria spp. were detected in
18 of 74 (24.3%) samples of frozen raw shrimp imported from 10 different countries
between July and October of 1987 (see Table 1). L. monucytugenes also was isolated from
4 of 74 imported samples of frozen raw shrimp, with all positive samples originating from
Central or South American countries. Subsequently, three lots of raw shrimp, imported
from Ecuador and Honduras, were found to contain 103-105L. monocytogenes or L. inno-
cua CFU/g [76]. However, since shrimp are normally not consumed raw in the United
States, FDA officials did not request the recall of any of these contaminated lots.
In the second FDA survey, domestic and imported samples of cooked, frozen, and
refrigerated crabmeat (i.e., picked or extracted) were examined for the presence of L.
monocytogenes, Staphylococcus aureus, Vibrio cholera, V. parahaemolyticus, V. vulniJ-
cus, and Yersinia enterocolitica and numbers of E. coli [3]. Again, samples of imported
crabmeat from as many different countries as possible were collected. As of January 1988,
6 of 98 (6.1 %) domestic samples of cooked crabmeat contained Listeria, with L. monocyto-
genes and L. innocua being recovered from 4 and 2 samples, respectively (Table 2). Simi-
larly, Listeria spp. were detected in 3 of 24 (12.5%) imported samples of cooked crabmeat,
with L. monocytogenes being discovered in 2 of 24 (8.3%) samples of product marketed
in the United States.
Weagant et al. [106] formally published the first results of a survey dealing with
the incidence of Listeria spp. in imported/domestic frozen seafood products analyzed at
the FDA District Laboratory in Seattle during the second half of 1987; 31 of 50 (62%)
imported and 4 of 7 (57%) domestic samples of frozen seafood tested positive for Listeria
spp. using the FDA method [69]. The only Listeria spp. detected were L. monocytogenes
(15 of 57,26.3%) and L innocua (26 of 57,45.6%); both L. monocytogenes and L. innocua
were isolated from several of the samples. Although the number of samples examined
from the various product categories was limited, results suggested that frozen seafood
more frequently contains L. innocua than L. monocytogenes. Hence, as was true for raw
milk, meat, and poultry products, both organisms also likely occupy similar niches in
seafood-processing environments. Therefore, the presence of L. innocua in raw and partic-
ularly in cooked seafood should not be ignored but rather should be viewed as an indicator
of possible contamination with L. monocytogenes.
TABLE
2 Results from an FDA Survey of Domestic/lmported
Refrigerated or Frozen Cooked Crabmeat, July 1987 to January
1988
No. of positive samples (%)
No. of Other
samples L. mono- Listeria
Country of origin analyzed cytogenes SPP.
~~
Discovery of Listeria in raw shrimp, crabmeat, and other seafood products, coupled
with an increased concern about the general safety of seafood, prompted FDA officials
in October 1987 to include analysis for L. monocytogenes in a compliance program for
domestic/imported shrimp [7] and to increase testing of many other domestically produced
seafoods for Listeria spp. under the program for pathogen monitoring of select high-risk
foods (CPGM 7303.030) [ 11,15,40]. This increased sampling effort sought to determine
the geographical distribution of Listeria in domestic/imported seafood and to identify the
incidence of Listeria spp. in such products. In March 1988, a processed seafood assignment
was issued [42]. The purpose of the processed seafood compliance program (CPGM
7303.036) was to test for several bacterial pathogens, including Listeria, in imported and
domestic processed seafood that receives minimal to no processing by the consumer. Prod-
ucts selected for Listeria analyses under the processed seafood compliance program in-
cluded crabmeat (cooked or pasteurized), crayfish/crawfish, lobster, langostinos (cooked,
parboiled), molluscan shellfish, processed imitation seafood (surimi), seafood salads,
shrimp (cooked), smoked or salted fish, and other processed seafood. In addition, the
National Advisory Committee on Microbiological Criteria for Foods (NACMCF) in April
1988 began the laborious task of developing microbiological criteria for cooked shrimp
and crabmeat [8]. During the FDA surveys from October 1988 through September 1990,
L. monocytogenes was recovered from domestic samples of crabmeat, lobster, shrimp,
smoked salmon, and surimi. Imported fish, lobster, shellfish, shrimp, smoked fish, squid,
and surimi also tested positive for L. monocytogenes during the same time period [42].
Three FDA compliance programs in effect since 1991 have surveyed the incidence
of Listeria in fish and seafood products and reported analytical data into the FDA Microbi-
ological Information System. The Domestic Fish and Fisheries Products Compliance Pro-
gram (CPGM 7303.842) covers investigations and sampling of domestic fish and fishery
products [45]. Imported seafood and seafood products are surveyed for presence of Listeria
under the Import Seafood Products Compliance Program (CPGM 7303.844) [44]. The
Processed Seafood Compliance Program (CPGM 7303.036) was in existence through 1994
and covered both domestic and imported processed seafood and seafood products [42,43].
Since 1994 the items covered by this program have been incorporated into the CPGM
7303.842 and CPGM 7303.844 programs for domestic and imported products, respec-
tively. Ready-to-eat food products that require no further or minimal processing by the
consumer or products collected as a follow-up to suspected cases of foodborne illness are
identified for Listeria analyses in each of these compliance programs. The domestic fish
and fisheries product compliance program also includes analyses for Listeria of in-line
and swab samples collected during processor establishment investigation reviews.
Among these three compliance programs a total of 7158 samples of fish, seafood
products or seafood processing in-line samples were analyzed by the FDA between 1991
and 1996. Listeria monocytogenes was detected in 622 of these samples for an overall
incidence of 8.7%. The breakdown of samples analyzed and those in which L. monocyto-
genes was detected is given by year and origin (domestic or import) in Table 3. There is
no significant difference based on a heteroscedastic T-test ( P = .OS) between the incidence
of L. monocytogenes in imported compared with domestic products. The 8.7% incidence
found by the FDA is comparable to the 4-12% incidence of L. monocytogenes in seafood
and seafood products from temperate areas as reported by Embarek [32]. Surveys of other
food products have indicated a 4-60% incidence in raw meat, 23-60% in fresh poultry,
and 2.2% in raw milk [32,39,64].
606 Jinneman et al.
TABLE4 Fish and Seafood Products from which L. monocytogenes Was Isolated
by FDA from 1991 to 1996.
Product/Sample 1991 1992 1993 1994 1995 1996 Total
Crustacean
crab 12 34 37 28 25 6 142
shrimplprawns 7 1 7 11 2 4 32
lobster 8 9 9 7 4 1 38
crawfish 1 0 2 1 2 0 6
Shelljish
mussels 0 1 0 1 0 2 4
oysters 0 1 0 0 0 0 1
clams 1 0 3 1 3 0 8
scallops 0 0 0 0 1 0 1
snails 0 0 1 0 0 0 1
Fin Fish
smoked 16 32 38 33 23 22 164
other 2 12 10 25 11 7 67
Other seafood products
squid/calamari 1 0 1 0 1 0 3
eel 0 5 4 0 0 0 9
roelcaviar 1 1 9 2 5 1 19
imitation seafood 0 2 7 2 2 4 17
seafood (salad, spread, 0 4 3 4 1 1 13
pit& mousse)
processor in-line or swabs 3 12 22 19 28 10 94
not specified 0 0 0 1 0 0 1
Source: Data compiled from the FDA Microbiological Information System. Samples collected from the following
Compliance Programs (CPGM) [42-451: CPGM 7303.842 Domestic Fish and Fisheries Products (1 99 I - 1996);
CPGM 7303.843 Import Seafood Products (1992- 1996); CPGM 7303.036 Processed Seafood (1991-1994).
TABLE
5 Crab and Smoked Fin Fish Samples Analyzed for L. monocytogenes by
FDA from 1991 Through 1996
1991 1992 1993 1994 1995 1996 Total
Crab
positive 12 34 37 28 25 6 142
negative 248 324 363 320 272 217 1744
total 260 358 400 348 297 223 1886
% positive 4.6% 9.5% 9.3% 8.0% 8.4% 2.7% 7.5%
Smoked fin fish
positive 16 32 38 33 23 22 164
negative 117 175 193 23 1 154 176 1046
total 133 207 233 264 177 198 1210
% positive 12.0% 15.5% 16.3% 12.5% 13.0% 11.1% 13.6%
~ ~~
Source: Data compiled from the FDA Microbiological Information System. Samples collected from the following
Compliance Programs (CPGM) [42-451: CPGM 7303.842 Domestic Fish and Fisheries Products (1 99 I - 1996);
CPGM 7303.844 Import Seafood Products (1 992- 1996); CPGM 7303.036 Processed Seafood ( I 99 1- 1994).
608 Jinneman et al.
TABLE
6 Class I Recalls Issued i n the United States for Domestic and Domestic/
Import Ready-to-Eat Seafood Products Contaminated with L. monocytogenes Since
1987
No. of Class I
recalls since
Product 1987 lb affected Location of manufacturer
Crustacean
crab 46 >141197 AL, FL, GA, ME, NC, OR, TX,
VA, WA, Chile, Mexico
shrimp 7 >3 1332 FL, GA, MA, NY, WA
lobster 2 >264 Canada
ShellBsh
mussels (marinated) 1 Unknown MA
mussels (smoked) 1 Unknown New Zealand
snails 1 1455 CO, FL, GA, IL, KS, LA, NH, NJ,
NY, OR, PA, TX, WA
Fin Fish
hot smoked 6 >253 KY, MD, ME, NY, WA
cold smoked 22 >93722 CA, MA, ME, NJ, NY, OR, WA,
United Kingdom
smokeda 16 >9292 CA, FL, IL, MD, ME, NC, NY, SC,
TN, VA, WA
salted 1 Unknown Canada
Other
imitation seafood 5 > 1773 ID, NV, OR, UT, VA, WY, Japan,
Korea
seafood salad or spread 3 >42 FL, ME, WA
with hot smoked 8.8% (19 of 215) samples [57]. Several other investigations have evalu-
ated the prevalence of L. monocytogenes in smoked fin fish products, with incidence rates
ranging from 0 to 75% [32]. In surveys with over 100 samples, the incidence of L. monocy-
togenes also tended to be greater in cold smoked fin fish products (1 1.3-24.0%) [60-621
compared with hot-smoked fin fish products (8.4-8.9%) [61,62]. Similar to ready-to-eat
crab products, postprocess contamination likely accounts for the presence of L. rnonocyto-
genes when it occurs in hot-smoked fin fish products. However, with cold-smoked fin
fish, the process may not eliminate L. monocytogenes present on the raw product; neverthe-
less, it is also possible that contamination could occur during or after processing of the
product [29].
Overall, there have been 112 Class I recalls for domestic and domestichm-
ported ready-to-eat seafood products resulting from presence of L. monocytogenes
during 1987 through August 1998 in the United States [14,41]. Recalls only have been
issued when L. monocytogenes was found in seafood or seafood products which are ready-
to-eat and would therefore receive no subsequent or minimal heat treatment by the
consumer before consumption. The number of recalls by product category is shown in
Table 6. Products which resulted in the greatest number of recalls reflect the types of
products which were most frequently identified as being positive for L. monocytogenes
Listeria monocytogenes in Fish and Seafood 609
in compliance program surveys. Crab accounted for 47 and smoked fin fish for 16 of the
112 recalls.
Crustaceans
Listeria spp., including L. monocytogenes, have been recovered from cooked and picked,
ready-to-eat crabmeat, and, as noted earlier, this product has been the subject of several
recalls. Since crab meat is heat processed to eliminate or reduce microorganisms, the
presence of L. monocytogenes on the finished product most likely represents postpro-
cessing contamination. Several surveys have included small numbers of crab samples. L.
monocytogenes was detected in 7 and L. innocua in 12 of 24 cooked imported crab prod-
ucts [ 1061. Although L. monocytogenes was not detected in another study, L. welshirneri
was found in one of two cooked crab samples [20]. Listeria spp. were recovered in two
of five crab samples collected as part of a survey in Alexandria, Egypt [30], and L. monocy-
togenes was isolated from one of seven crab samples from the United States and China
[37]. Two larger U.S. studies of cooked and processed crab also have been published. In
the first, 3 1 of 138 (22.5%) processed crab samples contained Listeria spp., identified only
to the genus level [84]. In the second study which examined 126 cooked and picked blue
crab samples, 10 samples (7.9%) were positive for L. monocytogenes and 3 samples (2.4%)
TABLE
7 Incidence o f Listeria spp. in Fresh, Frozen, and Processed Seafood
% positive for
No. of L. mono-
Product (country) samples Listeria spp. cytogenes Ref.
Crab
crab (c) (multiple countries) 24 29.2 106
crab (c) (USA) 2 50 0 20
crab (r) (China) 7 14 37
crab (c or p) (USA) 138 22.5 84
crab (QY Pt 1 5 40 0 30
blue crab (c) (USA) 126 10.3 7.9 94
Shrimp/prawn s
shrimp (f), (multiple countries) 7 28.6 106
shrimp (f and fr) (USA) 4 25 0 20
shrimp (f) (Japan) 70 8.6 1.4 75
shrimp (f) (Trinidad) 41 5 1
shrimp (raw, fr) (France) 17 23.5 11.8 93
shrimp (c and p) (multiple countries) 8 25 106
610 Jinneman et al.
TABLE
7 Continued
% positive for
No. of L. mono-
Product (country) samples Listeria spp. cytogenes Ref.
shrimp (r) (multiple countries) 49 8.2 37
shrimp (f and p) (India) 19 10.5 0 73
shrimp (Canada) 20 20 37
shrimp in brine (r) (Norway) 16 18 97
shrimp (c and f ) (Iceland) 11 9 9 56
prawn (r) (Japan) 38 15.8 2.6 99
shrimp (Egypt) 5 40 20 30
prawns/shrimp/cockles (c) (UK) 40 0 95
Lobster
lobster tail (fr) (multiple countries) 2 50 106
Mussels
mussels (sm) (New Zealand) 14 35.7 59
mussels (f) (Spain) 40 22.5 7.5 101
mussels (f) (Australia) 15.4 102
Oysters
oysters (fr) (multiple countries) 1 0 0 106
oysters (p) (USA) 2 0 0 20
oysters (f) (Japan) 84 0 0 75
oysters (f) (Egypt) 2 0 0 30
oysters (f) (Australia) 15.4 102
Clams
clam (f) (India) 1 0 0 49
clam (f) (USA) 1 0 0 20
Scallops
scallops (fr) (multiple countries) 2 50 0 106
scallops (raw) (USA) 1 0 0 20
Other shellfish and invertabrates
shellfish (c) (Iceland) 11 0 0 56
non-oyster shellfish (f) (Japan) 147 11.6 1.4 75
shellfisha 25 44 25 59
Donax spp. (coquina) (f) (Egypt) 6 16.7 16.7 30
Ruditapes spp. (clam) (f) (Egypt) 4 50 25 30
Fin Fish
fish (f) (USA) 4 50 50 20
catfish (f) (USA) 1 100 0 20
fish (f) (Trinidad) 61 14.8 2 1
minced fish (f) (Norway) 8 12 97
fish (f) (Japan) 382 12.6 2.4 75
fish (f) (India) 51 3.9 0 73
fish (f) (Egypt) 39 25.6 12.8 30
fish (fr) (Egypt) 17 17.6 5.9 30
fish (f) (India) 4 25 49
fish (fr) (India) 10 20 49
fish (r) (New Zealand) 25 52 32 59
fish minced (raw, r) (Japan) 37 43.2 8.1 99
fish (trout) (f) (Iceland) 2 0 0 56
Listeria monocytogenes in Fish and Seafood 611
TABLE7 Continued
% positive for
No. of L. mono-
Product (coutitry) samples Listeria spp. cytogenes Ref.
fish (dried haddock) (Iceland) 5 0 0 56
fish (fr) (multiple countries) 4 25 106
fish (ceviche) (Peru) 32 75 9 48
fish (lightly pickled) (Switzerland) 89 25.8 61
fish (gravad) (Iceland) 22 63.6 22.7 56
fish (cold-sm, salmon) (Switzerland) 100 24 60
fish (cold-sm, salmon) (Switzerland) 64 6.3 52
fish (cold-sm, fish) (Switzerland) 324 13.6 61
fish (cold-sin, fish) (Switzerland) 434 11.3 62
fish (cold-sin, salmon) (Norway) 33 9 97
fish (sm,-salmon) (Iceland) 31 29 3.2 56
fish (cold-sin, salmon) (Canada) 32 31.2 37
fish (cold-srn, salmon) (Italy) 37 0-80 0 104
fish (cold-srn, salmon) (New Zealand) 12 75 59
fish (cold-sm, salmon) (USA) 61 78.7 29
fish (sm)-(New Zealand) 12 66.7 59
fish (sm fish) (Canada) 71 11.3 27
fish (sm fish) (Canada) 20.4 4.4 25
fish (hot-sm fish) (Switzerland) 496 8.9 61
fish (hot-sm fish) (Switzerland) 69 1 8.4 62
fish (sm and/or salted) (Egypt) 11 18.2 5.6 30
fish (f) (Denmark) 232 14.2 2
fish (cold-sm, cured) (Denmark) 335 10.8 2
Other fish and seafood products
seafood (squid, langostinos) (multiple countries) 2 I00 0 106
seafood (f arid fr) (Taiwan) 57 10.5 105
seafood (f and p) (Iceland) 26 3.9 3.9 56
seafood (India) 200 8 0 65
seafood ( f ) (USA) 59 49.2 84
seafood (p) (USA) 14 0 84
seafood (other) (Iceland) 5 20 20 56
seafood raw, (r) (Japan) 28 7.1 10.7 99
seafood (r) (New Zealand) 50 48 26 59
seafood (c) (Japan) 5 0 0 99
seafood (other) (Japan) 6 0 0 99
seafood salad (p) (USA) 2 0 0 20
fish salads (r) (Iceland) 37 32 16 56
seafood (past ) (pasta with minced fish) (Iceland) 3 0 0 56
seafood (surirni) (multple countries) 7 28.6 106
seafood (surirni) (USA) 1 0 0 20
seafood (surirni) (Canada) 46 2 37
~ ~-
Included in the 25 ready-to-eat fish samples in the New Zealand study listed above.
Source: Adapted in part from Ref. 32.
672 Jinneman et al.
positive for L. innocua [94]. Very few studies have enumerated L. monocytogenes in
naturally contaminated cooked and processed crab. Among the 10 samples positive for
L. monocytogenes in one study [94], one sample contained 1100 Listerialg, but in the
remaining samples, <100 Listeridg were detected, indicating a generally low level of
contamination.
Listeria spp. have been detected in fresh or raw shrimp. Two of seven samples
contained L. monocytogenes [ 1061; one of four samples contained Listeria innocua 1201
in two U.S. studies. In a survey of fresh shrimp in Japan, Listeria spp. were recovered
from 6 of 70 samples (8.6%), with one (1.4%) being positive for L. monocytogenes [70].
In France, L. monocytogenes was isolated from 2 of 17 (1 13%) uncooked shrimp samples
and Listeria spp. from 4 samples (23.5%) [93]. In Trinidad where shrimp are consumed
in a nearly raw state, Listeria spp. were recovered from 2 of 41 ( 5 % ) fresh, uncooked
shrimp samples [I].
Despite cooking and other heat-processing steps which should eliminate Listeria
spp. present on raw product, several investigators have recovered Listeria spp. from
cooked and ready-to-eat shrimp/prawns. L. monocytogenes was detected in 2 of 8 (25%)
cooked and processed shrimp in a U.S. study [106], 4 of 49 (8.2%) ready-to-eat shrimp
in Canada [37], and 1 of 38 (2.6%) ready-to-eat shrimp products in Japan 1993. No L.
monocytogenes was recovered from 40 retail samples of cooked prawns, shrimp, and cock-
les sold in England and Wales between 1987 and 1989 [95]. Ready-to-eat shrimp in brine
had L. monocytogenes in 3 of 18 (16.7%) samples, tested in Norway [97].
As with the surveys of cooked and processed crab, there have been few studies where
numbers of L. monocytogenes were determined in cooked shrimp products. However, low
levels of L. monocytogenes in three lots of naturally contaminated ready-to-eat shrimp
(0.54,5.5, and 0.04 most probable number (MPN)/g)and lobster (2.0,0.23, and 0.4 MPN/
g) were reported in a Canadian study [37].
She1Ifish
Smoked mussels have been associated with several listeriosis cases in Australia and New
Zealand [35,77] and with recalls in the United States. L. monocytogenes was isolated from
5 of 14 smoked mussel samples in a survey [59] in New Zealand, whereas fresh Spanish
mussels yielded L. monocytogenes from 3 and other Listeria spp. from 9 of 40 samples
[loll.
The incidence of Listeria in other shellfish products, including oysters, clams, and
scallops has been remarkably low. No Listeria spp. were isolated from oysters in two
studies in the United States and one in Egypt which included one or two samples
[20,30,106]. In a study in Japan, no Listeria spp. were recovered from 84 oyster samples
[75]. No Listeria spp. were found in a single clam sample in each of two studies [20,49];
however, in a study in Egypt, Listeria spp. were found in two of four Ruditapes spp.
(clam) samples, with L. monocytogenes being present in one of these samples [30]. Only
a limited number of samples of scallops have been tested; however L. innocua was isolated
from one of two frozen U.S. samples [ 1061, but no Listeria spp. were recovered from a
single sample in a later study [20]. No Listeria were found in 11 Icelandic cooked shellfish
samples [56]. In a survey of fresh seafood samples purchased from markets in Alexandria,
Egypt, one of six Donax spp. (coquina) was positive for L. monocytogenes [28]. In Japan,
L. monocytogenes was isolated from 2 samples and Listeria spp. were recovered from 17
samples of a total of 147 non-oyster shellfish samples [75,81].
Listeria monocytogenes in Fish and Seafood 613
Fin Fish
In the United States, raw fresh or frozen fish are generally not consumed without further
processing; therefore, surveys for Listeria spp. have included very few fresh or frozen
fish samples. In surveys conducted in the United States, L. monocytogenes was isolated
from two of four fresh fish samples, L. innocua from one catfish sample [20] and L.
monocytogenes from one of four frozen fish samples [106]. In India, Fuchs and Surrendan
[49] reported Listeria spp. in 1 of 4 fresh and 2 of 10 frozen fish samples. In a larger
survey of fresh fish in India, Listeria spp. were isolated from 2 of 51 (3.9%) samples, but
L. monocytogenes was not recovered [73]. In Alexandria, Egypt, Listeria spp. were present
in 10 of 39 (25.6%) fresh and 3 of 17 (17.6%) frozen fish samples, with L. monocytogenes
being isolated from 5 of 39 (12.8%) and 1 of 17 (5.9%) of these fresh and frozen fish
samples, respectively [30]. In Norway, L. monocytogenes was isolated from one of eight
(12.5%) minced fresh fish samples [97]. The practice of consuming fresh seafood in an
almost raw state is common in Trinidad where Listeria spp. were detected in 9 of 61
(14.8%) fresh fish samples [I]. In Japan, 3 of 37 (8.1%) raw ready-to-eat minced fish
samples were positive for L monocytogenes and 16 of 37 (43.2%) for Listeria spp. [99].
In a similar Japanese survey, L. monocytogenes was found in 9 (2.4%) and Listeria spp.
in 48 of 382 (12.6%) samples [75]. However, it is not clear if all these samples were
ready-to-eat.
processed fish-based product from which L. monocytogenes was found, with an incidence
of 28.6% in the United States [I061 and 2.2% in Canada. No Listeria were recovered
from a single sample in a second U.S. survey [20].
togenes in ready-to-eat foods have been based on the ability of L. monocytogenes to grow
in a given food product [38].
There still is not complete agreement among the countries which compose the Euro-
pean Economic Community (EEC) for criteria regarding L. monocytogenes in various
foods [ 1031. Criteria for L. monocytogenes developed by the EEC are only within the Milk
Hygiene Directive. Several public health approaches to food safety including addressing
industry groups about HACCP-based hygiene plans and educating the most susceptible
groups, (e.g., pregnant women and immunocompromised individuals) have been used by
individual European countries. A quantitative approach setting limits at the point of sale
or at the end of product shelf life, also has been explored by several countries. For example,
German regulations categorize foods into four risk levels and set specific L. monocyto-
genes action levels based on the risk category. Group I foods have the most restrict-
ive limit (absence of L. monocytogenes in 25 g or 25 mL of food). Within the German
approach, seafood products like heat-treated shrimp or prawns would be categorized
as Group 111. For products in this category, low-level contamination (<loo L.
monocytoguneslg) would require a food establishment hygiene check. Higher levels of
Contamination would cause the product to be classified as unfit for human consumption
or a health hazard in substantiated cases [ 1031.
Modes of Transmission
Current data point to cross contamination as the major source of Listeria in cooked or
otherwise processed seafood, as evidenced by the recovery of healthy, noninjured cells of
L. monocytogenes from the surface of many heat-processed/ready-to-eat seafood products.
However, a small percentage of aquatic creatures may become contaminated through
direct/indirect contact with Listeria in their natural environment. This appears even more
plausible when one recalls the salt-tolerant nature of L. monocytogenes and that this patho-
gen has been isolated from sewage effluent entering the North Sea [24] and also from
crustaceans that were harvested from stream water in which L. monocytogenes was previ-
ously identified [86]. In 1989, Fuad et al. [47] evaluated the ability of L. monocytogenes
to survive in the estuarine environment. Since there appears to be a higher incidence of
Listeria in chitinous seafood (i.e., shrimp, crab, lobster), samples of filtered and unfiltered
seawater with and without chitin and chitin-free filtered and unfiltered stream water were
inoculated with various strains of L. monocytogenes, many of which possessed chitinase
activity. Although Listeria populations decreased in chitin-free filtered and unfiltered sea-
water, adding chitin to both types of seawater stimulated growth of Listeria. Moreover,
the pathogen grew in filtered stream water.
These findings, along with those from a report in which L. monocytogenes was found
on the exoskeleton but not in the digestive tract of shrimp that were exposed to high
levels of L. monocytogenes in aquaculture tanks [ 1051, suggest that this pathogen may be
676 Jinneman et al.
ecologically adapted to chitin. If this is true, then it is imperative that holding tanks for
chitinous marine animals be properly set up and maintained to avoid potential microbiolog-
ical problems involving L. monocytogenes and other foodborne pathogens including Vibrio
spp. and Aeromonas hydrophilia.
It is well established that Listeria spp. are often associated with wild animals and
birds which can serve as reservoir hosts. Fenlon et al. [46] demonstrated the role that
scavenging birds can play in the Listeria cycle. In that study, a definite association between
gulls feeding on sewage and fecal carriage of Listeria (26.3% positive) was shown, which
compared with a carriage rate of only 8% for gulls feeding in less polluted areas. This
higher carrier rate suggests that L. monocytogenes is a part of the normal microflora of
the near estuarine environment [ 131. Two studies of estuarine waters, shrimp, and oysters
along the northern Gulf of Mexico [80] and of freshwater tributaries, sediments, bay water,
and oysters in the Humboldt-Arcata Bay of Northern, California 1221, reached similar
conclusions. In one study [79], 5% of 78 saltwater samples were positive for Listeria spp.
In comparison, 11% of 74 shrimp samples were positive for L. monocytogenes and no
Listeria were isolated from oysters. Listeria species and L. monocytogenes were found in
8 1 and 62% (37 samples), respectively, of freshwater or low-salinity waters in tributaries
draining into Humboldt-Arcata Bay. The incidence of Listeria spp. and L. monocytogenes
in sediment (46 samples) from the same tributaries was 30.4 and 17.4%, respectively. One
of three bay water samples contained Listeria spp. (including L. monocytogenes), whereas
L. innocua was recovered from only 1 of 35 oyster samples [22].Both of these studies indicate
that the estuarine environments are continuously subjected to potential contamination with
Listeria spp. from, for example, processing effluents, agricultural runoff, and sewage efflu-
ents. Listeria spp. can be recovered from nonpolluted environments, and the source of these
bacteria may very well be from avian species, especially sea-gulls [79].
erally decreasing by approximately I log after 2 I days of storage in an ice chest. When
catfish were stored at 4C the L. monocytogenes population increased slowly ( 1 .O- 1.5
log,,,) during the first 12 days and then decreased I .5 log,,, by day 16 [68]. During storage,
psychrotrophic populations increased from 1O3 to > 1 O7 CFU/g, thus reinforcing the notion
that L. monocytogenes can readily survive in refrigerated raw foods even when greatly
outnumbered by other natural contaminants. Since L. monocytogenes was recovered from
laboratory-contaminated shrimp (initial inoculum 2 10' CFU/g) after 90 days at -20C
[76], it is evident that this pathogen also is fairly resistant to subfreezing temperatures.
Unlike the aforementioned products, preliminary results from Kaysner et al. [66]
suggest that L. monocytogenes was unable to grow in artificially contaminated oysters,
with Listerill populations remaining constant in shucked oysters after 21 days at 4C.
Apparent inability of Listeria to grow in raw oysters may be related to difficulties in
isolating Listeria from retail raw oysters. According to Farber [37], L. monocytogenes
(inoculum level of 2 X 103CFU/mL) grew fairly well on cooked lobster, shrimp, crab,
and smoked fish and in most instances increased about 2-3 log,,, within 7 days at 4C.
When these same products were temperature abused for a short time (6 h) at room tempera-
ture, levels of L. monocytogenes increased by 1 log on shrimp. crab, and lobster, and only
0.2 log on smoked salmon. In a survey for the incidence of this pathogen on shrimp and
lobster meat at the wholesale level, 13 of 113 samples were positive and were contami-
nated at a level of < 10 MPN/g. Storage of these naturally contaminated products at 4C
resulted in L. monocytogenes populations of <I00 MPN/g after 1 week, with numbers
increasing to 2.5 X 103MPN/g after 2 weeks [37]. In these studies, the growth rate of
different strains of L. monocytogenes was comparable in shrimp, crab, and cooked lobster.
However, the difference in growth rate of two strains in smoked salmon probably reflected
a greater tolerance of one strain to sodium chloride and/or the antimicrobial compounds
present in smoke.
The behavior of L. monocytogenes in cold-smoked salmon has been studied at sev-
eral laboratories. Guyer and Jemmi [53] and Jemmi [62] determined growth of L. monocy-
togenes during fabrication and storage of cold-smoked salmon. During the preparation of
cold-smoked salmon, L. rnonocytogenes did not change when inoculated onto the surface
levels. Populations increased to 2.3 X 106 MPN/g at 10C and up to I .5 X 10s at 4C
during 20 days of storage. After 30 days, L. monocytogenes had increased to 107MPN/
g in samples stored at both temperatures. A pH of 5.8 and a, of 0.93 did not prevent
growth of L. nionocytogenes. Eklund et al. [29] demonstrated that with injection of recircu-
lated brines into the interior of cold-smoked salmon, L. monocytogenes also was inoculated
into these sites. During processing at 17.2-2 1.1 "C, L. rnonocytogenes increased 2- to
6-f0ld, and at 22.2-30.6"C this pathogen increased up to 100-fold. These authors thus
emphasized the importance of eliminating or reducing the L. rnonocytogenes population
on the outside of the fish before they are filleted. In addition, they also recommended that
brines that drain off fillets during the injection process not be collected, recirculated, and
injected into the product.
In a similar study by Hudson and Mott [ 5 8 ] ,L. rnonocytogenes populations increased
rapidly on colcl-smoked salmon within the shelf life of the product. At 10C, L. monocyto-
genes growth was comparable in samples packaged in either oxygen-permeable or oxygen-
impermeable films. However, at 5 C the lag phase was longer in vacuum-packaged prod-
uct, but once growth was established, generation times were again comparable. The effect
of inoculum level on the growth of L. monocytogenes in cold-smoked salmon stored at
4C was reported by Rorvik et al. [96]. Starting with an initial population of 6 or 600
CFU/g, levels increased about 4.8 log,(]for the higher inoculum and 2.1 log,,, for the 6
6 78 Jinneman et al.
CFU/g inoculum. In these same studies, the growth rate of L. monocytogenes also was
determined in products with different initial bacterial counts. When the inoculum level
was 6 CFU/g, L. monocytogenes populations increased faster in samples with the lower
rather than higher initial total bacterial populations.
Inhibition
Of the processes used to prepare smoked fishery products, the cold-smoking operation has
been of special interest, because the temperatures used are not lethal to L. monocytogenes.
Hence, the following interventions have been recommended to reduce the risks associated
with L. monocytogenes in these products: (a) eliminate or reduce numbers of L. monocyto-
genes on the outside surfaces of frozen or fresh fish before filleting, (b) prevent recontamina-
tion and growth of L. monocytogenes during all stages of processing, and (c) inhibit growth
of any possible survivors or recontaminants during processing and distribution 1291.
Several papers have been published on inhibition of L. monocytogenes in cold-
smoked fish processed with sodium chloride, sodium nitrite and sodium lactate. In these
studies, smoke was not applied to the products, so that the efficacy of the different forms
of inhibition could be addressed. Peterson et al. [91] studied behavior of L. monocytogenes
(150 CFUI15 g) in cold-processed salmon containing 3.5 or 6.0% water phase sodium
chloride. The products were packaged in either oxygen-permeable film or vacuum sealed
in impermeable film and stored at 5 and 10C. After the second week at 1OOC, L. monocytu-
genes populations increased to the range of 106-108 CFU/g, with no difference being
attributed to the sodium chloride concentration. Vacuum packaging suppressed growth of
L. monocytogenes by 10- to 100-fold in samples with 3 or 5% sodium chloride. Inhibition
related to salt concentration was most apparent at 5"C, with L. rnonocytogenes populations
being held below 10' CFU/g by 6% water phase salt, but increased to 104CFU/g in
products with 5% water phase salt and to 10' CFU/g with 3% water phase salt. Brown
sugar is often used in processing of cold-smoked salmon; use of the sugar in the product,
however, did not influence growth of L. monocytogenes. In these same studies, growth
of the clinical isolate Scott A and two L. monocytogenes strains isolated from salmon
were comparable in cold-smoke salmon stored at 5 and 10C.
Given the salt tolerance of L. monocytogenes and consumer unacceptability of
smoked fish products with water phase NaCl concentrations much above 3 or 4%, it was
concluded that other inhibitors, in addition to NaCl, were needed to control growth of
this bacterium. Pelroy et al. [90] therefore studied the behavior of L. monocytogenes (150
or 4.9 X 103CFU/15-g sample) in relation to sodium nitrite (190-200 ppm) combined
with sodium chloride in cold-processed salmon stored at 5 and 10C. The combination
of NaCl and NaN0, was most effective at 5C. With an initial inoculum of 150 CFU/l5 g,
L. monocytogenes was held below IS CFU/g by a combination of 190-200 ppm NaN02
and 3% water phase salt and below 20 CFU/g with NaNO, and 5% NaC1. Packaging in
oxygen-permeable or oxygen-impermeable (vacuum-sealed) films had little effect on
growth of L. monocytogenes when NaN02 was included in the process. Increasing the
storage temperature to 10C markedly reduced the efficacy of both NaCl and NaCl +
NaN0, treatments. There was little difference in inhibition between 3 or 5% water phase
NaCl at 10C, and the combined effect of NaCl and NaNO, was only slightly greater than
than that of NaCl alone. However, the packaging method had the most pronounced effect
on growth of L. monocytogenes at 10C. Growth was consistently greater in samples
packaged in oxygen-permeable film than in the vacuum-sealed impermeable film pack-
Listeria monocytogenes in Fish and Seafood 619
ages. L. monocytogenes populations had increased from 10 CFU/g to about 108 CFU/g
in samples packaged in oxygen-permeable film and 106 CFU/g in impermeable film.
Differences in inhibition attributable to inoculum level and NaCl + NaN02concentrations
were obvious in products stored at 5C. In comparison, when the initial inoculum level
was increased to 327 CFU/g, L. monocytogenes reached a population of l 06- 107CFU/g
after 20 days of storage.
Of the different inhibitors studied by Pelroy et al. [89], sodium lactate used in combi-
nation with salt or salt plus sodium nitrite was most effective in controlling growth of L.
monocytogenes (150 CFU/l5 g) on vacuum packaged cold-smoked salmon stored at 5
and 10C [89]. A concentration of 3% lactate in combination with 3% salt or 3% salt +
125 ppm nitrite prevented any increase in L. monocytogenes populations during 40-50
days of storage at 5 or 10C (Fig. 1). Addition of 2% sodium lactate prevented growth
of L. rnonocytogenes in all samples stored at 5C. At 10C, however, a combination of
sodium lactate (2%) and NaCl (3%) inhibited growth for 14 days, but then the pathogen
reached populations about 1-2 logs less than those of the control samples with NaCl only.
When the products contained NaN02 (125 ppm), sodium lactate (2%), and NaCl (3%),
growth of L. monocytogenes was totally suppressed at 10C except in one of the four
samples where populations reached 9.3 X 102 CFU/g.
Certain spices and herbs also can be used to minimize growth of Listeria, as previ-
ously discussed in Chapter 6. According to Rorvik et al. [98], L. monocytogenes popula-
tions remained unchanged on vacuum-packaged gravad salmon (i.e., an unsmoked salmon
product containing dill with a similar pH, salt content, and a, to that of smoked salmon)
10C 5C
7 E 3% NaCl
$ 7
0 No Lactate
A 2% Lactate
Days stored
during 4 weeks of storage at 4C. Since addition of as little as 0.5% dill prevented growth
of Listeria in laboratory media, dill was most likely responsible for inhibiting L. monocyto-
genes in gravad salmon during extended cold storage.
Fermentation products from lactic acid bacteria have been studied to determine their
efficacy in controlling L. monocytogenes growth in blue crab meat [23]. In these studies,
steam-sterilized crab meat was inoculated to contain 5.5 log,, CFU/g of a three strain
mixture of L. monocytogenes and then washed with various lactic acid bacteria fermenta-
tion product at levels of 2000-20,000 arbitrary units (AU) per milliliter of wash water.
L. monocytogenes populations remained relatively constant in control samples stored for
6 days at 4C. In comparison, numbers of L. monocytogenes on crabmeat washed with
Perlac 1911 or Micro-Gard (10,000-20,000 AU) initially decreased (0.5-1 .O log,, unit/g)
and then recovered to original levels within 6 days. When crab meat was washed with
10,000-20,000 AU of Alta 234 1, enterocin 1083, or nisin per milliliter, L. monocytogenes
populations initially decreased by I .5-2.7 log units/g. Thereafter, counts increased by
0.5- 1.6 log 1o units within 6 days.
Degnan et al. [23] also showed that when the crabmeat was washed with food-grade
sodium acetate (4 M), sodium diacetate (0.5 to 1 M), and sodium lactate ( I M) or sodium
nitrate (1.5 M), there was only a modest reduction in L. monocytogenes population (0.4-
0.8 log,, unit/g). However, Listeria counts decreased 2.6 logl0/gwithin 6 days when the
sodium diacetate concentration was increased to 2 M. Trisodium phosphate (1 M) also
reduced L. monocytogenes counts from 1.7 to >4.6 log 10/gwithin 6 days. Hence, L. mono-
cytogenes populations on crabmeat can be reduced by washing with selected antimicrobial
agents.
Potential use of certain strains of Enterococcus faecium to control L. monocytogenes
was suggested by Embarek et al. [34]. E. faecium isolates from sous-vide cooked fish
fillets were tested on different strains of L. monocytogenes and other pathogenic bacteria
using Brain Heart Infusion broth with added CaC03 to avoid decreases in pH during
growth of E. faecuim. Of the 19 isolates tested, 14 produced proteinaceous substances
inhibitory to different strains of L. monocytogenes. An inoculum of 107CFU E. faecium/
mL reduced L. monocytogenes populations of 102CFU/mL to lO/mL after 14 days at
3C and to I CFU/mL after 35 days. With a lower inoculum of 1 O4 CFU/mL, L. monocyto-
genes was only slightly inhibited at 15C and not at 3 or 5C. However, after 11 days at
15"C, spontaneous resistance was observed and L. monocytogenes reached I X I O8 CFU/
mL. All of these L. monocytogenes isolates were resistant to E. faecium.
The antibacterial effects of 209 psychrotrophic Pseudoinonas strains isolated from
spoiled iced fish and newly caught fish were assessed by screening L. monocytogenes and
other organisms using agar diffusion assays [51]. Only eight strains inhibited growth of
L. monocytogenes. Inhibitory action was most pronounced among Pseudomonas strains
producing siderophores which chelate iron; addition of iron sometimes eliminated the
antibacterial effect. Some strains of Pseudomonas enhanced growth of L. monocytogenes,
with dense growth being observed around wells containing these Pseudomonas strains.
These strains may have created a more advantageous nutritional environment for L. mono-
cytogenes by increasing the supply of iron or the availability of low molecular weight
nutrients.
Inactivation
As was true for dairy, meat, and poultry products, the rash of Class I recalls during the
past decade involving ready-to-eat seafoods has prompted concerns about the adequacy
Listeria monocytogenes in Fish and Seafood 621
of thermal processing treatments used for raw seafood. Hence, in early 1988, Pace et al.
[88] reported results from a study in which freshly shucked oysters were exposed to 150
ppm chlorine for 30 min, pasteurized at an internal temperature of 72-74C for 8 min,
and then periodically examined for major bacterial groups during 5 months of refrigerated
storage. According to these authors, chlorination reduced initial aerobic plate counts of
4.5 X 1O5 C'FU/g by 40-90%, with pasteurization reducing the population by an additional
99.9%. Despite these large reductions in microbial flora, survivors classified in eight differ-
ent genera of aerobic or facultatively anaerobic bacteria were present in the product. How-
ever, at this point, the oysters were unfit for consumption, as evidenced by profuse gas
production and swelling of plastic pouches in which the product was pasteurized.
In response to public and industry concerns, the FDA 1761 also examined the thermal
resistance of Listeria in raw shrimp tails that were inoculated internally to contain approxi-
mately 104--1OS L. rnonocytogenes CFU/g. Using a combination of cold (with/without
broth) enrichment and warm (selective) enrichment, these investigators failed to recover
the pathogen from shrimp tails that were boiled longer than 5 min. Although appreciable
numbers of heat-stressed cells were detected in inoculated shrimp tails that were boiled
for 3 min, frozen storage of the product at -20C eventually led to complete inactivation
of the pathogen. More important, when this study was repeated using naturally contami-
nated frozen shrimp from Ecuador and Honduras in which 1 03-1OS L. monocytogenes or
L. innocua CFU/g were presumably present only on the chitinous exoskeleton, all Listeria
were eliminated after 1 min of boiling. Hence, since shrimp are more likely to be contami-
nated externally than internally with relatively low levels of Listeria, these findings sug-
gest that present cooking methods are adequate to eliminate these organisms from raw
shrimp.
Since these initial studies, the thermal death time of L. monocytogenes has been
determined in different seafoods and fish. The results of these studies are summarized in
Table 8. In an effort to determine if the presence of L. rnonocytogenes in processed lobster
could be the result of undercooking or postcooking contamination, Budu-Amaoko et al.
[21] determined the thermal death time of 107cells of L. monocytogeneslg of product
using 25-g samples. The observed D-values at 51.6, 54.4, 57.2, 60.0, and 62.7"C were
97.0, 55.0, 8.3, 2.39, and 1.06 min, respectively, with a z-value of 5.0"C (Table 8). After
isolating this pathogen from plants using good manufacturing practices, the authors specu-
lated that the presence of L. monocytogenes in the final product was probably the result
of underprocessing .
Blue crab is a popular seafood item, with more than 50% of picked meat being
pasteurized to offset seasonal fluctuations in some regions. The lack of information about
the growth and survival of this pathogen in blue crabmeat prompted Harrison and Huang
[55] to determine the heat resistance of the Scott A strain in this product. The crabmeat
was inoculated with 107 cells/g before distributing 7.5 g into sausage casings (1.6 X 4
cm) for thermal processing. D-values were 40.43, 12.0, and 2.6 I min at 50, 55, and 6OoC,
respectively, with a z-value of 8.40"C as determined by using Trypticase Soy Agar (see
Table 8). Use of Vogel Johnson agar, a selective plating medium that is less able to support
repair and subsequent growth of sublethally injured Listericz, yielded lower D-values
(34.48, 9.18, and 1.31 min) and a lower z-value (6.99"C) at the same temperatures.
Heightened interest in foodborne listeriosis has also led to intense efforts toward
determining whether current industry practices for cooking crawfish are adequate to inacti-
vate L. rnonocytogenes. In 1993, Dorsa et al. [28] first determined the growth rate of L.
monocytogenes in crawfish tail meat stored at 0.6 and 12C. Exponential growth began
with no apparent lag phase and 109CFU/g were observed after 10 and 4 days at 6 and
622 Jinneman et al.
ACKNOWLEDGMENTS
The authors thank Cecilia Wolyniak (FDA, CFSAN), Pat Pinkerton (FDA, Seattle Dis-
trict), Stephanie Dalgliesh (FDA, Seattle District), and Daryl Thompson (FDA, Atlanta
Regional Office) for retrieval and assistance with the FDA recall information. Maxine
Heinitz (FDA, Midwest Laboratory for Microbiological Investigations, FDA Microbiolog-
ical Information Systems Manager) and Jan Johnson (FDA, Seattle District) provided valu-
able assistance with accessing data from the FDA Microbiological Information System.
Literature searches were conducted by Walter E. Hill and Jim Hungerford (FDA, Seattle
District) for which we are grateful. We would also like to thank Walter E. Hill and Nancy
Hill (FDA, Seattle District) for assistance with building and finalizing a data base for the
reference list.
REFERENCES
1. Adesiyun, A.A. 1993. Prevalence of Listeria spp., Campylobacter spp., Salmonella spp.,
Yersinia spp. and toxigenic Escherichia coli on meat and seafoods in Trinidad. Food Micro-
biol. 10:395-403.
2. Anderson, J.K., and B. Nerrrung. 1995. Occurrence of Listeria monocytogenes in Danish
retail foods. Proc. XI1 Int. Symposium on Problems of Listeriosis. Perth, Australia, Oct. 2-
6, p. 241 -244.
3. Anonymous. 1987. FDA checking imported, domestic shrimp, crabmeat for Listeria. Food
Chem. News 29(24):15-17.
4. Anonymous. 1987. First Listeria finding in crabmeat confirmed by FDA. Food Chem. News
29( 14):38.
5. Anonymous. 1987. Mexican crabmeat, Greek pasta automatically detained. Food Chem.
News 29( 19):43.
6. Anonymous. 1988. FDA regional workshops to discuss microbial concerns in seafoods. Food
Chem. News 30(43):27-3 1 .
7. Anonymous. 1988. FDA to sample shrimp for Salmonella, Listeria. Food Chem. News 30:
9-10.
8. Anonymous. 1988. Hot dogs, shrimp, crab targeted for microbiological criteria. Food Chem.
News 30(6):43-45.
626 Jinneman et al.
9. Anonymous. 1988. Micro criteria for crabmeat, shrimp would have 3-class attributes. Food
Chem. News 30(24):25-27.
10. Anonymous. 1989. Listeria, Salmonella zero tolerance in cooked crab, shrimp proposed.
Food Chem. News 31(1):11-14.
11. Anonymous. 1989. Monitoring of changes to listeriosis problem urged. Food Chem. News
3 l(20): 13-1 7.
12. Anonymous. 1989. Seafood micro group publishes pathogen criteria. Food Chem. News
31( 19):31-34.
13. Anonymous. 1991. Recommendations by The National Advisory Committee on Microbio-
logical Criteria for Foods. Int. J. Food Microbiol. 14:185-246.
14. Anonymous. 1995. Misdeclaration and Recall of Smoked Salmon from the United Kingdom.
U.S. FDA Import Bull. 16-B86.
15. Archer, D.L. 1988. Review of the latest FDA information on the presence of Listeria in
foods. WHO Working Group on Foodborne Listeriosis, Geneva, Feb. 15-19.
16. Brackett, R.E., and L.R. Beuchat. 1990. Changes in the pathogenicity of Listeria monocyto-
genes grown in crabmeat. Appl. Environ. Microbiol. 56: 12 16- 1220.
17. Bremer, P.J., and C.M. Osborne. 1995. Efficacy of marinades against Listeria rnonocytogenes
cells in suspension or associated with green shell mussels (Perna canaliculus). Appl. Environ.
Microbiol. 6 1:1514- 1519.
18. Bremer, P.J., and C.M. Osborne. 1995. Thermal-death times of Listeria rnonocytogenes in
green shell mussels (Perna canaliculus) prepared for hot smoking. J. Food Prot. 58:604-
608.
19. Brett, M., P. Short, and J. McLauchlin, 1995. Listeriosis associated with smoked mussels.
Proceedings of XI1 International Symposium on Problems of Listeriosis. Perth, Australia.
Oct. 2-6. p. 467.
20. Buchanan, R.L., H.G. Stahl, M.M. Bencivengo, and R. del Corral. 1989. Comparison of lithium
chloride-phenylethanol-moxalactamand modified Vogel Johnson agars for detction of Listeria
spp. in retail-level meats, poultry and seafood. Appl. Environ. Microbiol. 55:599-603.
21. Budo-Amako, E., S. Toora, C. Walter, R.F. Ablett, and J. Smith. 1992. Thermal death times
for Listeria rnonocytogenes in lobster meat. J. Food Prot. 55:211-213.
22. Colburn, K.G., C.A. Kaysner, C. Abeyta Jr., and M.M. Wekell. 1990. Listeria species in a
California coast estuarine environment. Appl. Environ. Microbiol. 56:2007-2011.
23. Degnan, A.J., C.W. Kaspar, W.S. Otwell, M.L. Tamplin, and J.B. Luchansky. 1994. Evalua-
tion of lactic acid bacterium fermentation products and food-grade chemicals to control Liste-
ria monocytogenes in blue crab (Callinectes sapidus) meat. Appl. Environ. Microbiol. 60:
3 198-3203.
24. Dijkstra, R.G. 1982. The occurrence of Listeria monocytogenes in surface water of canals
and lakes, in ditches of one big polder and in the effluents and canals of a sewage treatment
plant. Zbl. Bakteriol. Hyg., I Abt. Orig. B 176:202-205.
25. Dillion, R., and T. Patel. Isolation of Listeriu from commercially produced smoked fish. 1lth
International Symposium on Problems of Listeriosis, May 1 1- 14, 1992. Copenhagen, Abst.
146.
26. Dillon, R.M., and T.R. Patel. 1992. Listeria in seafoods: a review. J. Food Prot. 55:1009-
1015.
27. Dillon, R., T. Patel, and S. Ratnam. 1992. Prevalence of Listeria in smoked fish. J. Food
Prot. 55:866-870.
28. Dorsa, W.J., D.L. Marshall, M.W. Moody, and C.R. Hackney. 1993. Low temperature growth
and thermal inactivation of Listeria monocytogenes in precooked crawfish tail meat. J. Food
Prot. 56: 106- 109.
29. Eklund, M.W., F.T. Poysky, R.N. Paranjpye, L.C. Lashbrook, M.E. Peterson, and G.A.
Pelroy. 1995. Incidence and sources of Listeria rnonocytogenes in cold-smoked fishery prod-
ucts and processing plants. J. Food Prot. 58502-508.
Listeria monocytogenes in Fish and Seafood 627
30. El-Shenawy, M.A., and M.A. El-Shenawy. 1995. Incidence of Listeria rnonocytogenes in
seafood enhanced by prolong enrichment. J. Med. Res. Inst. Alex. Univ. ARE 16:32-40.
31. Embarek, P.K.B. 1995. Survival and growth potential of Listeria rnonocytogenes in sous-
vide cooked fish fillets. Proc. XI1 Int. Symposium on Problems of Listeriosis. Perth, Australia,
Oct, 2-6, pp. 245-249.
32. Embarek, P.K.B. 1994. Presence, detection and growth of Listeria rnonocytogenes in sea-
foods: a review. Int. J. Food Microbiol. 23:17-34
33. Embarek, P.K.B., and H.H. Huss. 1993. Heat resistance of Listeria rnonocytogenes in vacuum
packaged pasteurized fish fillets. Int. J. Food Microbiol, 20:85-95.
34. Embarek, P.K.B., V.F. Jeppesen, and H.H. Huss. 1994. Antibacterial potential of Enterococ-
cus fcreciurn strains isolated from sous-vide cooked fish fillets. Food Microbiol. 1 I 525-536.
35. Eyles, M.J. 1994. Australian perspective on Listeria rnonocytogenes. Dairy Food Environ.
Sanit 14:205-206.
36. Facinelli, B., P.E. Varaldo, M. Toni, C. Casolari, and U. Fabio. 1989. Ignorance about Liste-
ria. Br. Med. J 299:738.
37. Farber, J.M. 199I . Listeria rnonocytogenes in fish products. J. Food Prot. 54:922-924.
38. Farber, J.M. 1993. Current research on Listeria rnonocytogenes in foods: an overview. J.
Food Prot. 56:640-643.
39. Farber, J.M. and P.I. Peterkin. 1991. Listeria rnonocytogenes, a food-borne pathogen. Micro-
biol. Rev. 55:476-5 1 1.
40. FDA. 1987. Program 7303.030 Pathogen monitoring of selected high risk foods (FY 88/89).
In Department of Health and Human Services. Public Health Service. FDA (ed.), U.S. Food
and Drug Administration Compliance Program Guidance Manual. U.S. Government Printing
Office, Pittsburgh, PA.
41. FDA. Recalls and Field Corrections. (In FDA Enforcement Reports for June 10, 1987; Dec.
16, 1987; Dec, 23, 1987; July 20, 1988; Aug. 10, 1988; Oct. 5 , 1988; Oct. 20, 1988; Dec.
28, 1988; Jan 18, 1989; May 24, 1989; Oct. 4, 1989; Nov. 1, 1989; Nov. 8, 1989; Jan. 24,
1990; July 1 1, 1990; Aug. 8, 1990; Oct. 31, 1990; Nov. 28, 1990; Dec. 19, 1990; Jan. 9,
1991; Mar. 27, 1991; Aug. 14, 1991; Sept. 4, 1991; Nov. 6, 1991; Dec. 4, 1991; Dec. 11,
1991; Feb. 19, 1992; June 10, 1992; July 2, 1992; July 29, 1992; Aug. 5 , 1992; Aug. 19,
1992; Sept. 2, 1992; Sept. 9, 1992; Sept. 23, 1992; Sept. 30, 1992; Oct. 7, 1992; Oct. 28,
1992; Nov. 25, 1992; Jan. 27, 1993; Feb. 17, 1993; Mar. 10, 1993; May 19, 1993; June 9,
1993; Aug. 25, 1993; July 28, 1993; Aug. 4, 1993; Aug. 25, 1993; Oct. 27, 1993; Nov. 10,
1993; Nov. 17, 1993; Dec. 15, 1993; Jan. 19, 1994; April 13, 1994; June 1, 1994; July 20,
1994; July 27, 1994; Sept. 21, 1994; Oct. 5 , 1994; Dec. 7, 1994; Feb. 22, 1995; Mar. 8,
1995; Mar. 15, 1995; April 5 , 1995; April 12, 1995; April 26, 1995; Aug. 9, 1995; Aug. 30,
1995; Sept. 27, 1995; Dec. 13, 1995; May 8, 1996; June 12, 1996; July 24, 1996; Sept. 4,
1996; Oct. 23, 1996; Jan. 2, 1997; Mar. 12, 1997; April 30, 1997; Aug. 6, 1997; Sept. 2,
1997; Sept. 17, 1997; Nov. 5 , 1997; April 1, 1998; July 2, 1998. U.S. Gov. Printing Office,
Pittsburgh, PA.)
42. FDA. 1988. Program 7303.036 Processed Seafood Program. In Department Human Health
Services. Public Health Service. FDA (ed.), U.S. Food and Drug Administration Compliance
Program Guidance Manual. U S . Government Printing Office, Pittsburgh, PA.
43. FDA. 1992. Program 7303.036 Processed Seafood Program. In Department of Health and
Human Services. Public Health Services. FDA (ed.), U.S. Food and Drug Administration
Compliance Program Guidance Manual. U.S. Government Printing Office, Pittsburgh, PA.
44. FDA. 1994. Program 7303.844 Import Seafood Products Compliance Program (FY 95/96/
97). In Department of Health and Human Services. Public Health Service. FDA (ed.), U.S.
Food and Drug Administration Compliance Program Guidance Manual. U.S. Government
Printing Office, Pittsburgh, PA.
45. FDA. 1996. Program 7303.842 Domestic Fish and Fishery Products Inspection Program
(FY96). In Department Health and Human Services. Public Health Service. FDA (ed.), U.S.
628 Jinneman et al.
Food and Drug Administration Compliance Guidance Program Manual. U.S. Government
Printing Office, Pittsburgh, PA.
46. Fenlon, D.R. 1985. Wild birds and silage as reservoirs of Listeriu in the agricultural environ-
ment. J. Appl. Bacteriol. 59537-543,
47. Fuad, A., S. Weagant, M. Wekell, and J. Liston. 1989. Ignorance about Listeria rnonocyto-
genes in the estuarine environment. Annual Meeting of American Society for Microbiology,
New Orleans, May 14- 18, Abst. Q-243.
48. Fuchs, R.S., and S. Sirvas. 1991. Incidence of Listeria monocytogenes in an acidified fish
product, cerviche. Lett. Appl. Microbiol. 12:88-90.
49. Fuchs, R.S., and P.K. Surendran. 1989. Incidence of Listeria in tropical fish and fishing
products. Lett. Appl. Microbiol. 9:49-5 I .
50. Garland, C.D. 1995. Microbiological quality of agriculture products with special reference
to Listeria rnonocytogenes in Atlantic salmon, Proceedings of XI1 International Symposium
on Problems of Listeriosis, Perth, Australia. Oct. 2-6. p. 261-275.
51. Gram, L. 1993. Inhibitory effect against pathogenic and spoilage bacteria of Pseudomonas
strains isolated from spoiled and fresh fish. Appl. Environ. Microbiol. 59:2 197-2203.
52. Guyer, S., and T. Jemmi. 1990. Betriebsuntersuchungen zum Vorkommen von Listeriu rnono-
cytogenes in gerauchertem Lachs. Arch. Lebensmittelhyg. 41 :144- 146.
53. Guyer, S., and T. Jemmi. 1991. Behavior of Listeria monocytogenes during fabrication and
storage of experimentally smoked salmon. Appl. Environ. Microbiol. 57: 1523- 1527.
54. Harrison, M.A., Y. Huang, C. Chao, and T. Shineman. 1991. Fate of Listeria rnonocytogenes
on packaged, refrigerated, and frozen seafood. J. Food Prot. 54524-527.
55. Harrison, M.A., and Y.-W. Huong. 1990. Thermal death times for Listeria rnonocytogenes
(Scott A) in crabmeat. J. Food Prot. 53:878-880.
56. Hartemink, R., and F. Georgsson. 1991. Incidence of Listeria species in seafood and seafood
salads. Int. J. Food Microbiol. 12:189- 196.
57. Heinitz, M.L., and J.M. Johnson. 1998. The incidence of Listeria, Sulrnonella and Clostrid-
iurn botzrlinum in smoked fish and shellfish. J. Food Prot. 61 :318-323.
58. Hudson, J.A., and S.J. Mott. 1993. Growth of Listeria monocytogenes, Aerornonas hydrophilu
and Yersinia enterocolitica on cold-smoked salmon under refrigeration and mild temperature
abuse. Food Microbiol. 10:61-68.
59. Hudson, J.A., S.J. Mott, K.M. Delacy, and A.L. Edridge. 1992. Incidence and coincidence
of Listeria spp., motile aeromonads and Yersinia enterocolitica on ready-to-eat foods. Int.
J. Food Microbiol. 16:99-108.
60. Jemmi, T. 1990. Actual knowledge of Listeria in meat and fish products. Mitt. Gebiete Leb-
ensm. Hyg. 8 1 :144- 157.
61. Jemmi, T. 1990. Zum Vorkommen von Listeriu monocytugenes in importierten geraucherten
and fermentierten Fischen (Occurrence of Listeria rnonocytogenes in imported smoked and
fermented fish). Arch. Lebensmittelhyg. 41 :107- 109.
62. Jemmi, T. 1993. Listeria monocytogenes in smoked fish: an overview. Arch. Lebensmittel-
hyg. 44: 1-24.
63. Jemmi, T., and A. Keusch. 1992. Behavior of Listeria rnonocytogenes during processing and
storage of experimentally contaminated hot-smoked trout. Int. J. Food Microbiol. 15:339-
346.
64. Johnson, J.L., M.P. Doyle, and R.G. Cassens. 1990. Listeria rnonocytogenes and other Liste-
ria spp. in meat products. A review. J. Food Prot. 53:81-91.
65. Karunasugar, I., K. Segar, I. Karunasugar, and W. Goebel. Incidence of Listeria spp. in
tropical seafoods. 1 1th International Symposium on Problems of Listeriosis, 1 1 - 14 May
1992. Copenhagen, Abst. 155.
66. Kaysner, C., K. Colburn, C. Abeyta, and M. Wekell. 1990. Survival of Listeria monocyto-
genes in shellstock and shucked oysters, Crussostrea gigas, stored at 4C. Annual Meeting
of American Society for Microbiology, Anaheim, CA May 13- 17, Abstr. P-52.
Listeria monocytogenes in Fish and Seafood 629
67. Kvenberg, J.E. 1988. Occurrence of Listeria monocytogenes in seafood. Society for Industrial
Microbiology-Comprehensive Conference on Listeria rnonocytogenes, Rohnert Park, CA
Oct. 2-5.
68. Leung, C.-K., Y.W. Huang, and M.A. Harrison. 1992. Fate of Listeria rnonocytogenes and
Aerornonas hydrophila on packaged channel catfish fillets stored at 4C. J. Food Prot. 55:
728-730.
69. Lovett, J. 1987. Listeria isolation. In FDA (ed.), U.S. Food and Drug Administration Bac-
teriological Analytical Manual. 6th ed. Association of Analytical Chemists, Washington, DC.
70. Lovett, J., D.W. Francis, and J.G. Bradshaw. 1988. Outgrowth of Listeria rnonocytogenes
in foods. In Foodborne Listeriosis, A.L. Miller, J.L. Smith, and G.A. Somkuti, eds., Elsevier,
Amsterdam, p. 183- 187.
71. Macrae, M., and D.M. Gibson. 1990. Processing and quality of foods. p. 3-168-3-174. In
P. Zeuthen, J.C. Cheftel, C. Eriksson, T.R. Gormley, P. Linko, and K. Paules (eds.), Pro-
cessing and Quality of Foods, 3 . Chilled Foods: Revolution in Freshness. Proc. Final COST
91 bis Final Seminar, 2-5 October, 1989. Goteborg. Elsevier, London.
72. Madden, J.M. I 994. Concerns regarding the occurrence of Listeria monocytogenes, Carnpylo-
bacterjejuni and Eschrrichia coli 0157:H7 in foods regulated by the U.S. Food and Drug
Administration. Dairy Food Environ. Sanit. 14:262-267.
73. Manjo, Y.B., G.M. Rosalind, I. Karunasugar, and 1. Karunasuga. 1991. Listeria spp. in fish
and fish-handling areas, Mangalore, Indian. Asian Fish. Sci. 4: 119-122.
74. Maple, P.C. 1995. Listeria monocytogenes in fish and fish product exports. Proceedings of XI1
International Symposium on Problems of Listeriosis. Perth, Australia. Oct. 2-6, pp. 279-283.
75. Masuda, T., M. Iwaya, H. Miura, Y. Kokubo, and T. Marayama. 1992. Occurrence of Listeria
species in fresh seafood. J. Food Hyg. Soc. Jpn. 33599-602.
76. McCarthy, S.A., M.L. Motes, and M. McPhearson. 1990. Recovery of heat-stressed Listeria
monocytogenes from experimentally and naturally contaminated shrimp. J. Food Prot. 53:
22-2s.
77. Misrachi, S., A.J. Watson, and D. Coleman. 1991. Listeria in smoked mussels in Tasmania.
Commun. Dis. Intell. 15:427.
78. Mosst:l, D.A.A., and C.B. Struijk. 1991. Public health implications of refrigerated pasteurized
(souc-vide) foods. Int. J. Food Microbiol. 13:187-206.
79. Motes, M. 1990.Recovery of Listeria spp. from seafoods and their ambient environments. Annual
Meeting of American Society for Microbiology, Anaheim, CA May 13- 17, Abstr. Q-76.
80. Motes, M.L.J. 199I . Incidence of Listeria in shrimp, oysters, and estuarine waters. J. Food
Prot. 54: 170- 173.
81. Nakania, A., T. Maruyama, Y. Kokubo, T. Iida, and F. Umeki. 1992. Incidence of Listeria
monocvtogenes in foods in Japan. 1 1 th International Symposium on Problems of Listeriosis,
May 1 I - 14, 1992, Copenhagen.
82. National Health and Medical Research Council. 1992. Listeria. Advice to Medical Praction-
ers. National Health and Medical Research Council, Canberra, Australia.
83. National Health and Medical Research Council. 1992. Listeria. Special Dietary Advice. Na-
tional Health and Medical Research Council, Canberra, Australia.
84. Noah, C.W., J.C. Perez, N.C. Ramos, C.R. McKee, and M.V. Gipson. 1991. Detection of
Listerici spp. in naturally contaminated seafoods using four enrichment procedures. J. Food
Prot. 54: 174- 177.
85. Noel, I)., G.E. Rodrick, W.S. Otwell, and J. Bacus. 1989. Lactic acid use in seafood microbial
control. Annual Meeting of Institute of Food Technologists, Chicago, June 25-29; Abstr.
355.
86. Olsufjew, N.G., and V.G. Petrow. 1959. Detection of Erysipelothrix and Listeria in stream
water. Zh. Mikrobiol. Epidemiol. Immunobiol. 30:89-94.
87. Osborne, C.M., and P.G. Bremer. 1995. Core temperatures obtained by greenshell mussels
(Perna canaliculus) during processing and the implications for Listeria monocytogenes con-
630 Jinneman et al.
ROBERTE. BRACKETT
The University of Georgia, Griffin, Georgia
INTRODUCTION
Use of adequate isolation procedures, enough time, and a little perseverance by investiga-
tors makes it possible to isolate Listeria spp., including L. rnonocytogenes, from most
forms of animal life. A similar situation also exists with products of plant origin. Chapter
3 describes the apparent association between consumption of silage and occurrence of an
illness resembling listeriosis in ruminants, which was observed as early as 1922; however,
this link between silage consumption and listeriosis in domestic livestock was not con-
firmed until 1960 [43]. Although several papers published during the next 15-year period
documented the presence of L. rnonocytogenes in vegetation grown primarily for consump-
tion by animals [75-771 (see Chap. 2), scientists at the time were generally unconcerned
about the incidence of listeriae in produce destined for human consumption, primarily
since such products had not been positively linked to human listeriosis. In fact, the only
instance in which listeriae were recovered from raw retail produce before the 1981 listeri-
osis outbreak in Canada involving coleslaw occurred in 1975 when successful isolation
of three untypable Listeria strains from lettuce marketed in Brazil was reported [50].
Although 18 of 41 Canadians died of listeriosis in 1981 after consuming coleslaw
from which L. rnonocytogenes was isolated and positively identified [69], it was not until
631
632 Brackett
the 1985 cheese-related listeriosis outbreak in California that researchers began to develop
more than a passive interest in the public health significance of L. monocytogenes in
vegetables, fruits, and other products of plant origin. Nevertheless, with the exception of
one isolated listeriosis case in Finland involving homemade salted mushrooms [53] and
a recent cluster of five cases traced to frozen broccoli and cauliflower in Texas [70], no
additional cases of listeriosis have been positively linked to consumption of plant products
produced in North America or elsewhere.
This chapter is devoted to food products and it will specifically address the incidence
of Listeria spp. in raw retail vegetables and fruits. As in earlier chapters, information
concerning behavior of L. monocytogenes in fresh produce and related products (orange
juice/serum, soy milk, pasta, beet pigment) will also be presented along with some possible
means by which listeriae can be inactivated in some of these products.
FIGURE 1 Mechanism b y which fresh produce can become contaminated with patho-
genic microorganisms and serve as vehicles of human disease. (Adapted from Ref.
11.)
Listeria monocytogenes in Products of Plant Origin 633
When one considers the enormous variety and quantity of produce being marketed
annually, routine microbiological examination of raw vegetables (and fruits) seems highly
impractical and probably unnecessary if good agricultural practices are used in growing
crops along with acceptable hygienic practices while harvesting, packaging, and trans-
porting raw produce to market. Although consumption of coleslaw prepared from contami-
nated cabbage was linked to a large Canadian outbreak of listeriosis in 1981 [68], in
retrospect, it appears that this outbreak could have been easily avoided if the coleslaw
manufacturer had realized that the cabbage farmer had fertilized the cabbage with sheep
manure from a flock that was previously diagnosed as having listeriosis. However, van
Renterghem et al. [74] suggested that L. rnonocytogenes dies quickly in fecal matter, and
therefore animal manure may not be as important in the spread of L. monocytogenes as
once thought. (The role of manure and sewage as vehicles for listeriae is discussed in
Chapter 2. ) However, they were able to demonstrate that L. rnonocytogenes could be trans-
ferred from contaminated soil to vegetables. In these experiments, carrots and radishes
were planted in soil which had been inoculated with L. rnonocytogenes (10 CFU/g soil).
They found that three of six radishes but none of the carrots grown in the inoculated soil
contained L. monocytogenes.
In spite of the Canadian outbreak, which resulted in 18 fatalities, it is not surprising
that, unlike dairy, meat, poultry, and seafood, no surveillance and/or regulatory programs
have been initiated until relatively recently to assess the incidence of listeriae in raw
vegetables (or fruits) marketed in the United States, Canada, or elsewhere. Nevertheless,
inadvertent isolation of L. rnonocytogenes from potato salad in April 1990 prompted a
Virginia-based manufacturer to recall 5700 pounds of product that had been distributed
in the southeastern United States [2]. During 1997 and the first half of 1998, six Class 1
recalls were issued for fresh frozen coconut [2c], hummus with red peppers and vegetables
[2a,2d,2e], sprouts [2f], and potato salad [2b], the last of which involved over 5.5 million
pounds of product. Although the risk of contracting listeriosis from such products is gener-
ally thought to be quite low, lack of routine microbiological analysis of raw produce
should not be interpreted to mean that fresh vegetables and fruits will always be free of
listeriae, including L. monocytogenes.
United States
In response to heightened concern about foodborne listeriosis, several small surveys were
conducted after the 1985 outbreak in California to determine the extent of Listeria contam-
ination in raw fresh and frozen vegetables destined for human consumption. During 1986
and 1987, Petran et al. [65] used the U.S. Food and Drug Adniinistration (FDA) procedure
in an attempt to isolate Listeria spp., including L. rnonocytogenes, from 23 retail samples
of vegetables, including fresh beet peels, broccoli, cabbage (outer leaves), carrot peels,
cauliflower sterns, corn husks, head lettuce, leaf lettuce, mushroom stems, potato peels,
and spinach as well as frozen green beans, pea pods, green peas, and spinach. Using
FDA and Centers for Disease Control and Prevention (CDC) procedures along with direct
plating, officials at the CDC [3] tried to isolate listeriae from 22 samples of broccoli,
carrots, celery, lettuce, green peppers, and potatoes in conjunction with several clusters
of listeriosis cases in Los Angeles County, California, and Philadelphia, Pennsylvania.
Finally, as part of a much larger survey dealing with the incidence of Listeria spp. in
retail meat, poultry, and seafood products, Buchanan et al. 1231 used an MPN (most proba-
ble number) method to examine two samples of potato salad for listeriae. As already
634 Brackett
implied, no Listeria spp. were recovered from any samples examined in the three surveys
just described. However, when one considers the small number of samples examined,
these findings cannot assure consumers that these products will always be free of listeriae.
In the first truly definitive survey reported, Heisick et al. [48] used the FDA proce-
dure to determine the incidence of various Listeria spp., including L. monocytogenes, in
10 different varieties of raw unwashed vegetables (total of 1000 samples) obtained from
two Minneapolis-area supermarkets between October 1987 and August 1988. As shown
in Table 1, Listeria spp. were detected in one or more samples of cabbage, cucumbers,
lettuce, mushrooms, potatoes, and radishes, but were never found in broccoli, carrots,
cauliflower, or tomatoes. Although L. monocytogenes, L. innocua, L. welshimeri, and L.
seeligeri were recovered from 5.0,2.6,0.8, and 1.3% of all raw produce examined, respec-
tively, with 41 of 50 (82%) and 9 of 50 (18%) L. monocytogenes strains classified as
serotypes l a and 4a/4ab, respectively, the overall incidence of Listeria spp. as well as L.
monocytogenes was markedly higher in radishes and potatoes than in other types of vegeta-
bles. Given that carrots recently were shown to possess some inherent antilisterial activity
[6,14,19,59,60], it appears that root crops such as potatoes and radishes more frequently
carry viable listeriae than other vegetables because of their close association with soil.
Interestingly, contamination rates for most raw vegetables were fairly consistent through-
out the year, and this reinforces the belief that listeriae populations remain relatively con-
stant in soil. These findings also are supported by those from a similar study in which
Heisick et al. [48] used four procedures to ultimately identify Listeria spp. in 19 of 70
(27.1%) and 25 of 68 (36.8%) potato and radish samples, respectively, with no listeriae
being detected in mushrooms, carrots, cabbage, broccoli, cauliflower, lettuce, tomatoes,
or cucumbers obtained from the same two Minneapolis supermarkets.
Use of an adequate isolation procedure and sufficient time to examine large numbers
of samples, has made it clear that a small percentage of raw vegetables marketed in
the United States is likely to harbor Listeria spp., including L. monocytogenes, with the
incidence of this pathogen being highest in root crops. Hence, the inability to detect
listeriae in raw vegetables examined in the three aforementioned surveys probably resulted
because insufficient numbers of samples were examined. In support of this observation,
Steinbruegge et al. [72] isolated L. monocytogenes from only 2 of 43 retail samples of
head lettuce purchased in Nebraska. Although the occasional presence of listeriae in retail
raw vegetables should not be viewed with alarm, careful handling and washing of all
produce to be consumed raw is recommended, particularly for pregnant women, the
elderly, and other individuals at greater than normal risk of developing listeriosis.
Others in the United States have similarly found L. monocytogenes to be an infre-
quent contaminant of fresh vegetables. Lin et al. [57] determined occurrence of L. monocy-
togenes and other foodborne pathogens in vegetable salads served in 3 1 food service estab-
lishments in Florida. Of the 63 vegetable salad samples tested, only one was contaminated
with L. monocytogenes. The vegetable salad from which the bacterium was isolated con-
sisted of iceberg lettuce, red cabbage, carrots, cucumbers, and tomatoes. Interestingly, this
salad and others yielding potentially pathogenic bacteria other than Listeria were pur-
chased from only 5 of the 31 establishments. Moreover, several of these implicated facili-
ties were apparently guilty of selling contaminated salads on more than one occasion,
with contamination most likely a result of product mishandling by workers.
The importance of proper sanitation and handling in minimizing L. monocytogenes
contamination of salads and vegetables was mentioned by Harvey and Gilmour [47]. They
suggested that systematic contamination of vegetable salads by L. monocytogenes was
Listeria monocytogenes in Products of Plant Origin 635
TABLE1 incidence of Various Listeria spp. in Unwashed Raw Retail Vegetables Marketed in the Minneapoiis, Minnesota, Area
Between October 1987 and August 1988
No. of positive samples (%)
No. of
samples L. rnono-
Type of vegetable analy zed cytogenes L. innocua L. welshimeri L. seeligeri Total
Broccoli 92 0 0 0 0 0
Cabbage 92 1 (1.1) 0 0 1 (1.1) 2 (2.2)
carrots 92 0 0 0 0 0
Cauliflower 92 0 0 0 0 0
Cucumbers 92 2 (2.2) 5 (5.4) 2 (2.2) 0 9 (9.8)
Lettuce 92 0 1 (1.1) 0 0 1 (1.1)
Mushrooms 92 0 11 (12.0) 0 0 1 1 (12.0)
Potatoes 132 28 (21.2) 5 (3.8) 1 (0.8) 0 34 (25.8)
Radishes 132 19 (14.4) 4 (3.0) 5 (3.8) 12 (9.1) 40 (30.3)
Tomatoes 92 0 0 0 0 0
Total 1,000 50 (5.0) 26 (2.6) 8 (0.8) 13 (1.3) 97 (9.7)
more likely a result of improper handling by food service workers rather than from natural
contamination of the raw product.
Canada
Despite the Canadian coleslaw outbreak of 1981, the incidence of listeriae in fresh produce
has received relatively little attention in Canada, with only one formal Canadian publica-
tion on the subject presently recorded in the scientific literature. Using the original FDA
procedure, Farber et al. [37] failed to recover any Listeria spp. from lettuce (50 samples),
celery (30 samples), or tomatoes (20 samples) purchased in Ottawa during 1988. However,
L. innocua was detected in 1 of 10 radish samples, which again suggests that the incidence
of listeriae may be somewhat higher in root crops than in other vegetables.
Western Europe
When the first edition of this book appeared in 1991, knowledge concerning the incidence
of listeriae in raw vegetables marketed in western Europe was confined to a few scattered
reports. Since then much more information has been published.
An increase in the number of listeriosis cases in England, along with the possibility
that some of these cases may have been food-related, prompted several surveys to deter-
mine the incidence of listeriae in various foods including dairy, meat, poultry and seafood
products, raw vegetables, and prepackaged salads. Working at Cambridge, Sizmur and
Walker [71] examined 10 different varieties of prepackaged salads obtained from two
leading area supermarkets. Overall, L. monocytogenes serotype 1/2 was isolated from 4
of 60 (6.7%) samples, with L. monocytogenes serotype 4b also being present in one of
these positive samples. Prepackaged salads from which the pathogen was recovered con-
sisted of two varieties that contained either (a) cabbage, celery, sultanas, onions, and car-
rots or (b) lettuce, cucumbers, radishes, fennel, watercress, and leeks. Both of these salad
varieties contained cabbage, cucumbers, and/or radishes-three of four raw vegetables
from which L. monocytogenes (predominantly serotype la) was isolated in the United
States (see Table 1). Although no Listeria spp. were recovered from plain bean sprout
salads or those that contained nuts, possibly because of a low pH, L. innocua was detected
in 13 of 60 (21.7%) samples representing five different varieties of mixed vegetables and/
or fruit salad. In addition to these findings, English investigators [41] also have isolated
L. monocytogenes from coleslaw. Thus, raw salad vegetables can serve as a potential
source of L. monocytogenes in the human diet.
Bending and Strangeways 171 proposed that a 74-year-old postoperative patient in
a London hospital may have acquired listerial septicemia and meningitis from consuming
contaminated lettuce. Although different serotypes of L. monocytogenes (1/2a and 1/2c)
were isolated from the patient and 1 of 11 (9.1 %) samples of washed English round
lettuce, recovery of the pathogen from washed lettuce prepared in the hospitals kitchen,
but not from 44 other food samples examined, suggests that consumption of washed raw
vegetables may pose a potential health threat to hospital patients, many of whom are
debilitated and/or immunocompromised.
Consumption of homemade uncooked salted mushrooms containing 1O6 L. monocy-
togenes serotype 4b CFU/g has been positively linked to a nonfatal case of listerial septice-
mia in an 80-year-old apparently healthy Finnish man [53] (see Chap. 10). (Working in
the Netherlands, van Netten et al. [73] also isolated L. monocytogenes from 2 of 20 raw
mushroom samples obtained from area markets.) During this investigation, low levels
Listeria monocytogenes in Products of Plant Origin 637
vegetables and found L. monocytogenes present in 12.2% of vegetables sampled; the or-
ganism was only recovered from lettuce, Chinese cabbage, and green onions. On further
characterization, all isolates obtained from vegetables were of serotypes other than 1 and
4. In contrast, more than 90% of isolates from turkey and beef were serotype 1, with these
strains and those from seafood having greater hemolytic activity than isolates obtained
from vegetables. Consequently, these authors hypothesized that characteristics of L. mono-
cytogenes might be related to their food origins.
A similar survey of occurrence of L. monocytogenes in foods sold in Tokyo was
published by Ryu et al. in 1992 [66]. Their survey also included a variety of plant products,
including fresh vegetables, potato salad, and pickled vegetables. L. monocytogenes was
frequently isolated from meat (34%) and fish products (6.1%); however, no listeriae were
recovered from vegetable products or from ready-to-eat vegetable foods such as fermented
soybeans, cooked bamboo shoots, or coleslaw.
Arumugaswamy et al. [4] surveyed various fresh and ready-to-eat foods in Malaysia
and found the highest incidence of L. monocytogenes contamination yet reported. About
22% of leafy vegetables analyzed contained the bacterium, with similar proportions of
bean cakes and peanut sauces also reported as being positive. Although these values are
higher than those reported for other countries, they were lower when compared with other
Malaysian vegetable products tested. Eighty percent of ready-to-eat cucumber slices and
80% of bean sprouts tested positive for L. monocytogenes. The authors suggested that a
high percentage of positive samples may have resulted because many of the samples came
from street vendors or small processors, which often employed less than desirable sanita-
tion practices. They also suggested that the results indicate a need for health agencies to
put greater priority on street-vended foods.
Salamah [67] conducted an extensive survey for the presence of L. rnonocytogenes
in various fresh market vegetables sold in Riyadh, Saudi Arabia. A summary of their
results is given in Table 2. In general, all samples analyzed contained L. monocytogenes,
with incidence rates varying from 1.3 to 16.3%. Salamah confirmed the observations of
Heisick et al. [46] that root crops were more frequently contaminated with L. monocyto-
genes than vegetables grown above ground.
Finally, Gohil et al. [42] examined 183 imported and locally grown vegetables in
the United Arab Emirates for the presence of L. monocytogenes. Unlike other surveys,
however, they were unable to detect any L. monocytogenes in vegetables. However, they
isolated L. innocua from two samples each of imported and locally grown vegetables.
lations decreasing from approximately 107to 1O4 or 1O5 CFU/g during 42 days of refriger-
ated storage.
This apparent inability of L. monocytogenes to grow on heat-sterilized cabbage at
5C suggests that heating either decreases the availability of essential nutrients or leads
to development of toxic and/or inhibitory constituents in cabbage. In sharp contrast, L.
monocytogenes competed well with the normal aerobic flora and lactic acid bacteria of
raw cabbage, with Listeria populations increasing approximately 4 orders of magnitude on
raw cabbage during the first 25 days of refrigerated storage (Fig. 2). Thereafter, numbers of
listeriae failed to decrease appreciably on raw cabbage stored up to 64 days. Similar results
also were observed in subsequent studies by Hao et al. [44] and Lovett et al. [58]. Thus,
these findings demonstrate that L. rnonocytogenes can grow under conditions normally
encountered during shipping and distribution of cabbage. Although both Listeria strains
failed to grow in autoclaved cabbage juice containing >5% NaCl during 2 weeks of stor-
age at 3OoC, L. monocytogenes increased to 106CFU/g in the aforementioned homemade
salted mushrooms (-7.5% NaCl) during 5 months of cold storage [53]. Hence, behavior
of listeriae in raw vegetables appears to be greatly affected by incubation temperature as
well as the concentration of salt and various growth constituents [64].
Subsequently, Conner et al. [29] more closely examined the influence of tempera-
ture, NaCI, and pH on growth of L. monocytogenes in autoclaved (121"C/ I5 min) clarified
and unclarified cabbage juice. As in the previous study, salt-free unclarified cabbage juice
again was an excellent growth medium for L. monocytogenes, with initial populations of
- -
104CFU/mL increasing to I09CFU/mL after 8 days of incubation at 30C. Beyond
8 days, populations in cabbage juice containing low levels of salt decreased rapidly and
viable cells were no longer detected after 20 days of incubation at 30C. Growth rates of
M
0, 6-1 I
el
0 16 32 48 61
Days
FIGURE2 Behavior of L. monocytogenes (O), lactic acid bacteria (m), and total
aerobic microorganisms (A)o n raw cabbage incubated at 5C. (Adapted from
Ref. 18.)
Listeria monocytogenes in Products of Plant Origin 64 7
both Listeria strains at 30C decreased markedly in cabbage juice containing low levels
of salt, with inactivation of strains Scott A and LCDC 81-861 occurring in the presence
of 1 1 . 5 and 12.5% NaCI, respectively. As expected, behavior of both strains was strongly
influenced by acid production, with the pH of samples in which growth had occurred
decreasing from 5.6 to 5 4 . 3 after 8 days of incubation at 30C. Although numbers of
both Listeria strains failed to increase in salted and unsalted cabbage juice when the experi-
ment was repeated at 5"C, populations remained relatively stable, generally decreasing
only 10- to 100-fold during 70 days of refrigerated storage of cabbage juice contain-
ing 3.5-5.0% NaCI. Results from another study [28] suggest that viability of
Listeria in similar samples of salted and unsalted cabbage juice can be reduced by adding
extracts from several Chinese medicinal plants.
Interestingly, growth patterns for L. monocytogenes differed dramatically in clarified
cabbage juice, with both strains growing well at 30C in the presence of up to 2% NaCl.
Since similar changes were observed between pH and growth of listeriae in clarified and
unclarified cabbage juice, these findings suggest that particulate matter in unclarified cab-
bage juice may be partly inhibitory to L. monocytogenes in the presence of salt.
Using pH-adjusted unclarified cabbage juice, these researchers [25] further demon-
strated that L. monocytogenes failed to initiate growth at pH values <5 when inoculated
samples were incubated at 30C. Although more acidic environments (pH 14.8) were
lethal, complete inactivation of both Listeria strains did not occur until the pH was reduced
to -4.1. When the incubation temperature was lowered to 5"C, L. monocytogenes popula-
tions gradually decreased in cabbage juice adjusted to pH (5.2, with the pathogen surviv-
ing >63, 49, <21, and <21 days in samples adjusted to pH values of 5.2, 5.0, 4.8, and
4.6, respectively. In contrast, numbers of Listeria remained relatively constant during ex-
tended cold storage of cabbage juice adjusted to pH values >5.2. Since inactivation rates
were markedly slower at 5 than at 3OoC, it appears that lower temperatures help protect
L. monocytogenes from the harmful effects of low pH, as noted in Chapter 6.
Concern about behavior of L. rnonocytogenes in fresh produce has extended beyond
cabbage and now includes an ever increasing variety of fresh salad vegetables. In 1988,
Steinbruegge et al. [72] first reported results of a study which examined the ability of L.
monocytogenes to survive and grow on inoculated ( 103- l O5 CFU/g) samples of washed
retail head lettuce during storage in sealed and unsealed plastic bags at 5, 12, and 25C.
Although behavior of L. monocytogenes on lettuce was somewhat variable, the pathogen
generally grew under conditions simulating proper refrigeration, normal handling, and
ambient serving temperatures, with the pathogen increasing 1-4 orders of magnitude fol-
lowing 2 weeks of storage. Similar results were observed when inoculated samples of fresh
lettuce juice were held at 5C for 2 weeks. Salamah [67] later reported similar increases in
lettuce juices held at 26C and up to a 2-log increase at 4C.
Several more recent reports have appeared regarding the fate of L. monocytogenes
on various types of salad greens. According to Carlin and Nguyen-the [24], butterhead
lettuce (Lnctuca sativa L.) supported growth of L. monocytagenes better than did endive
(Cichoriumendivia L.), but the bacterium was unable to grow on lamb's lettuce (Valeria-
nella olitoria L.). In contrast, populations of total aerobic microorganisms were unaffected
by salad type. The authors offered no hypothesis as to why lamb's lettuce failed to support
growth of L. monocytogenes.
Carlin et al. [25] followed up on their previous work by more closely examining
several factors that affected growth of L. monocytogenes on endive, with emphasis on
storage temperature, age, and quality of the endive leaves, role of epiphytic microflora,
642 Brackett
and the strains and initial concentration of L. monocytogenes present. Overall, the growth
rate of L. monocytogenes was essentially the same as that of the natural aerobic microflora
at 10 and 20C but slower than the native microflora at 30 and 6C. Furthermore, the
bacterium grew faster when initially present at lower (10-1000 CFU/g) rather than at
higher ( 105CFU/g) populations. In accord with earlier results of Beuchat and Brackett
[ 151, Carlin et al. [25] detected no differences in growth among the various strains tested.
Carlin and coworkers [26] subsequently investigated in detail the role of indigenous
microflora on growth of L. monocytogenes in unsanitized endive leaves and on leaves
treated with 10% hydrogen peroxide to reduce or eliminate the indigenous microflora.
These investigators also challenged L. monocytogenes with individual strains of pseu-
domonads and Enterobacteriaceae isolated from endive. The authors observed that reduc-
ing the native microflora by disinfection resulted in higher populations of L. monocyto-
genes on endive leaves. Moreover, they also observed that high populations ( 106- 107
CFU/g) of some strains of indigenous microorganisms reduced growth of L. monocyto-
genes on endive. A complex mixture of various microorganisms isolated from endive
completely inhibited growth of L. monocytogenes in a medium composed of endive leave
exudate.
In addition to lettuce, tomatoes are among the most popular ingredients in fresh
salads. Beuchat and Brackett [16] demonstrated that tomatoes are not a good substrate
for growth of L. monocytogenes, probably because of their acidity. Although some growth
of Listeria was evident on whole tomatoes after 10-2 1 days of storage at 10 and particu-
larly 21"C, the pathogen was inactivated in chopped tomatoes (-pH 4.1) held at these
same temperatures. Additionally, when commercial tomato products were inoculated to
-
contain 1O6 L. monocytogenes CFU/g, populations remained reasonably stable in tomato
sauce and tomato juice during 14 days of storage at 21 and particulary 5C. However,
the pathogen survived only 4 and 8 days in samples of ketchup held at 21 and 5"C, respec-
tively, with Listeria inactivation being attributed to higher levels of acetic acid in ketchup
as compared with the other tomato products.
Information regarding the fate of L. monocytogenes in or on other types of salad
vegetables also is limited; however, results from several studies indicate that with the
exception of raw carrots [ 14,221, fennel, red cabbage, and Savoy cabbage [22], and beets
[2 11, this pathogen will grow and/or survive on most other types of fresh produce including
asparagus [9], broccoli [9], cauliflower [9], corn [52], green beans [52], lettuce [15,52],
certain types of cabbage [22,29], celery [21], potato juice [67], and radishes [58] during
the normal refrigerated shelf life of the product. Gianfranceschi and Aureli [40] similarly
noted that survival of L. monocytogenes in spinach was essentially unaffected by freezing
at -50C and extended storage at - 18C. In addition, Sizmur and Walker [7 11 reported
that L. monocytogenes populations in several naturally contaminated vegetable salads pur-
chased from two supermarkets in England increased approximately twofold after 4 days
of refrigerated storage. Given the apparent ability of L. monocytogenes to survive and/or
grow on most raw salad vegetables and the possible presence of this pathogen on many
types of raw produce, health officials need to consider raw vegetables as another possible
source of listerial infections.
The observation that L. monocytogenes can thrive in various fresh vegetables also
led to questions regarding its growth and survival in products prepared from these vegeta-
bles. In 1995, Lee et al. [56] published a study on growth and survival of L. monocytogenes
in kimchi, a traditional Korean fermented vegetable product. This product can be prepared
from various ingredients, but the most common type contains Chinese cabbage and various
Listeria monocytogenes in Products of P h t Origin 643
flavoring agents such as red pepper, garlic, ginger, NaCl, and pickled seafood. These
ingredients are mixed together and subjected to a natural lactic acid fermentation, with
the product ultimately reaching a mildly acidic pH [4-51. The authors found that popula-
tions of L. monocytogenes Scott A increased during the first 2 days of kimchi fermentation
but then decreased. Although the bacterium was eventually inactivated by kimchi ingredi-
ents and low pH, the pathogen still persisted after 10 days of fermentation. However, the
authors concluded that kimchi could be safely produced by using ingredients of good
microbiological quality.
at 5C. However, the bacterium only achieved populations of about 105-106 CFU/g in
tomatoes regardless of storage atmosphere.
The term modijied atmosphere usually refers to systems where atmosphere in which
a product is stored is intentionally changed to the desired gas composition. One such
example is vacuum packaging of produce. This action has the effect of reducing O2 and
thereby slowing respiration and senescence. Aytac and Gorris IS] investigated the effect
of a moderate vacuum on growth of L. monocytogenes in chicory endive and mung-bean
sprouts; the organism responded differently depending on the product in question. Growth
of L. monocytogenes at 5.6"C was enhanced by 400 mB of vacuum in the endive but was
repressed in sprouts. The authors pointed out the need for additional barriers when such
techniques are used to extend the shelf life of fresh vegetables.
The atmosphere also can be changed as a result of metabolic processes of fresh
fruits and vegetables. In this instance, gas-permeability characteristics of the packaging
material can often drastically affect the atmosphere within the package and, consequently,
the microflora in the food. Omary et al. [6 1 ] investigated the influence of packaging mate-
rial on growth of L. rnonocytogenes in shredded cabbage packaged in films having oxygen
transmission ratios (OTR) of 5.6, 1500, 4000, and 6000 cc 02/m2/24h. They found that
the type of packaging material used had a significant effect on growth of L. innocua (as
a substitute for L. monocytogenes) in cabbage. Populations of L. innocua decreased in all
samples after 14 days of storage regardless of packaging film used. However, populations
of L. innocua then increased 3.5 logs CFU/g in cabbage packaged in all but the film with
the highest ORT. In that instance, populations only increased by about 2 logs CFU/g. In
the latter sample, CO2 concentrations had equilibrated at near ambient concentrations,
whereas concentrations reached from 30 to 90% when films of lower OTR were used.
Although most publications to date indicate that only low populations of L. monocy-
togenes infrequently contaminate fresh produce, the chance of this pathogen multiplying
in products with extended shelf life is significant. Therefore, it appears prudent for handlers
of raw produce to store their product in a modified atmosphere and institute sanitation
and quality control programs that will decrease the incidence of listeriae in incoming raw
vegetables. It also may be necessary to shorten the marketable period for such products
even though the food may appear to be acceptable.
Inactivation
As afollow-up to the aforementionedstudies dealing with growth and survivalof L. monocyto-
genes in raw vegetables, scientists also examined different methods by which this pathogen
can be eliminatedfrom raw vegetables. Although these methods, which will now be discussed,
primarily involve use of heat, chlorine, and lysozyme, information concerning the effect of
other methods such as ozone, chlorine dioxide, and irradiation should be forthcoming.
In response to the 1981 listeriosis outbreak in Canada involving consumption of
contaminated coleslaw, Beuchat et al. [ 181 investigated thermal inactivation of L. monocy-
togenes in cabbage juice. Flasks of sterile, clarified cabbage juice adjusted to pH 4.0, 4.6,
and 5.6 were inoculated to contain approximately 4 X 106 L. monocytogenes CFU/mL;
placed in a shaking water bath at 50, 52, 54, 56, or 58C; and sampled for listeriae at
10-min intervals for up to 60 min. As expected, thermal inactivation rates for Listeria in
cabbage juice at 50, 52, 54, and 56C were faster at lower pH values, with D-values of
25, 14, 6.7, and 3.6 min at pH 4.6 as compared with D-values of 60, 34, 8.4, and 6.8 min
at pH 5.6, respectively. No viable cells were detected in cabbage juice held at 58C for
Listeria monocytogenes in Products of Plant Origin 645
after 12 days of refrigerated storage (Fig. 3). Although lysozyme alone was listeriostatic,
use of EDTA alone failed to prevent growth of L. monocytogenes, with the pathogen
eventually attaining levels only slightly lower than those observed in untreated lettuce.
Listeria behaved similarly on fresh green beans and sweet corn with two exceptions: (a)
growth occurred on lysozyme-treated sweet corn and (b) combined use of lysozyme and
EDTA never completely eliminated the pathogen from either product. Unlike fresh lettuce,
green beans, and sweet corn, numbers of listeriae on EDTA-and lysozyme-treated raw
cabbage increased during the first 20 days of refrigerated storage and then decreased
4-5 orders of magnitude during 28 days of incubation at 5C. Although combined use
of lysozyme and EDTA again was most listericidal, 41 days of refrigerated storage were
required to rid this lettuce of listeriae. Unlike other fresh vegetables, the pathogen was
eliminated within 9 days from untreated raw carrots as well as from those that were treated
with lysozyme alone or in combination with EDTA (Fig. 4). Hence, these findings support
the notion that carrots probably contain one or more naturally occurring listericidal sub-
stances [ 141.
In contrast to fresh vegetables, numbers of listeriae remained relatively constant
on previously frozen green beans and corn that were treated with lysozyme alone or in
"1
3i
\ 0 EDTA
Control
2i\
0 5 15
Days
J K 0 Control
combination with EDTA. This apparent failure of lysozyme to inactivate listeriae on frozen
vegetables may be related to loss of certain lysis-enhancing substances during processing
of vegetables. These findings, together with the current use of lysozyme to prevent growth
of gas-producing spore-forming bacteria in certain European cheeses, suggest that com-
mercial use of lysozyme in combination with other previously discussed measures should
help to inhibit Listeria and other foodborne pathogens on fresh vegetables.
Various plant components have long been known to possess antimicrobial proper-
ties. Specifically, the essential oils of herbs and spices have been studied most extensively
in this regard. Kim et al. [55] demonstrated the antilisterial activity of various essential
oil components in vitro and suggested that these components might also be incorporated
into foods as barriers to microbial growth. Hao and Brackett [45] and Hao et al. [46]
tested this suggestion by determining the efficacy of plant extracts in inhibiting growth of
L. monocytogenes in beef and chicken, respectively. They found that eugenol and pimento
extracts significantly inhibited growth of the bacterium on cooked chicken breasts during
refrigerated storage [46]. However, they also noted that the type of food in which extracts
were used was important. Unlike chicken, none of the spice extracts tested effectively
inhibited growth of L. monocytogenes in refrigerated, cooked beef [45]. Moreover, some
extracts contributed strong odors and flavors which would need consumer approval if
actually used for commercial products.
As mentioned previously, carrots reportedly possess some inhibitory antilisterial
factor(s). Beuchat and Brackett [ 141 were the first to document this when they attempted
to artificially inoculate carrots by dipping in suspensions of L. monocytogenes. They noted
that populations of the bacterium decreased on both raw whole and shredded carrots but
not cooked carrots. Moreover, numbers of L. monocytogenes decreased in the inoculating
648 Brackett
suspensions after dipping of shredded carrots. Based on these results, they suggested that
the antilisterial component(s) of carrots is heat sensitive and released when carrot tissue
is damaged. The authors suggested that phytoalexin 6-methoxymellein might be one poten-
tial compound responsible for the antilisterial action. The results of Beuchat and Brackett
were later confirmed by Nguyen-the and Lund [59], who also noted that maceration of
carrots in a high-speed blender or in liquid nitrogen likewise destroyed the antilisterial
activity.
The obvious potential for using carrot juice or its antilisterial component(s) as a
natural antimicrobial agent in foods prompted the same two groups of researchers to inves-
tigate the mechanism of antilisterial activity. Nguyen-the and Lund [60] found that the
antilisterial effect of carrots was suppressed by anaerobiosis, thiol compounds, bovine
serum, and the free-radical scavengers histidine and diazabenzocyclooctane. However, the
activity was not affected by sodium ascorbate, propyl gallate, catalase, superoxide dismu-
tase, or chelating agents but was enhanced by Tween 20. Despite these results, these
authors were unable to determine a specific compound or compounds responsible for the
antilisterial activity.
Beuchat et al. [ 171 likewise attempted to characterize the antilisterial component(s)
of carrots. They found that the lethal and inhibitory effects were greatest in the pH range
of 5.0-6.4 and that the optimum concentration of carrot juice needed to inhibit L. monocy-
togenes growth was about 10%. In addition, they observed that NaCl at concentrations
up to 5% protected the listeriae from the antimicrobial action of carrot juice, especially
in 10% juice incubated at 5 or 12C. Despite these observations, they were unable to
identify the compound(s) responsible for antilisterial activity or predict the effectiveness
of carrot juice as an antilisterial ingredient in food. However, Babic et al. [6] suggested
that dodecanoic acid and methyl esters of dodecanoic and pentadecanoic acids identified
in purified active extracts of carrots may be responsible for antimicrobial activity.
Looking at the potential of using carrot juice as an antilisterial treatment for foods,
Beuchat and Doyle [19] determined the influence of dipping shredded lettuce in 20 or
50% carrot juice or adding up to 10% carrot juice to Brie cheese and frankfurter homoge-
nates. Overall, both concentrations of carrot juice significantly repressed growth of L.
monocytogenesin shredded lettuce stored at 5 and 12C but not at 20C. It is also notewor-
thy that the carrot juice was rather specific in its activity in that it had no discernible effect
on growth of other aerobic microorganisms. In contrast to lettuce, addition of carrot juice
to Brie cheese was less effective and was completely ineffective in frankfurter homoge-
nates. Hence, it appears that carrot juice may be of value as an antilisterial agent in some
foods.
ing was used to link consumption of pickled olives with a sporadic neonatal case of liste-
riosis [27].
Although scientific evidence is lacking, two observations, namely, (a) infrequent
association between consumption of fruit and listeriosis and (b) most fruits grow well
above ground and are therefore not subject to frequent contact with Listeria-contaminated
soil or feces, lead one to speculate that the incidence of listeriae on fruit may well be as
low or lower than that observed for raw vegetables.
Given the probable low incidence of listeriae on raw fruits, it may seem somewhat
surprising to learn that FDA officials prompted an Oregon firm to issue a Class I recall
for over 500,000 flavored frozen ice and juice bars that were contaminated with L. monocy-
togenes during the latter stages of manufacture [ 11, which suggests postpasteurization
contamination. Since raw milk also was routinely processed into frozen dairy products at
this same facility, L. monocytogenes was most likely introduced into the factory environ-
ment through the raw milk supply rather than fruit juice. If this is true, then it follows
that the incidence of listeriae in highly acidic fruit juice is likely to be extremely low.
This view is further supported by results from a recent survey in which Parish and Higgins
[62] failed to detect any Listeria spp. in 100 retail samples of reconstituted single-strength
orange juice (pH 3.63-3.84) that were pasteurized at 30 geographically distinct dairy and
nondairy facilities located across the United States and Canada.
10 20 30 40 50 60 70 80 90
Days
42 days of refrigerated storage. These findings appear to be consistent with those from
the previous study involving orange serum.
refrigeration temperatures suggests that this product should not be overlooked as a possible
vehicle of human listerial infections.
Concern about behavior of Listeria in delicatessen products marketed in England
and the United States prompted Beuchat and Brackett [13] to investigate viability and
thermal inactivation of L. monocytogenes in commercially prepared meat, cheese, and egg
ravioli purchased from Atlanta-area delicatessens. The growth portion of this study in-
volved quantitation of L. monocytogenes in inoculated (- 104and 1 O6 CFU/g) samples
of ravioli during 14 days of incubation at 5C. For thermal inactivation tests, the three
types of ravioli were inoculated to contain 3 X 105L. monocytogenes CFU/g, stored 0
or 9 days at 5OC, and then boiled up to 7 min using cooking procedures that might be
practical in the home. Overall, numbers of viable listeriae decreased < 10-fold during the
9-day refrigerated shelf life of the three types of ravioli. Results of thermal inactivation
studies indicated that normal cooking procedures (7 min of boiling) were adequate to
-
destroy L. monocytogenes populations of 105CFU/g in all three types of ravioli regard-
less of whether or not ravioli was refrigerated 0 or 9 days before cooking. Although this
study provides valuable information concerning behavior of Listeria in ravioli, there ap-
pears to be an urgent need for more work of this type to address the microbiological safety
of precooked and/or ready-to-eat delicatessen products such as sandwiches, filled rolls,
pizza, garlic bread, desserts, confectionery products, and chocolate, since work in England
[41] and elsewhere has shown that all of these products can harbor L. monocytogenes.
Increased use of plant-based food colorants prompted El-Gazzar and Marth [35] to
investigate behavior of Listeria in a commercial aqueous extract from the red beet root
(Beta vulgaris) to which vitamin C, citric acid, and sodium propionate ( 51.5%) were
added as preservatives. As in their previous work with milk coagulants [33,34] and annatto
colorants [32], samples of beet extract were inoculated to contain 103-107L. monocyto-
genes strain CA, V7, or Scott A CFU/mL and examined for numbers of survivors during
prolonged storage at 7C. Not surprisingly, the combined effect of a relatively low pH
of 4.3-4.8 and sodium propionate prevented growth of listeriae in all samples of beet
colorant. However, although 42-56 days of incubation at 7C was sufficient to rid these
extracts of 10'-104 strain CA CFU/mL, this strain was still detected in 56-day-old samples
that contained larger initial populations. In contrast, strains V7 and Scott A were far more
resistant to the listericidal action of beet extract, with both strains being recovered at levels
of 10'-104 CFU/mL, depending on initial inoculum, following 56 days of storage. Hence,
unlike highly alkaline annatto extracts in which L. monocytogenes was inactivated almost
instantaneously (see Chap. 12), prolonged survival of listeriae in beet colorants makes it
imperative that these extracts be processed and handled carefully to prevent their contami-
nation with this pathogen.
The sporadic nature of listeriosis suggests that L. monocytogenes can be an infre-
quent problem in unusual foods of plant origin as well as fruits and vegetables. Whereas
this viewpoint is supported by one sporadic case of listeriosis in Canada that was linked
to alfalfa tablets [30,36], the fact that L. monocytogenes can be present in virtually any
ecological niche suggests that occasional cases of listeriosis are likely to be associated
with unusual as well as common plant-based foods.
REFERENCES
1. Anonymous. 1987. Frozen ice, juice and fudge bars recalled. FDA Enforcement Report,
June 3,
652 Brackeff
2. Anonymous. 1990. Chicken, potato salads recalled by Campbell unit due to Listeria. Food
Chem. News 32(9):61.
2a. Anonymous. 1997. Hummus with roasted peppers recalled. FDA Enforcement Report,
Aug. 13.
2b. Anonymous, 1997. Potato salad recalled. FDA Enforcement Report, Sept. 2.
2c. Anonymous. 1998. Fresh frozen coconut recalled. FDA Enforcement Report, Jan. 14.
2d. Anonymous. 1998. Hummus dips and salads recalled. FDA Enforcement Report, June 12.
2e. Anonymous. 1998. Vegetable hummus recalled. FDA Enforcement Report, April 29.
2f. Anonymous. 1998. Sprouts recalled. FDA Enforcement Report, Sept. 5.
3. Archer, D.L. 1988. Review of the latest FDA information on the presence of Listeria in foods.
WHO Working Group on Foodborne Listeriosis. Geneva, Feb. 15-19.
4. Arumugaswamy, R.K., G.R.R. Ali, and S.N.B.A. Hamid. 1994. Prevalence of Listeria monocy-
togenes in foods in Malaysia. Int. J. Food Microbiol. 23:117-121.
5. Aytac, S.A., and L.G.M. Gorris. 1994. Survival of Aeromonas hydrophilu and Listeria mono-
cytogenes on fresh vegetables stored under moderate vacuum. World J. Microbiol. Biotechnol.
10:670-672.
6. Babic, I.C., C. Nguyen-the, M.J. Amiot, and S. Aubert. 1994. Antimicrobial activity of shred-
ded carrot extracts on food-borne bacteria and yeast. J. Appl. Bacteriol. 76:135-141.
7. Bendig, J.W.A., and J.E.M. Strangeways. 1989. Listeria in hospital lettuce. Lancet 1:616-
617.
8. Bennik, M.H.J., E.J. Smid, F.M. Rombouts, and L.G.M. Gorris. 1995. Growth of psychro-
trophic foodborne pathogens in a solid surface model system under the influence of carbon
dioxide and oxygen. Food Microbiol. 12509-5 19.
9. Berrang, M.E., R.E. Brackett, and L.R. Beuchat. 1989. Growth of Listeria monocytogenes on
fresh vegetables stored under controlled atmosphere. J. Food Prot. 52:702-705.
10. Beuchat, L.R. 1996. Listeria monocytogenes: incidence on vegetables. Food Control 7(4/5):
223-228.
11. Beuchat, L.R. 1996. Pathogenic microorganisms associated with fresh produce. J. Food Prot.
59:204-216.
12. Beuchat, L.R., M.E. Berrang, and R.E. Brackett. 1990. Presence and public health implications
of Listeria monocytogenes on vegetables. In A.J. Miller, J.L. Smith, and G.A. Somkuti. Food-
borne Listeriosis. Elsevier, New York, pp. 175- 181.
13. Beuchat, L.R., and R.E. Brackett. 1989. Observations on survival and thermal inactivation of
Listeria monocytogenes in ravioli. Lett. Appl. Microbiol. 8: 173- 175.
14. Beuchat, L.R., and R.E. Brackett. 1990. Inhibitory effects of carrots on Listeria monocyto-
genes. Appl. Environ. Microbiol. 56: 1734-1742.
15. Beuchat, L.R., and R.E. Brackett. 1990. Survival and growth of Listeria monocytogenes on
lettuce as influenced by shredding, chlorine treatment, modified atmosphere packaging and
temperature. J. Food Sci. 55:755-758, 870.
16. Beuchat, L.R., and R.E. Brackett. 1991. Behavior of Listeriu monocytogenes inoculated into
raw tomatoes and processed tomato products. Appl. Environ. Microbiol. 57: 1367-1 37 1.
17. Beuchat, L.R., R.E. Brackett, and M.P. Doyle. 1994. Lethality of carrot juice to Listeria mono-
cytogenes as affected by pH, sodium chloride and temperature. J. Food Prot. 57:470-474.
18. Beuchat, L.R., R.E. Brackett, D.Y.-Y. Hao, and D.E. Conner. 1986.Growth and thermal inactiva-
tion of Listeria monocytogenes in cabbage and cabbage juice. Can. J. Microbiol. 32:791-795.
19. Beuchat, L.R., and M.P. Doyle. 1995. Survival and growth of Listeria monocytogenes in foods
treated or supplemented with carrot juice. Food Microbiol, 12:73-80.
20. Brackett, R.E. 1987. Antimicrobial effect of chlorine on Listeria monocytogenes. J. Food Prot.
50~999-1003.
21. Breer, C. 1988. Occurrence of Listeria spp. in different foods. WHO Working Group on
Foodborne Listeriosis, Geneva, Feb. 15- 19.
22. Breer, C., and A.A. Baumgartner. 1992. Vorkommen und Verhalten von Listeria monocyto-
Listeria monocytogenes in Products of Plant Origin 653
genes auf Salaten und Gemusen sowie in frischgepresten Gemusesaften. Archiv. Lebensmittel-
hyg. 43:97- 120.
23. Buchanan, R.L., H.G. Stahl, M.M. Bencivengo, and F. del Corral. 1989. Comparison of lithium
chloride-phenylethanol-moxalactamand modified Vogel Johnson agars for detection of Liste-
ria spp. in retail-level meats, poultry and seafood. Appl. Environ. Microbiol. 55599-603.
24. Carlin, F., and C. Nguyen-the. 1994. Fate of Listeria monocytogenes on four types of mini-
mally processed green salads. Lett. Appl. Microbiol. 18:222-226.
25. Carlin, F., C. Nguyen-the, and A. Abreu da Silva. 1995. Factors affecting the growth of Listeria
monocytogenes on minimally processed fresh endive. J. Appl. Bacteriol. 778:636-646.
26. Carlin, F., C. Nguyen-the, and C.E. Morris. 1996. Influence of background microflora on Liste-
ria monocytogenes on minimally processed fresh broad-leaved endive (Cichorium endivia var.
Zatifolia). J. Food Prot. 59:698-703.
27. Casolari, C., R. Neglia, M. Malagoli, and U. Fabio. 1994. Foodborne sporadic neonatal listeri-
osis confirmed by DNA fingerprinting. Annual Meeting of the American Society of Microbiol-
ogists, Las Vegas, NV, May 23-27, p. 382, Abstr. P-77.
28. Chung, K.-T., W.R. Thomasson, and C.D. Wu-Yuan. 1990. Growth inhibition of selected
food-borne bacteria by plant extracts. J. Appl. Bacteriol, 69:498-503.
29. Conner, D.E., R.E. Brackett, and L.R. Beuchat. 1986. Effect of temperature, sodium chloride,
and pH on growth of Listeria monocytogenes in cabbage juice. Appl. Environ. Microbiol. 52:
59-63.
30. Czajka, J., and C.A. Bott. 1994. Verification of causal relationships between Listeria monocy-
togenes isolates implicated in food-borne outbreaks of listeriosis by randomly amplified DNA
patterns. J. Clin. Microbiol. 32:1280- 1287.
31. De S i m h , M., C. Tarrag6, and M.D. Ferrer. 1992. Incidence of Listeria monocytogenes in
fresh foods in Barcelona (Spain). Int. J. Food Microbiol. 16:153-156.
32. El-Gazzar, F.E., and E.H. Marth. 1989. Fate of Listeria monocytogenes in some food colorants
and starter distillate. Lebensm. Wiss. Technol. 22:406-4 10.
33. El-Gazzar, F.E., and E.H. Marth. 1989. Loss of viability by Listeria monocytogenes in com-
mercial bovine-pepsin rennet extract. J. Dairy Sci. 72: 1098-1 102.
34. El-Gazzar, F.E., and E.H. Marth. 1989. Loss of viability by Listeria monocytogenes in com-
mercial microbial rennet. Milchwissenschaft 44:83-86.
35. El-Gazzar, F.E., and E.H. Marth. 1991. Survival of Listeria monocytogenes in food colorant
derived from red beets. J. Dairy Sci. 74:81-85.
36. Farber, J.M., A.O. Carter, P.V. Varughese, F.E. Ashton, and E.P. Ewan. 1990. Listeriosis
traced to the consumption of alfalfa tablets and soft cheese. N. Engl. J. Med. 322:338.
37. Farber, J.M., G.W. Sanders, and M.A. Johnston. 1989. A survey of various foods for the
presence of Listeria species. J. Food Prot. 52:456-458.
38. Ferguson, R.D., and L.A. Shelef. 1990. Growth of Listeria monocytogenes in soy milk. Food
Microbiol. 7:49-52.
39. Garcia-Gimeno, R.A., G. Zurera-Cosano, and M. Amaro-L6pez. 1996. Incidence, survival and
growth of Listeria monocytogenes in ready-to-use-mixed vegetable salads in Spain. J. Food
Safety 16:75-86.
40. Gianfranceschi, M., and P. Aureli. 1996. Freezing and frozen storage on the survival of Listeria
monocytogenes in different foods. Ital. J. Food Sci. 4:303-309.
41. Gilbert, R.J. 1990. Personal communication.
42. Gohil, V.S., M.A. Ahmed, R. Davies, and R.K. Robinson. 1985. Incidence of Listeria spp.
in retail foods in the United Arab Emirates. J. Food Prot. 48:102-104.
43. Gray, M.L. 1960. A possible link in the relationship between silage feeding and listeriosis.
J. Am. Vet. Med. Assoc. 136:205-208.
44. Hao, D. Y.-Y., L.R. Beuchat, and R.E. Brackett. 1989. Comparison of media and methods
for detecting and enumerating Listeria monocytogenes in refrigerated cabbage. Appl. Environ.
Microbiol. 53:955-957.
654 Brackett
45. Hao, Y .-Y., and R.E. Brackett. 1995. Efficacy of plant extracts and cultured whey of antagonis-
tic organisms to inhibit Listeria monocytogenes in refrigerated, cooked poultry. Annual Meet-
ing of American Society of Microbiology, Washington, DC, May 21-25, p 388., Abstr.
P-37.
46. Hao, Y.-Y., R.E. Brackett, and M.P. Doyle. 1994. Efficacy of plant extracts to inhibit psychro-
trophic pathogens in refrigerated, cooked beef. Annual Meeting of American Society of Micro-
biology, Las Vegas, NV, May 23-27, p 379., Abstr. P-58.
47. Harvey, J., and A. Gilmore. 1993. Occurrence and characteristics of Listeria in foods produced
in Northern Ireland. Int. J. Food Microbiol. 19:193-205.
48. Heisick, J.E., F.M. Harrell, E.H. Peterson, S. McLaughlin, D.E. Wagner, I.V. Wesley, and J.
Bryner. 1989. Comparison of four procedures to detect Listeria spp. in foods. J. Food Prot.
52:154- 157.
49. Heisick, J.E., D.E. Wagner, M.L. Nierman, and J.T. Peeler. 1989. Listeria spp. found on fresh
market produce. Appl. Environ. Microbiol. 55: 1925-1927.
50. Hofer, E. 1975. Study of Listeria spp. on vegetables suitable for human consumption. VI
Congress0 Brazil de Microbiologia, Salvador, July 27-3 I , Abstr. K- 1 1. Cited in Ralovich, B.
1984. Listeriosis Research, Present Situation and Perspective, Akadimiai Kiado, Budapest,
p. 73.
51. Hughey, V.L., and E.A. Johnson. 1987. Antimicrobial activity of lysozyme against bacteria
involved in food spoilage and food-borne disease. Appl. Environ. Microbiol. 53:2 165-
2170.
52. Hughey, V.L., P.A. Wilger, and E.A. Johnson. 1989. Antibacterial activity of hen egg white
lysozyme against Listeria monocytogenes Scott A in foods. Appl. Environ. Microbiol. 55:
63 1-638.
53. Junttila, J., and M. Brander. 1989. Listeria monocytogenes septicemia associated with con-
sumption of salted mushrooms. Scand. J. Infect. Dis. 21 :339-342.
54. Kallander, K.D., A.D. Hitchens, G.A. Lancette, J.A. Schmieg, G.R. Garcia, H.M. Solomon,
and J.N. Sofos. 1991. Fate of Listeria monocytogenes in shredded cabbage stored at 5 and
25C under a modified atmosphere. J. Food Prot. 54:302-304.
55. Kim, J.M., M.R. Marshall and C.-I. Wei. 1995. Antibacterial activity of some essential oil
components against five foodborne pathogens. J. Agric. Food Chem. 43:2839-2845.
56. Lee, S.-H., M.K. Kim, and J.F. Frank. 1995. Growth of Listeria monocytogenes Scott A dur-
ing kimchi fermentation and in the presence of kimchi ingredients. J. Food Prot. 58:1215-
1218.
57. Lin, C.-M., S.Y. Fernando, and C.-i. Wei. 1996. Occurrence of Listeria monocytogenes, Salmo-
nella spp., E. coli and E. coli 0157:H7 in vegetable salads. Food Control 7(3):135-140.
58. Lovett, J., D.W. Francis, and J.G. Bradshaw. 1988. Outgrowth of Listeria monocytogenes in
foods. In A.J. Miller, J.L. Smith, and G.A. Somkuti. Foodborne Listeriosis. Elsevier, New
York, pp. 183-187.
59. Nguyen-the, C., and B.M. Lund. 1991. The lethal effect of carrot on Listeria species. J. Appl.
Bacteriol. 70:479-488.
60. Nguyen-the, C., and B.M. Lund. 1992. An investigation of the antibacterial effect of carrot
on Listeria monocytogenes. J. Appl. Bacteriol. 73:23-30.
61. Omary, M.B., R.F. Testin, S.F. Barefoot, and J.W. Rushing. 1993. Packaging effects on growth
of Listeria innocua in shredded cabbage. J. Food Sci. 58:623-626.
62. Parish, M.E., and D.P. Higgins. 1989. Extinction of Listeria monocytogenes in single-strength
orange juice: Comparison of methods for detection in mixed populations. J. Food Safety 9:
267-277.
63. Parish, M.E., and D.P. Higgins. 1989. Survival of Listeria monocytogenes in low pH model
broth systems. J. Food Prot. 52: 144-147.
64. Petran, R., and E. Zottola. 1989. A study of factors affecting growth and recovery of Listeria
monocytogenes Scott A. J. Food Sci. 54:458-460.
Listeria monocytogenes in Products of Plant Origin 655
65. Petran, R.L., E.A. Zottola, and R.B. Gravani. 1988. Incidence of Listeria monocytogenes in
market samples of fresh and frozen vegetables. J. Food Sci. 53: 1238-1240.
66. Ryu, C.-H., S. Igimi, S. Inoue, and S. Kumagai. The incidence of Listeria species in retail
foods in Japan. Int. J. Food Microbiol. 16:157-160.
67. Salamah, A.A. 1993. Isolation of Yersinia enterocolitica and Listeria monocytogenes from
fresh vegetables in Saudi Arabia and their growth behavior in some vegetable juices. J. Univ.
Kuwait (Sci.) 20:283-290.
68. Schlech, W.F. 1996. Overview of listeriosis. Food Control 7: 183- 186.
69. Schlech, W.F., P.M. Lavigne, R.A. Bortolussi, A.C. Allen, E.V. Haldane, A.J. Wort, A.W.
Hightower, S.E. Johnson, S.H. King, E.S. Nichols, and C.V. Broome. 1983. Epidemic listeri-
osis: evidence for transmission by food. N. Engl. J. Med. 308:203-206.
70. Simpson, D.M. 1996. Microbiology and epidemiology in foodborne disease outbreaks: the
whys and why nots. J. Food Prot. 59:93-95.
71. Sizmur, K.I., and C.W. Walker. 1988. Listeria in prepackaged salads. Lancet i: 1167.
72. Steinbruegge, E.G., R.B. Maxcy, and M.B. Liewen. 1988. Fate of Listeria monocytogenes on
ready to serve lettuce. J. Food Prot. 5 1596-599.
73. Van Netten, P., I. Perales, A. van de Moosdijk, G.D.W. Curtis, and D.A.A. Mossel. 1989.
Liquid and solid selective differential media for the detection and enumeration of L. monocyto-
genes and other Listeria spp. Int. J. Food Microbiol. 8:299-3 16.
74. Van Renterghem, B., F. Huysman, R. Rygole, and W. Verstraete. 1991. Detection and preva-
lence of Listeria monocytogenes in the agricultural ecosystem. J. Appl. Bacteriol. 71 :211-
217.
75. Weis, J. 1975. The incidence of Listeria monocytogenes on plants and in soil. In M. Woodbine,
ed. Problems of Listeriosis. Surrey, UK: Leicester University Press, pp. 61-65.
76. Weis, J., and H.P.R. Seeliger. 1975. Incidence of Listeria monocytogenes in nature. Appl.
Microbiol. 30:29-32.
77. Welshimer, W.J. 1968. Isolation of Listeria monocytogenes from vegetation. J. Appl. Bacte-
ri01. 95:300-303.
78. Willcox, F., P. Tobback, and M. Hendrickx. 1994. Microbial safety assurance of minimally
processed vegetables by implementation of the hazard analysis critical control point (HACCP)
system. Acta Aliment. 23:221-238.
79. Wong, H.-C., W.-L. Chao, and S.-J. Lee. 1990. Incidence and characterization of Listeria
monocytogenes in foods available from Taiwan. Appl. Environ. Microbiol. 56:3 101-3 104.
80. Zhang, S., and J.M. Farber. 1996. The effects of various disinfectants against Listeria monocy-
togenes on fresh-cut vegetables. Food Microbiol. 13:31 1-321.
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17
Incidence and Control of Listeria
in Food-Processing Facilities
ROBERTGRAVANI
Cornell University, Ithaca, New York
INTRODUCTION
Overwhelming evidence indicates that when L. monocytogenes and other Listeria spp. are
present in commercially processed foods, this happens primarily because the product was
contaminated after processing rather than because these organisms survived heat treat-
ments that normally render the product safe. This view is strongly supported by the lack
of scientific evidence indicating that minimum required heat treatments given to dairy,
meat, poultry, seafood, and other products are inadequate to inactivate levels of listeriae
that might be reasonably expected to occur in such products before heat processing. Al-
though L. monocytogenes is clearly more heat tolerant than most other non-spore-forming
foodborne pathogens, to date, no recalls of commercially prepared, Listeria-contaminated
products have been unequivocally linked to the inadequacy of minimum required heat
treatments. However, the clearest indication that L. monocytogenes and other Listeria spp.
enter commercially processed foods as postprocessing contaminants comes from the fact
that apparently healthy, non-thermally injured cells have been routinely recovered from
many thermally processed dairy, meat, poultry, and seafood products and that these organ-
isms have been found in the working environments of virtually all processing facilities
that have produced foods involved in Listeria-related recalls.
L. monocytogenes is a particularly difficult organism to control in food-processing
facilities [61d]. Refrigerated food plants, in particular, provide conditions which allow for
657
658 Gravani
L. rnonocytogenes survival and growth. The organism can adhere to food-contact surfaces
and form a biofilm or coating which impedes the effectiveness of sanitation procedures
[4 lc]. The refrigerated, moist environment, coupled with organic soil deposition, allows
L. rnonocytogenes to survive and grow. L. rnonocytogenes is also a frequent contaminant
of raw materials used in processing plants, so there is constant reintroduction of the organ-
ism into the plant environment [41b]. To control this pathogen, every potential avenue of
entry and cross contamination must be controlled.
This final chapter has been specifically designed for plant managers, sanitation work-
ers, and quality control/quality assurance personnel employed in the food industry. In
keeping with the format of Chapters 11 through 16, results from recent American and
European surveys concerning the incidence of listeriae in environments of dairy-, meat-,
poultry-, seafood-, vegetable-, and fruit-processing facilities as well as household kitchens
will be described first. This will be followed by some general guidelines for reducing
levels of listeriae and other microbial contaminants in working areas that are common to
virtually all food-processing facilities.
The wide variations in microbial load and types of microorganisms present in similar
raw and finished products manufactured at different facilities along with the fact that no
two factories are exactly alike in terms of design, equipment, maintenance, product flow,
sanitation practices and procedures, distribution patterns, and managerial policies suggest
that a discussion of current cleaning and sanitation programs used at particular food-pro-
cessing facilities would be of little benefit. Instead specific problem areas within pro-
cessing plants such as pasteurizers, fillers, sausage peelers etc., associated with the manu-
facture of particular products will be identified. Then a brief discussion of how good
manufacturing practices (GMPs), prerequisite programs, and the Hazard Analysis Critical
Control Point (HACCP) programs can all be used to decrease sharply the microbial load
in any food, thereby reducing the possibility of producing a product contaminated with
L. rnonocytogenes or any other foodborne pathogen.
along with in-depth inspection of fluid milk factories and eventually all types of dairy-
processing facilities located throughout the United States. With the subsequent discovery
of L. monocytogenes in cooked, ready-to-eat meat, poultry, and seafood products by FDA
and U.S. Department of Agriculture (USDA) officials, manufacturers of foods other than
dairy products also became concerned about the incidence of listeriae in their products
and processing facilities.
Once FDA and USDA officials announced their plans to review GMPs that were
presumably being used by most American firms, the food industry launched a Herculean
effort to identify Listeria spp. and eliminate such problems within the food-processing
environment before governmental inspectors arrived. Considering the adverse publicity
and potential monetary losses that could result from discovery of L. monocytogenes in the
finished product and the factory environment, it is not surprising that very little information
concerning the incidence of listeriae and other microbial contaminants in food (except
Class I recalls) and food-processing facilities has been released to the scientific commu-
nity. Hence, although vast amounts of data have been generated since 1985 by local, state,
and federal government inspectors as well as private microbiological testing and con-
sulting laboratories and the food manufacturers themselves, much of the information which
now follows is either of a general nature describing particular niches within food-pro-
cessing facilities from which listeriae have been isolated or consists of limited results
from academic surveys which describe the actual incidence of Listeria contamination in
a relatively small number of food-processing facilities.
Dairy-Processing Facilities
Following several dairy-related outbreaks of salmonellosis and listeriosis, FDA officials
in cooperation with state governments and the dairy industry intensified surveillance of
various types of dairy-processing facilities under the Dairy Safety Initiative Program
which began April 1, 1986 [57]. Under this program, state officials were requested to
sponsor a series of statewide meetings to discuss foodborne illness associated with Grade
A and non-Grade A dairy products and to intensify their surveillance and inspection
efforts in dairy-processing facilities. Nationally, FDA officials vowed to (a) conduct inten-
sified check ratings in every interstate milk shipment (IMS) milk pasteurization plant, (b)
conduct similar inspections at non-Grade A (non-IMS) milk pasteurization plants, (c)
initiate a microbiological surveillance program designed to detect pathogenic microorgan-
isms in finished product (see Chaps. 11 and 12), (d) intensify and upgrade training and
standardization practices for federal and state milk specialists, rating officers, and sanitari-
ans, and (e) regularly prepare national reports which summarize the status of the United
States dairy industry.
In the first of these reports [4] covering the 6-month period from April to September
1986, 9 of 357 (2.5%) milk pasteurization factories examined produced various dairy
products contaminated with L. monocytogenes. A subsequent report in February 1987
indicated generally similar contamination rates with 16 of 620 (2.6%) and 3 of 620 (0.48%)
dairy-processing facilities manufacturing finished products containing L. monocytogenes
and L. innocua, respectively [ 5 ] .Eight months later, FDA officials reported that 11 of
604 (1 3%)IMS and 18 of 412 (4.4%) non-IMS milk pasteurization factories had produced
products contaminated with Listeria spp., principally L. rnonocytogenes [6].
Extensive follow-up efforts in milk-processing plants producing Listeria-contami-
nated products uncovered various defects in factory design and pasteurization equipment.
660 Gravani
Nevertheless, FDA officials have maintained that listeriae entered these products as post-
pasteurization contaminants. This view is strongly supported by FDAs success in isolating
Listeria from numerous floor drains in processing and other areas, wooden (porous) walls,
floors and ceilings, wooden pallets, external surfaces of milk cartons, and sweetwater
(refrigerated water) from leaking pasteurizer plates. Although not clearly identified in
FDAs list of environmental samples that harbored Listeria, FDA officials [75] noted
the following problem areas related to environmental, postpasteurization contamination
of dairy products with listeriae: (a) improperly operating high-temperature short-time
(HTST) and/or vat pasteurizers, (b) leaking and/or cracked storage tanks, jacketed vessels,
and valves, (c) inadequate sanitizing regimens, (d) cross-connecting pipes which allow
commingling of raw and pasteurized product, (e) use of contaminated rags and sponges,
(f) exposure to contaminants in unfiltered air and condensate, (g) filling and packaging
operations, (h) conveyor belts, (i) use of returned product and reclaiming operations, (i)
walls, floors, and ceilings particularly in walk-in refrigerators, (k) formation of aerosols,
(1) traffic patterns within the factory, (m) entrances and floor mats, and (n) personal cleanli-
ness of employees and others in the factory. In reality, L. monocytogenes and other food-
borne pathogens have been detected in environmental samples from many of these problem
areas as indicated in the following surveys of dairy factories in California and Vermont.
In response to these federal programs, officials of the Milk and Dairy Foods Control
Branch of the California Department of Food and Agriculture published [38] results from
a statewide survey in which 597 environmental samples were collected from 156 milk-
processing facilities during the first half of 1987 and analyzed for listeriae. Overall, Liste-
ria spp. were identified in the working environment of 46 (29.5%) milk-processing facili-
ties with 31 of these 46 (67.4%) Listeria-positive factories being contaminated with L.
monocytogenes (Table 1). Furthermore, L. monocytogenes and other Listeria spp. were
most frequently observed in factories producing fluid milk products followed by those
that manufactured frozen dairy products (i.e., ice cream and novelty desserts) and cultured
milk products (i.e., yogurt and cottage cheese), with lowest contamination rates being
associated with production of miscellaneous products and cheese. In all likelihood, this
unusually low incidence of listeriae in California cheese factories was a direct result of
TABLE
1 Incidence of Listeria in Various Types of Milk-Processing
Facilities in California, January to July 1987
massive clean-up efforts that were instituted following the 1985 listeriosis outbreak in the
Los Angeles area involving Mexican-style cheese.
A comparison of the incidence of listeriae in different milk-processing areas and
sample sites (Table 2) supports the widespread belief that listeriae most frequently enter
products after rather than before pasteurization, with the prevalence of these organisms
in the factory environment increasing as the product passes through processing, filling,
packaging, and storage areas. This apparent movement of listeriae through milk-processing
facilities is most readily seen in results obtained from sampling conveyor belts and floor
drains. However, sporadic isolation of listeriae from condensate as well as wooden blocks,
pallets, case dollies, and utility tables points to additional routes by which these organisms
can be disseminated in dairy processing plants. Although the low incidence of listeriae
in raw milk receiving rooms as compared to other areas of the factory may at first seem
surprising, these findings most likely reflect difficulties encountered in adequately cleaning
and sanitizing equipment in processing, filling, and packaging areas of factories rather
than what could be interpreted as a near absence of listeriae in California raw milk.
In an environmental survey of 39 frozen milk product plants in California, Walker
et al. [77a] collected 922 samples and found 111 (12%) positive for Listeria spp. Listeria
monocytogenes was the only species recovered from 5 (12.8%) plants and L. innocua was
the only species recovered from 13 (33.3%) plants. Both species were isolated from 9
(23.1%) plants. The highest recovery rates of Listeria were found in the batch flavoring,
freezing, ingredient blending, and packaginglfilling areas of plants surveyed.
Working at the University of Vermont, Klausner and Donnelly [56] made a large-
TABLE
2 Incidence of Listeriae in Different Milk-Processing Areas
and Sample Sites
No. of No. (%) of positive samples
Facility working area samples
and sample site examined L. monocytogenes ,411 Listeria spp.
Raw milk receiving room
Drain 30 1 (3.3) I (3.3)
Condensate 32 0 1 (4.5)
Othei 1 0 0
Processing room
Drain 150 4 (2.7) 14 (9.3)
Condensate 76 1 (1.3) 3 (3.9)
Other" 21 3 (14.3) 6 (28.6)
Filling/Packaging room
Drain 60 7 (11.7) 12 (20.0)
Condensate 36 1 (2.8) 1 (2.8)
Conveyor 15 5 (33.3) 7 (20.0)
Othei 10 0 2 (20.0)
Cold storage room
Drain 105 12 (11.4) 17 (16.2)
Condensate 44 0 1 (2.3)
Conveyor 14 4 (28.6) 9 (64.3)
a Includes wooden blocks, pallets, case dollies, and utility tables.
scale survey to identify sources of Listeria (and Yersinia) contamination in fluid milk-,
cheese-, and non-cheese-processing facilities. Overall 66.7, 9.5, and 23.8% of samples
collected from floors and other non-food-contact surfaces at 34 fluid milk, cheese, and
non-cheese factories were positive for Listeria spp., with L. rnonocytogenes and L. inno-
cua being identified in 1.4 and 16.1%, respectively, of 361 samples examined. As ex-
pected, the percentage of Listeria-positive samples was higher among those from floors
(12.0-27.9%) than from other non-food-contact surfaces (8.1%) (Table 3) and wet
(85.7%) rather than dry (14.3%) areas. According to these investigators, paper filler beds,
whey drainage pans on cheese presses, and case-washing areas were particularly prone
to contamination with Listeria and Yersinia. The fact that 20.9% of all positive samples
contained both Listeria and Yersinia suggests that yersiniae might be somewhat useful as
a potential indicator of Listeria contamination within the dairy-processing environment.
As noted, L. rnonocytogenes, Yersinia spp., and most other foodborne pathogens are
more commonly found in wet than dry processing areas. However, the fact that listeriae
(a) were recovered from whey drainage pans, (b) were routinely shed in whey during
cheese-making experiments, (c) grew in samples of refrigerated milk and whey, and (d)
survived the typical spray-drying process used to manufacture nonfat dry milk suggests
that these organisms should be of concern to manufacturers of dry dairy products.
In the light of these concerns, Gabis et al. [44] determined the incidence of Listeria
in the working environment of 18 dry milk- and whey-processing facilities throughout
the United States. The authors supplied environmental sampling kits containing sterile
cellulose sponges, fabric-tipped swabs, and other necessities to all firms participating in
the survey along with instructions as to how and where to collect samples. All samples
were then sent to a central laboratory and within 48 h of collection were analyzed for
listeriae according to the FDA procedure. Overall, only 2 of 410 (0.24%) samples exam-
ined were positive for Listeria spp., with L. monocytogenes and a species other than L.
rnonocytogenes being isolated from floor drains in a raw milk receiving area and from a
composite sample from several floor drains and trenches in a powder production area,
respectively (Table 4). Allowing factory employees to choose specific sampling sites as
well as the number of samples to be analyzed may have somewhat biased these results;
TABLE
3 Incidence of Listeria and Yersinia on Floors and Non-
Food-Contact Surfaces of 34 Fluid Milk, Cheese, and Non-Cheese
Factories i n Vermont
however, the incidence of listeriae and hence the risk of postprocessing contamination
appears to be many times lower in dry rather than wet dairy-processing facilities. Neverthe-
less, since manufacturers of nonfat dry milk and dry whey are not immune to the Listeria
problem, they should take appropriate action to eliminate this organism from the pro-
cessing environment, thereby greatly reducing the chance of producing a contaminated
product.
In a follow-up study, Pritchard et al. [67a] sampled 30 dairy processing plants in
Vermont. Of the 346 sites tested, 122 (35.3%) contained one or more species of Listeria.
Coolers and freezers had the highest rate, with 14 of 30 sites (46.7%) being positive for
Listeria (Table 5). Other sites that resulted in high positive rates included dry storage
areas (39.6%) and raw milk receiving and storage areas (39.4%). Pritchard et al. [67a]
a Includes areas such as common hallways, testing laboratories, and wheels of fork-
lifts.
Source: Adapted from Ref. 67a.
664 Gravani
also noted that plants producing dairy ingredients, frozen milk products or fluid milk, had
significantly higher incidence rates of Listeria than expected. Facilities producing cultured
dairy foods or a combination of cultured dairy foods and fluid milk were found to have
significantly lower incidence rates of Listeria than expected. These researchers also ob-
served that when dairy farms were contiguous to the processing facilities, these plants
were more likely to be contaminated than plants without on-site dairy farms.
Meat-Processing Facilities
Unlike dairy-processing facilities in which raw milk is pumped into the factory, pasteur-
ized, and then either packaged immediately or pumped to closed vats for processing into
cream, butter, ice cream, cheese, or other dairy products, meat-processing factories are in
actuality open-air disassembly line operations in which animals are slaughtered, eviscer-
ated, and broken down to obtain various cuts of meat, hides for leather, and other items
of commercial value. Considering that domestic cattle, sheep, and pigs frequently shed
L. monocytogenes asymptomatically in fecal material, it is not surprising that surveys have
shown this pathogen to be not only ubiquitous but also endemic to slaughterhouses and
meat-packing facilities.
Initiation of the USDA-FSIS testing program for listeriae in cooked and ready-to-
eat meat products in September of 1987 (see Chap. 13) prompted immediate action by the
meat industry. However, even before government testing began, meat processors became
concerned about the incidence of listeriae in the working environment. In June 1987,
results from a large-scale survey were reported in which nearly 2300 environmental sam-
ples were collected from over 40 meat-processing facilities nationwide and analyzed for
listeriae [9]. Fourteen processing areas within these factories yielded evidence of being
contaminated with L. monocytogenes or other Listeria spp. Overall, listeriae were recov-
ered from -21% of all environmental samples examined. (These results also compare
favorably with those of a much smaller survey [15] in which Listeria spp. were detected
in 9 of 27 (33%) meat-processing environmental samples.) Problem areas in which 220%
of the samples were positive included drains, trenches, floors, exhaust hoods, cleaning
aids (sponges, brooms, hoses, and mops), product-contact surfaces (peelers, conveyors,
and slicers), and wash areas. Sampling of surfaces in contact with sliced luncheon meat
revealed Listeria contamination rates of 9.3, 32.3, and 23.6% before, during, and after
production, respectively. Similarly, listeriae were recovered from 2.8, 14.5, and 25.5%,
respectively, of food-contact surfaces examined before, during, and after production of
frankfurters.
From September 1987 to October 1991, USDA-FSIS inspectors sampled over
15,000 processed meat products, including cooked beef, sliced hams from cans, cooked
sausage, jerky, cooked poultry, salads and spreads, and imported meats [74a]. The overall
incidence of L. monocytogenes during this sampling period was 1.6%, with 235 products
testing positive for L. monocytogenes This led to 25 recalls of product from the market
during 1989-1991 [74a].
Data from the USDAs microbiological monitoring of over 13,000 lots of ready-to-
eat meat products, from January 1, 1993 to September 30, 1997, indicated an incidence
of 2.9% of the lots testing positive for L. monocytogenes (Levine, personal communication
1998). Results from a large-scale 1987 survey sponsored by the American Meat Institute
[2,15] support the notion that Listeria spp. are widely distributed within the environment
of many meat-processing facilities, and as in the earlier study by Flowers [9], also point
Lister ia in Food-Processing Facilities 665
to floors, drains, cleaning aids, wash areas, sausage peelers, and food-contact surfaces as
significant problem areas, with between 20 and 37% of such samples harboring listeriae
(Table 6). With the identification of listeriae in condensate and compressed air and on
walls and ceilings, there can be no doubt that these organisms are ubiquitous in at least
some meat-processing facilities.
Recognizing the potential opportunity for Listeria to contaminate meat during pack-
aging, one major manufacturer of processed meat products attempted to obtain near-
operating room conditions in its packaging room by cleaning the area for 3 days and
then fogging the entire packaging room with 200 ppm quaternary ammonium compound
[8,9]. In spite of these efforts, listeriae were still detected in 1 of 19 environmental samples
obtained from the packaging room. After this exercise, the firm packaged processed meat
products in this room over a 2-week period. Despite adherence to normal cleaning and
sanitizing procedures at the end of each working day, the overall incidence of listeriae in
the packaging room increased, with 3 of 20 (15%), 6 of 20 (30%), and 8 of 20 (40%)
samples being positive for Listeria spp. 3,6, and 8 days after the room was initially cleaned
and fogged, respectively.
Owing to the increased concern for L. monocytogenes in meat products, there has
been a concerted effort to minimize the risk of postprocess contamination during the pro-
duction of processed meats. In one study, cited by Tompkin et al. [74a], swab samples
were collected from packaging lines and floors where exposed ready-to-eat product was
transported, chilled, stored, or packaged. The incidence of Listeria at these locations, from
August 1989 to January 1992, is summarized in Figure 1. The overall trend is toward
improved control of Listeria with fewer positive samples being evident following the
inception of a Listeria control program. The results show a strong seasonal effect for the
presence of Listeria in the finished product environments, with fewer positive samples
being detected during the winter months. In addition, Tompkin et al. [74a] also reported
results of a three-year study in which about 100 packaging lines were tested for Listeria
(Table 7). The percentage of Listeria-negative samples increased from 44 in 1989 to 64
in 1991. The percentage of lines that exceeded the companys established criterion of 5%
of samples positive for Listeria decreased from 29 in 1989 to 13 in 1991. As improvements
were made, attention was given to chronically positive lines. Examples of contaminated
TABLE
6 Incidence of Listeria spp. in
Post-Heat-Processing Areas of 41 Meat
Factories Examined in the United States
During 1987
Area Positive samples (%)
Floors 37
Drains 37
Cleaning aids 24
Wash areas 24
Sausage peelers 22
Food contact surfaces 20
Condensate 7
Walls and ceilings 5
Compressed air 4
30
25
%
20
P
0
15
I
T
I
v 10
E
n
D J D J D J D
E U E U E U E
C N C N C N C
1989 1990 1991
sites on packaging lines included hollow rollers for conveyors, on/off valves and switches,
rubber seals around doors, fibrous conveyor belts, and areas of equipment which were
inaccessible to cleaning. The authors also noted that occasional lapses in cleaning and
sanitizing procedures resulted in a fairly rapid loss of control. They observed that a certain
sequence of events can lead to periodic contamination of packaging lines. The floor is
particularly difficult to render Listeria-negative, and this situation provides a ready source
of organisms to contaminate the packaging line during production or while cleaning,
allowing the establishment of sites for microbial multiplication. This sequence of events
can be prevented by striving for Listeria-negative floors, effectively cleaning and sanitiz-
ing the packaging lines at the end of each days production, eliminating inaccessible sites
in the equipment, and by providing adequate preventive maintenance of the equipment.
TABLE
7 Listeria Contamination on Packing Lines
from 1989 to 1991
% of Lines positive for Listeria at
No. of
Year lines 0% 15% >5%a
1989 96 44 27 29
I990 I06 59 27 14
1991 97 64 23 13
aExceeds company guideline.
Source: Adapted from Ref. 74a.
Listeria in Food-Processing Facilities 667
The authors summarized their report with the comment, ". . . for the present, it must be
concluded that existing technology cannot eliminate Listeria from the cooked product
environment of processing plants."
Since Listeria spp., including L. monocytogenes, have been found in up to 50% of
raw beef, pork, and lamb marketed in the United States, complete elimination of listeriae
from meat-processing environments appears highly improbable. However, the American
Meat Institute has developed a series of interim guidelines [2], which, if followed, will
reduce the incidence of listeriae and decrease the overall microbial load in the working
environment. A detailed description of these guidelines appears later in this chapter.
Poultry-Processing Facilities
Reports have shown that up to 50% of all raw poultry sold in the United States contains
various Listeria spp., including L. monocytogenes, with fecal material from infected flocks
cited most frequently as the source of contamination. Unfortunately, information concern-
ing the incidence of listeriae in American poultry-processing facilities is presently limited
to results from two California surveys. In these studies, researchers at the University of
California-Davis investigated the prevalence of listeriae in processing samples from one
chicken [46] and one turkey slaughterhouse [47] during three or four separate visits. Ac-
cording to these investigators, no Listeria spp. were isolated from feathers, incoming
chiller water, or scalding water, the latter of which aids in feather removal (Table 8).
Nonetheless, L. monocytogenes and L. innocua were identified in samples of overflow
chiller water and feather picker drip water obtained from the chicken slaughterhouse, with
both organisms being detected in recycled water used to clean gutting equipment. Inci-
dence rates for L. monocytogenes in chicken- and turkey-processing facilities were gener-
ally similar, with the percentage of Listeria-positive samples increasing approximately
2- to 2.5-fold during the latter stages of processing. However, L. welshimeri and L. innocua
were absent from most chicken- and turkey-processing samples, respectively. Although
only two poultry slaughterhouses were examined in this survey, inability of these research-
ers routinely to detect L. welshimeri in fresh chicken meat and L. innocua in fresh turkey
meat processed at these facilities suggests that L. welshimeri and L. innocua might be
able preferentially to colonize the gastrointestinal tract of turkeys and chickens, respec-
tively. These findings, along with the ability of these investigators to further demonstrate
an increasing incidence of Listeria spp. on the gloves and hands of poultry workers from
the beginning to the end of processing (Table 9) confirms that these contaminants move
along the processing line with the raw product.
Unfortunately, neither the USDA nor the poultry industry have released any data
regarding the incidence of listeriae within the general working environment of poultry-
processing facilities. However, considering the fecal carriage rate for listeriae in domestic
birds, the current assembly line methods for processing poultry, and the fact that Listeria
spp. (including L. monocytogenes) and salmonellae have be.en isolated from up to about
half of all raw chickens marketed in the United States, one can speculate that the poultry
and meat industries face similar problems in controlling the spread of listeriae and other
organisms in the work environment. If one draws a parallel between methods used to
process meat and poultry, then floors, drains, cleaning aids, wash areas, and food-contact
surfaces emerge as likely niches for Listeria spp., including I,. monocytogenes, in poultry-
processing facilities. These predictions may be supported by published scientific data in
the future.
668 Gravani
TABLE
8 Incidence of Listeria spp. in One Chicken and One Turkey Slaughterhouse in California
No. of
chickenhurkey No. (%) of positive samples
slaughterhouse
Sample samples analyzed L. monocytogenes L. innocua L. welshimeri Total
o/o o/o o/o
~~~~~~~ ~ ~ ~ ~
TABLE
9 Incidence of L. rnonocytogenes and L. innocua on the Hands and Gloves of Poultry Meat Processors Assigned to Three
Different Stations in a Slaughterhouse
No. of
chickedturkey No. (%) of positive samples
slaughterhouse
Sample samples analyzed L. monocytogenes L. innocua L. welshimeri Total
Postchilling handlers 20/30 2 (10.0)/3 (10.0) 2 (10.0)/0 012 (6.7) 4 (20.0)/5 (16.7)
Leg/wing cutters 11/30 4 (36.4)/3 (10.0) 1 (9.1)/0 0/7 (23.3) 5 (45.5)/10 (33.3)
Leg/wing packers 44/30 20 (45.5)/5 (16.7) 1 1 (25.0)/0 0/7 (23.3) 31 (70.5)/12 (40.0)
Source: Adapted from Refs. 46 and 47.
670 Gravani
Egg-Processing Facilities
The discovery of L. innocua and, to a lesser extent, L. monocytogenes in 15 of 42 (36%)
samples of frozen, raw, commercial liquid whole egg obtained from 6 of 11 manufacturers
located throughout the United States suggests that listeriae-as well as salmonellae-contam-
inated poultry feces may contaminate the surface of eggs before breaking, and that these
organisms in turn may be spread to various areas within the egg-processing environment.
Fortunately, the Egg Products Inspection Act of 1970 led to regulations which now require
that all egg products be pasteurized to eliminate salmonellae (and L. monocytogenes).
However, as is true for fluid milk, there is ample opportunity for recontamination of liquid
egg products with listeriae, salmonellae, and nonpathogenic organisms after pasteurization
which can greatly decrease the shelf life and/or microbial quality of the finished product.
Although Listeria spp. have not yet been recovered from commercially prepared pasteur-
ized egg products or the associated manufacturing environment, prudent producers of such
products should be certain that floors, drains, cleaning aids, wash areas, and food-contact
surfaces as well as egg-breaking and egg-separating, pasteurization, and packaging equip-
ment are thoroughly cleaned and sanitized on a regular basis to eliminate potential prob-
lems involving listeriae, salmonellae, and high levels of spoilage organisms.
nated L. monocytogenes from the processing line and equipment, but recontamination
occurred soon after processing was resumed. They also identified the external surfaces of
fresh and frozen fish as the primary source of L. monocytogenes in cold-smoked fish-
processing plants (Table 10). During the filleting, rinsing, and brining operations, the
bacterium is transferred to the exposed flesh, and as the product moves through the pro-
cessing steps, the equipment, personnel, and other surfaces which the product contacts
become contaminated and these then serve as secondary sources of contamination.
Destro et al. traced the transmission of L. monocytogenes in a shrimp-processing
plant [41a 1, using two molecular typing methods: random amplified polymorphic DNA
(RAPD) analysis and pulsed-field gel electrophoresis (PFGE). Of the 115 L. monocyto-
genes isolates examined, 25 were recovered from the plant environment (floors, walls,
and pipes); 15 were from equipment and utensils, including,tables, plastic boxes, knives,
and trays; 9 were found in water used in shrimp processing; 7 were isolated from the
hands of employees; and 59 were from the shrimp. The results from this interesting study
indicated that environmental strains all fell into composite groupings unique to the envi-
ronment, whereas strains from both water and utensils shared another composite profile
group. The L. monocytogenes isolates from fresh shrimp belonging to one profile group
were found in different areas of the processing line. This same profile group was also
present on the hands of employees from the processing and packaging areas of the plant.
This study showed that there were many different sources of L. rnonocytogenes in the
shrimp-processing plants. Information on preventing postprocessing contamination of fish,
seafood, and other fishery products is presented in the second half of this chapter.
TABLE
10 incidence of Listeria in a Cold-Smoked
Sal mon-Processi n g Pia nt
Area in Plant L. monocytogenes L. innocua
~~~~ ~ ~~
listeriae in raw vegetables and fruits and particularly the prevalence of these organisms
in work environments of vegetable- and fruit-processing facilities have received relatively
little attention. Nevertheless, the long-recognized association of listeriae with soil and the
discovery of Listeria spp., including L. monocytogenes on raw vegetables suggest that
these organisms are almost certainly in vegetable- and fruit-processing facilities. Unfortu-
nately, the extent of Listeria contamination in such facilities in the United States is cur-
rently unknown. However, soil and production-area samples from one potato-processing
factory in the Netherlands have yielded L. monocytogenes, L. innocua, and L. seeligeri
(see Tables 14 and 15).
Western Europe
Interest in the incidence of listeriae within European food-processing facilities has devel-
oped in parallel with the discovery of these organisms in foods destined for human con-
sumption. As noted in Chapter 12, large quantities of French Brie cheese were contami-
nated with L. monocytogenes in 1986. Therefore, emphasis was first placed on determining
the prevalence of listeriae in cheese factories. The results of one small-scale environmental
survey of French cheese factories [32] identified L. monocytogenes in one floor sample
and L. innocua was recovered from boards, wheels, and equipment (7 of 22 samples),
brushes (1 of 6 samples), and filtered air (1 of 19 samples). From 1988 to 1990, a French
cheese factory was sampled for Listeria contamination [53a]. Of the 344 samples collected
and analyzed for Listeria, 61 strains (44 L. monocytogenes and 17 L. innocua) were iso-
lated from four varieties of cheese, cheese brines, processing equipment, and the plant
environment. The L. monocytogenes strains were recovered from the ripening and rind
washing stages and not before, so Jacquet et al. [53a] theorized that the cheese contamina-
tion occurred at these points in the manufacturing process. During a survey of German
factories producing soft smear-ripened cheese, Terplan 1741 also isolated nonpathogenic
Listeria spp. from smear liquid, various pieces of machinery (especially smearing ma-
chines), and floor drains, with L. monocytogenes being detected far less frequently than
other listeriae (Table 11). Hence, opportunity exists for contamination of both mold and
bacterial surface-ripened cheese during the latter stages of manufacture and storage.
Although such published information is limited, some unpublished data are available
on the prevalence of listeriae in other Western European cheese factories. As mentioned
in Chapter 12, Swiss officials who were tracing the source of contamination in the 1987
listeriosis outbreak involving consumption of Vacherin Mont dOr soft-ripened cheese
Listeria in Food-Processing Facilities 673
recovered the epidemic strain of L. monocytogenes from smear brine, curing brine, waste-
water sinks, wooden cheese hoops, and wooden boards used in 10 different cheese facto-
ries that manufactured Listeria-contaminated cheese [37]. Additionally, nearly half of the
12 cellars used to ripen cheese contained listeriae, with the pathogen being detected on
6.8% of the wooden shelves and 19.8% of the brushes used in the ripening cellars. Al-
though not noted in the report, one would suspect that L. mc,nocytogenes also was present
in commonly recognized environmental niches such as drains, floors, stagnant water, and
various food-contact surfaces within cheese factories and ripening cellars. Thus brushing
cheese with saltwater and ripening hooped cheese on wooden shelves appear to be two
important means for dissemination of listeriae within cheese factories.
In 1988, Cox [39,40] presented some preliminary data concerning the prevalence
of Listeria spp. within one blue and six soft cheese factories in Western Europe as well
as in one ice cream factory and eight chocolate factories. As expected, listeriae generally
occupied similar environmental niches in both soft and blue cheese factories; however,
Listeria contamination was far more common in ripening than production areas of the
one blue cheese factory examined (Table 12). Ripening practices for blue cheese, including
maintenance of a relatively moist environment, appear to be the likely reason for higher
rates of Listeria contamination in ripening than production areas. Although some environ-
mental niches in this blue cheese factory were not sampled, results for soft cheese factories
point to walls, air coolers, stagnant water, and condensate as possible problem areas in
blue cheese factories as well.
During a similar investigation, samples from at least half of the drains, conveyors,
stagnant water, floors, and residue and waste products from one Western European ice
cream factory contained populations of Listeria spp. ranging from 10 to >106 CFU/g or
mL (Table 13). This factory manufactured all of its ice cream from commercially produced
reconstituted powdered milk (a product from which Listeria has not yet been isolated)
rather than fresh milk. Hence, these findings strongly suggest that Listeria contamination
in dairy-processing facilities is not always linked to incoming raw milk or milk haulers.
Listeria spp., including L. monocytogenes, also have been detected in commercially
produced chocolate that was marketed in England [48]. Furthermore, a 1988 report by
Cox [39] indicated that 8 of 32 (25%) and 10 of 59 (17%) samples obtained from damp,
wet, and dry areas of eight Western European chocolate factories were positive for Listeria
spp. Although growth of listeriae in chocolate is very unlikely, contamination of the fin-
674 Gravani
TABLE
12 Incidence of Listeria spp. in Several Western
European Blue and Soft Cheese Factories
~~~ ~
Ripening areas.
Not analyzed.
Source: Adapted from Refs. 39 and 40.
ished product during packaging is clearly possible. The relatively low risk of producing
Listeria-contaminated chocolate can be further reduced by development of adequate clean-
ing and sanitation programs and by maintaining production and packaging areas as dry
as possible.
In one of the largest European surveys reported thus far, Cox et al. [41], during the
latter half of 1986, investigated the incidence of Listeria spp. in the processing environ-
ment of 17 establishments in the Netherlands that produced fluid dairy products, ice cream,
Italian-style cheese, frozen food, potato products, and dry culinary foods. A total of 608
samples were collected from drains, floors, condensed and stagnant water, residues, pro-
cessing equipment, and/or other areas and were analyzed for listeriae using the original
USDA or FDA method with or without modification. All presumptive Listeria isolates
were then speciated according to results from conventional biochemical tests.
Despite use of GMPs in these factories, Listeria spp. were recovered from all types
of food-processing facilities examined with the exception of two that produced dry culi-
nary products. Overall, 181 of 608 (29.8%) samples yielded Listeria spp. with L. innocua,
L. monocytogenes, and L. seeligeri being identified in 87.3, 14.9, and 0.5% of all positive
samples, respectively. Although only five samples contained both L. monocytogenes and
L. innncua, the actual number of such samples is probably somewhat greater, since a
limited number of presumptive Listeria isolates from each sample were chosen for con-
firmation. As shown in Table 14, L. innocua was most prevalent in establishments that
produced processed potato products followed by those that produced ice cream, frozen
food, Italian-style cheese, and fluid dairy products, with the organism generally being
isolated most frequently from drains, floors, and condensed and stagnant water.
In contrast, L. monocytogenes was detected in 11.8% of all environmental samples
obtained from one ice cream factory but was found in 2.9, 3.0, 3.3, and 3.7% of similar
samples from establishments that manufactured fluid dairy products, potato products, fro-
zen food, and Italian-style cheese, respectively (Table 15). Although only one ice cream
factory was examined in this survey, the results are as expected when one recalls that
Cox [39,40] previously found that listeriae were widespread in another Western European
ice cream factory and also were present in very large numbers, particularly in floor drains
(Table 13). Given such populations of listeriae in ice cream factories and the current
extruding, niolding, and freezing methods used to produce ice cream, and particularly ice
cream novelties, one can easily postulate many routes whereby listeriae may recontaminate
the finished product, as has been reported in the United States.
Results concerning the incidence of Listeria spp. as well as L. innocua and L. muno-
cytogenes in various work environments of all 15 food-processing facilities are summa-
rized in Table 16. Overall, these findings are comparable to what has been previously
noted for similar food-processing facilities in the United States; for example, Listeria spp.
and L. innocw were most frequently recovered from drains followed by condensed and
stagnant water, floors, residues, and processing equipment. With a few minor exceptions,
which probably resulted from the number of samples analyzed, this same trend is readily
apparent for all five types of food-processing facilities listed in Table 14. Thus a logical
pattern emerges in which L. innocua moves from floor drains to pools of condensed and
stagnant water, which then come into direct contact with floors and residues. Once present
in open areas of the work environment, L. innocua is spread by employees to processing
equipment that comes into direct contact with the product. Unlike L. innocua, L. monocyto-
genes was far less prevalent in all types of food-processing facilities and was distributed
fairly evenly within the factory environment with incidence rates ranging between 2.3 and
7.7%. Although L. innocua is by definition nonpathogenic, the fact that L. innocua and
L. monocyto,qenes (and possibly other Listeria spp.) occupy similar environmental niches
indicates that detection of listeriae anywhere within the manufacturing environment should
prompt immediate corrective action, the details of which will be discussed shortly.
In one of the remaining few Western European surveys reported, Hudson and Mead
[511 determined the incidence of Listeria spp. at 10 different sites within one large English
poultry-processing facility. According to these authors, scald water, feathers, and chill
water as well as swab samples from defeathering machines and conveyors leading to
the chiller were free of listeriae; however, L. monocytogenes was routinely isolated from
automatic carcass openers and also was present in samples from evisceration-line drains,
676 Gravani
TABLE
14 Incidence of L. innocua in Working Environments of 15 Food-Processing Facilities in the Netherlands
No. of positive samples/No. of samples analyzed (%)
Italian-style Frozen food Potato-processing
Fluid dairy factory Ice cream factory cheese factory factory factory
Environmental sample na = 5 n = l n= 5 n = 3 n = l
Drains 2/4 (50.0) 4/4 (100.0) 19/42 (45.2) 2/3 (66.7) 7/13 (53.8)
Condensed/stagnant water 2/5 (40.0) 4/8 (50.0) 7/20 (35.0) NA 7/10 (70.0)
Floors 0/2 8/16 (50.0) 14/44 (31.8) 2/4 (50.0) 9/13 (69.2)
Residues NAb 4/12 (33.3) 16/71 (22.5) NA 5/15' (33.3)
Processing equipment o/ 10 7/20 (35.0) 6/68 (8.8) 1/6 (16.7) NA
Miscellaneous 0/13 2 B d (25.0) 12/103' (11.7) 15/78 (19.2) 4/ 17' (23.5)
Total 4/34 (11.8) 29/68 (42.6) 74/348 (2 1.3) 20/91 (22.0) 32/68 (47.1)
Not analyzed.
Includes one sample positive for L. seeligeri.
Conveyor belt (two of two positive).
Raw milk (two of two positive), untreated effluent.
Potato delivery soil (two of three positive), sand from effluent treatment (two of two positive).
Source: Adapted from Ref. 4 1.
Lister ia in Food- Processing Facilities 677
TABLE
16 Overall Incidence of Listeria spp. in Working Environments
of 15 Food-Processing Facilities in the Netherlands
neck-skin trimmers, and conveyors on which carcasses travel to the packing area (Table
17). Although only one to three samples from each site were analyzed in three successive
visits, the areas from which L. monocytogenes was recovered in this poultry-processing
facility are generally similar to those observed by Genigeorgis et al. [46,47] for chicken
and turkey slaughterhouses in California (see Table 8 ) .
Australia
Information concerning the prevalence of listeriae in food-processing facilities located in
other parts of the world is currently limited to a few Australian studies. Following the
isolation of L. monocytogenes from ricotta cheese in 1987, the Victorian Dairy Industry
Authority and the Department of Agriculture and Rural Affairs conducted a joint survey
to determine the extent of Listeria contamination in the working environments of 5 2 Mel-
bourne-area factories producing pasteurized milk and different types of cheese [76]. Over-
all, various Listeria spp. were detected in 141 of 763 ( 1 8.5%) environmental samples from
21 of 5 2 (40.4%) factory environments, with L. monocytogenes, L. seeligeri, and L. iva-
novii being identified in 132 (93.6%), 8 (5,7%), and 1 (0.7%) of these Listeria-positive
samples, respectively. More important, L. rnonocytogenes was present in all but one of
the ListeriLz-positive factories. As expected from other surveys conducted in the United
States and Western Europe, factory sites most frequently contaminated with listeriae once
again included drains and floors in coolers, surfaces of manufacturing and packaging
equipment, and conveyors. Even though strict cleaning and sanitizing programs were im-
plemented at many of these facilities, Listeria spp. were very difficult to eliminate from
the working environment, with these organisms being continuously isolated from one fac-
tory over a period of 5 months.
Sutherland and Porritt [73a] conducted a 3-year study in 12 Australian dairy-pro-
cessing facilities to assess the environmental diversity and identify the major environmen-
tal niches for L. rnonocytogenes. A total of 565 environmental samples were collected
and tested. The overall incidence of Listeria-positive samples was 21% (Table 18). Ap-
proximately half of these samples (12%) were positive for L. monocytogenes.
Cheese, ice cream, and mixed-product plants all had similar incidences of L. rnono-
cytogenes and Listeria spp. The incidence of L. rnonocytogenes in mixed-product factories
(18%) was comparable to the higher levels found in milk factories. Sutherland and Porritt
[73a] also highlighted four major ways that L. rnonocytogenes enters a dairy-processing
facility, including:
Once L. rnonocytogenes is inside the processing plant, these authors [73a] found numerous
areas in which this pattern can survive, grow, and potentially contaminate product. Con-
veyor systems, drains, and floors were the most common isolation sites. Other areas of
concern related to were traffic flow, cooking units, and internal air quality.
Complete elimination of listeriae from dairy-processing facilities may, in some in-
stances, be nearly impossible; however, the likelihood of producing Listeria-contaminated
products can be greatly reduced by following GMPs, which include implementation of
rigorous cleaning and sanitizing programs for equipment used at critical points during
manufacture and packaging of the foods in question.
TABLE
18 Three-Year Study in 12 Australian Dairy-Processing Plants to Determine Environmental Diversity and Identify Major
Environmental Niches of L. rnonocytogenes
areas as drains, U-tubes, and drain boards. If this is true, then garbage disposal systems
could conceivably lead to problems from production of aerosols. Although further work
is needed to clarify the public health significance of listeriae in the kitchen environment,
you may recall from Chapter 13 that L. rnonocytogenes was found in many refrigerated
foods belonging to an Oklahoma woman who contracted listeriosis after consuming con-
taminated turkey frankfurters that were eventually recalled nationwide.
Centers for Disease Control and Prevention (CDC) officials also isolated L. rnonocy-
togenes from 15 of 25 (60%)refrigerators that were used by apparent victims of foodborne
listeriosis [25]. Hence, consumers should regularly clean and sanitize kitchen areas, sinks,
and refrigerators. Such efforts should help prevent potential problems involving listeriosis
and other forms of foodborne illness in the home.
General Guidelines
Factory Design
Every food processor should be firmly committed to the long-term production of safe,
wholesome food. The first step toward such a goal is an adequately designed factory
to produce the particular product. Although newly constructed buildings offer countless
advantages in that they can be designed for production of specific products, existing build-
ings also can be used for safe production of food provided that such facilities have been
properly modified to meet certain basic requirements.
Design features that are widely considered to be essential for all types of food-
processing facilities include (a) a raw product receiving area that is completely isolated
from processing and packaging areas of the factory; (b) tight-fitting exterior windows and
doors that will prevent animals and insects from entering processing and packaging areas;
(c) easily cleaned and sanitized walls, floors, and ceilings that are constructed of tile,
metal, or concrete and not porous materials such as wood; (d) floors designed to drain
rapidly and prevent pooling of water; (e) floor drains located away from packaging equip-
ment, especially if processed foods are exposed to factory air; (f) proper screens, debris
baskets, and traps on floor drains; (g) a quality control and/or quality assurance laboratory
that is well isolated from other areas of the factory; and (h) proper means of waste disposal
outside the factory to discourage congregation of insects, rodents, birds, and other animals
that may harbor Listeria and other pathogenic microorganisms.
In addition to these concerns, the heating, ventilating and air-conditioning (HVAC)
system also must be properly designed to minimize airborne contamination [68]. Features
considered to be essential for such a system include (a) intake air vents on the roof of
the building that are located upwind from prevailing air currents but away from dumpsters,
raw product receiving areas, and vents that are discharging factory air; (b) installation of
screens and filters inside incoming air vents to remove particulate matter and condensate;
(c) easily cleanable HVAC systems; and (d) proper location of dehumidifiers and air-
conditioning systems so that these units drain away from processing and packaging areas.
Most important, all HVAC systems must be designed to produce a higher positive air
pressure in processing and packaging rather than in receiving areas. This design readily
prevents movement of airborne contaminants from raw product areas to the cleanest areas
of the factory where foods are processed and packaged.
Factory Environment
Various bacteria, yeasts, and molds can be found in most food-processing areas other than
those associated with aseptic packaging, with populations normally being many times
higher in receiving than in processing and packaging areas. Furthermore, most of these
microorganisms will grow in the factory environment if given a suitable temperature and
enough time along with an adequate supply of nutrients and water. Although microbial
contamination will always occur in food-processing facilities, eliminating microbial
growth by altering (a) temperature, (b) time that the organism is present in the environ-
ment, (c) availability of nutrients, and/or (d) availability of water will sharply decrease
the incidence of L. rnonocytogenes and other foodborne pathogens as well as spoilage
organisms in the factory environment. Hence, production of a safe food product with a
long shelf life depends largely on control of timehemperature constraints and elimination
of available nutrients and/or water through the concerted effort of everyone involved.
Since air, water, waste products, and anything else that comes in contact with the
Listeria in Food-Processing Facilities 683
quality of finished products. In establishments such as those that produce fluid milk and
ice cream, adherence to good cleaning and sanitation practices that involve both equipment
and the factory environment is the only means of preserving product quality beyond initial
pasteurization of ingredients.
Each food-processing facility needs to institute and enforce an effective cleaning and
sanitizing program that will ensure production of safe products. As part of this program,
management personnel need to develop standard operating procedures for every job in
the factory along with master schedules with the frequency of cleaning and sanitizing
procedures so that the workers will recognize their individual responsibilities and will
maintain accurate records regarding routine sanitation practices. Management personnel
also need to instill in their employees the great importance of good cleaning and sanitizing
practices through the use of continuing education programs that deal with current issues
such as Listeria. Such cleaning and sanitizing responsibilities should never be assigned
to new untrained employees.
Floors, drains, walls, ceilings, and each piece of equipment in the factory should
be cleaned and/or sanitized on a regular basis with the frequency of cleaning and sanitizing
being dependent on the extent to which the particular item becomes contaminated during
normal operation and whether or not a product is likely to come in contact with the item
during processing and/or packaging. All food-contact surfaces such as tables, peelers,
slicers, collators, overhead shielding, conveyors, conveyor belts, chain rollers, supports,
and other intricate equipment directly associated with processing, filling, and packaging
operations need to be cleaned and sanitized daily and in some instances more often, partic-
ularly around filling and packaging operations. A regular cleaning and sanitizing schedule
also must be adopted for non-food-contact surfaces such as floors, walls, ceilings, floor
drains, pipes, blowers, HVAC ducts, coils and pans from dehumidifying and air-condition-
ing units, light fixtures, material handling equipment, and wet and dry vacuum canisters.
As indicated in the first half of this chapter, Listeria spp., including L. monocytogenes,
have been most frequently isolated from floor drains and floors, thus suggesting that these
areas may function as reservoirs for listeriae in food-processing facilities. Although all
floors and drains, including drain covers and baskets, in production and refrigerated storage
areas should be thoroughly cleaned and sanitized daily, high-pressure hoses should never
be used in these areas, since such practices readily promote the spread of listeriae to nearby
equipment and other areas of the factory through splashing and the production of aerosols.
Managers of food-processing facilities must be sure that proper equipment is avail-
able for daily cleaning and sanitizing operations. Absorbent articles such as sponges and
rags should never be used in the factory environment, since these items function as virtual
microbial zoos. Various types of metal scrappers can be used for removing hard mineral
deposits, with disposable paper towels being best suited for eliminating excess moisture
and accidental spills. Unlike sponges and rags, brushes are readily cleaned and sanitized
and are therefore suitable for widespread use in the factory. However, to avoid cross
contamination, separate color-coded brushes with nonporous plastic or metal handles
should be used for scrubbing (a) exterior and interior surfaces of equipment, (b) raw and
finished product areas, (c) food-contact and non-food-contact equipment surfaces, and
(d) floor drains. Brushes, particularly those used to scrub floor drains, are best cleaned
and stored in sanitizing solution after use.
Sanitizing is the final step in eliminating L. monocytogenes, other foodborne patho-
gens, and the myriad of spoilage organisms present in the production environment. Since
the presence of organic debris, particularly if proteinaceous, readily decreases the effec-
Listeria in Food-Processing Facilities 685
pathogenic microorganisms from such samples unless the laboratory is in a separate build-
ing and completely removed from the factory. Although analysis of environmental and,
if necessary, food samples for microbial pathogens is best left to outside commercial
testing laboratories that are FDA approved or otherwise certified, coliform and standard
aerobic plate counts should be obtained for samples from the factory environment and
the food during all stages of production to monitor the extent of postprocessing contamina-
tion and thus to quickly identify any problems associated with inadequate cleaning and
sanitizing. Coliform organisms are commonly regarded as being indicators of postpro-
cessing contamination and the possible presence of pathogens; however, the presence or
absence of coliforms in food or environmental samples does not guarantee the presence
or absence of foodborne pathogens. In fact, often little if any correlation has been observed
between the presence of coliforms and Listeria in finished product. Therefore, routine
testing of environmental samples for Listeria spp. and other foodborne pathogens by out-
side laboratories remains a critical component of any sanitation verification program.
Traffic Patterns
Employee movement within food-processing facilities also can have a major impact on
the microbiological quality of finished products. Therefore, traffic patterns need to be
developed that restrict or preferably eliminate movement of workers between raw, pro-
cessing, filling, packaging, and shipping areas. Managers need to educate employees about
the spread of Listeria and other microbial contaminants from clothing, boots, and tools
to all areas of the factory, and they need to situate locker rooms, changing areas, and
lunch and break rooms to minimize traffic through production areas. Issuing different-
colored outer garments to workers in various areas of the factory has proven helpful in
monitoring employee movement. Since L. monocytogenes and other microbial pathogens
are commonly associated with raw products of both plant and animal origin, employees
working in raw product receiving areas (including maintenance personnel) and individuals
who deliver raw products, particularly milk haulers, should be denied access to all pro-
cessing areas. When necessary, employee movement between raw product and processing
areas of the factory should only be allowed after completely changing outer garments as
well as scrubbing and disinfecting boots. All workers should be encouraged to use disinfec-
tant-containing footbaths that should be placed in all doorways leading into the factory
as well as between raw product and production areas. These footbaths need to be monitored
daily for sanitizer strength and cleanliness. Since a great variety of microorganisms are
carried on street clothing, it also may be prudent for managers to consider limiting the
number of visitors and tour groups going through the factory. Large glass observation
windows provide ample opportunity for visitors to view processing areas while at the
same time prevent introduction of additional microbial contaminants.
Personnel Clean Iiness
Factory managers and supervisors must stress good employee hygiene and also set a good
example for other workers. All individuals with obvious illnesses, infected cuts, or abra-
sions need to be excluded from working in processing areas or from doing other tasks
that may lead to contamination of food, food-contact surfaces, or packaging materials or
equipment. Furthermore, the use of tobacco and chewing gum as well as the consumption
of food should be banned in processing areas along with the wearing of hairpins, rings,
earrings, watches, and other jewelry. Above all, employees should always wash their hands
thoroughly before starting work, on returning to work, and after touching floors, walls,
Listeria in Food-Processing Facilities 687
light switches, any other unclean surface, and garbage. To further promote their use, hand-
washing facilities should be properly designed and conveniently located near work sta-
tions. All factory workers need to be provided with hair and/or beard nets as well as clean
clothes, suitable footwear, and disposable gloves. Special attention also is needed to assure
that street clothes do not enter processing areas and that factory clothing, including foot-
wear, remain inside the factory. All factory clothing should be changed daily or more
often if soiled, with the responsibility of laundering being left to the employer. Although
these recommendations may, in some instances, be difficult for food processors to follow
and enforce, this task will be made much easier if management can instill in workers the
conviction that each employee is personally responsible for both the quality and safety
of the foods that are produced and ultimately consumed by the public.
Dairy Industry
Farm Environment
Since listeriae are widespread in the environment, any quality control program should first
contain a plan to minimize contamination of raw milk with Listeria and other microorgan-
isms on the dairy farm. Along with good animal husbandry practices, including the use
of only high-quality feed and silage, farm workers also should give attention to cleanliness
of the milkhouse and milking equipment. Most important, teats and udders of all cows
should be properly sanitized and dried before milking equipment is attached. Bulk tanks
in which raw milk is stored also need to be properly maintained and inspected regularly.
CIa rif iers and Separat0 rs
All raw milk should be filtered and subsequently clarified and separated by centrifugation
to remove extraneous matter and somatic cells (i.e., leukocytes) before pasteurization.
Since L. monocytogenes is sometimes found in leukocytes, clarifiers and separators should
be well isolated from the pasteurizer and all finished product areas of the factory. Sealed
containers should be used to dispose of all clarifier and separator waste, both of which
may contain high levels of listeriae. Special care also should be used in cleaning and
sanitizing separators, clarifiers, and surrounding areas.
Pasteurization
Proper pasteurization using a vat or high-temperature short-time (HTST) pasteurizer is
the only commercially practical means by which all non-spore-forming pathogens, includ-
688 Gravani
ing L. monocytogenes, can be inactivated in raw milk. Thus it is imperative that all pasteur-
ization equipment be designed, installed, maintained, and operated properly.
Although continuous-flow HTST pasteurization is used to process virtually all fluid
milk and ice cream mix, vat (or batch) pasteurization is employed by many smaller firms,
particularly those involved in cheesemaking, when the volume of incoming raw milk is
too small to justify the use of a continuous-flow HTST system. If vat pasteurization is
used, raw milk must be heated to a minimum of 623C (145F) and then held at that
temperature for at least 30 min. In theory, vat pasteurization is a relatively simple process
with raw milk being pumped into a steam- or hot water-jacketed vat and held for the
prescribed time. However, FDA inspections conducted as part of the Dairy Initiative Pro-
gram mentioned earlier have uncovered numerous problems with vat pasteurizers, includ-
ing improper equipment design, the absence of proper outlet valves and air space thermom-
eters, and improperly operated air space heaters. The latter problem is particularly critical,
since the air space temperature above the product in the vat must be at least 23C (5F)
higher than that of the product at all times to assure proper Pasteurization. Operators of
such pasteurizers should be made accountable for proper performance as well as proper
cleaning and sanitizing of the equipment. In addition, recording charts showing time and
temperature relationships along with other data for each vat of product pasteurized should
be kept for at least 3 months.
As mentioned earlier, continuous-flow HTST pasteurization at 71.7"C (161OF) for
a minimum of 15 s is the principal method for processing raw milk. Although an in-depth
discussion of the many intricate problems associated with HTST pasteurization equipment
is beyond the scope of this book, a basic knowledge of HTST pasteurization is essential
to appreciate the seriousness of some of the recently identified problems that have been
linked to faulty maintenance and/or operation of the equipment. Interested readers may
consult the HTST Pasteurizer Operation Manual [50] for more detailed information on
HTST pasteurization.
All HTST pasteurizers consist of five basic components, as shown in Figure 2:
(a) plate heat exchanger-a series of thin, gasketed stainless steel plates divided into
three sections (heater, regenerator, and cooler) for heating incoming raw milk and cooling
outgoing pasteurized milk; (b) constant level tank-provides a constant level of raw milk
to the HTST system; (c) timing pump-a positive displacement pump that establishes the
holding time of the time and temperature relationship for pasteurization; (d) holding
tube-a length of pipe in which fully heated milk is held for the required holding time;
and (e) flow diversion valve-a three-way valve that will allow properly pasteurized milk
to enter the regenerator section of the plate heat exchanger or divert improperly pasteurized
milk to the constant level tank for repasteurization. In addition to these five components,
a source of steam and/or hot water is required to heat incoming raw milk, a safety thermal
limit recorder is needed to activate the flow diversion value in the event of improper
pasteurization, and a cold milk recorder is required to record the temperature of outgoing
pasteurized milk. Finally, auxiliary components that may be added to HTST units for
additional processing of milk or milk products include a booster pump, homogenizer as
a timing pump, stuffing pump, and flavor treatment or vacuum units.
Inspections of HTST pasteurizers conducted in conjunction with the FDA Dairy
Initiative Program uncovered numerous problems relating to proper installation and main-
tenance of these units. Problems most commonly associated with HTST pasteurization
equipment have included stress cracks and/or pinholes in the heat exchanger plates, leak-
ing gaskets, improper flow diversion valves, and inadequate cleaning and sanitizing of
Listeria in Food-Processing Facilities 689
Raw Milk
Pasteurized Milk - --- Diversion Line
Flow-Diversion valve
Cold Past,
-I
Holding Tube
the pasteurization unit. Although not a strict regulatory requirement, positive pressure
should be maintained between the product and heating medium as well as the product
and cooling medium (sweetwater) to prevent Listeria-contaminated raw milk or sweetwa-
ter from mixing with pasteurized product in the event that some of the heat exchanger
plates contain stress cracks or pinholes. Operators should examine all pasteurization plates
for defects every 6 months using the standard dye test. Sweetwater and glycol solutions
also should be routinely examined for microbial contaminants, since these coolants may
harbor L. monocytogenes, Yersinia enterocolitica, and Salmonella typhimurium for ex-
tended periods along with large populations of psychrotrophs [ 18,661; the latter are particu-
larly detrimental to product shelf life. As was true for vat pasteurization, operators of
HTST pasteurizers must be responsible for proper operation of these units and retain accu-
rate records and chart recordings for each lot of pasteurized product for at least 3 months.
Although the inability of L. monocytogenes to survive the minimum allowable HTST heat
treatment given to commercially available raw milk ( 7 1 . 7 W 1 5 s) is now generally ac-
cepted, most fluid milk processors in the United States are pasteurizing milk at -76.7"C
for 20 s, which is well above the minimum requirements established in the Pasteurized
Milk Ordinance. This more severe heat treatment markedly extends the shelf life of the
finished product by inactivating larger numbers of spoilage organisms than does minimal
HTST pasteurization. However, the psychrotrophic nature of L. monocytogenes increases
the need to prevent introduction of listeriae into the product after pasteurization.
Pipeline and Cross Connections
Many large dairy processors have installed up to several miles of pipeline in the factory
to handle movement of raw milk from storage tanks to the pasteurizer and pasteurized
milk from the pasteurizer to various holding tanks, mixing tanks, and product areas located
690 Gravani
throughout the factory. Considering the enormous quantities of product that can be manu-
factured at such facilities during one production period, careful attention must be given
to each stage of manufacture, since an error made during these operations could adversely
affect thousands of people, as was true for the 1985 outbreak of milkborne salmonellosis
in Chicago.
FDA inspections have uncovered numerous violations related to pipelines, including
cross connections between raw and pasteurized milk lines and/or storage and holding
tanks as well as cross connections between CIP and product lines and other potentially
hazardous circuits. Since many of these lines allow easy bypass of raw product around
the pasteurizer thus permitting postpasteurization contamination in the event of equipment
failure or operator error, factory managers, engineers, or other qualified people need to
walk through the factory and construct an up-to-date detailed blueprint of raw and pasteur-
ized product flow throughout the entire factory. Once the blueprint is constructed, any
unwanted piping, dead ends, illegal cross connections, or unauthorized changes made to
initial installations should be promptly identified and eliminated. Most important, all pas-
teurized product lines need to be separated from raw and CIP lines by a physical break.
In many plants, pipes are physically labeled with the type of product (raw or pasteurized)
that flows through them. To be of continued use, blueprints must be routinely updated
and reviewed for accuracy by walking the blueprints through the factory. Finally, no
piping changes should ever be made without prior review by qualified authorities.
Filling a n d Packaging
Postpasteurization contamination frequently occurs during filling and packaging opera-
tions when products are exposed to difficult-to-clean surfaces on equipment, the manufac-
turing environment, and airborne contaminants [54]. Areas associated with product con-
tamination have included mandrels, drip shields, bottom and top breakers, prefilling coding
equipment, deflecter bars, and cutting blades as well as overhead shielding, conveyors,
conveyor belts, chain rollers, supports, and lubricants. Product extruder heads are particu-
larly prone to contamination and therefore should be sanitized frequently during filling
operations. Such practices will lead to the production of safe products with markedly
increased shelf lives.
Reclaimed a n d Reworked Product
Salvage programs, by their nature, are high-risk operations that can put an entire company
in jeopardy if not done in a sanitary manner. Potential hazards associated with such salvage
operations include (a) failure to repasteurize returned product before reuse; (b) inadver-
tently pumping returned but not repasteurized product through pasteurized product lines
without proper cleaning and sanitizing between use; (c) accidental reuse of outdated prod-
uct; (d) reuse of product returned from retail stores that may have been temperature abused,
tampered with, or exposed to chemical or biological contamination; and (e) the use of
product from contaminated, leaking, or otherwise damaged containers. Therefore, any
product that left the possession and control of the processor or has been mishandled,
inadequately protected from Contamination, or exposed to temperatures of 27.2C (45F)
should be discarded. Dairy processors also should seriously consider confining the use of
reclaimed and repasteurized milk to dairy products prepared from non-Grade A milk.
According to the Pasteurized Milk Ordinance, American dairies involved in re-
claiming programs now must have separate areas or rooms isolated from Grade A milk
operations for receiving, handling, and storing all returned products. Outdated products
Listeria in Food-Processing Facilities 691
and those which have left the control of the processor and later are returned to the dairy
for disposal should never reenter the factory. Given the recent isolation of L. monocyto-
genes and other microbial contaminants from the external surface of cartons containing
returned product, along with the proven ability of L. monocytogenes and Salmonella spp.
to survive up to 14 days on the external surface of both waxed cardboard and plastic-type
milk containers [72], the process of opening containers and emptying reclaimed product
into vats for reprocessing will likely introduce many new unwanted microbial contami-
nants into the factory environment. Therefore, it is imperative that all returned products
be handled similar to raw milk and be repasteurized, preferably using times and tempera-
tures well above the required minima. After reprocessing, all equipment including tanks,
pumps, and pipelines used in the reclaiming operation should be thoroughly cleaned and
sanitized. In view of the problems associated with salvage operations, each dairy processor
needs to reevaluate the advantages and disadvantages involved in reclaiming products and
then decide whether or not the monetary benefits gained by such practices will outweigh
the potential public health and other risks.
Frozen Dairy Products
Although few bacterial species can grow at temperatures below OOC, most microorgan-
isms, including listeriae, can survive for long times in frozen dairy products such as ice
cream, ice cream novelties, and sherbet. Unlike fluid milk, frozen dairy products are partic-
ularly susceptible to microbial contamination during freezing and filling operations. All
barrel freezers used to make frozen dairy products should be thoroughly sanitized before
use, since hand assembly of the many intricate freezer parts is likely to introduce numerous
contaminants. The source of air for the barrel freezer is another likely source of contamina-
tion. Hence, in addition to maintaining positive air pressure in this area and keeping the
surrounding area as clean and sanitary as possible, all air lines connected to the barrel
freezer should be equipped with dryers and bacterial filters to prevent airborne contami-
nants from entering the product.
Ingredient feeders are perhaps the greatest source of Contaminants in frozen dairy
products. Therefore, fruits, nuts, candy, and other ingredients that are added directly to
frozen ice cream mix need to be closely monitored for coliforms, pathogens, and other
microbial contaminants. Exposure of ingredients to the factory environment also should
be minimized. Strict adherence to GMPs is necessary during the production of molded,
extruded, and/or dipped ice cream novelties, since many such products have been recalled
because of contamination with L. monocytogenes (see Table 5 in Chapter 11). Condensate
in and around hardening rooms as well as conveyor belts appears to be a likely source
for such contaminants.
Finally, handling of product rerun exiting the freezer needs to be assessed at each
factory. Although rerun product should never be added directly back to tanks containing
unfrozen mix, frozen rerun product can be reclaimed by blending it with fresh mix, which
is then repasteurized. Any rerun that is not reclaimed should be clearly separated from
reclaimable material and properly disposed.
Fermented Dairy Products
Fortunately, the incidence of Listeria contamination in yogurt, cultured cream, cultured
buttermilk, and other fermented fluid milk products appears to be quite low with very
few recalls being issued for these products. The species of lactic acid bacteria used in
manufacturing these products as well as the bacteriostatic and bactericidal effects of vari-
692 Gravani
ous organic acids produced during fermentation and the resultant lowering of pH are un-
doubtedly responsible for the near-absence of such recalls. However, since bacterial patho-
gens (including L. monocytogenes) and various spoilage organisms may inadvertently
contaminate fermented milk products during any stage of manufacture, producers of such
products need to follow GMPs and be readily aware of potential problems regarding im-
proper cleaning and sanitizing of equipment and the processing plant environment as well
as potential sources of postpasteurization contamination (e.g., filling and packaging areas)
discussed earlier in this chapter.
The production of Listeria-free cheese, particularly soft and semisoft varieties
surface-ripened with mold (e.g., Brie, Camembert) or bacteria (e.g., brick, Limburger), is
difficult, since environmental conditions required for proper cheese ripening also promote
the growth of L. monocytogenes and other unwanted organisms. Swiss officials who inves-
tigated the 1987 listeriosis outbreak involving consumption of Vacherin Mont d' Or soft-
ripened cheese (see Chapter 12) eventually isolated the epidemic strain of L. monocyto-
genes from wooden shelves and cheese hoops found in over half of the caves used to
ripen the tainted cheese. Thus the basic problem associated with soft cheese manufacture
is to prevent postprocessing contamination by eliminating L. monocytogenes from the
ripening room and particularly the shelves on which such cheese must be ripened. Consid-
ering the ability of L. monocytogenes to grow very rapidly both inside and on the surface
of Brie, Camembert, brick, Limburger, and other similar cheeses during ripening, manu-
facturers of such products should test a portion of each lot for listeriae before releasing
the product for sale.
In addition to these concerns, several studies have demonstrated that L. monocyto-
genes can survive well beyond 60 days in brick, Cheddar, and other varieties of cheese
that were prepared from pasteurized milk inoculated with the pathogen. Certain cheeses,
primarily hard and semihard varieties, can be manufactured from raw milk in the United
States and elsewhere if the finished product is aged a minimum of 60 days at or above
1.7"C (35F) to eliminate pathogenic microorganisms. However, since experimental evi-
dence has indicated that this process is inadequate to free contaminated cheese from viable
cells of L. monocytogenes, cheesemakers should consider preparing cheese from pasteur-
ized milk whenever possible.
Meat Industry
Since Listeria spp., including L. rnonocytogenes, are virtually endemic to slaughterhouse
environments, meat processors are faced with an almost impossible challenge of producing
Listeria-free raw meats. Direct application of lactic and/or acetic acid to animal carcasses
is one of the few economically feasible means by which meat processors can effectively
reduce populations of listeriae and other surface contaminants, including common spoilage
organisms [ 16,28,62]. Nevertheless, although adoption of this procedure and following the
general guidelines for controlling listeriae in food-processing establishments will benefit
slaughterhouse operators, it appears unlikely that rigid enforcement of even the most strin-
gent slaughter, dressing, cleaning, and sanitizing procedures will completely eliminate L.
monocytogenes from wholesale and retail cuts of raw beef, pork, and lamb. Therefore,
consumers of such products need to understand the potential health hazards associated
with consumption of less than thoroughly cooked meats and also must follow appropriate
hygienic practices in the kitchen to prevent the spread of listeriae from raw meats to ready-
to-eat foods.
Firms producing processed meat products must assume that all incoming raw meat
Listeria in Food-Processing Facilities 693
cooked using steam or hot water and then hung for smoking. To obtain skinless frankfurt-
ers, the artificial casing must be mechanically peeled from the congealed meat mixture.
Although prompt attention to cleaning, sanitizing, and cross-contamination problems is
required during all stages of frankfurter production, the product is particularly vulnerable
to contamination with listeriae and other microorganisms during the peeling process. It
is imperative to keep the area around peeling machines as dry and as free from meat
scraps and juices as possible. Peeling machine operators also need to change protective
garments and gloves frequently. Hoods on peeler machines have been cited as a source
of listeriae and should therefore be eliminated if at all possible.
Manufacturing practices also should be reviewed to ensure that losses from floor
contamination and reworked product are minimized. Although unpeeled frankfurters that
touch the floor or other unclean surfaces can be reworked (i.e., washed and peeled after
all other frankfurters have been peeled), any peeled frankfurters that come in contact with
the floor or other unclean surfaces should be destroyed. This latter recommendation is
supported by data indicating that L. monocytogenes is difficult to destroy on the surface
of frankfurters during cooking without making the product organoleptically unacceptable
[ 131. In addition to these concerns, brine chillers also have been cited as a potential source
of listeriae, thus leading to contamination of casings and product surfaces. Finally, all
packaging and heat-shrinkmg equipment should be cleaned and sanitized daily to avoid
spreading contaminants from steam and water to packaging lines.
Luncheon Meats
Concerns regarding control of listeriae and other contaminants in luncheon meats are
generally similar to those just discussed for frankfurters and other link products. However,
in addition, slicing equipment should be kept dry and free of scraps and juices that may
serve as potential nutrients for microbial contaminants, including listeriae.
Poultry Industry
Potential sources of listeriae contamination during processing of raw poultry are in many
ways similar to those just discussed for the meat industry. Since a substantial percentage
of birds harbor Listeria spp. (including L. monocytogenes) and Salmonella in their intesti-
nal tract, enforcement of proper clean-up (i.e., elimination of water, condensate, and waste)
and cleaning and sanitizing programs will likely decrease the incidence of contamination
but will never completely eliminate these pathogens from raw poultry-processing facilities
or the raw product.
Most modern poultry-processing facilities are continuous line operations in which
incoming birds are shackled, electrically stunned, bled, scalded to facilitate feather re-
moval, plucked of feathers, eviscerated, inspected, washed, chilled, dried, and packaged
for sale. Processing steps during which L. rnonocytogenes, Salmonella spp., and other
pathogens are most likely to contaminate the product include scalding, defeathering, evis-
ceration, and chilling [63,64]. In 1988, USDA officials proposed processing changes that
may be helpful in decreasing the incidence of Salmonella (and presumably Listeria) in
raw poultry [ 191. These changes included (a) segregating and processing pathogen-infected
flocks at different times from noninfected flocks; (b) examining the potential benefits of
adding bactericidal concentrations of organic acids to chill water tanks; (c) experimenta-
tion with different scalding methods (e.g., hot water sprays, steam scalders, or scald addi-
tives); (d) routine sanitizing of all equipment and utensils with hot water or bactericidal
Listeria in Food-Processing Facilities 695
agents; (e) reemphasis of employee hygiene programs with routine handwashing and sani-
tizing required by all evisceration line workers; (f) elimination of off-line processing; and
(g) installation of equipment designed automatically to transfer carcasses from the picking
line to the evisceration line. Additional work is needed to streamline further processing
of poultry carcasses and minimize cross contamination during their processing.
With increasing consumption of poultry both in and outside the home, persons pre-
paring these products must take special precautions to prevent the spread of L. monocyto-
genes, Salmonella spp., and other foodborne pathogens from raw poultry to other products
(e.g., fruits and vegetables) that are frequently consumed without heating. The common
practice of washing and rinsing raw poultry before coohng has been questioned, since
this step fails to reduce microbial populations markedly on poultry skin and also leads to
increased contamination of kitchen sinks, faucets, and other food preparation areas [79].
Since all foodborne pathogens commonly associated with raw poultry (including L. mono-
cytogenes) are readily susceptible to heat, thorough cooking appears to be the best means
of assuring that such products are free of hazardous microorganisms.
An Oklahoma breast cancer patient contracted listeriosis in December of 1988 after
consuming Listeria-contaminated turkey frankfurters. Thus producers of processed poultry
products (e.g., turkey and chicken frankfurters and rolls) need to take precautions similar
to those previously described for the manufacture of roast beef, corned beef, frankfurters,
link sausage, and luncheon meats with special attention being given to the cleanliness of
rebagging operations and sausage peelers.
Egg Industry
As stated earlier, the contents of intact whole eggs are normally sterile unless the laying
hen infects the yolk with Salmonella enteritidis. Foodborne pathogens, including L. mono-
cytogenes and S. enteritidis, have frequently been isolated from commercially broken,
raw liquid whole egg, with contamination most likely resulting from the presence of the
organisms in the manufacturing environment or on eggshells. Although pasteurization as
required for commercially broken, raw liquid whole egg is likely sufficient to eliminate
normally encountered populations of L. monocytogenes and salmonellae in raw liquid egg,
all egg-breaking operations need to be well isolated from pasteurization and filling and
packaging areas to minimize recontamination of finished product. Since L. monocytogenes
and other foodborne pathogens probably enter egg-processing facilities as eggshell con-
taminants, egg receiving and washing sections of the factory also should be segregated
from other processing areas. Considering the potential for postpasteurization contamina-
tion, many of the previously described guidelines for cleaning and sanitizing dairy facto-
ries also appear to be applicable to manufacturers of pasteurized liquid egg products.
importers need to evaluate the kinds of hazards that could affect their products, institute
controls to keep these hazards from occurring, or significantly to minimize their occur-
rence, monitor the performance of those controls, and maintain records of this monitoring
as a matter of routine practice.
Limiting postharvest contamination of freshly caught fish and seafood is the first
step toward producing a safe, high-quality endproduct. Adherence to good sanitation and
hygienic practices aboard fishing vessels is imperative. Contact between freshly caught
seafood and waterfowl such as pelicans and seagulls should be minimized, because these
birds are intestinal carriers of L. monocytogenes and other foodborne pathogens. All sea-
food should be either frozen or refrigerated immediately after harvest to stop or retard
growth of microbial contaminants, including spoilage organisms.
Two observations, namely, (a) the routine recovery of healthy rather than thermally
or otherwise injured listeriae from processed seafood and (b) the discovery of L. monocyto-
genes in the manufacturing environment of all American factories that have been involved
in Listeria-related recalls, indicate that this pathogen enters the product primarily after
processing through improper handling. Inadequate separation between raw and finished
product resulting from faulty factory design and indifferent attitudes of employees toward
proper sanitation have been most frequently cited as factors that promote postprocessing
contamination.
The general guidelines that were discussed previously regarding factory design, pro-
cessing environment, proper cleaning and sanitizing, employee traffic patterns, and person-
nel cleanliness also are valid for the seafood industry. In addition to these recommenda-
tions, seafood processors also are urged to (a) eliminate processing waste, pooled water,
and condensate from walls, floors, and ceilings as well as from processing and refrigerated
areas; (b) eliminate the use of high-pressure sprays; (c) reduce airborne contamination;
(d) cover outside dumpsters to decrease problems involving seagulls and other wildlife;
(e) assign specific equipment (i.e., product totes) for use in either raw or cooked product
areas of the factory; and (f) if possible, replace wooden totes with fiber totes, which can
be easily cleaned and sanitized.
Listeria spp., including L. monocytogenes, have been isolated most frequently from
crabmeat and cooked and peeled shrimp (see Table 4 in Chapter 15). This observation is
not surprising if one considers how these products are processed and packaged for the con-
sumer. Processing of Dungeness crab generally begins by immersing and cooking either
sections of or the entire crab in boiling water for approximately 7-9 or 17-20 mins, re-
spectively. Although current information indicates that such a heat treatment is sufficient
to destroy listeriae [43], underprocessing may lead to survivors. After cooking, the crab
is cooled in a water bath and either picked immediately or iced and refrigerated in a
walk-in cooler until the meat can be hand picked from the shell. Extensive handling of the
product by workers during picking, subsequent inspection, and packaging affords many
opportunities for postprocessing contamination. Although lactic acid dips appear to be
somewhat useful in reducing populations of L. monocytogenes and other microorganisms on
the surface of crabmeat as well as fresh and frozen shrimp, such treatments will not com-
pletely eliminate listeriae from the finished product [43]. Therefore, strict adherence to
GMPs, which include proper employee hygiene and cleaning and sanitizing of picking equip-
ment, must be observed in and among picking areas to avoid negating the benefits of cooking.
Unfortunately, crab processing varies widely with the species of crab-Dungeness,
blue, stone, king, and golden crab. Hence, some of the critical control points discussed
for Dungeness crab are not applicable to other species. For example, blue crabmeat is
Listeria in Food-Processing Facilities 697
typically removed from the animal in the raw state, placed in sealed containers, and then
pasteurized (85"C/ 1 min) to eliminate L. monocytogenes and other microbial pathogens.
Since pasteurization of blue crabmeat becomes a critical control point in processing, it
may be prudent to certify and/or license crabmeat pasteurization operators or their supervi-
sors, as has been required for operators of retorts in the canning industry for many years.
Problems regarding postprocessing contamination also are encountered during the
production of cooked and peeled shrimp. After shrimp are cooked, those destined for
breading are mechanically peeled and sometimes deveined by splitting and removing the
vein-like intestine. Unfortunately, many mechanical shrimp peelers have design flaws
which necessitate almost continuous movement of the operator between both raw and
cooked sides of the equipment, thus affording ample opportunity for postprocessing con-
tamination. Proper cleaning and sanitizing of the equipment (particularly protective covers
over flumes and gutters) and the surrounding area are essential for producing high-quality
microbiologically safe products.
Even when handled under the best possible conditions, raw seafood such as crab,
shrimp, lobster, clams, oysters, and the myriad of finfish currently available to consumers
probably will never be completely free of L. monocytogenes or other foodborne pathogens.
Considering that many individuals are not ' 'seafood-smart,' ' processors and marketers of
seafood have an obligation to educate the general public and provide consumers with
proper haridling and cooking instructions. Individuals who insist on consuming unpro-
cessed fish (e.g., sushi) and seafood (e.g., oysters) also should be made aware of potential
health problems associated with consumption of such products.
hazardous microorganisms to other foods. Finally, all knives, cutting boards, and other
food-contact surfaces should be thoroughly cleaned and sanitized after use to inactivate
organisms inadvertently introduced into the kitchen environment during preparation of
raw produce.
cal hazards that could cause illness or injury. After much research and evaluation, the
HACCP concept was developed and first presented to the scientific community at the 1971
Conference for Food Protection [74b]. The HACCP concept was first used in the acidified
and low-acid canned food industry and was then adopted by a number of companies during
the 1970s and early 1980s. After an important 1985 National Academy of Sciences publi-
cation strongly recommended the HACCP concept, the food industry expressed consider-
able interest in the application of HACCP.
The National Advisory Committee on Microbiological Criteria for Foods
(NACMCF) was established and embraced the HACCP concept. In 1989, the committee
developed a HACCP document as a guide for maintaining uniformity of the principles
and definitions of terminology [61a]. Since then, the NACMCF has made several refine-
ments and improvements in the HACCP concept and published revisions in 1992 [61b]
and 1997. The 1997 document, entitled HACCP Principles and Guidelines [61c], contains
many additions and includes a section on prerequisite programs. Prerequisite programs
are essential to the successful development and implementation of a HACCP plan [70b]
and form the foundation upon which a HACCP plan is built. Many of the prerequisite
programs are based on the current GMPs in the Code of Federal Regulations [43a] and
in the Codex Alimentarius General Principles of Food Hygiene [53b] for foods intended
for international trade. In addition to specific items in the GMPs, prerequisite programs
can include other activities such as ingredient specifications, supplier approval programs,
ingredient-to-product traceability, and consumer complaint management programs. A
summary of prerequisite program activities is presented in Table 19 [70b].
Prerequisite programs are not part of the formal HACCP system and are established
and maintained separately. There are some circumstances where the existence of a prereq-
uisite program does not preclude the use of specific activities with a HACCP system [70b].
For example, although sanitation procedures are normally part of a prerequisite program,
some manufacturers manage selected sanitation procedures as critical control points
(CCPs) in their HACCP systems. This has been done frequently in the meat and dairy
industries where sanitation procedures for meat slicers, ice cream fillers, and other pieces
of equipment were established as CCPs to help prevent recontamination of processed
products by L. rnonocytogenes [70b].
The existence and effectiveness of prerequisite programs should be assessed during
the design and implementation of each HACCP plan. Well-developed and consistently
performed prerequisite programs can simplify the HACCP plan, so it is imperative that
all food processors establish, document, and maintain effective prerequisite programs to
support their HACCP plans [70b].
HACCP is a management system that is designed for use in all segments of the
food industry from production agriculture to consumption of the finished product. The
HACCP approach for controlling biological hazards in food is based on seven principles
[61c]:
TABLE
19 Summary of Prerequisite Program Activities
-~
Facilities
Adjacent properties
Building exterior
Building interior
Traffic flow patterns
Ventilation
Waste disposal facilities
Sanitary handwashing facilities
Water, ice, culinary steam
Lighting
Raw Materials Controls
Specifications
Supplier approval
Receipt and storage
Temperature control
Testing procedures
Sanitation
Master schedules
Pest control program
Environmental surveillance activities
Chemical control programs
Training
Personal safety
GMPs
HACCP
Production Equipment
Sanitary design and installation
Cleaning and sanitation
Preventive maintenance
Calibration of equipment
Production Controls
Product zone controls
Foreign material control
Metal protection program
Allergen control
Glass control
Storage and Distribution
Temperature control
Transport vehicle cleaning and inspection
Product Controls
Labeling
Product traceability
Customer and consumer complaint investigations
humidity, moisture level, water activity (aw), pH, titratable acidity, salt concentration,
available chlorine, viscosity, or preservatives. Critical limits must be scientifically based
and may be obtained from regulatory standards and guidelines, the scientific literature,
experimental results, and experts.
Principle 4: Establish Monitoring Procedures
Monitoring is a planned sequence of observations or measurements to assess whether a
CCP is under control and to produce an accurate record for future use in verification.
Monitoring facilitates the tracking of an operation and is used to keep the process in
control. Monitoring is also used to determine when there is loss of control and a deviation
occurs at a CCP (i.e., exceeding or not meeting a critical limit). Monitoring procedures
must be effective to determine deviations and then corrective actions must be taken. Most
monitoring procedures need to be rapid and often include visual observations and measure-
ment of temperature, time, pH, and moisture level. Microbial tests are seldom effective
for monitoring owing to their time-consuming nature and problems with assuring detection
of contaminants.
Principle 5: Establish Corrective Actions
When there is a deviation from an established critical limit, corrective actions are neces-
sary. Through the establishment of corrective actions, foods that may be hazardous are
prevented from reaching consumers. When there is a deviation from critical limits, correc-
tive actions are needed to:
Determine and correct the cause of noncompliance.
Determine the disposition of noncompliant product.
Record the corrective actions that are taken.
Specific corrective actions should be developed for each CCP and included in the HACCP
plan.
Principle 6: Establish Verification Procedures
Verification determines the validity of the HACCP plan and is used in evaluating whether
the facilitys HACCP system is functioning according to the HACCP plan. An effective
HACCP system requires little endproduct testing, since sufficient validated safeguards are
built in early in the process. Firms should rely on frequent reviews of their HACCP plan,
verification that the plan is being correctly followed, and review of CCP monitoring and
corrective action records.
Another important aspect of verification is the initial validation of the HACCP plan
to determine that the plan is scientifically and technically sound, that all hazards have
been properly identified, and that if the HACCP plan is properly implemented, these haz-
ards will be effectively controlled. Subsequent validations are performed and documented
by the HACCP team or independent expert as needed. Validations are conducted when
there is an unexplained system failure, when a significant product, process, or packaging
change occurs, or when new hazards are recognized.
Principle 7: Establish Record-Keeping and
Documentation Procedures
The establishment of an effective record-keeping system is an integral part of a HACCP
system. Records are the only reference available to trace the production history of a fin-
Listeria in Food-Processing Facilities 703
ished product. If questions arise concerning the safety of a product, a review of records
may be the only way to prove that the product was prepared and handled in a safe manner
in accordance with the company's HACCP plan [51a].
A well-developed and implemented HACCP plan built on a strong foundation of
GMPs and prerequisite programs can reduce the level of L. monocytogenes in food-
processing facilities and in cooked, ready-to-eat foods.
Many varieties of Listeria-contaminated cheese also have been identified since 1985,
with contamination most frequently being reported in surface-ripened cheeses. Since L.
monocytogenes can grow rapidly in Brie and Camembert cheese during the late stages of
ripening, a two-class attribute sampling program should be considered if such cheeses are
destined for consumption by pregnant women, immunocompromised adults, or the elderly.
However, since no sampling program can ensure that such products are completely free
of L. monocytogenes, public health interests are far better served by application of HACCP
principles during cheese manufacture and ripening. Although the ICMSF recognized that
raw vegetables also may become contaminated with L. monocytogenes, routine testing of
raw vegetables is unlikely to markedly reduce the risk of contracting listeriosis. Hence,
consumers of raw vegetables are urged to wash all such products vigorously before con-
sumption.
Endproduct-sampling programs are not the answer to protecting consumers from
listeriosis or other types of foodborne illness. However, microbiological sampling is
recommended by many regulatory agencies and the World Health Organization as part
of the HACCP approach to prevent opportunities for contamination by, survival, and
growth of L. monocytogenes as well as other microbial pathogens in raw materials,
factory environments, and food products during manufacture, storage, distribution, sale,
and use.
REFERENCES
1. Adams, C.E. 1990. Use of HACCP in meat and poultry inspection. Food Technol. 44(5):
169- 170.
2. American Meat Institute. 1987. Interim guideline: microbial control during production of
ready -to-eat meat products. Controlling the incidence of Listeria monocytogenes. American
Meat Institute, Washington, DC.
3. Anonymous. 197 I . Workshop 2, Prevention of contamination of commercially processed
foods. In Proceedings of the 1971 National Conference on Food Protection, U.S. Government
Printing Office, Washington, DC, p. 56.
4. Anonymous. 1986. Food and Drug Administration dairy product safety initiatives-Prelimi-
nary status report. FDA Center for Food Safety and Applied Nutrition-Milk Safety Branch.
Washington, DC, September 22.
5. Anonymous. 1987. FDA continues to find Listeria during dairy plant inspections. Food
Chem. News 29( 1):47-48.
6. Anonymous. 1987. FDA convinced dairy industry can avoid Listeria contamination. Food
Chem. News 29(39):3-4.
7. Anonymous. 1987. FSIS to give firms 5 days for clean-up before resampling for Listeria.
Food Chem. News 29(32):7-9.
8. Anonymous. 1987. Ice cream industry seeks parity with meat industry on Listeria policy.
Food Chem. News 29(23): 15.
9. Anonymous. 1987. Meat industry research shows Listeria widespread, control difficult. Food
Chem. News 29( 17):27-29.
10. Anonymous. 1988. FDA regional workshops to discuss microbial concerns in seafoods. Food
Chem. News 30(43):27-3 1.
706 Gravani
11. Anonymous. 1988. Hot dogs, shrimp, crab targeted for microbiological criteria. Food Chem.
News 30(6):43-45.
12. Anonymous. 1988. International Dairy Federation: Group E64-Detection of Listeria mono-
cytogenes-sampling plans for Listeria monocytogenes in foods, Feb. 9. IDF, Brussels.
13. Anonymous. 1988. Listeria destruction in cooked meat products ineffective: Hormel. Food
Chem. News 30( 15):32-34.
14. Anonymous. 1988. Micro criteria for crabmeat, shrimp would have 3-class attributes. Food
Chem. News 30(24):25-27.
15. Anonymous. 1988. Meat industry workshop warned about Listeria threat. Food Chem. News
29(47):54-56.
16. Anonymous. 1988. Meat scientists exchange views in Wyoming. National Provisioner
199(11):6-10.
17. Anonymous. 1988. Recommandations pour la lutte contre la contamination dans les laiteries.
Rev. Lait. Franc. 47357-62.
18. Anonymous. 1988. Recommended guidelines for controlling environmental contamination
in dairy plants. Dairy Food Sanit. 852-56.
19. Anonymous. 1988. USDA to check chicken process changes to lower contamination levels.
Food Chem. News 29(49):34-35.
20. Anonymous. 1989. Appropriations committee tells USDA to draft fish inspection plan. Food
Chem. News 3 1(22):45-46.
21. Anonymous. 1989. Controlling Listeria-the Danish solution. Dairy Ind. Intern. 54(5):3 1-
32, 35.
22. Anonymous. 1989. HACCP programs are in new draft for micro criteria meeting. Food
Chem. News 30(47):53-55.
23. Anonymous. 1989. HACCP working definition assignment taken on by micro subgroup.
Food Chem. News 30(49):55-57.
24. Anonymous. 1989. Micro committee approves HACCP scheme: First major document. Food
Chem. News 3 1(40):45-47.
25. Anonymous. 1989. Monitoring of changes leading to listeriosis problem urged. Food Chem.
News 3 l(20): 13-17.
26. Anonymous. 1989. NFI board votes to seek HACCP-type seafood inspection legislation.
Food Chem. News 3 1(9):34-35.
27. Anonymous. 1989. Three agencies woo Congress on fish inspection. Food Chem. News
31(29):53-54.
28. Anonymous. 1990. Changes in Listeria regulatory strategy recommended by AMI. Food
Chem. News 32(2):58-59.
29. Anonymous. 1990. Fish inspection bill by end of year predicted by NFIs Weddig. Food
Chem. News 3 1(48):50-5 1.
30. Anonymous. 1990. USDA monitoring finds Listeria in ready-to-eat products at 78 plants.
Food Chem. News 32(7):7 1-73.
30a. Australian Dairy Authorities Standards Committee. 1994. Australian Manual for Control of
Listeria in the Dairy Industry, pp. 1-35.
31. Ball, H.R., M. Hamid-Samimi, P.M. Foegeding, and K.R. Swartzel. 1987. Functionability
and microbial stability of ultrapasteurized, aseptically packaged refrigerated whole egg. J.
Food Sci. 52:1212-1218.
32. Barnier, E., J.P. Vincent, and M. Catteau. 1988. Listeria et environnement industriel. Sci.
Aliment. 8:239-242.
32a. Bauman, H.E. 1992. In: M.D. Pierson and D.A. Corlett, eds. Introduction to HACCP. In
HACCP: Principles and Applications. New York: Van Nostrand Reinhold, pp. 1-5.
33. Best, M., M.E. Kennedy, and F. Coates. 1990. Efficacy of a variety of disinfectants against
Listeria spp. Appl. Environ. Microbiol. 56:377-380.
34. Boyle, D.L., J.N. Sofos, and G.R. Schmidt. 1990. Growth of Listeria monocytogenes inocu-
lated in waste fluids collected from a slaughterhouse. J. Food Prot. 53:102-104, 118.
Listeria in Food-Processing Facilities 707
35. Boyle, D.L., G.R. Schmidt, and J.N. Sofos. 1990. Growth of Listeria monocytogenes inocu-
lated in waste-fluids from clean-up of a meat grinder. J. Food Sci. 55:277-278.
36. Bradley, R.L., Jr. 1986. The hysteria of Listeria. Dairy Field 169(11):37, 57.
37. Breer, C. 1988. Occurrence of Listeria spp. in different foods. WHO Working Group on
Foodborne Listeriosis, Geneva, Switzerland, Feb. 15- 19.
38. Charlton, B.R., H. Kinde, and L.H. Jensen. 1990. Environmental survey for Listeria species
in California milk processing plants. J. Food Prot. 43: 198-201.
39. Cox, L.J. 1988. Listeria monocytogenes-a European viewpoint. General Assembly of
IOCCC, Hershey, PA, April 28-30.
40. Cox, L.J. 1988. Prevention of foodborne listeriosis-the role of the food processing industry.
WHO Informal Working Group on Foodborne Listeriosis, Geneva, Switzerland, February
15-19.
41. Cox, L.J., T. Kieiss, J.L. Cordier, C. Cordellana, P. Konkel, C. Pedrazzini, R. Beumer, and
A. Siebenga. 1989. Listeria spp. in food processing, non-food and domestic environments.
Food Microbiol. 6:49-6 1.
41a. Destro, M.T., M.F.F. Leitao, and J.M. Farber. 1996. Use of molecular typing methods to trace
the dissemination of Listeria monocytogenes in a shrimp processing plant. Appl. Environ.
Microbiol. 62:705-7 1 1.
41b. Doyle M.P. 1988. Effect of environmental and processing conditions on Listeria monocyto-
genes. Food Technol. 42: 169- 171.
41c. Eckner, K.F. 1990. Biofilms and food sanitation. Silliker Tech. Bull., SCOPE 5 : 1-4.
41d. Eklund, M.E., F.T. Poysky, R.N. Paranjpye, L.C. Lashbrook, M.E. Peterson, and G.A. Pelroy .
1995. Incidence and sources of Listeria monocytogenes in cold-smoked fishery products and
processing plants. J. Food Prot. 58502-508.
42. Facinelli, B., P.E. Varaldo, M. Toni, C. Casolari, and V. Fabio. 1989. Ignorance about Liste-
ria. Hr. Med. J. 299:738.
42a. Farber, J.M., and J. Harwig. 1996. The Canadian position on Listeria monocytogenes in
ready-to-eat Foods. Food Control. 7(4/5):253-258.
42b. Flickinger, B. 1996. Plant sanitation comes to light: evaluation of ATP-bioluminescence sys-
tems for hygiene monitoring. Food Quality March:22-36.
42c. Flickinger, B. 1997. Light up your plant, part 11: into the laboratory. Food Quality June/
July:20-22.
42d. Flowers, R., L. Milo, E. Myers, and M.S. Curiale. 1997. An evaluation of five ATP biolumi-
nescence systems. Food Quality June/July:23-33
43. Food and Drug Administration. 1988. Proceedings of the National Meeting on Cooked/
Processed Seafood, Food and Drug Administration, Center for Food Safety and Applied
Nutrition, Washington, DC, December 16.
43a. Food and Drug Administration. 1997. Current Good Manufacturing Practices in Manufactur-
ing, Packing or Holding Human Food. Code of Federal Regulations. No. 21, Part 110. U.S.
Government Printing Office, Washington, DC.
44. Gabis, D.A., R.S. Flowers, D. Evanson, and R.E. Faust. 1989. A survey of 18 dry dairy
product processing plant environments for Salmonella, Listeria and Yersinia. J. Food Prot.
52: 122- 124.
45. Garrett, S.E., and M. Hudak-Roos. 1990. Use of HACCP for seafood surveillance and certi-
fication. Food Technol. 44(5): 159-165.
46. Genigeorgis, C.A., D. Dutulescu, and J.F. Garayzabal. 1989. Prevalence of Listeria spp.
in poultry meat at the supermarket and slaughterhouse level. J. Food Prot. 52:618-624,
630.
47. Genigeorgis, C.A., P. Oanca, and D. Dutulescu. 1990. Prevalence of Listeria spp. in turkey
meat at the supermarket and slaughterhouse level. J. Food Prot. 53:282-288.
48. Gilbert, R.J. 1990. Personal communication.
49. Goff, H.D. 1988. Hazard analysis and critical control point identification in ice cream plants.
Dairy Food Sanit. 8: 131- 135.
708 Gravani
50. HTST Pasteurizer Operation Manual. 1987. Oregon Association of Milk, Food and Environ-
mental Sanitarians, Oregon State University, Corvallis, OR.
51. Hudson, W.R., and G.C. Mead. 1989. Listeria contamination at a poultry processing plant.
Lett. Appl. Microbiol. 9:2 1 1-2 14.
51a. Humm, B.J., K.E. Stevenson, and J.H. Humber. 1995. Record Keeping and Verification Pro-
cedures. In: K.E. Stevson and D.T. Bernard, eds. HACCP-Establishing Hazard Analysis Criti-
cal Control Point Programs: A Workshop Manual. 2nd ed. Food Processors Institute. Wash-
ington, DC, 10.1- 10.10.
52. International Commission on Microbiological Specifications for Foods. 1986. Microorgan-
isms in Foods. 2. Sampling for Microbiological Analysis: Principles and Specific Applica-
tions, 2nd ed., University of Toronto Press, Toronto.
53. Jacobs, M. 1988. Personal communication.
53a. Jacquet, C., J. Rocourt, and A. Reynard. 1993. Study of Listeria monocytogenes in a dairy
plant and characterizations of strains isolated. Int. J. Food Microbiol. 20: 13-22.
53b. Joint FAO/WHO Codex Alementarius Commission, Supplement to Volume IB, 2nd ed.
1997. General Principles of Food Hygiene. CAC/RCP 1-1969, REV. 3, pp. 1-26.
54. Kang, Y.-J., and J.F. Frank. 1989. Biological aerosols: A review of airborne contamination
and its measurement in dairy processing facilities. J. Food Prot. 5 2 5 12-524.
55. Kang, Y.-J., and J.F. Frank. 1990. Characteristics of biological aerosols in dairy processing
plants. J. Dairy Sci. 73:621-626.
56. Klausner, R., and C.W. Donnelly. 1989. Personal communication.
57. Kozak, J.J. 1986. FDAs dairy program initiatives. Dairy Food Sanit. 6: 184- 185.
58. Lacey, R.W., and K.G. Kerr. 1989. Listeriosis-the need for legislation. Lett. Appl. Micro-
biol. 8:121-122.
59. Lecos, C. 1986. Of microbes and milk: probing Americas worst Salmonella outbreak. FDA
Consumer 20( I): 18-2 1.
60. Llabris, C.M., and B.E. Rose. 1989. Antibacterial properties of retail sponges. J. Food Prot.
52:49-50, 54.
61. Lopes, J.A. 1986. Evaluation of dairy and food plant sanitizers against Salmonella typhimu-
rium and Listeria monocytogenes. J. Dairy Sci. 69:279 1-2796.
61a. National Advisory Committee on Microbiological Criteria for Foods. 1989. HACCP Princi-
ples for Food Production.
61b. National Advisory Committee on Microbiological Criteria for Foods. 1992. HACCP System.
61c. National Advisory Committee on Microbiological Criteria for Foods. 1997. HACCP Princi-
ples and Application Guidelines.
61d. National Advisory Committee on Microbiological Criteria for Foods (NACMCF). 1991. Lis-
teria rnonocytogenes: recommendations by the National Advisory Committee on Microbio-
logical Criteria for Foods. Intern. J. Food Microbiol. 14:185-246.
61e. Nelson, J.H. 1990. Where are Listeria likely to be found in dairy plants? Dairy Food Environ.
Sanit. 10:344-354.
62. Netten, P. van, and D.A.A. Mossel. 1981. The ecological consequences of decontaminating
raw meat surfaces with lactic acid. Arch. Lebensmittelhyg. 3 1 : 190- 191.
63. Notermans, S., R.J. Terbijhe, and M. van Schothorst. 1980. Removing faecal contamination
of broilers by spray-cleaning during evisceration. Br. Poultry Sci. 2 I : 1 15- 12 1.
64. Notermans, S., J. Dufrenne, and W.J. van Leeuwen. 1982. Contamination of broiler chickens
by Staphylococcus aureus during processing; incidence and origin. J. Appl. Bacteriol. 52:
275-280.
65. Orth, R., and H. Mrozek. 1989. Is the control of Listeriu, Campylobacter, and Yersinia a
disinfection problem? Fleischwirtschaft 69: 1575- 1576.
66. Petran, R., and E.A. Zottola. 1988. Survival of Listeria monocytogenes in simulated milk
cooling systems. J. Food Prot. 5 I : 172- 175.
Listeria in Food-Processing Facilities 709
67. Prentice, G.A. 1989. Living with Listeria. J. Soc. Dairy Technol. 4255-58.
67a. Pritchard, T.J., C.M. Beliveau, K.J. Flanders, and C.W. Donnelly. 1994. Increased incidence
of Listeria species in dairy processing plants having adjacent farm facilities. J. Food Prot.
57:770-775.
68. Radmore, K., W.H. Holzapfel, and H. Luck. 1988. Proposed guidelines for maximum accept-
able air-borne microorganism levels in dairy processing and packaging plants. Intern. J. Food
Microbiol. 6:91-95.
69. Rank, R.J. 1989. Liquid eggs deviating from the standard of identity; temporary permit for
rnarket testing. Fed. Reg. 54: 1794- 1795.
69a. Ryser, E.T. 1998. Personal communication.
69b. Scott, V.N. 1998. Personal communication.
70. Shapton, N. 1988. Hazard analysis applied to control of pathogens in the dairy industry. J.
Soc. Dairy Technol. 41 :62-63.
70a. Skinner, R. 1996. Listeria: UK Governments approach. Food Control. 7(4/5):245-247.
70b. Sperber, W.H., K.E. Stevenson, D.T. Bernard, K.E. Deibel, L.J. Moberg, L.R. Hontz, and
V.N. Scott. 1998. The role of prerequisite programs in managing a HACCP system. J. Dairy
Food Environ. Sanit. 18:4 18-423.
71. Spurlock, A.T., E.A. Zottola, and R.K.L. Petran. 1989. The survival of Listeria monocyto-
genes in aerosols. J. Food Prot. 52:751 (Abstr.).
72. Stanfield, J.T., C.R. Wilson, W.H. Andrews, and G.J. Jackson. 1987. Potential role of refrig-
erated milk packaging in the transmission of listeriosis and salmonellosis. J. Food Prot. 50:
730-732.
73. Surak. J.G., and S.F. Barefoot. 1987. Control of Listeria in the dairy plant. Vet. Hum. Toxicol.
29:241-249,
73a. Sutherland, P. and R. Porritt. 1995. Dissemination and ecology of Listeria monocytogenes
in Australian dairy factory environments. Proceedings of the XI1 International Symposium
on Problems of Listeriosis Perth, Australia. pp. 29 1-297.
74. Terplan, G. 1988. Listeria in the dairy industry-situation and problems in the Federal Re-
public of Germany. Foodborne Listeriosis-Proceedings of a Symposium, Wiesbaden, West
Germany. Sept. 7, pp. 52-70.
74a. Tompkin, R.B., L.N. Christiansen, A.B. Shaparis, R.L. Baker, and J.M. Schroeder. 1992.
Control of Listeria monocytogenes in processed meats. Food Australia 44:370-376.
74b. U.S. Department of Health, Education and Welfare. 1972. Proceedings of the 1971 Confer-
ence on Food Protection. U.S. Government Printing Office, Washington, DC.
75. U.S. Food and Drug Administration and Milk Industry Foundation-International Ice Cream
Association. 1988. Recommended guidelines for controlling environmental contamination in
dairy plants. Dairy Food Sanit. 852-56.
75a. U.S. Food and Drug Administration. 1995. Procedures for the safe and sanitary processing
and importing of fish and fishery products. Final Rule 2 1 CFR 123 and 1240. Federal Register
60 CFR, pp. 65095-65202.
76. Venables, L.J. 1989. Listeria rnonocytogenes in dairy products-the Victorian experience.
Food .4ustralia 4 1 :942-943.
77. Wagner, L.R. Van. 1989. FDA takes action to combat seafood contamination. Food Proc.
50(2):8-12.
77a. Walker, R.L., L.H. Jensen, H. Kinde, A.V. Alexander, and L.S. Owens. 199 1. Environmental
survey for Listeria species in frozen milk products plants in California. J. Food Prot. 54:
178- 182.
78. Wimpfeimer, L., N.S. Altman, and J.H. Hotchkiss. 1990. Growth of Listeria rnonocytogenes
Scott A, serotype 4 and competitive spoilage organisms in raw chicken packaged under modi-
fied atmospheres and in air. Intern. J. Food Microbiol. 1 I :205-214.
79. Woodhurn, M. 1989. Myth: wash poultry before cooking. Dairy Food Environ. Sanit. 9:65-66.
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Appendix
Media to Isolate and Cultivate
Listeria monocytogenes
and Lisferia spp.
Fraser broth
Proteose peptone
Tryptone
Lab-Lemco powder
Yeast extract
NaCl
KH2IO,
71 1
7 12 Appendix
Na2HP04 12 g
Esculin 1g
Nalidixic acid 20 mg
Lithium chloride 3g
Acriflavine 25 mg
Ferric ammonium citrate 0.5 g
Distilled H 2 0 1000 ml
L-PALCAMY broth
Special peptone (Oxoid)
Yeast extract
Lab-Lemco powder
Peptonized milk (Oxoid)
NaCl
D-Mannitol
Esculin
Appendix 713
UVM
Proteose peptone 5 g
Tryptone 5g
Lab-Lemco powder 5 g
Yeast extract 5 g
NaCl 20 g
Disodium phosphate-7-hydrate 12 8
Potassium phosphate, monobasic 1.35 g
Esculin 1g
Nalidixic acid 40 mg
Acriflavine - HC1 12 mg
Distilled H 2 0 1000 ml
* Commercially available from Difco Laboratories, Detroit, MI; BBL, Cockeysville, MD; Oxoid Ltd., Basing-
stoke, Hampshire, England; Merck, Darmstadt, Germany.
ALPAMY agar
Columbia blood agar base (Oxoid) 39 g
Lithium chloride 15 g
D-Mannitol 10 g
2-Phenylethanol 2.5 g
Ferric ammonium citrate 0.5 g
Esculin 0.5 g
Acriflavine 10 mg
Phenyl red 80 mg
Egg yolk emulsion (Oxoid) 25 ml
Distilled H 2 0 1000 ml
ARS-modified MMLA
Phenylethanol agar (Difco) 35.5 g
Lithium chloride 0.5 g
Glycine anhydride 10 g
Cycloheximide 0.2 g
Nalidixic acid 50 mg
Appendix 715
Moxalactam 5 mg
Bacitracin 20 mg
Distilled H 2 0 1000 ml
LPM agar"
Phenylethanol agar 35.5 g
Glycine anhydride 10.0 g
Lithium chloride 5.0 g
Moxalactam 20 mg
Distilled H 2 0 1000 ml
McBride Listeria agar
Phenylethanol agar 35.5 g
Glycine 10.0 g
Lithium chloride 0.5 g
Sheep blood 50 ml
Distilled H 2 0 1000 ml
RAPAMY agar
Columbia blood agar base 39 g
D-Mannitol 10 g
2-Phenylethanol 2.5 g
D-Glucose 1g
Ferric ammonium citrate 0.5 g
Esculin 0.5 g
Appendix 717
Nalidixic acid 40 mg
Acriflavine 10 mg
Phenol red 80 mg
Egg yolk emulsion (Oxoid) 25 ml
Distilled H 2 0 1000 ml
Low-fat milk, 371, 372, 374, 391 Mettwutst, 5 18, 520, 535, 539, 555
Low pH Mexican-style cheese, 302,307,308,309,
growth, 148 310, 324, 325, 333, 344, 345, 359,
survival, 150 360, 370, 389, 390,411,412,413,
Lubricants, conveyor chain, 204, 205 4 14, 4 15, 468,484, 566, 639, 658,
Luncheon meats, 334,506,508,513,519, 66 1
521,531,532,536,694,695 Microbacterium thermosphactum, 3
Lung, 522 Microbial rennet, 345, 450, 451, 452
Lupus erythematosus, 337 Microbiological Surveillance Program 370,
Lymph nodes, 522,523 371,373
Lymphocytes, 141, 142 Micrococci, 236
Lys Bleu cheese, 436 Micrococcus spp., 235,459,525
Lysozyme, 167, 176, 178, 182, 183, 193,383, Micro-Gard, 620
447, 455,478,487, 535, 536, 592, Microwave radiation, 583, 584, 585, 586
639,644,645,646,647 Milano salami, 541
Milk, 301, 314
Maasdam cheese, 462,463,367 Minas Frescal cheese, 43 1
Macrolides, 282 Mint, 175
Macrophages, 97, 100, 103, 104, 105, 107, Modified atmosphere (packaging), 187, 188,
113, 114, 115, 117, 141, 142,227, 189,479, 531, 532, 545, 548, 549,
265,384 577, 578, 579, 617, 619, 639, 643,
Malic acid, 158, 469 644
Manchego cheese, 150,385,473 Moist heat, 538, 584, 585, 586
Manouri cheese, 479 Mold-ripened cheeses, 453
MAP kinase phosphatase, 116 Monitoring programs, 506, 508, 566, 567
Margarine, 453 Monitoring sample, 506, 507, 567
Marinated seafoods, 624 Monoacylglycerols, 167, 168, 169
Mascarpone cheese, 434 Monocaprin, 166, 168, 169
Mayonnaise Monocaprylin, 166, 169
cholesterol-freereduced-calorie, 592 Monocin, 281, 282
low calorie, 592 Monoclonal antibody, 27 1,272,274, 327
real, 592 Monoglycerides, 460
reduced calorie, 592 Monolaurin, 166, 167, 168, 169, 193, 551
Meatballs, 549, 550 Monolimolein, 166
Meat industry, 692 Monomyristin, 166
frankfurters and other link products, 693 Monoolein, 165
luncheon meats, 694 Monopalmitin, I66
roast beef, corned beef and other rebagged Monostearin, 166
products, 693 Monterey Jack cheese, 325,412,414, 417,
Meat processing environmental problems, 484,485
664,665,666 Morbier Rippoz cheese, 420,422
Meat processing facilities, 664, 665,666,667 Mortality rate, 304, 317, 322, 329, 343
Meat products, 326, 507, 508,664 Moxalactam, 229,230,233,234,236
Meat salads, 508, 509, 5 17, 5 18 Mozzarella cheese, 150, 4 14, 4 17, 429,434,
Meat slicers, 517 443,445,46 1,462,479,482,484
Meat spreads, 508, 509 Mucor miehei, 450
Menaquinones, 7 Muenster cheese, 4 12,419,436,45 1,484,
Meningitis, 3 485
Mercury compounds, 203 Multilocus enzyme electrophoresis, 282, 290,
Mesophilic starter cultures, 44 1, 462, 474 292,313,318,321,330
Metalloprotease, 100, 106, 107, 108, 110, 291 Multitest assays, commercial, 11
Methyl sulfide, 460 Murein Rydrolase, 102
Methyl trisulfide, 460 Murraya grayi, 7
Index 733