Microbial Forensics
in Food Safety
1
Biological Sciences Department, California Polytechnic State University, San Luis Obispo, CA 93407
ABSTRACT Foodborne diseases represent a significant public
health burden to the United States, considering that they cause
illness in 1 in 6 people annually, which amounts to ∼48 million
people (E. Scallan, R. M. Hoekstra, F. J. Angulo, R. V. Tauxe,
M. A. Widdowson, S. L. Roy, J. L. Jones, and P. M. Griffin,
Emerg Infect Dis 17:7–15, 2011). The average national cost of
illness associated with 30 foodborne pathogens is estimated to
be $55.5 to $93.2 billion based on two cost-of-illness models
(R.L. Scharff, J Food Prot 78:1064–1071, 2015). Predominately,
foodborne illnesses are the result of accidental contamination or
unintentional mishandling of food materials during the farm-to-
table continuum. Nevertheless, principles and methodologies
derived from microbial forensics are applied in foodborne
outbreaks investigation to determine the source of the
pathogen. Drawing from multiple real-life examples and case
studies, this review discusses how the current food industry
practice, demography, and consumer preference are shaping
the landscape of food safety. The approaches to source tracking,
or traceback, are described, with a focus on bacterial pathogens
associated with food-producing animals. Current challenges
and opportunities in microbial forensics in food safety are also
addressed.
CURRENT STATUS OF FOOD SAFETY
Changing Landscape and a Global Issue
Food production practice has changed substantially in
the last few decades in the United States. The number
of small farms has decreased, giving way to larger
operations. For example, according to U.S. Department
of Agriculture (USDA) data, the number of dairy farms
with dairy cows fell from 648,000 operations in 1970
to 75,000 in 2006 (1). While the numbers of farms de-
creased, the size grew, with herd size rising from 19 to
120 cows per farm in those years. Centralization of food
production is becoming a common practice in the United
States for many food commodities. In addition, global
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MARIE YEUNG1
food trade is also changing the landscape of food pro-
duction and distribution patterns. With global income
level on the rise, there is a growing consumer demand
for a variety of food. Facilitated by improved transpor-
tation networks, global food trade is expected to in-
crease. Demand for minimally processed food has also
been trending up. Minimally processed food is often
characterized by high water activity and mild acidic to
neutral pH and is not subjected to high-heat treatment.
This includes ready-to-eat food, fruits, and vegetables.
As a result, a different set of food safety issues emerges:
for example, tracking implicated food ingredients in a
widely distributed network is increasingly difficult, new
foodborne pathogens are discovered from imported
food, and common foodborne pathogens are associated
with new niches of food products. With new issues
and challenges, the regulatory agencies responsible for
protecting our food supply have been working hard to
ensure public health.
Food Safety System and
Programs in the United States
In the United States, 15 federal agencies collectively
administer at least 30 food-related laws related to
food safety (2). As suggested by the cited report, higher
Received: 18 November 2013, Accepted: 16 September 2014,
Published: 12 August 2016
Editors: Raúl J. Cano, California Polytechnic State University,
San Luis Obispo, CA; Gary A. Toranzos, University of Puerto Rico-
Río Piedras, San Juan, Puerto Rico.
Citation: Yeung M. 2016. Microbial forensics in food safety.
Microbiol Spectrum 4(4):EMF-0002-2013. doi:10.1128
/microbiolspec.EMF-0002-2013.
Correspondence: Marie Yeung, pmyeung@calpoly.edu
© 2016 American Society for Microbiology. All rights reserved.
1
Yeung
efficiency may be achieved by more integration among
agencies. Nevertheless, it is obvious that the U.S. gov-
ernment has given food safety a high priority and con-
sequently allocates a significant amount of resources
to tackle the complex food safety issues. Among the
agencies, the Food and Drug Administration (FDA)
oversees the majority of domestic and imported food,
including bottled water and shell eggs. On the other
hand, meat, poultry, and related products, such as egg
products, are under the jurisdiction of the USDA-Food
Safety and Inspection Service (FSIS). Therefore, the FDA
and FSIS are the two primary agencies overseeing U.S.
food safety and regulating the food industry with in-
spection and enforcement. During outbreaks of food-
borne illness, they have the responsibility to determine
where the source is and why it occurs and to take steps to
control the current outbreak and prevent future ones.
They also work closely with the Centers for Disease
Control and Prevention (CDC), state agencies, and local
officials during foodborne outbreak investigations.
These agencies have taken a proactive approach in
controlling and preventing foodborne illnesses. Several
surveillance networks and databases were established to
facilitate active monitoring of foodborne diseases caused
by common etiological agents. The Foodborne Diseases
Active Surveillance Network, or FoodNet, is an active
surveillance program that has been tracking foodborne
diseases in selected states since 1996. PulseNet is another
prime example of a surveillance program. Coordinated
by the CDC, PulseNet is the national molecular sub-
typing network that contains molecular fingerprinting
data of microbial isolates associated with foodborne
illnesses (3). The molecular subtyping data are generated
using pulsed-field gel electrophoresis (PFGE). PulseNet
is particularly invaluable during foodborne investiga-
tions. By looking at the database, epidemiologists and
health officials can identify clusters of illnesses caused
by microbes that share the same fingerprint. With other
data acquired through the investigation, such as the
food consumption record, the outbreak investigators
can more rapidly track the transmission of the patho-
gens and narrow down the source. Having the ability
to identify the implicated food more quickly can help
prevent additional cases and potentially save lives. The
positive impact of PulseNet on tracking diseases is ex-
emplified by a 2011 outbreak associated with Listeria
monocytogenes and cantaloupe, in which a national
warning could be issued days, not months, after the
outbreak was detected. The rapid response is notewor-
thy because it is typically a daunting challenge to detect
an outbreak and to identify the source when foodborne
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illnesses occur in multiple states. The coordinated effort
by the CDC, FDA, and state and local health depart-
ments, aided by the information provided by PulseNet,
likely saved many lives.
Common Etiological Agents
of U.S. Foodborne Illnesses
The molecular subtyping database is extremely valuable
because many foodborne illnesses are caused by micro-
biological agents. Food safety hazards in the United
States are less commonly associated with pesticide or
other chemical contamination, although food recall
due to allergens is on the rise. Using the Foodborne
Outbreak Online Database (FOOD Tool) developed
by the CDC, which contains descriptive summaries
of outbreak data, an estimated 18,211 outbreaks were
reported from 1998 to 2014. Among the ∼11,800
outbreaks that had the etiological agents identified,
only ∼180 were associated with heavy metals, cleaning
agents, pesticides, and other chemicals. The rest of the
outbreaks were associated with viruses, bacteria, para-
sites, and biological toxins. In other countries where
chemical hazard is the predominant factor behind food-
borne illnesses, databases concerning the physical and
chemical properties (e.g., chromatography profiles and
radioisotopes) are necessary to track the source.
It is estimated that 48 million people in the United
States contract foodborne illness annually, resulting in
128,000 hospitalizations and 3,000 fatalities (4). The
public health systems track many pathogens responsible
for these illnesses. Of the illnesses caused by known
foodborne agents, 91% were contributed by norovirus,
nontyphoidal Salmonella, Campylobacter spp., Clos-
tridium perfringens, and Staphylococcus aureus. The
first three pathogens, together with Toxoplasma gondii,
Escherichia coli O157, and Listeria monocytogenes,
contributed to >88% of hospitalization and fatality
cases. Pathogens contributing to other cases include
Bacillus cereus, Brucella spp., Clostridium botulinum,
enterotoxigenic E. coli, Mycobacterium bovis, Shigella
spp., Streptococcus spp., Vibrio cholerae, Vibrio vul-
nificus, Vibrio parahaemolyticus, Yersinia enterocoli-
tica, Cryptosporidium spp., Cyclospora cayetanensis,
Giardia intestinalis, Trichinella spp., astrovirus, hepati-
tis A virus, rotavirus, and sapovirus.
FOOD-PRODUCING ANIMALS AS
IMPORTANT RESERVOIRS OR CARRIERS
Painter et al. (5) found that though most foodborne
illnesses were attributed to plant commodities, most

deaths were associated with land animal commodities.
It is not surprising that many foodborne pathogens are
associated with food-producing animals, as they are
the natural inhabitants in or on the animal hosts, and
some require the animal host to reproduce. These ani-
mal-associated foodborne pathogens do not necessarily
cause disease in animals. For instance, cattle are known
asymptomatic carriers of E. coli O157. While the mi-
crobes do not harm the animal hosts, they may induce
severe and long-lasting illness in infected human in-
dividuals upon consuming the tainted meat. Meat that
is not effectively treated by a “kill step” or that is con-
taminated postprocessing has the highest risk. For
example, raw and undercooked pork is often the impli-
cated food behind outbreaks associated with Yersinia
enterocolitica and Trichinella spp., as pigs serve as a re-
servoir or carrier for these microbes. Salmonella spp. and
Campylobacter are also prevalent in food-producing ani-
mals, especially poultry, and therefore are often the cul-
prits behind implicated chicken meat and egg products.
Some animals harbor foodborne pathogens as a result
of their interaction with the environment. Microbes
ubiquitous in the farm environment, such as L. mono-
cytogenes, Clostridium botulinum, and Bacillus cereus,
can be easily transferred to the exterior surface of ani-
mals, from which it can be subsequently transmitted
to the food supply. Finished food products can be con-
taminated by these pathogens in part when animal car-
casses are not effectively cleansed. Similarly, raw milk
produced from healthy cows can harbor high microbial
counts from dirty udders, which frequently come in con-
tact with bedding materials, soil, and fecal matter.
Fecal matter, which contains a plethora of enteric
microbes, constitutes a major source of contamination.
Notably, members of the family Enterobacteriaceae,
such as Salmonella and virulent types of E. coli, make
up a significant portion of the microbial population in
fecal matter. Highly contaminated raw meat can dis-
tribute these microbes to the entire lot of food produc-
tion. Thus, a considerable number of outbreaks has been
traced back to animal farms. In some instances, produce
farms can be contaminated with fecal matter shed by
non-food-producing animals wandering on or near the
farms. The following real-life event highlights the im-
portance of keeping our food supply free from fecal
matter.
A multistate outbreak of E. coli O157:H7 occurred in
late 1996. During the early phase of the investigation,
among the 28 confirmed cases, 12 patients—many were
infants or toddlers—developed hemolytic-uremic syn-
drome (6). All patients had consumed Odwalla juice
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Microbial Forensics in Food Safety
within 10 days before illness onset. When the outbreak
was over, 65 illnesses had been reported and a 16-
month-old girl had died. Molecular fingerprint patterns
between clinical and juice isolates were indistinguishable
(7). At that time, apple juice was not subjected to the
pasteurization requirement because it was not thought
to be a common vehicle of foodborne pathogens. Fruit
juice products typically have a low pH and are kept at
cold temperatures. Both of these conditions are known
to deter microbial growth. In the wake of the outbreak,
the processing plant was inspected, but no evidence
of E. coli contamination was found in the facility. The
source of contamination was never definitively found.
Several speculations were made by FDA officials, in-
cluding fecal contamination by orchard workers or by
apples that had fallen on ground topped with cow or
deer feces (8). This outbreak highlights the need to
monitor the presence of fecal microflora in raw materials
and finished products. As with water quality assessment,
it is a mandate or an advisory for many food commod-
ities to test for the presence of fecal bacterial indicators
in food products to ensure safety. However, this ap-
proach generally does not offer critical information in
source tracking.
Recently, an apparent surge of antibiotic-resistant
pathogens has sparked rigorous discussion on the role of
food-producing animals as reservoirs of these patho-
gens. Using Salmonella spp. as an example, some spec-
ulated that infection by multidrug-resistant Salmonella
enterica serovar Typhimurium DT104 in humans is the
result of consumption of animal products (9, 10). This
is conceivable, as it is a common practice for many farms
to administer antibiotics to animals. The presence of
antibiotics in an animal host may impose a selective
pressure facilitating the transfer of antibiotic resistance
genes between microbes residing in the host. As a result,
the viability of antibiotic-resistant enteric pathogens is
enhanced. On the other hand, Mather et al. (11) ana-
lyzed a national collection of both human and animal
S. Typhimurium DT104 isolates by whole-genome se-
quencing (WGS). They concluded that antibiotic resis-
tance genes were largely maintained separately within
animal and human populations, with limited transmis-
sion occurring between them. Further, on a large spatial
and temporal scale, the phylogenetic relationship be-
tween the bacterium and its antibiotic resistance genes
appears to show different patterns for these populations.
However, this study cannot omit the possibility that
antibiotic-resistant pathogens in certain outbreaks were
indeed derived from contaminated animal products.
Once we have the tools to trace the implicated food to a
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Yeung
particular farm, we will have the ability to address this
issue more effectively. If antibiotic-resistant pathogens
are found in animal herds, the industry will need to
modify animal care and management practices.
FOODBORNE OUTBREAK INVESTIGATION
As defined by the CDC, a foodborne disease outbreak is
a “cluster of two or more infections caused by the same
agent (pathogen or toxin) which upon investigation
are linked to the same food.” The scope of outbreaks
can be confined to local regions and within a state, but
many extend to multiple states and nationwide. With a
few exceptions, all foodborne outbreaks are uninten-
tional or accidental. Since there is no motive to inflict
damage, microbial forensics in the context of food safety
is not a bona fide forensic investigation. Nevertheless,
the approach of the investigation, techniques involved,
and data analyses share great similarities with inten-
tional and accidental outbreaks.
Steps in Foodborne Outbreak Investigation
Foodborne outbreak investigation involves several steps.
Since the nature of the investigation is highly dynamic and
collaborative, the steps in the procedure described below
may happen simultaneously instead of sequentially.
In the first step, a cluster of illnesses is reported and
a possible outbreak is detected. The early detection is
often enabled as a result of a public health surveillance
program. The state laboratory may conduct subtyping
tests to determine if the microbial isolates collected from
patients are of the same subtype. In order to include
more subjects in the ongoing investigation to determine
the source, it is important to define what a case is. Case
definition may include the features of the illnesses,
symptoms, time range, geographic region, and the etio-
logical agent, if known. It is possible to have several
case definitions to serve different purposes. To determine
if the illness is a result of consuming a particular food
item(s), the health officials interview ill persons to col-
lect information about what and where they have eaten.
Based on the interview and other additional data, a
hypothesis about the likely source of outbreak is made.
Two main methods are used by the health officials
to test the hypothesis: analytical epidemiological studies
and food testing. Epidemiologists often conduct case-
control studies in larger-scale or multistate outbreaks
in which the attack rate is low. On the other hand,
cohort studies are more applicable to highly localized
outbreaks, such as when all ill persons are associated
with a single event or a food-serving facility. During the
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epidemiological investigation, more interviews are con-
ducted with both ill and well persons to calculate the
strength of the association between illness and food
items. If the suspected food items are available, they are
tested for the presence of microbes and toxins.
Once the probable implicated food is identified, the
next step is to investigate the origins of the food and the
point of contamination. If the outbreak is confined to a
small number of facilities, an environmental assessment
is done to evaluate how the food preparation practice,
sanitation procedure, health status and hygiene practice
of food handlers contribute to the foodborne illnesses.
If the contamination happens along the farm-to-table
continuum, the health officials would attempt to trace
the implicated food to the producers. The U.S. food in-
dustry has played a significant role in developing a
traceability program. Currently, most traceability is at
the “case-level,” which means that the codes on finished
products can be used to trace food products in bulk (12).
The ultimate goal is to trace back to a particular grower.
Some companies have been adopting a new technology
to implement a QR (quick response) code, with the idea
that the consumers can get information about the origins
of the raw materials after scanning the code with their
mobile devices. The consumers can also receive alerts
should the products fall under foodborne outbreak in-
vestigation. Though a very appealing idea, the imple-
mentation of this level of trackback is very challenging
for many food commodities.
The ultimate goals for foodborne outbreak investi-
gations are to control current outbreaks and prevent
further ones. Therefore, as soon as the suspect food
is identified, actions are taken immediately. These in-
clude food recall, closing of the implicated facility,
and public announcement. Depending on the severity of
the outbreaks, health officials sometimes must take ac-
tion based on preliminary results, i.e., without the more
definitive evidence. This may create a huge economic
setback to some food companies and commodities that
are initially tied to the outbreak based on preliminary
data but are later found to be not responsible. On 3 June
2008, the FDA and public health partners issued public
advisories to recommend that consumers in Texas and
New Mexico avoid eating certain types of tomatoes.
The advisory was expanded nationwide on 7 June in the
wake of a multistate outbreak of Salmonella Saintpaul.
This resulted in a widespread public refusal to purchase
tomatoes. A few weeks later, the tomato advisory was
lifted, as the source was instead found to be jalapeño
peppers grown in Mexico (13). Despite the lawful action
by the FDA, the tomato growers suffered significant

losses. Seventeen growers from different states filed a
claim to get compensation for a total of ∼$40 million
and to include the possibility of a class action (14). This
is yet another example illustrating the difficulties and
challenges faced by food safety officials.
MICROBIAL FORENSIC TOOLS IN
FOODBORNE OUTBREAK INVESTIGATION
As described in the above outbreak investigation pro-
cess, the ability to link a microbial isolate in a patient’s
stool to an implicated food or the source partly hinges
on the resolving power of the analytical test. The food
industry has been using traditional methods to isolate,
detect, and identify microbes on a routine basis for
quality assurance and quality control purposes. These
methods include microscopic observation, culturing on
selective and/or differential media, biochemical testing,
immunoassay, and species-specific PCR. These methods
generally are not used to identify microbes at the sub-
species level. In fact, in many cases, it is sufficient to
detect the presence of a particular group of microbes to
meet quality and safety guidelines and regulations. As
an example, the bacteriological standard of the FDA’s
Grade “A” Pasteurized Milk Ordinance specifies pas-
teurized milk not to exceed 20,000 and 10 per ml for
total aerobic bacterial and coliform counts, respectively.
It is not necessary to identify the microbes at the species
or sub-species level to meet this rule.
On the other hand, for the purpose of microbial
forensics or source tracking, a higher level of sensitivity
is required to differentiate among isolates or strains.
Traditional subtyping techniques include serotyping,
ribotyping, biotyping, phage typing, antibiotic suscep-
tibility typing, plasmid profile analysis, and multilocus
enzyme electrophoresis. Some of these techniques are
still commonly used in conventional identification of
major foodborne pathogens. For example, serotypes are
often accompanied in the identification of Salmonella
spp., Vibrio parahaemolyticus, Vibrio cholerae, entero-
hemorrhagic E. coli, Listeria monocytogenes, etc. Epi-
demiological studies suggested that certain serotypes,
such as E. coli O157:H7 and V. parahaemolyticus
O3:K6, are associated with enhanced virulence and
are therefore more frequently implicated in human dis-
ease. However, most of the other traditional subtyping
techniques have become obsolete in foodborne outbreak
investigation due to the lack of sensitivity to distinguish
closely related strains. To this end, appropriate molec-
ular techniques must be adopted to provide the desirable
resolution.
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Microbial Forensics in Food Safety
DNA Polymorphism and Microbial Forensics
Conventional subtyping methods can fall into PCR-
based, restriction fragment length polymorphism (RFLP)-
based, and sequencing-based categories. The basis of
discrimination of these methods depends on DNA poly-
morphism among different isolates. There are several
types of DNA polymorphism, including single nucleotide
polymorphisms (SNPs), insertions and deletions (indels),
variations in length of repeated sequences, and large-scale
genome rearrangements (e.g., inversions).
PCR-based methods include amplified fragment length
polymorphism, random amplification of polymorphic
DNA, arbitrarily primed PCR, and repetitive-element
PCR. Some of these methods combine PCR amplification
and restriction enzyme digestion. The output of these
methods is a pattern reflecting the number and size of
amplicons. Though PCR-based methods have been shown
to be successful in subtyping some foodborne pathogens,
they may be better suited as an initial screening tool due
to inadequate resolving power. To more definitively dis-
criminate different isolates, current standard strategies rely
largely on an RFLP-based approach and DNA sequencing.
Ribotyping and PFGE were among the first molecu-
lar subtyping methods developed based on the prin-
ciple of RFLP. In general, genomic DNA is cut with one
or several restriction enzymes generating fragments of
different sizes. Following electrophoresis, fragments are
separated according to their sizes, yielding a profile re-
sembling a “fingerprint” for each strain. Patterns gen-
erated by ribotyping and PFGE reflect RFLP in rRNA
operons and the whole genome, respectively. Commer-
cially available automated systems were developed for
ribotyping (15), making this method appealing to some
food companies that need subtyping information for
quality assurance purposes in a more rapid and con-
venient manner. For outbreak investigation purposes,
PFGE is considered the gold standard in bacterial sub-
typing by food safety agencies, despite the foreseeable
takeover by WGS, as discussed below. In PFGE, an
infrequently cutting enzyme(s) is employed to gener-
ate a manageable number of fragments. The digitized
PFGE patterns are submitted to PulseNet. CryptoNet,
on the other hand, is the tracking system for a common
foodborne parasite, Cryptosporidium. Instead of PFGE,
CryptoNet is based on PCR of the 18S ribosomal
RNA (rRNA) gene, followed by RFLP. Out of the ∼30
Cryptosporidium species, Cryptosporidium hominis,
C. parvum, and C. meleagridis are subtyped by se-
quencing the GP60 gene.
Sequence determination has been used regularly as a
tool in genotying. For example, sequencing of the entire
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or a partial region of 16S rRNA genes is a common
detection and identification method for food-related
bacteria. Although 16S rRNA gene sequencing is tech-
nically possible to resolve microbes at the subspecies
level, its most common usage is confined to identification
to the genus or species level. On the other hand, se-
quence determination of a partial region of the capsid
gene, such as region D, of norovirus is the basis of
CaliciNet, a database developed by the CDC for track-
ing norovirus implicated in foodborne illnesses. With the
increasing knowledge of microbial genomes and the
advancement of sequencing technology, several newer
sequence-based methods have been developed. They
have shown promise in providing higher resolution,
enhanced interlaboratory agreement, and improved
portability potential.
One of the developed methods is the sequencing of
multiple regions or loci on the chromosome. In multi-
locus sequence typing, partial sequences of six or seven
housekeeping genes are amplified and sequenced. Each
fragment size is ∼400 to 600 bp, which is chosen as the
read length on a single run of the gel-based automated
sequencing platform in the mid-1990s (16). The number
of housekeeping genes is chosen based on studies with
invasive meningococci (17, 18). For each locus, a unique
number is assigned to a different sequence or allele frag-
ment. The numbers from several loci are combined to
yield a code, which is referred to as an allelic profile or
sequence type of a bacterial isolate.
Emerging and Future Microbial
Forensic Tools in Food Safety
A newer-generation subtyping method is multiple-locus
VNTR analysis (MLVA), which is based on variable-
number tandem repeats (VNTR). According to pub-
lished complete bacterial genomes, we have learned that
much of the bacterial genome consists of repeated short
nucleotide sequences. The repeats show variability in
their sizes as well as numbers. These repeating arrays
are called VNTR. To enhance the discriminatory power,
Keim et al. (19) modified the VNTR-based method by
combining multiple alleles at several marker loci to de-
termine the genetic relationship using Bacillus anthracis
as the model organism. Since then, this method has been
used on other potential bioterrorism agents, such as
Yersinia pestis (20) and Francisella tularensis (21).
Owing to its higher reproducibility and discriminatory
power to differentiate individual isolates, this method
was used to complement PFGE in some recent food-
borne outbreak investigations (22). In addition, progress
has been made to standardize MLVA protocols on
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Shiga-toxin producing E. coli O157, S. Enteritidis, and
S. Typhimurium among laboratories domestically and
internationally (23, 24).
In other areas of microbial forensics, such as bio-
terrorism, nongenetic information can be crucial to
the investigation. Using Bacillis subtilis cells and spores
as the test organism, Kreuzer-Martin et al. (25) sug-
gested that stable isotope ratio analysis is a useful
tool to trace the geographic point of origin for micro-
bial products. This method helped investigators to
link the bioterrorism agent to putative culture media
seized by the officials (26), although it took them sev-
eral years. However, many foodborne pathogens are
ubiquitous in nature and are distributed widely by
anthropical causes. Furthermore, a variety of food ma-
trices support the growth of most microbes. Taken
together, geolocation based on stable isotope ratios is
unlikely to have broad application in foodborne out-
break investigations.
Will WGS Be the Next Gold Standard?
With the ongoing improvement made to the technical
and cost aspects of high-throughput sequencing tech-
nologies, and more user-friendly bioinformatics tools
are developed, sequencing-based subtyping methods will
likely become the mainstream in microbial forensics.
Since a complete sequence of the genome is the ultimate
level of genetic resolution, WGS in theory has the max-
imum discriminatory power and hence stands out as
the potential gold standard in the near future. In addi-
tion to subtyping, WGS can be used to determine the
identities of pathogens that are difficult to culture (27).
Further, it can also be potentially used to detect geneti-
cally engineered organisms by identifying signatures
of engineering such as selectable makers or altered
sequences. These signatures can be overlooked by con-
ventional sequencing strategies that detect polymor-
phism in limited regions. Comparative genomic analyses
can also identify unique SNPs that can be used in routine
typing (28). The following case studies examine the
feasibility of the WGS approach in foodborne disease
investigations.
Gilmour et al. (29) sequenced the genomes of two
L. monocytogenes isolates that were involved in a large,
multiprovince outbreak in Canada in 2008, and they
found genetic variations previously unable to be re-
solved by PFGE. Using the restriction enzyme AscI,
PFGE revealed similar patterns between these isolates,
with 11 identical bands. The only difference was a band
shift of approximately 32 kbp. The authors acquired
draft genome sequences (>99% genome coverage) of

both isolates in 3 days, allowing them to begin prelimi-
nary comparative analyses. In the next 8 weeks or more,
they obtained more accurate determination of evolu-
tionary lineages and all genetic variations at the SNP
level. The cost of sequencing genomes, preparing fosmid
libraries, and resequencing for confirmation was re-
ported to be ∼$10,000. Though an expensive mission,
this study highlighted that WGS could reveal new in-
formation: one isolate harbored 28 SNPs and 3 indels,
including a 33-kbp prophage that accounted for the
observed difference in PFGE patterns. Using these
observed differences as markers, the authors further
assessed the distribution and transmission of the out-
break-associated isolates derived from different sources;
they deduced that three distinct but highly related strains
were likely involved in that outbreak. Using WGS, the
authors were also able to discover a new 50-kbp putative
mobile genomic island.
Salmonella is a major foodborne pathogen involved
in many large-scale outbreaks. However, S. Enteritidis, a
major serotype involved in foodborne disease, cannot be
accurately discriminated by PFGE because only 5 PFGE
types were observed from 85% isolates (30). The fact
that many isolates yielded identical PFGE types is likely
due to their overall genetic homogeneity, yet significant
nucleotide variations occur outside of the restriction
enzyme sites. Two previous studies sought to determine
the practicality of using WGS to cluster S. Enteritidis
isolates more accurately, which would aid in real-time
outbreak surveillance and investigation. Allard et al.
(31) and den Bakker et al. (30) each characterized >100
S. Enteritidis isolates previously typed by a standard
subtyping method such as PFGE and/or MLVA. Each
team employed different sequencing platforms and
bioinformatics programs. The former team found the
presence of several phages and plasmids and discovered
several mobile elements in some isolates. All isolates of
the serotype Enteritidis, which yielded PFGE pattern
JEGX01.0004, were clustered into nine clades or line-
ages following phylogenetic analyses of their genomes.
In their study, genetic variation within 100 bp was seen
within the same lineage, whereas genetic variation be-
tween lineages spanned from 100 to 600 nucleotide
changes. Outbreak-associated clinical isolates were clus-
tered into four lineages, whereas isolates unrelated to the
outbreak were in different lineages. However, isolates
harboring prophage were phylogenetically distributed
across many clades. Similarly, den Bakker et al. (30)
conducted cluster analysis following WGS. As part of
their experimental design, they sequenced 35 S. Enter-
itidis isolates that were different from those in the work
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of Allard et al. (31). These isolates yielded an identical
PFGE pattern, JEGX01.0004, but three different MLVA
types. Seven of the isolates were epidemiologically linked
to an outbreak tied to a long-term care facility, whereas
the remaining isolates were considered to be from spo-
radic outbreaks. Analysis of the SNP matrix grouped the
epidemiologically defined outbreak isolates in a single
clade. The average pairwise SNP difference among iso-
lates within this clade is <1 versus 78 SNPs relative to
the nearest related clade. Based on this “whole-genome
cluster analysis,” the authors identified nine additional
isolates that should have been grouped with the out-
break cluster, indicating the lack of resolving power of
PFGE and MLVA.
Though WGS has advantages over current subtyping
methods, the biggest hurdle for the food safety agencies to
readily adopt the WGS approach is probably the curation
of massive sequencing data and metadata. Nonetheless,
building on the success of PulseNet, it is foreseeable that
food safety agencies will develop a centralized database
to house and analyze WGS data, once a standardized
protocol is established. In fact, the FDA has been testing
the efficacy of WGS in a limited scope of foodborne
outbreak investigations since 2008 (http://www.fda.gov
/Food/FoodScienceResearch/WholeGenomeSequencing
ProgramWGS/ucm403550.htm). A few pilot studies have
been conducted to understand and address the practical
issues brought forth by WGS approach, including the
quality control of sequencing data and the need to es-
tablish a new definition of a clone. Unlike biological
agents with bioterrorism potential, foodborne microbes
are common and sometimes persist in diverse environ-
ments. They also exhibit relatively greater genetic fluidity
and variation. Circulating persistent clones may con-
found sequence analysis and thus mask the source of
outbreak. Hence, as with any microbial forensic analyses,
it is critically important to build a high-quality WGS
database.
ROADBLOCKS OF SOURCE
TRACKING IN FOOD SAFETY
Though technologies are advancing at a rapid pace, it
is not possible to definitively link a food sample to the
source based on molecular subtyping method alone. The
obstacles are inherent not only to the molecular tech-
niques but also to other factors intimately involved in the
outbreak investigations, such as the interview process.
Many times, foodborne outbreaks could not warrant
full investigation due to the nontimely detection of an
illness cluster or the lack of resources.
7
Yeung
Challenges in the Interview Process
As mentioned above, interviews are among the first steps
to gather information before generating a hypothesis.
The accuracy of the information is highly dependent on
the memories of the interviewees. It is not uncommon
that individuals fail to remember their food consump-
tion history, especially when the incubation time is
long or the implicated food is an ingredient. Interviewees
are also unlikely to remember stealth food such as con-
diments. To circumvent some of the problems associated
with memory issues, investigators may take advantage
of the shopper cards program, conduct a personal visit
to the interviewee’s home to identify possible food items,
and/or reinterview patients. A multistate outbreak as-
sociated with a raw scraped ground tuna product in
2012 illustrates how difficult it was to identify the
culprits, Salmonella Bareilly and S. Nchanga, when the
food source was a common ingredient of a product,
sushi in this case. This outbreak was further facilitated
by the lack of standard terminology for this tuna prod-
uct and inadequate paper trails. Some business owners
might also lie about using low-quality ingredients in
their sushi. In this incident, the investigators were able
to test the food source and establish a link. However,
in many other investigations, food items are no longer
available at the firm, plant, or farm following the trace-
back process due to the short shelf life of fresh and
raw food items. In some situations where the source
is a common ingredient or multiple food products are
contaminated by the same source (e.g., water or a hu-
man carrier), the statistical analysis would likely yield
no significance. As a result, no implicated food can be
linked to the outbreak. For this reason, many outbreak
investigations have ended up with no food vehicles
identified, and therefore, the etiological agent has been
declared “unknown.”
Currently, conducting and completing timely inter-
views using a standardized format remains a critical de-
ficiency during multistate outbreak investigations. The
lack of an electronic data submission system further
hinders rapid analysis. To facilitate more rapid dissem-
ination of information and close collaborative work
among agencies, the FDA established a pilot program,
FoodCORE—Foodborne Disease Centers for Outbreak
Response Enhancement. With support from the USDS-
FSIS and CDC, the ten FoodCORE centers, which
are located in major cities, address gaps in foodborne
disease outbreak responses by enhancing laboratory,
epidemiological, and environmental health capacity.
Currently, the centers focus on illnesses caused by Sal-
monella, Shiga toxin-producing E. coli, and Listeria.
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The activities or actions (surveillance, detection, inter-
view, response, control, and prevention) are docu-
mented in detail in order to be assessed according to the
performance metrics (http://www.cdc.gov/foodcore/pdfs
/244137_foodcore-salmonella-stec-listeria-report_508
.pdf).
Limitation in Technology
Whenever possible, molecular subtyping should be done
on microbial isolates recovered from a patient’s stool
and any food samples collected. The current subtyping
methods chosen are based on the best scientific knowl-
edge. It is understandable that each method has its own
limitation. Besides, all DNA-based subtyping methods
rely on the genetic structure and sequence of the mi-
crobes and thus are subject to the influence of genetic
mutation. Naturally occurring mutation or mutation
under the selective pressure inside of the human host
may give rise to different subtypes if the polymorphism
occurs at the restriction enzyme recognition site (for
PFGE) or specific locus (for multilocus sequence typing).
To alleviate the limitation of one method, more than one
subtyping methods should be employed. A combination
of omics approaches may be the future trend (32).
While well-developed and standardized subtyping
methods are currently established for many bacterial
and some viral pathogens, it is more difficult to optimize
a molecular subtyping method for protozoan para-
sites. As with Cryptosporidium spp., isolation and initial
detection of Cyclospora cayetanensis are through mi-
croscopic analyses. Species-level detection can be ac-
complished by nested multiplex PCR to differentiate it
from other closely related nonhuman pathogenic para-
sites, such as Eimeria spp. Rigorous development, how-
ever, has not been made on optimizing a subtyping
strategy for Cyclospora cayetanensis. It is unclear if
WGS can feasibly be performed on these parasites in a
cost-effective manner. The lack of discriminative power
by current technology to subtype this species was partly
responsible for the lengthy investigation of an outbreak
that sickened 631 persons from June to August 2013. It
was later determined that more than one outbreak had
occurred in this period.
Challenges in Whole-Chain Traceability
Undoubtedly, a comprehensive traceability system will
significantly help foodborne investigations to trace back
the implicated food to its source. Consumers’ awareness
on animal welfare and sustainability issues, coupled
with health concerns regarding food ingredients, has
prompted them to demand “whole-chain traceability.”

In addition to meeting consumer demand, having the
ability to trace back to the origins will ensure that farms
continue to engage in legitimate and responsible prac-
tices. Driven by these factors, the FDA asked the Insti-
tute of Food Technologists to conduct pilot studies on
product tracing in 2011. Collaborating with more than
100 volunteering organizations—including USDA, state
departments, consumer and industry groups—the stud-
ies focused on identifying and gathering information on
ways to improve product tracing and to explore and
evaluate ideas to rapidly and effectively identify the food
recipient. As expected, the process of conducting a step-
wise product tracing investigation was complicated and
confusing due to the diversity and complexity of the
food supply chain. The costs associated vary widely de-
pending on the size of business and the format or method
of product tracing. Several recommendations were made
to the FDA, including standardization of recordkeeping
format and electronic mechanisms for reporting (33).
To help address the gaps in research, development, and
implementation of whole-chain traceability system, the
Institute of Food Technologists’ Global Food Trace-
ability Center was launched in 2013. This center is
poised to become a central entity that brings key stake-
holders of many food businesses together to provide
product tracing solutions.
Historically, partial-chain traceability programs have
been routinely performed by the food industry. The
goal to achieve whole-chain traceability is ambitious and
will require many resources. Nonetheless, given the ad-
vancement of technology and growing consumer de-
mand, it is expected to become a reality in the near
future for at least some food products. According to
Crandall et al. (34), Maverick Ranch Beef (Denver, CO)
has an extensive system to trace their products marked
as “Natural Beef.” They use retinal scanning to identify
calves at various stages from birth to slaughter. They
use trolley tracking and bar code tagging of individual
cuts. However, these are not common practices for
many beef operations. It is more expensive to use retinal
scanning or radio frequency identification than ear tag-
ging. At the slaughter operation, the process is slowed
down and hence has a lower throughput if an individual
carcass or the trolley carrying multiple ones must be
retagged. At the processing facility, most beef is pro-
cessed into ground meat or hamburger. The conven-
tional practice is to comingle beef trim from multiple
sources in the initial grinder. In the context of trace-
ability, this implies that a mechanism must be in place to
identify each cut of meat. Crandall et al. (34) contended
that even Maverick Ranch Beef could not manage to
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Microbial Forensics in Food Safety
maintain identity of beef trimmings. Currently, the USDA
requires ground beef production to be traceable to the
boneless beef lots (35). At the retail level, most beef
can be traced back to the processor or slaughter plant,
since USDA inspection numbers for the processing plant
must be shown on the labels. DNA-based technologies
have been proposed to trace ground beef mixtures
to individual animal contributors (36). In fact, DNA-
based traceability method has been commercialized.
IdentiGen claims that their “DNA TraceBack provides
meat packers, processors, grocery retailers and the food
service sector with the ability to trace the origin of meat
products from point of sale to the animal of origin”
(http://www.identigen.com/products-services/dna
-traceback). Unrelated to the safety issue, there is also a
consumer demand for authentic food that is correlated to
a perception of good quality. Thus, a successful trace-
ability system not only will enhance the source tracing
capacity during foodborne outbreak investigations but
also will greatly benefit food authenticity validation.
PROACTIVE STRATEGY TO
ENHANCE FOOD SAFETY
There are fewer estimated cases of foodborne illness in
the United States than there were 15 years ago. Never-
theless, foodborne illnesses still represent a significant
public health and economic burden. While the immedi-
ate goal of foodborne outbreak investigation is to curb
the ongoing outbreak, the long-term goal is to reduce
and prevent illnesses. This is evident by the organiza-
tional structure of FoodCORE, which is divided into
three teams, responsible for signals and surveillance,
response, and postresponse. The signals and surveillance
team works closely with the CDC and other agencies
to identify emerging outbreaks linked to FDA-regulated
products. The response team coordinates response ef-
forts such as conducting interviews, testing, and recall.
The postresponse team is dedicated to learning from
the outbreak and making suggestions to improve future
investigations and/or to prevent illnesses. As the out-
break investigation is winding down, the postresponse
team begins to follow up issues or roadblocks that arose
during the investigation period. For example, the team
may include follow-up inspections or recommend a mod-
ification to industry practice. This team also works with
individuals who have the responsibility of implementing
the Food Safety Modernization Act (FSMA).
Signed into effect on 4 January 2011, the FSMA is
the nation’s newest food safety law. Under this law,
the FDA has a legislative mandate to require compre-
9
Yeung
hensive, prevention-based controls across the whole
food chain for the first time. It is evident that the spirit
of the FSMA is to control and prevent foodborne ill-
nesses. Under this law, the FDA now has (i) greater re-
sponsibility to improve training of food safety officials,
(ii) new tools to ensure that imported food meet U.S.
safety standards, (iii) greater potential to maximize in-
spection effectiveness, and (iv) the mandatory authority
to issue recall.
Before the FSMA, many regulations and recommen-
dations were already in place, with the goal of prevent-
ing illnesses. It was long recognized that it is more
important to take a proactive approach than to rely
solely on sampling and analyzing the finished products
to identify unsafe food. To this end, several major reg-
ulations have been enforced, including the Current
Good Manufacturing Practice (CGMPs) and Hazard
Analysis and Critical Control Point (HACCP). The im-
plementation of CGMP ensures proper design, moni-
toring, and control of manufacturing processes and
facilities. HAACP constitute a risk-based assessment
approach to manage or minimize food safety risk
through the analysis and control of hazards from raw
material production, procurement, and handling to
manufacturing, distribution, and consumption of the
finished product. In conjunction with the mandates,
many guidance documents are available to encourage
good handling practices. A notable example is the Good
Agricultural & Good Handling Practices audit program,
which is a voluntary, audit-based program that verifies
the fruit and vegetable industry’s conformance to gen-
erally recognized good practice outlined in the FDA’s
Guide to Minimize Microbial Food Safety Hazards for
Fresh Fruits and Vegetables (37).
At the far end of the farm-to-table continuum, the re-
sponsibility for food safety falls onto the shoulders of
consumers and food handlers. According to Healthy
People 2020 (https://www.healthypeople.gov/2020/topics
-objectives/topic/food-safety/objectives), which provides
science-based, 10-year national objectives for improving
the health of citizens, only <40% of consumers follow the
food safety practice “Cook: cook to proper tempera-
tures” in 2010. The number projected for 2020 is 50%,
although the target is 76%. Approximately 72.6% of
consumers follow the directive “Clean: wash hands and
surfaces often.” About 90.8% and 83.7% of consumers
follow the directives “Separate: don’t cross-contaminate”
and “Chill: refrigerate promptly,” respectively. Evidently,
the data suggest that we must continue to educate our-
selves and fellow consumers to exercise common food
safety practices.
10
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CONCLUSIONS
Microbes are ubiquitous on our planet and therefore
are bound to be present in our food products. A multi-
tude of food processing technologies is in place to im-
prove food safety and quality. However, the constantly
evolving food habit and consumer demand in our society
inevitably increase our risk of contracting foodborne
illnesses. It is unrealistic to set a goal of a 100% safe
food supply, but food safety regulatory agencies strive to
reduce illness incidence via improved surveillance, out-
break investigation, and follow-up measures. Microbial
forensic tools will continue to play an important role in
foodborne outbreak investigations. In the near future,
WGS will likely be the standard tool in microbial source
tracking. The development of a comprehensive whole-
chain traceability program is in progress. As consumers,
we must better educate ourselves about the risk associ-
ated with consuming raw and minimally processed food,
especially in the context of our and our family’s health
status. We must also do our part to exercise good food
safety practice.
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