FOODBORNE PATHOGENS AND DISEASE

Volume 0, Number 0, 2015 Original Article

ª Mary Ann Liebert, Inc.
DOI: 10.1089/fpd.2014.1895

Extended-Spectrum b-Lactamase (ESBL)–Producing

Klebsiella pneumoniae in Bulk Tank Milk
from Dairy Farms in Indonesia

Mirnawati Sudarwanto,1 Ömer Akineden,2 Sabrina Odenthal,2 Madeleine Gross,2 and Ewald Usleber 2

Abstract
Bulk tank milk from 80 dairy farms located in the West Java Region of Indonesia was analyzed for the
presence of extended-spectrum b-lactamase (ESBL)–producing Enterobacteriaceae. Isolates from seven dairy
farms were ESBL positive, and all were identified as Klebsiella pneumoniae. The isolates showed ESBL-
characteristic antibiotic resistance patterns. Further analysis revealed that all K. pneumoniae isolates harbored
the blaSHV gene, and two isolates were additionally positive for the blaTEM-1 and blaCTX-M-15 genes. Isolates
from different farms were clonally diverse according to macrorestriction analysis. The results indicate that the
relatively high frequency of ESBL-producing K. pneumoniae in bulk tank milk implies the risk that milk is both
a source of local exposure and a vector contributing to the supraregional spread of antibiotic-resistant bacteria
by trade.

Introduction (Morey, 2011; Nugroho, 2012). In some regions, antibiotic

residue testing of milk is done only sporadically. The lack of

W est Java is one of the most important dairy re-

gions in Indonesia, with about 25,000 dairy farmers.
Dairy milk production in 2010 reached 270,616 tons, up from
201,885 tons in 2005, an average annual increase of 6.8%
over 5 years. Over 97% of all Indonesian dairy cows are
located in Java (Morey, 2011). The overall milk quality is
poor, which may be attributed to the specific production sit-
uation (Nugroho, 2012). The majority of farms are owned by
individual farmers. Many of them are smallholders who
distribute their milk via local milk collection centers,
koperasi unit desa (KUD). From these KUDs, the bulk milk is
further transported to milk processors, but larger coopera-
tives may also process and distribute some of the milk di-
rectly (Morey, 2011). Raw milk cooling at 4C at the KUD
site is standard currently, but dairy farmers usually have no
cooling facilities. Therefore, time delay between milking and
transport to the KUD increases the risk of bacterial growth,
especially in a tropical climate. Local transport and decanting
of raw, unrefrigerated milk from many small sources also
present some risk of bacterial contamination at the KUD level
(Morey, 2011).
Milk payment in Java is dependent on quality parameters
such as protein, fat, and total bacteria (maximum 1 million
per milliliter), but stringent routine testing for inhibitors
(antibiotic residues) is only implemented in some regions
efficient veterinary drug control increases the risk of drug
residues in milk, and enhanced selection of b-lactam-
resistant bacteria may be the result (Sudarmono, 2013;
Morey, 2011).
Extended-spectrum b-lactamases (ESBL) are enzymes
produced by bacteria that can degrade and confer resistance
to some of the most commonly used group of antibiotics, the
b-lactams, which comprises the penicillins, the cephalospo-
rins, and the monobactams (Falagas and Karageorgopoulos,
2009; Ewers et al., 2011). Recent studies in Indonesia indi-
cated that antibiotic resistance patterns from several hospital
isolates have spread, especially in Gram-negative bacteria
such as Escherichia coli, Klebsiella pneumoniae, Pseudo-
monas aeruginosa, and Acinetobacter baumannii. Most of
these were resistant both to third-generation cephalosporins
and to fluoroquinolones (Moehario et al., 2009; Kuntaman
et al., 2011; Radji et al., 2011; Karuniawati et al., 2013).
K. pneumoniae, in particular ESBL-producing strains, is
an important cause of nosocomial infections in humans
worldwide (Podschun and Ullmann, 1998; Marra et al., 2006;
EFSA, 2011; Sumer et al., 2014). For Indonesia, an increase
of ESBLs within clinical isolates of K. pneumoniae has been
reported in recent years (Herwana et al., 2008, Kuntaman
et al., 2011; Parwati et al., 2012; Saharman and Lestari,
2013). In dairy farming, K. pneumoniae is one of the most

1
Laboratory of Veterinary Public Health, Faculty of Veterinary Medicine, Bogor Agricultural University, Bogor, West Java, Indonesia.
2Dairy Sciences, Institute of Veterinary Food Science, Justus-Liebig University Giessen, Giessen, Germany.

1
2 SUDARWANTO ET AL.
important causative agents for clinical mastitis in lactating
cows, together with Escherichia coli (Hogan and Smith,
2003), and b-lactam antibiotics are the drugs of choice for
treatment (Carattoli, 2008; Smet et al., 2010). Improper and
excessive use of intramammary or parenteral b-lactam anti-
biotics may provoke bacterial adaptation. In Indonesia, there
are few restrictions concerning the use of antibiotics, neither
in human medicine nor in veterinary medicine. Thus, it may
be suspected that the rapidly increasing incidence of multi-
drug-resistant bacteria could be a result of the unconscious
and extensive use of antibiotics in this country (Sudarmono,
2013).
So far, no information concerning the dimension of the risk
from emerging antibiotic-resistant bacteria in milk is avail-
able for West Java. Therefore, the aim of this study was to
assess the frequency of ESBL-producing Enterobacteriaceae
in bulk tank milk samples collected from dairy farms located
in the West Java Region of Indonesia.
Materials and Methods
Milk samples
A total of 80 randomly selected raw milk samples, re-
presenting bulk tank milk of dairy farms located in the West
Java region of Indonesia, were collected from November to
December 2011. Farms were either smallholders (3–8 lac-
tating cows) or medium-sized dairy farms (10–15 lactating
cows), and none of these had on-site milk-cooling facilities.
One sample per farm was collected directly from the milk
tank. All samples were then immediately put into an ice-
refrigerated box ( < 8C), and reached the laboratory within
1–4 h after collection.
Microbiological analysis
For screening of ESBL-producing bacteria, 10-mL milk
samples were supplemented with cefotaxime (Sigma-
Aldrich, Munich, Germany) at a final concentration of 1 mg/L
and incubated for 24 h at 37C. This approach is an adapta-
tion of a procedure recently recommended for ESBL isola-
tion from other matrices (EFSA, 2011). Then 0.1 mL was
streaked onto MacConkey agar (Oxoid, Wesel, Germany)
containing 1 mg/L cefotaxime and incubated at 37C for 24 h.
For each morphologically distinct type of colony, 1 isolate
was selected and subcultivated on tryptone soy (CASO) agar
(Oxoid) at 37C for 24 h.
All isolates were tested for colony characteristics, Gram
staining, and oxidase reaction. Gram-negative and oxidase-
negative colonies were further characterized by using standard
bacteriological procedures, including colony morphology,
motility, lactose fermentation, malonat utilization, the pro-
duction of catalase and indole, and by API20E rapid test kit
(BioMérieux, Marcy l’Etoile, France). Isolates were stored in
trypticase soy broth containing 20% glycerol at - 20C until
further workup.
ESBL confirmation and antimicrobial susceptibility
All cefotaxime-resistant, and oxidase-negative, isolates
were screened for ESBL production by the combined-disk
method according to the Clinical Laboratory Standards In-
stitute guidelines (CLSI, 2013). Zones of inhibition were
determined for each isolate, using antibiotic disks each
containing 30 mg of cefotaxime, ceftazidime, or cefpodox-
ime, either alone or in combination with 10 mg of clavulanic
acid (MAST Group Ltd., Reinfeld, Germany). Results were
confirmed in a microdilution assay (CLSI, 2013) by using the
Micronaut-S b-lactamase VI test plates (Merlin Diagnostika,
Bornheim-Hersel, Germany), which claim the detection of
isolates showing a cephalosporinase phenotype (ESBL,
AmpC) or a carbapenemase phenotype (K. pneumoniae car-
bapenemase [KPC], metallo-b-lactamase [MBL]). The isolates
in which ESBL production was confirmed by phenotypical
methods, the minimum inhibitory concentration (MIC) for
11 antimicrobials commonly used to treat mastitis (Table 1),
were determined by the CLSI broth microdilution method
using a custom-designed microtiter panel (Merlin Diagnostika).
MICs were interpreted according to CLSI guidelines. A sus-
ceptible strain (E. coli ATCC 25922) and a blaSHV-positive
strain (K. pneumoniae ATCC 700603) were included to
monitor the performance of ESBL detection agents and as
quality controls for all susceptibility tests.
Characterization of b-lactamases by polymerase chain
reaction (PCR)
ESBL-positive isolates were further analyzed for the
presence of bla genes of the ESBL subtypes TEM, SHV, and
CTX-M (group 1, 2, 8, 9, or 25) by PCR using primers and
conditions as previously reported (Pitout et al., 1998;
Batchelor et al., 2005; Woodford et al., 2006). Bacterial
DNA was isolated with the DNeasy blood and tissue kit
(Qiagen, Hilden, Germany) according to the manufacturer’s
recommendations. Two strains, K. pneumoniae ATCC
700603 (harboring a blashv gene), and K. pneumoniae isolate
(harboring both blactx-m and blatem genes) from the strain
collection of the chair of dairy science, were used as standard
ESBL-positive strains. A non-ESBL-producing organism (E.
coli ATCC 25922) was used as negative control. PCR
products were determined by electrophoresis in a 2% agarose
gel (Biozym, Hessisch-Oldendorf, Germany). The molecular
marker GeneRuler 100-bp DNA ladder (MBI Fermentas, St.
Leon-Roth, Germany) was used.
Sequencing of bla genes
The ESBL-encoding genes blaTEM, blaSHV, and blaCTX-M
of the ESBL-positive isolates were amplified with primers
and PCR conditions as described previously (Pitout et al.,
1998; Batchelor et al., 2005). Resulting amplicons were pu-
rified using the PCR Purification Kit (Qiagen). Sequencing
was performed at SeqLab (Goettingen, Germany). Results
were evaluated using the BLAST algorithm available at
http://blast.ncbi.nlm.nih.gov/Blast.cgi.
Analysis of chromosomal DNA restriction patterns
by pulsed-field gel electrophoresis (PFGE)
All ESBL-positive isolates were characterized after di-
gestion of the chromosomal DNA with the restriction enzyme
XbaI by PFGE using the Chef-Dr II pulsed-field electro-
phoresis system (Bio-Rad, Munich, Germany) according to
the PulseNet standard protocol for Gram-negative bacteria by
the Centers for Disease Control and Prevention. A dendro-
gram was created using the Bionumerics software package
version 5.1 (Applied Maths, Kortrijk, Belgium), with the
ESBL–K. PNEUMONIAE IN BULK TANK MILK IN INDONESIA 3

For all isolates, identical MICs were obtained with the following substances: cefoxitin ( < 4), ampicillin ( > 16), cefazolin ( > 32), cefoperazon ( > 16), erythromycin ( < 4), oxacillin ( > 4),
< 0.25
< 0.25
< 0.25

< 4/0.4 < 0.25

< 4/0.4 < 0.25

clavulanic acid; CTX, cefotaxime; CTB, cefotaxime/3-APB; C/C, cefotaxime/clavulanic acid; MER, meropenem; MEB, meropenem/3-APB; MEE, meropenem/clavulanic acid; AMC,
3-APB, 3-aminophenylboronic acid; CEP, cefepime; CMC, cefepime (with/without) clavulanic acid; CAZ, ceftazidime; CZB, ceftazidime (with/without) 3-APB; CZC, ceftazidime/
DICE coefficient and the unweighted-pair group method with

MAF

0.5
Table 1. Identification, bla Genes and Antibiotic Resistance Profiles (Minimum Inhibitory Concentration [MIC], lg/mL) of Extended-Spectrum arithmetic means with a band position tolerance of 1%.

>2

32/3.2 > 2
< 4/0.4
< 4/0.4
< 4/0.4
32/3.2

< 4/0.4
Results and Discussion
K/C
After the initial screening of bulk milk, a total number of 20
morphologically different, cefotaxime-resistant colonies from
CEQ
8
8
>8
>8

>8
>8
>8

>8
16 milk samples were obtained on cefotaxime-supplemented
MacConkey agar plates. Concerning the origin of ESBL-
32/16

32/16
suspect isolates, there was no obvious geographic pattern; all
AMC
16/8
8/4
8/4

8/4
8/4

8/4
regions under study yielded ESBL-suspect samples. The
screening method, employing pre-enrichment of ESBL from
milk in the presence of cefotaxime, has been successfully used
< 0.25 < 0.25
< 0.25 < 0.25
< 0.25 < 0.25

< 0.25 < 0.25

< 0.25 < 0.25
< 0.25 < 0.25

< 0.25 < 0.25

MEE

0.5

for other matrices (EFSA, 2011) and consistently yielded pos-

itive results for inoculated milk samples at approximately 1
colony-forming unit (CFU)/mL (data not shown). The validity
< 0.25
MEB

of the selective isolation method was routinely determined by

recovery experiments, in which either raw milk or ultra-high-
temperature-processed milk was inoculated with one of four
MER
<1
<1
<1
32

<1
<1
<1

<1

different ESBL-positive strains of Enterobacteriaceae (K.

pneumoniae ATCC 700603 and three E. coli from our Institutes’
b-Lactamase–Producing Klebsiella pneumoniae

strain collection). It was therefore considered as sufficient for

< 0.25/4
< 0.25/4
< 0.25/4
< 0.25/4

< 0.25/4
< 0.25/4
< 0.25/4

< 0.25/4
C/C

the rapid isolation of ESBL-suspect Enterobacteriaceae for

further identification.
The isolates were then biochemically identified as A.
2
2
4

2
1
> 32 > 32

8 < 0.25/4 > 32 > 32

2
CTX CTB

baumannii (n = 9), K. pneumoniae (n = 8), Enterobacter clo-

acae (n = 2), and Enterobacter aerogenes (n = 1). Expression
of ESBL was considered a threefold concentration decrease
2
4
4

4
4

in the MIC ( ‡ 2 mg/mL) of either cephalosporins in the

amoxicillin/clavulanic acid 2:1; CEQ, cefquinom; K/C, kanamycin/cephalexin; MAF, marbofloxacin.

presence of clavulanic acid compared to its MIC when tested

2 < 0.25/4
2 < 0.25/4
4 < 0.25/4

2 < 0.25/4
2 < 0.25/4

2 < 0.25/4

alone (MIC ratio ‡ 4) (Table 1).

CZC

2/4

Number and location of individual farm; FB, Fapet-Bogor; C, Cipanas; KB, Kunak-Bogor.

ESBL production, however, was phenotypically confirmed

only in the K. pneumoniae isolates from seven different bulk
milk tanks. For three Enterobacter spp. isolates, the pro-
SHV-11, TEM-1, > 32 < 0.25/4 > 32 > 32
CAZ CZB

duction of another b-lactamase (AmpC) could be affirmed.

The nine isolates of A. baumannii were negative for ESBL-
2
1
2

2
4
32

< 1 < 0.25/4 < 1

and AmpC-production. One K. pneumoniae isolate per

sample was further characterized, except for sample IM-12
from which two K. pneumoniae isolates with different mor-
0.5 < 0.25/4
< 0.25/4
< 0.25/4

< 0.25/4
< 0.25/4
SHV-28, TEM-1, > 32 < 0.25/4
CMC

phology were selected. The MICs of the eight ESBL-positive

K. pneumoniae isolates for various antibiotics are summa-
rized in Table 1. All isolates were resistant to many b-lactams
of importance in veterinary mastitis therapy (penicillins,
CEP

cefquinome, cefazolin, cefoperazone), as well as to lincosa-

1
1

1
2

For all isolates, identical T-value (1.0) was obtained.

mides (pirlimycin) and macrolides (erythromycin). Although

the members of the genus Klebsiella can on occasion contain
different resistance markers (Severin et al., 2010; Calbo
bla genes

et al., 2011), the broad resistance patterns of these isolates are

CTX-15

CTX-15

remarkable.
SHV-36
SHV-36

SHV-2a
SHV-2a

SHV-2a
SHV-1

Two isolates (IM-35 and IM-36) were simultaneously re-

penicillin G ( > 8), pirlimycin ( > 4).

sistant to marbofloxacin, kanamycin/cephalexin, and amox-

icillin/clavulanic acid. One isolate (IM-36) showed a high
Origina ID (%)b
api20E

resistance to meropenem, a carbapenem antibiotic used in

97.6
97.6
97.6
97.6

69.9
70.9
97.6

97.6

human medicine. This isolate was phenotypically confirmed

to produce MBL enzymes and KPC, although carbapenem
resistance and the encoding genes were not further charac-
1, KB
2, KB

3, KB
4, FB
4, FB
5, C
7, C

6, C

terized. Carbapenem resistance has been increasingly reported

among Enterobacteriaceae, often isolated from clinical speci-
mens, mainly attributed to MBL and KPC (Nordmann et al.,
IM-12b
IM-12a

2009).
Isolate

IM-26
IM-36

IM-35

IM-8b
IM-5a
IM-7a

Based on PCR analysis, all eight isolates harbored SHV

b
a

genes (Fig. 1). When these were sequenced, three were

4 SUDARWANTO ET AL.

FIG. 1. Pulsed-field gel electrophoresis (PFGE) pattern of eight Klebsiella pneumoniae isolates from seven different
farms in the West Java region of Indonesia, digested with the XbaI restriction enzyme. The dendrogram was constructed
with the BioNumerics software 5.1 (Applied Maths NV, Sint-Martens-Latem, Belgium) choosing the Dice coefficient
setting both tolerance and optimization at 1%. The horizontal scale on the left side (100–40) indicates the level of similarity
in percent among fingerprints. The 95% of similarities indicates the minimal levels for defining clusters and subtypes,
respectively.

identified as SHV-2a, two as SHV-36, and one each as SHV-1,
SHV-11, and SHV-28, respectively. Two isolates (IM-35 and
IM-36), which were SHV-28 and SHV-11 positive, respec-
tively, both co-produced TEM-1 penicillinase and CTX-M-
15. Although the SHV group of genes has most frequently
been reported for ESBL-producing K. pneumoniae isolates,
co-occurrence of TEM and SHV enzymes has also been de-
scribed for ESBLs from dairy herds (Hammad et al., 2008;
Locatelli et al., 2009). More recently, a rapidly growing
number of reports on positive results for the third group of
ESBL, the CTX-M enzymes, have been observed (Woodford,
2010). CTX-M-positive Enterobacteriaceae isolates have
been found both in companion animals and in livestock
(Carattoli, 2008; Smet et al., 2010; Ewers et al., 2011). CTX-
M-15 is considered to have spread worldwide, especially in
Europe (Carattoli, 2008; Rogers et al., 2011), and to be the
most common source of resistance to broad-spectrum ceph-
alosporins in E. coli, and to a lesser extent in K. pneumoniae
(Poirel et al., 2013). A relative increase of the percentage of
CTX-M-15-positive isolates in sick animals, as compared to
healthy animals, has been pointed out by Smet et al. (2010).
The two CTX-M-15 isolates, which were both from farms
in the Cipanas region, but were otherwise unrelated to each
other, indicate that this type of ESBL-producing K. pneu-
moniae requires specific attention. Strikingly similar findings
were reported in clinical isolates from Javanese hospitals. For
clinical isolates collected at two governmental teaching
hospitals in Java, Indonesia, E. coli and K. pneumoniae were
the dominant Enterobacteriaceae, and CTX-M-15 was the
most frequent ESBL type in both species. For K. pneumoniae
isolates, the blaSHV-type (54.3%) and the blaCTX-M type en-
zymes were each detected in more than 50% of all isolates
(Severin et al., 2010, 2012). Studies from other countries
confirm the importance of blaCTX-M genes in K. pneumoniae
(Locatelli et al., 2010; Dahmen et al., 2013; Ohnishi et al.,
2013; Schmid et al., 2013; Randall et al., 2014).
Although a direct linkage between ESBL-producing
K. pneumoniae in bulk tank milk and in hospitals in Java
seems to be unlikely, further investigations on the possible
interrelation or interaction between both environments via
milk production and distribution should be done in the future,
either to confirm or to exclude implications of dietary ESBLs
on human health.
PFGE analysis of ESBL-producing K. pneumoniae isolates
from the seven different dairy farms yielded a markedly
different pulsotype for each bulk milk tank. This indicates
that the ESBL isolates have contaminated the bulk milk in-
dependently, each one in its own environment (Fig. 1). In
contrast, the two isolates obtained from the same bulk tank
milk sample (IM-12a and IM-12b) showed a closely related
restriction profile. Whether or not this is the result of a clonal
spread of K. pneumoniae–producing ESBLs in this farm
remains unclear. Members of the genus Klebsiella are fre-
quently isolated from environment and food samples, par-
ticularly from cattle with mastitis, soil, feces, and water
among several sources within a dairy herd, and, together with
E. coli, are probably the most important raw milk–related
Enterobacteriaceae worldwide (Paulin-Curlee et al., 2008;
Locatelli et al., 2010; Verbist et al., 2011).
Nevertheless, it remains remarkable that all ESBL pro-
ducers were K. pneumoniae and not E. coli. On a worldwide
basis, E. coli and nontyphoidal Salmonella are by far the most
frequently reported ESBL producers in food-producing ani-
mals and food of animal origin in general, and specifically in
cattle (Smet et al., 2010; EFSA, 2011). Although ESBL en-
zymes are frequently found in K. pneumoniae, this species
has a small share in the overall number of ESBLs in En-
terobacteriaceae recovered from food-producing animals or
food of animal origin (EFSA, 2011; Dahmen et al., 2013). On
the other hand, our results are not fully unprecedented. For
bovine mastitis isolates from Japan, monitored over a period
of 5 years, Ohnishi et al. (2013) reported that CTX-M-2–
positive K. pneumoniae alone accounted for 63% of all
ESBLs, and other Klebsiella spp. added another 12%. In
contrast, ESBL-producing E. coli were found with a fre-
quency of only 20%. Locatelli et al. (2010) studied isolates of
ESBL–K. PNEUMONIAE IN BULK TANK MILK IN INDONESIA
K. pneumoniae from Italian mastitis cases and also found a
high number of ESBLs, with blaSHV and blaTEM being the
predominant genes.
Because our study is the first one for ESBLs in bulk tank
milk from a tropical country, this could indicate that there is a
different situation for Indonesia than has been reported for
dairy environments from other countries, in particular Eu-
ropean countries. So far, most studies dealing with ESBL-
producing Enterobacteriaceae in cattle and bovine products
had a primary focus on E. coli (Smet et al., 2010; Schmid
et al., 2013), and this may have contributed to a relative
underestimation of the relevance of K. pneumoniae.
Most of the domestically produced milk in Indonesia is
thermally processed in dairy plants (Morey, 2011; Vanzetti
et al., 2013). Such products should be essentially free from
Enterobacteriaceae, if recontamination can be avoided.
However, some of the bulk milk is pasteurized locally, using
high-temperature short time (HTST, 72–75C, 15–30 s)
conditions (Slette and Meylinah, 2012; Vanzetti et al., 2013).
Considering the risk of exceptionally high total bacteria
numbers in bulk tank milk ( > 106 CFU/mL) in Indonesia, it is
questionable whether or not HTST treatment is always suf-
ficient, under real-life conditions, to fully eliminate En-
terobacteriaceae. Therefore, pasteurized milk could at least
be an occasional source of exposure with ESBLs.
If ESBL-producing K. pneumoniae from raw milk could
find habitats at the farm site, at the KUDs, and in the dairy
processing plant, this could contribute to a spread of resistant
isolates not only among all those working in such environ-
ments. Furthermore, this presents a permanent risk of re-
contamination of pasteurized milk with these bacteria.
In conclusion, the relatively high frequency (8.75%) of
ESBL-producing K. pneumoniae in raw bulk tank milk from
West Java imply the risk that milk is both a source of local
exposure and a vector contributing to the supraregional
spread of antibiotic-resistant bacteria by trade.
Acknowledgments
The technical expertise of Cornelia Borchardt and Annette
Schaus-Früauff is gratefully acknowledged.
Disclosure Statement
No competing financial interests exist.
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Address correspondence to:
Dr. Ömer Akineden
Dairy Sciences
Institute of Veterinary Food Science
Justus-Liebig University
Ludwigstrasse 21
35390 Giessen, Germany
E-mail: oemer.akineden@vetmed.uni-giessen.de