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2015 sudarwanto 2015 Extended-Spectrum β-Lactamase (ESBL) -Producing Klebsiella pneumoniae in Bulk Tank Milk from Dairy Farms in Indonesia
2015 sudarwanto 2015 Extended-Spectrum β-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.
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 containing 30 mg of cefotaxime, ceftazidime, or cefpodox-
cows, together with Escherichia coli (Hogan and Smith, ime, either alone or in combination with 10 mg of clavulanic
2003), and b-lactam antibiotics are the drugs of choice for acid (MAST Group Ltd., Reinfeld, Germany). Results were
treatment (Carattoli, 2008; Smet et al., 2010). Improper and confirmed in a microdilution assay (CLSI, 2013) by using the
excessive use of intramammary or parenteral b-lactam anti- Micronaut-S b-lactamase VI test plates (Merlin Diagnostika,
biotics may provoke bacterial adaptation. In Indonesia, there Bornheim-Hersel, Germany), which claim the detection of
are few restrictions concerning the use of antibiotics, neither isolates showing a cephalosporinase phenotype (ESBL,
in human medicine nor in veterinary medicine. Thus, it may AmpC) or a carbapenemase phenotype (K. pneumoniae car-
be suspected that the rapidly increasing incidence of multi- bapenemase [KPC], metallo-b-lactamase [MBL]). The isolates
drug-resistant bacteria could be a result of the unconscious in which ESBL production was confirmed by phenotypical
and extensive use of antibiotics in this country (Sudarmono, methods, the minimum inhibitory concentration (MIC) for
2013). 11 antimicrobials commonly used to treat mastitis (Table 1),
So far, no information concerning the dimension of the risk were determined by the CLSI broth microdilution method
from emerging antibiotic-resistant bacteria in milk is avail- using a custom-designed microtiter panel (Merlin Diagnostika).
able for West Java. Therefore, the aim of this study was to MICs were interpreted according to CLSI guidelines. A sus-
assess the frequency of ESBL-producing Enterobacteriaceae ceptible strain (E. coli ATCC 25922) and a blaSHV-positive
in bulk tank milk samples collected from dairy farms located strain (K. pneumoniae ATCC 700603) were included to
in the West Java Region of Indonesia. monitor the performance of ESBL detection agents and as
quality controls for all susceptibility tests.
Materials and Methods
Milk samples Characterization of b-lactamases by polymerase chain
reaction (PCR)
A total of 80 randomly selected raw milk samples, re-
presenting bulk tank milk of dairy farms located in the West ESBL-positive isolates were further analyzed for the
Java region of Indonesia, were collected from November to presence of bla genes of the ESBL subtypes TEM, SHV, and
December 2011. Farms were either smallholders (3–8 lac- CTX-M (group 1, 2, 8, 9, or 25) by PCR using primers and
tating cows) or medium-sized dairy farms (10–15 lactating conditions as previously reported (Pitout et al., 1998;
cows), and none of these had on-site milk-cooling facilities. Batchelor et al., 2005; Woodford et al., 2006). Bacterial
One sample per farm was collected directly from the milk DNA was isolated with the DNeasy blood and tissue kit
tank. All samples were then immediately put into an ice- (Qiagen, Hilden, Germany) according to the manufacturer’s
refrigerated box ( < 8C), and reached the laboratory within recommendations. Two strains, K. pneumoniae ATCC
1–4 h after collection. 700603 (harboring a blashv gene), and K. pneumoniae isolate
(harboring both blactx-m and blatem genes) from the strain
Microbiological analysis collection of the chair of dairy science, were used as standard
ESBL-positive strains. A non-ESBL-producing organism (E.
For screening of ESBL-producing bacteria, 10-mL milk coli ATCC 25922) was used as negative control. PCR
samples were supplemented with cefotaxime (Sigma- products were determined by electrophoresis in a 2% agarose
Aldrich, Munich, Germany) at a final concentration of 1 mg/L gel (Biozym, Hessisch-Oldendorf, Germany). The molecular
and incubated for 24 h at 37C. This approach is an adapta- marker GeneRuler 100-bp DNA ladder (MBI Fermentas, St.
tion of a procedure recently recommended for ESBL isola- Leon-Roth, Germany) was used.
tion from other matrices (EFSA, 2011). Then 0.1 mL was
streaked onto MacConkey agar (Oxoid, Wesel, Germany) Sequencing of bla genes
containing 1 mg/L cefotaxime and incubated at 37C for 24 h.
For each morphologically distinct type of colony, 1 isolate The ESBL-encoding genes blaTEM, blaSHV, and blaCTX-M
was selected and subcultivated on tryptone soy (CASO) agar of the ESBL-positive isolates were amplified with primers
(Oxoid) at 37C for 24 h. and PCR conditions as described previously (Pitout et al.,
All isolates were tested for colony characteristics, Gram 1998; Batchelor et al., 2005). Resulting amplicons were pu-
staining, and oxidase reaction. Gram-negative and oxidase- rified using the PCR Purification Kit (Qiagen). Sequencing
negative colonies were further characterized by using standard was performed at SeqLab (Goettingen, Germany). Results
bacteriological procedures, including colony morphology, were evaluated using the BLAST algorithm available at
motility, lactose fermentation, malonat utilization, the pro- http://blast.ncbi.nlm.nih.gov/Blast.cgi.
duction of catalase and indole, and by API20E rapid test kit
(BioMérieux, Marcy l’Etoile, France). Isolates were stored in Analysis of chromosomal DNA restriction patterns
trypticase soy broth containing 20% glycerol at - 20C until by pulsed-field gel electrophoresis (PFGE)
further workup.
All ESBL-positive isolates were characterized after di-
gestion of the chromosomal DNA with the restriction enzyme
ESBL confirmation and antimicrobial susceptibility
XbaI by PFGE using the Chef-Dr II pulsed-field electro-
All cefotaxime-resistant, and oxidase-negative, isolates phoresis system (Bio-Rad, Munich, Germany) according to
were screened for ESBL production by the combined-disk the PulseNet standard protocol for Gram-negative bacteria by
method according to the Clinical Laboratory Standards In- the Centers for Disease Control and Prevention. A dendro-
stitute guidelines (CLSI, 2013). Zones of inhibition were gram was created using the Bionumerics software package
determined for each isolate, using antibiotic disks each 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
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
0.5
<1
<1
<1
<1
< 0.25/4
< 0.25/4
< 0.25/4
< 0.25/4
C/C
2
1
> 32 > 32
2
CTX CTB
4
4
2 < 0.25/4
2 < 0.25/4
2 < 0.25/4
2/4
Number and location of individual farm; FB, Fapet-Bogor; C, Cipanas; KB, Kunak-Bogor.
2
4
32
< 0.25/4
< 0.25/4
SHV-28, TEM-1, > 32 < 0.25/4
CMC
1
2
CTX-15
remarkable.
SHV-36
SHV-36
SHV-2a
SHV-2a
SHV-2a
SHV-1
69.9
70.9
97.6
97.6
3, KB
4, FB
4, FB
5, C
7, C
6, C
2009).
Isolate
IM-26
IM-36
IM-35
IM-8b
IM-5a
IM-7a
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, seems to be unlikely, further investigations on the possible
SHV-11, and SHV-28, respectively. Two isolates (IM-35 and interrelation or interaction between both environments via
IM-36), which were SHV-28 and SHV-11 positive, respec- milk production and distribution should be done in the future,
tively, both co-produced TEM-1 penicillinase and CTX-M- either to confirm or to exclude implications of dietary ESBLs
15. Although the SHV group of genes has most frequently on human health.
been reported for ESBL-producing K. pneumoniae isolates, PFGE analysis of ESBL-producing K. pneumoniae isolates
co-occurrence of TEM and SHV enzymes has also been de- from the seven different dairy farms yielded a markedly
scribed for ESBLs from dairy herds (Hammad et al., 2008; different pulsotype for each bulk milk tank. This indicates
Locatelli et al., 2009). More recently, a rapidly growing that the ESBL isolates have contaminated the bulk milk in-
number of reports on positive results for the third group of dependently, each one in its own environment (Fig. 1). In
ESBL, the CTX-M enzymes, have been observed (Woodford, contrast, the two isolates obtained from the same bulk tank
2010). CTX-M-positive Enterobacteriaceae isolates have milk sample (IM-12a and IM-12b) showed a closely related
been found both in companion animals and in livestock restriction profile. Whether or not this is the result of a clonal
(Carattoli, 2008; Smet et al., 2010; Ewers et al., 2011). CTX- spread of K. pneumoniae–producing ESBLs in this farm
M-15 is considered to have spread worldwide, especially in remains unclear. Members of the genus Klebsiella are fre-
Europe (Carattoli, 2008; Rogers et al., 2011), and to be the quently isolated from environment and food samples, par-
most common source of resistance to broad-spectrum ceph- ticularly from cattle with mastitis, soil, feces, and water
alosporins in E. coli, and to a lesser extent in K. pneumoniae among several sources within a dairy herd, and, together with
(Poirel et al., 2013). A relative increase of the percentage of E. coli, are probably the most important raw milk–related
CTX-M-15-positive isolates in sick animals, as compared to Enterobacteriaceae worldwide (Paulin-Curlee et al., 2008;
healthy animals, has been pointed out by Smet et al. (2010). Locatelli et al., 2010; Verbist et al., 2011).
The two CTX-M-15 isolates, which were both from farms Nevertheless, it remains remarkable that all ESBL pro-
in the Cipanas region, but were otherwise unrelated to each ducers were K. pneumoniae and not E. coli. On a worldwide
other, indicate that this type of ESBL-producing K. pneu- basis, E. coli and nontyphoidal Salmonella are by far the most
moniae requires specific attention. Strikingly similar findings frequently reported ESBL producers in food-producing ani-
were reported in clinical isolates from Javanese hospitals. For mals and food of animal origin in general, and specifically in
clinical isolates collected at two governmental teaching cattle (Smet et al., 2010; EFSA, 2011). Although ESBL en-
hospitals in Java, Indonesia, E. coli and K. pneumoniae were zymes are frequently found in K. pneumoniae, this species
the dominant Enterobacteriaceae, and CTX-M-15 was the has a small share in the overall number of ESBLs in En-
most frequent ESBL type in both species. For K. pneumoniae terobacteriaceae recovered from food-producing animals or
isolates, the blaSHV-type (54.3%) and the blaCTX-M type en- food of animal origin (EFSA, 2011; Dahmen et al., 2013). On
zymes were each detected in more than 50% of all isolates the other hand, our results are not fully unprecedented. For
(Severin et al., 2010, 2012). Studies from other countries bovine mastitis isolates from Japan, monitored over a period
confirm the importance of blaCTX-M genes in K. pneumoniae of 5 years, Ohnishi et al. (2013) reported that CTX-M-2–
(Locatelli et al., 2010; Dahmen et al., 2013; Ohnishi et al., positive K. pneumoniae alone accounted for 63% of all
2013; Schmid et al., 2013; Randall et al., 2014). ESBLs, and other Klebsiella spp. added another 12%. In
Although a direct linkage between ESBL-producing contrast, ESBL-producing E. coli were found with a fre-
K. pneumoniae in bulk tank milk and in hospitals in Java quency of only 20%. Locatelli et al. (2010) studied isolates of
ESBL–K. PNEUMONIAE IN BULK TANK MILK IN INDONESIA 5
K. pneumoniae from Italian mastitis cases and also found a Dahmen S, Métayer V, Gay E, Madec JY, Haennim M. Char-
high number of ESBLs, with blaSHV and blaTEM being the acterization of extended-spectrum beta-lactamase (ESBL)-
predominant genes. carrying plasmids and clones of Enterobacteriaceae causing
Because our study is the first one for ESBLs in bulk tank cattle mastitis in France. Vet Microbiol 2013;162:793–799.
milk from a tropical country, this could indicate that there is a [EFSA] European Food Safety Authority. Scientific Opinion on
different situation for Indonesia than has been reported for the public health risks of bacterial strains producing extended-
dairy environments from other countries, in particular Eu- spectrum b-lactamases and/or AmpC b-lactamases in food and
ropean countries. So far, most studies dealing with ESBL- food-producing animals. EFSA J 2011;9:2322.
producing Enterobacteriaceae in cattle and bovine products Ewers C, Grobbel M, Bethe A, Wieler LH, Guenther S.
had a primary focus on E. coli (Smet et al., 2010; Schmid Extended-spectrum beta-lactamases-producing gram-negative
bacteria in companion animals: Action is clearly warranted!
et al., 2013), and this may have contributed to a relative
Berl Munch Tierarztl 2011;124:94–101.
underestimation of the relevance of K. pneumoniae.
Falagas ME, Karageorgopoulos DE. Extended-spectrum beta-
Most of the domestically produced milk in Indonesia is lactamase-producing organisms. J Hosp Infect 2009;73:345–
thermally processed in dairy plants (Morey, 2011; Vanzetti 354.
et al., 2013). Such products should be essentially free from Hammad AM, Ahmed AM, Ishida Y, Shimamoto T. First
Enterobacteriaceae, if recontamination can be avoided. characterization and emergence of SHV-60 in raw milk of a
However, some of the bulk milk is pasteurized locally, using healthy cow in Japan. J Vet Med Sci 2008;70:1269–1272.
high-temperature short time (HTST, 72–75C, 15–30 s) Herwana Y, Pudjiadi L, Surjawidjaja JE, Lesmana M. Pre-
conditions (Slette and Meylinah, 2012; Vanzetti et al., 2013). valence of extended spectrum beta-lactamase in Klebsiella
Considering the risk of exceptionally high total bacteria pneumoniae. Univ Med 2008;27:98–105.
numbers in bulk tank milk ( > 106 CFU/mL) in Indonesia, it is Hogan J, Smith KL. Coliform mastitis. Vet Res 2003;34:507–
questionable whether or not HTST treatment is always suf- 519.
ficient, under real-life conditions, to fully eliminate En- Karuniawati A, Saharman YS, Lestari DC. Detection of car-
terobacteriaceae. Therefore, pasteurized milk could at least bapenemase encoding genes in Enterobacteriaceae, Pseudo-
be an occasional source of exposure with ESBLs. monas aeruginosa, and Acinetobacter baumanii isolated from
If ESBL-producing K. pneumoniae from raw milk could patients at Intensive Care Unit Cipto Mangunkusumo Hos-
find habitats at the farm site, at the KUDs, and in the dairy pital in 2011. Acta Med Indones 2013;45:101–106.
processing plant, this could contribute to a spread of resistant Kuntaman K, Santoso S, Wahjono H, Mertaniasih NM, Lestari
isolates not only among all those working in such environ- ES, Farida H, Hapsari R, Firmanti SC, As N, Santosaningsih
ments. Furthermore, this presents a permanent risk of re- D, Purwono PB, Kusumaningrum D. The sensitivity pattern
contamination of pasteurized milk with these bacteria. of extended spectrum beta lactamase-producing bacteria
against six antibiotics that routinely used in clinical setting. J
In conclusion, the relatively high frequency (8.75%) of
Indones Med Assoc 2011;61:482–486.
ESBL-producing K. pneumoniae in raw bulk tank milk from
Locatelli C, Caronte I, Scaccabarozzi L, Migliavacca R, Pagani
West Java imply the risk that milk is both a source of local L, Moroni P. Extended-spectrum beta-lactamase production
exposure and a vector contributing to the supraregional in E. coli strains isolated from clinical bovine mastitis. Vet
spread of antibiotic-resistant bacteria by trade. Res Commun 2009;33:141–144.
Locatelli C, Scaccabarozzi L, Pisoni G, Moroni P. CTX-M1
Acknowledgments
ESBL-producing Klebsiella pneumoniae subsp. pneumoniae
The technical expertise of Cornelia Borchardt and Annette isolated from cases of bovine mastitis. J Clin Microbiol
Schaus-Früauff is gratefully acknowledged. 2010;48:3822–3823.
Marra AR, Wey SB, Castelo A, Gales AC, Cal RG, Filho JR,
Disclosure Statement Edmond MB, Pereira CA. Nosocomial bloodstream infec-
tions caused by Klebsiella pneumoniae: Impact of extended-
No competing financial interests exist. spectrum beta-lactamase (ESBL) production on clinical
outcome in a hospital with high ESBL prevalence. BMC
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