RAW BOVINE MILK AS A RESERVOIR OF MULTI-DRUG RESISTANT, BETA-LACTAMASE-PRODUCING Klebsiella

. The transmission of zoonotic bacteria through consumption of raw milk is complicated by the dissemination of antimicrobial-resistant bacteria. The present study was conducted to detect the occurrence of antimicrobial-resistant bacteria (ESBL-/AmpC-producing Klebsiella spp.) in cow’s milk originating from healthy or infected (mastitis) cattle in India. In total, 450 milk samples were collected from apparently healthy cattle and cattle suffering from clinical or sub-clinical mastitis. Out of 455 Klebsiella spp., 67 (14.73%) isolates were found to be ESBL producers in the double-disc diffusion test. The occurrence of ESBL-producing Klebsiella spp. was significantly (p < 0.05) higher in milk samples collected from cattle suffering with mastitis than in healthy cattle. Among the ESBL-producing Klebsiella spp., 56 (83.6%) isolates were also detected that produced AmpC β - lactamases. All the ESBL and AmpC-producing Klebsiella spp. possessed bla CTX-M (100%) and bla AmpC (100%), respectively. The present study revealed a higher occurrence of class 1 integron in ESBL-producing Klebsiella spp. isolates. All ESBL-producing-Klebsiella spp. isolates were multi-drug resistant. The ciprofloxacin-and/or levofloxacin-resistant Klebsiella spp. isolates possessed the quinolone resistance gene ( qnrS ). The co-trimoxazole-resistant


Raw bovine milk as a reservoir of multi-drug resistant, beta-lactamase-producing Klebsiella
Food items such as milk function as ideal media for bacterial growth due to the presence of nutrients in optimum proportions.The microbes in raw milk originate from the bovine udder during intra-mammary infection (IMI) or diverse external sources such as air, contaminated hands of milkers, milking equipment, bulk tanks etc.The poor animal husbandry conditions associated with contaminated feed and drinking water, as well as the hind quarter hygiene of milch animals, play a significant role in the development of IMI, as environmental pathogens can enter the udder through the teat canal (Parekh and Subhash, 2008).The transmission of zoonotic bacteria through the consumption of raw milk and/or milk products is complicated by the dissemination of mobile genetic elements carrying antimicrobial resistance genes.The therapeutic efficacy and short withdrawal period of third-and fourth-generation cephalosporin β-lactam antibiotics make antimicrobial resistance a critical factor in dairy farming, especially during the treatment of mastitis, the most significant multi-etiological intramammary infection compromising the microbiological quality and quantity of produced milk.Overuse or misuse of antibiotics produces selection pressure that can generate commensal Enterobacteriaceae, including Klebsiella pneumoniae, that produce extendedspectrum β-lactamase (ESBL) or aminopenicillin-inactivating cephalosporinase (AmpC) (Liebana et al., 2013).Data on the occurrence of ESBL-/AmpC-producing Enterobacteriaceae, particularly Klebsiella pneumoniae, in raw cattle milk are inconsistent (Gundogan and Yakar, 2007).Most of the studies published thus far have focused on milk samples collected from cattle suffering with mastitis, so their results cannot be extrapolated to apparently normal milk entering the food chain, collected from healthy cattle reared in the same geographical location (Dahmen et al., 2013;Ohnishi et al., 2013).
In the Enterobacteriaceae group of bacteria, including Klebsiella spp., Escherichia coli and Salmonella spp., extended-spectrum beta-lactamases (ESBLs) are major antimicrobial resistance determinants which are transmitted through horizontal gene transfer.The ESBL enzyme can produce resistance to a variety of β-lactam antibiotics, such as penicillins, highergeneration cephalosporins and aztreonam.Classical ESBL varieties include TEM (except TEM-1), SHV (except SHV-1 and 2) and CTX-M (EFSA, 2011).AmpC β-lactamaseproducing organisms (ACBL) can generate resistance against β-lactamase inhibitors, such as clavulanic acid, in addition to cephalosporins, penicillins, cephamycins and monobactams.Overexpression of the chromosomal AmpC gene (bla AmpC ) or possession of plasmid-mediated (CITM or bla CMY-2 ) genes are found associated with the generation of resistance by ACBL producers (Schmid et al., 2013).The emergence and spread of carbapenemresistant Enterobacteriaceae (CRE) is a cause of serious clinical and public health concerns, as carbapenem is considered as a last-resort therapeutic option against ESBL-producing bacteria.Carbapenem resistance in Klebsiella spp. is associated with the production of enzymes such as metallo-β-lactamase and K. pneumoniae carbapenemase (KPC) (Birgy et al., 2012).Moreover, possession of ESBL determinants in bacterial plasmids is often associated with resistance to unrelated classes of antibiotics, for example, fluoroquinolones, aminoglycosides and sulfmethoxazole-trimethoprim (Coque et al., 2008).Mobile genetic elements, such as class 1 integrons, aid in the transmission of ESBL-/ACBLproducing organisms (EFSA, 2011).
The Indian dairy sector is mostly unorganized, and farmers are the major stakeholders.The ownership of milch animals (cattle and water buffalo) is fragmented, and large numbers of farmers rear only a few animals (0.6-2.0 animals per holding) for milking and draught purposes (Landes et al., 2017).About 35% of the milk produced is collected by local co-operatives for processing and distribution to consumers, whereas the majority of the milk (40%) produced is consumed within the farmer's household or distributed locally without processing.During milk processing in dairy plants, the majority of bacteria, including Enterobacteriaceae, are destroyed at pasteurization temperature.The consumption of raw milk is still a traditional practice in Indian villages, and this practice has increased recently among urban youth, who believe in the beneficial health effects of raw milk.As the local food animals were found to harbour ESBL-/ACBL-producing Enterobacteriaceae (Bandyopadhyay et al., 2015;Kar et al., 2015;Samanta et al., 2015 ), animal products such as milk may constitute an important reservoir of ESBL-/AmpC-producing Enterobacteriaceae.The present study was conducted to detect the occurrence of antimicrobial-resistant bacteria (ESBL-/AmpC-producing Klebsiella spp.) in cow's milk originating from healthy or infected (mastitis) cattle in all agro-climatic zones of West Bengal, one of the major milk-producing states in India (Landes et al., 2017).The study also intended to reveal the occurrence of integron, production of carbapenemase and metallo-beta-lactamase enzymes, MIC of antimicrobials and phylogenetic relationships among the Klebsiella spp.isolates.

Sampling
In the study, 450 milk samples were collected from apparently healthy (n = 168, without 1990).Cattle without visible clinical signs and with a negative CMT were considered as 'apparently healthy'.A pooled milk sample was prepared by mixing all four quarter milk samples, and the pooled sample was brought into the laboratory, maintaining the cold chain.
Most of the cattle were reared in backyard farming systems where the farmers kept 2-3 cattle per household.All the cattle were milked by hand by the farmers.In cattle suffering with clinical mastitis, mostly higher generation cephalosporins (ceftizoxime) and sometimes enrofloxacin and tetracycline were used for therapy during milk sample collection.The data were collected from farmers during milk sample collection using the 'drug bag' method, where the bags containing the foils/vials of common antibiotics were displayed to the farmers to identify the antibiotics used in his/her cattle.Cattle suffering with sub-clinical mastitis were not treated, as no clinical signs were present.

Isolation, Identification and PCR-Based Confirmation of Klebsiella sp.
Klebsiella-selective agar (HiMedia, India) was used for isolation of the bacteria from the collected milk samples.More than one characteristic purple-magenta-coloured colony was selected and streaked on a nutrient agar slant (HiMedia, India) for morphological and biochemical confirmation.Morphological confirmation was performed by Gram staining and biochemical confirmation by indole, methyl-red, Voges-Proskauer and citrate utilization tests (Quinn et al., 1994).PCR was used to confirm the isolates as Klebsiella spp.following the cycling conditions reported earlier, with some modifications (Brisse and Verhoef, 2001).The annealing temperature was modified using the positive control (ATCC-BAA-1705) in a gradient thermal cycler (Mastercycler Nexus, Eppendorf).

Detection of Beta-Lactamase (Bla ctx-M , Bla tem , Bla shv ), Chromosomal Bla ampc , Plasmid-Mediated Ampc Β-Lactamase (CITM) and Integron Genes in the Klebsiella spp. Isolates
PCR was conducted for detection of bla CTX-M , bla TEM and bla SHV in Klebsiella spp.isolates showing a positive reaction in the double-disc diffusion test (Cao et al., 2002;Weill et al., 2004) Standard PCR for detection of bla AmpC and CITM genes was also conducted in Klebsiella spp.isolates showing phenotypical AmpC production (Féria et al., 2002;van et al., 2008).Class I integron was detected by PCR in all the ESBL-producing Klebsiella spp.isolates (Mazel et al., 2000).

Detection of Minimum Inhibitory Concentration of Cefotaxime, Ceftazidime, Ceftriaxone and Ampicillin
The MIC of cefotaxime, ceftazidime, ceftriaxone and ampicillin was determined against ESBL-producing Klebsiella spp.isolates using HiComb™ MIC Strip (HiMedia, India) and Ezy MIC strips (HiMedia, India) as per the guidelines of the manufacturer.
The MIC of ciprofloxacin was determined against Klebsiella spp.isolates possessing qnr genes using Ezy MIC paper strips (HiMedia, India) as per the guidelines of manufacturer.

Phylogeny of ESBL-Producing Klebsiella spp. Isolates
All ESBL-producing Klebsiella spp.isolates were typed by RAPD-PCR (Lim et al., 2009), and images were analysed by using Doc-itLs image analysis software (UVP, UK).The phylogeny of the isolates was established by comparing the differences in the RAPD banding pattern.The neighbour joining method was used to construct an unrooted phylogenetic tree.

Statistical Analysis
The occurrence of ESBL-producing Klebsiella spp. in milk samples collected from apparently healthy and infected cattle was compared by chi-square test and a descriptive analysis was performed using SPSS software (SPSS Inc.).

RESULTS AND DISCUSSION
The present study detected the occurrence of Klebsiella spp. in 238 (238/450, 52.8%) milk samples collected from healthy and diseased cattle.From the 238 samples, a total of 455 Klebsiella spp.isolates were identified phenotypically and confirmed by specific PCR.The isolation rate of Klebsiella spp.(38.09%-64.57%)varies according to the health status of the studied animals.Significant (p < 0.05) differences in the occurrence of Klebsiella spp.were detected between the three groups of cattle according to their health status (Table 1).More than one colony was selected from each composite milk sample; therefore, the isolated strains outnumbered the collected samples.The isolation rate (20%-60%) of Klebsiella spp.varied widely in raw milk samples collected from farmers, bulk tank milk and milk products (cheese, khoa) in Jordan, Sudan, Sri Lanka, Egypt and Turkey (Ahmed et al., 2016;Badri et al., 2017;El-Sukhon, 2003;Jayaweera et al., 2018;Tepeli and Zorba, 2018).The existence of Klebsiella in the bovine udder is facilitated by the lac and fec iron-enterobactin operon, which helps Klebsiella spp. to utilize milk lactose more effectively than other commensal bacteria and might explain the high occurrence of the studied bacteria in the collected milk samples (Holt et al., 2015).
Out of 455 Klebsiella sp.isolates, 67(67/455, 14.73%) were found to be phenotypical ESBL producers in the double-disc diffusion test.The frequency of ESBL-producing Enterobacteriaceae in raw milk from dairy farms varies from 0 to 9.5% throughout the world depending on husbandry conditions, hind quarter hygiene, the use of milking equipment and the use of antimicrobials on farms (Tepeli et al., 2018;Geser et al., 2012;Skočková et al., 2015;Odenthal et al., 2016).The studies with detection of ESBL-producing Klebsiella spp. in raw milk described a highly variable occurrence between 0 and 45% worldwide, which is concurrent with the findings of the present study (Gundogan and Yakar, 2007;Badri et al., 2017;Özpınar et al., 2017;Diab et al., 2017).The occurrence of ESBL-producing Klebsiella spp. was significantly (p < 0.05) higher in milk samples collected from cattle suffering with mastitis (54/334, 16.1%) than in samples from healthy ones (13/121, 10.7%, Table 1).More samples were collected from infected cattle (clinical/sub-clinical mastitis) than from apparently healthy cattle, which may be one of the reasons, other than exposure to antibiotics, why more ESBL producers were obtained from infected milk samples.However, cattle with sub-clinical mastitis did not undergo any antibiotic therapy as they had no clinical signs.Earlier studies, including some form from India, also revealed mastitis milk as a potential source of ESBL-producing organisms due to high therapeutic antimicrobial exposure of the infected animals (Bandyopadhyay et al., 2015;Locatelli et al., 2010).Earlier studies detected faecal carriage of ESBL/ACBLproducing Enterobacteriaceae by food animals in India (Kar et al., 2015;Samanta et al., 2015).Moreover, Klebsiella spp.isolated from human patients in West Bengal was detected to possess resistance determinants against higher-generation cephalosporins depicting the picture of the community carriage (Saha et al., 2014), which indicates the milker's hands as another probable source of infection.Indeed, a limitation of the study is the lack of sampling from the milkers, which could successfully establish the hypothesis on the origin and transmission of K. pneumoniae clones.
Among the ESBL-producing Klebsiella spp., 56 isolates (56/67, 83.6%) were also confirmed as AmpC βlactamase producers.The AmpC βlactamase producing bacteria are although less common in nature but coexistence of ESBL and AmpC βlactamase is a significant concern, as the co-existence of both resistance types exhibits a broader resistance profile (EFSA, 2011).AmpC β-lactamaseproducing Enterobacteriaceae were detected earlier in raw milk and milk products (cheese) in different countries (Endimiani et al., 2012;Özadam and Özpinar, 2016).
The present study revealed a higher occurrence of class 1 integron in ESBLproducing Klebsiella spp.isolates (53.7%), depicting their high transmission potential.
Five out of the nine ciprofloxacin and/or levofloxacin resistant Klebsiella spp.isolates possessed plasmidmediated quinolone resistance gene (qnrS).The MIC of ciprofloxacin against qnr positive Klebsiella spp.isolates was found to be >32 µg/ml (Table 2).Similarly, earlier studies showed an MIC of 33.3 µg/ml produced by qnrS1 possessing Enterobacteriaceae isolates (van der Putten et al., 2019).Among co-trimoxazole-resistant (n = 37) ESBL-producing Klebsiella sp.isolates, seven and two isolates were positive for the sul1and sul2 genes, respectively, whereas, 28 isolates possessed both the sul1and sul2, but none of the isolates were positive for sul3 in PCR.
All 67 ESBL-producing Klebsiella spp.isolates were typeable with the primers used in RAPD-PCR.The amplified fragment size ranged from 170 bp to 4178 bp (calculated by Doc-itLs image analysis software, UVP, UK).The phylogenetic analysis of the studied isolates revealed that the strains isolated from same district were grouped in same cluster, indicating their phylogenetic relationship (K6 and K7; K49 and K50; K8 and K14; K39 and K40; K37 and K44; KP7, KP8 and KP10; K9, K10, KP13 and K17; KP9 and KP11).

CONCLUSIONS
The present study revealed a moderately higher occurrence of multidrug resistant ESBL-/AmpC-producing Klebsiella spp. in raw milk collected from healthy as well as infected cattle.The occurrence of ESBL-producing Klebsiella spp. was significantly higher in milk samples collected from cattle suffering with mastitis than in those from healthy ones.Among the ESBLproducing Klebsiella spp., 56 isolates were also detected to produce AmpC βlactamases.The present study detected the occurrence of class 1 integron in ESBL-producing Klebsiella spp.Isolates,87 depicting their high transmission possibility.The phylogenetic analysis of the studied isolates revealed that the strains isolated from same location had clonal relationship.The study made the consumers aware to avoid the raw milk consumption to prevent the spread of antimicrobial resistance Author Contributions: AM, JB, SG collected the samples and conducted the methodology part.SNJ and IS supervised the study.IS conceptualized the study and wrote the primary manuscript.SB and TKD edited the manuscript.All authors declare that they have read and approved the publication of the manuscript in the present form.

Table 2 -
Antimicrobial resistance profile, bla genotyping and MIC values of different antibiotics against ESBL-producing Klebsiella isolated from milk samples in West Bengal, India