Molecular signature of extended spectrum β-lactamase producing Klebsiella pneumoniae isolated from bovine milk in eastern and north-eastern India

    Molecular signature of extended spectrum β-lactamase producing Klebsiella pneumoniae isolated from bovine milk in eastern and north-eastern India S. Koovapra, S. Bandyopadhyay, G. Das, D. Bhattacharya, J. Banerjee, A. Mahanti, I. Samanta, P.K. Nanda, A. Kumar, R. Mukherjee, U. Dimri, R.K. Singh PII: DOI: Reference:

S1567-1348(16)30322-7 doi: 10.1016/j.meegid.2016.07.032 MEEGID 2861

To appear in: Received date: Revised date: Accepted date:

23 March 2016 22 July 2016 24 July 2016

Please cite this article as: Koovapra, S., Bandyopadhyay, S., Das, G., Bhattacharya, D., Banerjee, J., Mahanti, A., Samanta, I., Nanda, P.K., Kumar, A., Mukherjee, R., Dimri, U., Singh, R.K., Molecular signature of extended spectrum β-lactamase producing Klebsiella pneumoniae isolated from bovine milk in eastern and north-eastern India, (2016), doi: 10.1016/j.meegid.2016.07.032

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ACCEPTED MANUSCRIPT Molecular signature of extended spectrum β-lactamase producing Klebsiella pneumoniae

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isolated from bovine milk in eastern and north-eastern India

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S Koovapra , S Bandyopadhyay*, G Das1, D Bhattacharya, J Banerjee, A Mahanti, I Samanta, P K Nanda, A Kumar, R Mukherjee, U Dimri, R K Singh

ICAR-Indian Veterinary Research Institute, Eastern Regional Station,

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37 Belgachia Road, Kolkata – 700 037

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1. CAU. Selesiah, Aizwal, Mizoram

*[email protected], [email protected] Dr. Samiran Bandyopadhyay Scientist, ICAR-IVRI Both S Koovapra and S Bandyopadhyay contributed equally and should be considered as co-first authors

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ACCEPTED MANUSCRIPT Summary The present study reports on 23 extended spectrum lactamase producing Klebsiella

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pneumoniae (KP), isolated from milk samples (n=340) of healthy cows (n=129) and cows with subclinical (n=159) and clinical (n=52) mastitis, from three different states of India viz. West

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Bengal, Jharkhand and Mizoram. Seven of them were AmpC type β- lactamase producers, as

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well. The ESBL producing KP were significantly (P =0.006, χ2= 10.04, df= 2) and more

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frequently detected in milk samples of mastitic cows than healthy ones. The β-lactamase genes blaCTX-M, blaTEM and blaSHV were detected in 19, 8 and 3 isolates, respectively. In all but

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one CTX-M positive isolates, the genetic platform- ISEcp1-blaCTX-M-orf477 was detected. Ten of the isolates carried plasmid mediated quinolone resistance gene – qnrS and 1 isolate possessed

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qnrB. Again 11 of them were found to have sulfonamide resistance gene - sul1 and 12 possessed class I integron. Sequencing of the class 1 integron revealed the presence of dfrA12 /dfrA17 and

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aadA2/ aadA5 gene cassettes conferring resistance to trimethoprim and aminoglycosides, respectively. All the isolates, characterized by enterobacterial repetitive intergenic consensus (ERIC) PCR, yielded distinct fingerprinting profile. However, most of the isolates from Jharkhand were clustered along with two isolates each from West Bengal and Mizoram indicating their clonal relatedness even though isolated from geographically different areas. Isolation of ESBL producing KP from bovine milk samples implies its public health significance; as such pathogens may enter the human food chain causing severe health hazards. Keywords: ACBL, Bovine, ESBL, Klebsiella pneumoniae, Mastitis, Milk

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1. Introduction:

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Extended spectrum β-lactamases (ESBLs) are the plasmid mediated enzymes that confer

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resistance to 3rd and 4th generation cephalosporins (oxy-imino β-lactam) and monobactam

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(aztreonam) groups of drugs except carbapenems and cephamycins. ESBLs are the most commonly detected among Enterobacteriaceae like Klebsiella pneumoniae (KP) and Escherichia coli and

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can be inhibited by β-lactamase inhibitors like clavulanic acid, sulbactam and tazobactam

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(Paterson and Bonomo, 2005). Since their emergence in early eighties, ESBLs are continuously being reported as hospital-borne pathogens (Gupta et al., 2003) and thereafter disseminating to

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cause community acquired infections. Livestock acts as a major reservoir of multi-drug resistant

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(MDR) pathogens including the ESBL producers. Recently, we have reported ESBL-producing E. coli in cows with clinical and subclinical mastitis, broiler birds and healthy pigs in West

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Bengal and Odisha, India (Bandyopadhyay et al., 2015; Kar et al., 2015; Samanta et al., 2015). Studies in UK (Timofte et al., 2014), France (Locatelli et al., 2010) and Japan (Saishu et al., 2014) have also reported the occurrence of ESBL producing KP in bovine mastitis indicating the possibility of intra-mammary infection by ESBL producers. Furthermore, KP is one of the major causes of coliform or environmental mastitis in high yielding cows (Radostits et al., 2000). The presence of ESBL producing KP in bovine milk may be a significant threat to the consumer as this group of pathogens have been recorded to cause obstinate infections with increased morbidity and mortality (Bhattacharjee et al., 2010; Warjri et al., 2015).

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ACCEPTED MANUSCRIPT Taking a clue from our previous findings, the present work was carried out to investigate the occurrence of ESBL producing KP in milk samples obtained from healthy cows and cows

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suffering from clinical and subclinical mastitis cows from three different states of India viz. West

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Bengal, Jharkhand and Mizoram and their further characterization such as drug resistance profile

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and clonal diversity using enterobacterial repetitive intergenic consensus (ERIC) PCR.

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2. Materials and methods 2.1. Sample collection

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A total of 340 bovine milk samples were aseptically collected from three states of India West Bengal (159), Jharkhand (78) and Mizoram (103) and transported to the laboratory

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maintaining cold chain. All the milk samples were subjected to California Mastitis and Somatic Cell Count Tests. Of the total, 129 milk samples were from healthy cows without any evidence

(159).

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of mastitis whereas 211 samples were from cows with clinical (52) and subclinical mastitis

2.2. Isolation and identification of Klebsiella spp. from milk samples All the samples were inoculated into nutrient broth (BD BBL, Difco,

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incubated at 37°C for overnight and thereafter streaked into MacConkey agar (BD BBL, Difco, USA). The pink, mucoid colonies were only taken for overnight incubation at 37°C in Klebsiella Selective Agar Base (Himedia, India) supplemented with carbenicillin. Finally, magenta coloured individual colonies were picked up for processing by standard biochemical tests (Quinn et al., 1994) and their further molecular confirmation as Klebsiella spp. based on gyrA gene (Chander et al., 2011). Details with regard to the oligonucleotide primers and PCR conditions are 4

ACCEPTED MANUSCRIPT described in Table 1. Again, all the confirmed Klebsiella isolates were subjected to PCR for detection of rpoB and pehX genes for species-specific confirmation of KP (Chander et al., 2011).

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2.3. Phenotypic detection of extended spectrum β-lactamase production

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To confirm the KP isolates phenotypically as ESBL producers, we followed a three step

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procedure. First of all, the confirmed isolates were subjected to drug sensitivity assay against cefpodoxime (10 μg), ceftriaxone (30 μg), cefotaxime (30 μg), ceftazidime (30 μg), cefepime (30

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μg) and aztreonam (30 μg) [BD BBL, Difco, USA]. The isolates with reduced susceptibility to

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any of these drugs were again screened by combination disc diffusion test (CDT) and ESBL Etest for further phenotypic confirmation (CLSI, 2014). CDT is based on the principle that the

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ESBL producing isolate will exhibit an expanded zone of inhibition against 3rd or 4th generation

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cephalosporins in the presence of β-lactamase inhibitors like clavulanic acid (Andrews, 2012; Kar et al., 2015). In CDT, two cephalosporins were tested simultaneously – at one side

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ceftazidime or cefotaxime (30 μg) was placed alone whereas a combination of the same antibiotic with clavulanic acid (10 μg) was placed at the other side. Following overnight incubation at 37°C, an increase by 5 mm or more in the zone of inhibition (≥50%) in the disc containing the antibiotic along with clavulanic acid than the disc containing the antibiotic alone was considered as an indicative of ESBL production. In ESBL E-test, commercially available ESBL detection strips coated with a mixture of ceftazidime, cefotaxime and cefepime at one end and these antibiotics with clavulanic acid and tazobactam at the other end were used in a concentration gradient manner as per the manufacturer’s guidelines (Himedia, India). Following 18-24 h incubation at 35-37°C, a decrease in minimum inhibitory concentration (MIC) values by 8 fold or more in the presence of β-lactamase inhibitors indicated ESBL production.

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ACCEPTED MANUSCRIPT 2.4. Antibiotic sensitivity profile of the ESBL producing K. pneumoniae All the phenotypically confirmed ESBL producing KP isolates were subjected to

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antibiotic sensitivity test using the following commercially available antibiotic discs (BD, BBL

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Difco, USA / Himedia, India) - sulpha/trimethoprim (1.25/23.75 μg), chloramphenicol (30 μg),

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tetracycline (30 μg), gentamicin (10 μg), amikacin (30 μg), cefixime (5μg), ceftizoxime (30 μg), cefoxitin (30 μg), gatifloxacin (5 μg), tobramycin (10 μg), ciprofloxacin (5 μg), imipenem (10

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μg), meropenem (10 μg), piperacillin-tazobactam (100/10 μg) and cefepime-tazobactam (80/10

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μg). The isolates were categorized as sensitive, intermediate or resistant as per the standard guidelines (CLSI, 2014). MIC values for the drugs like ceftriaxone, ceftazidime and cefotaxime

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for all the confirmed ESBL producers was determined using broth dilution method as described

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previously (Andrews, 2001).

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2.5. Phenotypic confirmation of AmpC type β-lactamase production Phenotypic confirmation of AmpC type -lactamase (ACBL) production by the ESBL producing KP isolates was carried out by cefoxitin-cloxacillin double disc synergy (CC-DDS) test following previously described protocols (Kar et al., 2015; Polsfuss et al., 2011). Here the isolates were tested simultaneously using the disc containing cefoxitin (30 μg) alone or in combination with cloxacillin (200 μg). An increase in 4 mm or more in the zone of inhibition in the presence of cloxacillin and cefoxitin combination than cefoxitin alone was considered as an indicative of ACBL production. For further confirmation, all the isolates were subjected to ACBL E-test following manufacturer’s protocol (Himedia, India).

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ACCEPTED MANUSCRIPT 2.6. Phenotypic detection of metallo-β-lactamase or carbapenemase production For detection of carbapenemase activity of the ESBL producing KP isolates exhibiting

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reduced sensitivity to carbapenem, modified Hodge test (MHT) and imipenem – EDTA disc

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2.7. PCR based detection of drug resistance genes

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synergy test were conducted following the method described previously (Birgy et al., 2012).

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The KP isolates, phenotypically confirmed as ESBL producers, were examined by PCR for the presence of β-lactamase and carbapenemase genes – blaAmpC, blaSHV, blaTEM,

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blaCTX-M, blaNDM, blaVIM, blaIMP, blaOXA-48-type and blaKPC, class 1 and 2 integrons (int1, int2), plasmid mediated quinolone resistance (PMQR: qnrS, qnrB, qnrA, qep and aac),

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sulfonamide resistance (sul-1) genes (Ferjani et al., 2015; Kar et al., 2015; Kashif et al., 2013; Mendes et al., 2007; Monteiro et al., 2012; Poirel et al., 2011; Weill et al., 2004). The details of

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PCR reaction are mentioned in Table 1.

2.8. Enterobacterial repetitive intergenic consensus PCR All the ESBL producing KPs were subjected to ERIC-PCR following the method of (Bandyopadhyay et al., 2012).

The oligonucleotide primers used and the PCR conditions

followed for ERIC-PCR are described in Table 1. The amplified products were checked by 2% gel electrophoresis and images taken by gel documentation system were analyzed using the DocitLs image analysis software (UVP, USA). By comparing the differences in the banding pattern, phylogenetic relationship among the isolates was established to illustrate the degree of genetic divergence and similarity among them.

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ACCEPTED MANUSCRIPT 2.9. Cloning, sequencing and anlysis The purified PCR products were cloned in pGMET-Easy vector (Promega, Madison, WI,

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USA) or pTZ57/R (Fermentas, USA). The plasmids with the expected insert were sequenced

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commercially using standard pUC/M13 universal primers and homology searches were made,

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thereafter, using BLAST algorithm available at http://blast.ncbi.nlm.nih. gov/Blast.cgi.

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3. Results:

From 340 bovine milk samples, 291 Klebsiella spp. were isolated including 211 KP of

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which only 53 (25.1%) exhibited reduced susceptibility to at least one of the oxy-imino β-lactam antibiotics tested. Finally, 23 KP were confirmed as ESBL producers by CDT and ESBL E-test

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(Tables 2 & 3). Of them, 10 isolates were from Jharkhand, 7 from West Bengal and 6 from Mizoram. All the ESBL producing KPs, except 2, were isolated from cows with subclinical (12)

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and clinical mastitis (9). The frequency of isolation of such pathogens was significantly higher (P =0.006, χ 2= 10.04, df= 2) in bovine clinical (13.4%) and subclinical mastitis (7.5 %) than that from the healthy cows (1.5 %) [Table 2]. Out of the 23 ESBL producers, seven were also positive for ACBL production (J6MK2, MZKP189, JLF3K1, J6MK2, WM2K1, WBKP17 and NA1K2) by CC-DDS and ACBL E-test. In drug sensitivity assay, the isolates (23) were resistant to ceftriaxone, ceftazidime, cefotaxime, aztreonam, cefpodoxime, ceftizoxime, cefexime (100% each), gentamicin (78%), tetracycline (74%), sulpha/trimethoprim combination (70%), cefepime (61%), ciprofloxacin and piperacillin / tazobactam (52% each). However, the isolates were mostly sensitive to meropenem (100%), imipenem and chloramphenicol (78% each) and gatifloxacin (60%). The MIC values of the isolates ranged from 32- 256 μg/ml for ceftazidime, whereas values were 128- 256 μg/ml in case of cefotaxime and ceftriaxone. Among various β8

ACCEPTED MANUSCRIPT lactamase genes screened, blaCTX-M was predominant, being present in 19 (82.6 %) isolates (Table 3). Other genes like blaTEM and blaSHV were detected in 8 (34.8%) and 3 (13%)

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isolates, respectively. Eight isolates harbored more than one β-lactamase genes (blaCTX-M and

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blaTEM in six isolates; blaSHV and blaCTX-M, blaSHV and blaTEM in one isolate each,). However, none of the isolates carried all the three genes. The gene blaAmpC was detected in 20

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(82.6%) isolates, but only 7 of them were positive phenotypically (KR080208). Sequencing of

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the plasmid with desired insert using universal primer and blast analysis revealed that the blaCTX-M gene of 17 isolates had 100% similarity with CTX-M-15 where as one isolate from

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Jharkhand (JRH4K1) exhibited 98% similarity with CTX-M-15 (KR812384). The phylogenetic analysis from predicted amino acid sequences of KR812384 and other closely related CTX-M

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genes (available in the database) and the corresponding sequence alignment are depicted in Fig. 1 and 2. For another isolate from West Bengal (GF4K3), amplified CTX-M gene showed

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similarity with CTX-M-63 (KT314168). In 18 isolates, CTX-M gene carried insertion element ISEcp1 in the upstream and orf477 in the downstream region. For the isolate GF4K3 no such insertion element could be detected. Sequencing of the amplified SHV and TEM genes revealed that they belong to SHV-180 group (KR080209) and TEM-1 (KT314169, KR080210), respectively. Among the various PMQR genes screened, qnrS was detected in 10 and qnrB in one isolate only. Further, 11 isolates of the 23 isolates carried sul1 and 12 (52.2%) possessed class I integron. Sequencing of the amplified class I integron revealed that these carried aad2/aad5 and dfrA12/dfrA17 gene cassettes mediating resistance to aminoglycoside and sulfonamide groups of drugs (KR261577.1, KT425373, KT425374 and KT425375). All the isolates, except three, characterized by ERIC-PCR exhibited amplified fragments of size ranging from 150 bp to 3800 bp and were distributed in 8 clusters (Table 3). Eleven 9

ACCEPTED MANUSCRIPT isolates were grouped in cluster 5 of which 6 were from Jharkhand. Besides, three isolates from West Bengal (WM2K1, CTWK1 and WBKP17) and two from Mizoram (1SH7K2 and 3L18K1)

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were also placed in cluster 5 with Jharkhand isolates.

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4. Discussion:

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The present study reports the occurrence of ESBL producing KP in bovine milk from Indian subcontinent for the first time, although such pathogens are not uncommon in human patients

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(Ali et al., 2014; Dhara and Tripathi, 2014; Rao et al., 2014). Several studies have also documented that food animals may act as important reservior for spreading drug resistant

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bacteria or ESBL genes to human (Bandyopadhyay et al., 2015; Saishu et al., 2014; Timofte et

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al., 2014). There are few studies describing clear evidence of such cross-transmission of ESBL

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producing Enterobacteriaceae between human and food producing animals/food (Bezanson et al., 1983; Leverstein-van Hall et al., 2011). Existence of similar kind of clones of ESBL- and/or AmpC-producing enterobacteriaceae in both animals and food animals further provides indirect evidence of such cross-transmission (EFSA, 2011). Out of 340 milk samples screened, 154 (45.2%) samples were detected to harbor Klebsiella spp., and 23 KP were confirmed as ESBL producers. The frequency of isolation of such pathogens was significantly (P =0.006) higher in milk samples of cows with clinical and subclinical mastitis than that of the healthy ones. Bovine mastitis has been reported as an important predisposing factor for colonization of these pathogens by researchers from United Kingdom, France and Japan (Locatelli et al., 2010; Saishu et al., 2014; Timofte et al., 2014). Nevertheless, 1.5% of the milk samples from healthy cows were also detected with ESBL 10

ACCEPTED MANUSCRIPT producing KP. Similarly, Hammad et al. (2008) have previously reported KP with blaSHV-60 gene in milk of healthy cows. The existence of acquired lac operon and fec iron-enterobactin

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operon in KP may provide additional advantage for their exuberant growth over other bacterial

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pathogens with utilization of lactose in cow milk and help to invade the udder and thrive in the mammary epithelial cells as noted by previous workers (Holt et al., 2015). Further, milk is

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considered as a convenient vehicle for transmission of such kind of pathogens into human food

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chain (Bandyopadhyay et al., 2012; Hammad et al., 2008; Osman et al., 2014). Detection of ESBL producers in cow milk in this study, therefore assumes significance, as people may

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unknowingly consume the milk resulting in asymptomatic colonization which may even lead to

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complicated systemic infection.

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There was no significant difference in the state wise occurrence of the ESBL producing KP in bovine milk. The presents finding could not be compared because of unavailability of any

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such reports from these regions. Nevertheless, ESBL producing E. coli from bovine milk were reported from West Bengal and Odisha, previously (Bandyopadhyay et al., 2015; Kar et al., 2015). Again reports on ESBL producing KP in human patients from different states of India including Mizoram and West Bengal are on the rise (Chatterjee et al., 2012; Warjri et al., 2015). Thus, detection of such pathogens in bovine milk from these regions may possibly be due to cross transmission between the human and animal population along with their environment. Most of the ESBL producing isolates (82.6%) of the present study possessed blaCTX-M gene. Other β-lactamase genes like blaTEM and blaSHV were detected in 34.8% and 13% of the ESBL producing isolates, respectively. However, this is in contrast to the previous findings of Timofte et al., (2014) wherein all the isolates from cattle mastitis were positive for blaTEM and blaSHV, however, none of them carried blaCTX-M gene. Likewise, in another study from 11

ACCEPTED MANUSCRIPT France, only 2 of the 9 ESBL producing KP isolated from bovine mastitis were positive for CTX-M and rest were positive for blaSHV (Locatelli et al., 2010). In our previous studies,

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CTX-M was predominantly detected among the ESBL producing E. coli in cattle, poultry and

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pigs (Bandyopadhyay et al., 2015; Kar et al., 2015; Samanta et al., 2015). Furthermore, all the ESBL producers reported from bovine mastitis in West Bengal also possessed blaCTX-M gene

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(Bandyopadhyay et al., 2015; Kar et al., 2015). This is in agreement with the observations of

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previous workers (Canto and Coque 2006; Warjri et al., 2015) who pointed out that ESBL producing Enterobacteriaceae having blaCTX-M became pandemic in Asian countries,

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particularly in Indian subcontinent and are, therefore, more frequently detected than blaTEM and blaSHV genes. Quite similar to our previous study, the sequence analysis of amplified CTX-M

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gene (KR812384) revealed its 100% similarity with CTX-M-I cluster (CTX-M-15) in all but two isolates. The extrapolated amino acid sequence from the amplified CTX-M gene of one isolate

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from Jharkhand (JRH4K1) revealed only 98% similarity with CTX-M-15 with two amino acid substitutions (E-130-K + R-144-W) [Fig. 2]. This may be a unique variant of CTX-M gene emerged in these areas. CTX-M-15/CTX-M class-1 is the most common ESBL found in KP in India (Mohamudha Parveen et al., 2012; Tripathi et al., 2012). Previously, Ohnishi et al. (2013) also reported CTX-M-2-producing KP, and CTX-M-15 producing E.coli in bovine mastitis from Japan. Such wide dissemination of CTX-M-15 producing KP all over the world including India may be due to the horizontal transfer of the gene from E. coli by conjugation of IncFII plasmids (Upadhyay et al., 2015). In this study, the association of ISEcp1-like element in upstream and orf477 in downstream region of blaCTX-M gene was recorded in all the CTX-M positive isolates but one. Such genetic arrangement was frequently noted in human isolates from India (Shahid et al., 12

ACCEPTED MANUSCRIPT 2012; Upadhyay et al., 2015). This also gives indirect evidence of cross-transmission of ESBL producers in human and food animals. Thus chance of human infection from consumption of

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such infected milk cannot be ruled out. Furthermore, reports also indicate that efficient mobile

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elements could play an important role in spreading of ESBL genes (Chong et al., 2011). The present findings, therefore, suggest the possible role of ISEcp1-like element in spread and

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dissemination of blaCTX-M-15 gene. Further, one isolate (2L9K1) from Mizoram was positive

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for ESBL production despite the fact that none of the -lactamase genes except blaAmpC was detected in it. This could probably be due to the possession of other gene(s) encoding ESBL(s),

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which were not included in this study. Although 20 ESBL producers carried blaAmpC gene, only seven of them were positive in phenotypic detection method. Altogether, 13 isolates of KP

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were phenotypically categorized as ACBL non-producers despite harbouring blaAmpC gene. These types of ACBL non-producers were also previously reported in human patients from India

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(Shahid et al., 2012). Such weak expression of blaAmpC gene could be due to hyper-production of ESBL by these isolates which might have masked the phenotypic expression of ACBL in CCDDS and ACBL E-tests (Bandyopadhyay et al., 2015; Kar et al., 2015). Although five of the KP isolates (JRH13K2, JRF12K1, WM2K1, HFK5 and 1SH7K2) were resistant to imipenem, none of them carried genes (blaNDM, blaVIM, blaIMP, blaOXA48-type and blaKPC), for carbapenemase enzymes and were also negative in imipenem/EDTA disc synergy test and MHT which ruled out the possibility of KPC or MBL production. In our previous study, many of the ESBL producing E. coli from cattle and poultry were resistant to carbapenem, despite the fact they were not MBL or KPC producers (Kar et al., 2015). Earlier studies indicated that overexpression of various β-lactamases like ACBL or ESBL (Baroud et al., 2013; Birgy et al., 2012) and presence of inhibitor resistant TEM β-lactamase (Chaibi et al., 13

ACCEPTED MANUSCRIPT 1999) may also cause carbapenem resistance. Twelve ESBL producers in the present study were positive for class 1 integron. Such association of integron with ESBL producers is not

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uncommon and has been previously recorded (Akram et al., 2011; Bhattacharjee et al., 2010; Kar

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et al., 2015). Sequence analysis of class I integron of the isolates revealed two kinds (dfrA12/dfrA17 and aadA2/aadA5) of gene cassette arrangements (encoding for dihydrofolate

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reductase and aminoglycoside – 3’ adenyl transferase) responsible for resistance to sulfonamide

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and aminoglycoside groups of drugs (KT425373, KT425374, KT425375, KR812385, KR261577.1). This could have led to high rate of phenotypical resistance as noticed among the

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present ESBL producers to drugs like gentamicin (78%) and sulpha-trimethoprim (70%). Similar gene cassette arrangements were also detected in our previous study (Kar et al., 2015).

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Further, the isolates were resistant to all -lactam drugs tested and most of the isolates with

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blaCTX-M genes exhibited even higher MICs to the 3rd generation cephalosporins. This could

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possibly be due to high phenotypic expression of CTX-M gene as recorded earlier (Edelstein et al., 2003). It is noteworthy to mention that these CTX-M-15 positive isolates also exhibited resistance to both ceftazidime and cefotaxime. In general, blaCTX-M enzyme is more active against cefotaxime but not against ceftazidime. Therefore, the present CTX-M-15 variant must have evolved over the years, to exhibit resistance to ceftazidime as well (Baraniak et al., 2002). Moreover, 56% of the ESBL producers showed resistance to -lactamase inhibitor (tazobactam) potentiated -lactams, probably mediated either by overexpression of ESBL, or co-expression of ESBL and ACBL, or by inhibitor resistant beta-lactamase. Emergence of such resistance to lactamase inhibitor further narrows the available therapeutic options to tackle ESBL infection. Apart from -lactams, all the isolates exhibited resistance to drugs of divergent groups like

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ACCEPTED MANUSCRIPT aminoglycosides, tetracyclines and fluorquinolones. These MDR features among CTX-M type ESBL producing KP in bovine milk were earlier reported from Japan by Ohnishi et al., (2013).

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The present work revealed that about 47.8% of the ESBL producing KP were positive for

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sul-1 gene. Previous studies also suggested such high prevalence of sulfonamide resistance among ESBL producers in foods of animal origin (Schwaiger et al., 2013; Vogt et al., 2014).

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Likewise, ten ESBL producers in the present study carried qnrS gene and one for qnrB gene.

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Similar to our observations, Timofte et al. (2014) reported three ESBL producing KP from bovine mastitis and all of them carried qnrS gene. A recent report by a group of workers

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indicates that PMQR is instrumental for fluroquinolone resistance among the CTX-M type ESBL producing E. coli isolates from Mongolia (Kao et al., 2016). In our study, of the 11 ESBL

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producing KP possessing qnr genes (qnrS :10 and qnrB :1), 8 were also positive for blaAmpC gene. Liao et al. (2015) reported such co-existence of qnr allele and blaAmpC among

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Citrobacter freundii isolates from different origins. One isolate (HFK6) was found resistant to quinolones although, it did not harbour any of the qnr resistant determinants. Such resistance could have been mediated by the drug efflux pump or mutations in the gyrA and gyrB genes encoding DNA gyrase or mutations of parC and parE genes encoding topoisomerase IV (Nam et al., 2013). In contrast, some isolates (JRFIK2, JKP5 and MZKP23) were found to be quinolone sensitive despite having qnr. Although, qnr proteins confer only low level of quinolone resistance in vitro, they can facilitate the emergence of high level of resistance in vivo in the presence of quinolone drugs at therapeutic concentration (Strahilevitz et al., 2009). Genotyping of the ESBL producing KP by ERIC-PCR generated twenty distinct fingerprinting patterns separated in eight different clusters. It indicated high discriminatory potential of ERIC-PCR and heterogeneity of the isolates, except in three from Mizoram. The 15

ACCEPTED MANUSCRIPT isolates from Jharkhand (JLF8K1, JLF4K1, J5K2, J6MK2, JKP5 and JRF12K1), West Bengal

(CTWK1, WM2K1 and WBKP17) and Mizoram (3L18K1 and 1SH7K2) were found in one

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cluster indicating their genetic relatedness. Of them, two isolates (JLF8K1 and JLF4K1) from

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Jharkhand had almost identical ERIC profile exhibiting a high degree of genetic relatedness. However, the isolates (GF4K3 and NA1K2) originated from two distinctly located corners of

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West Bengal were placed in different lineage. Although, most of the isolates from Jharkhand

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were genetically related, they were grouped together in cluster no. 5 with other isolates from West Bengal and Mizoram, geographically located far away from Jharkhand. This shows wide

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dissemination of the isolates from a single lineage and existence of genetic inter-relationship, even though isolated from different geographical locations. However, no single clone was found

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to colonize the udder of milching cattle and no specific trend was observed on the basis of virulence or resistance characteristics of the pathogens. Such clonal diversity was noted by

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Ohnishi et al., (2013) even among the ESBL producing strains from single farm. To the best of our knowledge, this is the first ever attempt to study the occurrence and characterization of ESBL producing KP in food animals from eastern and north-eastern India where animals and human live in a close proximity. The present investigation points out the predominance of blaCTX-M producing KP in bovine milk which indicates possibility of ISEcp1 insertion element mediated pandemic dissemination of CTX-M clone beyond the health-care premises to community and livestock. Moreover, 20% of the isolates exhibited resistance to imipenem which is a cause of great concern, as carbapenems are the last resort of treatment for ESBL infections in critical human patients. From this study, it can be concluded that bovine milk, particularly from cows with mastitis, is an important reservoir of ESBL and ACBL producing KP. Their occurrence in milk can be a significant risk to consumers and animal 16

ACCEPTED MANUSCRIPT handlers who may get such infection easily. On the other hand, persistence of such pathogen in udder or milk may complicate the therapeutic regimen of intra-mammary infection and clinical

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recovery of the animals. The pathogens may also disseminate to other in-contact animals via

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infected milk and contaminate the surrounding environment aggravating the situation further, if adequate precautionary measures are not taken. This is the first ever systematic study on

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occurrence of ESBL and ACBL producing KP in bovine milk from India.

5. Acknowledgements

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The work was carried out with the funds received from ICAR -“Outreach programme

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on Zoonotic diseases” project. The help received from Dr. B Hembram, PI, AICRP on ADMAS, Jharkhand unit for collection of samples from Jharkhand is thankfully acknowledged.

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References for table:

(Ferjani et al., 2015; Kashif et al., 2013; Mendes et al., 2007; Monteiro et al., 2012; Poirel et al., 2011; Weill et al., 2004) References

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Kar, D., Bandyopadhyay, S., Bhattacharyya, D., Samanta, I., Mahanti, A., Nanda, P.K., Mondal, B., Dandapat, P., Das, A.K., Dutta, T.K., 2015. Molecular and phylogenetic characterization of multidrug resistant extended spectrum beta-lactamase producing Escherichia coli isolated from poultry and cattle in Odisha, India. Infect., Genet. Evol. 29, 82-90*. Kashif, J., Buriro, R., Memon, J., Yaqoob, M., Soomro, J., Dongxue, D., Jinhu, H., Liping, W., 2013. Detection of Class 1 and 2 Integrons, β-Lactamase genes and molecular characterization of sulfonamide resistance in Escherichia coli Isolates recovered from poultry in China. Pakistan Veterinary Journal 33, 321-324. Leverstein-van Hall, M.A., Dierikx, C.M., Cohen Stuart, J., Voets, G.M., van den Munckhof, M.P., van EssenZandbergen, A., Platteel, T., Fluit, A.C., van de Sande-Bruinsma, N., Scharinga, J., Bonten, M.J., Mevius, D.J., 2011. Dutch patients, retail chicken meat and poultry share the same ESBL genes, plasmids and strains. Clinical Microbiology Infection 17, 873-880. Liao, X., Fang, L., Li, L., Sun, J., Li, X., Chen, M., Deng, H., Yang, Q., Liu, Y., 2015. Characterization of chromosomal qnrB and ampC alleles in Citrobacter freundii isolates from different origins. Infect., Genet. Evol. 35, 214-220. Locatelli, C., Scaccabarozzi, L., Pisoni, G., Moroni, P., 2010. CTX-M1 ESBL-producing Klebsiella pneumoniae subsp. pneumoniae isolated from cases of bovine mastitis. J. Clin. Microbiol. 48, 3822-3823. Mendes, R.E., Kiyota, K.A., Monteiro, J., Castanheira, M., Andrade, S.S., Gales, A.C., Pignatari, A.C., Tufik, S., 2007. Rapid detection and identification of metallo-beta-lactamase-encoding genes by multiplex real-time PCR assay and melt curve analysis. J. Clin. Microbiol. 45, 544-547. Mohamudha Parveen, R., Manivannan, S., Harish, B.N., Parija, S.C., 2012. Study of CTX-M Type of extended spectrum -Lactamase among nosocomial isolates of Escherichia coli and Klebsiella pneumoniae in south India. Indian J. Microbiol. 52, 35-40. Monteiro, J., Widen, R.H., Pignatari, A.C., Kubasek, C., Silbert, S., 2012. Rapid detection of carbapenemase genes by multiplex real-time PCR. J. Antimicrob. Chemother. 67, 906-909. Nam, Y.S., Cho, S.Y., Yang, H.Y., Park, K.S., Jang, J.H., Kim, Y.T., Jeong, J.W., Suh, J.T., Lee, H.J., 2013. Investigation of mutation distribution in DNA gyrase and topoisomerase IV genes in ciprofloxacin-non-susceptible Enterobacteriaceae isolated from blood cultures in a tertiary care university hospital in South Korea, 2005-2010. Int. J. Antimicrob. Agents 41, 126-129. Ohnishi, M., Okatani, A.T., Harada, K., Sawada, T., Marumo, K., Murakami, M., Sato, R., Esaki, H., Shimura, K., Kato, H., 2013. Genetic characteristics of CTX-M-type extended-spectrum-β-lactamase (ESBL)-producing Enterobacteriaceae involved in mastitis cases on Japanese dairy farms, 2007 to 2011. J. Clin. Microbiol. 51, 31173122. Osman, K.M., Hassan, H.M., Orabi, A., Abdelhafez, A.S., 2014. Phenotypic, antimicrobial susceptibility profile and virulence factors of Klebsiella pneumoniae isolated from buffalo and cow mastitic milk. Pathogens and Global Health 108, 191-199. Paterson, D.L., Bonomo, R.A., 2005. Extended-spectrum beta-lactamases: a clinical update. Clin. Microbiol. Rev. 18, 657–686. Poirel, L., Walsh, T.R., Cuvillier, V., Nordmann, P., 2011. Multiplex PCR for detection of acquired carbapenemase genes. Diagn. Microbiol. Infect. Dis. 70, 119-123. Polsfuss, S., Bloemberg, G.V., Giger, J., Meyer, V., Bottger, E.C., Hombach, M., 2011. Practical approach for reliable detection of AmpC beta-lactamase-producing Enterobacteriaceae. J. Clin. Microbiol. 49, 2798-2803. Quinn, P.J., Carter, M.E., Markey, B., Carter, G.R., 1994. Clinical Veterinary Microbiology. Wolfe Publishing, London Radostits, O., Gay, C., Blood, D., Hinchcliff, K., 2000. Veterinary Medicine: A Textbook of the Diseases of Cattle, Sheep, Pigs, Goats and Horses, WB Saunders. New York, USA. Rao, S.P., Rama, P.S., Gurushanthappa, V., Manipura, R., Srinivasan, K., 2014. Extended spectrum -Lactamases producing Escherichia coli and Klebsiella pneumoniae: A multi-centric study across Karnataka. Journal of Laboratory Physicians 6, 7-13. Saishu, N., Ozaki, H., Murase, T., 2014. CTX-M-type extended-spectrum lactamase-producing Klebsiella pneumoniae isolated from cases of bovine mastitis in Japan. J. Vet. Med. Sci. 76, 1153-1156. Samanta, I., Joardar, S.N., Mahanti, A., Bandyopadhyay, S., Sar, T.K., Dutta, T.K., 2015. Approaches to characterize extended spectrum -lactamase producing Escherichia coli in healthy organized vis-a-vis backyard farmed pigs in India. Infect., Genet. Evol. 36, 224-230.

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Figure legends:

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Schwaiger, K., Bauer, J., Holzel, C.S., 2013. Selection and persistence of antimicrobial-resistant Escherichia coli including extended-spectrum beta-lactamase producers in different poultry flocks on one chicken farm. Microb. Drug Resist. 19, 498-506. Shahid, M., Sobia, F., Singh, A., Khan, H.M., 2012. Concurrent occurrence of blaampC families and blaCTX-M genogroups and association with mobile genetic elements ISEcp1, IS26, ISCR1, and sul1-type class 1 integrons in Escherichia coli and Klebsiella pneumoniae isolates originating from India. J. Clin. Microbiol. 50, 1779-1782. Strahilevitz, J., Jacoby, G.A., Hooper, D.C., Robicsek, A., 2009. Plasmid-mediated quinolone resistance: a multifaceted threat. Clin. Microbiol. Rev. 22, 664-689. Timofte, D., Maciuca, I.E., Evans, N.J., Williams, H., Wattret, A., Fick, J.C., Williams, N.J., 2014. Detection and molecular characterization of Escherichia coli CTX- M-15 and Klebsiella pneumoniae SHV-12 producing βlactamases isolated from cases of bovine mastitis in the United Kingdom. Antimicrob. Agents Chemother. 58, 789794. Tripathi, A., Dutta, S.K., Majumdar, M., Dhara, L., Banerjee, D., Roy, K., 2012. High prevalence and significant association of ESBL and QNR genes in pathogenic Klebsiella pneumoniae isolates of patients from Kolkata, India. Indian J. Microbiol. 52, 557-564. Upadhyay, S., Hussain, A., Mishra, S., Maurya, A.P., Bhattacharjee, A., Joshi, S.R., 2015. Genetic environment of plasmid mediated CTX-M-15 extended spectrum -lactamases from clinical and food borne bacteria in north-eastern India. PLoS One 10, e0138056. Vogt, D., Overesch, G., Endimiani, A., Collaud, A., Thomann, A., Perreten, V., 2014. Occurrence and genetic characteristics of third-generation cephalosporin-resistant Escherichia coli in Swiss retail meat. Microb. Drug Resist. 20, 485-494. Warjri, I., Dutta, T., Lalzampuia, H., Chandra, R., 2015. Detection and characterization of extended-spectrum βlactamases (blaCTX-M-1 and blaSHV) producing Escherichia coli, Salmonella spp. and Klebsiella pneumoniae isolated from humans in Mizoram. Veterinary World 8, 599-604. Weill, F.X., Demartin, M., Fabre, L., Grimont, P.A., 2004. Extended-spectrum-beta-lactamase (TEM-52)-producing strains of Salmonella enterica of various serotypes isolated in France. J. Clin. Microbiol. 42, 3359-3362.

Fig. 1: Phylogenetic analysis of the predicted amino acid sequences from he genbank KR812384 and other closely related CTX-M genes. Fig. 2: Predicted amino acid sequences of the genbank KR812384 and other closely related CTX-M genes. The red under line denotes amino acid substitutions - glutamic acid in place of lysine at 130th and arginine in place of tryptophan at 143rd positions.

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Fig. 1

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Fig. 2

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22

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Oligonucleotide sequences (5’— 3’) gyrA F: CGC GTA CTA TAC GCC ATG AAC GTA R: ACC GTT GAT CAC TTC GGT CAG G rpoB F: CAA CGG TGT GGT TAC TGA CG R: TCT ACG AAG TGG CCG TTT TC pehX F: GAT ACG GAG TAT GCC TTT ACG GTG R:TAG CCT TTA TCA AGC GGA TAC TGG blaTEM F: ATA AAA TTC TTG AAG ACG AAA R: GAC AGT TAC CAA TGC TTA ATC A blaCTXM F: TTT GCG ATG TGC AGT ACC AGT AA R: CGA TAT CGT TGG TGG TGC CAT A blaSHV F: AGG ATT GAC TGC CTT TTT G R: ATT TGC TGA TTT CGC TCG

PT

Table 1: PCR conditions and oligo nucleotide primers used for detection of different antibiotic resistance and virulence genes for characterization of ESBL producing Klebsiella spp. isolated from milk of the healthy cows and cows with clinical and subclinical mastitis

PCR sequence

1

RI

Sl.no Genes

blaampC

F: CCC CGC TTA TAG AGC AAC AA R: TCA ATG GTC GAC TTC ACA CC

8

qnrS

F: GCA AGT TCA TTG AAC AGG GT R: TCT AAA CCG TCG AGT TCG GCG

9

qnrB

10

qnrA

F: GAT CGT GAA AGC CAG AAA GG R:ATG AGC AAC GAT GCC TGG TA F: ATT TCT CAC GCC AGG 23 ATT TG F: GAT CGG CAA AGG TTA

95x5 m / 95X1 m 56x1 m -72x1 m (35Cycles) / 72x10 m 95x5 m / 95X1 m 56x1 m -72x1m (35Cycles) / 72x10

6

AC CE P

5

TE

D

4

NU

3

MA

2

SC

7

95x5 m / 95x30 s 55x1m -72x2 m (35Cycles) / 72x10 m 95x3 m / 95x30 s 55x1 m -72x2 m (35Cycles) / 72x10 m 95x3 m / 95x30 s 55x1 m -72x2 m (35Cycles) / 72x10 m 94x5 m / 94x1 m 55x1 m -72x1 m (40Cycles) / 72x10 m 94x3 m / 95x20 s 51x30 s -72x30 s (35Cycles) / 72x10 m 94x5 m / 94x30 s 60x30 s -72x30 s (20Cycles) / 94x30 s -58x30 s -72x30 s (15Cycles) / 72x7 m 94x5 m / 94x30 s 58x2 m -72x60 s (10Cycles) / 94x30 s -55x1 m -72x60 s (15Cycles) / 94x30 s -50x30 s -72x1 m (10Cycles) 72x10 m 95x5 m / 95X1 m 56x1 m -72x1 m (35Cycles) / 72x10 m

Amplicon References size 441 bp

108 bp

Chander et al., 2011

343 bp

1080 bp

Weill et al., 2004

544 bp

Edelstein et al., 2003

393 bp

Kar et al., 2015

634 bp

Kar et al., 2015

428 bp

Kar et al., 2015

476 bp

516 bp

ACCEPTED MANUSCRIPT GGT CA

aac

13

Sul1

14

Int1

15

Int2

16.

blaKPC

17.

blaNDM

18.

blaOXA48

19.

blaIMP

20.

blaVIM

218 bp

Ferjani et al., 2015

509 bp

Ferjani et al., 2015

433 bp

Kar et al., 2015

481 bp

Kashif et al., 2013

250 bp

Kashif et al., 2013

785 bp

Monteiro et al., 2012

621 bp

Poirel et al., 2011

177 bp

Monteiro et al., 2012

120 bp

Mendes et al., 2007

382 bp

Mendes et al., 2007

RI

12

94x5 m / 94x30 s 50x30 s -72x60 s (35Cycles) / 72x10 m 94x5 m / 94x1 m 56x1 m -72x1 m (10Cycles) / 94x1 m -54x1 m -72x1 m (15Cycles) / 72x10m F: CGG CGT GGG CTA CCT 95x5 m / 95X30 s GAACG 65x1 m -72x1 m R: GCC GAT CGC GTG AAG (35Cycles) / 72x10 TTC CG m F: 94×5 m / 94×30 s GGTCAAGGATCTGGATTTCG 60×20 s - 72×1 m R: (35Cycles) / 72×10 GGTCGCGAGTGACGGCTTTGT m F: TTATTGCTGGGATTAGGC 94×5 m / 94×30 s R: ACGGCTACCCTCTGTTATC 60×30 s - 72×1 m (35Cycles) / 72×10 m F: TCGCTAAACTCGAACAGG 94x5 m / 94X1 m R: 60x1 m -72x1m (35Cycles) / 72x10 TTACTGCCCGTTGACGCCCAATCC m F: GTTTGGCGATCTGGTTTTC 94x5 m / 94X0.5 m52x40s-72x1m R: CGGAATGGCTCATCACGATC (35Cycles) / 72x10 m 95x5 m /95x20sF: TGTTTTTGGTGGCATCGAT 55°C X45 s/72°C X R: GTAAMRATGCTTGGTTCGC 30 s(35Cycles) / 72x10 m 95x5 m /95x20sF: GAGTGGCTTAATTCTCRATC 55°C X45 s/72°C X R: AACTAYCCAATAYRTAAC 30 s(35Cycles) / 72x10 m 95x5 m /95x20sF: GTTTGGTCGCATATCGCAAC 55°C X45 s/72°C X R: AATGCGCAGCACCAGGATAG 30 s(35Cycles) / 72x10 m

PT

F: GCA GGT CCA GCA GCG GGT AG 3’ R: CTT CCT GCC CGA GTA TCG TG 3’ F: TGA CCT TGC GAT GCT CTA TG R: TTA GGC ATC ACT GCG TGT TC

SC

qepA

AC CE P

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11

m

24

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PT

Table 2: Risk factors influencing the frequency of ESBL producing Klebsiella pneumoniae isolated from milk of healthy cows and cows with clinical and subclinical mastitis

Samples positive for Klebsiella spp. (%)

Subclinical mastitis

159

109 (68.5)

Clinical mastitis

52

31 (59.6)

Healthy

129

340

No. of Samples with ESBL producing K. pneumoniae (%)

No. of ESBL producing K. pneumoniae

181

12 (7.5)*

12

74

7 (13.4)*

9

14 (10.8)

36

2 (1.5)

2

154 (45.3)

291

23 (6.7)

23

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Total

No. of Klebsiella spp. isolated

NU

No. of samples

MA

Clinical status of the animal

SC

*The frequency of isolation of such pathogens was significantly higher (P =0.006, χ 2= 10.04,

df= 2) in milk of cows with bovine clinical (13.4 %) and subclinical (7.5 %) mastitis than that of the healthy cows (1.5 %).

25

ACCEPTED MANUSCRIPT Table 3: Details of resistance of ESBL producing Klebsiella pneumoniae isolated from bovine milk samples from three states of India. Drug MIC (μg/ml) sensitivity CAZ CTX profile

1

JLF8K1

JH

blaTEM, blaCTXM, int1, sul1, blaampC

FOX, IMP, MEM, C. CIP, GAT, PTZ

2

JRH13K2

JH

blaCTXM , sul1, blaampC, qnrS

3

JLF3K1

JH

blaCTXM , sul1, blaampC

-

4

JRFIK2

JH

blaCTXM , blaampC, qnrS

-

5

JRH4K1

JH

blaTEM, blaCTXM, int1, qnrS,

dfrA12attC— gcuF-

MEM, GEN, TOB, SULTM, C , PTZ IMP, MEM, C, CIP, GAT, PTZ, FEP FOX, FEP, IMP, MEM, C, TOB, SULTM, CIP, GAT, PTZ FOX, IMP, MEM,

dfrA12attC— gcuFattCaadA2bqattC3’CS -

PT

Resistance Gene determinants cassettes in class I Integron

CRO

ERIC cluster

128

256

256

5

64

256

256

6

128

256

256

3

128

256

256

7

128

256

256

4

RI

State

SC

Isolate name

AC CE P

TE

D

MA

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Sl. no

26

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JH

blaTEM, blaCTXM, int1, sul1, blaampC,

7

J6MK2

JH

blaCTXM, qnrS, blaampC

8

J5K2

JH

blaCTXM qnrS, blaampC

9

JRF12K1

JH

blaCTXM , blaampC

-

10

JKP5

JH

blaTEM, blaSHV, sul1, int1, qnrS

dfrA17attCaadA5attC

11

WM2K1

WB

blaSHV

-

D

MA

-

-

TE

AC CE P

FOX, FEP, IMP, MEM, C, GEN, TOB, SULTM, NOR, GAT FOX, IMP, MEM, C, TOB FOX, IMP, MEM, C, GEN, PTZ, SUL-TM MEM, NOR, TOB, C, CIP, GAT FOX, IMP, MEM, C, CIP, GAT, TE MEM, TOB, C, CIP, FEP,

27

128

256

256

5

128

256

256

5

32

256

256

5

64

128

128

5

4

256

256

5

124

256

256

5

PT

JLF4K1

NU

6

PTZ, C

RI

attCaadA2bqattC3’CS dfrA12attC— gcuFattCaadA2bqattC3’CS

SC

blaampC,

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HFK6

WB

blaTEM, int1, sul1, blaampC,

14

NA1K2

WB

blaCTXM, int1, sul1, blaampC

15

GF4K3

16

17

124

256

256

2

PT

13

dfrA12attC— gcuFattCaadA2bqattC3’CS dfrA12attC— gcuFattCaadA2bqattC3’CS dfrA17attCaadA5attC

RI

blaCTXM, int1, sul1, blaampC

SC

WB

IMP, MEM, GEN

64

256

256

2

IMP, MEM, C, CIP, GAT, TE IMP, MEM, PTZ, FEP, C

256

256

256

1

32

128

256

8

256

256

5

64

64

5

NU

HFK5

AC CE P

TE

D

MA

12

GAT, PTZ, SULTM, TE MEM, C, FEP, GAT

WB

blaTEM, blaCTXM, int1, sul1, qnrS blaampC

CTWK1

WB

blaCTXM, int1, sul1, blaampC

WBKP17

WB

blaCTXM, blaampC,

dfrA12attC— gcuFattCaadA2bqattC3’CS dfrA12attC— gcuFattCaadA2bqattC3’CS -

28

IMP, 256 MEM, C, GAT

FEP, IMP,

4

ACCEPTED MANUSCRIPT

1SH7K2

MZ

blaCTXM , blaSHV, blaampC, qnrS

20

2L9K1

MZ

blaampC, int1

21

MZKP23

MZ

blaCTXM , blaampC, qnrS, int1

22

MZKP76

MZ

blaTEM, blaCTXM, qnrS,

23

MZKP189 MZ

blaTEM, blaCTXM, blaampC, int1

TE

AC CE P

256

256

5

PT

19

64

RI

blaCTXM, sul1, blaampC

128

256

256

5

128

256

256

6

128

256

256

NT

32

16

64

NT

128

256

256

NT

SC

MZ

NU

3L18K1

D

18

MEM, PTZ, C IMP, MEM, C, CIP, GAT, TE MEM, PTZ, GEN, TOB, SULTM, C dfrA17FOX, attCFEP, aadA5IMP, attC MEM, C, CIP, GAT dfrA12IMP, attC— MEM, gcuFC, CIP, attCGAT, aadA2bq- SULattCTM, TE 3’CS IMP, MEM, C, FEP, PTZ, SULTM, TE dfrA17IMP, attCMEM, aadA5PTZ, attC CIP, GAT

MA

qnrB

29

ACCEPTED MANUSCRIPT

AC CE P

TE

D

MA

NU

SC

RI

PT

CAZ: ceftazidime, CTX: cefotaxime, CRO: ceftriaxone, FOX: cefoxitin, MEM: meropenem, CIP: ciprofloxacin, FEP: cefepime, TOB: tobramycin, GEN: gentamicin, SUL-TM: sulpha/trimethoprim, TE: tetracycline, Nor: Norfloxacin, PTZ: piperacillin/ tazobactam, GAT: gatifloxacin, C: chloramphenicol, IMP: imipenem. JH: Jharkhand, WB: West Bengal, MZ: Mizoram, ERIC: Enterobacterial Repetitive Intergenic Consensus, MIC: Minimum Inhibitory Concentration, NT: Non Typable.

30

ACCEPTED MANUSCRIPT Highlights We report multi-drug resistant Klebsiella pneumoniae in bovine milk.



The isolates were ESBL & ACBL producers & mostly carried ISEcp1-blaCTX-Morf477



Class I integron carried dfrA12/dfrA17 and aadA2/aadA5 gene cassettes.



Isolation rate was significantly higher in mastitic bovine milk



Emergence of such pathogen may be disastrous for herd economy and public

SC

RI

PT



AC CE P

TE

D

MA

NU

health.

31