Characterization of Multidrug-Resistant Biofilm-Producing Escherichia coli and Klebsiella pneumoniae in Healthy Cattle and Cattle with Diarrhea

Abstract

This study describes comparative occurrence and characterization of multidrug-resistant (MDR) Escherichia coli and Klebsiella pneumoniae (KP) in healthy cattle (HC) and cattle with diarrhea (DC) in India. During 2018–2020, 72 MDR isolates, including 35 E. coli (DC: 27; HC 8) and 37 K. pneumoniae (DC: 34; HC: 3), from 251 rectal swabs (DC: 219; HC: 32) were investigated for extended-spectrum beta-lactamase (ESBL), AmpC type β-lactamase and carbapenemase production, antimicrobial susceptibility profile, biofilm production, and efflux pump activity. Fifty-five MDR isolates were ESBL producers (ESBLPs) (DC: 50; HC: 5) and ESBLPs from DC were coresistant to multiple antibiotics. The blaCTX-M gene (50) was the most frequently detected β-lactamases followed by blaAmpC (22), blaTEM1 (13), blaCMY-6 (6), blaOXA1 (5), blaPER (2), blaDHA, and blaFOX and blaSHV12 (1 each). Plasmid-mediated quinolone resistance determinants qnrB, qnrS, qnrA, and qepA were detected in 18, 16, 2, and 3 isolates, respectively. Twenty three isolates revealed mutation in gyrA and parC genes. Tetracycline-resistance markers tetA, tetB, tetC, and tetE were detected in 33, 10, 3, and 2 isolates, respectively. Only one of the 41 imipenem-resistant isolates harbored blaNDM-5 and two were colistin-resistant. Altogether, 20 MDR isolates were strong biofilm producers and 19 harbored different virulence factors. This is the first ever report from India on the presence of MDR Enterobacteriaceae with resistance to even last-resort antimicrobials in the bovine diarrhea.

Introduction

Human civilization is at risk of zoonotic infection via direct contact because of proximity with food animals, environmental contamination, and intake of contaminated food items causing enormous economic losses associated with loss of man-days. The situation becomes more complicated due to the transmission of antimicrobial-resistant bacteria such as extended-spectrum beta-lactamase (ESBL)-producing bacteria into the human food chain from the animals. Cattle are considered as a major source of cheap protein as well as an important source of biological fertilizer throughout the world.¹ Escherichia coli and Klebsiella pneumoniae are enlisted as critical sentinel organisms for antimicrobial resistance in food animals, human and environment due to ubiquitous presence as commensal or pathogen and horizontal transfer of antibiotic resistance determinants such as ESBLs between the strains.²

ESBLs are essential resistance determinants transmitted through horizontal gene transfer in the Enterobacteriaceae group of commensal or pathogens.³ The production of the enzyme is associated with resistance to a variety of β-lactam antibiotics, including penicillins, higher-generation cephalosporins, and monobactams (e.g., aztreonam), but usually not with carbapenems or cephamycins (e.g., cefoxitin). Moreover, AmpC β-lactamase-producing organisms can produce resistance against cephalosporins, penicillins, cephamycins, and monobactams and cannot be inhibited by β-lactamase inhibitors such as clavulanic acid. The chromosomal blaAmpC of E. coli is repressed or weakly expressed, although mutation in the promoter region may cause constitutive overexpression. There are three classical ESBLs, that is, TEM (except TEM-1), SHV (except SHV-1 and 2), and CTX-M.⁴ Among them, CTX-M is observed as the most prevalent type in clinically infected human patients world wide.⁵

Carbapenem is the last resort antibiotic to treat β-lactamase-producing Enterobacteriaceae.⁶ Carbapenem-resistant E. coli/K. pneumoniae (CRE) are emerging concern associated with the production of metallo-β-lactamase (VIM), imipenemase (IPM), New Delhi metallo-β-lactamase (NDM-1), oxacillinase (OXA-48), and K. pneumoniae carbapenemase (KPC).⁷ On the contrary, colistin (COL) has gained the confidence of the clinicians as a typical drug of choice in the infections caused by CRE. Currently, gram-negative bacteria (GNB) from food animals showing resistance to carbapenem and COL has emerged as a serious concern in several countries, including India.^8,9 Multidrug-resistant (MDR) K. pneumoniae and E. coli isolates also possess quinolone resistance genes (PMQR, qnrA, qnrB, qnrC, qnrD, and qnrS), efflux pumps (qepA), sulfonamide resistance (sul1, sul2, and sul3), aminoglycoside-resistance (ArmA, RmtA, RmtB, RmtC, RmtD, and NpmA), and tetracycline-resistance (TC-r) genes (tetA, tetB, tetC, tetD, and tetE).^10–14

Resistance genes are often associated with biofilm formation among clinical isolates of K. pneumoniae and E. coli.¹⁵ Biofilms are microbial communities adhered to biotic or abiotic surfaces and act as a major site for horizontal gene transfer. The phenotypic transformation from planktonic cells to immobile stage is a coordinated process regulated by environment and putative factors.¹⁶

Food animals such as cattle are considered as reservoirs of MDR bacteria, including ESBL/AmpC-producing K. pneumoniae and E. coli.¹⁷ Although there is no direct evidence of MDR—Enterobacteriaceae transmission into human food chain from cattle, a few studies elucidated the pseudovertical transmission from parent broiler flocks to hatcheries.¹⁸ This kind of transmission can be further extended into the community through the farm handlers. Generation of MDR bacteria in food animals is associated with direct exposure to antibiotics, which might be more in adult cattle and calves suffering from diarrhea due to frequent therapeutic intervention. Neonatal calf diarrhea (NCD) is a multietiological complex, in which enterotoxigenic or enteropathogenic E. coli plays a vital role.¹⁹ The studies associated with occurrence of ESBL/AmpC-producing Klebsiella and E. coli in cattle are limited,^20,21 and no study is apparently available to depict a comparative profiling of MDR bacteria from the healthy cattle (HC) and from diarrheic cattle or calves with NCD. Thus, the present study was conducted to detect and characterize MDR K. pneumoniae and E. coli isolates from rectal swabs of diarrheic and asymptomatic cattle from five agro-climatic zones of West Bengal (India), the state with one of the largest cattle populations in the country with an increasing trend.²²

Materials and Methods

Sample collection, isolation, and identification

Rectal swab samples (n: 219) were aseptically collected from adult cattle (n: 46) and calves with diarrhea (n: 173) from different districts of five agro-climatic zones of West Bengal, India—Vindhyan Alluvial Zone (n: 38), Gangetic Alluvial Zone (n: 67), Undulating Red and Laterite Zone (n: 36), Caostal Saline Zone (n: 51), and Terai-Teesta Alluvial Zone (n: 29). The animals were maintained in the semi-intensive stall-feeding system with an average herd size of 10–30. The calves suffering with NCD were treated with gentamicin, potentiated amoxicillin, tetracycline, and cephalosporins. Few adult cattle earlier suffered from mastitis and were treated with intramammary infusion of cefoperazone and cefquinome.

Following overnight enrichment, the samples were plated on MacConkey agar, from which 3–5 lactose fermenting colonies were picked up randomly for incubation in nutrient broth to run standard biochemical tests for presumptive identification. Furthermore, the isolates were subjected to molecular tests, a pentaplex PCR covering five genes (lacY, lacZ, cydA, uidA, and phoA) and a duplex PCR for two genes (gyrA and pehX) for confirmation of E. coli²³ and K. pneumoniae,²⁴ respectively. Likewise, rectal swab samples were also collected from healthy or asymptomatic cattle (n: 32) and processed similarly.

Antibiotic susceptibility testing

Confirmed E. coli and K. pneumoniae isolates were subjected to antimicrobial susceptibility testing (AST) for 11 classes of antimicrobials namely, penicillin (ampicillin, AMP), potentiated penicillin (amoxicillin-clavulanic acid, AMC), aminoglycosides (gentamicin, GEN), cephalosporin [ceftiofur (EFT 30 μg); cefpodoxime (CPD); ceftazidime (CAZ); cefotaxime (CTX); ceftriaxone (CTR)], monobactam [aztreonam (AZT)], fluoroquinolone (FQ) [nalidixic acid (NAL); enrofloxacin (ENR 30 μg)], carbapenem (IPM), dihydrofolate reductase inhibitor, and sulfonamide [sulfamethoxazole with trimethoprim (SXT, 23.75/1.25 μg)], tetracyclines [tetracycline (tet)], phenicols [chloramphenicol (C, 30 μg)], and polymixin (COL). While disc diffusion test was conducted for AST against ENR, EFT, C, and SXT, broth dilution assay was used for COL-resistance determination. For other antibiotics, agar dilution assay was used. The antibiotic susceptibility of the isolates were determined following the breakpoints prescribed by Clinical and Laboratory Standards Institute (CLSI).²⁵ Isolates resistant to more than three different classes of antimicrobials were categorized as MDR. For each isolate, the multiple antibiotic resistance index (MARI) was calculated as the ratio of the number of antibiotics, to which the isolate exhibited resistance to the number of antibiotics, to which the isolate had been tested.²⁶

ESBL and AmpC enzyme detection

All the MDR isolates, which were resistant to any of the extended-spectrum cephalosporins (ESCs) or monobactam, were evaluated for ESBL and AmpC type β-lactamase production using three phenotypic tests—combination disc (CD) test, cefoxitin-cloxacillin double-disc synergy test (CC-DDS), and Ezy MIC™ ESBL-AmpC coexistence detection test (HiMedia, India). In CD test, pairs of discs containing ESCs (cefotaxime or ceftazidime 30 μg) alone or with CA (30/10 μg) were placed at a distance of 25 mm, center to center, on a cation-adjusted Mueller Hinton agar (MHA) plate inoculated with the test isolates. An increase in the inhibition zone diameter of 5 mm or more for an ECS+CA disc versus ESC disc alone was indicative of ESBL production.³ Modified Three Dimensional Tests (M3DT) was performed following the way prescribed by Shahid et al.²⁷ with little modification. A cefoxitin disc (30 μg FOX) was placed at the center of an MHA plate inoculated with a lawn of E. coli ATCC 25922. Multiple linear slits of 3 cm were made radially, 3 mm away from the edge of FOX disc with a well at end of each slit. The enzyme extract (40 μL) of the test isolates prepared by repeated freeze-thawing was put into the wells. Following 18–24 hr of incubation at 37°C, isolates showing clear distortion at the edge of the slit were noted. To confirm the AmpC production, the test was repeated after incubating the extract with a 5 μg cloxacillin disc for 30 min at 37°C before being discharged in the well. Inhibition of zone distortion following cloxacillin treatment confirmed AmpC production. Ezy MIC ESBL-AmpC coexistence was determined using improved ESBL and AmpC detection Ezy MIC strips following the manufacturer's guideline (HiMedia).

Metallo-β-lactamase detection

To confirm carbapenemase/metallo-β-lactamases (MBL) production, the isolates with reduced IPM susceptibility were further subjected to modified carbapenem inactivation method (mCIM)²⁸ and Carba NP direct tests (CNPt-direct).²⁹ In mCIM assay, a meropenem disc (MEM, 10 μg) was coincubated with test bacteria in 2 mL trypticase soy broth at 35°C for 4 hr and then placed on MHA plate, previously streaked with 0.5 McFarland suspension of E. coli ATCC^® 25922, for further overnight incubation at 35°C. A zone diameter of 6–15 mm or presence of colonies within a 16–18 mm zone (indicative of inactivated MEM) was considered positive for MBL. For CNPt-direct test, 1 μL loop-full bacterial mass, scraped off from its overnight growth, was suspended in two separate 1.5-mL microfuge tubes—(1) reaction tube containing 100 μL of aqueous indicator solution comprising 0.1% (v/v) of Triton X-100, 0.05% phenol red with 0.1 mmol/L ZnSO4, (pH 7.8) and 12 mg/mL IMP-cilastatin (equivalent to 6 mg of IPM) and (2) control tube having the above-mentioned solution without IPM-cilastatin. The suspension was vigorously mixed and incubated at 35°C for 2 hr for any appreciable color change (red to orange/yellow) in the reaction tube indicating MBL production.

MEM carbonyl cyanide-m-chlorophenylhydrazone disc synergy test

All the IMP-R isolates were further incubated on MH agar with two MEM discs placed 20 mm apart—one with 30 μg of MEM and another with 30 μg of MEM and 200 μg CCCP (carbonyl cyanide-m-chlorophenylhydrazone) for 24 hr at 37°C. An increase of 5 mm or more in zone diameter around MEM-CCCP disc than that of the MEM disc alone was interpreted as positive for EP-mediated carbapenem- resistance.³⁰

Assessment of efflux pump activity

To investigate the role of the EP in FQ and polymixin resistance, all the ENR/NAL- and COL- nonsusceptible isolates were screened for MIC_CIP and MIC_COL using the EP inhibitors at fixed subinhibitory concentrations—CCCP and phenylalanine arginine β-naphthylamide (PAβN) (Sigma-Aldrich) as prescribed previously.^31,32

Biofilm detection by tissue culture plate method

All the MDR isolates were evaluated for biofilm production using tissue culture plate (TCP) assay,³³ where 100 μL of 100-fold diluted inoculum of 0.5 McFarland units of all the isolates were dispensed in a 96-well TCP containing 100 μL brain–heart infusion broth and incubated for 72 hr at 37°C. After washing the nonadherent cells, the adherent cells were fixed (with 2% sodium acetate) and stained with 0.1% crystal violet for 30 min. Following removal of the excess stain, the dye with the adherent cells was solubilized in ethanol for spectrophotometric measurement of biofilm at 595 nm (Multiskan ELISA Reader, Thermo).

Molecular characterization

All the MDR isolates were screened for the presence of the genes encoding different β-lactamases—blaCTX-M, blaTEM, blaSHV, blaVEB, blaAmpC, different plasmid-mediated AmpC type β-lactamases (pMAmpC), genes conferring carbapenem resistance (CR)- blaKPC, blaNDM, blaOXA-48-like, and blaVIM by PCR^34–36 following extraction of genomic and plasmid DNA. Further, the isolates were screened for the presence of plasmid-mediated quinolone resistance (PMQR) determinants [qnrA, qnrB, qnrS, qnrC, qnrD, aac(6¢)-Ib-cr], quinolone EPs (qepA, and oqxA and B), genes mediating tetracycline- (tetA, tetB, tetC, tetD, and tetE), and sulfonamide resistance (sul1, sul2, and sul3).^37–40 The quinolone-resistant determinant regions (QRDRs) of gyrA and parC were also amplified and sequenced.⁴¹ The COL-R isolates (MIC_COL ≥4 μg/mL) were screened by multiplex PCR for detection of plasmid-based COL resistance genes⁴² and involvement of chromosomal modification if any, as described previously.⁴³ Further, the MDR E. coli isolates were screened for the presence of Shiga toxin-producing genes⁴⁴ and extraintestinal pathogenic factors.⁴⁵ Details regarding oligonucleotides and PCR conditions are described in Table 1. Relative sensitivity and specificity of the PCR used in the study are illustrated in Supplementary Table S1.

Table 1.

Details of the Oligonucleotides and PCR Used in the Study

Primer	Oligonucleotide sequence(5′–3′)	Amplicon size (bp)	PCR cycle	Reaction details	Internal control^a	Reference	GenBank
TEM_for	CATTTCCGTGTCGCCCTTATTC	800	94 × 10 min/94 × 40 sec—60 × 40 sec—72 × 60 sec (30 cycles)/72 × 10 min	0.2 mM dNTPs, 1.5 mM MgCl₂, 0.5 μM each primer	Escherichia coli strain—ivkdr6	³⁶	MT174046
TEM_rev	CGTTCATCCATAGTTGCCTGAC	800			Escherichia coli strain—ivkdr6	³⁶	MT174046
SHV_for	AGGATTGACTGCCTTTTTG	393	94 × 5 min/94 × 30 sec—60 × 30 sec—72 × 30 sec (20 cycles)/94 × 30 sec—58 × 30 sec—72 × 30 sec (10 cycles)/72 × 10 min		Klebsiella pneumoniae strain-HOMBK11	³⁴	MT174044
SHV_rev	ATTTGCTGATTTCGCTCG	393			Klebsiella pneumoniae strain-HOMBK11	³⁴	MT174044
OXA_rev	GGCACCAGATTCAACTTTCAAG	564	94 × 10 min/94 × 40 sec—60 × 40 sec—72 × 60 sec (30 cycles)/72 × 10 min		K. pneumoniae 700063 E. coli strain 15121/b	³⁶	MT371583
OXA_rev	GACCCCAAGTTTCCTGTAAGTG	564			K. pneumoniae 700063 E. coli strain 15121/b		MT371583
CTXMGp1_for	TTAGGAARTGTGCCGCTGYA	688	94 × 10 min/94 × 40 sec—60 × 45 sec—72 × 60 sec (35 cycles)/72 × 10 min	0.25 mM dNTPs, 1.5 mM MgCl₂, 0.3 μM each primer, 0.2 mM dNTPs, 2 mM MgCl₂, 0.25 μM each primer	K. pneumoniae strain JRH4K1, E. coli strain IVRI ERS DK 46 E. coli (Gp1); E. coli strain IVKD91(Gp2); K. pneumoniae strain GF4K3 (8/25); E. coli strain SL-8C (CTX-M 9)		MT174045 MT371584
CTXMGp1_rev	CGATATCGTTGGTGGTRCCAT	688
CTXMGp2_for	CGTTAACGGCACGATGAC	404
CTXMGp2_rev	CGATATCGTTGGTGGTRCCAT	404
CTXMGp9_for	TCAAGCCTGCCGATCTGGT	561
CTXMGp9_rev	TGATTCTCGCCGCTGAAG	561
CTX-Mg8/25_for	AACRCRCAGACGCTCTAC	326
CTX-Mg8/25_rev	TCGAGCCGGAASGTGTYAT	326
ACC_for	CACCTCCAGCGACTTGTTAC	346	94 × 10 min/94 × 40 sec—60 × 40 sec—72 × 60 sec (30 cycles)/72 × 10 min	0.2 mM dNTPs, 2 mM MgCl₂, 0.5 μM each primer	K. pneumoniae strain HOMBK11 (MOX); E. coli strains ivkd6/(CMY-6), E. coli strain ivkd74 (CMY-2); ivkd 105, ivkd 117 (FOX Gr); ivkd132 (ACC Gr); ivkd98 (DHA)	³⁶	NA
ACC_rev	GTTAGCCAGCATCACGATCC	346					NA
FOX_for	CTACAGTGCGGGTGGTTT	162					MT154086
FOX_rev	CTATTTGCGGCCAGGTGA	162					MT154086
MOX_for	GCAACAACGACAATCCATCCT	895					NA
MOX_rev	GGGATAGGCGTAACTCTCCCAA	895					NA
DHA_for	TGATGGCACAGCAGGATATTC	997					MT174047
DHA_rev	GCTTTGACTCTTTCGGTATTCG	997					MT174047
CIT_for	CGAAGAGGCAATGACCAGAC	538					MT154088
CIT_rev	ACGGACAGGGTTAGGATAGT	538					MT154088
EBC_for	CGGTAAAGCCGATGTTGCG	683					NA
EBC_rev	AGCCTAACCCCTGATACA	683					NA
GES_for	AGTCGGCTAGACCGGAAAG	399	94 × 10 min/94 × 40 sec—60 × 40 sec—72 × 60 sec (30 cycles)/72 × 10 min	0.2 mM dNTPs, 2 mM MgCl₂, 0.5 μM each primer	K. pneumoniae strain BBSCM-1 (VEB), E. coli strains ivrikoldentrh57a (VEB); ivkd89 (GES); ivkd 144(PER)	³⁶	NA
GES_rev	TTTGTCCGTGCTCAGGAT	399					NA
PER_for	GCTCCGATAATGAAAGCGT	520					MT154087
PER_rev	TTCGGCTTGACTCGGCTGA	520					MT154087
VEB_for	CATTTCCCGATGCAAAGCGT	648					NA
VEB_rev	CGAAGTTTCTTTGGACTCTG	648					NA
GES_for	AGTCGGCTAGACCGGAAAG	399					NA
GES_rev	TTTGTCCGTGCTCAGGAT	399					NA
OXA-48_for	GCTTGATCGCCCTCGATT	281	94 × 10 min/94 × 40 sec—60 × 40 sec—72 × 60 sec (30 cycles)/72 × 10 min	0.3 mM dNTPs, 1.5 mM MgCl₂, 0.5 μM each primer	E. coli strain ivkd205	³⁶	NA
OXA-48_rev	GATTTGCTCCGTGGCCGAAA	281		0.3 mM dNTPs, 1.5 mM MgCl₂, 0.5 μM each primer	E. coli strain ivkd205	³⁶	NA
IMP_for	TTGACACTCCATTTACDG	139		0.5 mM dNTPs, 2 mM MgCl₂, 0.5 μM each primer	E. coli strains-VMC/E. coli/2017/74 (KPC); ivkdr6 (NDM); ivkd139 (IMP); ikd124 (VIM)	³⁶	NA
IMP_rev	GATTGAGAATTAAGCCACTCT	139					NA
VIM_for	GATGGTGTTTGGTCGCATA	390					NA
VIM_rev	CGAATGCGCAGCACCAG	390					NA
KPC_for	CATTCAAGGGCTTTCTTGCTGC	538					NA
KPC_rev	ACGACGGCATAGTCATTTGC	538					NA
blaAmpC for	CCCCGCTTATAGAGCAACAA	631	94 × 5 min/94 × 30 sec—57 × 30 sec—72 × 30 sec (30 cycles)/72 × 10 min	0.25 mM dNTPs, 2 mM MgCl₂, 0.2 μM each primer	K. pneumoniae strain JRFIK2	³⁵	MT174049
blaAmpC rev	TCAATGGTCGACTTCACACC	631		0.25 mM dNTPs, 2 mM MgCl₂, 0.2 μM each primer	K. pneumoniae strain JRFIK2	³⁵	MT174049
qnrA for	ATTTCTCACGCCAGGATTTG	516	95 × 5 min/95 × 30 sec—56 × 1 min—72 × 1 min (35 cycles)/72 × 10 min	0.2 mM dNTPs, 2 mM MgCl₂, 0.2 μM each primer	E. coli strain JSD/14552/a (qnrA), K. pneumoniae strain-WBKP17 (qnrB), MUMBK2 (qnrS)	^38,39	MT371576
qnrA rev	GATCGGCAAAGGTTAGGTCA	516					MT371576
qnrB for	GATCGTGAAAGCCAGAAAGG	469					MT371577
qnrB rev	ACGATGCCTGGTAGTTGTCC	469					MT371577
qnrS for	ACGACATTCGTCAACTGCAA	417					MT050524
qnrS rev	TAAATTGGCACCCTGTAGGC	417					MT050524
qepA for	GCAGGTCCAGCAGCGGGTAG	218	96 × 5 min/96 × 1 min—60 × 1 min—72 × 1 min (35 cycles)/72 × 7 min	0.2 mM dNTPs, 2 mM MgCl₂, 0.5 μM each primer	K. pneumoniae strain-NAMBK6		MT174048
qepA rev	CTTCCTGCCCGAGTATCGTG	218		0.2 mM dNTPs, 2 mM MgCl₂, 0.5 μM each primer	K. pneumoniae strain-NAMBK6		MT174048
sul1 for	CGG CGT GGG CTA CCT GAACG	436	95 × 5 min/95 × 30 sec—65 × 1 min—72 × 1 min (35 cycles)/72 × 10 min	0.3 mM dNTPs, 1 mM MgCl₂, 1 μM each primer	K. pneumoniae strain—JLF8K1	^40,41	MW116081
sul1 rev	GCC GAT CGC GTG AAG TTC CG	436		0.3 mM dNTPs, 1 mM MgCl₂, 1 μM each primer	K. pneumoniae strain—JLF8K1		MW116081
sul 2 for	GCGCTCAAGGCAGATGGCATT	293	95 × 5 min/95 × 30 sec—69 × 1 min—72 × 1 min (35 cycles)/72 × 10 min	0.3 mM dNTPs, 3 mM MgCl2, 1 μM each primer	VMC/E. coli/2014/47		NA
sul 2 rev	GCGTTTGATACCGGCACCCGT	293			VMC/E. coli/2014/47		NA
sul 3 for	GAG CAAGAT TTT TGG AAT CG	750	94 × 5 min/95 × 30 sec—53 × 30 min—72 × 1 min (30 cycles)/72 × 10 min		VMC/E. coli/2014/93		NA
sul3 rev	CTA ACC TAG GGC TTTGGA TAT	750			VMC/E. coli/2014/93		NA
gyrA for	AAATCTGCCCGTGTCGTTGGT	344	95 × 10 min/95 × 1 min—55 × 1 min—72 × 1 min (35 cycles)/72 × 10 min	0.2 mM dNTPs, 2 mM MgCl₂, 0.5 μM each primer	E. coli strain ATCC 25922	^40,41	MT371578
gyrA rev	GCCATACCTACGGCGATACC	344		0.2 mM dNTPs, 2 mM MgCl₂, 0.5 μM each primer			MT371578
ParC for	CTGAATGCCAGCGCCAAATT	168	95 × 10 min/95 × 1 min—55 × 1 min—72 × 1 min (35 cycles)/72 × 10 min	0.5 mM dNTPs, 2 mM MgCl₂, 1 μM each primer			MT371582
ParC rev	GCGAACGATTTCGGATCGTC	168		0.5 mM dNTPs, 2 mM MgCl₂, 1 μM each primer			MT371582
blaNDM-5 for	ATGGAATTGCCCAATATTATGCAC	813	95 × 5 min/95 × 30 sec—49 × 30 sec—72 × 30 sec (35 cycles)/72 × 10 min	0.2 mM dNTPs, 1.5 mM MgCl₂, 0.5 μM each primer	ivkdr6	⁷⁹	MT050525
blaNDM-5 rev	TCAGCGCAGCTTGTCGGC	813		0.2 mM dNTPs, 1.5 mM MgCl₂, 0.5 μM each primer	ivkdr6	⁷⁹	MT050525
tetA for	GCT ACA TCC TGC TTG CCT TC	210	95 × 5 min/95 × 30 sec—55 × 60 sec—72 × 60 sec (35 cycles)/72 × 10 min	0.2 mM dNTPs, 2 mM MgCl₂, 0.75 μM each primer	K. pneumoniae strain-MUMBK1(tetA); E. coli strains ivrikoldec16b (tetB); ivrikoldrh6a (tetC); 6390/a (tetD); 14372/GR/b (tetE)	³⁷	MT371586
tetA rev	CAT AGA TCG CCG TGA AGA GG	210					MT371586
tetB for	TTG GTT AGG GGC AAG TTT TG	659					MT371580
tetB rev	GTA ATG GGC CAA TAA CAC CG	659					MT371580
tetC for	CTT GAG AGC CTT CAA CCC AG	418					MT371585
tetC rev	ATG GTC GTC ATC TAC CTG CC	418					MT371585
tetD for	AAA CCA TTA CGG CAT TCT GC	787					NA
tetD rev	GAC CGG ATA CAC CAT CCA TC	787					NA
tetE for	AAA CCA CAT CCT CCA TAC GC	278					MT371579
tetE rev	AAA TAG GCC ACA ACC GTC AG	278					MT371579
Stx 1 for	ATAAATCGCCATTCGTTGACTAC	180	95 × 5 min/95 × 30 sec—65 × 1 min–72 × 1 min (15 cycles)/95 × 30 sec—60 × 1 min–72 × 1 min (20 cycles)/72 × 10 min	0.25 mM dNTPs, 1.5 mM MgCl₂, 0.3 μM each primer	E. coli strains JK1 and JK2 (positive for stx1, stx2, eaeA and hlyA)	⁴⁴	MW384886
Stx 1 rev	AGAACGCCCACTGAGATCATC	180
Stx 2 for	GGCACTGTCTGAAACTGCTCC	255
Stx 2 rev	TCGCCAGTTATCTGACATTCTG	255
eaeA for	GACCCGGCACAAGCATAAGC	384
eaeA rev	CCACCTGCAGCAACAAGAGG	384
hlyA for	GCATCATCAAGCGTACGTTCC	534
hlyA rev	AATGAGCCAAGCTGGTTAAGCT	534
lacY for	CTA CCG GTG AAC AGG GTA CG	289	95 × 5 min/95 × 30 sec—60 × 30 sec—72 × 1 min (35 cycles)/72 × 10 min	0.2 mM dNTPs, 2 mM MgCl₂, 1.5 U TAQ, 0.5 μM for ÿacy, lacZ, cydA and 1 μM for uidA, phoA	E. coli strain ATCC 25922 for all the genes	²³	NA
ÿacy rev	GTC GCT GAA AAA CGC ACT TC	289
lacZ for	ATG AAA GCT GGC TAC AGG AAG G	517
lacZ rev	CTC CAC ACA GTT TCG GGT TTT C	517
cydA for	CCG TAT CAT GGT GGC GTG TGG	398
cydA rev	GCC GGC TGA GTA GTC GTG GAA G	398
uidA for	CGC CGA TGC AGA TAT TCG	603
uidA rev	GCT GTG ACG CAC AGT TCA TAG	603
phoA for	GGT AAC GTT TCA CCG CAG AGT TG	468
phoA rev	CAG GGT TGG TAC ACT GTC ATT ACG	468
gyrA for	CGC GTA CTA TAC GCCATG AAC GTA	441	95 × 5 min/95 × 30 sec—55 × 1 min—72 × 2 min (35 cycles)/72 × 10 min	0.25 mM dNTPs, 2 mM MgCl₂, 0.5 μM each primer, 0.3 mM dNTPs, 2 mM MgCl₂, 0.5 μM each primer	Klebsiella oxytoca strain H4T4 (gyrA and pehX); K. pneumoniae strains HOMBK9, ATCC 700603 (gyrA and rpoB)	²⁴	NA
gyrA rev	ACC GTT GAT CAC TTC GGT CAG G	441
rpoB for	CAA CGG TGT GGT TACTGA CG	108	95 × 3 min/95 × 30 sec—55 × 1 min—72 × 2 min (35 cycles)/72 × 10 min
rpoB rev	TCT ACG AAG TGG CCG TTT TC	108
pehX for	GAT ACG GAG TAT GCCTTT ACG GTG	343
pehX rev	TAG CCT TTA TCA AGC GGA TAC TGG	343
papC for	ACGGCTGTACTGCAGGGTGTGGCG	328	94 × 5 min/94 × 1 min—63 × 1 min—72 × 3 min (30 cycles)/72 × 10 min	0.25 mM dNTPs, 2.5 mM MgCl₂, 0.15 μM each primer	E. coli strain ivkd66	⁴⁵	MW384885
papC rev	ATATCCTTTCTGCAGGGATGCAATA	328		0.25 mM dNTPs, 2.5 mM MgCl₂, 0.15 μM each primer	E. coli strain ivkd66		MW384885
iucD for	AAA ACTGACATCGGATGGC	253	94 × 5 min/94 × 1 min—60 × 1 min—72 × 1 min (35 cycles)/72 × 10 min	0.5 mM dNTPs, 2.5 mM MgCl₂, 1 μM each primer	E. coli strain ivkd73		MW384884
iucD rev	GTATTTGTGGCAACGCAGAA	253		0.5 mM dNTPs, 2.5 mM MgCl₂, 1 μM each primer	E. coli strain ivkd73		MW384884
astA for	CCATCAACACAGTATATCCGA	119	95 × 5 min/95 × 1 min—55 × 1 min—72 × 1 min (35 cycles)/72 × 10 min	0.5 mM dNTPs, 2.5 mM MgCl₂ 0.5 μM each primer	E. coli strain ivrikoldec19a
astA rev	GGTCGCGAGTGACGGCTTTGT	119		0.5 mM dNTPs, 2.5 mM MgCl₂ 0.5 μM each primer	E. coli strain ivrikoldec19a

Genes/groups for which the internal controls used in multiplex reactions are given in parenthesis. Otherwise the internal controls indicate the corresponding primers in the same row.

dNTPs, deoxynucleotide triphosphates; NA, not applicable; TAQ, Taq polymerase.

Cloning and sequencing

Amplicons were purified with Nucleospin gel and PCR purification kit (Takara Bio, Inc., Japan) and cloning vector pMD20-T (Mighty TA cloning Kit, Takara Bio Inc.) or pTZ57/R (Fermentas) were used to clone some selective amplicons as insert. The plasmids containing the expected insert were sequenced using the Big Dye Terminator_ v3.1 Cycle Sequencing Kit (Applied Biosystems, Inc., Foster City, CA) on an automated sequencer (Applied Biosystems 3130 Genetic Analyzer) following the manufacturer's instructions. After sequencing, homology searches were made using BLAST algorithm available at http://blast.ncbi.nlm.nih.gov/Blast.cgi.

The study was conducted following the guidelines prescribed by Institutional Animal Ethical Committee.

Results

All the 219 rectal swab samples from animals with diarrhea yielded characteristic E. coli colonies; however, large, shiny, pink, and mucoid colony characteristic for Klebsiella were detected only in 142 samples. From each sample, two characteristic colonies for each bacterium (E. coli and Klebsiella [if positive]) were processed for biochemical confirmation and specifies specific PCR. Finally, one confirmed E. coli and one confirmed K. pneumoniae (if positive) from each sample—in total, 219 E. coli and 111 K. pneumoniae isolates—were further included for AST. Likewise, 30 E. coli and 19 K. pneumoniae were isolated from 32 HC and processed. Sixty-one isolates from bovine diarrhea, that included 27 E. coli (caec1–caec27) and 34 K. pneumoniae (cakp1–cakp34), were found MDR (Table 2). Of them, forty isolates were coresistant to aminoglycoside (GEN), penicillin (Amp), potentiated penicillin (AMC), cephalosporins (any of the drugs—EFT, CPD, AT, CAZ, CTX, and CTR), tetracycline (Tet), and quinolones (NAL/ENR) and all the forty but seven were not susceptible to imipenem. For all these isolates, MARI ranged between 0.6 and 1.0. On the contrary, 11 isolates including 8 E. coli (caec1-caec8) and 3 K. pneumoniae (cahkp1, cahkp2, cahkp3) from asymptomatic animals demonstrated MDR feature with MARI ranging between 0.25 and 0.625. All these 11 isolates were resistant to quinolones (NAL/ENR), 9 to cefpodoxime, 8 to ampicillin, 7 to amoxyclav, 4 to ceftazidime and 6 to each tetracycline and ceftiofur.

Table 2.

Characteristics of Multidrug-Resistant Isolates from Healthy Cattle and Cattle with Diarrhea

Agro-climatic zone	Multidrug-resistant isolates with resistance characteristics
Vindhyan Alluvial Zone	Diarrhea group
	caec1-AmpC-qnrB-tetA (ACBL)^a; cakp13-CTXM1-tetA-sul1 (ESBL); cakp14-qnrS-sul1 (ESBL)^a; cakp22-CTXM1-tetA (ESBL); cakp26-CTXM1-tetA-sul1 (ESBL); caec9-CTXM1 (ESBL); caec10-AmpC-tetA (ACBL)^a; caec14-CTXM1 (ESBL); caec15- CTXM1 (ESBL); cakp33-CTXM1-tetB (ESBL)^a; cakp34-CTXM1-qnrB (ESBL); caec22-TEM-OXA-CTXM1-CMY-FOX-AmpC-tetA-tetB (ESBL)
	Healthy group
	cahec1- AmpC-qnrB-tetA (ACBL)
Gangetic Alluvial Zone	Diarrhea group
	caec2-CTXM1-tetA-sul1 (ESBL & ACBL); caec4-CTXM1-tetB-sul1 (ESBL & ACBL)^a; caec6-AmpC-tetA-sul1 (ACBL)^a; cakp1-CTXM1-qnrS (ESBL); cakp5-TEM-CTXM1-CMY-AmpC (ESBL); cakp9-CTXM1-qnrS (ESBL); cakp11-CTXM1-qnrS (ESBL)^a; cakp15-TEM-CTXM1-qnrS-tetA (ESBL & ACBL); cakp16-TEM-CTXM1-qnrS-tetA (ESBL); cakp18-TEM-CTXM1-qnrB (ESBL); cakp20-CTXM1-qnrB-sul1 (ESBL); cakp25-CTXM1-tetA (ESBL); caec8-CMY-AmpC (ACBL); cakp27-CTXM1 (ESBL)^a; cakp28-CTXM1-qnrB-tetA (ESBL); cakp29-CTXM1-qnrB-AAC(6’)-Ib-cr-tetA (ESBL); caec13-AmpC-tetA (ACBL)^a; cakp32-SHV-CTXM1-tetA (ESBL); caec17-CTXM1-tetA (ESBL); caec18-TEM-CMY-NDM-tetA-tetB (MBL); caec20-OXA-CTXM1-AmpC-tetA -sul1 (ESBL & ACBL)^a; caec23-PER-CTXM1-qepA-tetA (ESBL & ACBL); caec24-PER-CTXM1-qepA-tetB (ESBL & ACBL)
	Healthy group
	cahec2-OXA-CTXM1 (ESBL); cahec3-tetB; cahec5- AmpC-tetA; cahec7-CTXM1-qnrS (ESBL)^a
Costal Saline Zone	Diarrhea group
	caec3-TEM-AmpC-qnrB-tetB (ACBL)^a; caec5-AmpC-qnrB-tetA (ACBL)^a; caec7-CTXM1-tetA-ESBL (ACBL); caec11-AmpC-tetA (ACBL)^a; cakp2-TEM-CTXM1-qnrS (ESBL); cakp3-CTXM1-qnrS (ESBL); cakp4-TEM-CTXM1-qnrS (ESBL)^a; cakp8-CTXM1-qnrS (ESBL)^a; cakp17-CTXM1-DHA-AmpC-qnrB-sul1 (ESBL); cakp23-CTXM1-qnrS (ESBL); cakp30-TEM-CTXM1-tetA (ESBL); cakp31-TEM-CTXM1 (ESBL)
	Healthy group
	cahkp1-AmpC-qnrB-tetB (ACBL); cahec4-OXA-CTXM1-AmpC-qnrB (ESBL & ACBL); cahec6-CTXM1-tetA (ESBL & ACBL);
	cahec8-TEM-CTXM1-qnrS (ESBL)^a
Terai-Teesta Alluvial Zone	Diarrhea group
	cakp6-AAC(6’)-Ib-cr (ESBL); cakp19-qnrB (ESBL); caec19-CTXM1-AmpC-qepA-tetA-sul1 (ESBL & ACBL); caec26-CTXM9-qnrA-qnrB-tetB-tetE-sul1 (ESBL)
	Healthy group
	cahkp2-AmpC-tetC; cahkp3-AmpC (ACBL)
Undulating Red and Laterite Zone	Diarrhea group
Undulating Red and Laterite Zone	cakp7-TEM-CTXM1-qnrS-tetA (ESBL & ACBL); cakp10-CTXM1-qnrS (ESBL)^a; cakp12-CTXM1-CMY-AmpC-tetA-tetC (ESBL); cakp21- CTXM1-qnrB-tetA (ESBL); cakp24-qnrB (ESBL)^a; caec12-AmpC-tetA (ACBL)^a; caec16-AmpC-qnrS-tetA (ACBL); caec21-OXA-CTXM1-CMY-qnrA-tetA-tetC-tetE-sul1 (ESBL); caec25-qnrB-sul1 (ESBL); caec27-CTXM1-AmpC-qnrB-tetB (ESBL).

Isolate names and the animal groups are kept bold.

Isolates with strong biofilm producers.

ACBL, isolates are AmpC type β-lactamase producers; ESBL, isolates are extended-spectrum β-lactamase producers; MBL, isolates are metallo-β-lactamase producers.

Interestingly, 50 of the 61 MDR isolates (∼82%) from bovine diarrhea were ESBL producers (ESBLPs) and these ESBLPs exhibited resistance to fluoroquinolone and tetracycline (100% each), aminoglycosides, and carbapenem (76% each) with MARI ranging from 0.6 to 1.0 (Table 2). Only 12 ESBLPs were strong biofilm producers with MARI ranging from 0.7 to 1.0. Eleven non-ESBLPs (caec1, caec3, caec5, caec6, caec8, caec10, caec11, caec12, caec13, caec16, and caec18) exhibited resistance to tetracycline and nalidixic acid/enrofloxacin in addition to one or more drugs screened in this study with MARI ranging from 0.2 to 0.6 and majority (8) of them were strong biofilm producers. Nineteen of the 61 MDR isolates produced AmpC type β-lactamase. Five (7) of 11 MDR isolates from asymptomatic animals were ESBLs and were mostly resistant to amoxyclav (∼64%), ampicillin (∼73%), cefpodoxime (∼82%), tetracycline and ceftiofur (54%), and ceftazidime (∼37%). Eight of these isolates were AmpC type β-lactamase producer. None of these isolates was resistant to IPM, COL and chloramphenicol.

Of all the 72 MDR isolates investigated for the role of EP in quinolone resistance, only 9 isolates—2 E. coli and 7 K. pneumoniae showed a characteristic reduction in MIC_CIP in the presence of EP inhibitor.

The blaCTX-M gene (45) (MT174045) (CTX-M 1 in 44 and CTX-M 9 (MT371584) in 1 isolate) was more frequently detected among the isolates from bovine diarrhea followed by blaTEM 1 (12) (MT174046), blaOXA-1 (3) (MT371583), blaPER (2) (MT154087), and blaSHV12 (1) (MT174044) genes. Other β-lactamase genes, blaAmpC (MT174049) was detected in 16 isolates, blaCMY-6 (MT154085, MT154088) in 6 isolates, and blaDHA (MT174047) and blaFOX (MT154086) in one isolate each. PMQR determinants, qnrS (MT050524), qnrB (MT371577), and qnrA (MT371576), were detected in 14, 15, and 2 isolates, respectively. Three isolates carried qepA gene (MT174048). Of all the ENR/NAL-resistant K. pneumoniae (12) and E. coli (14) isolates, wherein no PMQR gene could be detected, 10 K. pneumoniae and 11 isolates E. coli revealed mutations in gyrA (MT371578) and ParC gene (MT371582) indicating the role of QRDR-mutation in ENR/NAL-resistance. No other β-lactamase gene except blaCTX-M 1 (5) and blaAmpC (6) was detected in 9 MDR isolates from healthy animals; however, few carried qnrB (3) and qnrS (2) genes. Two isolates from healthy had mutations in gyrA and ParC gene.

All the MDR isolates from diarrheic animals were tet-resistant with Tc-r markers—tetA (MT371586), tetB (MT371580), tetC (MT371585), and tetE (MT371579) in 33, 10, 3, and 2 isolates, respectively. In contrast, only four of the MDR isolates from the healthy group were tet-resistant and the genes tetA and tetB were detected in three and two isolates, respectively. Of 20 sul-resistant isolates from bovine diarrhea, 13 harbored sul1 gene (MW116081). None of the MDR isolates from the healthy group had sul1 gene.

Although 41 isolates were IPM-resistant/nonsusceptible, only one of them (caec18) harbored blaNDM-5 gene (MT050525) and exhibited carbapenemase production in carba NP and mCIM tests. Apart from β-lactams, the isolate was resistant to fluoroquinolone, tet, and aminoglycoside. However, it was susceptible to COL and chloramphenicol. Eleven of these IPM-R isolates have MARI ranging from 0.7 to 1.0 with coresistance to NAL, ENR, TET, and β-lactams. Ten of the IPM-R isolates were strong biofilm producers. Efflux pump-mediated CR was noticed in three isolates when IPM-R isolates were tested against MEM in the presence of CCCP. Further, many of these IPM-R isolates were ESBL (38) or AmpC type β-lactamase producers (8).

In total, two isolates from bovine diarrhea, one K. pneumoniae (cakp34) and one E. coli (caec21), were COL-resistant and five were chloramphenicol-resistant (caec8, cakp34, caec21, caec25, and caec26). Upon PCR-screening, none of the COL-resistant isolates had mcr gene(s). However, PCR could not amplify mgrB gene in cakp34. We could not detect any change in MIC_COL when the two COL-R isolates were tested in the presence of EPI. Of various virulence factors, estA, iucD, and papC were detected among 11, 4, and 3 isolates from bovine diarrhea (MW384885, MW384884). Only two isolates from this group harbored Shiga toxin-producing E. coli virulence genes stx2 (MW384886); however, none of the isolates from healthy animals had any virulence factors.

Discussion

This study attempts to detect and characterize MDR isolates from rectal swab of the adult cattle and calves from the five agro-climatic zones of West Bengal, India. In total, 61 MDR isolates were recovered from 219 rectal swab samples of diarrheic animals (61/219, 27.8%), which included both E. coli (n = 27) and K. pneumoniae (n = 34). These isolates were resistant or nonsusceptible to three or more antimicrobial classes- aminoglycosides (GEN), penicillin (AMP), potentiated penicillin (AMC), cephalosporins (EFT, CPD, AT, CAZ, CTX, and CTR), tetracycline (TET), fluoroquinolones (NAL and ENR), and carbapenem (IPM). Previous studies conducted at different parts of the world noted such MDR E. coli strains (to ampicillin, tetracycline, streptomycin, and sulfonamide) in healthy or diarrheic cattle.^20,46–48 Of late, there was a report of imipenem-resistant E. coli in slurry samples from dairy farms²¹; yet, CR in bovine is largely considered incidental. We observed some difference in antimicrobial susceptibility profile of the MDR isolates from asymptomatic animals than that from the animals with diarrhea. Interestingly, while all the MDR isolates from diarrheic animals were tetracycline resistant, only 6 of the 13 isolates from healthy animals exhibited TC-r. Similarly, gentamicin resistance rate of the MDR isolates from diarrheic cattle stood at 73%, much higher than that (36.3%) of the isolates from healthy animals. In contrast to 27.8% SXT resistance of the MDR isolates from diarrheic animals, none of the isolates from healthy animals exhibited SXT resistance. We were unable to find any such comparative study to support our finding.

About 22.8% of the diarrheic animals (50/219) carried ESBLPs in contrast to 15.6% of the HC (5/32); this is quite higher than what we observed in cow and buffalo milk in our previous studies.^24,49,50 The occurrence of ESBL-producing E. coli in healthy or diarrheic cattle varies widely (3–63%) in different geographical locations with diverse antibiotic exposure, husbandry practices, and isolation techniques.^51–53

Most of the ESBL (+ve) isolates from bovine diarrhea cases exhibited resistance to nalidixic acid/enrofloxacin, tetracycline (100% each), gentamicin (76%), and imipenem (80%) with MARI ranging from 0.6 to 1.0. All five ESBLPs from healthy animals were nalidixic acid/enroloxacin resistant; however, two of them were not susceptible to gentamicin and tetracycline. ESBL-producing E. coli strains from bovine displayed similar resistance feature indicating the involvement of multiresistance plasmid in spreading extensive drug resistance.^21,54,55 Although we had a very small size of ESBL (+ve) isolates from healthy animals, resistance feature of the ESBLPs from two (healthy and diarrheic) population was conspicuously different. Many of the farm owners usually treat the diarrheic animals with antimicrobials, antiparasitic drugs, or home-made ethnoveterinary practices. Prior antimicrobial application of the diarrheic animals may be a possible reason. Nonetheless, such a conclusion requires complete and authentic information on antimicrobial exposure. Most of these samplings were conducted in unorganized sector, and we could not trace the link as many of the animal owners failed to provide meticulous record.

Among various drug-resistant genes, blaCTX-M 1 was the predominantly detected β-lactamase gene followed by blaTEM1, blaOXA1, blaPER, blaSHV12, blaAmpC, blaCMY-6, and blaDHA in bovine diarrhea. In healthy animals, blaCTX-M 1 blaOXA, and blaAmpc genes were detected. Cattle are considered as reservoir of E. coli strains possessing CTX-M 1, -14, and -15 in Europe^56,57 and Asia.⁵¹ Even though previous studies from Europe and Africa in bovine rarely reported blaSHV gene,^21,52 it is not an uncommon observation in Asia.⁵⁸ Similar to our findings, chromosomal AmpC and blaOXA were detected in bovine E. coli strains.²¹ A recent study conducted with human K. pneumoniae and E. coli strains from four tertiary care hospitals of India indicated the predominance of blaTEM, blaCTX-M 15, and blaOXA-1 genes.⁵⁹ The gene CTX-M 9 detected in one isolate from bovine diarrhea was reported in neonatal stool samples by a group of workers from Kolkata, West Bengal.⁶⁰ It is noteworthy that we were unable to trace any of the β-lactamase genes in five MDR isolates—four K. pneumoniae (cakp6, cakp14, cakp19, and cakp24) and one E. coli (caec25), which produced ESBL in phenotypic assays. In our previous study too, we noted similar findings among a few ESBL-producing K. pneumoniae strains from bovine milk.²⁴ Such findings emphasized on the need for further investigation with such isolates circulating in food animals in this region to explore the hidden mechanism of β-lactam resistance.

Remarkably, except one isolate, we could not detect metallo-β-lactamase/carbapenemase in any of the 43 imipenem nonsusceptible isolates. Only in three isolates, we observed CCCP-mediated potentiation of MEM-inhibition zone indicating the role of efflux-pumps like AcrA in CR.⁶¹ Further, strong biofilm production observed in 10 imipenem-resistant isolates might have a role in CR.⁶² Previous studies indicated that chromosomal and plasmid-mediated cephalosporinase combined with decreased drug permeability might lead to imipenem resistance among the noncarbapenemase producers.⁶³ Of late, Webb et al.⁶⁴ reported the role of blaCMY-2 in CR among CRE from dairy cattle in the United States. In this study also, we detected pMAmpC-β-lactamase genes like CMY-2, FOX, and CMY-6 in some of the imipenem-resistant isolates. Although the blaNDM-5-producing GNB were reported in human patients from tertiary care hospitals in the Indian subcontinent, such report in food animals is scarce; Ghatak et al., 2013 reported such pathogen in bovine mastitis.⁹ Further, blaNDM-5-positive K. pneumoniae were reported from dairy cows in Jiangsu, China.⁶⁵ Carbapenems are not used in food animals in India, however, emergence of CR is a serious concern to public health. The present NDM-5-producing strain is also coresistant to other drugs such as fluoroquinolones, tetracycline, and aminoglycosides harboring the corresponding resistance genes and use of any of these antibiotics could have facilitated the coacquisition of these resistance genes together with blaNDM-5 due to possible genetic linkage. On the contrary, overuse of carbapenem in human could have driven the spread of such pathogens in animals via environmental contamination.⁶⁶

Recently, plasmid-mediated COL resistance mechanism emerged among various GNB pathogens in food animals. However, we were unable to detect such a resistance mechanism in any of the two COL-R isolates in this study. In the COL-resistant K. pneumoniae—cakp34, mgrB could not be amplified with two different sets of primers indicating the absence of mgrB gene. Deletion or inactivation of the mgrB gene was described responsible for acquired COL resistance in Klebsiella, as it upregulates PhoQ-PhoP and activates the Pmr system responsible for modification of the lipopolysaccharide.^43,67 However, we could not retrieve such a report in Klebsiella sp of animal origin.

None of the cefotaxime-resistant CTX-R (56) or ceftazidime-resistant (55) isolates from this study was sensitive to quinolone. In total, ∼72.2% of the isolates were coresistant to cefotaxime/ceftazidime and enrofloxacin/nalidixic acid. Further, 28 of the 50 (56%) blaCTX-M harboring ESBLPs carried any of the PMQR genes investigated in this study. This finding reiterated the reports of the previous workers describing a strong correlation between ESBL production and quinolone resistance in human⁶⁸ and animals²⁴ possibly due to their carriage on the same plasmid.

Abundance of PMQR genes documented in this study is in corroboration with our previous observations with the ESBL-producing Enterobacteriaceae from bovine and bubaline milk.^24,49,50 The drug efflux pump-qepA was detected in three isolates; all of which exhibited considerably higher MIC to nalidixic acid (results not shown). The gene qepA is an antiporter quinolone EP system having considerable similarity to the MFS-type efflux pumps, which actively excrete the drugs from the cytosol to the exterior of bacterial cells.⁶⁹ On the other hand, the gene AAC(6′)-Ib-cr, detected in two of the quinolone-resistant isolates, was reported to encode the aminoglycoside acetyltransferase that conferred reduced susceptibility to fluoroquinolone by N-acetylation of its piperazinyl amine. Mutation in the QRDR (gyrA and ParC) that was detected in 21 MDR isolates from bovine diarrhea (34.4%, 21/61) and 4 isolates from asymtomatic animals had been reported to cause fluoroquinolone-resistance.⁷⁰ Only 12.8% of the MDR isolates exhibited efflux pump mediated fluoroquinolone-resistance when tested for MIC_CIP in the presence of EPI (CCCP); this could have additionally contributed to FQ-R.³¹ In general, efflux pump-mediated fluoroquinolone resistance is more common among the nonfermenter than Enterobacteriaceae.⁷¹ Moreover, expression of EP might not have been evident in some of the fluoroquinolone-resistant isolates where other resistance mechanisms (like a mutation in QRDR) were involved.⁷²

All but five isolates in this study were tetracycline-resistant (93%, 67/72) and tetA gene (33) was predominantly detected followed by tetB (10), tetC (3) tetD, and tetE (two each). Such a high frequency of TC-r markers was previously reported among the enterohemorrhagic E. coli isolates from bovine in United States.¹³ Das et al.⁷³ reported GNB harboring tetA and tetB genes in the milk of the cows with subclinical mastitis from India. Further, a study conducted in Sweden revealed that tetracycline-resistant E. coli were more frequently associated with diarrheic calves, possibly due to coselection of TC-r in E. coli in calves for some “beneficial mutation.”²⁰ Moreover, because of broad-spectrum activity, tetracycline is often used in veterinary practice and that may be an important driver for selection of tetracycline-resistant strains in bovine.⁷⁴ A study conducted in dairy farms of Czech Republic revealed the abundance of TC-r genes in the in fresh excrements of calves and soils in farm proximity and such abundance was linked to the prophylactic use of chlortetracycline in the farms.⁷⁵ In contrast to our observation, Wilkerson et al.¹³ reported predominance of tetB gene among E. coli isolates from cattle with no trace of tetD and tetE genes. The predominance of tetA and tetB genes among commensal E. coli isolates from cattle farms was also reported in recent past.⁷⁶ Lack of tet genes in many of the tet-resistant strains in the present study indicated that they might harbor other unexplored resistance mechanisms as reported recently by a group of workers from Tunisia.⁷⁷

Apart from COL (all but 2 were sensitive), the isolates were relatively sensitive to chloramphenicol (83.3%, 60/72) and sulfamethoxazole and trimethoprim (76.38%, 55/72). Chloramphenicol was once the drug of choice for treating typhoid and other enteric fever; however, its use has been discontinued after the introduction of safer drugs. Ban of chloramphenicol in food animals, coupled with its restricted use in human beings probably facilitated the bacterial population to become sensitive to it. This finding was also observed in our previous studies.^24,78

Among various virulence factors, none of the MDR E. coli isolates except two carried stx, eaeA, and exhA genes. Two isolates, caec3 and caec16, were detected with stx2 and stx2-eaeA genes. Although these two isolates were non-ESBL producing, they had PMQR (qnrB and qnrS) and TC-r (tetA and tetB) genes. Previously we could not find Shiga toxin-producing gene among the isolates harboring ESBL, PMQR, or Tet-r determinants. Nevertheless, various ExPEC-specific virulence factors were detected among many of the isolates from diarrheic animals as reported previously.⁵⁰ Existence of virulent and resistant determinants in the same isolate in food animals may cause serious food-borne infection in human beings in absence of adequate precaution.

The present study described a comparative higher occurrence of MDR E. coli and K. pneumoniae in bovine diarrhea than healthy group reflecting the antibiotics usage pattern in livestock. The frequency of resistance determinants, including ESBLs (cattle with diarrhea [DC]: 82% vs. HC: 45%), was found to be higher among the MDR isolates from diarrheic cattle than that from the healthy counterparts. The β-lactamase (CTX-M, AmpC, TEM) was the most common mechanism of resistance; however, coresistance to tetracycline, quinolone, and imipenem was also evident. Isolation of carbapenem- and COL-resistant isolates is worrisome as these are the last resort antibiotics in human health care.

Footnotes

Acknowledgments

The authors are thankful to the Director, ICAR-IVRI, Izatnagar for providing the facilities for carrying out the work. The fund received from NAHEP, CAAST-ACLH program of ICAR, is thankfully acknowledged.

Disclosure Statement

No competing financial interests exist.

Funding Information

The work was carried out from the fund received under ICAR-National Agricultural Higher Education Project (NAHEP), CAAST-ACLH (Advanced Centre for Livestock Health).

Supplementary Material

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