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

The present study reports on 23 extended spectrum ß-lactamase producing Klebsiella 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 Bengal, Jharkhand and Mizoram. Seven of them were AmpC type ß-lactamase producers, as well. The ESBL producing KP were significantly (P=0.006, χ2=10.04, df=2) and more frequently detected in milk samples of mastitic cows than healthy ones. The ß-lactamase genes - blaCTX-M, blaTEM and blaSHV were detected in 19, 8 and 3 isolates, respectively. In all but 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 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 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.

Molecular characterization and antibiotic susceptibility pattern of caprine Shiga toxin producing-Escherichia coli (STEC) isolates from India.

The present study was conducted to detect the occurrence, serotype, genotype, phylogenetic relationship and antimicrobial resistance pattern of STEC from healthy goats of West Bengal, India. From the 125 faecal samples collected from healthy goats, 245 isolates were identified as Escherichia coli. The E. coli harbouring any gene for Shiga toxins (stx 1/stx 2) was detected in 36 (14.7%) of the 245 E. coli isolates. These STEC strains belonged to 22 different serogroups (O2, O5, O20, O21, O22, O25, O41, O44, O45, O60, O71, O76, O84, O85, O87, O91, O103, O112, O113, O120, O156, and O158) and three were untypeable. The stx 1 and stx 2 was detected in 26 (72.2%) and 21 (58.3%) of Shiga toxin producing-E. coli (STEC) isolates, respectively. Further, E. coli harbouring eaeA only (Enteropathogenic E. coli) and ehxA was detected in 22 (61.1%) and 28 (77.7%) isolates, respectively. Whereas the saa was present in 8 (22.2%) E. coli isolates. The subtyping of the 26 E. coli strains possessing stx 1 showed that 73.% (19/26) of these isolates were positive for stx 1C subtype. Of the 21 isolates with the stx 2 gene, 42.8% (9/21) were positive for stx 2C, and 38.1% (8/21) were positive for stx 2d gene. The phylogenetic analysis of STEC strains after RAPD reveals eight major clusters. However, no serogroup specific cluster was observed. Resistance was observed most frequently to erythromycin (80.5%), amikacin (52.7%), cephalothin (50%), kanamycin (41.6%), neomycin (36.1%) and gentamycin (36.1%) and less frequently to norfloxacin (2.7%), enrofloxacin (2.7%), and ciprofloxacin (2.7%). Multidrug resistance was observed in eleven STEC isolates.

Isolation, molecular characterization and antibiotic resistance of Shiga Toxin-Producing Escherichia coli (STEC) from buffalo in India.

In total, 363 Escherichia coli were isolated from 165 faecal samples of healthy buffaloes in West Bengal, India. Twenty-four of these isolates (6·61%) were found to carry at least one gene characteristic for Shiga toxin-producing Escherichia coli (STEC). These STEC strains belonged to 13 different O-serogroups. The stx1 gene was present in 23 (95·8%) of total STEC isolates, whereas 20 (83·3%) STEC isolates carried the gene stx2. Twelve strains of E. coli (50% of total STEC isolates) possessed enterohaemolysin (ehxA) gene in combination with others. Fourteen (58·33%) isolates found to possess saa gene. However, no E. coli was detected harbouring gene for intimin protein (eaeA). Of 23 stx1 -positive isolates, seven (30·43%) were positive for genes of the stx1C subtype. Of the 20 isolates with the stx2 gene, 25% (5/20) possessed stx2C and 10% (2/20) possessed stx2d gene. The phylogenetic analysis after RAPD of STEC strains revealed six major clusters. The isolated STEC strains were resistant most frequently to erythromycin (95·83%), cephalothin (62·5%), amikacin (54·17%), kanamycin (45·83%) and gentamicin (41·67%) group of antibiotics. No ESBL-producing (blaCTXM , blaTEM , blaSHV ) or quinolone resistance gene (qnrA) was detected in the STEC isolates.

Characterization of shiga toxin producing (STEC) and enteropathogenic Escherichia coli (EPEC) in raw yak (Poephagus grunniens) milk and milk products.

Thirty-one shiga toxin-producing (STEC) and 6 enteropathogenic Escherichia coli (EPEC) were isolated from 87 raw yak milk and 63 'churpi' samples. Of 18 stx(1) positive isolates (48.6%), 14 carried stx(1c) (77.7%). Subtyping of 28 stx(2) positive isolates (75.7%) revealed the presence of stx(2c) (9, 32.1%), stx(2d) (3, 10.7%), stx(2e) (1, 3.57%) and stx(2f) (3, 10.7%) variants. Furthermore, intimin (eaeA), enterohaemolysin (ehxA), autoagglutinating adhesin (saa), iha (adherence conferring protein), efa1 (EHEC factor for adherence), bundle forming pilli (bfpA) and toxB (type III secreted protein encoded on LEE Island, similar to toxin B of Clostridium difficile) genes were detected in 14, 16, 12, 4, 3, 2 and 2 isolates, respectively. Univariate and multivariate analysis depicted that both stx(1) and stx(2) or their variants were more likely to occur in isolates from Arunachal Pradesh (p<0.04) rather than Sikkim. Dendogram constructed on the basis of RAPD and ERIC PCR profile distributed the STEC and EPEC isolates in separate clusters irrespective of their sources and serotypes. The STEC and EPEC isolates exhibited resistance against erythromycin, amikacin, azithromycin, amoxicillin, ampicillin+cloxacillin, cephalothin, furazolidone, gentamicin, kanamycin, streptomycin and tetracycline. This is the first ever report on occurrence and characterization of STEC and EPEC isolated from yak milk and milk products.