Borah, ProbodhDAS, LEENA2022-07-012022-07-012019-07https://krishikosh.egranth.ac.in/handle/1/5810185029Extended-spectrum beta-lactamase producing Enterobacteriaceae has become a major threat to both animals and human health globally. The present study was undertaken to isolate and identify ESBL producing Escherichia coli and Klebsiella pneumoniae from various sources, to study their resistant gene profile, to detect insertion sequences, to genogroup the isolates and to compare the efficacy of REP-PCR and PFGE to discriminate ESBL producing E. coli and K. pneumoniae isolates. Out of 385 samples from various sources, 31 (8.05%) were positive for ESBL producing E. coli. Such isolates could be isolated from 10.05, 8.33, 15.63, 6.67 and 4.35 per cent of cattle milk, curd, chicken, pork and cattle faeces samples, respectively. However, no ESBL producing E. coli could be isolated from goat milk, goat faeces and beef samples. A total of 59 (15.32%) samples were positive for ESBL producing K. pneumoniae, which could be isolated from 14.35, 6.25, 21.43 and 34.78 per cent samples of cattle milk, chicken, beef and cattle faeces, respectively. No ESBL producing K. pneumoniae isolates could, however, be isolated from goat milk and faeces, curd and pork. In-vitro drug susceptibility assay against 3rd and 4th generation cephalosporins showed resistance of all the 90 ESBL isolates to at least one antibiotic. In CDT, 93.55% of E. coli and 88.14% K. pneumoniae and in ESBL –E test, 96.77% E. coli and 88.14% K. pneumoniae showed positive results. Antibiogram of the ESBL producing E. coli and K. pneumoniae showed resistance of 74.19% and 69.49%, respectively to ceftizoxime, 25.81% and 23.73% to both co-trimoxazole and tetracycline, 19.35% and 25.42% to ciprofloxacin, 9.68% and 16.95% to chloramphenicol, 3.23% and 5.08% to pipercillin-tazobactam, and 3.23% and 3.39% to gentamicin. Resistance gene profiling showed blaCTX-M gene to be present in all the 90 (100%) ESBL isolates. The blaTEM gene was found in 54.84% and 55.93%, blaSHV gene in 90.32% and 77.97%, Sul 1 gene in 90.32% and 86.44% isolates. The Int1 gene was detected in 70.97% and 62.71% isolates, while qnrB gene was found in 3.23% and 10.17% of E. coli and K. pneumoniae isolates, respectively. Out of the insertion sequences under study, ISEcp1 was found to be present in all the 90 (100%) ESBL producing isolates, followed by IS26 (100% and 90.32%) and ISCR1 (80.65% and 45.76%) in E. coli and K. pneumoniae isolates, respectively. All the 90 ESBL producing isolates were subjected to PCR for detection of CTX-M genogroups. All the 90 (100%) ESBL producing isolates were found to be positive for group 1 gene. A total of 80.65% and 55.93% E. coli and K. pneumoniae isolates, respectively showed presence of group 2 genes. The corresponding percentages for group 25 gene were 27.27% and 67.8%. However, group 9 gene could be detected in 5.08% of K. pneumoniae isolates only. None of the E. coli isolates were found be positive for group 8 and 9 genes, while no isolate of K. pneumoniae was found to be positive for group 8 gene. The two molecular typing methods, REP-PCR and PFGE were found to show similar discriminatory power and could distinctly differentiate the ESBL producing E. coli and K. pneumoniae isolates. As both the methods were found equally competent, REP-PCR may be recommended as the preferred method of typing for epidemiological investigations owing to its advantages over PFGE in terms of rapidity, simplicity and ease of performance.EnglishThesis