Characterization of Escherichia coli isolates from diarrhoeic calves for transferable drug resistance, colicinogeny and virulence associated genes
Choudhary, Vandana (2006) Characterization of Escherichia coli isolates from diarrhoeic calves for transferable drug resistance, colicinogeny and virulence associated genes. Masters thesis, College of Veterinary Science and Animal Husbandry, Anand Agricultural University, Anand, Gujarat, INDIA.
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The present study was undertaken to analyze plasmid profile of E.coli isolates, to ascertain transferable nature of antibiotic resistance, colicinogeny and unusual biochemical characters among the isolates from calf diarrhoea cases. Further PCR based detection of virulence associated genes was also carried out. On conjugation 19 transconjugates were obtained which were tested for in vitro transferable nature of antibiotic resistance. In eighteen transconjugates en bloc transfer of Cephalexin, Cephalodrine and Kanamycin was seen. In 7 transconjugates en bloc transfer of Amikacin, Cephalexin, Cephaloridine, Kanamycin, Tetracycline and Enrofloxacin was observed. In 2 isolates, en bloc transfer of Amikacin, Cephalexin, Cephaloridine, Kanamycin and Tetracycline, to their respective transconjugates was achieved. In one transconjugate, Cephalexin, Cephaloridine, Enrofloxacin, Kanamycin and Tetracycline was transferred while in another one transfer of Amikacin, Cephalexin, Cephaloridine, Enrofloxacin and Kanamycin was seen. Transfer of Cephalexin, Cephaloridine, Kanamycin was seen in seven transconjugates. Cephalexin and Kanamycin were found to be transferred in JU13T. In 11 transconjugates Ampicillin resistance was not transferred, similarly in 2 transconjugates Co-trimoxazole resistance was not transferred. Sixteen transconjugates, whose wild strains exhibited colicin production, were tested for transfer of colicinogeny to the recipient strain. Out of 16, ten transconjugates (62.5percent) were detected for the ability to transfer colicinogeny. Out of 19 transconjugates obtained eleven were assayed for transferability of unusual biochemical character of the wild parent strain. The biochemical tests viz. Urea hydrolysis, production of H2S, Citrate utilization and Carbohydrate fermentation were assayed. Only three wild strains could transfer ability for raffinose fermentation to their respective transconjugates while no other unusual biochemical character could be transferred from wild to recipient strain. Plasmid profile analysis of wild isolates revealed the possession of single to multiple plasmids (1to4) and that of transconjugates revealed the plasmid profile of their parent strain indicating that all the plasmid carried by wild strain was transferable except for 2 transconjugates which did not reveal the transfer of 2 small sized plasmid of 5.5kbp and 5.8kbp from their respective wild strain. Eight wild strains contained large plasmid of molecular weight ranging from 54kbp to 56kbp. Medium sized plasmids ranging from 17kbp to 32kbp is found in eight strains. Small sized plasmids ranging from 3kbp to 8kbp is isolated from maximum of thirteen strains. Eight isolates showed the presence of one plasmid. In seven wild strains three plasmids were isolated. In three isolates two plasmids were isolated and in only one isolate four plasmids were found. Multiplex PCR based detection of virulence associated genes among 91 E.coli isolates showed the presence of amplified product size of 815bp in 15 (16.8%) isolates indicating the presence of intimin (eae) gene. Eighteen (19.78%) isolates possessed bfp gene of 597bp and 62 (68.13%) isolates harbored EAST1 gene of 111bp and in 15 (16.48%) isolates amplification of 382bp product size indicated the presence of invE gene. Out of 91 isolates, 15 isolates which were positive for intimin gene were, typed for 6 intimin variant types by PCR using the total 6 sets of primer viz. eaeβ1(811bp), eaeγ1(804bp), eaeγ2(808bp), eaeδ (833bp), eaeζ (206bp), and eaeξ (468bp). Eight out of 15 isolates could be typed for intimin variant types, however in seven isolates no intimin variant type was detected. Two out of 15 (13.3%) were positive for eaeβ1 (811bp) primer. One isolate (6.67%) was positive for eaeγ1 primer (804bp). Five isolates (33.3%) were positive for eaeγ2 (808bp) and no isolate was positive for other three primer viz., eaeδ (833bp), eaeζ (206bp), and eaeξ (468bp).
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