PHENOTYPIC AND GENOTYPIC CHARACTERISATION OF ANTIMICROBIAL RESISTANCE IN ENTEROCOCCUS SPECIES OF DAIRY ORIGIN
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Date
2022
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ICAR-NDRI, KARNAL
Abstract
Prevalence of antibiotic resistance among food-based enterococci is matter of concern because of its opportunistic pathogenicity as well as its ability to disseminate AMR genes to other bacteria in the food chain. The present study was undertaken to characterize antimicrobial resistance in Enterococcus species of dairy origin. A total of 64 dairy samples comprising of traditional Dahi and raw milk samples were collected from the districts of Karnal, Kaithal, Sirsa, Bhiwani and Jind of Haryana region. The sample were processed for isolation of enterococci by pour plate technique using selective media viz. Citrate Azide and Bile Esculin Agar and further identified by phenotypic and genotypic approach. A total of 235 isolates were initially shortlisted based on Gram’s staining and catalase test. The genotypic identification of isolates was done by using genus specific PCR. Finally, a total of 140 isolates were genotypically confirmed as enterococci. A total of 84 Enterococcus isolates were further tested for antimicrobial susceptibility against major antibiotic classes as per CLSI guidelines. The highest resistance in the enterococci isolates were observed against Cefuroxime (50%), Rifampicin (21.43%), Cefepime (19.05%), Erythromycin (15.48%), Fosfomycin (9.52%), Cefotaxime (8.33%), as well certain isolates showing resistance against Tetracycline, High Level Streptomycin, Gentamycin, Norfloxacin, Chloramphenicol and Levofloxacin. Extended Spectrum β Lactamases production was confirmed in 9 out of 24 isolates. PCR for antibiotic resistance genes in respective resistant isolates showed positive results for Aminoglycoside resistance genes such as aac(6’)-Ie- aph(2”)-Ia, aph(3’)-IIIa, ant(6)-Ia, Tetracyclines resistance genes [tet(M), tet(O)], Chloramphenicol resistance genes (catA8) and also certain isolates with Macrolide resistance genes such as ermA, ermB, ermC, ermF and msrA. Among the 43 resistant isolates, 69.76% of isolates were positive for multi-drug transporter gene (emeA), while 25.58% and 30.23% were positive for Transposons family, Tn-5397 (tndX) and Tn-916/Tn-1545 (Int-Tn), respectively. Further, 4 isolates also showed the presence of class-1 Integron gene, while 12 isolates were positive for class-3 Integron gene. A total of 43 resistance isolates were tested for virulence traits. Among these only 2 (4.6%) isolates demonstrated Gelatinase production (also positive for gelE gene), 17 (39.53%) for Hemolysin production (6 showed positive for cylA gene) and none for DNase production. No significant correlation between the virulence and antibiotic resistance traits were observed in the isolates. Further, the resistant isolates have been sent for 16S rRNA partial sequencing for species identification and the most resistant isolate B1(C) for Whole Genome Sequencing to study the organization of AMR genes in the isolate.