Molecular assessment of antimicrobial resistance and virulence in multi drug resistant ESBL-producing Escherichia coli and Klebsiella pneumoniae from food fishes, Assam, India

The present study investigated the prevalence of Extended-Spectrum Beta Lactamase (ESBL) -producing E. coli and K. pneumoniae from the food fishes in retail markets in Assam, India. A total of 54 ESBL-producing E. coli and 12 K. pneumoniae isolates were recovered from 79 fish samples and were analyzed for antimicrobial resistance genes (ARGs) and virulence genes. E. coli isolates were categorized as multi drug resistant with resistance up to 12 different antibiotics with multiple antibiotic resistances (MAR) index ranging from 0.26 to 0.63. In E. coli, 100% resistance to cefotaxime along with 6% resistance to ceftazidime (third-generation cephalosporins) was observed. Moreover, 85% of the E. coli isolates were resistant to cefepime, a fourth-generation cephalosporin. K. pneumoniae showed resistance to 11 different antibiotics with MAR index value ranging from 0.21 to 0.57. All K. pneumoniae isolates showed 100% resistance to cefotaxime, 67% resistance to ceftazidime and 75% resistance to cefepime. Molecular characterization of ARGs revealed the presence of CTX-M group 1(CTX-M-15) in almost all E. coli isolates (98%, n = 53) and 100% in K. pneumoniae. A combination of uniplex and multiplex PCRs revealed fewer ARGs in E. coli isolates, with each isolate carrying 3 to 5 genes (tetA, dfrA1, sul1, sul2, qnrB, qnrS, aac(6')-Ib-cr). Majority of the E. coli were assigned to low-virulence phylogroup B1 and A while 8% of them belonged to pathogenic phylogroup D. 31 unique genetic profiles were identified for E. coli isolates by Pulsed-Field Gel Electrophoresis (PFGE) typing. K. pneumoniae isolates were highly diverse with 11 unique genetic profiles and a substantial ARG profile (bla(TEM), bla(SHV), bla(OXA-1-like), tetA, strA, strB, dfrA1, sul1, sul2, qnrB, qnrS, aac (6 ')-Ib-cr, oqxA, oqxB). The frequency of ARGs ranged between 4 and 11. All K. pneumoniae isolates belonged to capsular serotype with wzi gene. Virulence gene iutA was prominent in all isolates while ybtS and kfu were confirmed in two isolates. Our findings raise concerns that fishes bought for consumption may serve as potential reservoirs of AMR genes and pose serious threat to public health. The study emphasizes the need for extensive surveillance of resistant strains in aquaculture and related settings, their in-depth analysis of population structure and transmission dynamics.