Molecular Characterization of Multidrug-Resistant Escherichia Coli from Cultured Fish: Co-Occurrence of β-Lactamase and Plasmid-Mediated Resistance Genes

Abstract

This study investigated the characterization of antimicrobial resistance (AR) profiles and beta-lactamase production in Escherichia coli isolates recovered from cultured fish, with particular emphasis on plasmid-mediated resistance (PMR) determinants, including quinolone resistance (qnr), colistin resistance (mcr-1 to mcr-5) genes, class 1 integrons (intI1), and phylogenetic backgrounds, using PCR-based methods. From 64 cultured fish samples collected between 2024 and 2025, 34 E. coli isolates were obtained following cultivation on Harlequin (R) E. coli/Coliform Agar and subsequently confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and Sanger sequencing. Antimicrobial susceptibility testing against 22 agents representing 11 antibiotic classes revealed resistance frequencies ranging from 5.88% to 50.00%, with the highest resistance rates observed for penicillins (67.65%) and cephalosporins (23.53%). Multidrug resistance was identified in 11.76% of the isolates. All isolates exhibited extended-spectrum beta-lactamase (ESBL) activity, predominantly associated with bla(TEM) (91.18%), bla(SHV) (58.82%), and bla(CTX-M) (23.53%). Carbapenemase-encoding genes were detected in 38.23% of the isolates, mainly bla(IMP) (14.71%) and bla(OXA-48) (11.76%), while plasmid-borne AmpC genes (CITM and DHA) were identified in 5.88% of the isolates each. Co-carriage of ESBL and carbapenemase genes occurred in 38.23% of isolates, whereas concurrent detection of ESBL and AmpC genes was observed in 8.82%. Plasmid-borne quinolone resistance genes, primarily qnrS and qnrB, were detected in 64.70% of the isolates, and intI1 was identified in 38.23%; however, no mcr genes were detected. Phylogenetic analysis showed that most isolates (73.53%) belonged to phylogroup B2(3). Notably, phenotypic resistance profiles did not fully correspond with PCR-based genotypic findings, highlighting the complexity of AR expression in aquatic E. coli. In conclusion, cultured fish were found to harbor a high proportion of multidrug-resistant and beta-lactamase-producing E. coli. The simultaneous detection of ESBL, carbapenemase, AmpC, and PMR genes suggests that aquaculture systems may contribute to the persistence and spread of AR. Continuous monitoring within a One Health framework is therefore essential to reduce risks to food safety and public health.