Molecular characterization of extended-spectrum β-lactamase (ESBL) and virulent genes in multidrug-resistant Escherichia coli isolated from pharmaceutical and environmental Wastewaters in Dhaka, Bangladesh

Multidrug resistance poses a significant challenge to the efficacy of microbial infection treatment. Pharmaceuticals are significant reservoirs of antibiotic residues, which play a major role in inducing antibiotic-resistant microorganisms in wastewater. Antimicrobial resistance (AMR) among microorganisms present in pharmaceutical wastewater increases the threat of multidrug-resistant (MDR) bacteria, particularly in developing countries such as Bangladesh. This study investigates AMR in Escherichia coli isolated from effluent treatment plants (ETPs) and surrounding environmental water near six pharmaceutical sites in Dhaka, Bangladesh. Ninety (90) E. coli isolates were subjected to antibiotic susceptibility testing to determine their resistance. E. coli was confirmed via 16 S rRNA sequencing. PCR was employed for molecular analysis to screen extended-spectrum beta-lactamase (ESBL) genes and virulent genes linked to diarrheagenic E. coli. Genetic relatedness was assessed using Enterobacterial Repetitive Intergenic Consensus PCR (ERIC-PCR) to evaluate genomic diversity and potential clonal relationships. ETP isolates exhibited higher resistance than environmental isolates, with 93.10% showing β-lactam resistance (including 86.21% resistance to ampicillin) and 58.62% resistance to macrolides. In comparison, environmental isolates showed 67.21% β-lactam resistance and 57.38% macrolide resistance. Colistin and meropenem remained highly effective, with resistance detected in only 3.45% and 0% of ETP isolates, respectively, and 1.64% and 0% among environmental isolates. The blaNDM gene was most predominant in ETP isolates (79.31%), while the blaTEM gene was more frequent in environmental isolates (54.10%). Environmental isolates also carried ETEC virulence genes (eLT - 75.76%, eSTh - 63.64%) and the EPEC gene (eae - 45.45%). No virulence genes were found in ETP isolates. Phylogenetic analysis via ERIC-PCR revealed genetic clustering by location and resistance profile, suggesting localized resistance dissemination. Overall, this study reveals that pharmaceutical wastewater in Dhaka serves as a critical reservoir for MDR E. coli, with effluent treatment plant isolates showing higher antibiotic resistance, particularly to β-lactams, and harboring dominant resistance genes such as blaNDM. Moreover, environmental isolates carried key virulence genes such as eLT, eSTh, and eae, indicating localized dissemination and potential environmental spread of antimicrobial resistance.