Tracking ESBL-Producing <em>Escherichia coli</em> Across Municipal Wastewater and Farm Ecosystems: A One Health Investigation

Extended-spectrum β-lactamase–producing E. coli (ESBL-EC) threatens public health by driving widespread antimicrobial resistance transmission in environmental and agricultural settings. This study examined the prevalence, genetic determinants, and phylogenetic relationships of ESBL-EC isolated from municipal wastewater treatment plants (WWTPs) and farm environments in southeastern North Carolina. A cross-sectional survey was conducted between May and September 2025 across two WWTPs and two farms (cattle and poultry). We sampled influent and effluent wastewater, plus fecal and water specimens collected from chickens, ducks, and cattle. Antimicrobial susceptibility testing was performed using Kirby–Bauer disk diffusion method against nine drugs, while PCR and sequencing were used for genotypic characterization. Phylogenetic analysis assessed genetic relatedness among isolates. ESBL-EC was detected in 27.4% (n = 124) of 452 samples, with the highest prevalence in chickens (31.5%), followed by WWTP influents (28.2%), ducks (18.5%), and cattle (12.1%). Dominant resistance genes included blaCMY-2 (71.8%), blaCTX-M-1 and blaOXA (54% each), and blaSHV (29.8%). Co-occurrence of blaCMY-2 with blaCTX-M-1 and blaOXA was observed in poultry isolates. Phylogenetic analysis revealed clonal relatedness between poultry and cattle isolates. These findings highlight poultry as a key reservoir and emphasize the need for One Health surveillance to mitigate cross-reservoir transmission of resistant E. coli.