Virulence Signatures and Biofilm-Forming Capacity of ESBL-Producing Escherichia coli in Wastewater and Agricultural Environments
Abstract
The persistence of extended-spectrum β-lactamase (ESBL)-producing E. coli in livestock and municipal wastewater systems represent a key environmental pathway for antimicrobial resistance (AMR) and virulence determinants with direct public-health implications. This study investigated the distribution of virulence genes and biofilm-forming capacity of ESBL-producing E. coli from two municipal WWTPs, a poultry farm, and a cattle farm. A total of 452 environmental samples were collected between May 2025 and May 2026. ESBL-producing E. coli were identified using culture-based and biochemical methods. Virulence genes were characterized by polymerase chain reaction (PCR) and whole-genome sequencing (WGS). Biofilm formation was assessed using the crystal violet microtiter plate assay. Of the 452 samples, 120 (26.5%) yielded ESBL-producing E. coli. PCR analysis showed that poultry farm isolates exhibited the highest prevalence of virulence genes, predominantly traT (64/66), ompA (61/66), fimH (54/66), and chuA (39/66), followed by isolates from WWTP-A, the cattle farm, and WWTP-B. Significant differences among sampling sources were observed for traT (p = 0.0398) and stx1 (p = 0.0224). WGS analysis identified significant source-dependent associations for several virulence genes, with the strongest associations observed for esp (FDR = 3.47 × 10−6) and fimH (FDR = 2.36 × 10−5). Most isolates were non-biofilm producers (52/120, 43.3%). Among biofilm-forming isolates, weak biofilm production predominated across all sampling locations, with the highest prevalence observed in poultry farm isolates (21/120, 17.5%), followed by isolates from the cattle farm and WWTP-A (10/120, 8.3% each) and WWTP-B (7/120, 5.8%). These findings identify poultry farms and wastewater environments as important reservoirs of virulence-associated ESBL-producing E. coli and support integrated One Health surveillance to limit the environmental spread of AMR.