Integrated Genotypic and Phenotypic Determinants Underpin the Environmental Persistence of Escherichia coli in Treated Sewage in Japan

ABSTRACT Escherichia coli poses global health risks, affecting human, animal, and environmental systems. To investigate the determinants underpinning its environmental persistence, we conducted an integrated genotypic and phenotypic analysis of 365 E . coli isolates recovered from treated sewage in Hiroshima, Japan. Isolates were confirmed by uidA ‐PCR and screened for virulence, antimicrobial resistance (AMR), phylogroup, and siderophore‐associated genes using multiplex PCR. Phenotypic assays assessed biofilm formation on microtiter plates and motility on soft agar. Among the isolates, 13.7% carried one or more virulence genes, with astA being the most prevalent (70.0%), followed by eaeA (8.0%), bfpA (6.0%), and the aggR ‐ astA combination (4.0%). AMR genes were detected in 50.0% of isolates, predominantly bla TEM . The major phylogroups were D (36.0%) and B1 (24.0%). All isolates carried entA–F and tonB , while fecA and chuA were the predominant accessory iron‐acquisition genes (52.0%). Phenotypic analysis showed that 92.0% of isolates formed biofilms and 100.0% were motile. aggR ‐positive isolates exhibited the highest biofilm production, whereas astA ‐, bfpA ‐, and eaeA ‐positive isolates demonstrated greater motility than ipaH ‐positive isolates. Principal component analysis indicated that biofilm formation was associated with aggR , whereas motility showed an opposing trend. Together, these traits contribute to the environmental persistence of E . coli in treated sewage.