Genome-informed monitoring and characterization of Escherichia coli, including shiga toxin-producing strains, in a river with diverse land uses

Escherichia coli is used as an indicator of fecal contamination in river water, yet its geographic variation associated with land use and virulence potential of these strains remain insufficiently characterized. In particular, Shiga toxin (Stx)-producing E. coli (STEC) represents a highly virulent subset capable of causing severe human diseases, while its quantification and virulence assessment are hindered by low abundance in river water. Fixed-point sampling at 10 stations in the Oyodo River, Japan, revealed E. coli counts reaching up to 1.70 × 10 4 CFU/100 mL and correlating positively with water quality parameters. The counts spiked in residential areas lacking sewer systems and adjacent livestock farms. Four stations where segments characteristics changed were investigated for STEC using the foam concentration method. STEC strains were detected at all locations, with a maximum of 3.98 cells/100 mL, and 13 isolates were recovered, with higher levels downstream of unsewered residences and livestock farms. Given the low infectious dose of STEC, this concentration indicates potential human infection risk. Whole-genome sequencing (WGS) revealed that three isolates (O103:H2 or O111:H8) harbored stx1a and/or stx2a with eae, a combination linked to severe human infections. Ten isolates, including the two stx2a- and eae -positive strains, exhibited multidrug resistance genotypically and phenotypically. WGS of 219 non-STEC E. coli strains isolated across sampling periods revealed consistent shifts in phylogenetic diversity and antimicrobial resistance gene profiles after the river passed through unsewered residences and livestock farms, suggesting persistent contamination. Overall, combining efficient concentration strategies with genome-based analyses enables robust evaluation of STEC occurrence, virulence, and contamination sources in human-impacted river environments.