Characterization of Pathogenic Escherichia coli in River Water by Simultaneous Detection and Sequencing of 14 Virulence Genes

The occurrence of pathogenic Escherichia coli in environmental waters increases the risk of waterborne disease. In this study, 14 virulence genes in 669 E. coli isolates (549 isolates from the Yamato River in Japan, and 30 isolates from each of the following hosts: humans, cows, pigs, and chickens) were simultaneously quantified by multiplex PCR and dual index sequencing to determine the prevalence of pathogenic E. coli. Among the 549 environmental isolates, 64 (12%) were classified as extraintestinal pathogenic E. coli (ExPEC) while eight (1.5%) were classified as intestinal pathogenic E. coli (InPEC). Only ExPEC-associated genes were detected in human isolates and pig isolates, and 12 (40%) and five (17%) were classified as ExPEC, respectively. A high proportion (63%) of cow isolates possessed Shiga-toxin genes (stx1 or stx2) and they were classified as Shiga toxin-producing E. coli (STEC) or enterohemorrhagic E. coli (EHEC). Among chicken isolates, 14 (47%) possessed iutA, which is an ExPEC-associated gene. Unlike other assays, this method can determine the sequences as well as the presence/absence of the virulence genes. By comparing the sequences of virulence genes, we identified that sequences of iutA were different among sources and may be useful for discriminating isolates, although further studies including larger numbers of isolates are needed. Results indicate that humans are a likely source of ExPEC strains in the river.