Fecal Source Tracking in Water by Next-Generation Sequencing Technologies Using Host-Specific <i>Escherichia coli</i> Genetic Markers

High levels of fecal bacteria are a concern for the aquatic environment, and identifying sources of those bacteria is important for mitigating fecal pollution and preventing waterborne disease. <i>Escherichia coli</i> has been used as an indicator of fecal pollution, however less success has been achieved using this organism for library-independent microbial source tracking. In this study, using next-generation sequencing technology we sequenced the whole genomes of 22 <i>E. coli</i> isolates from known sources (9 from humans, 2 from cows, 6 from pigs, and 5 from chickens) and identified candidate host-specific genomic regions. Specificity testing on the candidate regions was performed using 30 <i>E. coli</i> isolates from each source. Finally, we identified 4 human-, 2 cow-, 3 pig-, and 4 chicken-specific genetic markers useful for source tracking. We also found that a combination of multiplex PCR and dual index sequencing is effective for detecting multiple genetic markers in multiple isolates at one time. This technique was applied to investigating identified genetic markers in 549 <i>E. coli</i> isolates obtained from the Yamato River, Japan. Results indicate that humans constitute a major source of water contamination in the river. However, further work must include isolates obtained from geographically diverse animal hosts to make this method more reliable

Fecal Source Tracking in Water by Next-Generation Sequencing Technologies Using Host-Specific <i>Escherichia coli</i> Genetic Markers

High levels of fecal bacteria are a concern for the aquatic environment, and identifying sources of those bacteria is important for mitigating fecal pollution and preventing waterborne disease. <i>Escherichia coli</i> has been used as an indicator of fecal pollution, however less success has been achieved using this organism for library-independent microbial source tracking. In this study, using next-generation sequencing technology we sequenced the whole genomes of 22 <i>E. coli</i> isolates from known sources (9 from humans, 2 from cows, 6 from pigs, and 5 from chickens) and identified candidate host-specific genomic regions. Specificity testing on the candidate regions was performed using 30 <i>E. coli</i> isolates from each source. Finally, we identified 4 human-, 2 cow-, 3 pig-, and 4 chicken-specific genetic markers useful for source tracking. We also found that a combination of multiplex PCR and dual index sequencing is effective for detecting multiple genetic markers in multiple isolates at one time. This technique was applied to investigating identified genetic markers in 549 <i>E. coli</i> isolates obtained from the Yamato River, Japan. Results indicate that humans constitute a major source of water contamination in the river. However, further work must include isolates obtained from geographically diverse animal hosts to make this method more reliable

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

The occurrence of pathogenic <i>Escherichia coli</i> in environmental waters increases the risk of waterborne disease. In this study, 14 virulence genes in 669 <i>E. coli</i> 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 potentially pathogenic <i>E. coli</i>. Among the 549 environmental isolates, 64 (12%) were classified as extraintestinal pathogenic <i>E. coli</i> (ExPEC) while eight (1.5%) were classified as intestinal pathogenic <i>E. coli</i> (InPEC). Only ExPEC-associated genes were detected in human isolates and pig isolates, and 11 (37%) and five (17%) isolates were classified as ExPEC, respectively. A high proportion (63%) of cow isolates possessed Shiga-toxin genes (<i>stx1</i> or <i>stx2</i>) and they were classified as Shiga toxin-producing <i>E. coli</i> (STEC) or enterohemorrhagic <i>E. coli</i> (EHEC). Among the chicken isolates, 14 (47%) possessed <i>iutA</i>, which is an ExPEC-associated gene. This method can determine the sequences as well as the presence/absence of virulence genes. By comparing the sequences of virulence genes, we determined that sequences of <i>iutA</i> 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