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Five human-specific markers were detected in 59-74% of 27 human fecal-source samples collected in Yamanashi Prefecture, Japan. Similarly, potential human-specific markers, crAssphage, pepper mild mottle virus (PMMoV), and tobacco mosaic virus were detected in 96-100% of samples, with crAssphage showing the maximum concentration of 12.03 log copies/L. However, these markers were detected in 100% (3/3) of pig fecal-source samples, suggesting their applicability as general fecal pollution markers. Microbial source tracking analysis demonstrated that the rivers are contaminated by human and pig fecal sources. CrAssphage showed higher marker concentrations in river water samples than PMMoV, suggesting the preference of crAssphage to PMMoV as a marker of fecal pollution.

Shiga toxin-producing Escherichia coli (STEC) is one of the most commonly heard sources of foodborne outbreaks but presently lacks studies of in the Kathmandu Valley. This study explored the presence of STEC in river water (n = 17), groundwater (n = 83), feces (n = 50), and manure (n = 20) in the Kathmandu Valley, Nepal. Samples that were confirmed to be E. coli–positive using the Colilert assay in previous studies were selected to examine the presence of the sfmD, stx1, and stx2 genes using quantitative polymerase chain reaction. Based on Colilert/sfmD gene ratios, the percentage of viable cells of E. coli in the fecal (2.0% ± 2.5%), manure (0.1% ± 0.1%), river water (3.4% ± 3.1%), and groundwater samples (3.9% ± 5.8%) were determined. The presence of the stx1 gene was observed in the fecal (6%), river water (53%), and groundwater (2%) samples, whereas the stx2 gene was detected in the fecal (8%), manure (5%), and river water (71%) samples. Interestingly, the stx/sfmD gene ratios in the groundwater samples were greater than 100%, suggesting the presence of other stx1/stx2-harboring microorganisms. These findings indicated the importance of continuing investigation into waterborne sources of STEC contamination within the Kathmandu Valley.