CrAssphage as a Potential Human Sewage Marker for Microbial Source Tracking in Southeast Asia

The human gut bacteriophage crAssphage has been proposed as a human-specific microbial source tracking (MST) marker for impacted water bodies. However, its global use as a human-specific MST marker requires validation in a tropical region. In this study, a crAssphage qPCR marker (CPQ_056) was detected in 21 sewage samples in Thailand with 100% sensitivity. The marker was detected in sewage from hospitals and residential buildings at 5.28-7.38 log(10) copies/100 mL and in four influent and four effluent samples of municipal wastewater treatment plants at 4.23-6.19 and 3.78-4.89 log(10) copies/100 mL, respectively. Furthermore, a 99.2% specificity (n = 127) was observed using feces from swine, cattle, chicken, duck, goat, sheep, buffalo, and fish, with cross-detection only occurring for one composite swine sample. The crAssphage marker was present in 56.25% (27 out of 48) of river samples at 3.20-7.29 log(10) copies/100 mL. The concentrations of the crAssphage marker and a prevalidated human-specific Bacteroidales marker (HF183/BFDrev) did not differ significantly in any of the sewage or wastewater samples, whereas the crAssphage marker abundance was higher in river samples. This initial validation of the crAssphage gene as a human-specific MST marker in Southeast Asia will promote its inclusion in an MST toolbox.