Occurrence and distributions of human-associated markers in an impacted urban watershed

Numerous genetic markers for microbial source tracking (MST) have been evaluated by testing a panel of target and nontarget faecal samples. However, the performance of MST markers may vary between faecal and water samples, thereby resulting in inaccurate water quality assessment. In this study, a 30-day sampling study was conducted in an urban river impacted by human- and sewage-associated pollution to evaluate the performance of different human-associated markers in environmental water. Additionally, marker decay was assessed via a microcosms approach. Overall, Bacteroidales 16sRNA and crAssphage markers exhibited higher prevalence in the study area, and their detection frequencies exceeded 90%. In contrast, Bacteroidales protein markers exhibited poor detection frequencies compared to other markers, with the prevalence of Hum2 and Hum163 reaching only 63% and 84%, respectively. Regarding marker abundance, there was no significant difference in the detection concentrations between Bacteroidales 16sRNA and crAssphage markers (p > 0.05); however, the concentrations of Bacteroidales protein markers were nearly 1 order of magnitude lower than those of other MST markers. The microcosm experiments indicated that the decay rate of crAssphage markers was significantly lower than that of other bacterial target markers, which may improve their detectability when the pollution source is located far from the sampling site. Due to the observed differences in performance and decay patterns among Bacteroidales 16sRNA, crAssphage, and Bacteroidales protein markers, we recommend the simultaneous use of multiple markers from different target microorganisms to obtain a more comprehensive understanding of the pollution sources. This approach would also provide an accurate assessment of pollution levels and health risks.