Validation of crAssphage microbial source tracking markers and comparison with Bacteroidales markers for detection and quantification of faecal contaminations in surface water

Human-specific faecal contamination has been affecting surface water and is a threat to both the environment and public health due to its potential co-occurrence with pathogens. Extended studies were conducted to detect and quantify faecal contamination using microbial source tracking (MST) markers targeting bacteria and viruses. The prototypical crAssphage, a presumed Bacteroides-infecting phage discovered in 2014, showed superior specificity to human faeces and high abundance in untreated sewage water. This study evaluated the applicability of crAssphage markers, CPQ_056 and CPQ_064, as MST tools for detecting domestic sewage contamination in surface water in China. Validation tests based on domestic sewage and animal faecal samples demonstrated high sensitivity/specificity of 100%/96.7% for CPQ_056 and 100%/100% for CPQ_064 within the scope of this study, surpassing the performance of traditional Bacteroidales markers such as HF183 (100%/80.4% against sewage). MST markers targeting different hosts and validated in the Taihu watershed (CPQ_056, CPQ_064, BacUni, HF183 TaqMan, Pig-2-Bac, and GFD) were quantified in water samples collected from the inflow rivers of Taihu Lake in summer and winter 2020. The results showed the dominance of sewage/wastewater as the source of contamination in all faecal pollution. Spatial analysis revealed higher contamination levels in northwest rivers, which were those most impacted by human activities. There was also a diluting pattern downstream of some rivers. Correlations with water quality parameters indicated the co-occurrence of nutrient-related pollution and faecal contamination, particularly in areas with industrial, low-density residential, green space, and municipal service land uses. The findings established the efficacy of crAssphage markers in enhancing precision and accuracy in monitoring faecal contamination, offering valuable tools for policymakers and environmental managers.