Critical Evaluation of CrAssphage as a Molecular Marker for Human-Derived Wastewater Contamination in the Aquatic Environment

The discharge of human-derived wastewater represents a major threat to water quality with the potential for waterborne disease outbreaks mainly associated with enteric viruses. To prevent illnesses, indicators associated with fecal contamination are monitored in polluted areas, however, their prevalence often does not correlate well with viral pathogens. In this study, we used crAssphage, a recently discovered human-specific gut-associated bacteriophage, for the surveillance of wastewater-derived viral contamination. Untreated and treated wastewater, surface water, sediment and mussel samples were collected monthly over 1 year from the Conwy River and estuary (UK) and were analyzed for crAssphage marker by quantitative PCR. This is the first long-term catchment-to-coast scale study of environmental crAssphage concentrations. CrAssphage was detected in all sample types and showed no distinct seasonal pattern. CrAssphage concentrations were 2 × 105-109 genome copies (gc)/L in all untreated wastewater influent and 107-108 gc/L in secondary treated effluent samples, 3 × 103 gc/L-3 × 107 gc/L in surface water samples (94% positive) and 2 × 102-104 gc/g sediment (68% positive) and mussel digestive tissue (79% positive). CrAssphage concentrations were 1-5 log10 higher than human enteric virus titers (norovirus, sapovirus, adenovirus, polyomavirus). Our results indicate that crAssphage is well suited to tracking human wastewater contamination and pollution risk assessment in aquatic environments.