crAssphage as a human molecular marker to evaluate temporal and spatial variability in faecal contamination of urban marine bathing waters

Bathing water quality may be negatively impacted by diffuse pollution arising from urban and agricultural activities and wildlife, it is therefore important to be able to differentiate between biological and geographical sources of faecal pollution. crAssphage was recently described as a novel human-associated microbial source tracking marker. This study aimed to evaluate the performance of the crAssphage marker in designated bathing waters. The sensitivity and specificity of the crAss_2 marker was evaluated using faecal samples from herring gulls, dogs, sewage and a stream impacted by human pollution (n = 80), which showed that all human impacted samples tested positive for the marker while none of the animal samples did. The crAss_2 marker was field tested in an urban marine bathing water close to the discharge point of human impacted streams. In addition, the bathing water is affected by dog and gull fouling. Analysis of water samples taken at the compliance point every 30 min during a tidal cycle following a rain event showed that the crAss_2 and HF183 markers performed equally well (Spearman correlation ρ = 0.84). The levels of these marker and faecal indicators (Escherichia coli, intestinal enterococci, somatic coliphages) varied by up to 2.5 log₁₀ during the day. Analysis of a high-tide transect perpendicular to the shoreline revealed high levels of localised faecal contamination 1 km offshore, with a concomitant spike in the gull marker. In contrast, both the crAss_2 and HF183 markers remained at a constant level, showing that human faecal contamination is homogenously distributed, while gull pollution is localised. Performance of the crAss_2 and HF183 assay was further evaluated in bimonthly compliance point samples over an 18-month period. The co-occurrence between the crAss_2 and HF183 markers in compliance sampling was 76%. A combination of both markers should be applied in low pollution impacted environments to obtain a high confidence level.