Comparative evaluation of decay characteristics of commonly used genetic faecal markers crAssphage and Bacteroides in complex river water microcosms

Comparative information on decay characteristics of genetic faecal markers including crAssphage is very limited for water resources, although PCR-based indicator-diagnostics are increasingly used worldwide. To our knowledge, we performed the first comparative microcosm experiments to assess the decay behaviour of commonly used qPCR-based genetic faecal markers (crAssphages, HF183/BacR287, BacHum) in a large European river. In addition, cultivation-based standards (E. coli, intestinal enterococci, Clostridium perfringens spores, somatic coliphages) were enumerated to allow for a cross-comparison between novel and established targets. River water was spiked with raw sewage from two combined municipal sewer systems and incubated at 8 °C and 20 °C for up to 66 days. The focus of the experimental work was put on genetic faecal markers. Two different filtration methods targeting either virus/phages and bacteria (using mixed cellulose esters, MCE) or solely bacteria (using polycarbonate filters, PC) were also evaluated. Results indicated biphasic decay for all targets (i.e., initial fast followed by secondary slow decay), with Bacteroides markers showing lower persistence under all conditions when compared to crAssphage. In addition, a tendency for higher long-term persistence of crAssphage based on MCE filtration as compared to PC filtration was observed. Finally, a meta-data analysis was conducted, comparing the results obtained here with those from recent case studies. This analysis further substantiated the comparatively higher persistence and lower decay of crAssphage relative to Bacteroides markers and established FIO, with the sole exception of C. perfringens spores.