Discrimination between human, pig and ruminant fecal contaminations in a river catchment by real-time PCR using host-specific markers

The microbiological quality of coastal waters and shellfish harvesting areas in Brittany (France) can be affected by faecal pollutions from human activities and animal breeding (especially pigs and cattle). To discriminate among faecal pollution of human and animal origin, a library-independent microbial source tracking method was selected: Bacteroidales host-specific 16S rRNA gene markers by real-time Polymerase Chain Reaction (PCR). A human-specific Bacteroidales marker (Hum-1-Bac) was designed. Tested on faecal samples, the Hum-1-Bac marker showed 95 % sensitivity and 95 % specificity (n= 80). Average values (± STD) of the Hum-1-Bac marker were found to be 7.3 ± 1.4 16S rRNA gene copies per g wet faeces in human faeces samples (n=10) and 5.7 ± 1.3 log10 copies per 100 ml water in Sewage Treatment Plant (STP) water samples (n=10). These results highlight that the human-specific marker present in individual faeces was still present in the effluents.
The human-specific marker developed in this study (Hum-1-Bac) and the human- (HF183), pig- (Pig-2-Bac) and ruminant- (Rum-2-Bac) specific Bacteroidales markers previously described by Seurinck et al. (2005) and Mieszkin et al. (2009, 2010) were then applied to river water samples (n=33) collected in 2008-2009 from the catchment of the Daoulas river estuary (Brittany, France), upstream of shellfish harvesting areas. For all sites, at least two host-specific markers were detected. The ruminant-specific Bacteroidales marker was more often quantified (60.6 %) than the human- or pig-specific Bacteroidales markers (Hum-1-Bac, 45.5 %; HF183, 48.5 % and Pig-2-Bac, 30.3 %) in river water samples. These results show that faecal pollution came generally from multiple origins and that host-specific Bacteroidales markers are promising as a quantitative microbial source tracking method to determine sources of faecal pollution in environmental water.