Identification and Application of Phocaeicola-Specific Conserved Signature DNA Markers for Human Fecal Source Tracking

A major goal of fecal pollution monitoring in the environment is to identify point sources of fecal contamination that may pose potential health risks due to animal- and human-specific pathogens. Ideal source tracking markers should have high host specificity and can be employed for the unambiguous identification of the host/fecal point sources. Conserved signature proteins (CSPs) are a class of unique, phylogenetically coherent indicators that are specific to a given taxon (e.g., genus or species). In this study, we report the identification and characterization of a new CSP, whose gene (designated as CSP-DV) is present in a single copy, and for whom homologs showing a high degree of sequence similarity are found only in genomes of Phocaeicola dorei and Phocaeicola vulgatus, two commensal species commonly found in the human gut and feces. We developed a qPCR method targeting this CSP gene to explore its usefulness as a human source tracking marker. We confirmed that the CSP-DV marker showed an absolute human sensitivity (100%) but some cross-reactivities in chicken, cats, dogs, rabbits, and rodents. In recreational water, the CSP-DV marker gene levels were well correlated with those of HF183, a well-validated human marker that predominantly targets the 16S rRNA gene of P. dorei, suggesting that it can be a new potential source tracking tool for human fecal contamination in specific environmental waters. In summary, our CSP-DV marker targets Phocaeicola clade-specific microbes and can provide an additional approach independent of the 16S rRNA gene to detect human sources of fecal pollution.