Comparison of propidium monoazide and total-DNA based qPCR and long-read sequencing for microbial source tracking in an estuarine river.

Pathogen pollution is a widespread water quality issue motivating fecal source tracking efforts to inform mitigation strategies. Biomolecular approaches including qPCR and library-based methods have been widely applied for fecal source tracking but can result in false positives given the persistence of DNA in aquatic systems. The potential for viable-cell DNA (i.e., propidium monoazide, PMA-DNA) to improve correlations with cultivable fecal microbes has received less attention than total-DNA based biomolecular approaches. The objectives of this study were to compare the results from total and PMA-DNA for human fecal marker HF183 qPCR and long-read library-based fecal source tracking. Biomolecular results were compared to enterococci observations generated through civic-science monitoring efforts sampling non-bathing beaches along the Lower Raritan River, NJ. Lower HF183 gene copy concentrations were seen in the PMA- compared to the total-filter concentrated DNA in this study, confirming the potential for total DNA to overestimate the contribution of human fecal indicator in this watershed. Nonetheless, no correlations were observed between human fecal gene marker HF183 and enterococci for PMA- or total-DNA. The source attributions identified via the long-read library-based method using SourceTracker varied by sampling site and DNA preparation method. More and stronger correlations were observed between putative fecal sources and enterococci observations for the PMA-DNA compared to the total-DNA samples. Interestingly, source tracking results also varied by total-DNA preparation method: total-centrifuge concentrated DNA source attributions to wastewater correlated with enterococci concentrations, not total filter-concentrated DNA. Overall, these results provide insight into the utility of PMA-qPCR and PMA-sequencing via the Nanopore platform for source tracking. Comparison of the additional costs and labor required for PMA-DNA preparation method is provided along with discussion of research needs.