Comparing viable cell and total DNA using qPCR and long read 16S rRNA gene sequencing for fecal source identification on the Raritan River

Fecal pollution in waterways poses a threat to human health worldwide. Fecal indicator bacteria are commonly used to assess water quality and the risk associated with recreational use of the water. However, traditional fecal indicator bacteria such as E. coli and enterococci are shed in the feces of many mammals and birds, underscoring the need for methods aimed at identifying the sources of contamination for targeted mitigation of fecal pollution in the waterway. Several microbial source tracking methods have been developed to date. Among these methods are quantitative PCR (qPCR) for genetic markers shed in the feces of specific animals as well as library-based approaches such as 16S rRNA gene sequencing. In this study, both methods were used on total DNA extracts from water and compared to results from viable cell DNA. Surface water samples were collected from six sites along the Raritan River in central New Jersey from May through October 2022 and 2023 by civic scientists for pathogen monitoring (i.e., cultivation of enterococci). Sample aliquots were preserved for total DNA analysis or treated with propidium monoazide dye for viable cell DNA analysis. Selected water samples from three sites representing a range of enterococci concentrations and rainfall conditions were analyzed via qPCR for a human fecal marker gene HF183 and 16S rRNA gene and sequenced using long-read 16S rRNA gene Nanopore sequencing. Fecal samples from wastewater influent and agricultural, wild, and domestic animals were sequenced for the construction of a fecal source library. The microbial communities of total and viable cell DNA from surface water were compared to the microbial communities of the source feces for diversity, structure, and using SourceTracker. The results of the two source tracking methods were compared to the culturable enterococci levels reported for each site and sampling day. The results of the qPCR analysis showed no correlation between human fecal gene marker HF183 and enterococci for viable or total cell DNA at any of the sampling sites. Results from SourceTracker showed that wastewater, swine, and bird were potential sources of feces in the watershed. Wastewater and bird source contributions both had a significant positive correlation with enterococci levels for viable cell DNA but not total DNA extracts from surface water samples. This demonstrates the potential utility of focusing on viable cell rather than total DNA when performing library-based fecal source tracking. Potential sources identified by SourceTracker, including wastewater, bird, and pig, highlight the complexity of the microbial contamination observed in the Lower Raritan River. Fecal contamination from wastewater suggests a potential need for improved wastewater management, and track down efforts to pinpoint whether leaking sanitary sewers, cross connections, unhoused populations, etc. are the primary contributors. Contamination from bird and pig indicate possible non-point pollution caused by agricultural runoff and wildlife in the watershed. Understanding these sources and pathways of entry into the river is crucial for developing interventions to improve water quality and protect public health. Differences in the results from viability qPCR and long-read amplicon sequencing emphasize the importance of multiple lines of microbial source tracking to narrow down potential sources of fecal contamination in a mixed land use watershed.