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Estuarine water quality is declining worldwide due to increased tourism, coastal development, and a changing climate. Although well-established methods are in place to monitor water quality, municipalities struggle to use the data to prioritize infrastructure for monitoring and repair and to determine sources of contamination when they occur. The objective of this study was to assess water quality and prioritize sources of contamination within Town Creek Estuary (TCE), Beaufort, North Carolina, by combining culture, molecular, and geographic information systems (GIS) data into a novel contamination source ranking system. Water samples were collected from TCE at ten locations on eight sampling dates in Fall 2021 (n = 80). Microbiological water quality was assessed using US Environmental Protection Agency (U.S. EPA) approved culture-based methods for fecal indicator bacteria (FIB), including analysis of total coliforms (TC), Escherichia coli (EC), and Enterococcus spp. (ENT). The quantitative microbial source tracking (qMST) human-associated fecal marker, HF183, was quantified using droplet digital PCR (ddPCR). This information was combined with environmental data and GIS information detailing proximal sewer, septic, and stormwater infrastructure to determine potential sources of fecal contamination in the estuary. Results indicated FIB concentrations were significantly and positively correlated with precipitation and increased throughout the estuary following rainfall events (p < 0.01). Sampling sites with FIB concentrations above the U.S. EPA threshold also had the highest percentages of aged, less durable piping materials. Using a novel ranking system combining concentrations of FIB, HF183, and sewer infrastructure data at each site, we found that the two sites nearest the most aged sewage infrastructure and stormwater outflows were found to have the highest levels of measurable fecal contamination. This case study supports the inclusion of both traditional water quality measurements and local infrastructure data to support the current need for municipalities to identify, prioritize, and remediate failing infrastructure. [ABSTRACT FROM AUTHOR]

Sewage contamination of environmental waters is increasingly assessed by measuring DNA from sewage-associated microorganisms in microbial source tracking (MST) approaches. However, DNA can persist through wastewater treatment and reach surface waters when treated sewage/recycled water is discharged, which may falsely indicate pollution from untreated sewage. Recycled water discharged from an advanced wastewater treatment (AWT) facility into a Florida stream elevated the sewage-associated HF183 marker 1,000-fold, with a minimal increase in cultured Escherichia coli . The persistence of sewage-associated microorganisms was compared by qPCR in untreated sewage and recycled water from conventional wastewater treatment (CWT) and AWT facilities. E. coli (EC23S857) and sewage-associated markers HF183, H8, and viral crAssphage CPQ&#95;056 were always detected in untreated sewage (6.5-8.7 log 10 GC/100 mL). Multivariate analysis found a significantly greater reduction of microbial variables via AWT vs CWT. Bacterial markers decayed ~4-5 log 10 through CWT, but CPQ&#95;056 was ~100-fold more persistent. In AWT facilities, the log 10 reduction of all variables was ~5. In recycled water, bacterial marker concentrations were significantly correlated ( P ≤ 0.0136; tau ≥ 0.44); however, CPQ&#95;056 was not correlated with any marker, suggesting varying drivers of decay. Concentrations of cultured E. coli carrying the H8 marker (EcH8) in untreated sewage were 5.24-6.02 log 10 CFU/100 mL, while no E. coli was isolated from recycled water. HF183 and culturable EcH8 were also correlated in contaminated surface waters (odds ratio β 1 = 1.701). Culturable EcH8 has a strong potential to differentiate positive MST marker signals arising from treated (e.g., recycled water) and untreated sewage discharged into environmental waters., Importance: Genes in sewage-associated microorganisms are widely accepted indicators of sewage pollution in environmental waters. However, DNA persists through wastewater treatment and can reach surface waters when recycled water is discharged, potentially causing false-positive indications of sewage contamination. Previous studies have found that bacterial and viral sewage-associated genes persist through wastewater treatment; however, these studies did not compare different facilities or identify a solution to distinguish sewage from recycled water. In this study, we demonstrated the persistence of bacterial marker genes and the greater persistence of a viral marker gene (CPQ&#95;056 of crAssphage) through varying wastewater treatment facilities. We also aim to provide a tool to confirm sewage contamination in surface waters with recycled water inputs. This work showed that the level of wastewater treatment affects the removal of microorganisms, particularly viruses, and expands our ability to identify sewage in surface waters., Competing Interests: The authors declare no conflict of interest.