Cross-method assessment of coliphage detection methods in the framework of EPA regulatory standards.

This study aimed to understand the performance and utility of US EPA-approved coliphage methods in comparison to fecal indicator bacteria (FIB) and molecular microbial source tracking (MST) methods in recreational waters. We used US EPA Method 1642 to quantify concentrations of coliphage along with culture- and molecular-based enumeration of E. coli and Enterococcus sp, and human fecal source marker HF183. We also conducted a feasibility assessment to determine the utility of US EPA Method 1642 for application to routine recreational water monitoring. Ten sampling events were conducted at three sampling sites over the duration of a year. Average concentrations of somatic (SC) (log10 1.48 PFU/100 mL) and male-specific (MSC) coliphages (log10 1.00 PFU/100 mL) at all sites were low with SC found across a broader range (0.3-3.1 log10 PFU/100 mL) of concentrations compared to MSC (non-detect-1.7 log10 PFU/100 mL). A feasibility assessment was conducted across US EPA Method 1642 for coliphage enumeration, culture-based FIB, defined substrate technology (DST) approaches Enterolert™ and Colilert®, and quantitative microbial source tracking (qMST) US EPA Method 1696. US EPA Method 1642 had the longest processing times, but also was moderate in cost, compared to the DST and qMST molecular methods. Given the poor correlations between MSC and SC with FIB and qMST markers in this study and the cumbersome nature of US EPA Method 1642, the method may not be the most applicable method for use in systems impacted predominantly by stormwater and other non-point source pollution. Findings from this study, however, provide guidance on the application of fecal indicator virus in ambient coastal surface waters.

Integrating culture and molecular quantification of microbial contaminants into a predictive modeling framework in a low-lying, tidally-influenced coastal watershed.

Examinations of stormwater delivery in the context of tidal inundation are lacking. Along the coastal plains of the southeastern United States, tidal inundation is increasing in frequency and severity, often with dramatic adverse impacts on timely stormwater discharge, coastal flooding hazards, and even "sunny day flooding". Therefore, a comprehensive study was conducted to examine tidally-influenced stormwater outfalls discharging to Taylor's Creek, an estuary proximal to Beaufort, NC used regularly for recreation and tourism. Over a wide range of meteorological conditions, water samples were collected and analyzed for fecal indicator bacteria (FIB, used for water quality management) and previously published quantitative microbial source tracking (qMST) markers. Nineteen sampling events were conducted from July 2017-June 2018 with samples classified according to tidal state and defined as either inundated, receding, or transition. A first-of-its-kind multiple linear regression model was developed to predict concentrations of Enterococcus sp. by tidal cycle, salinity and antecedent rainfall. We demonstrated that the majority of variability associated with the concentration of Enterococcus sp. could be predicted by E. coli concentration and tidal phase. FIB concentrations were significantly (<0.05) influenced by tide with higher concentrations observed in samples collected during receding (low) tides (EC: log 3.12 MPN/100 mL; ENT: 2.67 MPN/100 mL) compared to those collected during inundated (high) (EC: log 2.62 MPN/100 mL; ENT: 2.11 MPN/100 mL) or transition (EC: log 2.74 MPN/100 mL; ENT: 2.53 MPN/100 mL) tidal periods. Salinity, was also found to significantly (<0.05) correlate with Enterococcus sp. concentrations during inundated (high) tidal conditions (sal: 17 ppt; ENT: 2.04 MPN/100 mL). Tide, not precipitation, was shown to be a significant driver in explaining the variability in Enterococcus sp. concentrations. Precipitation has previously been shown to be a driver of Enterococcus sp. concentrations, but our project demonstrates the need for tidal parameters to be included in the future development of water quality monitoring programs.

Use of viral pathogens and indicators to differentiate between human and non-human fecal contamination in a microbial source tracking comparison study.

Assays for the detection and typing of adenoviruses, enteroviruses and F+ specific coliphages were performed on samples created as part of a national microbial source tracking methods comparison study. The samples were created blind to the researchers, and were inoculated with a variety of types of fecal contamination source (human, sewage, dog, seagull and cow) and mixtures of sources. Viral tracer and pathogen assays demonstrated a general ability to discriminate human from non-human fecal contamination. For example, samples inoculated with sewage were correctly identified as containing human fecal contamination because they contained human adenovirus or human enterovirus. In samples containing fecal material from individual humans, human pathogen analysis yielded negative results probably because the stool samples were taken from healthy individuals. False positive rates for the virus-based methods (0-8%) were among the lowest observed during the methods comparison study. It is suggested that virus-based source tracking methods are useful for identification of sewage contamination, and that these methods may also be useful as an indication of the public health risk associated with viral pathogens. Overall, virus-based source tracking methods are an important approach to include in the microbial source tracking 'toolbox'.