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Somatic and F+ coliphage methods are under consideration as potential routine surface water quality monitoring tools to identify unsafe levels of fecal pollution in recreational waters. However, little is known about the cooccurrence of these virus-based fecal indicators and host-associated genetic markers used to prioritize key pollution sources for remediation. In this study, paired measurements of cultivated coliphage (somatic and F+) and bacterial (E. coli and enterococci) general fecal indicators and genetic markers indicative of human (HF183/BacR287 and HumM2), ruminant (Rum2Bac), canine (DG3), and avian (GFD) fecal pollution sources were assessed in 365 water samples collected from six Great Lakes Basin beach and river sites over a 15-week recreational season. Water samples were organized into groups based on defined viral and bacterial fecal indicator water quality thresholds and average log 10 host-associated genetic marker fecal score ratios were estimated to compare pollutant source inferences based on variable routine water quality monitoring practices. Eligible log 10 fecal score ratios ranged from -0.051 (F+ coliphage, GFD) to 2.08 (enterococci, Rum2Bac). Using a fecal score ratio approach, findings suggest that general fecal indicator selection for routine water quality monitoring can influence the interpretation of host-associated genetic marker measurements, in some cases, prioritizing different pollutant sources for remediation. Variable trends were also observed between Great Lake beach and river sites suggesting disparate management practices may be useful for each water type., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021. Published by Elsevier Ltd.)

Though Lake Michigan beaches in Chicago are not impacted by stormwater or wastewater outfalls, several of those beaches often exceed USEPA Beach Action Values (BAVs). We investigated the role of microbial source tracking (MST) as a complement to routine beach monitoring at Chicago beaches. In summer 2016, water samples from nine Chicago beaches were analyzed for E. coli by culture and enterococci by qPCR. A total of 195 archived samples were then tested for human (HF183/BacR287, HumM2), canine (DG3, DG37), and avian (GFD) microbial source tracking (MST) markers. Associations between MST and general fecal indicator bacteria (FIB) measures were evaluated and stratified based on wet and dry weather definitions. Among the 195 samples, HF183/BacR287 was quantifiable in 4%, HumM2 in 1%, DG3 in 6%, DG37 in 2%, and GFD in 23%. The one beach with a dog area was far more likely to have DG3 present in the quantifiable range than other beaches. Exceedance of general FIB BAVs increased the odds of human, dog and avian marker detection. MST marker weighted-average fecal scores for DG3 was 2.4 times, DG37 was 2.1 times, and GFD was 1.6 times higher during wet compared to dry weather conditions. HF183/BacR287 weighted-average fecal scores were not associated with precipitation. Associations between FIB BAV exceedance and MST marker detection were generally stronger in wet weather. Incorporating MST testing into routine beach water monitoring can provide information that beach managers can use when developing protection plans for beaches not impacted by point sources., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: The authors have no financial stake in the products described or in this research. The employer of two of the authors (AS and SD), the University of Illinois at Chicago, is contracted by the Chicago Park District to conduct beach monitoring, and that monitoring is managed by those authors. The beaches managed by the Chicago Park District are the subject of the manuscript., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

There is a growing interest for the use of coliphage as an alternative indicator to assess fecal pollution in recreational waters. Coliphage are a group of viruses that infect Escherichia coli and are considered as potential surrogates to infer the likely presence of enteric viral pathogens. We report the use of a dead-end hollow fiber ultrafiltration single agar layer method to enumerate F+ and somatic coliphage from surface waters collected from three Great Lake areas. At each location, three sites (two beaches; one river) were sampled five days a week over the 2015 beach season (n = 609 total samples). In addition, culturable E. coli and enterococci concentrations, as well as 16 water quality and recreational area parameters were assessed such as rainfall, turbidity, dissolved oxygen, pH, and ultra violet absorbance. Overall, somatic coliphage levels ranged from non-detectable to 4.39 log 10 plaque forming units per liter and were consistently higher compared to F+ (non-detectable to 3.15 log 10  PFU/L), regardless of sampling site. Coliphage concentrations weakly correlated with cultivated fecal indicator bacteria levels (E. coli and enterococci) at 75% of beach sites tested in study (r = 0.28 to 0.40). In addition, ultraviolet light absorption and water temperature were closely associated with coliphage concentrations, but not fecal indicator bacteria levels suggesting different persistence trends in Great Lake waters between indicator types (bacteria versus virus). Finally, implications for coliphage water quality management and future research directions are discussed., (Published by Elsevier Ltd.)

Human fecal pollution of recreational waters remains a public health concern worldwide. As a result, there is a growing interest in the application of human-associated fecal source identification quantitative real-time PCR (qPCR) technologies for water quality research and management. However, there are currently no standardized approaches for field implementation and interpretation of qPCR data. In this study, a standardized HF183/BacR287 qPCR method was combined with a water sampling strategy and a novel Bayesian weighted average approach to establish a human fecal contamination score (HFS) that can be used to prioritize sampling sites for remediation based on measured human waste levels. The HFS was then used to investigate 975 study design scenarios utilizing different combinations of sites with varying sampling intensities (daily to once per week) and number of qPCR replicates per sample (2-14 replicates). Findings demonstrate that site prioritization with HFS is feasible and that both sampling intensity and number of qPCR replicates influence reliability of HFS estimates. The novel data analysis strategy presented here provides a prescribed approach for the implementation and interpretation of human-associated HF183/BacR287 qPCR data with the goal of site prioritization based on human fecal pollution levels. In addition, information is provided for future users to customize study designs for optimal HFS performance., (Published by Elsevier Ltd.)

Understanding the decomposition of microorganisms associated with different human fecal pollution types is necessary for proper implementation of many water quality management practices, as well as predicting associated public health risks. Here, the decomposition of select cultivated and molecular indicators of fecal pollution originating from fresh human feces, septage, and primary effluent sewage in a subtropical marine environment was assessed over a six day period with an emphasis on the influence of ambient sunlight and indigenous microbiota. Ambient water mixed with each fecal pollution type was placed in dialysis bags and incubated in situ in a submersible aquatic mesocosm. Genetic and cultivated fecal indicators including fecal indicator bacteria (enterococci, E. coli, and Bacteroidales), coliphage (somatic and F+), Bacteroides fragilis phage (GB-124), and human-associated genetic indicators (HF183/BacR287 and HumM2) were measured in each sample. Simple linear regression assessing treatment trends in each pollution type over time showed significant decay (p ≤ 0.05) in most treatments for feces and sewage (27/28 and 32/40, respectively), compared to septage (6/26). A two-way analysis of variance of log 10 reduction values for sewage and feces experiments indicated that treatments differentially impact survival of cultivated bacteria, cultivated phage, and genetic indicators. Findings suggest that sunlight is critical for phage decay, and indigenous microbiota play a lesser role. For bacterial cultivated and genetic indicators, the influence of indigenous microbiota varied by pollution type. This study offers new insights on the decomposition of common human fecal pollution types in a subtropical marine environment with important implications for water quality management applications., (Published by Elsevier Ltd.)

Urban runoff can carry a variety of pollutants into recreational beaches, often including bacterial pathogens and indicators of fecal contamination. To develop complete recreational criteria and risk assessments, it is necessary to understand conditions under which human contamination could be present at beaches solely impacted by urban runoff. Accurately estimating risk requires understanding sources, concentrations, and transport mechanisms of microbial contaminants in these environments. By applying microbial source tracking methods and empirical modeling, we assessed the presence and level of human contamination at urban runoff impacted recreational beaches. We also identified environmental parameters and pollution sources that can influence the concentration and transport of culturable and molecular fecal indicator bacteria (FIB) in systems impacted solely by urban runoff. Water samples and physico-chemical parameters were collected from shoreline locations from three South Carolina (SC) beaches (five locations per beach) and two Florida (FL) beaches (three locations per beach). Each SC beach was directly impacted by swashes or tidal creeks receiving stormwater runoff from the urbanized area and therefore were designated as swash drain associated (SDA) beaches, while FL beaches were designated as non-swash drain associated (NSDA). Sampling in swash drains (SD; three sites per SD) directly impacting each SC beach was also conducted. Results indicate that although culturable (enterococci) and real-time quantitative polymerase chain reaction (qPCR) (EC23S857, Entero1, and GenBac3) FIB concentrations were, on average, higher at SD locations, SDA beaches did not have consistently higher molecular FIB signals compared to NSDA beaches. Both human-associated markers (HF183 and HumM2) were concomitantly found only at SDA beaches. Bacteroidales species-specific qPCR markers (BsteriF1 and BuniF2) identified differences in the Bacteroidales community, depending on beach type. The marker for general Bacteroidales was most abundant at SD locations and exhibited a high correlation with both culturable and other molecular markers. Combining molecular information with predictive modeling allowed us to identify both alongshore movement of currents and SD outflow as significant influences on the concentration of molecular and culturable indicators in the bathing zone. Data also suggests that combining methodologies is a useful and cost effective approach to help understand transport dynamics of fecal contamination and identify potential sources of contamination at marine beaches., (Published by Elsevier Ltd.)

The State of California has mandated the preparation of a guidance document on the application of fecal source identification methods for recreational water quality management. California contains the fifth highest population of cattle in the United States, making the inclusion of cow-associated methods a logical choice. Because the performance of these methods has been shown to change based on geography and/or local animal feeding practices, laboratory comparisons are needed to determine which assays are best suited for implementation. We describe the performance characterization of two end-point PCR assays (CF128 and CF193) and five real-time quantitative PCR (qPCR) assays (Rum2Bac, BacR, BacCow, CowM2, and CowM3) reported to be associated with either ruminant or cattle feces. Each assay was tested against a blinded set of 38 reference challenge filters (19 duplicate samples) containing fecal pollution from 12 different sources suspected to impact water quality. The abundance of each host-associated genetic marker was measured for qPCR-based assays in both target and non-target animals and compared to quantities of total DNA mass, wet mass of fecal material, as well as Bacteroidales, and enterococci determined by 16S rRNA qPCR and culture-based approaches (enterococci only). Ruminant- and cow-associated genetic markers were detected in all filters containing a cattle fecal source. However, some assays cross-reacted with non-target pollution sources. A large amount of variability was evident across laboratories when protocols were not fixed suggesting that protocol standardization will be necessary for widespread implementation. Finally, performance metrics indicate that the cattle-associated CowM2 qPCR method combined with either the BacR or Rum2Bac ruminant-associated methods are most suitable for implementation., (Published by Elsevier Ltd.)

The characteristics of fecal sources, and the ways in which they are measured, can profoundly influence the interpretation of which sources are contaminating a body of water. Although feces from various hosts are known to differ in mass and composition, it is not well understood how those differences compare across fecal sources and how differences depend on characterization methods. This study investigated how nine different fecal characterization methods provide different measures of fecal concentration in water, and how results varied across twelve different fecal pollution sources. Sources investigated included chicken, cow, deer, dog, goose, gull, horse, human, pig, pigeon, septage and sewage. A composite fecal slurry was prepared for each source by mixing feces from 6 to 22 individual samples with artificial freshwater. Fecal concentrations were estimated by physical (wet fecal mass added and total DNA mass extracted), culture-based (Escherichia coli and enterococci by membrane filtration and defined substrate), and quantitative real-time PCR (Bacteroidales, E. coli, and enterococci) characterization methods. The characteristics of each composite fecal slurry and the relationships between physical, culture-based and qPCR-based characteristics varied within and among different fecal sources. An in silico exercise was performed to assess how different characterization methods can impact identification of the dominant fecal pollution source in a mixed source sample. A comparison of simulated 10:90 mixtures based on enterococci by defined substrate predicted a source reversal in 27% of all possible combinations, while mixtures based on E. coli membrane filtration resulted in a reversal 29% of the time. This potential for disagreement in minor or dominant source identification based on different methods of measurement represents an important challenge for water quality managers and researchers., (Published by Elsevier Ltd.)

Many PCR-based methods for microbial source tracking (MST) have been developed and validated within individual research laboratories. Inter-laboratory validation of these methods, however, has been minimal, and the effects of protocol standardization regimes have not been thoroughly evaluated. Knowledge of factors influencing PCR in different laboratories is vital to future technology transfer for use of MST methods as a tool for water quality management. In this study, a blinded set of 64 filters (containing 32 duplicate samples generated from 12 composite fecal sources) were analyzed by three to five core laboratories with a suite of PCR-based methods utilizing standardized reagents and protocols. Repeatability (intra-laboratory variability) and reproducibility (inter-laboratory variability) of observed results were assessed. When standardized methodologies were used, intra- and inter-laboratory %CVs were generally low (median %CV 0.1-3.3% and 1.9-7.1%, respectively) and comparable to those observed in similar inter-laboratory validation studies performed on other methods of quantifying fecal indicator bacteria (FIB) in environmental samples. ANOVA of %CV values found three human-associated methods (BsteriF1, BacHum, and HF183Taqman) to be similarly reproducible (p > 0.05) and significantly more reproducible (p < 0.05) than HumM2. This was attributed to the increased variability associated with low target concentrations detected by HumM2 (approximately 1-2 log10copies/filter lower) compared to other human-associated methods. Cow-associated methods (BacCow and CowM2) were similarly reproducible (p > 0.05). When using standardized protocols, variance component analysis indicated sample type (fecal source and concentration) to be the major contributor to total variability with that from replicate filters and inter-laboratory analysis to be within the same order of magnitude but larger than inherent intra-laboratory variability. However, when reagents and protocols were not standardized, inter-laboratory %CV generally increased with a corresponding decline in reproducibility. Overall, these findings verify the repeatability and reproducibility of these MST methods and highlight the need for standardization of protocols and consumables prior to implementation of larger scale MST studies involving multiple laboratories., (Copyright © 2013 Elsevier Ltd. All rights reserved.)