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_056 were always detected in untreated sewage (6.5-8.7 log10 GC/100 mL). Multivariate analysis found a significantly greater reduction of microbial variables via AWT vs CWT. Bacterial markers decayed ~4-5 log10 through CWT, but CPQ_056 was ~100-fold more persistent. In AWT facilities, the log10 reduction of all variables was ~5. In recycled water, bacterial marker concentrations were significantly correlated (P ≤ 0.0136; tau ≥ 0.44); however, CPQ_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 log10 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 β1 = 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.
OBJECTIVE: 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_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.

We evaluated the potential impacts from using a rapid same-day quantitative polymerase chain reaction (qPCR) monitoring method for beach posting outcomes at two Toronto beaches.
In total, 228 water samples were collected at Marie Curtis Park East and Sunnyside Beaches over the 2021 summer season. Water samples were processed using the USEPA 1609.1 Enterococcus qPCR-based method. Escherichia coli (E. coli) culture data and daily beach posting decisions were obtained from Toronto Public Health.
No significant correlation was observed between previous-day and same-day (retrospective) E. coli enumeration results at any Sunnyside Beach transect, and only relatively low (R = 0.41-0.56) or no significant correlation was observed at sampling transects for Marie Curtis Park East Beach. Comparing our same-day Enterococcus qPCR data to Toronto\'s 2-day E. coli geometric mean beach posting decisions, we noted the need for additional postings for 1 (2%) and 3 (8%) missed health-risk days at Sunnyside and Marie Curtis Park East Beaches, respectively. The qPCR data also pointed to incorrect postings for 12 (31%) and 6 (16%) lost beach days at Sunnyside and Marie Curtis Park East Beaches, respectively.
Application of a rapid Enterococcus qPCR method at two Toronto beaches revealed 5% of beach posting decisions were false negatives that missed health-risk days, while 23% of decisions were false positives resulting in lost beach days. Deployment of the rapid same-day qPCR method offers the potential to reduce both health risks and unnecessary beach postings.
RéSUMé: OBJECTIFS: Nous avons évalué, à deux plages de Toronto, l’effet possible de l’utilisation d’une méthode de surveillance rapide par PCR quantitative (qPCR) le même jour sur les avis de fermeture ou d’ouverture des plages. MéTHODE: En tout, 228 échantillons d’eau ont été prélevés aux plages Marie Curtis Park East et Sunnyside au cours de la saison estivale 2021. La présence d’Enterococcus dans les échantillons a été détectée par la méthode USEPA 1609.1, utilisant la qPCR. Les données sur les cultures d’Escherichia coli (E. coli) et les avis quotidiens de fermeture ou d’ouverture des plages ont été obtenus auprès du Bureau de santé de Toronto. RéSULTATS: Aucune corrélation significative n’a été observée entre les résultats (rétrospectifs) du dénombrement de E. coli obtenus la veille et le même jour dans les transects de la plage Sunnyside, et une corrélation significative faible (R = 0,41–0,56) ou nulle a été observée dans les transects d’échantillonnage de la plage Marie Curtis Park East. En comparant nos données sur Enterococcus obtenues le même jour par qPCR à la moyenne géométrique des avis de fermeture ou d’ouverture des plages sur deux jours liés à E. coli émis par le Bureau de santé de Toronto, nous avons remarqué qu’il aurait fallu émettre des avis de fermeture pour 1 jour de risques pour la santé manqué (2 %) à la plage Sunnyside et pour 3 jours de risques pour la santé manqués (8 %) à la plage Marie Curtis Park East. Les données de la qPCR ont aussi fait état d’avis de fermeture incorrects ayant entraîné la perte de 12 jours de plage (31 %) à Sunnyside et de 6 jours de plage (16 %) à Marie Curtis Park East. CONCLUSION: L’application d’une méthode de surveillance rapide d’Enterococcus par qPCR à deux plages de Toronto a montré que 5 % des avis étaient des faux négatifs qui n’ont pas détecté des jours de risques pour la santé, et que 23 % étaient des faux positifs qui ont entraîné des jours de plage perdus. Le déploiement de la méthode rapide par qPCR le même jour offre la possibilité de réduire à la fois les risques pour la santé et les avis de fermeture de plages inutiles.

Viral indicators of human-fecal contamination in wastewaters and environmental waters have been getting much attention in the past decade. Cross-assembly phage (crAssphage) is the most abundant DNA virus in human feces. Recently, the usefulness of crAssphage as a microbial source tracking and water quality monitoring tool for human-fecal contamination has been highlighted. Here, we conducted a comprehensive review on crAssphage in water, focusing on detection methodology, concentration range in various waters and wastewaters, specificity to human-fecal contamination, and reduction in wastewater treatment systems. This review highlights that crAssphage is globally distributed in wastewaters and various fecal-contaminated water bodies at high concentrations without seasonal fluctuations. CrAssphage is highly specific to human-fecal contamination and is rarely found in animal feces. It also has a good potential as a performance indicator to ensure virus reduction in wastewater treatment systems. Accordingly, crAssphage could be an effective tool for monitoring of human-fecal contamination and potential presence of fecal pathogenic microbes in environmental waters. Bridging the research gaps highlighted in this review would make crAssphage a powerful tool to support the control of water-related health risks.

Despite the widely acknowledged public health impacts of surface water fecal contamination, there is limited understanding of seasonal effects on (i) fate and transport processes and (ii) the mechanisms by which they contribute to water quality impairment. Quantifying relationships between land use, chemical parameters, and fecal bacterial concentrations in watersheds can help guide the monitoring and control of microbial water quality and explain seasonal differences. The goals of this study were to (i) identify seasonal differences in Escherichia coli and Bacteroides thetaiotaomicron concentrations, (ii) evaluate environmental drivers influencing microbial contamination during baseflow, snowmelt, and summer rain seasons, and (iii) relate seasonal changes in B. thetaiotaomicron to anticipated gastrointestinal infection risks. Water chemistry data collected during three hydroclimatic seasons from 64 Michigan watersheds were analyzed using seasonal linear regression models with candidate variables including crop and land use proportions, prior precipitation, chemical parameters, and variables related to both wastewater treatment and septic usage. Adaptive least absolute shrinkage and selection operator (LASSO) linear regression with bootstrapping was used to select explanatory variables and estimate coefficients. Regardless of season, wastewater treatment plant and septic system usage were consistently selected in all primary models for B. thetaiotaomicron and E. coli. Chemistry and precipitation-related variable selection depended upon season and organism. These results suggest a link between human pollution (e.g., septic systems) and microbial water quality that is dependent on flow regime. IMPORTANCE In this study, a data set of 64 Michigan watersheds was utilized to gain insights into fecal contamination sources, drivers, and chemical correlates across seasons for general E. coli and human-specific fecal indicators. Results reaffirmed a link between human-specific sources (e.g., septic systems) and microbial water quality. While the importance of human sources of fecal contamination and fate and transport variables (e.g., precipitation) remain important across seasons, this study provides evidence that fate and transport mechanisms vary with seasonal hydrologic condition and microorganism source. This study contributes to a body of research that informs prioritization of fecal contamination source control and surveillance strategy development to reduce the public health burden of surface water fecal contamination.

With a focus on five sites in an impaired, densely populated area in the New Orleans area, we investigated the temporal and spatial variability of standard FIB and a marker of human-associated pollution (Bacteroides HF183). With all sites combined, only a weak positive correlation (r = 0.345; p = 0.001) was observed between E. coli and HF183. Also, specific conductivity (r = - 0.374; p < 0.0001) and dissolved oxygen (r = - 0.390; p < 0.0001) were observed to show a weak moderate correlation with E. coli. These correlations increased to moderately negative when HF183 was correlated with specific conductivity (r = - 0.448; p < 0.0001) and dissolved oxygen (r = - 0.455; p < 0.0001). E. coli contamination was generally highest at the sites in the canal that are situated in the most densely populated part of the watershed while HF183 was frequently detected across all sites. E. coli concentrations were significantly higher (p < 0.05) when HF183 was present. HF183 was detected at significantly higher concentrations in samples that exceeded the EPA water quality standard (WQS) than those that did not (p < 0.05). Dissolved oxygen and specific conductivity were significantly lower when E. coli WQS was exceeded or when HF183 was present (p < 0.05). Rainfall impacted E. coli concentrations and HF183 differently at the study sites. While HF183 and E. coli concentrations levels were significantly higher (p < 0.05) if the days prior to sampling had been wet, the frequency of detection of HF183 was unimpacted, as comparable detection rates were recorded during wet and dry weather conditions. Without testing for HF183, it would have been assumed, based on testing for E. coli alone, that human fecal pollution was only associated with densely populated areas and rainfall events. E. coli alone may not be an effective indicator of sewage pollution at the study sites across all weather conditions and may need to be complemented with HF183 enumeration to optimize human fecal pollution identification and management at the watershed level.

Fecal source tracking (FST) may be useful to assess pathways of fecal contamination in domestic environments and to estimate the impacts of water, sanitation, and hygiene (WASH) interventions in low-income settings. We measured two nonspecific and two human-associated fecal indicators in water, soil, and surfaces before and after a shared latrine intervention from low-income households in Maputo, Mozambique, participating in the Maputo Sanitation (MapSan) trial. Up to a quarter of households were impacted by human fecal contamination, but trends were unaffected by improvements to shared sanitation facilities. The intervention reduced Escherichia coli gene concentrations in soil but did not impact culturable E. coli or the prevalence of human FST markers in a difference-in-differences analysis. Using a novel Bayesian hierarchical modeling approach to account for human marker diagnostic sensitivity and specificity, we revealed a high amount of uncertainty associated with human FST measurements and intervention effect estimates. The field of microbial source tracking would benefit from adding measures of diagnostic accuracy to better interpret findings, particularly when FST analyses convey insufficient information for robust inference. With improved measures, FST could help identify dominant pathways of human and animal fecal contamination in communities and guide the implementation of effective interventions to safeguard health.

Rapid economic development has caused industrial expansion into residential communities, leading to higher fecal pollution loads that could be discharged into aquatic environments. However, little is known regarding the potential microbial impact on human health. This study investigated microbial contamination from coastal industrial-residential community areas in nine sampling sites in waterways during three dry events. A general microbial source tracking (MST) marker, GenBac3, was detected in all samples from all three events, indicating continuing fecal pollution in the area, mostly from human sewage contamination. This was shown by the human-specific genetic marker crAssphage (88.9%) and human polyomavirus (HPyVs; 92.6%) detection. Enteric human adenovirus (HAdV40/41) showed three positive results only from residential sites in the first event. No spatial difference was observed for MST markers and traditional fecal indicators (total coliforms and Escherichia coli) in each event. Still, a significantly lower abundance of GenBac3, HPyVs, and total coliforms in the first sampling event was detected. Spearman\'s rho analysis indicated a strong correlation among certain pairs of microbial parameters. Multivariate analysis revealed two clusters of samples separated by land use type (industrial vs. residential). According to factor analysis of mixed data, the land use parameter was more associated with physicochemical parameters (i.e., salinity, conductivity, water temperature, and dissolved oxygen). A Quantitative Microbial Risk Assessment (QMRA) was then conducted to estimate the annual infection risks of HAdV40/41 for non-potable water reuse purposes using predicted concentrations from crAssphage and HPyVs. The highest risks (95th percentiles) were ranked by food crop irrigation, aquaculture, and toilet flushing, at 10-1, 10-2, and 10-3 per person per year (pppy). Required treatment levels to achieve a 10-4 pppy annual infection risk were estimated. QMRA-based water treatment scenarios were suggested, including chlorination for toilet flushing reuse and depth filtration prior to chlorination for aquaculture and food crop irrigation. Microbial monitoring combined with a QMRA could provide better insights into fecal pollution patterns and the associated risks, facilitating effective water quality management and appropriate prior treatments for water reuse.

The ribbed mussel has been demonstrated to tolerate high levels of urban pollution and inhabits intertidal regions of the New York City estuary. The ability of this bivalve to filter bacteria raises the question of whether it can remove from the water column the fecal bacteria introduced to urban waterways by septic system leakage or sewer overflow. The study here addresses the hypothesis that ribbed mussel filters bacteria introduced by combined sewer overflow (CSO) discharge. Mussels and water were collected from a highly polluted region of the NYC estuary in order to conduct two sets of five trials for filtration of coliform and coccoid fecal indicator bacteria, respectively, Escherichia coli and Enterococcus species. Mussels and water samples were collected in proximity to a major CSO outfall within 1-2 days of a rainfall event to ensure high baseline values of bacterial contamination for filtration trials. For any given Enterococcus or E. coli trial, equal volume water samples were serially distributed across aerated tanks either containing a mussel or not. Comparison of with-mussel versus no-mussel tank water contamination across pooled trials showed significant (P < 0.05) reduction in water exposed to mussel filtration for both, Enterococcus and E. coli trials. For Enterococcus trials, measures of turbidity (suspended particle density) were taken concurrently with measures of bacterial contamination. Regression of contamination against turbidity, with measures standardized across trials, yielded a significant positive association (n = 50, P < 0.0001) across all tank water with a mussel. Thus, contamination reduction was associated with particle removal by mussel filtration.

Fecal indicator bacteria (FIB) are typically used to monitor microbial water quality but are poor representatives of viruses due to different environmental fate. Viral fecal indicators have been proposed as alternatives to FIB; however, data evaluating the persistence of emerging viral fecal indicators under realistic environmental conditions is necessary to evaluate their potential application. In this study, we examined the persistence of five viral fecal indicators, including crAssphage and pepper mild mottle virus (PMMoV), and three bacterial fecal indicators (E. coli, enterococci and HF183/BacR287) in large-scale experimental ponds and freshwater mesocosms. Observed inactivation rate constants were highly variable and ranged from a minimum of -0.09 d-1 for PMMoV to a maximum of -3.5 d-1 for HF183/BacR287 in uncovered mesocosms. Overall, viral fecal indicators had slower inactivation than bacterial fecal indicators and PMMoV was inactivated more slowly than all other targets. These results demonstrate that bacterial fecal indicators inadequately represent viral fate following aging of sewage contaminated water due to differential persistence, and that currently used fecal indicator monitoring targets demonstrate highly variable persistence that should be considered during water quality monitoring and risk assessment.

Human and animal feces are important sources of various types of microbial contamination in water. Especially, enteric viruses, the major agents of waterborne infection, can attain long-term survival in water environments due to their strong resistance to various environmental factors including pH, salinity, and temperature. Coliphages are promising viral indicators for fecal contamination in water environments. Here, we investigated the seasonal and spatial distribution of male-specific and somatic coliphages in surface water and seawater at three major aquaculture areas, including Goseong Bay, Aphae Island, and Gomso Bay, in Republic of Korea over a period of 1 year. We selected 6 surface water and 14 seawater sampling sites for each study area and collected a total of 480 water samples from March 2014 to February 2015. Overall, surface water samples contained higher occurrences of coliphages than seawater samples. The high coliphage concentrations were detected in spring (March to May 2014). The differences in geographical features and patterns in land usage of the three aquaculture areas may have affected the coliphage concentration and occurrence. Moreover, environmental factors such as cumulative precipitation were strongly correlated with coliphage concentrations. Therefore, we suggest that further longitudinal studies on coliphage concentrations and distributions should be performed to support the application of coliphages in tracking fecal contamination in water.