Viral and Bacterial Fecal Indicators in Untreated Wastewater across the Contiguous United States Exhibit Geospatial Trends.

Cultivated fecal indicator bacteria such as Escherichia coli and enterococci are typically used to assess the sanitary quality of recreational waters. However, these indicators suffer from several limitations, such as the length of time needed to obtain results and the fact that they are commensal inhabitants of the gastrointestinal tract of many animals and have fate and transport characteristics dissimilar to pathogenic viruses. Numerous emerging technologies that offer same-day water quality results or pollution source information or that more closely mimic persistence patterns of disease-causing pathogens that may improve water quality management are now available, but data detailing geospatial trends in wastewater across the United States are sparse. We report geospatial trends of cultivated bacteriophage (somatic, F+, and total coliphages and GB-124 phage), as well as genetic markers targeting polyomavirus, enterococci, E. coli, Bacteroidetes, and human-associated Bacteroides spp. (HF183/BacR287 and HumM2) in 49 primary influent sewage samples collected from facilities across the contiguous United States. Samples were selected from rural and urban facilities spanning broad latitude, longitude, elevation, and air temperature gradients by using a geographic information system stratified random site selection procedure. Most indicators in sewage demonstrated a remarkable similarity in concentration regardless of location. However, some exhibited predictable shifts in concentration based on either facility elevation or local air temperature. Geospatial patterns identified in this study, or the absence of such patterns, may have several impacts on the direction of future water quality management research, as well as the selection of alternative metrics to estimate sewage pollution on a national scale.IMPORTANCE This study provides multiple insights to consider for the application of bacterial and viral indicators in sewage to surface water quality monitoring across the contiguous United States, ranging from method selection considerations to future research directions. Systematic testing of a large collection of sewage samples confirmed that crAssphage genetic markers occur at a higher average concentration than key human-associated Bacteroides spp. on a national scale. Geospatial testing also suggested that some methods may be more suitable than others for widespread implementation. Nationwide characterization of indicator geospatial trends in untreated sewage represents an important step toward the validation of these newer methods for future water quality monitoring applications. In addition, the large paired-measurement data set reported here affords the opportunity to conduct a range of secondary analyses, such as the generation of new or updated quantitative microbial risk assessment models used to estimate public health risk.

Longitudinal Assessment of the Dynamics of Escherichia coli, Total Coliforms, Enterococcus spp., and Aeromonas spp. in Alternative Irrigation Water Sources: a CONSERVE Study.

As climate change continues to stress freshwater resources, we have a pressing need to identify alternative (nontraditional) sources of microbially safe water for irrigation of fresh produce. This study is part of the center CONSERVE, which aims to facilitate the adoption of adequate agricultural water sources. A 26-month longitudinal study was conducted at 11 sites to assess the prevalence of bacteria indicating water quality, fecal contamination, and crop contamination risk (Escherichia coli, total coliforms [TC], Enterococcus, and Aeromonas). Sites included nontidal freshwater rivers/creeks (NF), a tidal brackish river (TB), irrigation ponds (PW), and reclaimed water sites (RW). Water samples were filtered for bacterial quantification. E. coli, TC, enterococci (∼86%, 98%, and 90% positive, respectively; n = 333), and Aeromonas (∼98% positive; n = 133) were widespread in water samples tested. Highest E. coli counts were in rivers, TC counts in TB, and enterococci in rivers and ponds (P < 0.001 in all cases) compared to other water types. Aeromonas counts were consistent across sites. Seasonal dynamics were detected in NF and PW samples only. E. coli counts were higher in the vegetable crop-growing (May-October) than nongrowing (November-April) season in all water types (P < 0.05). Only one RW and both PW sites met the U.S. Food Safety Modernization Act water standards. However, implementation of recommended mitigation measures of allowing time for microbial die-off between irrigation and harvest would bring all other sites into compliance within 2 days. This study provides comprehensive microbial data on alternative irrigation water and serves as an important resource for food safety planning and policy setting.IMPORTANCE Increasing demands for fresh fruit and vegetables, a variable climate affecting agricultural water availability, and microbial food safety goals are pressing the need to identify new, safe, alternative sources of irrigation water. Our study generated microbial data collected over a 2-year period from potential sources of irrigation (rivers, ponds, and reclaimed water sites). Pond water was found to comply with Food Safety Modernization Act (FSMA) microbial standards for irrigation of fruit and vegetables. Bacterial counts in reclaimed water, a resource that is not universally allowed on fresh produce in the United States, generally met microbial standards or needed minimal mitigation. We detected the most seasonality and the highest microbial loads in river water, which emerged as the water type that would require the most mitigation to be compliant with established FSMA standards. This data set represents one of the most comprehensive, longitudinal analyses of alternative irrigation water sources in the United States.

Uneven genotypic diversity of Escherichia coli in fecal sources limits the performance of a library-dependent method of microbial source tracking on the southwestern French Atlantic coast.

To develop a library-dependent method of tracking fecal sources of contamination of beaches on the Atlantic coast of southwestern France, a library of 6368 Escherichia coli isolates was constructed from samples of feces, from 40 known human or animal sources collected in the vicinity of Arcachon Bay in 2010, and in French Basque Country, Landes, and Béarn, between 2017 and 2018. Different schemes of source identification were tested: use of the complete or filtered reference library; characterization of the isolates by genotypic or proteomic profiling based on ERIC-PCR or MALDI-TOF mass spectrometry, respectively; isolate by isolate assignment using either classifiers based on the Pearson similarity or SVM (support vector machine). With the exception of one source identification scheme, which was discarded since it used self-assignment, all tested schemes resulted in low rates of correct classification (<35%) and significant rates of incorrect classification (>15%). The heterogeneous coverage of E. coli genotypic diversity between sources and the uneven distribution of E. coli genotypes in the library likely explain the difficulties encountered in identifying the sources of fecal contamination. Shannon diversity index of sources ranged from 0 for several wildlife species sampled once to 3.03 for sewage treatment plant effluents sampled on various occasions, showing discrepancies between sources. The uneven genotypic composition of the library was attested by the value of the Pielou index (0.54), the high proportion of nondiscriminatory genotypes (>91% of the isolates), and the very low proportion of discriminatory genotypes (<3%). Since efforts made to constitute such a library are not affordable for routine analyses, the results question the relevance of developing such a method for identifying sources of fecal contamination on such a coastline.

Verification of Bacteroidales 16S rRNA markers as a complementary tool for detecting swine fecal pollution in the Yangtze Delta.

To correctly assess and properly manage the public health risks associated with exposure to contaminated water, it is necessary to identify the source of fecal pollution in a watershed. In this study, we evaluated the efficacy of our two previously developed real time-quantitative PCR (qPCR) assays for the detection of swine-associated Bacteroidales genetic markers (gene 1-38, gene 3-53) in the Yangtze Delta watershed of southeastern China. The results indicated that the gene 1-38 and 3-53 markers exhibited high accuracy (92.5%, 91.7% conditional probability, respectively) in detecting Bacteroidales spp. in water samples. According to binary logistic regression (BLR), these two swine-associated markers were well correlated (P < 0.05) with fecal indicators (Escherichia coli and Enterococci spp.) and zoonotic pathogens (E. coli O157: H7, Salmonella spp. and Campylobacter spp.) in water samples. In contrast, concentrations of conventional fecal indicator bacteria (FIB) were not correlated with zoonotic pathogens, suggesting that they are noneffective at detecting fecal pollution events. Collectively, the results obtained in this study demonstrated that a swine-targeted qPCR assay based on two Bacteroidales genes markers (gene 1-38, gene 3-53) could be a useful tool in determining the swine-associated impacts of fecal contamination in a watershed.

Occurrence of Traditional and Alternative Fecal Indicators in Tropical Urban Environments under Different Land Use Patterns.

This study evaluated the geospatial distribution of fecal indicator bacteria (FIB) (i.e., Escherichia coli, Enterococcus spp.) and the alternative fecal indicator pepper mild mottle virus (PMMoV) in tropical freshwater environments under different land use patterns. Results show that the occurrence and concentration of microbial fecal indicators were higher for urban than for parkland-dominated areas, consistent with land use weightage. Significant positive correlations with traditional FIB indicate that PMMoV is a suitable indicator of fecal contamination in tropical catchments waters (0.549 ≤ rho ≤ 0.612; P < 0.01). PMMoV exhibited a strong significant correlation with land use weightage (rho = 0.728; P < 0.01) compared to traditional FIB (rho = 0.583; P < 0.01). In addition, chemical tracers were also added to evaluate the potential relationships with microbial fecal indicators. The relationships between diverse variables (e.g., environmental parameters, land use coverage, and chemical tracers) and the occurrence of FIB and PMMoV were evaluated. By using stepwise multiple linear regression (MLR), the empirical experimental models substantiate the impact of land use patterns and anthropogenic activities on microbial water quality, and the output results of the empirical models may be able to predict the sources and transportation of human fecal pollution or sewage contamination. In addition, the high correlation between PMMoV data obtained from quantitative real-time PCR (qPCR) and viral metagenomics data supports the possibility of using viral metagenomics to relatively quantify specific microbial indicators for monitoring microbial water quality (0.588 ≤ rho ≤ 0.879; P < 0.05).IMPORTANCE The results of this study may support the hypothesis of using PMMoV as an alternative indicator of human fecal contamination in tropical surface waters from the perspective of land use patterns. The predictive result of the occurrence of human fecal indicators with high accuracy may reflect the source and transportation of human fecal pollution, which are directly related to the risk to human health, and thereafter, steps can be taken to mitigate these risks.

Utilizing novel Escherichia coli-specific conserved signature proteins for enhanced monitoring of recreational water quality.

Escherichia coli serves as a proxy indicator of fecal contamination in aquatic ecosystems. However, its identification using traditional culturing methods can take up to 24 h. The application of DNA markers, such as conserved signature proteins (CSPs) genes (unique to all species/strains of a specific taxon), can form the foundation for novel polymerase chain reaction (PCR) tests that unambiguously identify and detect targeted bacterial taxa of interest. This paper reports the identification of three new highly-conserved CSPs (genes), namely YahL, YdjO, and YjfZ, which are exclusive to E. coli/Shigella. Using PCR primers based on highly conserved regions within these CSPs, we have developed quantitative PCR (qPCR) assays for the evaluation of E. coli/Shigella species in water ecosystems. Both in-silico and experimental PCR testing confirmed the absence of sequence match when tested against other bacteria, thereby confirming 100% specificity of the tested CSPs for E. coli/Shigella. The qPCR assays for each of the three CSPs provided reliable quantification for all tested enterohaemorrhagic and environmental E. coli strains, a requirement for water testing. For recreational water samples, CSP-based quantification showed a high correlation (r > 7, p < 0.01) with conventional viable E. coli enumeration. This indicates that novel CSP-based qPCR assays for E. coli can serve as robust tools for monitoring water ecosystems and other critical areas, including food monitoring.

Occurrence of recreational water quality monitoring general fecal indicator bacteria and fecal source identification genetic markers in gray seal scat.

The number of gray seals (Halichoerus grypus) observed along the United States Northwest Atlantic region has been increasing for decades. These colonial animals often haul-out on beaches seasonally in numbers ranging from a few individuals to several thousands. While these larger aggregations are an important part of gray seal behavior, there is public concern that haul-outs could lead to large amounts of fecal waste in recreational areas, potentially resulting in beach closures. Yet, data to confirm whether these animals contribute to beach closures is lacking and minimal information is available on the occurrence of key water quality monitoring genetic markers in gray seal scat. This study evaluates the concentration of E. coli (EC23S857), enterococci (Entero1a), and fecal Bacteroidetes (GenBac3) as well as six fecal source identification genetic markers (HF183/BacR287, HumM2, CPQ_056, Rum2Bac, DG3, and GFD) measured by qPCR in 48 wild gray seal scat samples collected from two haul-out areas in Cape Cod (Massachusetts, U.S.A.). Findings indicate that FIB genetic markers are shed in gray seal scat at significantly different concentrations with the Entero1a genetic marker exhibiting the lowest average concentration (-0.73 log10 estimated mean copies per nanogram of DNA). In addition, systematic testing of scat samples demonstrated that qPCR assays targeting host-associated genetic markers indicative of human, ruminant, and canine fecal pollution sources remain highly specific in waters frequented by gray seals (>97 % specificity).

Identification of potential microbial risk factors associated with fecal indicator exceedances at recreational beaches.

BACKGROUND: Fecal bacterial densities are proxy indicators of beach water quality, and beach posting decisions are made based on Beach Action Value (BAV) exceedances for a beach. However, these traditional beach monitoring methods do not reflect the full extent of microbial water quality changes associated with BAV exceedances at recreational beaches (including harmful cyanobacteria). This proof of concept study evaluates the potential of metagenomics for comprehensively assessing bacterial community changes associated with BAV exceedances compared to non-exceedances for two urban beaches and their adjacent river water sources. RESULTS: Compared to non-exceedance samples, BAV exceedance samples exhibited higher alpha diversity (diversity within the sample) that could be further differentiated into separate clusters (Beta-diversity). For Beach A, Cyanobacterial sequences (resolved as Microcystis and Pseudanabaena at genus level) were significantly more abundant in BAV non-exceedance samples. qPCR validation supported the Cyanobacterial abundance results from metagenomic analysis and also identified saxitoxin genes in 50% of the non-exceedance samples. Microcystis sp and saxitoxin gene sequences were more abundant on non-exceedance beach days (when fecal indicator data indicated the beach should be open for water recreational purposes). For BAV exceedance days, Fibrobacteres, Pseudomonas, Acinetobacter, and Clostridium sequences were significantly more abundant (and positively correlated with fecal indicator densities) for Beach A. For Beach B, Spirochaetes (resolved as Leptospira on genus level) Burkholderia and Vibrio sequences were significantly more abundant in BAV exceedance samples. Similar bacterial diversity and abundance trends were observed for river water sources compared to their associated beaches. Antibiotic Resistance Genes (ARGs) were also consistently detected at both beaches. However, we did not observe a significant difference or correlation in ARGs abundance between BAV exceedance and non-exceedance samples. CONCLUSION: This study provides a more comprehensive analysis of bacterial community changes associated with BAV exceedances for recreational freshwater beaches. While there were increases in bacterial diversity and some taxa of potential human health concern associated with increased fecal indicator densities and BAV exceedances (e.g. Pseudomonas), metagenomics analyses also identified other taxa of potential human health concern (e.g. Microcystis) associated with lower fecal indicator densities and BAV non-exceedances days. This study can help develop more targeted beach monitoring strategies and beach-specific risk management approaches.

Simultaneous microbial capture and nucleic acid extraction from wastewater with minimal pre-processing and high recovery efficiency.

The COVID-19 pandemic accelerated research towards developing low-cost assays for automated urban wastewater monitoring assay that can be integrated into an environmental surveillance system for early warning of frequent disease outbreaks and future pandemics. Microbial concentration is one of the most challenging steps in wastewater surveillance, due to the sample heterogeneity and low pathogen load. Keeping in mind the requirements of large-scale testing in densely populated low- or middle-income countries (LMICs), such assays would need to be low-cost and have rapid turnaround time with high recovery efficiency. In this study, two such methods are presented and evaluated against commercially available kits for pathogen detection in wastewater. The first method utilizes paper dipsticks while the second method comprises of a PTFE membrane filter (PMF) integrated with a peristaltic pump. Both methods were used to concentrate and isolate nucleic acids from different microbes such as SARS-CoV-2, pepper mild mottle virus (PMMoV), bacteriophage Phi6, and E. coli from wastewater samples with minimal or no sample pre-processing. While the paper dipstick method is suitable for sub-milliliter sample volume, the PMF method can be used with larger volumes of wastewater sample (40 mL) and can detect multiple microbes with recovery efficiency comparable to commercially available kits.

Relationship between coliphage and Enterococcus at southern California beaches and implications for beach water quality management.

Coliphage have been suggested as an alternative to fecal indicator bacteria for assessing recreational beach water quality, but it is unclear how frequently and at what types of beaches coliphage produces a different management outcome. Here we conducted side-by-side sampling of male-specific and somatic coliphage by the new EPA dead-end hollow fiber ultrafiltration (D-HFUF-SAL) method and Enterococcus at southern California beaches over two years. When samples were combined for all beach sites, somatic and male-specific coliphage both correlated with Enterococcus. When examined categorically, Enterococcus would have resulted in approximately two times the number of health advisories as somatic coliphage and four times that of male-specific coliphage,using recently proposed thresholds of 60 PFU/100 mL for somatic and 30 PFU/100 mL for male-specific coliphage. Overall, only 12% of total exceedances would have been for coliphage alone. Somatic coliphage exceedances that occurred in the absence of an Enterococcus exceedance were limited to a single site during south swell events, when this beach is known to be affected by nearby minimally treated sewage. Thus, somatic coliphage provided additional valuable health protection information, but may be more appropriate as a supplement to FIB measurements rather than as replacement because: (a) EPA-approved PCR methods for Enterococcus allow a more rapid response, (b) coliphage is more challenging owing to its greater sampling volume and laboratory time requirements, and (c) Enterococcus' long data history has yielded predictive management models that would need to be recreated for coliphage.

Assessment of seasonality and normalization techniques for wastewater-based surveillance in Ontario, Canada.

Introduction: Wastewater-based surveillance is at the forefront of monitoring for community prevalence of COVID-19, however, continued uncertainty exists regarding the use of fecal indicators for normalization of the SARS-CoV-2 virus in wastewater. Using three communities in Ontario, sampled from 2021-2023, the seasonality of a viral fecal indicator (pepper mild mottle virus, PMMoV) and the utility of normalization of data to improve correlations with clinical cases was examined. Methods: Wastewater samples from Warden, the Humber Air Management Facility (AMF), and Kitchener were analyzed for SARS-CoV-2, PMMoV, and crAssphage. The seasonality of PMMoV and flow rates were examined and compared by Season-Trend-Loess decomposition analysis. The effects of normalization using PMMoV, crAssphage, and flow rates were analyzed by comparing the correlations to clinical cases by episode date (CBED) during 2021. Results: Seasonal analysis demonstrated that PMMoV had similar trends at Humber AMF and Kitchener with peaks in January and April 2022 and low concentrations (troughs) in the summer months. Warden had similar trends but was more sporadic between the peaks and troughs for PMMoV concentrations. Flow demonstrated similar trends but was not correlated to PMMoV concentrations at Humber AMF and was very weak at Kitchener (r = 0.12). Despite the differences among the sewersheds, unnormalized SARS-CoV-2 (raw N1-N2) concentration in wastewater (n = 99-191) was strongly correlated to the CBED in the communities (r = 0.620-0.854) during 2021. Additionally, normalization with PMMoV did not improve the correlations at Warden and significantly reduced the correlations at Humber AMF and Kitchener. Flow normalization (n = 99-191) at Humber AMF and Kitchener and crAssphage normalization (n = 29-57) correlations at all three sites were not significantly different from raw N1-N2 correlations with CBED. Discussion: Differences in seasonal trends in viral biomarkers caused by differences in sewershed characteristics (flow, input, etc.) may play a role in determining how effective normalization may be for improving correlations (or not). This study highlights the importance of assessing the influence of viral fecal indicators on normalized SARS-CoV-2 or other viruses of concern. Fecal indicators used to normalize the target of interest may help or hinder establishing trends with clinical outcomes of interest in wastewater-based surveillance and needs to be considered carefully across seasons and sites.

Spatial distribution of fecal pollution indicators in sewage sludge flocs and their removal and inactivation as revealed by qPCR/viability-qPCR during potassium ferrate treatment.

Sludge reuse and utilization is one of important routines of disseminating fecal pollution to surface water and groundwater. However, it remains unclear the spatial distribution of fecal pollution indicators in sludge flocs and their reductions during sludge treatment processes. In this study, the abundances of fecal pollution indicators including cross-assembly phage (crAssphage), JC and BK polyomavirus (JCPyV, BKPyV), human adenovirus (HAdV), the human-specific HF183 Bacteroides (HF183) and Escherichia coli (EC) in soluble extracellular polymeric substances (S-EPS), loosely-bound EPS (LB-EPS), tightly-bound EPS (TB-EPS), and pellets of sludge flocs were determined, and the effect of potassium ferrate (PF) treatment on their removal and inactivation was investigated by using both qPCR and viability-qPCR. Results showed that all investigated indicators were detected in each fraction of sludge flocs. The PF treatment led to a great migration of indicators from sludge pellets to sludge EPS and some extent of their inactivation in each fraction of sludge flocs. The overall reductions of human fecal indicators in sludge determined by qPCR were 0-1.30 logs, which were 0-2 orders of magnitude lower than those of 0.69-2.39 logs detected by viability-qPCR, implying their inactivation by PF treatment to potentially alleviate the associated human health risks.

Pepper Mild Mottle Virus as a Potential Indicator of Fecal Contamination in Influents of Wastewater Treatment Plants in Riyadh, Saudi Arabia.

Several indicators of fecal pollution in water resources are continuously monitored for their reliability and, of particular interest, their correlation to human enteric viruses-not justified by traditional bacterial indicators. Pepper mild mottle virus (PMMoV) has recently been proposed as a successful viral surrogate of human waterborne viruses; however, in Saudi Arabia there are no available data in terms of its prevalence and concentration in water bodies. The concentration of PMMoV in three different wastewater treatment plants (King Saud University (KSU), Manfoha (MN), and Embassy (EMB) wastewater treatment plants (WWTP)) was measured using qRT-PCR during a one-year period and compared to the human adenovirus (HAdV), which is highly persistent and considered an indicator for viral-mediated fecal contamination. PMMoV was found in ~94% of the entire wastewater samples (91.6-100%), with concentrations ranging from 62 to 3.5 × 107 genome copies/l (GC/l). However, HAdV was detected in 75% of raw water samples (~67-83%). The HAdV concentration ranged between 1.29 × 103 GC/L and 1.26 × 107 GC/L. Higher positive correlation between PMMoV and HAdV concentrations was detected at MN-WWTP (r = 0.6148) than at EMB-WWTP (r = 0.207). Despite the lack of PMMoV and HAdV seasonality, a higher positive correlation (r = 0.918) of PMMoV to HAdV was recorded at KSU-WWTP in comparison to EMB-WWTP (r = 0.6401) around the different seasons. Furthermore, meteorological factors showed no significant influence on PMMoV concentrations (p > 0.05), thus supporting the use of PMMoV as a possible fecal indicator of wastewater contamination and associated public health issues, particularly at MN-WWTP. However, a continuous monitoring of the PMMoV distribution pattern and concentration in other aquatic environments, as well as its correlation to other significant human enteric viruses, is essential for ensuring its reliability and reproducibility as a fecal pollution indicator.

Probabilistic fecal pollution source profiling and microbial source tracking for an urban river catchment.

We developed an innovative approach to estimate the occurrence and extent of fecal pollution sources for urban river catchments. The methodology consists of 1) catchment surveys complemented by literature data where needed for probabilistic estimates of daily produced fecal indicator (FIBs, E. coli, enterococci) and zoonotic reference pathogen numbers (Campylobacter, Cryptosporidium and Giardia) excreted by human and animal sources in a river catchment, 2) generating a hypothesis about the dominant sources of fecal pollution and selecting a source targeted monitoring design, and 3) verifying the results by comparing measured concentrations of the informed choice of parameters (i.e. chemical tracers, C. perfringensspores, and host-associated genetic microbial source tracking (MST) markers) in the river, and by multi-parametric correlation analysis. We tested the approach at a study area in Vienna, Austria. The daily produced microbial particle numbers according to the probabilistic estimates indicated that, for the dry weather scenario, the discharge of treated wastewater (WWTP) was the primary contributor to fecal pollution. For the wet weather scenario, 80-99 % of the daily produced FIBs and pathogens resulted from combined sewer overflows (CSOs) according to the probabilistic estimates. When testing our hypothesis in the river, the measured concentrations of the human genetic fecal marker were log10 4 higher than for selected animal genetic fecal markers. Our analyses showed for the first-time statistical relationships between C. perfringens spores (used as conservative microbial tracer for communal sewage) and a human genetic fecal marker (i.e. HF183/BacR287) with the reference pathogen Giardia in river water (Spearman rank correlation: 0.78-0.83, p < 0.05. The developed approach facilitates urban water safety management and provides a robust basis for microbial fate and transport models and microbial infection risk assessment.

SARS-CoV-2 monitoring at three sewersheds of different scales and complexity demonstrates distinctive relationships between wastewater measurements and COVID-19 case data.

Wastewater monitoring of SARS-CoV-2 presents a means of tracking COVID-19 community infection dynamics on a broader geographic scale. However, accounting for environmental and sample-processing losses may be necessary for wastewater measurements to readily inform our understanding of infection prevalence. Here, we present measurements of the SARS-CoV-2 N1 and N2 gene targets from weekly wastewater samples at three sites in Hamilton County, Ohio, during an increase and subsequent decline of COVID-19 infections. The concentration of N1 or N2 RNA in wastewater, measured over the course of six months, ranged from below the detection limit to over 104 gene copies/l, and correlated with case data at two wastewater treatment plants, but not at a sub-sewershed-level sampling site. We also evaluated the utility of a broader range of variables than has been reported consistently in previous work, in improving correlations of SARS-CoV-2 concentrations with case data. These include a spiked matrix recovery control (OC43), flow-normalization, and assessment of fecal loading using endogenous fecal markers (HF183, PMMoV, crAssphage). We found that adjusting for recovery, flow, and fecal indicators increased these correlations for samples from a larger sewershed (serving ~488,000 people) with greater industrial and stormwater inputs, but raw N1/N2 concentrations corresponded better with case data at a smaller, residential-oriented sewershed. Our results indicate that the optimal adjustment factors for correlating wastewater and clinical case data moving forward may not be generalizable to all sewersheds.

Bacterial and viral fecal indicator predictive modeling at three Great Lakes recreational beach sites.

Coliphage are viruses that infect Escherichia coli (E. coli) and may indicate the presence of enteric viral pathogens in recreational waters. There is an increasing interest in using these viruses for water quality monitoring and forecasting; however, the ability to use statistical models to predict the concentrations of coliphage, as often done for cultured fecal indicator bacteria (FIB) such as enterococci and E. coli, has not been widely assessed. The same can be said for FIB genetic markers measured using quantitative polymerase chain reaction (qPCR) methods. Here we institute least-angle regression (LARS) modeling of previously published concentrations of cultured FIB (E. coli, enterococci) and coliphage (F+, somatic), along with newly reported genetic concentrations measured via qPCR for E. coli, enterococci, and general Bacteroidales. We develop site-specific models from measures taken at three beach sites on the Great Lakes (Grant Park, South Milwaukee, WI; Edgewater Beach, Cleveland, OH; Washington Park, Michigan City, IN) to investigate the efficacy of a statistical predictive modeling approach. Microbial indicator concentrations were measured in composite water samples collected five days per week over a beach season (∼15 weeks). Model predictive performance (cross-validated standardized root mean squared error of prediction [SRMSEP] and R2PRED) were examined for seven microbial indicators (using log10 concentrations) and water/beach parameters collected concurrently with water samples. Highest predictive performance was seen for qPCR-based enterococci and Bacteroidales models, with F+ coliphage consistently yielding poor performing models. Influential covariates varied by microbial indicator and site. Antecedent rainfall, bird abundance, wave height, and wind speed/direction were most influential across all models. Findings suggest that some fecal indicators may be more suitable for water quality forecasting than others at Great Lakes beaches.

Surveillance of RNase P, PMMoV, and CrAssphage in wastewater as indicators of human fecal concentration across urban sewer neighborhoods, Kentucky.

Wastewater surveillance has been widely used as a supplemental method to track the community infection levels of severe acute respiratory syndrome coronavirus 2. A gap exists in standardized reporting for fecal indicator concentrations, which can be used to calibrate the primary outcome concentrations from wastewater monitoring for use in epidemiological models. To address this, measurements of fecal indicator concentration among wastewater samples collected from sewers and treatment centers in four counties of Kentucky (N = 650) were examined. Results from the untransformed wastewater data over 4 months of sampling indicated that the fecal indicator concentration of human ribonuclease P (RNase P) ranged from 5.1 × 101 to 1.15 × 106 copies/ml, pepper mild mottle virus (PMMoV) ranged from 7.23 × 103 to 3.53 × 107 copies/ml, and cross-assembly phage (CrAssphage) ranged from 9.69 × 103 to 1.85 × 108 copies/ml. The results showed both regional and temporal variability. If fecal indicators are used as normalization factors, knowing the daily sewer system flow of the sample location may matter more than rainfall. RNase P, while it may be suitable as an internal amplification and sample adequacy control, has less utility than PMMoV and CrAssphage as a fecal indicator in wastewater samples when working at different sizes of catchment area. The choice of fecal indicator will impact the results of surveillance studies using this indicator to represent fecal load. Our results contribute broadly to an applicable standard normalization factor and assist in interpreting wastewater data in epidemiological modeling and monitoring.

A 22-Site Comparison of Land-Use Practices, E-coli and Enterococci Concentrations.

Land-use practices can greatly impact water quality. Escherichia (E.) coli and Enterococcus are accepted water quality indicators. However, surprisingly little research has been conducted comparing both organisms' population density relationships to land use practices and water quality. Stream water grab samples were collected monthly (n = 9 months) from 22 stream monitoring sites draining varying land use practice types in a representative mixed-land-use watershed of the northeastern United States. E. coli and enterococci colony forming units (CFU per 100 mL) were estimated (n = 396) and statistically analyzed relative to land use practices, hydroclimate, and pH, using a suite of methods, including correlation analysis, Principal Components Analysis (PCA), and Canonical Correspondence Analysis (CCA). Correlation analyses indicated significant (p < 0.05) relationships between fecal indicator bacteria concentrations, water quality metrics and land use practices but emphasized significant (p < 0.05) negative correlations between pH and instream enterococci concentrations. PCA and CCA results indicated consistent spatial differences between fecal indicator bacteria concentrations, pH, and land use/land cover characteristics. The study showed that pH could be considered an integrated proxy variable for past (legacy) and present land use practice influences. Results also bring to question the comparability of E-coli and enterococci relative to dominant land use practices and variations in pH and provide useful information that will help guide land use practice and water pollutant mitigation decision making.

Total coliform and Escherichia coli in microplastic biofilms grown in wastewater and inactivation by peracetic acid.

Microplastics (MP) have been proposed as a vector for pathogenic microorganisms in the freshwater environment. The objectives of this study were (a) to compare the fecal indicator growth in biofilms on MP and material control microparticles incubated in different wastewater fractions and (b) to compare MP biofilm, natural microparticle biofilm, and planktonic cell susceptibility to disinfection by peracetic acid (PAA). Biofilms were grown on high-density polyethylene, low-density polyethylene, polypropylene MP, or wood chips (as a material control) and incubated in either wastewater influent or pre-disinfection secondary effluent. Reactors were disinfected with PAA, biofilms were dislodged, and total coliform and Escherichia coli were cultivated. Fecal indicators were quantifiable in both MP and wood biofilms incubated in the wastewater influent but only on the wood biofilms incubated in secondary wastewater effluent. More total coliform grew in the wood biofilms than MP biofilms, and the biofilms grown on MP and woodchips were more resistant to disinfection than planktonic bacteria. Thus, it may be possible to refer to the disinfection literature for fecal indicators in biofilm on other particles to predict behavior on MP. Treatments that remove particles in general would help reduce the potential for fecal indicator bypass of disinfection. PRACTITIONER POINTS: MP biofilm had lower concentrations of fecal indicators than wood biofilm Biofilm on MP was not more resistant to disinfection than wood biofilm Biofilms, regardless of substrate, were more resistant to disinfection than planktonic organisms.

Exploring prevalence of potential pathogens and fecal indicators in geographically distinct river systems through comparative metagenomics.

Microbial communities are considered as vital members to reflect the health of a riverine system. Among them, pathogenic and fecal indicators imply health risks involved with potability of river water. The present study explores the diverse microbial communities, distribution pattern of potential pathogens, and fecal indicators between the geographically distinct Himalayan and Peninsular river systems of India. It also inquires into the environmental factors associated with community variance and distribution pattern of microbial indicators. The application of high-throughput amplicon sequencing approach unveiled significant demarcation (p < 0.004, Anosim R = 0.62) of samples suggesting unique microbial diversities in these two river sediments. Random forest analysis revealed Desulfobulbulus, PSB_M_3, and Opitutus in Himalayan, while DA101, Bacillus, and Streptomyces in the Peninsular as significant contributors to develop overall dissimilarity between the river systems. Permutational multivariate analysis of variance and co-occurrence network analysis were used to study the relationships between microbial taxa and environmental factors. Amongst the various studied environmental parameters, pH, K, Ca, Mg, Ba, and Al in the Himalayan and salinity, Na, temperature, and Th in the Peninsular significantly influenced shaping of distinct microbial communities. Furthermore, the potential pathogenic genera, including Flavobacterium, Clostridium, Arcobacter, Pseudomonas, and Bacillus were highly prevalent in both the river systems. Arcobacter, Clostridium, Acinetobacter, Bacteroides, and Caloramator were the prominent fecal indicators in these river systems. Our findings provide salient information about the crucial role and interplay between various environmental factors and anthropogenic influences in framing the microbiome of the distinct river systems in India. Moreover, assessing potential pathogenic and fecal indicators suggest the public health risk associated with untreated sewage discharge into these water sources. The detection of various F/S indicators and potentially pathogenic bacteria in Himalayan and Peninsular river systems emphasize the urgent need for future monitoring and management of major riverine systems in India.