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.

Decay of infectious adenovirus and coliphages in freshwater habitats is differentially affected by ambient sunlight and the presence of indigenous protozoa communities.

BACKGROUND: Sanitary quality of recreational waters worldwide is assessed using fecal indicator bacteria (FIB), such as Escherichia coli and enterococci. However, fate and transport characteristics of FIB in aquatic habitats can differ from those of viral pathogens which have been identified as main etiologic agents of recreational waterborne illness. Coliphages (bacteriophages infecting E. coli) are an attractive alternative to FIB because of their many morphological and structural similarities to viral pathogens. METHODS: In this in situ field study, we used a submersible aquatic mesocosm to compare decay characteristics of somatic and F+ coliphages to those of infectious human adenovirus 2 in a freshwater lake. In addition, we also evaluated the effect of ambient sunlight (and associated UV irradiation) and indigenous protozoan communities on decay of somatic and F+ coliphage, as well as infectious adenovirus. RESULTS: Our results show that decay of coliphages and adenovirus was similar (p = 0.0794), indicating that both of these bacteriophage groups are adequate surrogates for decay of human adenoviruses. Overall, after 8 days the greatest log10 reductions were observed when viruses were exposed to a combination of biotic and abiotic factors (2.92 ± 0.39, 4.48 ± 0.38, 3.40 ± 0.19 for somatic coliphages, F+ coliphages and adenovirus, respectively). Both, indigenous protozoa and ambient sunlight, were important contributors to decay of all three viruses, although the magnitude of that effect differed over time and across viral targets. CONCLUSIONS: While all viruses studied decayed significantly faster (p < 0.0001) when exposed to ambient sunlight, somatic coliphages were particularly susceptible to sunlight irradiation suggesting a potentially different mechanism of UV damage compared to F+ coliphages and adenoviruses. Presence of indigenous protozoan communities was also a significant contributor (p value range: 0.0016 to < 0.0001) to decay of coliphages and adenovirus suggesting that this rarely studied biotic factor is an important driver of viral reductions in freshwater aquatic habitats.

A Constructed Wetland for Treatment of an Impacted Waterway and the Influence of Native Waterfowl on its Perceived Effectiveness.

A constructed, variable-flow treatment wetland was evaluated for its ability to reduce microbial loads from the Banklick Creek, an impacted recreational waterway in Northern Kentucky. For this study, levels of traditional (Escherichia coli and enterococci measured by culture and molecular techniques) and alternative fecal indicators (infectious somatic and F+ coliphage, Clostridium spp. and Clostridium perfringens by culture), potential pathogens (molecular signal of Campylobacter spp.) as well as various microbial source tracking (MST) markers (human fecal marker HF183 and avian fecal marker GFD) were monitored during the summer and early fall through five treatment stages within the Banklick Creek Wetland. No difference in concentrations of traditional or alternative fecal indicators were observed in any of the sites monitored. Microbial source tracking markers were employed to identify sources of fecal contamination within the wetland. Human marker HF183 concentrations at beginning stages of treatment were found to be significantly higher (P value range: 0.0016-0.0003) than levels at later stages. Conversely, at later stages of treatment where frequent bird activity was observed, Campylobacter and avian marker (GFD) signals were detected at significantly higher frequencies (P value range: 0.024 to <0.0001), and both signals were strongly correlated (P = 0.0001). Our study suggests constructed wetlands are an effective means for removal of microbial contamination in ambient waters, but reliance on general fecal indicators is not ideal for determining system efficacy or assessing appropriate remediation efforts.

Biotic interactions and sunlight affect persistence of fecal indicator bacteria and microbial source tracking genetic markers in the upper Mississippi river.

The sanitary quality of recreational waters that may be impacted by sewage is assessed by enumerating fecal indicator bacteria (FIB) (Escherichia coli and enterococci); these organisms are found in the gastrointestinal tracts of humans and many other animals, and hence their presence provides no information about the pollution source. Microbial source tracking (MST) methods can discriminate between different pollution sources, providing critical information to water quality managers, but relatively little is known about factors influencing the decay of FIB and MST genetic markers following release into aquatic environments. An in situ mesocosm was deployed at a temperate recreational beach in the Mississippi River to evaluate the effects of ambient sunlight and biotic interactions (predation, competition, and viral lysis) on the decay of culture-based FIB, as well as molecularly based FIB (Entero1a and GenBac3) and human-associated MST genetic markers (HF183 and HumM2) measured by quantitative real-time PCR (qPCR). In general, culturable FIB decayed the fastest, while molecularly based FIB and human-associated genetic markers decayed more slowly. There was a strong correlation between the decay of molecularly based FIB and that of human-associated genetic markers (r(2), 0.96 to 0.98; P < 0.0001) but not between culturable FIB and any qPCR measurement. Overall, exposure to ambient sunlight may be an important factor in the early-stage decay dynamics but generally was not after continued exposure (i.e., after 120 h), when biotic interactions tended to be the only/major influential determinant of persistence.

The comparison of decay rates of infectious SARS-CoV-2 and viral RNA in environmental waters and wastewater.

Understanding the decay characteristics of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in wastewater and ambient waters is important for multiple applications including assessment of risk of exposure associated with handling wastewater samples, public health risk associated with recreation in wastewater polluted ambient waters and better understanding and interpretation of wastewater-based epidemiology (WBE) results. We evaluated the decay rates of infectious SARS-CoV-2 and viral RNA in wastewater and ambient waters under temperature regimes representative of seasonal fluctuations. Infectious virus was seeded in autoclaved primary wastewater effluent, final dechlorinated wastewater effluent, lake water, and marine water at a final concentration of 6.26 ± 0.07 log10 plaque forming units per milliliter. Each suspension was incubated at either 4°, 25°, and 37 °C. Samples were initially collected on an hourly basis, then approximately every other day for 15 days. All samples were analyzed for infectious virus via a plaque assay using the Vero E6 cell line, and viral gene copy levels were quantified with the US CDC's N1 and N2 reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) assays. The infectious virus decayed significantly faster (p ≤ 0.0214) compared to viral RNA, which persisted for the duration of the study irrespective of the incubation conditions. The initial loss (within 15 min of seeding) as well as decay of infectious SARS-CoV-2 was significantly faster (p ≤ 0.0387) in primary treated wastewater compared to other water types, but viral RNA did not degrade appreciably in this matrix until day 15. Overall, temperature was the most important driver of decay, and after 24 h, no infectious SARS-CoV-2 was detected at 37 °C in any water type. Moreover, the CDC N2 gene assay target decayed significantly (p ≤ 0.0174) faster at elevated temperatures compared to CDC N1, which has important implications for RT-qPCR assay selection for WBE approach.

Quantitative fecal pollution assessment with bacterial, viral, and molecular methods in small stream tributaries.

Stream water quality can be impacted by a myriad of fecal pollution sources and waste management practices. Identifying origins of fecal contamination can be challenging, especially in high order streams where water samples are influenced by pollution from large drainage areas. Strategic monitoring of tributaries can be an effective strategy to identify conditions that influence local water quality. Water quality is assessed using fecal indicator bacteria (FIB); however, FIB cannot differentiate sources of fecal contamination nor indicate the presence of disease-causing viruses. Under different land use scenarios, three small stream catchments were investigated under 'wet' and 'dry' conditions (Scenario 1: heavy residential; Scenario 2: rural residential; and Scenario 3: undeveloped/agricultural). To identify fecal pollution trends, host-associated genetic targets HF183/BacR287 (human), Rum2Bac (ruminant), GFD (avian), and DG3 (canine) were analyzed along with FIB (Escherichia coli and enterococci), viral indicators (somatic and F+ coliphage), six general water quality parameters, and local rainfall. Levels of E. coli exceeded single sample maximum limits (235 CFU/100 mL) in 70.7 % of samples, enterococci (70 CFU/100 mL) in 100 % of samples, and somatic coliphage exceeded advisory thresholds (600 PFU/L) in 34.1 % of samples. The detection frequency for the human-associated genetic marker was highest in Scenario 3 (50 % of samples) followed by Scenario 2 (46 %), while the ruminant-associated marker was most prevalent in Scenario 1 (64 %). Due to the high proportion of qPCR-based measurements below the limit of quantification, a Bayesian data analysis approach was applied to investigate links between host-associated genetic marker occurrence with that of rainfall and fecal indicator levels. Multiple trends associated with small stream monitoring were revealed, emphasizing the role of rainfall, the utility of fecal source information to improve water quality management. And furthermore, water quality monitoring with bacterial or viral methodologies can alter the interpretation of fecal pollution sources in impaired waters.

Differential decay of enterococci and Escherichia coli originating from two fecal pollution sources.

Using in situ subtropical aquatic mesocosms, fecal source (cattle manure versus sewage) was shown to be the most important contributor to differential loss in viability of fecal indicator bacteria (FIB), specifically enterococci in freshwater and Escherichia coli in marine habitats. In this study, sunlight exposure and indigenous aquatic microbiota were also important contributors, whose effects on FIB also differed between water types.

Development and evaluation of EPA method 1615 for detection of enterovirus and norovirus in water.

The U.S. EPA developed a sample concentration and preparation assay in conjunction with the total culturable virus assay for concentrating and measuring culturable viruses in source and drinking waters as part of the Information Collection Rule (ICR) promulgated in 1996. In an effort to improve upon this method, the U.S. EPA recently developed Method 1615: Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. Method 1615 uses a culturable virus assay with reduced equipment and labor costs compared to the costs associated with the ICR virus method and introduces a new molecular assay for the detection of enteroviruses and noroviruses by reverse transcription-quantitative PCR. In this study, we describe the optimization of several new components of the molecular assay and examine virus recovery from ground, reagent-grade, and surface water samples seeded with poliovirus type 3 and murine norovirus. For the culturable virus and molecular assays, mean poliovirus recovery using the complete method was 58% and 20% in groundwater samples, 122% and 39% using low-titer spikes in reagent-grade water, 42% and 48% using high-titer spikes in reagent-grade water, and 11% and 10% in surface water with high turbidity, respectively. Murine norovirus recovery by the molecular assay was 30% in groundwater samples, less than 8% in both low- and high-titer spikes in reagent-grade water, and 6% in surface water with high turbidity. This study demonstrates the effectiveness of Method 1615 for use with groundwater samples and highlights the need for further research into its effectiveness with surface water.

The Effect of Protozoa Indigenous to Lakewater and Wastewater on Decay of Fecal Indicator Bacteria and Coliphage.

Fecal indicator bacteria (FIB: Escherichia coli and enterococci) are used to assess recreational water quality. Viral indicators (i.e., somatic and F+ coliphage), could improve the prediction of viral pathogens in recreational waters, however, the impact of environmental factors, including the effect of predatory protozoa source, on their survival in water is poorly understood. We investigated the effect of lakewater or wastewater protozoa, on the decay (decreasing concentrations over time) of culturable FIB and coliphages under sunlight and shaded conditions. FIB decay was generally greater than the coliphages and was more rapid when indicators were exposed to lake vs. wastewater protozoa. F+ coliphage decay was the least affected by experimental variables. Somatic coliphage decayed fastest in the presence of wastewater protozoa and sunlight, though their decay under shaded conditions was-10-fold less than F+ after 14 days. The protozoa source consistently contributed significantly to the decay of FIB, and somatic, though not the F+ coliphage. Sunlight generally accelerated decay, and shade reduced somatic coliphage decay to the lowest level among all the indicators. Differential responses of FIB, somatic, and F+ coliphages to environmental factors support the need for studies that address the relationship between the decay of coliphages and viral pathogens under environmentally relevant conditions.

Improved virus concentration methods for wash waters from decontamination of permeable and non-permeable surfaces.

Surface decontamination is a method of using wash water to decontaminated surfaces preventing transmission of biological contaminants that can pose potential health risks to responders and the public. However, the risks associated with handling used wash water are largely unknown due to the lack of effective methodology to screen for pathogenic microorganisms present in these samples, especially viral pathogens. This study adapted the dead-end hollow-fiber ultrafiltration (D-HFUF) system to wash waters, including a separate procedure for recovering particle attached viruses. Simulated wash water was created using dechlorinated tap water containing a mild surfactant (0.05 % Tween 80). To determine virus recovery efficiencies, measured amounts of somatic and F+ coliphage were spiked into 2-liter volumes of wash water under the following scenarios: (1) wash water was amended with a measured amount of sterile river sediment with no sediment separation prior to filter concentration; or (2) sediment added to wash water was allowed to settle prior to filter concentrating clarified liquid portions, while precipitated sediment was subjected to viral extraction techniques to recover particle attached virus; and (3) the optimized method was deployed on non-porous and porous surfaces to simulate a decontamination clean-up event. Separation of sediment prior to D-HFUF significantly increased recovery of coliphages, (P = <0.0001) versus filtration of sediment and liquids simultaneously. A tryptic soy broth (TSB) elution solution was significantly more effective (P = ≤0.010) for recovery of both somatic and F+ coliphage, (108 ± 9 % and 92 ± 9 %, respectively), compared to elution buffers containing various surfactants (sodium hexametaphosphate, Tween 80) for recovering particle attached virus. Simulating a biocontaminate clean-up event (using the optimized sediment separation and elution protocol) resulted in coliphage recoveries of 75-96 % (permeable surface) and 71-92 % (non-permeable surface). This procedure can be used to effectively detect viruses in used wash waters aiding in reducing risks to human health during site decontamination.