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.

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.

Assessment of two volumetrically different concentration approaches to improve sensitivities for SARS-CoV-2 detection during wastewater monitoring.

Wastewater monitoring for severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), the virus responsible for the global coronavirus disease 2019 (COVID-19) pandemic, has highlighted the need for methodologies capable of assessing viral prevalence during periods of low population infection. To address this need, two volumetrically different, methodologically similar concentration approaches were compared for their abilities to detect viral nucleic acid and infectious SARS-CoV-2 signal from primary influent samples. For Method 1, 2 L of SARS-CoV-2 seeded wastewater was evaluated using a dead-end hollow fiber ultrafilter (D-HFUF) for primary concentration, followed by the CP Select™ for secondary concentration. For Method 2, 100 mL of SARS-CoV-2 seeded wastewater was evaluated using the CP Select™ procedure. Following D-HFUF concentration (Method 1), significantly lower levels of infectious SARS-CoV-2 were lost (P value range: 0.0398-0.0027) compared to viral gene copy (GC) levels detected by the US Centers for Disease Control (CDC) N1 and N2 reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) assays. Subsamples at different steps in the concentration process were also taken to better characterize the losses of SARS-CoV-2 during the concentration process. During the centrifugation step (prior to CP Select™ concentration), significantly higher losses (P value range: 0.0003 to <0.0001) occurred for SARS-CoV-2 GC levels compared to infectious virus for Method 1, while between the methods, significantly higher infectious viral losses were observed for Method 2 (P = 0.0002). When analyzing overall recovery of endogenous SARS-CoV-2 in wastewater samples, application of Method 1 improved assay sensitivities (P = <0.0001) compared with Method 2; this was especially evident during periods of lower COVID-19 case rates within the sewershed. This study describes a method which can successfully concentrate infectious SARS-CoV-2 and viral RNA from wastewater. Moreover, we demonstrated that large volume wastewater concentration provides additional sensitivity needed to improve SARS-CoV-2 detection, especially during low levels of community disease prevalence.

Genomic Characterization and Wetland Occurrence of a Novel Campylobacter Isolate from Canada Geese.

Populations of resident, non-migratory Canada geese are rapidly increasing. Canada geese are known to transmit viral and bacterial diseases, posing a possible threat to human health. The most prevalent pathogens vectored by geese are Campylobacter species, yet the current understanding of the identity and virulence of these pathogens is limited. In our previous study, we observed a high prevalence of Campylobacter spp. in the Banklick Creek wetland-a constructed treatment wetland (CTW) located in northern KY (USA) used to understand sources of fecal contamination originating from humans and waterfowl frequenting the area. To identify the types of Campylobacter spp. found contaminating the CTW, we performed genetic analyses of Campylobacter 16s ribosomal RNA amplified from CTW water samples and collected fecal material from birds frequenting those areas. Our results showed a high occurrence of a Campylobacter canadensis-like clade from the sampling sites. Whole-genome sequence analyses of an isolate from Canada goose fecal material, called MG1, were used to confirm the identity of the CTW isolates. Further, we examined the phylogenomic position, virulence gene content, and antimicrobial resistance gene profile of MG1. Lastly, we developed an MG1-specific real-time PCR assay and confirmed the presence of MG1 in Canada goose fecal samples surrounding the CTW. Our findings reveal that the Canada goose-vectored Campylobacter sp. MG1 is a novel isolate compared to C. canadensis that possesses possible zoonotic potential, which may be of human health concern.

Effectiveness of two wastewater disinfection strategies for the removal of fecal indicator bacteria, bacteriophage, and enteric viral pathogens concentrated using dead-end hollow fiber ultrafiltration (D-HFUF).

Primary influent and final effluent samples were collected from wastewater treatment plants using either chlorination or ultraviolet (UV) disinfection biweekly for one year. Paired measurements were determined for fecal indicator bacteria (Escherichia coli and enterococci), cultivated bacteriophages (somatic, F+, and CB-390 coliphage and GB-124 Bacteroides phage), human-associated viral markers (human polyomavirus [HPyV] and crAssphage), enteric pathogens (adenovirus, noroviruses genogroups I and II) as well as total infectious enteric virus. To increase the probability of detecting low concentration targets, both primary (10L) and final effluent wastewater samples (40-100 L) were concentrated using a dead-end hollow-fiber ultrafilter (D-HFUF). Despite seasonal temperature fluctuations, concentration shifts of FIB, bacteriophages, human-associated viruses, and viral pathogens measured in primary influent samples were minimal, while levels of infectious enteric virus were significantly higher in the spring and fall (P range: 0.0003-0.0409). FIB levels measured in primary influents were 1-2 log10 higher than bacteriophage, human-associated viral markers (except crAssphage) and viral pathogens measured. FIB displayed the greatest sensitivity to chlorine disinfection, while crAssphage, adenoviruses and infectious enteric viruses were significantly less sensitive (P ≤ 0.0096). During UV treatment, bacteriophages F+ and GB-124 were the most resistant of the culturable viruses measured (P ≤ 0.001), while crAssphage were the most resistant (P ≤ 0.0124) overall. When UV lamps were inactive, infectious enteric viruses were significantly more resilient to upstream treatment processes than all other targets measured (P ≤ 0.0257). Similar to infectious enteric viruses and adenoviruses; GB-124, F+, and crAssphages displayed the highest resistance to UV irradiation, signaling a potential applicability as pathogen surrogates in these systems. The use of D-HFUF enhanced the ability to estimate removal of viruses through wastewater treatment, with the expectation that future applications of this method will be used to better elucidate viral behavior within these systems.

Performance evaluation of a dead-end hollowfiber ultrafiltration method for enumeration of somatic and F+ coliphage from recreational waters.

Dead-end hollow fiber ultrafiltration combined with a single agar layer assay (D-HFUF-SAL) has potential use in the assessment of sanitary quality of recreational waters through enumeration of coliphage counts as measures of fecal contamination. However, information on applicability across a broad range of sites and water types is limited. Here, we tested the performance of D-HFUF-SAL on 49 marine and freshwater samples. Effect of method used to titer the spiking suspension (SAL versus double agar layer [DAL]) on percent recovery was also evaluated. Average somatic coliphage recovery (72 % ± 27) was significantly higher (p < 0.0001) compared to F+ (53 % ± 19). This was more pronounced for marine (p ≤ 0.0001) compared to freshwaters (p = 0.0134). Neither method affected somatic coliphage, but DAL (28 % ± 12) significantly (p < 0.0001) underestimated F + coliphage recoveries compared to SAL (53 % ± 19). Overall, results indicate that, while D-HFUF-SAL performed well over a wide variety of water types, F + coliphage recoveries were significantly reduced for marine waters suggesting that some components unique to this habitat may interfere with the assay performance. More importantly, our findings indicate that choice of spike titer method merits careful consideration since it may under-estimate method percent recovery.

Development of a large volume concentration method for recovery of coronavirus from wastewater.

Levels of severe acute respiratory coronavirus type 2 (SARS CoV 2) RNA in wastewater could act as an effective means to monitor coronavirus disease 2019 (COVID-19) within communities. However, current methods used to detect SARS CoV 2 RNA in wastewater are limited in their ability to process sufficient volumes of source material, inhibiting our ability to assess viral load. Typically, viruses are concentrated from large liquid volumes using two stage concentration, primary and secondary. Here, we evaluated a dead-end hollow fiber ultrafilter (D-HFUF) for primary concentration, followed by the CP Select™ for secondary concentration from 2 L volumes of primary treated wastewater. Various amendments to each concentration procedure were investigated to optimally recover seeded OC43 (betacoronavirus) from wastewater. During primary concentration, the D-HFUF recovered 69 ± 18% (n = 29) of spiked OC43 from 2 L of wastewater. For secondary concentration, the CP Select™ system using the Wastewater Application settings was capable of processing 100 mL volumes of primary filter eluates in <25 min. A hand-driven syringe elution proved to be significantly superior (p = 0.0299) to the CP Select™ elution for recovering OC43 from filter eluates, 48 ± 2% compared to 31 ± 3%, respectively. For the complete method (primary and secondary concentration combined), the D-HFUF and CP select/syringe elution achieved overall 22 ± 4% recovery of spiked OC43 through (n = 8) replicate filters. Given the lack of available standardized methodology confounded by the inherent limitations of relying on viral RNA for wastewater surveillance of SARS CoV 2, it is important to acknowledge these challenges when interpreting this data to estimate community infection rates. However, the development of methods that can substantially increase sample volumes will likely allow for reporting of quantifiable viral data for wastewater surveillance, equipping public health officials with information necessary to better estimate community infection rates.

Antibiotic-Resistant Enterococcus Species in Marine Habitats: A Review.

Antibiotic-resistant Enterococcus (ARE) are among leading causes of nosocomial infections worldwide. Enterococcus spp. are ubiquitous in sewage, which can contaminate surface waters via many pathways, providing a route of exposure for humans. This review focuses on ARE in marine and estuarine habitats, including marine animals. Phylogenetic confirmation of the genus Enterococcus and intermediate or full resistance to clinically relevant antibiotics were inclusion criteria. The proportion of resistant isolates varied greatly among antibiotics, for example, 24.2% for ampicillin and 2.4% for vancomycin. The water column contained the highest proportion of ARE observations (18.8%), followed by animal feces and tissues (14.8%), sediment (9.4%), and sand (2.0%). The proportion of multidrug-resistant isolates was the greatest in animal tissue and fecal samples, followed by water and sediments. This review indicates that clinically relevant ARE are present in marine/estuarine habitats and that animals may be important reservoirs.

Comparison of virus concentration methods for the RT-qPCR-based recovery of murine hepatitis virus, a surrogate for SARS-CoV-2 from untreated wastewater.

There is currently a clear benefit for many countries to utilize wastewater-based epidemiology (WBE) as part of ongoing measures to manage the coronavirus disease 2019 (COVID-19) global pandemic. Since most wastewater virus concentration methods were developed and validated for nonenveloped viruses, it is imperative to determine the efficiency of the most commonly used methods for the enveloped severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Municipal wastewater seeded with a human coronavirus (CoV) surrogate, murine hepatitis virus (MHV), was used to test the efficiency of seven wastewater virus concentration methods: (A-C) adsorption-extraction with three different pre-treatment options, (D-E) centrifugal filter device methods with two different devices, (F) polyethylene glycol (PEG 8000) precipitation, and (G) ultracentrifugation. MHV was quantified by reverse-transcription quantitative polymerase chain reaction and the recovery efficiency was calculated for each method. The mean MHV recoveries ranged from 26.7 to 65.7%. The most efficient methods were adsorption-extraction methods with MgCl2 pre-treatment (Method C), and without pre-treatment (Method B). The third most efficient method used the Amicon® Ultra-15 centrifugal filter device (Method D) and its recovery efficiency was not statistically different from the most efficient methods. The methods with the worst recovery efficiency included the adsorption-extraction method with acidification (A), followed by PEG precipitation (F). Our results suggest that absorption-extraction methods with minimal or without pre-treatment can provide suitably rapid, cost-effective and relatively straightforward recovery of enveloped viruses in wastewater. The MHV is a promising process control for SARS-CoV-2 surveillance and can be used as a quality control measure to support community-level epidemic mitigation and risk assessment.

Extended persistence of general and cattle-associated fecal indicators in marine and freshwater environment.

Fecal contamination of recreational waters with cattle manure can pose a risk to public health due to the potential presence of various zoonotic pathogens. Fecal indicator bacteria (FIB) have a long history of use in the assessment of recreational water quality, but FIB quantification provides no information about pollution sources. Microbial source tracking (MST) markers have been developed in response to a need to identify pollution sources, yet factors that influence their decay in ambient waters are often poorly understood. We investigated the influence of water type (freshwater versus marine) and select environmental parameters (indigenous microbiota, ambient sunlight) on the decay of FIB and MST markers originating from cattle manure. Experiments were conducted in situ using a submersible aquatic mesocosm containing dialysis bags filled with a mixture of cattle manure and ambient water. Culturable FIB (E. coli, enterococci) were enumerated by membrane filtration and general fecal indicator bacteria (GenBac3, Entero1a, EC23S857) and MST markers (Rum2Bac, CowM2, CowM3) were estimated by qPCR. Water type was the most significant factor influencing decay (three-way ANOVA, p: 0.006 to <0.001), although the magnitude of the effect differed among microbial targets and over time. The presence of indigenous microbiota and exposure to sunlight were significantly correlated (three-way ANOVA, p: 0.044 to <0.001) with decay of enterococci and CowM2, while E. coli, EC23S857, Rum2Bac, and CowM3 (three-way ANOVA, p: 0.044 < 0.001) were significantly impacted by sunlight or indigenous microbiota. Results indicate extended persistence of both cultivated FIB and genetic markers in marine and freshwater water types. Findings suggest that multiple environmental stressors are important determinants of FIB and MST marker persistence, but their magnitude can vary across indicators. Selective exclusion of natural aquatic microbiota and/or sunlight typically resulted in extended survival, but the effect was minor and limited to select microbial targets.

Genome Sequences of Escherichia Bacteriophages Isolated from Raw Wastewater.

Somatic coliphages are alternative indicators of fecal pollution and attractive surrogates for viral pathogens. Here, we report the draft genome sequences of three replicate plaques from a novel Myoviridae bacteriophage isolated from raw wastewater. These genomes were similar to felix01virus phage and are predicted to contain up to 148 protein-coding genes.

Relationships between Microbial Indicators and Pathogens in Recreational Water Settings.

Fecal pollution of recreational waters can cause scenic blight and pose a threat to public health, resulting in beach advisories and closures. Fecal indicator bacteria (total and fecal coliforms, Escherichia coli, and enterococci), and alternative indicators of fecal pollution (Clostridium perfringens and bacteriophages) are routinely used in the assessment of sanitary quality of recreational waters. However, fecal indicator bacteria (FIB), and alternative indicators are found in the gastrointestinal tract of humans, and many other animals and therefore are considered general indicators of fecal pollution. As such, there is room for improvement in terms of their use for informing risk assessment and remediation strategies. Microbial source tracking (MST) genetic markers are closely associated with animal hosts and are used to identify fecal pollution sources. In this review, we examine 73 papers generated over 40 years that reported the relationship between at least one indicator and one pathogen group or species. Nearly half of the reports did not include statistical analysis, while the remainder were almost equally split between those that observed statistically significant relationships and those that did not. Statistical significance was reported less frequently in marine and brackish waters compared to freshwater, and the number of statistically significant relationships was considerably higher in freshwater (p < 0.0001). Overall, significant relationships were more commonly reported between FIB and pathogenic bacteria or protozoa, compared to pathogenic viruses (p: 0.0022⁻0.0005), and this was more pronounced in freshwater compared to marine. Statistically significant relationships were typically noted following wet weather events and at sites known to be impacted by recent fecal pollution. Among the studies that reported frequency of detection, FIB were detected most consistently, followed by alternative indicators. MST markers and the three pathogen groups were detected least frequently. This trend was mirrored by reported concentrations for each group of organisms (FIB > alternative indicators > MST markers > pathogens). Thus, while FIB, alternative indicators, and MST markers continue to be suitable indicators of fecal pollution, their relationship with waterborne pathogens, particularly viruses, is tenuous at best and influenced by many different factors such as frequency of detection, variable shedding rates, differential fate and transport characteristics, as well as a broad range of site-specific factors such as the potential for the presence of a complex mixture of multiple sources of fecal contamination and pathogens.

Comparison of somatic and F+ coliphage enumeration methods with large volume surface water samples.

Coliphages are alternative fecal indicators that may be suitable surrogates for viral pathogens, but majority of standard detection methods utilize insufficient volumes for routine detection in environmental waters. We compared three somatic and F+ coliphage methods based on a paired measurement from 1 L samples collected from the Great Lakes (n = 74). Methods include: 1) dead-end hollow fiber ultrafilter with single agar layer (D-HFUF-SAL); 2) modified SAL (M-SAL); and 3) direct membrane filtration (DMF) technique. Overall, D-HFUF-SAL outperformed other methods as it yielded the lowest frequency of non-detects [(ND); 10.8%] and the highest average concentrations of recovered coliphage for positive samples (2.51 ± 1.02 [standard deviation, SD] log10 plaque forming unit/liter (PFU/L) and 0.79 ± 0.71 (SD) log10 PFU/L for somatic and F+, respectively). M-SAL yielded 29.7% ND and average concentrations of 2.26 ± 1.15 (SD) log10 PFU/L (somatic) and 0.59 ± 0.82 (SD) log10 PFU/L (F+). DMF performance was inferior to D-HFUF-SAL and M-SAL methods (ND of 65.6%; average somatic coliphage concentration 1.52 ± 1.32 [SD] log10 PFU/L, no F+ detected), indicating this procedure is unsuitable for 1 L surface water sample volumes. This study represents an important step toward the use of a coliphage method for recreational water quality criteria purposes.

Concentration and quantification of somatic and F+ coliphages from recreational waters.

Somatic and F+ coliphages are promising alternative fecal indicators, but current detection methods are hindered by lower levels of coliphages in surface waters compared to traditional bacterial fecal indicators. We evaluated the ability of dead-end hollow fiber ultrafiltration (D- HFUF) and single agar layer (SAL) procedure to concentrate and enumerate coliphages from 1L and 10L volumes of ambient surface waters (lake, river, marine), river water with varying turbidities (3.74-118.7 NTU), and a simulated combined sewer overflow (CSO) event. Percentage recoveries for surface waters were 40-79% (somatic) and 35-94% (F+). The method performed equally well in all three matrices at 1L volumes, but percent recoveries were significantly higher in marine waters at 10L volumes when compared to freshwater. Percent recoveries at 1L and 10L were similar, except in river water where recoveries were significantly lower at higher volume. In highly turbid waters, D-HFUF-SAL had a recovery range of 25-77% (somatic) and 21-80% (F+). The method produced detectable levels of coliphages in diluted wastewater and in unspiked surface waters, emphasizing its applicability to CSO events and highlighting its utility in recovery of low coliphage densities from surface waters. Thus D-HFUF-SAL is a good candidate method for routine water quality monitoring of coliphages.