Somatic Coliphages Research Articles

Efficient quantification of somatic coliphages in water: Development and validation of the Enumera® Rapid kit.

The integration of somatic coliphage analysis into water quality regulations has driven the development of more streamlined, easier, and faster detection methods. These include the Bluephage method, initially designed for the qualitative assessment of coliphages in 100mL water samples. In the present study this technique was adapted for quantitative analysis using the most probable number method, enabling quantification of somatic coliphages in 100mL water samples within 6.5h of incubation. Early readings were optimised using an algorithm developed from an extensive dataset of over 400 environmental samples. This new quantitative method has been refined and commercialised as the Enumera® Rapid kit (BPF-SE, Bluephage S.L.), which includes a new reference somatic coliphage, strain GR8. After sequencing and further characterisation, it was confirmed that the new strain belongs to the same species as the reference somatic coliphage of the ISO 10705-2 method. The Enumera® Rapid kit was then validated by assessing its efficacy across 151 samples from various water matrices. The method provided a mean recovery rate of 103%, ranging from 95% for drinking water to 116% for wastewater, at concentrations from 0 to 300 PFU/100mL. In comparison, ISO 10705-2 applied after membrane filtration yielded a mean recovery rate of 63.8%, with the highest rate observed for wastewater (88.9%). The results obtained with the Enumera® Rapid kit did not differ significantly compared to the reference method after adjusting for potential losses during the concentration stage outlined in ISO 10705-3. The new method also demonstrated a robust accuracy of 0.16 logs, good linearity, and achieved a limit of detection and quantification of 1 MPN/100mL. The Enumera® Rapid kit enables rapid and straightforward quantification of somatic coliphages in 100mL water samples within a single working day. Its streamlined workflow serves as a useful tool for microbial water quality monitoring.

Somatic coliphages as bioindicators of contaminaution in Lake Guamuez, Colombia.

Lake Guamuez is the second largest lake in Colombia and economically supports hundreds of families in the area. The main activities carried out in the region have focused on tourism, agriculture, livestock, and rainbow trout production; however, these activities have been associated with contamination of the lake. This research aimed to evaluate the water quality of Lake Guamuez using somatic coliphages (SCs) as bioindicators. For this purpose, periodic sampling was carried out for 6 months at nine strategic points of the lake. For the detection of SCs, the method described in 9211 D of the Standard Methods for the Examination of Water and Wastewater was used. The genomic variability and presence of virulence genes in the isolated SCs were determined. Water contamination in the lake is evident, and the SCs titer is greater in areas with a high flow of anthropogenic activities. An important degree of genetic diversity and a high prevalence of virulence genes could be observed among the SCs analyzed. The results when compared with guidelines and water quality standards from various countries showed concentrations of SCs higher than those allowed. The high prevalence of gastrointestinal diseases in the region suggests a link to water contamination.

Molecular Typing of Somatic Coliphage Groups and Their Occurrence and Survival in Sewage

A conventional, group-specific PCR method was developed to identify each of the four previously defined major taxa (Myoviridae, Siphoviridae, Podoviridae and Microviridae) of somatic coliphages and used to classify isolates from sewage. Somatic coliphage infectivity detection, occurrence and survival in primary human sewage effluent was observed over time to further understand the presence and behavior of the groups of somatic coliphages at two environmental temperatures (4 and 25 °C). Over time, the taxonomic composition of the somatic coliphage population in sewage changed, with the Microviridae family becoming the most prevalent family in the sewage population after several weeks. Based on their persistence and prevalence in environmental waters, phages belonging to the Microviridae family provide supporting information on sewage contamination and possibly of human enteric viruses in sewage-contaminated water.

Distributions of Fecal Indicators at Aquaculture Areas in a Bay of Republic of Korea.

Aquaculture products, such as clams, scallops, and oysters, are major vectors of fecal-derived pathogens. Male-specific and somatic coliphages are strongly correlated with human noroviruses, the major enteric viruses worldwide. Geographic information system with local land-use patterns can also provide valuable information for tracking sources of fecal-derived pathogens. We examined distributions of four fecal indicator microorganisms, i.e., male-specific and somatic coliphage, total coliform, and Escherichia coli (E. coli) in three river and seawater sampling sites located on the coast of Gomso Bay in the Republic of Korea during the sampling period (from March 2015 to January 2016). Geospatial analyses of fecal indicators and correlation between environmental parameters and fecal indicators or among fecal indicators were also performed. Overall, river water samples showed highest concentrations of both types of coliphage in summer (July 2015). High concentrations of both total coliform and E. coli were detected in river water during the period from July to September 2015. High concentrations of all fecal indicators were found at site GL02, located in the innermost part of Gomso Bay, which has high-density agriculture and residential areas. Environmental factors related to precipitation-cumulative precipitation on and from 3 days before the sampling day (Prep-0 and Prep-3, respectively)-and salinity were strongly correlated with concentrations of all fecal indicators. The present results suggest that investigations of multiple fecal indicators with a systemic geospatial information are necessary for precisely tracking fecal contaminations of aquaculture products.

Biofilm Prevention and Removal in Non-Target Pseudomonas Strain by Siphovirus-like Coliphage.

Background/Objectives. Bacteriophages have gained significant interest as a potential solution to combat harmful bacteria, especially in the fight against antimicrobial resistance. With the rise in drug-resistant microorganisms, the medical community is increasingly exploring new alternatives to traditional antibiotics, and bacteriophages offer several advantages in this regard. However, phage applications still face some challenges, such as host specificity. Methods. In this study, a somatic Siphovirus-like coliphage (SOM7) was tested for inhibiting the biofilm-forming capacity of the non-target strain Pseudomonas aeruginosa (ATTC 10145). The phage-sensitive strain E. coli WG5 was used as a control. The selected microorganisms were first tested for growth in the presence of SOM7 at three different concentrations (105, 107, and 109 PFU/mL). Results. As expected, the phage-sensitive E. coli WG5 was fully inhibited by the coliphage, and no phage-related affection on the growth rate was observed for the SOM7-resistant P. aeruginosa. More notably, increasing concentrations of SOM7 significantly reduced both the biofilm-forming capacity and the amount of pre-established bacterial biofilm of the phage-insensitive P. aeruginosa (24.9% and 38.8% reduction in the biofilm-forming ability, and 18.8% and 28.0% biofilm degradation for 107 PFU/mL and 109 PFU/mL SOM7, respectively; p < 0.05). These results were supported by transmission electron microscopy (TEM) imaging, providing unprecedent evidence for the interaction of the somatic coliphage with the non-host strain. Conclusions. Although more studies in other biofilm models are necessary, our results show for the very first time that bacteriophages could potentially be used as an alternative to achieve desired anti-biofilm and biofilm-degrading activity in non-host bacterial strains.

Assessment of occurrence of somatic coliphages in drinking water in Poland

Assessment of occurrence of somatic coliphages in drinking water in Poland

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.

CrAss-like phages are suitable indicators of antibiotic resistance genes found in abundance in fecally polluted samples

Antibiotic resistance genes (ARGs) have been extensively observed in bacterial DNA, and more recently, in phage particles from various water sources and food items. The pivotal role played by ARG transmission in the proliferation of antibiotic resistance and emergence of new resistant strains calls for a thorough understanding of the underlying mechanisms. The aim of this study was to assess the suitability of the prototypical p-crAssphage, a proposed indicator of human fecal contamination, and the recently isolated crAssBcn phages, both belonging to the Crassvirales group, as potential indicators of ARGs. These crAss-like phages were evaluated alongside specific ARGs (blaTEM, blaCTX-M-1, blaCTX-M-9, blaVIM, blaOXA-48, qnrA, qnrS, tetW and sul1) within the total DNA and phage DNA fractions in water and food samples containing different levels of fecal pollution. In samples with high fecal load (>103 CFU/g or ml of E. coli or somatic coliphages), such as wastewater and sludge, positive correlations were found between both types of crAss-like phages and ARGs in both DNA fractions. The strongest correlation was observed between sul1 and crAssBcn phages (rho = 0.90) in sludge samples, followed by blaCTX-M-9 and p-crAssphage (rho = 0.86) in sewage samples, both in the phage DNA fraction. The use of crAssphage and crAssBcn as indicators of ARGs, considered to be emerging environmental contaminants of anthropogenic origin, is supported by their close association with the human gut. Monitoring ARGs can help to mitigate their dissemination and prevent the emergence of new resistant bacterial strains, thus safeguarding public health.

Capsid Integrity Detection of Enteric Viruses in Reclaimed Waters.

Climate change, unpredictable weather patterns, and droughts are depleting water resources in some parts of the globe, where recycling and reusing wastewater is a strategy for different purposes. To counteract this, the EU regulation for water reuse sets minimum requirements for the use of reclaimed water for agricultural irrigation, including a reduction in human enteric viruses. In the present study, the occurrence of several human enteric viruses, including the human norovirus genogroup I (HuNoV GI), HuNoV GII, and rotavirus (RV), along with viral fecal contamination indicator crAssphage was monitored by using (RT)-qPCR methods on influent wastewater and reclaimed water samples. Moreover, the level of somatic coliphages was also determined as a culturable viral indicator. To assess the potential viral infectivity, an optimization of a capsid integrity PMAxx-RT-qPCR method was performed on sewage samples. Somatic coliphages were present in 60% of the reclaimed water samples, indicating inefficient virus inactivation. Following PMAxx-RT-qPCR optimization, 66% of the samples tested positive for at least one of the analyzed enteric viruses, with concentrations ranging from 2.79 to 7.30 Log10 genome copies (gc)/L. Overall, most of the analyzed reclaimed water samples did not comply with current EU legislation and contained potential infectious viral particles.

Spatio-Temporal Dynamics of crAssphage and Bacterial Communities in an Algerian Watershed Impacted by Fecal Pollution

This study investigates the influence of urban pollution and climate dynamics on water quality and the bacterial communities in an Argelian watershed. Twenty-one sampling campaigns were conducted over two years at six sites along the Oued Boussellam, a river impacted by the effluent of a sewage treatment plant, from a low-polluted site to a water reservoir within a 50 km distance. Fecal indicators and the human fecal marker crAssphage were monitored. Illumina 16S rRNA amplicon sequencing was used to assess water microbial populations’ changes. Urban sewage discharge had an impact on the river quality and microbial ecosystem, which was attenuated along the river course. Significant reductions (&gt;4 log10 for E. coli and somatic coliphages, &gt;3 log10 for crAssphage) occurred, particularly during high-temperature periods. crAssphage correlated strongly with somatic coliphages downstream the river. Seasonal differences were observed in the diversity of the bacterial communities, with higher values during the high-temperature period. The genus-level community structure was similar at highly polluted river sites, also displaying seasonal differences. Despite high pollution levels, natural processes reduced fecal indicators to acceptable levels in the reservoir as well as shaped the bacterial communities along the river, highlighting the importance of understanding indicator persistence and microbial community resilience for effective water quality management within the context of the global warming scenario.

Urban wastewater-based epidemiology for multi-viral pathogen surveillance in the Valencian region, Spain

Wastewater-based epidemiology (WBE) has lately arised as a promising tool for monitoring and tracking viral pathogens in communities. In this study, we analysed WBE's role as a multi-pathogen surveillance strategy to detect the presence of several viral illness causative agents. Thus, an epidemiological study was conducted from October 2021 to February 2023 to estimate the weekly levels of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), Respiratory Syncytial virus (RSV), and Influenza A virus (IAV) in influent wastewater samples (n = 69). In parallel, a one-year study (October 2021 to October 2022) was performed to assess the presence of pathogenic human enteric viruses. Besides, monitoring of proposed viral fecal contamination indicators crAssphage and Pepper mild mottle virus (PMMoV) was also assessed, along with plaque counting of somatic coliphages. Genetic material of rotavirus (RV), human astrovirus (HAStV), and norovirus genogroup I (GI) and GII was found in almost all samples, while hepatitis A and E viruses (HAV and HEV) only tested positive in 3.77 % and 22.64 % of the samples, respectively. No seasonal patterns were overall found for enteric viruses, although RVs had a peak prevalence in the winter months. All samples tested positive for SARS-CoV-2 RNA, with a mean concentration of 5.43 log genome copies per liter (log GC/L). The tracking of the circulating SARS-CoV-2 variants of concern (VOCs) was performed by both duplex RT-qPCR and next generation sequencing (NGS). Both techniques reliably showed how the dominant VOC transitioned from Delta to Omicron during two weeks in Spain in December 2021. RSV and IAV viruses peaked in winter months with mean concentrations 6.40 and 4.10 log GC/L, respectively. Moreover, the three selected respiratory viruses strongly correlated with reported clinical data when normalised by wastewater physico-chemical parameters and presented weaker correlations when normalising sewage concentration levels with crAssphage or somatic coliphages titers. Finally, predictive models were generated for each respiratory virus, confirming high reliability on WBE data as an early-warning system and communities illness monitoring system. Overall, this study presents WBE as an optimal tool for multi-pathogen tracking reflecting viral circulation and diseases trends within a selected area, its value as a multi-pathogen early-warning tool stands out due to its public health interest.

Foodborne Viruses and Somatic Coliphages Occurrence in Fresh Produce at Retail from Northern Mexico.

Foodborne disease outbreaks linked to consumption of vegetables have been often attributed to human enteric viruses, such as Norovirus (NoV), Hepatitis A virus (HAV), and Rotavirus (RoV). Information about the occurrence of these viruses is scarce in many fresh-producing countries. Viral contamination detection of indicators, such as somatic coliphages, could indirectly reflect the presence of viral pathogens, being a valuable tool for better viral risk assessment in food industry. This study aimed to establish the occurrence and correlation of foodborne viruses and somatic coliphages in leafy greens in northern Mexico. A total of 320 vegetable samples were collected, resulting in 80 composite rinses, 40 of lettuce and 40 of parsley. Somatic coliphages were determined using the EPA 1602 method, while foodborne viruses (HAV, RoV, NoV GI, and GII) were determined by qPCR. The occurrence of RoV was 22.5% (9/40, mean 2.11 log gc/g) in lettuce and 20% (8/40, mean 1.91 log gc/g) in parsley. NoV and HAV were not detected in any samples. Somatic coliphages were present in all lettuce and parsley samples, with mean levels of 1.85 log PFU/100ml and 2.28 log PFU/100ml, respectively. Spearman analysis established the correlation of somatic coliphages and genomic copies of RoV, resulting in an r2 value of -0.026 in lettuce and 0.349 in parsley. Although NoV or HAV were undetected in the samples, the presence of RoV is a matter of concern as leafy greens are usually eaten raw, which poses a potential risk of infection.

Somatic Coliphages as an Operational Tool to Assess Loss of Bathing Water Quality after Heavy Rain Events

Rapid population growth and coastal development has led to increased fecal contamination of coastal surface waters worldwide, enhancing the potential risk of waterborne human pathogens in bathing areas. More frequent heavy rainfall events, attributed to global warming, have further exacerbated the problem by causing sometimes sewer overflows into recreational waters. As traditional bacterial indicators have limited accuracy for predicting health risks associated with waterborne viruses, the additional use of viral indicators such as coliphages is recommended. In this study, we compared the behavior of bacterial and viral indicators of water quality at 10 Barcelona beaches during three bathing seasons, in dry conditions, and after four rainstorms that caused specific pollution events due to rain runoff with combined sewer overflows (CSO). Levels of all target indicators increased after the rainstorms, but compared to Escherichia coli and intestinal enterococci, somatic coliphages exhibited a slower decline and higher environmental persistence following a rain event. Daily continuous sampling carried out during the days following a rainstorm allowed not only the determination of the decay kinetics of each target indicator but also the day when the water quality recovered the values established in the current European regulation in approximately 2 -3 days after each CSO. These observations indicate that the combined use of bacterial and viral indicators can enhance the surveillance of microbial quality of bathing waters. Moreover, coliphages can swiftly provide insights into transient fecal pollution linked to rainfall episodes, thanks to available analytical techniques that enable same-day recommendations. The management of urban wastewater and recreational water regulations should consistently employ microbial indicators to address rainwater runoff or sewer overflows resulting from heavy rainfall.

A new understanding of somatic coliphages belonging to the Microviridae family in urban wastewater

Somatic coliphages (SC) and F-specific RNA coliphages (FRNAPH) have been included in regulations or guidelines by several developed countries as a way of monitoring water safety and the microbiological quality of shellfish harvesting waters. SC are highly diverse in their morphology, size and genome. The Microviridae family contains three genera of phages (Alphatrevirus, Gequatrovirus, and Sinsheimervirus), all having a capsid of similar morphology (icosahedral) and size (25–30 nm in diameter) to that of common pathogenic enteric viruses. Three PCR assays specific for each genus of Microviridae were designed to study these phages in raw and treated wastewater (WW) in order to gain knowledge about the diversity and prevalence of Microviridae among SC, as well as their inactivation and removal during WW treatments. Among the four wastewater treatment plants (WWTPs) monitored here, two WWTPs applied disinfection by UV light as tertiary treatment. First, we noticed that Microviridae represented 10 to 30 % of infectious SC in both raw and treated WW. Microviridae appeared to behave in the same way as all SC during these WW treatments. As expected, the highest inactivation, at least 4 log10, was achieved for infectious Microviridae and SC in both WWTPs using UV disinfection. PCR assays showed that the highest removal of Microviridae reached about 4 log10, but the phage removal can vary greatly between WWTPs using similar treatments. This work forms the basis for a broader evaluation of Microviridae as a viral indicator of water treatment efficiency and WW reuse.

Detection and Quantification of Bacteriophages in Wastewater Samples by Culture and Molecular Methods.

Bacteriophages are promising tools for the detection of fecal pollution in water bodies and particularly for viral pathogen risk assessment. Having similar morphological and biological characteristics, bacteriophages are perfect surrogates for the study of the fate and transport of enteric viruses, generally better than any other group of indicators.Different groups of bacteriophages, such as somatic coliphages, F-specific RNA bacteriophages, and bacteriophages infecting selected strains of Bacteroides, have been comprehensively tested as indicators of fecal pollution. Somatic coliphages and F-specific RNA bacteriophages can be used as indicators of general fecal contamination, whereas Bacteroides phages can be used to detect a particular fecal source, for instance, human, bovine, porcine, or poultry fecal contamination.Feasible and cost-effective protocols standardized by the International Standardization Organization and the United States Environmental Protection Agency for the detection of infectious bacteriophages belonging to these three groups are available. Molecular methods for the detection of some particular phages have also been developed. Here we introduce those methods for the detection, enumeration, and isolation of bacteriophages in wastewater samples.

Enteric virus removal by municipal wastewater treatment to achieve requirements for potable reuse

Primary and secondary treatment of municipal wastewater contributes to virus removal upstream of advanced purification to produce water for potable reuse. In this study, virus occurrence by cultivable and molecular methods was measured over a 24-month period in raw wastewater influents and secondary effluents from two municipal wastewater treatment plants that together provide the recycled water source for an advanced water purification facility. Using a rank-paired, covariance-based statistical approach, virus log removal values were determined for four wastewater treatment processes that operate in parallel at the two facilities (two activated sludge processes, trickling filter process, and trickling filter/solids contactor process). The trickling filter process exhibited the lowest observed removal of cultivable enteric virus with a median removal of 1.0 log10 (or 90% removal) and a 5th percentile log removal of 0.73 (or 82%), compared to the greatest removal observed for one of the activated sludge processes (median log removal of 2.4 or 99.6% and 5th percentile of 2.1 or 99.2%). Median log removal observed for male-specific (MS) and somatic (SOM) coliphage was 1.8 (98.6% removal) and 0.5 (70%), respectively, for trickling filter and 2.9 (99.9%) and 2.0 (99%) for activated sludge. Thus, coliphage removal was fairly similar to removal observed for cultivable enteric virus. The cultivable enteric virus 5th percentile removal (0.7) from the trickling filter treatment process was proposed to the state regulator for credit towards state requirements for virus removal related to groundwater augmentation with purified recycled water. Receiving pathogen removal credits for secondary wastewater treatment would allow for an improved margin (safety factor) of credits beyond the minimum required; and in this case may also increase the number of viable future groundwater recharge sites closer to drinking water production wells by reducing the underground travel time otherwise required to obtain sufficient credits.

Optimizing Ozone Disinfection in Water Reuse: Controlling Bromate Formation and Enhancing Trace Organic Contaminant Oxidation.

The use of ozone/biofiltration advanced treatment has become more prevalent in recent years, with many utilities seeking an alternative to membrane/RO based treatment for water reuse. Ensuring efficient pathogen reduction while controlling disinfection byproducts and maximizing oxidation of trace organic contaminants remains a major barrier to implementing ozone in reuse applications. Navigating these challenges is imperative in order to allow for the more widespread application of ozonation. Here, we demonstrate the effectiveness of ozone for virus, coliform bacteria, and spore forming bacteria inactivation in unfiltered secondary effluent, all the while controlling the disinfection byproduct bromate. A greater than 6-log reduction of both male specific and somatic coliphages was seen at specific ozone doses as low as 0.75 O3:TOC. This study compared monochloramine and hydrogen peroxide as chemical bromate control measures in high bromide water (Br- = 0.35 ± 0.07 mg/L). On average, monochloramine and hydrogen peroxide resulted in an 80% and 36% decrease of bromate formation, respectively. Neither bromate control method had any appreciable impact on virus or coliform bacteria disinfection by ozone; however, the use of hydrogen peroxide would require a non-Ct disinfection framework. Maintaining ozone residual was shown to be critical for achieving disinfection of more resilient microorganisms, such as spore forming bacteria. While extremely effective at controlling bromate, monochloramine was shown to inhibit TrOC oxidation, whereas hydrogen peroxide enhanced TrOC oxidation.

Use of Somatic Coliphages in Biosensoring of Hygiene Safety of Recycled Wastewater

Outflows from secondary stages of conventional mechanical-biological wastewater treatment plants (WWTPs) still contain significant concentrations of faecal pollution indicators suggesting the potential presence of pathogenic organisms. The decrease in the concentration of somatic coliphages and bacterial indicators of faecal pollution during the technological stages (coagulation, sand filtration, membrane ultrafiltration, sorption on granular activated carbon, disinfection, accumulation) of a semi-operational plant designed for multi-stage tertiary treatment or rather recycling of treated wastewater was monitored and discussed. During the tertiary treatment, faecal bacteria indicators were better removed than somatic coliphages, hence the inclusion of somatic indicators among faecal pollution indicators in water quality control for reuse is entirely appropriate. Subsequent tertiary treatment, including disinfection, is essential for safe reuse of treated water.

Coliphages as indicators of primary wastewater treatment efficiency by constructed wetlands.

Constructed wetlands are an efficient and cost-effective system for the treatment of wastewater that can be reused for diverse purposes, including irrigation; however, few studies have determined the efficiency of microbial removal by constructed wetlands in tropical regions. Therefore, the present study aimed to determine the microbial quality of the influent and effluent of a constructed wetland in Puerto Rico, using traditional bacterial indicators (i.e., thermotolerant coliforms and enterococci), as well as somatic and male-specific (F+) coliphages. Results showed that over 99.9 and 97.7% of thermotolerant coliforms and enterococci were removed after treatment by constructed wetlands, respectively. Notably, approximately 84.0% of male-specific (F+) coliphages were removed, while somatic and total coliphages exhibited differing removal percentages at different steps during treatment by constructed wetlands. The potential risk of the presence of enteric viruses in treated wastewater by constructed wetlands may increase when considering traditional bacterial indicators exclusively. The present study may aid in the efforts to determine public health concerns associated with the exposure of bioaerosols resulting from wastewater treatment by constructed wetlands.

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.