Seasonal Detection of Human Viruses and Coliphage in Newport Bay, California

Recent studies have shown that the fecal indicator bacteria (FIB) currently used to indicate water quality in the coastal environment may be inadequate to reflect human viral contamination. Coliphage was suggested as a better indicator of human viral pollution and was proposed by the U.S. EPA as an alternative indicator for fecal pollution in groundwater. In this study, we investigated the occurrence and distribution of FIB, F+ coliphage, and PCR-detectable human adenovirus and enterovirus for an entire year at 15 locations around the Newport Bay watershed, an important southern California estuary for water recreation and an ecological reserve. Peak concentrations and prevalences of FIB and F+ coliphage were associated with winter storms (wet weather). Human adenoviruses and enteroviruses, however, were detected by PCR in ~5% of samples collected in the summer (dry weather) but only once in wet weather. These results demonstrated that FIB and coliphage have similar seasonal and freshwater-to-saltwater distribution patterns, while the detection of human viruses depends on a distribution pattern that is the opposite of that of FIB and coliphage. This research suggested that coliphage and FIB share similar environmental sources, while sources of human viruses in Newport Bay are perhaps different.     

Human enteric viruses–potential indicators for enhanced monitoring of recreational water quality

Recreational waters contaminated with human fecal pollution are a public health concern, and ensuring the safety of recreational waters for public use is a priority of both the Environmental Protection Agency (EPA) and the Centers for Disease Control and Prevention (CDC). Current recreational water standards rely on fecal indicator bacteria (FIB) levels as indicators of human disease risk. However present evidence indicates that levels of FIB do not always correspond to the presence of other potentially harmful organisms, such as viruses. Thus, enteric viruses are currently tested as water quality indicators, but have yet to be successfully implemented in routine monitoring of water quality. This study utilized enteric viruses as possible alternative indicators of water quality to examine 18 different fresh and offshore recreational waters on O‘ahu, Hawai‘i, by using newly established laboratory techniques including highly optimized PCR, real time PCR, and viral infectivity assays. All sample sites were detected positive for human enteric viruses by PCR including enterovirus, norovirus genogroups I and II, and male specific FRNA coliphage. A six time-point seasonal study of enteric virus presence indicated significant variation in virus detection between the rainy and dry seasons. Quantitative PCR detected the presence of norovirus genogroup II at levels at which disease risk may occur, and there was no correlation found between enteric virus presence and FIB counts. Under the present laboratory conditions, no infectious viruses were detected from the samples PCR-positive for enteric viruses. These data emphasize both the need for additional indicators for improved monitoring of water quality, and the feasibility of using enteric viruses as these indicators. Electronic Supplementary MaterialSupplementary material is available for this article at 10.1007/s12250-015-3644-x and is accessible for authorized users.     

Coliphage and indigenous phage in Mamala Bay, Oahu, Hawaii.

Public concern over the discharge of primarily treated sewage by two offshore outfalls in Mamala Bay, Oahu, prompted a multidisciplinary study to determine the impact of such activities on the water quality in the bay and at adjacent recreational beaches. As part of this study, we determined the abundance of coliphage as an indicator of fecal pollution along with total viral direct counts and phages infective for Vibrio parahaemoltyicus 16 at stations in Mamala Bay in four quarterly samplings over 13 months. Coliphage (< 1 to 1.2 x 10(3)/liter) were found during each quarterly sampling along an offshore transect to the Sand Island waste treatment facility outfall. The nonpoint coastal stations (Pearl Harbor, Ala Wai Canal, and Ke'ehi Lagoon) had high levels of coliphage during the storm event sampling in February 1994 but much lower levels or none when sampled during dry weather. Coliphage were absent at all samplings at Waikiki Beach and at the control station off Diamond Head. Viral direct counts in eutrophic coastal stations (Pearl Harbor, Ke'ehi Lagoon, Ala Moana Beach, and Ala Wai canal) averaged 10(9)/liter, while counts at offshore stations ranged from 9 x 10(7) to 1 x 10(9) viruses/liter, values similar to those for other marine environments. Vibriophage were found mainly in eutrophic coastal environments (Ala Wai Canal, Pearl Harbor, and Ke'ehi Lagoon) and at the Sand Island Transect stations D1 and D2. The greatest abundance was found during the storm event (February 1994) sampling. These results suggest that the Sand Island outfall influenced the water quality of the immediate surrounding waters but had little effect on the quality of the recreational beaches. Nonpoint discharge sources appeared to be more important in the distribution of fecal indicators in the coastal zone.     

应用肠道微生物示踪地表水粪便污染的研究进展

人和动物的粪便已成为水污染的重要污染源, 严重威胁着饮水安全和经济发展。水质污染微生物的传统检测指示菌是总大肠菌群、粪大肠菌群、埃希大肠菌、肠球菌和梭菌属。经过调查发现, 上述指示菌由于在体外能存活并繁殖, 并且不同宿主之间没有差异性, 不能准确用于追踪污染粪便的来源, 因此该指标难以直接说明粪便污染源和污染程度。最近的研究表明, Faecalibaterium作为水体粪便污染来源追踪的指示微生物具有很多优点。本文综述了粪便污染指示菌以及其相关替代方法在水质检测中的研究进展, 对各种指示菌进行了优、缺点比较, 展望了Faecalibaterium的应用前景。     

Selective accumulation may account for shellfish-associated viral illness

From 1991 through 1998, 1,266 cases of shellfish-related illnesses were attributed to Norwalk-like viruses. Seventy-eight percent of these illnesses occurred following consumption of oysters harvested from the Gulf Coast during the months of November through January. This study investigated the ability of eastern oysters (Crassostrea virginica) to accumulate indicator microorganisms (i.e., fecal coliforms, Escherichia coli, Clostridium perfringens, and F(+) coliphage) from estuarine water. One-week trials over a 1-year period were used to determine if these indicator organisms could provide insight into the seasonal occurrence of these gastrointestinal illnesses. The results demonstrate that oysters preferentially accumulated F(+) coliphage, an enteric viral surrogate, to their greatest levels from late November through January, with a concentration factor of up to 99-fold. However, similar increases in accumulation of the other indicator microorganisms were not observed. These findings suggest that the seasonal occurrence of shellfish-related illnesses by enteric viruses is, in part, the result of seasonal physiological changes undergone by the oysters that affect their ability to accumulate viral particles from estuarine waters.     

PCR detection of pathogenic viruses in southern California urban rivers

AIMS: To investigate human viral contamination in urban rivers and its impact on coastal waters of southern California, USA. METHODS AND RESULTS: Three types of human viruses (adeno, entero and hepatitis A) were detected using nested- and RT-PCR from 11 rivers and creeks. Faecal indicator bacteria as well as somatic and F-specific coliphage were also tested. Approximately 50% of the sites were positive for human adenoviruses. However, there was no clear relationship between detection of human viruses and the concentration of indicator bacteria and coliphage. Both faecal indicator bacteria and human viral input at beaches near river mouths were associated with storm events. The first storm of the wet season seemed to have the greatest impact on the quality of coastal water than following storm events. CONCLUSIONS: This study provides the first direct evidence that human viruses are prevalent in southern California urban rivers. Urban run-off impacts coastal water quality most significantly during the storm season. SIGNIFICANCE AND IMPACT OF THE STUDY: To protect human health during water recreational activities, it is necessary to develop effective strategies to manage urban run-off during storm events.     

Ecology of coliphages in southern California coastal waters

Aims: This study aims to investigate the ecology of coliphages, an important microbial pollution indicator. Specifically, our experiments address (i) the ability of environmental Escherichia coli (E. coli) to serve as hosts for coliphage replication, and (ii) the temporal and spatial distribution of coliphages in coastal waters. Methods and Results: Water samples from three locations in California’s Newport Bay watershed were tested for the presence of coliphages every 2 weeks for an entire year. A total of nine E. coli strains isolated from various sources served as hosts for coliphage detection. Coliphage occurrence was significantly different between freshwater, estuarine and coastal locations and correlated with water temperature, salinity and rainfall in the watershed. The coliphages isolated on the environmental hosts had a broad host‐range relative to the coliphages isolated on an E. coli strain from sewage and a US EPA recommended strain for coliphage detection. Conclusions: Coliphage occurrence was related to the temperature, rainfall and salinity within the bay. The adaptation to a broad host‐range may enable the proliferation of coliphages in the aquatic environment. Significance and Impact of the Study: Understanding the seasonal variation of phages is useful for establishing a background level of coliphage presence in coastal waters. The broad host‐range of coliphages isolated on the environmental E. coli host calls for investigation of coliphage replication in the aquatic environment.     

Relative Chlorine Resistance of Poliovirus I and Coliphages f2 and T2 in Water

Phage f2 is markedly more resistant to chlorine in water than either poliovirus or coliphage T(2). Its potential use as an indicator of viral pollution is suggested.     

Distribution of genetic marker concentrations for fecal indicator bacteria in sewage and animal feces

Very little is known about the density and distribution of fecal indicator bacteria (FIB) genetic markers measured by quantitative real-time PCR (qPCR) in fecal pollution sources. Before qPCR-based FIB technologies can be applied to waste management and public health risk applications, it is vital to characterize the concentrations of these genetic markers in pollution sources (i.e., untreated wastewater and animal feces). We report the distribution of rRNA genetic markers for several general FIB groups, including Clostridium spp., Escherichia coli, enterococci, and Bacteroidales, as determined by qPCR on reference collections consisting of 54 primary influent sewage samples collected from treatment facilities across the United States and fecal samples representing 20 different animal species. Based on raw sewage sample collection data, individual FIB genetic markers exhibited a remarkable similarity in concentration estimates from locations across the United States ranging from Hawaii to Florida. However, there was no significant correlation between genetic markers for most FIB combinations (P > 0.05). In addition, large differences (up to 5 log(10) copies) in the abundance of FIB genetic markers were observed between animal species, emphasizing the importance of indicator microorganism selection and animal source contribution for future FIB applications.     

Terrestrial Sources Homogenize Bacterial Water Quality During Rainfall in Two Urbanized Watersheds in Santa Barbara, CA

Microbiological contamination from runoff is a human health concern in urbanized coastal environments, but the contamination sources are often unknown. This study quantified fecal indicator bacteria and compared the distributions of human-specific genetic markers and bacterial community composition during dry and wet weather in urban creeks draining two neighboring watersheds in Santa Barbara, CA. In a prior study conducted during exclusively dry weather, the creeks were contaminated with human waste as indicated by elevated numbers of the human-specific Bacteroidales marker HF183 (Sercu et al. in Environ Sci Technol 43:293-298, 2009). During the storm, fecal indicator bacterial numbers and loads increased orders of magnitude above dry weather conditions. Moreover, bacterial community composition drastically changed during rainfall and differed from dry weather flow by (1) increased bacterial diversity, (2) reduced spatial heterogeneity within and between watersheds, and (3) clone library sequences more related to terrestrial than freshwater taxa. Finally, the spatial patterns of human-associated genetic markers (HF183 and Methanobrevibacter smithii nifH gene) changed during wet weather, and the contribution of surface soils to M. smithii nifH gene detection was suspected. The increased fecal indicator bacteria numbers during wet weather were likely associated with terrestrial sources, instead of human waste sources that dominated during dry weather flow.     

F+ RNA coliphage typing for microbial source tracking in surface waters

AIMS: The utility of coliphages to detect and track faecal pollution was evaluated using South Carolina surface waters that exceeded State faecal coliform standards. METHODS AND RESULTS: Coliphages were isolated from 117 surface water samples by single agar layer (SAL) and enrichment presence/absence (EP/A) methods. Confirmed F+ RNA coliphages were typed for microbial source tracking using a library-independent approach. Concentrations of somatic coliphages using 37 and 44.5 degrees C incubation temperatures were found to be significantly different and the higher temperature may be more specific for faecal contamination. The EP/A technique detected coliphages infecting Escherichia coli Famp in 38 (66%) of the 58 surface water samples negative for F+ coliphages by the SAL method. However, coliphages isolated by EP/A were found to be less representative of coliphage diversity within a sample. Among the 2939 coliphage isolates tested from surface water and known source samples, 813 (28%) were found to be F+ RNA. The majority (94%) of surface water F+ RNA coliphage isolates typed as group I. Group II and/or III viruses were identified from 14 surface water stations, the majority of which were downstream of wastewater discharges. These sites were likely contaminated by human-source faecal pollution. CONCLUSIONS: The results suggest that faecal contamination in surface waters can be detected and source identifications aided by coliphage analyses. SIGNIFICANCE AND IMPACT OF THE STUDY: This study supports the premise that coliphage typing can provide useful, but not absolute, information to distinguish human from animal sources of faecal pollution. Furthermore, the comparison of coliphage isolation methods detailed in this study should provide valuable information to those wishing to incorporate coliphage detection into water quality assessments.     

Basin-wide analysis of the dynamics of fecal contamination and fecal source identification in Tillamook Bay, Oregon

The objectives of this study were to elucidate spatial and temporal dynamics in source-specific Bacteroidales 16S rRNA genetic marker data across a watershed; to compare these dynamics to fecal indicator counts, general measurements of water quality, and climatic forces; and to identify geographic areas of intense exposure to specific sources of contamination. Samples were collected during a 2-year period in the Tillamook basin in Oregon at 30 sites along five river tributaries and in Tillamook Bay. We performed Bacteroidales PCR assays with general, ruminant-source-specific, and human-source-specific primers to identify fecal sources. We determined the Escherichia coli most probable number, temperature, turbidity, and 5-day precipitation. Climate and water quality data collectively supported a rainfall runoff pattern for microbial source input that mirrored the annual precipitation cycle. Fecal sources were statistically linked more closely to ruminants than to humans; there was a 40% greater probability of detecting a ruminant source marker than a human source marker across the basin. On a sample site basis, the addition of fecal source tracking data provided new information linking elevated fecal indicator bacterial loads to specific point and nonpoint sources of fecal pollution in the basin. Inconsistencies in E. coli and host-specific marker trends suggested that the factors that control the quantity of fecal indicators in the water column are different than the factors that influence the presence of Bacteroidales markers at specific times of the year. This may be important if fecal indicator counts are used as a criterion for source loading potential in receiving waters.     

Proposed modifications of Environmental Protection Agency Method 1601 for detection of coliphages in drinking water, with same-day fluorescence-based detection and evaluation by the performance-based measurement system and alternative test protocol validation approaches

Coliphages are microbial indicators specified in the Ground Water Rule that can be used to monitor for potential fecal contamination of drinking water. The Total Coliform Rule specifies coliform and Escherichia coli indicators for municipal water quality testing; thus, coliphage indicator use is less common and advances in detection methodology are less frequent. Coliphages are viral structures and, compared to bacterial indicators, are more resistant to disinfection and diffuse further distances from pollution sources. Therefore, coliphage presence may serve as a better predictor of groundwater quality. This study describes Fast Phage, a 16- to 24-h presence/absence modification of U.S. Environmental Protection Agency (EPA) Method 1601 for detection of coliphages in 100 ml water. The objective of the study is to demonstrate that the somatic and male-specific coliphage modifications provide results equivalent to those of Method 1601. Five laboratories compared the modifications, featuring same-day fluorescence-based prediction, to Method 1601 by using the performance-based measurement system (PBMS) criterion. This requires a minimum 50% positive response in 10 replicates of 100-ml water samples at coliphage contamination levels of 1.3 to 1.5 PFU/100 ml. The laboratories showed that Fast Phage meets PBMS criteria with 83.5 to 92.1% correlation of the same-day rapid fluorescence-based prediction with the next-day result. Somatic coliphage PBMS data are compared to manufacturer development data that followed the EPA alternative test protocol (ATP) validation approach. Statistical analysis of the data sets indicates that PBMS utilizes fewer samples than does the ATP approach but with similar conclusions. Results support testing the coliphage modifications by using an EPA-approved national PBMS approach with collaboratively shared samples.     

Basin-Wide Analysis of the Dynamics of Fecal Contamination and Fecal Source Identification in Tillamook Bay, Oregon

The objectives of this study were to elucidate spatial and temporal dynamics in source-specific Bacteroidales 16S rRNA genetic marker data across a watershed; to compare these dynamics to fecal indicator counts, general measurements of water quality, and climatic forces; and to identify geographic areas of intense exposure to specific sources of contamination. Samples were collected during a 2-year period in the Tillamook basin in Oregon at 30 sites along five river tributaries and in Tillamook Bay. We performed Bacteroidales PCR assays with general, ruminant-source-specific, and human-source-specific primers to identify fecal sources. We determined the Escherichia coli most probable number, temperature, turbidity, and 5-day precipitation. Climate and water quality data collectively supported a rainfall runoff pattern for microbial source input that mirrored the annual precipitation cycle. Fecal sources were statistically linked more closely to ruminants than to humans; there was a 40% greater probability of detecting a ruminant source marker than a human source marker across the basin. On a sample site basis, the addition of fecal source tracking data provided new information linking elevated fecal indicator bacterial loads to specific point and nonpoint sources of fecal pollution in the basin. Inconsistencies in E. coli and host-specific marker trends suggested that the factors that control the quantity of fecal indicators in the water column are different than the factors that influence the presence of Bacteroidales markers at specific times of the year. This may be important if fecal indicator counts are used as a criterion for source loading potential in receiving waters.     

Microbial Source Tracking for Identification of Fecal Pollution

Fecal pollution is a serious environmental problem that affects many coastal and inland waters worldwide. Both human and animal fecal pollution impose risks to human health from exposure to pathogenic bacteria, viruses, and protozoa. To assist authorities with the implementation of the changes suggested by more restricted legislation concering water quality in Europe, methods are needed which can identify the sources of fecal pollution. Management of fecal contamination of water would be improved if the origin of the fecal pollution could be correctly identified since remediation efforts could then be allocated in a more effective manner. The concept that the origin of fecal pollution can be traced has been termed microbial source tracking. In microbial source tracking (MST) endogenous markers of fecal sources are used for identification of the fecal pollution in aquatic environments. Chemical MST-methods can be used to trace mainly sewage pollution, but the used chemical targets have no direct relationship with pathogenic bacteria. This is not the case in microbial MST-methods where source-specific bacteria or viruses are cultured to identify fecal pollution sources. However, sometimes these microbial targets can be present in too low numbers to be detected. This is circumvented by using molecular assays for host-specific marker detection. Phenotypic and genotypic library-based methods can be used to discriminate among different fecal sources. However, the isolation step makes this procedure very labour-intensive, and issues as temporal and geographical variability remain unresolved. The underlying assumptions will be discussed and the methods mostly used in microbial source tracking will be described in more detail.     

Virus occurrence in municipal groundwater sources in Quebec, Canada

A 1 year study was undertaken on groundwater that was a source of drinking water in the province of Quebec, Canada. Twelve municipal wells (raw water) were sampled monthly during a 1 year period, for a total of 160 samples. Using historic data, the 12 sites were categorized into 3 groups: group A (no known contamination), group B (sporadically contaminated by total coliforms), and group C (historic and continuous contamination by total coliforms and (or) fecal coliforms). Bacterial indicators (total coliform, Escherichia coli, enteroccoci), viral indicators (somatic and male-specific coliphages), total culturable human enteric viruses, and noroviruses were analyzed at every sampling site. Total coliforms were the best indicator of microbial degradation, and coliform bacteria were always present at the same time as human enteric viruses. Two samples contained human enteric viruses but no fecal pollution indicators (E. coli, enterococci, or coliphages), suggesting the limited value of these microorganisms in predicting the presence of human enteric viruses in groundwater. Our results underline the value of historic data in assessing the vulnerability of a well on the basis of raw water quality and in detecting degradation of the source. This project allowed us to characterize the microbiologic and virologic quality of groundwater used as municipal drinking water sources in Quebec.     

Enterococci in the Environment

Summary: Enterococci are common, commensal members of gut communities in mammals and birds, yet they are also opportunistic pathogens that cause millions of human and animal infections annually. Because they are shed in human and animal feces, are readily culturable, and predict human health risks from exposure to polluted recreational waters, they are used as surrogates for waterborne pathogens and as fecal indicator bacteria (FIB) in research and in water quality testing throughout the world. Evidence from several decades of research demonstrates, however, that enterococci may be present in high densities in the absence of obvious fecal sources and that environmental reservoirs of these FIB are important sources and sinks, with the potential to impact water quality. This review focuses on the distribution and microbial ecology of enterococci in environmental (secondary) habitats, including the effect of environmental stressors; an outline of their known and apparent sources, sinks, and fluxes; and an overview of the use of enterococci as FIB. Finally, the significance of emerging methodologies, such as microbial source tracking (MST) and empirical predictive models, as tools in water quality monitoring is addressed. The mounting evidence for widespread extraenteric sources and reservoirs of enterococci demonstrates the versatility of the genus Enterococcus and argues for the necessity of a better understanding of their ecology in natural environments, as well as their roles as opportunistic pathogens and indicators of human pathogens.     

Selective Accumulation May Account for Shellfish-Associated Viral Illness

From 1991 through 1998, 1,266 cases of shellfish-related illnesses were attributed to Norwalk-like viruses. Seventy-eight percent of these illnesses occurred following consumption of oysters harvested from the Gulf Coast during the months of November through January. This study investigated the ability of eastern oysters (Crassostrea virginica) to accumulate indicator microorganisms (i.e., fecal coliforms, Escherichia coli, Clostridium perfringens, and F⁺ coliphage) from estuarine water. One-week trials over a 1-year period were used to determine if these indicator organisms could provide insight into the seasonal occurrence of these gastrointestinal illnesses. The results demonstrate that oysters preferentially accumulated F⁺ coliphage, an enteric viral surrogate, to their greatest levels from late November through January, with a concentration factor of up to 99-fold. However, similar increases in accumulation of the other indicator microorganisms were not observed. These findings suggest that the seasonal occurrence of shellfish-related illnesses by enteric viruses is, in part, the result of seasonal physiological changes undergone by the oysters that affect their ability to accumulate viral particles from estuarine waters.     

Incidence of coliphage in potable water supplies

Samples of drinking water from different sources in greater Cairo, Egypt, and bottled drinking water were tested for total coliform, fecal coliform, and coliphage populations. Of the 147 samples tested, 4 samples were positive for both total coliforms and coliphage, 65 samples were negative for total coliforms, fecal coliforms, and coliphage, and 78 samples were positive for coliphage and negative for total coliforms and fecal coliforms. The incidence of coliphage in these potable water supplies reflects the probability of human pathogenic virus survival in these waters also.