Decay of Bacteroidales genetic markers in relation to traditional fecal indicators for water quality modeling of drinking water sources

The implementation of microbial fecal source tracking (MST) methods in drinking water management is limited by the lack of knowledge on the transport and decay of host-specific genetic markers in water sources. To address these limitations, the decay and transport of human (BacH) and ruminant (BacR) fecal Bacteroidales 16S rRNA genetic markers in a drinking water source (Lake R?dasj?n in Sweden) were simulated using a microbiological model coupled to a three-dimensional hydrodynamic model. The microbiological model was calibrated using data from outdoor microcosm trials performed in March, August, and November 2010 to determine the decay of BacH and BacR markers in relation to traditional fecal indicators. The microcosm trials indicated that the persistence of BacH and BacR in the microcosms was not significantly different from the persistence of traditional fecal indicators. The modeling of BacH and BacR transport within the lake illustrated that the highest levels of genetic markers at the raw water intakes were associated with human fecal sources (on-site sewers and emergency sewer overflow). This novel modeling approach improves the interpretation of MST data, especially when fecal pollution from the same host group is released into the water source from different sites in the catchment.     

Application of an integrated community analysis approach for microbial source tracking in a coastal creek

High fecal indicator bacterial (FIB) concentrations signal urban coastal water quality impairments that can threaten public health. However, FIB (total and fecal coliform plus Enterococcus sp.) concentrations are not specific to human waste, and thus, microbial source tracking (MST) is employed to assess public health risks and remediation alternatives. Currently, water quality diagnosis requires several simultaneous MST assays. Relatively unexplored is a community analysis approach for MST where the overall microbial community composition is compared, through multivariate analysis, to link sources and sinks of microbial pollution. In this research, an urban coastal creek and drain sampling transect, previously diagnosed as human-waste-contaminated, were evaluated for bacterial community composition relative to fecal sources; a laboratory spiking study was also performed to assess method sensitivity and specificity. Multivariate statistical analysis of community profiles clearly distinguished different fecal sources, indicated a high sensitivity for sewage spikes, and confirmed creek contamination sources. This work demonstrates that molecular microbial community analysis combined with appropriate multivariate statistical analyses is an effective addition to the MST tool box.     

Detection of Bacteroidales fecal indicators and the zoonotic pathogens E. coli 0157:H7, salmonella, and campylobacter in river water

Bacteroidales host-specific PCR offers a rapid method of diagnosing fecal pollution in water and identifying sources of input. To assess human health risks from exposure to fecal pathogens, however, Bacteroidales markers should be detectable when pathogens are present. To determine if Bacteroidales general, human-, ruminant-, and swine-specific markers correlate with certain fecal pathogens, we conducted a retrospective study on water samples for which the presence of E. coli O157:H7, Salmonella spp., and Campylobacter spp. had been determined. We found a positive relationship between detection of the Bacteroidales general fecal marker and presence of the pathogens. Detection of ruminant-specific markers predicted E. coli O157: H7 occurrence. There was a significant increase in the likelihood of detecting Salmonella when a ruminant marker was present, and Campylobacter spp. when human markers were present. For pathogens such as E. coli O157: H7 that are strongly associated with particular hosts, Bacteroidales host-specific markers can estimate the likelihood of pathogen occurrence, enabling more accurate health risk assessments.     

Combining land use information and small stream sampling with PCR-based methods for better characterization of diffuse sources of human fecal pollution

Diffuse sources of human fecal pollution allow for the direct discharge of waste into receiving waters with minimal or no treatment. Traditional culture-based methods are commonly used to characterize fecal pollution in ambient waters, however these methods do not discern between human and other animal sources of fecal pollution making it difficult to identify diffuse pollution sources. Human-associated quantitative real-time PCR (qPCR) methods in combination with low-order headwatershed sampling, precipitation information, and high-resolution geographic information system land use data can be useful for identifying diffuse source of human fecal pollution in receiving waters. To test this assertion, this study monitored nine headwatersheds over a two-year period potentially impacted by faulty septic systems and leaky sanitary sewer lines. Human fecal pollution was measured using three different human-associated qPCR methods and a positive significant correlation was seen between abundance of human-associated genetic markers and septic systems following wet weather events. In contrast, a negative correlation was observed with sanitary sewer line densities suggesting septic systems are the predominant diffuse source of human fecal pollution in the study area. These results demonstrate the advantages of combining water sampling, climate information, land-use computer-based modeling, and molecular biology disciplines to better characterize diffuse sources of human fecal pollution in environmental waters.     

Microbial Source Tracking: A Tool for Identifying Sources of Microbial Contamination in the Food Chain

The ability to trace fecal indicators and food-borne pathogens to the point of origin has major ramifications for food industry, food regulatory agencies, and public health. Such information would enable food producers and processors to better understand sources of contamination and thereby take corrective actions to prevent transmission. Microbial source tracking (MST), which currently is largely focused on determining sources of fecal contamination in waterways, is also providing the scientific community tools for tracking both fecal bacteria and food-borne pathogens contamination in the food chain. Approaches to MST are commonly classified as library-dependent methods (LDMs) or library-independent methods (LIMs). These tools will have widespread applications, including the use for regulatory compliance, pollution remediation, and risk assessment. These tools will reduce the incidence of illness associated with food and water. Our aim in this review is to highlight the use of molecular MST methods in application to understanding the source and transmission of food-borne pathogens. Moreover, the future directions of MST research are also discussed.     

Potential use of a host associated molecular marker in Enterococcus faecium as an index of human fecal pollution

Several genotypic and phenotypic microbial source tracking (MST) methods have been proposed and utilized to differentiate groups of microorganisms, usually indicator organisms, for the purpose of tracking sources of fecal pollution. Targeting of host-specific microorganisms is one of the approaches currently being tested. These methods are useful as they circumvent the need to isolate individual microorganisms and do not require the establishment of reference databases. Several studies have demonstrated that the presence and distribution of Enterococcus spp. in feces seems to be influenced by the host species. Here, we present a method for detection of genetic sequences in culturable enterococci capable of identifying human sources of fecal pollution in the environment. The human fecal pollution marker designed in this study targets a putative virulence factor, the enterococcal surface protein (esp), in Enterococcus faecium. This gene was detected in 97% of sewage and septic samples but was not detected in any livestock waste lagoons or in bird or animal fecal samples. Epidemiological studies in recreational and groundwaters have shown enterococci to be useful indicators of public health risk for gastroenteritis. By identifying the presence of human fecal pollution, and therefore the possible presence of human enteric pathogens, this marker allows for further resolution of the source of this risk.     

Using flow cytometry and <Emphasis Type="Italic">Bacteroidales</Emphasis> 16S rRNA markers to study the hygienic quality of source water

Six source water fountains in the community of Berne, Switzerland were sampled monthly over the period of 1 year. The samples were tested for total counts by flow cytometry, and for fecal contamination by using the Bacteroidales 16S rRNA markers HF183, BacR and AllBac. The total counts varied considerably between the different fountains with a minimal value of 5115 counts/L and with a maximal count of 198,508 counts/L. The long-term patterns of total counts over 1 year were typical for each fountain. Comparison of rainfall data and data for the non-specific fecal marker AllBac was shown to be a suitable approach to highlight the vulnerability of sources to environmental influences. HF183, indicating contamination of human origin, occurred only sporadically and in insignificant amounts. Furthermore, as indicated by BacR, the studied fountains showed no evidence of contamination by ruminant feces. Further work is suggested in order to establish threshold values for molecular Bacteroidales markers, which could in future replace the currently used criteria for fecal indicator bacteria.     

Beach boundary layer: a framework for addressing recreational water quality impairment at enclosed beaches

Nearshore waters in bays, harbors, and estuaries are frequently contaminated with human pathogens and fecal indicator bacteria. Tracking down and mitigating this contamination is complicated by the many point and nonpoint sources of fecal pollution that can degrade water quality along the shore. From a survey of the published literature, we propose a conceptual and mathematical framework, the "beach boundary layer model", for understanding and quantifying the relative impact of beach-side and bay-side sources of fecal pollution on nearshore water quality. In the model, bacterial concentration in ankle depth water C(ankle) [bacteria L(-3)] depends on the flux m' [bacteria L(-2) T(-1)] of fecal bacteria from beach-side sources (bather shedding, bird and dog feces, tidal washing of sediments, decaying vegetation, runoff from small drains, and shallow groundwater discharge), a cross-shore mass transfer velocity k [L T(-1)] that accounts for the physics of nearshore transport and mixing, and a background concentration C(bay) [bacteria L(-3)] attributable to bay-side sources of pollution that impact water quality over large regions (sewage outfalls, creeks and rivers): C(ankle) = m'/k + C(bay). We demonstrate the utility of the model for identifying risk factors and pollution sources likely to impact shoreline water quality, and evaluate the model's underlying assumptions using computational fluid dynamic simulations of flow, turbulence, and mass transport in a trapezoidal channel.     

Influence of climate change, tidal mixing, and watershed urbanization on historical water quality in Newport Bay, a saltwater wetland and tidal embayment in southern California

Historical coliform measurements (n = 67,269; 32 years) in Newport Bay, a regionally important saltwater wetland and tidal embayment in southern California, have been compiled and analyzed. Coliform concentrations in Newport Bay decrease along an inland-to-ocean gradient, consistent with the hypothesis that this tidal embayment attenuates fecal pollution from inland sources. Nearly 70% of the variability in the coliform record can be attributed to seasonal and interannual variability in local rainfall, implying that stormwater runoff from the surrounding watershed is a primary source of coliform in Newport Bay. The storm loading rate of coliform from the San Diego Creek watershed--the largest watershed draining into Newport Bay--appears to be unaffected by the dramatic shift away from agricultural land-use that occurred in the watershed over the study period. Further, the peak loading of coliform during storms is larger than can be reasonably attributed to sources of human sewage, suggesting that nonhuman fecal pollution and/or bacterial regrowth contribute to the coliform load. Summer time measurements of coliform exhibit interannual trends, but these trends are site specific, apparently due to within-Bay variability in land-use, inputs of dry-weather runoff, and tidal mixing rates. Overall, these results suggest that efforts to improve water quality in Newport Bay will likely have greater efficacy during dry weather summer periods. Water quality during winter storms, on the other hand, appears to be dominated by factors outside of local management control; namely, virtually unlimited nonhuman sources of coliform in the watershed and global climate patterns, such as the El Nino Southern Oscillation, that modulate rainfall and stormwater runoff in southern California.     

Use of fecal steroids to infer the sources of fecal indicator bacteria in the Lower Santa Ana River Watershed, California: sewage is unlikely a significant source

The Santa Ana River (SAR), CA and adjacent wetlands have been identified as potential sources of fecal indicator bacteria (FIB) to the surf zone at Huntington Beach, CA. A suite of fecal steroids, including coprostanol (COP), epicoprostanol (eCOP), cholesterol (CHOE), cholestanol (CHOA), alpha-cholestanone (aONE), beta-cholestanone (bONE), beta-sitosterol (bSIT), stigmasterol (STIG), stigmastanol (STAN), and campesterol (CAM), were used as chemical markers to examine whether sewage was a significant source of FIB within the lower Santa Ana River watershed. A total of 54 water samples were collected from three locations in the intertidal zone near the mouth of the Santa Ana River at different tidal stages. Steroid ratios in SAR samples were different from those found in raw and treated sewage from a local wastewater treatment plant or in nearby effluent plume and did not appear to be influenced by the sampling location, daily tides, and spring/neap tidal cycle. The characteristics of steroid ratios suggested a diagenetic ratherthan a biogenic source forthe COP content of the samples. The log-based concentrations of COP and FIB in the SAR samples were not significantly correlated, inconsistent with sewage being the source of FIB in the study area. In addition, multivariate statistical analysis showed that the concentrations of FIB were better correlated with bird fecal steroids than with the typical sewage sterols. The results implied that sewage was not a significant source of fecal steroids, and therefore perhaps FIB to the study area. Instead, birds may be one possible source of the intermittently high levels of FIB observed in the lower Santa Ana River watershed and the nearby surf zone.     

Microbial source tracking: state of the science

Although water quality of the Nation's lakes, rivers and streams has been monitored for many decades and especially since the passage of the Clean Water Act in 1972, many still do not meet the Act's goal of "fishable and swimmable". While waterways can be impaired in numerous ways, the protection from pathogenic microbe contamination is most important for waters used for human recreation, drinking water and aquaculture. Typically, monitoring methods used for detecting potential pathogenic microorganisms in environmental waters are based upon cultivation and enumeration of fecal indicator bacteria (i.e. fecal coliforms, E. coli, and fecal enterococci). Currently, there is increasing interest in the potential for molecular fingerprinting methods to be used not only for detection but also for identification of fecal contamination sources. Molecular methods have been applied to study the microbial ecology of environmental systems for years and are now being applied to help improve our waters by identifying problem sources and determining the effect of implemented remedial solutions. Management and remediation of water pollution would be more cost-effective if the correct sources could be identified. This review provides an outline of the main methods that either have been used or have been suggested for use in microbial source tracking and some of the limitations associated with those methods.     

Spatial and annual variability in concentrations and sources of Escherichia coli in multiple watersheds

Nonpoint source fecal contamination is a concern for drinking water supplies worldwide. In this study, 4812 E. coli isolates were classified to source. Results of this experiment show that the fecal coliform (FC) counts varied by year, month, and site, for each of the watersheds sampled. For both years, the lowest FC counts tended to be at the highest elevation sites followed by the drinking water intake sites at the lowest elevation. The highest FC counts tended to be at the mid-elevation sites on BX, Deer, and Duteau Creeks. The sources of E. coli varied significantly with stream for 2003 and 2004 (P < 0.001, df = 39), although the main sources of E. coli (avian, deer/elk, canine, rodent, bovine, and bear) tended to be similar between watersheds. The dominant sources of E. coli changed from 2003 (avian, deer/elk, and canine) to 2004 (avian, bovine, and rodent). It is important to look at the results of more than 1 year of source tracking data to get a better picture of the dominant sources within a watershed. Overall, wildlife was the largest contributor of E. coli to the watersheds in both 2003 (> 84%) and 2004 (> 73%).     

Swimmer risk of gastrointestinal illness from exposure to tropical coastal waters impacted by terrestrial dry-weather runoff

This study used molecular methods to measure concentrations of four enteric viruses (adenovirus, enterovirus, norovirus GI, and norovirus GII) and fecal source tracking markers (human, ruminant, and pig Bacteroidales) in land-based runoff from 22 tropical streams on O'ahu, Hawai'i. Each stream was sampled twice in the morning and afternoon during dry weather. Viruses and human Bacteroidales were widespread in the streams. Watershed septic tank densities were positively associated with higher occurrence of human Bacteroidales and norovirus. There were no associations between occurrence of viruses and fecal indicator concentrations. Virus concentrations and previously reported culturable Salmonella and Campylobacter were used as inputs to a quantitative microbial risk assessment (QMRA) model to estimate the risk of acquiring gastrointestinal (GI) illness from swimming in tropical marine waters adjacent to discharging streams. Monte Carlo methods were used to incorporate uncertainties in the dilution of stream discharge with seawater, swimmer ingestion volumes, pathogen concentrations, and dose-response parameters into the model. Median GI illness risk to swimmers from exposure to coastal waters adjacent to the 22 streams ranged from 0 to 21/1000. GI illness risks from viral exposures were generally orders of magnitude greater than bacterial exposures. Swimming adjacent to streams positive for norovirus or adenovirus resulted in the highest risks. The median risk adjacent to each stream was positively, significantly correlated to the concentration of Clostridium perfringens in the stream. Although a number of important assumptions were made to complete the QMRA, results suggest land-based runoff in the tropics as a potential source of GI illness risk, with pathogens coming from both human and nonhuman nonpoint sources including septic tanks.     

Scaling and management of fecal indicator bacteria in runoff from a coastal urban watershed in southern California

This paper describes a series of field studies aimed at identifying the spatial distribution and flow forcing of fecal indicator bacteria in dry and wet weather runoff from the Talbert watershed, a highly urbanized coastal watershed in southern California. Runoff from this watershed drains through tidal channels to a popular public beach, Huntington State Beach, which has experienced chronic surf zone water quality problems over the past several years. During dry weather, concentrations of fecal indicator bacteria are highest in inland urban runoff, intermediate in tidal channels harboring variable mixtures of urban runoff and ocean water, and lowest in ocean water at the base of the watershed. This inland-to-coastal gradient is consistent with the hypothesis that urban runoff from the watershed contributes to coastal pollution. On a year round basis, the vast majority (>99%) of fecal indicator bacteria loading occurs during storm events when runoff diversions, the management approach of choice, are not operating. During storms, the load of fecal indicator bacteria in runoff follows a power law of the form L approximately Qn, where L is the loading rate (in units of fecal indicator bacteria per time), Q is the volumetric flow rate (in units of volume per time), and the exponent n ranges from 1 to 1.5. This power law and the observed range of exponent values are consistent with the predictions of a mathematical model that assumes fecal indicator bacteria in storm runoff originate from the erosion of contaminated sediments in drainage channels or storm sewers. The theoretical analysis, which is based on a conventional model for the shear-induced erosion of particles from land and channel-bed surfaces, predicts that the magnitude of the exponent n reflects the geometry of the stormwater conveyance system from which the pollution derives. This raises the possibility that the scaling properties of pollutants in stormwater runoff (i.e., the value of n) may harbor information about the origin of nonpoint source pollution.     

Microbial Hazards in Irrigation Water: Standards,Norms, and Testing to Manage Use of Water in Fresh Produce Primary Production

Accessibility to abundant sources of high‐quality water is integral to the production of safe and wholesome fresh produce. However, access to safe water is becoming increasingly difficult in many parts of the world, and this can lead to the production of fresh produce contaminated with pathogenic microorganisms, resulting in increased risk of human disease. Water, an important raw material in the fresh produce chain, is used in considerable amounts in many operations, including irrigation and application of pesticides and fertilizers, but also as a transport medium and for cooling and washing in postharvest practices. In several reported outbreaks related to uncooked fruit and vegetable products, water has been identified as a likely source of the outbreak. The present study, initiated by the ILSI Europe Emerging Microbiological Issues Task Force in collaboration with 8 other ILSI branches and support of WHO/FAO, was undertaken to review the status of, and provide suggestions for, consideration by different stakeholders on water and sanitation and its impact on food safety and public health. A limited number of guidelines and regulations on water quality for agricultural production are available, and many of them are still heavily based on microbial standards and (debated) parameters such as fecal coliforms. Data gaps have been identified with regard to baseline studies of microbial pathogens in water sources in many regions, the need for agreement on methods and microbial parameters to be used in assessing water quality, the fate of pathogens in water, and their transfer and persistence on irrigated/processed produce.     

Evaluation of the nifH gene marker of Methanobrevibacter smithii for the detection of sewage pollution in environmental waters in Southeast Queensland, Australia

This study aimed at evaluating the host-specificity and -sensitivity of the nifH gene marker of Methanobrevibacter smithii by screening 272 fecal and wastewater samples from 11 animal species including humans in Southeast Queensland (SEQ), Australia. In addition, environmental water samples (n = 21) were collected during the dry and wet weather conditions and tested for the presence of the nifH marker along with other sewage-associated markers, namely, enterococci surface protein (esp) found in Enterococci faecium, Bacteroides HF183, adenoviruses (AVs), and polyomaviruses (PVs). The overall host-specificity of the nifH marker to differentiate between human and animal feces was 0.96 (maximum value of 1), while the overall sensitivity of this marker in human sourced feces and wastewater was 0.81 (maximum value of 1). Among the 21 environmental water samples tested, 2 (10%), 3 (14%), 12 (57%), 6 (29%), and 6 (29%) were positive for the nifH, esp, HF183, AVs and PVs markers, respectively. The prevalence of the nifH marker in environmental water samples, however, was low compared to other markers, suggesting that the use of this marker alone may not be sensitive enough to detect fecal pollution in environmental waters. The nifH marker, however, appears to be sewage-specific in SEQ, Australia, and therefore, it is recommended that this marker should be used as an additional marker in combination with the HF183 or viral markers such as AVs or PVs for accurate and sensitive detection of fecal pollution in SEQ waterways.     

Identifying pollutant sources in tidally mixed systems: case study of fecal indicator bacteria from marinas in Newport Bay, southern California

This study investigates the contribution of several marinas to fecal indicator bacteria impairment in Newport Bay, a regionally important tidal embayment in southern California. Three different fecal indicator bacteria groups were assayed, including total coliform, Escherichia coli, and enterococci bacteria, all measured using the IDEXX Colilert and Enterolert system. To document temporal variability in the fecal indicator bacteria signal, water column samples (n = 4132) were collected from two marinas over time scales ranging from hours to months. To document spatial variability of the fecal indicator bacteria signal, water column and sediment samples were collected from a number of sites (n = 11 to 36, depending on the study) in and around the two marinas, over spatial scales ranging from meters to kilometers. To identify the dominant temporal and spatial patterns in these data a statistical approach--Empirical Orthogonal Function analysis--was utilized. Finally, to clarify the transport pathways responsible for the observed temporal and spatial patterns, fecal indicator bacteria data were compared to simultaneous measurements of tidal flow, temperature, and salinity. The results of this field effort collectively implicate runoff--both dry weather runoff at sampling sites located near some storm drains and wet weather runoff at all sites--as a primary source of fecal indicator bacteria in the water column and subtidal sediments. The results and analysis presented here reinforce the growing body of evidence that management of fecal indicator bacteria impairment in the coastal waters of southern California will require developing long-term strategies for treating nonpoint sources of both dry weather and stormwater runoff.     

农业源头污染对我国农产品质量安全的影响

通过分析影响农业源头污染的主要因素--化肥污染、农药污染、畜禽粪便污染以及饲料污染等对我国农产品质量安全产生的不利影响,说明我国农业源头污染问题已不容忽视,发展绿色、可持续发展农业应重视预防以及治理源头污染问题。     

Tracking anthropogenic inputs using caffeine, indicator bacteria, and nutrients in rural freshwater and urban marine systems

Our objective was to evaluate the hypothesis that measurements of caffeine, nutrients, and indicator bacteria can distinguish human versus non-human sources of surface water contamination in contrasting environments. A second objective was to determine if natural sources of caffeine were significant in unpopulated areas. Caffeine was measured in an isolated wetland, and a native plant source was identified. In two rural watersheds in southwest Georgia (U.S.), caffeine was detected in tributary creeks immediately below wastewater discharge sites and within towns. However, caffeine was not found in river main streams. Thus, although natural caffeine sources exist, background levels in stream drainage networks of these rural watersheds remained below detection. The presence of caffeine and elevated nitrate in streams was associated with anthropogenic inputs and population centers, whereas bacterial indicators did not correlate to these chemical indicators and appeared to have non-human sources. In contrast, caffeine in an urban coastal lagoon was generally linked to fecal coliform abundance. We observed sporadic relationships between caffeine and other water quality indicators, possibly due to differential rates of degradation. Creeks and bayous flowing into the lagoon contained the greatest caffeine concentrations and highest amounts of bacteria, nitrate, and radon, which is an indicator of groundwater discharge.