Characterizing fecal contamination in stormwater runoff in coastal North Carolina, USA

Microbial contaminants in stormwater runoff have the potential to negatively impact public health. Stormwater runoff to coastal waters is increasing in amount and rate of discharge due to loss of vegetated landscape and increasing coastal development. However, the extent and nature of microbial contamination of stormwater runoff in North Carolina (NC) has not been previously characterized. The aim of this study was to measure a range of fecal indicator bacteria (FIB) and molecular markers at three coastal sites. E. coli and Enterococcus sp. were measured in addition to molecular markers including Bacteroides Human-Specific Marker (HS) and fecal Bacteroides spp. Levels of FIB in stormwater far exceeded recreational water quality guidelines, frequently by several orders of magnitude. High concentrations of fecal Bacteroides spp. and the presence of HS indicated the presence of human fecal contamination in the stormwater runoff, but only during specific storms. Examinations of levels of fecal contamination in stormwater over multiple seasons and a range of storm conditions will allow managers to consider appropriate design of effective mitigation strategies necessary to maintain and restore coastal water quality.     

Rapid QPCR-based assay for fecal Bacteroides spp. as a tool for assessing fecal contamination in recreational waters

Concentrations of fecal indicator bacteria (FIB; e.g. Escherichia coli, and Enterococcus sp.) can only be used in limited ways for determining the source of fecal contamination in recreational waters because they cannot distinguish human from non-human fecal contamination. Several Bacteroides spp. have been suggested as potential alternative indicators. We have developed a rapid, culture-independent method for quantifying fecal Bacteroides spp. using quantitative PCR (QPCR) targeting the 16S rRNA gene. The assay specifically targets and quantifies the most common human Bacteroides spp. The details of the method are presented, including analyses of a wide range of fecal samples from different organisms. Specificity and performance of the QPCR assay were also tested via a laboratory experiment where human sewage and gull guano were inoculated into a range of environmental water samples. Concentrations of fecal Bacteroides spp., total Enterococcus sp., Enterococcus faecium, Enterococcus faecalis, and Enterococcus casseliflavus were measured using QPCR, and total Enterococcus sp. and E. coli were quantified by membrane filtration (MF). Samples spiked with gull guano were highly concentrated with total Enterococcus sp., E. coli, E. faecalis, and E. casseliflavus, demonstrating that these indicators are prominent in animal feces. On the other hand, fecal Bacteroides spp. concentrations were high in samples containing sewage and were relatively low in samples spiked with gull guano. Sensitivity and specificity results suggest that the rapid fecal Bacteroides spp. QPCR assay may be a useful tool to effectively predict the presence and concentration of human-specific fecal pollution.     

Bacterial pathogens in Hawaiian coastal streams--associations with fecal indicators, land cover, and water quality

This work aimed to understand the distribution of five bacterial pathogens in O’ahu coastal streams and relate their presence to microbial indicator concentrations, land cover of the surrounding watersheds, and physical-chemical measures of stream water quality. Twenty-two streams were sampled four times (in December and March, before sunrise and at high noon) to capture seasonal and time of day variation. Salmonella, Campylobacter, Staphylococcus aureus, Vibrio vulnificus, and V. parahaemolyticus were widespread —12 of 22 O’ahu streams had all five pathogens. All stream waters also had detectable concentrations of four fecal indicators and total vibrio with log mean ± standard deviation densities of 2.2 ± 0.8 enterococci, 2.7 ± 0.7 Escherichia coli, 1.1 ± 0.7 Clostridium perfringens, 1.2 ± 0.8 F⁺ coliphages, and 3.6 ± 0.7 total vibrio per 100 ml. Bivariate associations between pathogens and indicators showed enterococci positively associated with the greatest number of bacterial pathogens. Higher concentrations of enterococci and higher incidence of Campylobacter were found in stream waters collected before sunrise, suggesting these organisms are sensitive to sunlight. Multivariate regression models of microbes as a function of land cover and physical-chemical water quality showed positive associations between Salmonella and agricultural and forested land covers, and between S. aureus and urban and agricultural land covers; these results suggested that sources specific to those land covers may contribute these pathogens to streams. Further, significant associations between some microbial targets and physical-chemical stream water quality (i.e., temperature, nutrients, turbidity) suggested that organism persistence may be affected by stream characteristics. Results implicate streams as a source of pathogens to coastal waters. Future work is recommended to determine infectious risks of recreational waterborne illness related to O’ahu stream exposures and to mitigate these risks through control of land-based runoff sources.     

Relationships between Bacteroides 16S rRNA genetic markers and presence of bacterial enteric pathogens and conventional fecal indicators

Occurrence and prevalence of different bacterial enteric pathogens as well as their relationships with conventional (total and fecal coliforms) and alternative fecal indicators (host-specific Bacteroides 16S rRNA genetic markers) were investigated for various water samples taken from different sites with different degrees of fecal contamination. The results showed that a wide range of bacterial pathogens could be detected in both municipal wastewater treatment plant samples and in surface water samples. Logistic regression analysis revealed that total and human-specific Bacteroides 16S rRNA genetic markers showed significant predictive values for the presence of Escheriachia coli O-157, Salmonella, heat-labile enterotoxin (LT) of enterotoxigenic E. coli (ETEC), and heat-stable enterotoxin for human (STh) of ETEC. The probability of occurrence of these pathogenic bacteria became significantly high when the concentrations of human-specific and total Bacteroides 16S rRNA genetic markers exceeded 10(3) and 10(4) copies/100 mL. In contrast, Clostridium perfringens was detected at high frequency regardless of sampling sites and levels of Bacteroides 16S rRNA genetic markers. No genes related to Shigella spp., Staphylococcus aureus and Vibrio cholerae were detected in any samples analyzed in this study. Conventional indicator microorganisms had low levels of correlation with the presence of pathogens as compared with the alternative fecal indicators. These results suggested that real-time PCR-based measurement of alternative Bacteroides 16S rRNA genetic markers was a rapid and sensitive tool to identify host-specific fecal pollution and probably associated bacterial pathogens. However, since one fecal indicator might not represent the relative abundance of all pathogenic bacteria, viruses and protozoa, combined application of alternative indicators with conventional ones could provide more comprehensive pictures of fecal contamination, its source and association with pathogenic microorganisms.     

Persistence of fecal indicator bacteria in Santa Monica Bay beach sediments

Monitoring the water quality of recreational beaches is only one step toward understanding microbial contamination -- the primary cause of beach closings. The surf zone sediment reservoir is typically overlooked and may also be important. This study involved monitoring the fecal indicator bacteria (FIB) levels in water and sediment at three ocean beaches (two exposed and one enclosed) during a storm event, conducting laboratory microcosm experiments with sediment from these beaches, and surveying sediment FIB levels at 13 beaches (some exposed and some enclosed). Peaks in Escherichia coli and enterococci concentrations in water and sediment coincided with storm activity at the two exposed beaches, while levels of both FIB were consistently high and irregular at the enclosed beach. Results from microcosm experiments showing similar, dramatic growth of FIB in both overlying water and sediment from all beaches, as well as results from the beach survey, support the hypothesis that the quiescent environment rather than sediment characteristics can explain the elevated sediment FIB levels observed at enclosed beaches. This work has implications for the predictive value of FIB measurements, and points to the importance of the sediment reservoir.     

Concentrations of host-specific and generic fecal markers measured by quantitative PCR in raw sewage and fresh animal feces

We measured the concentrations of four host-specific (human, dog, cow, and horse Bacteroidales), four generic fecal (16S total Bacteroidales and Escherichia coli, 23S Enterococcus and uidA E. coli,) and two universal bacterial (16S universal and rpoB universal) DNA targets by qPCR in raw sewage and pooled fecal samples from dogs, cows, horses, and Canada Geese. A spiking protocol using the non-fecal bacterium Pseudomonas syringae pph6 was developed to estimate the recovery of DNA from fecal and environmental samples. The measured fecal marker concentrations were used to calculate baseline ratios and variability of host-specific to generic indicators for each host type. The host-specific markers were found in high concentrations (8-9 log₁₀ copies/g dry wt.) in their respective hosts' samples, which were equal to or greater than the concentrations of generic E. coli and Enterococcus markers, lending support to the use of host-specific and generic Bacteroidales as sensitive indicators of fecal pollution. The host-specific markers formed a consistent percentage of total Bacteroidales in target host feces and raw sewage, with human-specific comprising 82%, dog-specific 6%, cow-specific 4% and horse-specific 2%. Based on this limited data set, the measurement of host-specific indicators by qPCR has several promising applications. These applications include determining the percentage of total Bacteroidales contributed by a specific host type, using the ratios of host-specific markers to E. coli or Enterococcus to estimate the contribution of each source to these regulated fecal indicator bacteria, and estimating the mass of feces from each host type in environmental samples.     

Characterization of fecal concentrations in human and other animal sources by physical,culture-based,and quantitative real-time PCR methods

The characteristics of fecal sources, and the ways in which they are measured, can profoundly influence the interpretation of which sources are contaminating a body of water. Although feces from various hosts are known to differ in mass and composition, it is not well understood how those differences compare across fecal sources and how differences depend on characterization methods. This study investigated how nine different fecal characterization methods provide different measures of fecal concentration in water, and how results varied across twelve different fecal pollution sources. Sources investigated included chicken, cow, deer, dog, goose, gull, horse, human, pig, pigeon, septage and sewage. A composite fecal slurry was prepared for each source by mixing feces from 6 to 22 individual samples with artificial freshwater. Fecal concentrations were estimated by physical (wet fecal mass added and total DNA mass extracted), culture-based (Escherichia coli and enterococci by membrane filtration and defined substrate), and quantitative real-time PCR (Bacteroidales, E. coli, and enterococci) characterization methods. The characteristics of each composite fecal slurry and the relationships between physical, culture-based and qPCR-based characteristics varied within and among different fecal sources. An in silico exercise was performed to assess how different characterization methods can impact identification of the dominant fecal pollution source in a mixed source sample. A comparison of simulated 10:90 mixtures based on enterococci by defined substrate predicted a source reversal in 27% of all possible combinations, while mixtures based on E. coli membrane filtration resulted in a reversal 29% of the time. This potential for disagreement in minor or dominant source identification based on different methods of measurement represents an important challenge for water quality managers and researchers.     

Water quality prediction of marine recreational beaches receiving watershed baseflow and stormwater runoff in southern California, USA

Beach advisories are issued to the public in California when the concentration of fecal indicator bacteria (FIB), including total coliform, fecal coliform (or Escherichia coli), and Enterococcus, exceed their recreational water health standards, or when the amount of a rainfall event is above the pre-determined threshold. However, it is not fully understood about how and to what degree stormwater runoff or baseflow exerts impacts on beach water quality. Furthermore, current laboratory methods used to determine the FIB levels take 18-96 h, which is too slow to keep pace with changes in FIB levels in water. Thus, a beach may not be posted when it is contaminated, and may be posted under advisory when bacterial levels have already decreased to within water quality standards. The study was designed to address the above critical issues. There were large temporal and spatial variations in FIB concentrations along two popular State Beaches in San Diego, CA, USA. The rainstorm-induced runoff from the watersheds exerts significant impacts on the marine recreational water quality of the beaches adjacent to lagoons during the first 24-48 h after a rain event. The large volume of stormwater runoff discharging to beaches caused high FIB concentrations in beach water not only at the lagoon outlet channel and the mixing zone, but also at the locations 90 m away from the channel northward or southward along the shoreline. The geomorphology of beach shoreline, distance from the outlet channel, wind strength, wind direction, tide height, wave height, rainfall, time lapse after a rainstorm, or channel flow rate played a role in affecting the distribution of FIB concentrations in beach water. Despite the great temporal and spatial variability of FIB concentrations along a shoreline, the artificial neural network-based models developed in this study are capable of successfully predicting FIB concentrations at different beaches, different locations, and different times under baseflow or rainstorm conditions. The models are based on readily measurable variables including temperature, conductivity, pH, turbidity, channel water flow, rainfall, and/or time lapse after a rainstorm. The established models will help fill the current gap between beach posting and actual water quality and make more meaningful and effective decisions on beach closures and advisories.     

Identifying human and livestock sources of fecal contamination in Kenya with host-specific Bacteroidales assays

Microbial source tracking to distinguish between human, livestock and wildlife fecal pollution using molecular techniques is a rapidly evolving approach in many developed countries, but has not previously been applied on the African continent. DNA extracts from cow, donkey, and human fecal specimens and raw domestic sewage samples collected in Kenya were tested against five existing quantitative PCR assays designed to detect universal (2), human-specific (2), and cow-specific (1) fecal Bacteroidales genetic markers. Water samples from the River Njoro in Kenya were evaluated using the five tested Bacteroidales markers and a multi-species assay for Cryptosporidium in a preliminary exploration of fecal pollution sources and health risks in this watershed. Diagnostic sensitivity on the validation set varied from 18 to 100% for the five assays while diagnostic specificity was 100%. Of the 2 universal assays, Total Bacteroidales [Dick, L.K, Field, K.G., 2004. Rapid estimation of numbers of fecal Bacteroidetes by use of a quantitative PCR assay for 16S rRNA genes. Appl. Environ. Microbiol. 70, 5695-5697] showed lower generic fecal diagnostic sensitivity, at 55%, than BacUni-UCD, at 100%, in detecting fecal markers on the 42-sample validation set. Human-specific assay HF183 demonstrated 65% sensitivity overall, and 80% on the human sewage samples, compared to 18% overall and 0% sewage for human-specific assay BacHum-UCD. Cow-specific assay BacCow-UCD had 94% sensitivity. Testing of 18 water samples indicates cows are a likely predominant source of fecal contamination in the Njoro Watershed (78% prevailing rate). Probabilistic assessment of human assay results indicates at most three of the river water samples contained human Bacteroidales. Cryptosporidium spp. markers were detected in samples from nine of the 12 sampling locations. Evidence suggesting widespread contamination by cow feces and Cryptosporidium in the Njoro watershed raises serious concerns for human and animal health.     

Assessment of sources of human pathogens and fecal contamination in a Florida freshwater lake

We investigated the potential for a variety of environmental reservoirs to harbor or contribute fecal indicator bacteria (FIB), DNA markers of human fecal contamination, and human pathogens to a freshwater lake. We hypothesized that submerged aquatic vegetation (SAV), sediments, and stormwater act as reservoirs and/or provide inputs of FIB and human pathogens to this inland water. Analysis included microbial source tracking (MST) markers of sewage contamination (Enterococcus faecium esp gene, human-associated Bacteroides HF183, and human polyomaviruses), pathogens (Salmonella, Cryptosporidium, Giardia, and enteric viruses), and FIB (fecal coliforms, Escherichia coli, and enterococci). Bayesian analysis was used to assess relationships among microbial and physicochemical variables. FIB in the water were correlated with concentrations in SAV and sediment. Furthermore, the correlation of antecedent rainfall and major rain events with FIB concentrations and detection of human markers and pathogens points toward multiple reservoirs for microbial contaminants in this system. Although pathogens and human-source markers were detected in 55% and 21% of samples, respectively, markers rarely coincided with pathogen detection. Bayesian analysis revealed that low concentrations (<45 CFU × 100 ml⁻¹) of fecal coliforms were associated with 93% probability that pathogens would not be detected; furthermore the Bayes net model showed associations between elevated temperature and rainfall with fecal coliform and enterococci concentrations, but not E. coli. These data indicate that many under-studied matrices (e.g. SAV, sediment, stormwater) are important reservoirs for FIB and potentially human pathogens and demonstrate the usefulness of Bayes net analysis for water quality assessment.     

Comparing the partitioning behavior of Giardia and Cryptosporidium with that of indicator organisms in stormwater runoff

Microbial association with particles can significantly affect the fate and transport characteristics of microbes in aquatic systems as particle-associated organisms will be less mobile in the environment than their free phase (i.e. unattached) counterparts. As such, similarities or dissimilarities in the partitioning behavior of indicator organisms and pathogens may have an impact on the suitability of a particular indicator to act as a surrogate for a pathogen. This research analyzed the partitioning behavior of two pathogens (Cryptosporidium, Giardia) and several common indicator organisms (fecal coliform, Escherichia coli, Enterococci, Clostridium perfringens spores, and coliphage) in natural waters under both dry and wet weather conditions. Samples were taken from several streams in two distinct sampling phases: (i) single grab samples; and (ii) intrastorm samples obtained throughout the duration of four storms. Partitioning behavior varied by microbial type, with 15-30% of bacterial indicators (fecal coliform, E. coli, and Enterococci) associated with settleable particles compared to 50% for C. perfringens spores. Both pathogens exhibited similar levels of particle association during dry weather (roughly 30%), with increased levels observed during wet weather events (Giardia to 60% and Cryptosporidium to 40%). The settling velocities of particle-associated microbes were also estimated, with those of the bacterial indicators (fecal coliform, E. coli, and Enterococci), as well as C. perfringens spores, being similar to that of the Giardia and Cryptosporidium, suggesting these organisms may exhibit similar transport behavior. With respect to intrastorm analysis, the highest microbial concentrations, in both particle-associated and free phase, occurred during the earlier stages of a storm. The total loadings of both indicators and pathogens were also estimated over the course of individual storms.     

Mortality rates of autochthonous and fecal bacteria in natural aquatic ecosystems

Bacterial mortality has been investigated in freshwater (River Seine) and in marine (North Sea) systems using a method based on following the disappearance of radioactivity from the DNA of assemblages of bacteria previously labeled with tritiated thymidine. Measurement of bacterial mortality of autochthonous and various types of fecal bacteria allowed direct comparisons between their respective first-order mortality rates. Mortality rates obtained for the different types of bacteria in the River Seine were, respectively, 7.9-33.9 x 10(-3)h(-1) for Escherichia coli, 12.2-29.2 x 10(-3)h(-1) for S. faecium and 7.0-18.3 x 10(-3)h(-1) for the autochthonous bacteria. In the Belgian coastal waters, these rates were 4.6-27.3 x 10(-3)h(-1) for E. coli, 6.0-22.0 x 10(-3)h(-1) for S. typhimurium, 10.0-18.9 x 10(-3)h(-1) for S. faecium and 1.0-13.9 x 10(-3)h(-1) for autochthonous bacteria. In both environments, the overall mortality rates of autochthonous and the different fecal bacteria were in the same order of magnitude and overall mortality rates of E. coli were on average about twice as high for autochthonous bacteria. Grazing by protozooplankton was the dominant process of mortality for fecal and autochthonous bacteria in both environments. Except in a few situations, grazing by protozooplankton was responsible for more than 90% of the overall mortality rate of fecal and autochthonous bacteria in the river and in the coastal area.     

Sources and growth dynamics of fecal indicator bacteria in a coastal wetland system and potential impacts to adjacent waters

Coastal wetlands are receiving increased attention as a putative source of fecal indicator bacteria (FIB) in Southern California coastal waters. We examined temporal trends of water and sediment-associated FIB after rain events along with spatial sediment characteristics at two sites within the Santa Ana River wetlands and made comparisons to FIB levels observed in adjacent surf zone waters. During the first two rain events, total coliforms (TC), Escherichia coli (EC) and enterococci (ENT) in wetland water and sediment samples peaked either on the same day or within several days of the rain event, while the third resulted in elevated wetlands sediment TC levels only. TC in adjacent coastal waters consistently peaked on the same day as the rain event and decreased quickly thereafter (within 1 day). The TC/EC ratios of surf zone samples consistently fell below 10, indicating an increased probability of human fecal contamination whereas wetland TC/EC ratios were higher, averaging approximately 60 and 14 at each site. These results suggest sediment-associated FIB populations may be distinct from those found in the water samples, or at least have internal dynamics independent of water-borne populations. Increases in sediment-associated FIB may be due to in situ population growth and/or increased survival due to changes in environmental parameters (salinity, moisture and nutrient input) resulting from the rain events. Spatial differences in between the two sites may be due to sediment differences such as organic content and finer grain size and/or discrete sources of FIB.     

Survival dynamics of fecal bacteria in ponds in agricultural watersheds of the Piedmont and Coastal Plain of Georgia

Animal agriculture in watersheds produces manure bacteria that may contaminate surface waters and put public health at risk. We measured fecal indicator bacteria (commensal Escherichia coli and fecal enterococci) and manure pathogens (Salmonella and E. coli 0157:H7), and physical-chemical parameters in pond inflow, within pond, pond outflow, and pond sediments in three ponds in agricultural watersheds. Bishop Pond with perennial inflow and outflow is located in the Piedmont, and Ponds A and C with ephemeral inflow and outflow in the Coastal Plain of Georgia. Bromide and chloride tracer experiments at Bishop Pond reflected a residence time much greater than that estimated by two models, and indicated that complete mixing within Bishop Pond was never obtained. The long residence time meant that fecal bacteria were exposed to solar UV-radiation and microbial predation. At Bishop Pond outflow concentrations of fecal indicator bacteria were significantly less than inflow concentrations; such was not observed at Ponds A and C. Both Salmonella and E. coli 0157:H7 were measured when concomitant concentrations of commensal E. coli were below the criterion for surface water impairment indicating problems with the effectiveness of indicator organisms. Bishop Pond improved down stream water quality; whereas, Ponds A and C with ephemeral inflow and outflow and possibly greater nutrient concentrations within the two ponds appeared to be less effective in improving down stream water quality.     

Impact of urbanization and agriculture on the occurrence of bacterial pathogens and stx genes in coastal waterbodies of central California

Fecal pollution enters coastal waters through multiple routes, many of which originate from land-based activities. Runoff from pervious and impervious land surfaces transports pollutants from land to sea and can cause impairment of coastal ocean waters. To understand how land use practices and water characteristics influence concentrations of fecal indicator bacteria (FIB) and pathogens in natural waters, fourteen coastal streams, rivers, and tidal lagoons, surrounded by variable land use and animal densities, were sampled every six weeks over two years (2008 & 2009). Fecal indicator bacteria (FIB; Escherichia coli and Enterococci) and Salmonella concentrations, the occurrence of Bacteroidales human, ruminant, and pig-specific fecal markers, E. coli O157:H7, and Shiga toxin (stx) genes present in E. coli, were measured. In addition, environmental and climatic variables (e.g., temperature, salinity, rainfall), as well as human and livestock population densities and land cover were quantified. Concentrations of FIB and Salmonella were correlated with each other, but the occurrence of host-specific Bacteroidales markers did not correlate with FIB or pathogens. FIB and Salmonella concentrations, as well as the occurrence of E. coli harboring stx genes, were positively associated with the fraction of the surrounding subwatershed that was urban, while the occurrence of E. coli O157:H7 was positively associated with the agricultural fraction. FIB and Salmonella concentrations were negatively correlated to salinity and temperature, and positively correlated to rainfall. Areal loading rates of FIB, Salmonella and E. coli O157:H7 to the coastal ocean were calculated for stream and river sites and varied with land cover, salinity, temperature, and rainfall. Results suggest that FIB and pathogen concentrations are influenced, in part, by their flux from the land, which is exacerbated during rainfall; once waterborne, bacterial persistence is affected by water temperature and salinity.     

Fecal indicator bacteria and Salmonella in ponds managed as bird habitat, San Francisco Bay, California, USA

Throughout the world, coastal resource managers are encouraging the restoration of previously modified coastal habitats back into wetlands and managed ponds for their ecosystem value. Because many coastal wetlands are adjacent to urban centers and waters used for human recreation, it is important to understand how wildlife can affect water quality. We measured fecal indicator bacteria (FIB) concentrations, presence/absence of Salmonella, bird abundance, and physico-chemical parameters in two coastal, managed ponds and adjacent sloughs for 4 weeks during the summer and winter in 2006. We characterized the microbial water quality in these waters relative to state water-quality standards and examined the relationship between FIB, bird abundance, and physico-chemical parameters. A box model approach was utilized to determine the net source or sink of FIB in the ponds during the study periods. FIB concentrations often exceeded state standards, particularly in the summer, and microbial water quality in the sloughs was generally lower than in ponds during both seasons. Specifically, the inflow of water from the sloughs to the ponds during the summer, more so than waterfowl use, appeared to increase the FIB concentrations in the ponds. The box model results suggested that the ponds served as net wetland sources and sinks for FIB, and high bird abundances in the winter likely contributed to net winter source terms for two of the three FIB in both ponds. Eight serovars of the human pathogen Salmonella were isolated from slough and pond waters, although the source of the pathogen to these wetlands was not identified. Thus, it appeared that factors other than bird abundance were most important in modulating FIB concentrations in these ponds.     

Human and bovine adenoviruses for the detection of source-specific fecal pollution in coastal waters in Australia

In this study, the host-specificity and -sensitivity of human- and bovine-specific adenoviruses (HS-AVs and BS-AVs) were evaluated by testing wastewater/fecal samples from various animal species in Southeast, Queensland, Australia. The overall specificity and sensitivity of the HS-AVs marker were 1.0 and 0.78, respectively. These figures for the BS-AVs were 1.0 and 0.73, respectively. Twenty environmental water samples were collected during wet conditions and 20 samples were colleted during dry conditions from the Maroochy Coastal River and tested for the presence of fecal indicator bacteria (FIB), host-specific viral markers, zoonotic bacterial and protozoan pathogens using PCR/qPCR. The concentrations of FIB in water samples collected after wet conditions were generally higher compared to dry conditions. HS-AVs was detected in 20% water samples collected during wet conditions and whereas BS-AVs was detected in both wet (i.e., 10%) and dry (i.e., 10%) conditions. Both Campylobacter jejuni mapA and Salmonella invA genes detected in 10% samples collected during dry conditions. The concentrations of Salmonella invA ranged between 3.5 × 10² and 4.3 × 10² genomic copies per 500 ml of water Giardia lamblia β-giardin gene was detected only in one sample (5%) collected during the dry conditions. Weak or significant correlations were observed between FIB with viral markers and zoonotic pathogens. However, during dry conditions, no significant correlations were observed between FIB concentrations with viral markers and zoonotic pathogens. The prevalence of HS-AVs in samples collected from the study river suggests that the quality of water is affected by human fecal pollution and as well as bovine fecal pollution. The results suggest that HS-AVs and BS-AVs detection using PCR could be a useful tool for the identification of human sourced fecal pollution in coastal waters.     

Application of a rapid method for identifying fecal pollution sources in a multi-use estuary

We demonstrate the application of a new PCR assay to detect and differentiate human and ruminant sources of fecal pollution in natural water samples. We tested samples collected from Tillamook Bay, Oregon, which has a long history of fecal pollution levels that exceed acceptable standards. The most likely sources are from dairy operations and ineffective sewage treatment. Using a suite of three PCR primer pairs specific for human or ruminant bacterial 16S ribosomal DNA markers, we detected at least one marker in 17 of 22 samples. In general, host-specific fecal markers were detected in areas that are heavily impacted by anthropogenic activities. Nine out of 11 sites classified as either urban or near a sewage point source were positive for the human marker while only five of these same sites were positive for ruminant markers. Conversely, 12 out of 21 sites classified as rural or agricultural use were positive for ruminant markers, while only six of these sites were positive for human pollution. This suite of host-specific genetic markers holds promise for identifying non-point source fecal pollution in coastal waters.     

The application of a recently isolated strain of Bacteroides (GB-124) to identify human sources of faecal pollution in a temperate river catchment

Recent work has suggested that bacteriophages infecting Bacteroides are a potential tool for faecal source tracking, but that different host strains may be needed for different geographic areas. This study used a recently identified strain of Bacteroides (GB-124) to detect human sources of faecal pollution in a river catchment in southeast England (UK). A total of 306 river water, municipal wastewater and animal samples were obtained over a 16-month period. Bacteriophages capable of infecting GB-124 were present in all municipal wastewaters but were not detected in faecal samples from animals, and were detected at significantly lower levels (P< 0.001) in river waters directly downstream of a dairy farm. This last observation was despite the presence of high levels of faecal indicator bacteria at this site. The study suggests that GB-124 appears to be specific to human faeces. As such it may represent an effective and low-cost method of faecal source identification.