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.     

Detection of the human specific Bacteroides genetic marker provides evidence of widespread sewage contamination of stormwater in the urban environment

Human sewage contamination of surface waters is a major human health concern. We found urban stormwater systems that collect and convey runoff from impervious surfaces act as a conduit for sewage originating from breeches in sanitary sewer infrastructure. A total of 828 samples at 45 stormwater outfalls were collected over a four-year period and assessed by culture based methods, PCR, and quantitative PCR (qPCR) to test for traditional and alternative indicators of fecal pollution. All outfalls had the HF183 (human) Bacteroides genetic marker detected in at least one sample, suggesting sewage contamination is nearly ubiquitous in the urban environment. However, most outfalls were intermittently positive, ranging from detection in 11%-100% of the samples. Positive results did not correlate with seasonality, rainfall amounts, or days since previous rainfall. Approximately two-thirds of the outfalls had high (>5000 copy number, i.e. CN, per 100 ml) or moderate levels (1000-5000 CN per 100 ml) of the human Bacteroides genetic marker. Escherichia coli (E. coli) and enterococci levels did not correlate to human Bacteroides. A total of 66% of all outfall samples had standard fecal indicator levels above 10,000 CFU per 100 ml. A tiered assessment using this benchmark to identify high priority sites would have failed to flag 35% of the samples that had evidence of sewage contamination. In addition, high fecal indicators would have flagged 33% of samples as priority that had low or no evidence of sewage. Enteric virus levels in one outfall with high levels of the human Bacteroides genetic marker were similar to untreated wastewater, which illustrates stormwater can serve as a pathway for pathogen contamination. The major source of fecal pollution at four of five river sites that receive stormwater discharge appeared to be from sewage sources rather than non-human sources based on the ratios of human Bacteroides to total Bacteroides spp. This study shows the feasibility and benefits of employing molecular methods to test for alternative indicators of fecal pollution to identify sewage sources and potential health risks and for prioritization of remediation efforts.     

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.     

Prevalence of human pathogens and indicators in stormwater runoff in Brisbane,Australia

Elevated numbers of enteric pathogens in the receiving waters following a storm event can be a serious public health concern. The purpose of this study was to conduct a preliminary investigation into the presence of human pathogens of concern in urban stormwater runoff. The involvement of a human sewage as a potential source of contamination was also investigated by using microbial source tracking methods. Water samples (20 L) were collected after storm events and during the dry weather from six sites in Brisbane, Australia. Collected samples were analyzed for fecal indicator bacteria (FIB), and then concentrated using hollow fiber ultrafiltration followed by molecular detection of selected enteric pathogens. The levels of FIB were found to frequently exceed the upper limit of Australian guidelines for managing risks in recreational water, during the dry periods and by further several orders of magnitude in the stormwater runoff. Enterococcus spp. numbers as high as 3 × 10⁴ 100 mL^?1 were detected in the stormwater runoff at the Fitzgibbon site. Human adenovirus and polyomavirus were frequently detected from all six sampling sites during wet and dry weather conditions suggesting their wide spread presence in the urban aquatic environments. Campylobacter jejuni, Campylobacter coli and Salmonella enterica were also detected during both dry and wet weather conditions. Presence of human-specific HF183 Bacteroides marker in most of the samples tested suggests ubiquitous sewage contamination in the urban environment. Since stormwater runoff routinely contains high numbers of FIB and other enteric pathogens, some degree of treatment of captured stormwater would be required if it were to be used for non-potable purposes.     

Loading of fecal indicator bacteria in North Carolina tidal creek headwaters: Hydrographic patterns and terrestrial runoff relationships

In the New River Estuary (NRE) in eastern North Carolina (NC), fecal indicator bacteria (FIB) levels exceed water quality standards, leading to closure of estuarine waters for shellfishing and classification of parts of the estuary as “impaired” per the Clean Water Act section 303(d) list. As a means to investigate fecal contamination and loading of FIB to the NRE, a continuous automated sampler (ISCO) outfitted with flow modules and water quality probes was placed in four first-order tidal creek headwaters. Total storm discharge and bacterial load for Escherichia coli (EC) and Enterococcus spp. (ENT) were calculated using graphical volumetric flow calculations and interpolation of FIB measurements over each storm’s duration for 10 storms. Mean total load of 10⁹- 10¹² EC and ENT cells (MPN) occurred over the course of each storm. Total storm loading, averaged across all storms, was as much as 30 and 37 times greater than equivalent duration of baseflow loading for EC and ENT, respectively. Within the first 30% of creek storm volume for all storms and all creeks combined, a mean cumulative load of only 37% and 44% of the total EC and ENT cells, respectively, was discharged, indicating these creeks are not demonstrating a ‘first flush’ scenario for FIB. The median storm Event Mean Concentrations (EMCs) were 6.37 × 10² and 2.03 × 10² MPN/100 mL, for EC and ENT, respectively, compared with median baseflow concentrations of 1.48 × 10² and 4.84 × 10¹ for EC and ENT, respectively, and were significantly different between base and storm flow events. FIB was correlated with TSS (weak), flow rate (strong), and different stages (base, rising, peak, and falling) of the hydrograph (strong). Pollutographs indicate large intra-storm variability of FIB, and the need for more intensive sampling throughout a storm in order to attain accurate FIB contaminant estimates. Instream sediment concentrations ranged from 5 to 478 (MPN/g) and 13 to 776 (MPN/g) for EC and ENT, respectively, indicating sediment as a source, but a minor reservoir. This overall approach for calculating loading in headwater tidal creeks is detailed. Accurate loading characterization of FIB during storms and dry weather conditions, and understanding intra-storm FIB concentrations, is imperative for understanding patterns of water quality impairment, establishing management planning, and developing appropriate mitigation strategies.     

Addressing uncertainty in fecal indicator bacteria dark inactivation rates

Assessing the potential threat of fecal contamination in surface water often depends on model forecasts which assume that fecal indicator bacteria (FIB, a proxy for the concentration of pathogens found in fecal contamination from warm-blooded animals) are lost or removed from the water column at a certain rate (often referred to as an “inactivation” rate). In efforts to reduce human health risks in these water bodies, regulators enforce limits on easily-measured FIB concentrations, commonly reported as most probable number (MPN) and colony forming unit (CFU) values. Accurate assessment of the potential threat of fecal contamination, therefore, depends on propagating uncertainty surrounding “true” FIB concentrations into MPN and CFU values, inactivation rates, model forecasts, and management decisions. Here, we explore how empirical relationships between FIB inactivation rates and extrinsic factors might vary depending on how uncertainty in MPN values is expressed. Using water samples collected from the Neuse River Estuary (NRE) in eastern North Carolina, we compare Escherichia coli (EC) and Enterococcus (ENT) dark inactivation rates derived from two statistical models of first-order loss; a conventional model employing ordinary least-squares (OLS) regression with MPN values, and a novel Bayesian model utilizing the pattern of positive wells in an IDEXX Quanti-Tray^®/2000 test. While our results suggest that EC dark inactivation rates tend to decrease as initial EC concentrations decrease and that ENT dark inactivation rates are relatively consistent across different ENT concentrations, we find these relationships depend upon model selection and model calibration procedures. We also find that our proposed Bayesian model provides a more defensible approach to quantifying uncertainty in microbiological assessments of water quality than the conventional MPN-based model, and that our proposed model represents a new strategy for developing robust relationships between environmental factors and FIB inactivation rates, and for reducing uncertainty in water resource management decisions.     

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.     

Meteorological effects on the levels of fecal indicator bacteria in an urban stream: A modeling approach

Gwangju Creek (GJC) in Korea, which drains a highly urbanized watershed, has suffered from substantial fecal contamination, thereby limiting the beneficial use of the water in addition to threatening public health. In this study, to quantitatively estimate the sinks and sources of fecal indicator bacteria (FIB) in GJC under varying meteorological conditions, two FIB (i.e., Escherichia coli and enterococci bacteria) were monitored hourly for 24 h periods during both wet and dry weather conditions at four sites along GJC, and the collected data was subsequently used to develop a spatiotemporal FIB prediction model. The monitoring data revealed that storm washoff and irradiational die-off by sunlight are the two key processes controlling FIB populations in wet and dry weather, respectively. FIB populations significantly increased during precipitation, with greater concentrations occurring at higher rainfall intensity. During dry weather, FIB populations decreased in the presence of sunlight in daytime but quickly recovered at nighttime due to continuous point-source inputs. In this way, the contributions of the key processes (i.e., irradiational die-off by sunlight, settling, storm washoff, and resuspension) to the FIB levels in GJC under different meteorological conditions were quantitatively estimated using the developed model. The modeling results showed that the die-off by sunlight is the major sink of FIB during the daytime in dry weather with a minor contribution from the settling process. During wet weather, storm washoff and resuspension are equally important processes that are responsible for the substantial increase of FIB populations.     

Evaluation of host-specific Bacteroidales 16S rRNA gene markers as a complementary tool for detecting fecal pollution in a prairie watershed

Our ability to identify and eliminate fecal contamination of water, now and in the future, is essential to reduce incidences of waterborne disease. Bacterial source tracking is a recently developed approach for identifying sources of fecal pollution. PCR primers designed by Bernhard and Field [Bernhard, A.E., Field, K.G., 2000a. A PCR assay to discriminate human and ruminant feces on the basis of host differences in Bacteroides-Prevotella genes encoding 16S rRNA. Appl. Environ. Microbiol. 66(10), 4571-4574] and Dick et al. [Dick, L.K., Bernhard, A.E., Brodeur, T.J., Santo Domingo, J.W., Simpson, J.M., Walters, S.P., Field, K.G., 2005. Host distributions of uncultivated fecal Bacteroidales bacteria reveal genetic markers for fecal source identification. Appl. Environ. Microbiol. 71(6), 3184-3191] for the detection of human (HF183), pig (PF163) and ruminant (CF128) specific Bacteroidales 16s rRNA genetic markers were tested for their suitability in detecting fecal pollution in Saskatchewan, Canada. The sensitivity and specificity of these primers were assessed by testing eight raw human sewage samples and 265 feces from 12 different species in Saskatchewan. The specificity of each primer set was ≥94%. The accuracy of HF183 and PF163 to distinguish between the different species was 100%, whereas CF128 cross-reacted with 22% of the pig feces. Occurrence of the host-specific Bacteroidales markers and the conventional indicator Escherichia coli in relation to several enteropathogens was investigated in 70 water samples collected from different sites along the Qu'Appelle River (Saskatchewan, Canada). Human and ruminant fecal markers were identified in 41 and 14% of the water samples, respectively, whereas the pig marker was never detected in the river water. The largest concentrations in E. coli counts were concomitant to the simultaneous detection of HF183 and CF128. Thermotolerant Campylobacter spp., Salmonella spp. and Shiga toxin genes (stx1 and stx2)-positive E. coli (STEC) were detected in 6, 7 and 63% of the water samples, respectively. However, none of the stx positive water samples were positive for the E. coli O157:H7 gene marker (uidA). Odds ratios analysis suggests that CF128 may be predictive for the presence of Salmonella spp. in the river investigated. None of the fecal indicators were able to confidently predict the presence of thermotolerant Campylobacter spp. and STEC.     

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.     

Semi-quantitative evaluation of fecal contamination potential by human and ruminant sources using multiple lines of evidence

Protocols for microbial source tracking of fecal contamination generally are able to identify when a source of contamination is present, but thus far have been unable to evaluate what portion of fecal-indicator bacteria (FIB) came from various sources. A mathematical approach to estimate relative amounts of FIB, such as Escherichia coli, from various sources based on the concentration and distribution of microbial source tracking markers in feces was developed. The approach was tested using dilute fecal suspensions, then applied as part of an analytical suite to a contaminated headwater stream in the Rocky Mountains (Upper Fountain Creek, Colorado). In one single-source fecal suspension, a source that was not present could not be excluded because of incomplete marker specificity; however, human and ruminant sources were detected whenever they were present. In the mixed-feces suspension (pet and human), the minority contributor (human) was detected at a concentration low enough to preclude human contamination as the dominant source of E. coli to the sample. Without the semi-quantitative approach described, simple detects of human-associated marker in stream samples would have provided inaccurate evidence that human contamination was a major source of E. coli to the stream. In samples from Upper Fountain Creek the pattern of E. coli, general and host-associated microbial source tracking markers, nutrients, and wastewater-associated chemical detections—augmented with local observations and land-use patterns—indicated that, contrary to expectations, birds rather than humans or ruminants were the predominant source of fecal contamination to Upper Fountain Creek. This new approach to E. coli allocation, validated by a controlled study and tested by application in a relatively simple setting, represents a widely applicable step forward in the field of microbial source tracking of fecal contamination.     

Enterococcus and Escherichia coli fecal source apportionment with microbial source tracking genetic markers – Is it feasible?

Fecal pollution is measured in surface waters using culture-based measurements of enterococci and Escherichia coli bacteria. Source apportionment of these two fecal indicator bacteria is an urgent need for prioritizing remediation efforts and quantifying health risks associated with source-specific pathogens. There are a number of quantitative real-time PCR (QPCR) assays that estimate concentrations of source-associated genetic markers; however, their concentrations are not necessarily amenable to source apportionment because the markers may differ in prevalence across sources. Here we mathematically derive and test, under ideal conditions, a method that utilizes the ratios of fecal source-associated genetic markers and culture and molecular measurements of general fecal indicators to apportion enterococci and E. coli. The source contribution is approximately equal to the ratio of the source-associated and the general fecal indicator concentrations in a water sample divided by their ratio in the source material, so long as cross-reactivity is negligible. We illustrate the utility of the ratio method using samples consisting of mixtures of various fecal pollution sources. The results from the ratio method correlated well with the actual source apportionment in artificial samples. However, aging of contamination can confound source allocation predictions. In particular, culturable enterococci and E. coli, the organisms presently regulated in the United States and much of the world, decay at different rates compared to source-associated markers and as a result cannot be apportioned using this method. However, limited data suggest a similar decay rate between source-associated and QPCR-measured Enterococcus and E. coli genetic markers, indicating that apportionment may be possible for these organisms; however further work is needed to confirm.     

Isolation and characterization of Bacteroides host strain HB-73 used to detect sewage specific phages in Hawaii

Previous studies have shown that Escherichia coli and enterococci are unreliable indicators of fecal contamination in Hawaii because of their ability to multiply in environmental soils. In this study, the method of detecting Bacteroides phages as specific markers of sewage contamination in Hawaii’s recreational waters was evaluated because these sewage specific phages cannot multiply under environmental conditions. Bacteroides hosts (GB-124, GA-17), were recovered from sewage samples in Europe and were reported to be effective in detecting phages from sewage samples obtained in certain geographical areas. However, GB-124 and GA-17 hosts were ineffective in detecting phages from sewage samples obtained in Hawaii. Bacteroides host HB-73 was isolated from a sewage sample in Hawaii, confirmed as a Bacteroides sp. and shown to recover phages from multiple sources of sewage produced in Hawaii at high concentrations (5.2-7.3 × 10⁵ PFU/100 mL). These Bacteroides phages were considered as potential markers of sewage because they also survived for three days in fresh stream water and two days in marine water. Water samples from Hawaii’s coastal swimming beaches and harbors, which were known to be contaminated with discharges from streams, were shown to contain moderate (20-187 CFU/100 mL) to elevated (173-816 CFU/100 mL) concentrations of enterococci. These same samples contained undetectable levels (<10 PFU/100 mL) of F+ coliphage and Bacteroides phages and provided evidence to suggest that these enterococci may not necessarily be associated with the presence of raw sewage. These results support previous conclusions that discharges from streams are the major sources of enterococci in coastal waters of Hawaii and the most likely source of these enterococci is from environmental soil rather than from sewage.     

Performance of viruses and bacteriophages for fecal source determination in a multi-laboratory,comparative study

An inter-laboratory study of the accuracy of microbial source tracking (MST) methods was conducted using challenge fecal and sewage samples that were spiked into artificial freshwater and provided as unknowns (blind test samples) to the laboratories. The results of the Source Identification Protocol Project (SIPP) are presented in a series of papers that cover 41 MST methods. This contribution details the results of the virus and bacteriophage methods targeting human fecal or sewage contamination. Human viruses used as source identifiers included adenoviruses (HAdV), enteroviruses (EV), norovirus Groups I and II (NoVI and NoVII), and polyomaviruses (HPyVs). Bacteriophages were also employed, including somatic coliphages and F-specific RNA bacteriophages (FRNAPH) as general indicators of fecal contamination. Bacteriophage methods targeting human fecal sources included genotyping of FRNAPH isolates and plaque formation on bacterial hosts Enterococcus faecium MB-55, Bacteroides HB-73 and Bacteroides GB-124. The use of small sample volumes (≤50 ml) resulted in relatively insensitive theoretical limits of detection (10–50 gene copies or plaques × 50 ml⁻¹) which, coupled with low virus concentrations in samples, resulted in high false-negative rates, low sensitivity, and low negative predictive values. On the other hand, the specificity of the human virus methods was generally close to 100% and positive predictive values were ∼40–70% with the exception of NoVs, which were not detected. The bacteriophage methods were generally much less specific toward human sewage than virus methods, although FRNAPH II genotyping was relatively successful, with 18% sensitivity and 85% specificity. While the specificity of the human virus methods engenders great confidence in a positive result, better concentration methods and larger sample volumes must be utilized for greater accuracy of negative results, i.e. the prediction that a human contamination source is absent.     

Impact of population and latrines on fecal contamination of ponds in rural Bangladesh

A majority of households in Bangladesh rely on pond water for hygiene. Exposure to pond water fecal contamination could therefore still contribute to diarrheal disease despite the installation of numerous tubewells for drinking. The objectives of this study are to determine the predominant sources (human or livestock) of fecal pollution in ponds and examine the association between local population, latrine density, latrine quality and concentrations of fecal bacteria and pathogens in pond water. Forty-three ponds were analyzed for E. coli using culture-based methods and E. coli, Bacteroidales and adenovirus using quantitative PCR. Population and sanitation spatial data were collected and measured against pond fecal contamination. Humans were the dominant source of fecal contamination in 79% of the ponds according to Bacteroidales measurements. Ponds directly receiving latrine effluent had the highest concentrations of fecal indicator bacteria (up to 10⁶ Most Probable Number (MPN) of culturable E. coli per 100 mL). Concentrations of fecal indicator bacteria correlated with population surveyed within a distance of 30-70 m (p < 0.05) and total latrines surveyed within 50-70 m (p < 0.05). Unsanitary latrines (visible effluent or open pits) within the pond drainage basin were also significantly correlated to fecal indicator concentrations (p < 0.05). Water in the vast majority of the surveyed ponds contained unsafe levels of fecal contamination attributable primarily to unsanitary latrines, and to lesser extent, to sanitary latrines and cattle. Since the majority of fecal pollution is derived from human waste, continued use of pond water could help explain the persistence of diarrheal disease in rural South Asia.     

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.     

Efficacy of monitoring and empirical predictive modeling at improving public health protection at Chicago beaches

Efforts to improve public health protection in recreational swimming waters have focused on obtaining real-time estimates of water quality. Current monitoring techniques rely on the time-intensive culturing of fecal indicator bacteria (FIB) from water samples, but rapidly changing FIB concentrations result in management errors that lead to the public being exposed to high FIB concentrations (type II error) or beaches being closed despite acceptable water quality (type I error). Empirical predictive models may provide a rapid solution, but their effectiveness at improving health protection has not been adequately assessed. We sought to determine if emerging monitoring approaches could effectively reduce risk of illness exposure by minimizing management errors. We examined four monitoring approaches (inactive, current protocol, a single predictive model for all beaches, and individual models for each beach) with increasing refinement at 14 Chicago beaches using historical monitoring and hydrometeorological data and compared management outcomes using different standards for decision-making. Predictability (R²) of FIB concentration improved with model refinement at all beaches but one. Predictive models did not always reduce the number of management errors and therefore the overall illness burden. Use of a Chicago-specific single-sample standard—rather than the default 235 E. coli CFU/100 ml widely used—together with predictive modeling resulted in the greatest number of open beach days without any increase in public health risk. These results emphasize that emerging monitoring approaches such as empirical models are not equally applicable at all beaches, and combining monitoring approaches may expand beach access.     

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.     

Quantitative real-time PCR analysis of total and propidium monoazide-resistant fecal indicator bacteria in wastewater

A real-time quantitative PCR (qPCR) method and a modification of this method incorporating pretreatment of samples with propidium monoazide (PMA) were evaluated for respective analyses of total and presumptively viable Enterococcus and Bacteroidales fecal indicator bacteria. These methods were used in the analyses of wastewater samples to investigate their feasibility as alternatives to current fecal indicator bacteria culture methods for predicting the efficiency of viral pathogen removal by standard treatment processes. PMA treatment was effective in preventing qPCR detection of target sequences from non-viable cells. Concentrates of small volume, secondary-treated wastewater samples, collected from a publicly owned treatment works (POTW) under normal operating conditions, had little influence on this effectiveness. Higher levels of total suspended solids, such as those associated with normal primary treatment and all treatment stages during storm flow events, appeared to interfere with PMA effectiveness under the sample preparation conditions employed. During normal operating conditions at three different POTWs, greater reductions were observed in PMA-qPCR detectable target sequences of both Enterococcus and Bacteroidales than in total qPCR detectable sequences. These reductions were not as great as those observed for cultivable fecal indicator bacteria in response to wastewater disinfection. Reductions of PMA-qPCR as well as total qPCR detectable target sequences from enterococci and, to a lesser extent, Bacteroidales correlated well with reductions in infectious viruses during both normal and storm flow operating conditions and therefore may have predictive value in determining the efficiency at which these pathogens are removed.