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In sub-Saharan Africa, many families travel to collect water and store it in their homes for daily use, presenting an opportunity for the introduction of fecal contamination. One stored and one source water sample were each collected from 45 households in rural Kenya. All 90 samples were analyzed for fecal indicator bacteria ( E. coli and enterococci) and species-specific contamination using molecular microbial source tracking assays. Human (HF183), avian (GFD), and ruminant (BacR) contamination were detected in 52, two, and four samples, respectively. Stored water samples had elevated enterococci concentrations ( p < 0.01, Wilcoxon matched pairs test) and more frequent BacR detection (89% versus 27%, p < 0.01, McNemar's exact test) relative to source water samples. fsQCA (fuzzy set qualitative comparative analysis) was conducted on the subset of households with no source water BacR contamination to highlight combinations of factors associated with the introduction of BacR contamination to stored water supplies. Three combinations were identified: (i) ruminants in the compound, safe water extraction methods, and long storage time, (ii) ruminants, unsafe water extraction methods, and no soap at the household handwashing station, and (iii) long storage time and no soap. This suggests that multiple pathways contribute to the transmission of ruminant fecal contamination in this context, which would have been missed if data were analyzed using standard regression techniques.

Enterococci are used to monitor recreational water quality worldwide, so understanding their fate and transport in the environment is essential to the protection of human health. As such, researchers have documented enterococci inactivation under various exposure conditions and in diverse water matrices. However, the majority of studies have been performed using lab-cultured bacteria, which are distinct from indigenous, uncultured bacteria found in the environment. Here we investigate the photoinactivation of indigenous, uncultured enterococci from a range of sources, including wastewater treatment plants (WWTPs), marine beaches, urban streams, and a wastewater-influenced pond. We concentrated indigenous enterococci from their sources using filtration and centrifugation, placed them in a clear buffer solution, and then exposed them to simulated sunlight to measure their photoinactivation rates. First order decay rate constants (k) of indigenous, uncultured enterococci spanned an order of magnitude, from 0.3 to 2.3 m2 kJUVB-1. k values of indigenous enterococci from WWTPs tended to be larger than those from surface waters. The k value of lab-cultured Enterococcus faecalis was larger than those of indigenous, uncultured enterococci from most sources. Negative associations between the fraction of pigmented enterococci and sunlight susceptibility were observed. This work suggests that caution should be taken when extending results on bacterial photoinactivation obtained using lab-cultured bacteria to environmental bacteria, and that enterococci pigmentation may be a useful metric for estimating photoinactivation rate constants.

Fecal pollution of surface waters presents a global human health threat. New molecular indicators of fecal pollution have been developed to address shortcomings of traditional culturable fecal indicators. However, there is still little information on their fate and transport in the environment. The present study uses spatially and temporally extensive data on traditional (culturable enterococci, cENT) and molecular (qPCR-enterococci, qENT and human-associated marker, HF183/BacR287) indicator concentrations in marine water surrounding highly-urbanized San Francisco, California, USA to investigate environmental and anthropogenic processes that impact fecal pollution. We constructed multivariable regression models for fecal indicator bacteria at 14 sampling stations. The human marker was detected more frequently in our study than in many other published studies, with detection frequency at some stations as high as 97%. The odds of cENT, qENT, and HF183/BacR287 exceeding health-relevant thresholds were statistically elevated immediately following discharges of partially treated combined sewage, and cENT levels dissipated after approximately 1 day. However, combined sewer discharges were not important predictors of indicator levels typically measured in weekly monitoring samples. Instead, precipitation and solar insolation were important predictors of cENT in weekly samples, while precipitation and water temperature were important predictors of HF183/BacR287 and qENT. The importance of precipitation highlights the significance of untreated storm water as a source of fecal pollution to the urban ocean, even for a city served by a combined sewage system. Sunlight and water temperature likely control persistence of the indicators via photoinactivation and dark decay processes, respectively.

The decay of sewage-sourced Escherichia coli and enterococci was measured at multiple depths in a freshwater marsh, a brackish water lagoon, and a marine site, all located in California. The marine site had very clear water, while the waters from the marsh and lagoon contained colored dissolved organic matter that not only blocked light but also produced reactive oxygen species. First order decay rate constants of both enterococci and E. coli were between 1 and 2 d(-1) under low light conditions and as high as 6 d(-1) under high light conditions. First order decay rate constants were well correlated to the daily average UVB light intensity corrected for light screening incorporating water absorbance and depth, suggesting endogenous photoinactivation is a major pathway for bacterial decay. Additional laboratory experiments demonstrated the presence of colored dissolved organic matter in marsh water enhanced photoinactivation of a laboratory strain of Enterococcus faecalis, but depressed photoinactivation of sewage-sourced enterococci and E. coli after correcting for UVB light screening, suggesting that although the exogenous indirect photoinactivation mechanism may be active against Ent. faecalis, it is not for the sewage-source organisms. A simple linear regression model based on UVB light intensity appears to be a useful tool for predicting inactivation rate constants in natural waters of any depth and absorbance.

We evaluated the ability of chip-based digital PCR (dPCR) to quantify enterococci, the fecal indicator recommended by the United States Environmental Protection Agency (USEPA) for water-quality monitoring. dPCR uses Poisson statistics to estimate the number of DNA fragments in a sample with a specific sequence. Underestimation may occur when a gene is redundantly encoded in the genome and multiple copies of that gene are on one DNA fragment. When genomic DNA (gDNA) was extracted using two commercial DNA extraction kits, we confirmed that dPCR could discern individual copies of the redundant 23s rRNA gene in the enterococcal genome. dPCR quantification was accurate when compared to the nominal concentration inferred from fluorometer measurements (linear regression slope = 0.98, intercept = 0.03, R(2) = 0.99, and p value <0.0001). dPCR quantification was also consistent with quantitative PCR (qPCR) measurements as well as cell counts for BioBall reference standard and 24 environmental water samples. qPCR and dPCR quantification of enterococci in the 24 environmental samples were significantly correlated (linear regression slope =1.08, R(2) of 0.96, and p value <0.0001); the group mean of the qPCR measurements was 0.19 log units higher than that of the dPCR measurements. At environmentally relevant concentrations, dPCR quantification was more precise (i.e., had narrower 95% confidence intervals than qPCR quantification). We observed that humic acid caused a similar level of inhibition in both dPCR and qPCR, but calcium inhibited dPCR to a lesser degree than qPCR. Inhibition of dPCR was partially relieved when the number of thermal cycles was increased. Based on these results, we conclude that dPCR is a viable option for enumerating enterococci in ambient water.

Coastal groundwater has been implicated as a source of microbial pollution to recreational beaches. However, there is little work investigating the transport of fecal microbes through beach aquifers where waters of variable salinity are present. In this study, the potential for fecal indicator organisms enterococci (ENT) and F+ coliphage to be transported through marine beach aquifers was investigated. Native sediment and groundwaters were collected from the fresh and saline sections of the subterranean estuary at three beaches along the California coast where coastal communities utilize septic systems for wastewater treatment. Groundwaters were seeded with sewage and removal of F+ coliphage and ENT by the sediments during saturated flow was tested in laboratory column experiments. Removal varied significantly between beach and organism. F+ coliphage was removed to a greater extent than ENT, and removal was greater in saline sediments and groundwater than fresh. At one of the three beaches, a field experiment was conducted to investigate the attenuation of F+ coliphage and ENT down gradient of a septic leach field. ENT were detected up to 24 m from the leach field. The column study and field observations together suggest ENT can be mobile within native aquifer sediments and groundwater under certain conditions.

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., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Consistently high levels of bacterial indicators of fecal pollution rank Cowell Beach as the most polluted beach in California. High levels of fecal indicator bacteria (FIB), E. coli and enterococci, are measured throughout the summer, resulting in beach advisories with social and economic consequences. The source of FIB, however, is unknown. Speculations have been made that the wrack accumulating on the beach is a major source of FIB to the surf zone. The present study uses spatial and temporal sampling coupled with process-modeling to investigate potential FIB sources and the relative contributions of those sources. Temporal sampling showed consistently high FIB concentrations in the surf zone, sand, and wrack at Cowell Beach, and ruled out the storm drain, the river, the harbor, and the adjacent wharf as the sources of the high concentrations observed in the surf zone. Spatial sampling confirmed that the source of FIB to the beach is terrestrial rather than marine. Modeling results showed two dominant FIB sources to the surf zone: sand for enterococci and groundwater for E. coli. FIB from wrack represented a minor contribution to bacterial levels in the water. Molecular source tracking methods indicate the FIB at the beach is of human and bird origin. The microbial source tracking (MST) approach presented here provides a framework for future efforts.

Background: Surface-mediated transmission is a potential route for respiratory disease in child care centers, but evidence of its importance relative to other routes (eg, airborne) is limited., Methods: We tracked respiratory disease and monitored bacteria contamination on hands and fomites over 4 months during 64 visits at 2 child care centers. Staff monitored health daily by recording respiratory symptoms. We measured concentrations of Escherichia coli, Enterococcus spp, and fecal coliform in hand rinses and on select fomites., Results: We demonstrated that symptomatic respiratory illness was positively associated with microbial contamination on hands and fomites, as measured using Enterococcus spp. Enterococcus spp were 0.28 (95% confidence interval: 0.08-0.48)-log10 (colony-forming units per 2 hands) higher when an individual had symptomatic respiratory illness. Susceptible individuals were 1.62 (95% confidence interval: 1.06-2.46) times more likely to develop respiratory illness within 4 days with every log10 increase of Enterococcus spp on hands., Conclusion: The findings imply that hand contamination as measured using Enterococcus spp is a risk factor for onset of respiratory illness and highlight the utility of fecal indicator bacteria as a metric for hand and fomite contamination., (Copyright © 2013 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Mosby, Inc. All rights reserved.)

This study explores the transport of enterococci (ENT) from naturally contaminated beach sands to the groundwater table via infiltrating seawater using field, laboratory, and modeling experiments. ENT were readily mobilized and transported through the unsaturated zone during infiltration events in both the field and laboratory column experiments. Detachment mechanisms were investigated using a modified version of HYDRUS-1D. Three models for detachment kinetics were tested. Detachment kinetics that are first order with respect to the rate of change in the water content and attached surface bacterial concentrations were found to provide a best fit between predicted and observed data. From these experimental and model results we conclude that detachment mechanisms associated with the rapid increases in pore water content such as air-water interface scouring and thin film expansion are likely drivers of ENT mobilization in the investigated system. These findings suggest that through-beach transport of ENT may be an important pathway through which ENT from beach sands are transported to beach groundwater where they may be discharged to coastal waters via submarine groundwater discharge.

Enterococcus species composition was determined each hour for 72 h at a polluted marine beach in Avalon, Santa Catalina Island, CA. Species composition during the day was significantly different from that at night, based on an analysis of similarity. Enterococcus faecium and E. faecalis were more prevalent at night than during the day, while E. hirae and other Enterococcus species were more prevalent during the day than the night. Enterococcus spp. containing a yellow pigment were more common during the day than the night, suggesting that the pigmented phenotype may offer a competitive advantage under sunlit conditions. A laboratory microcosm experiment established that the pigmented E. casseliflavus isolate and a pigmented E. faecalis isolate recovered from the field site decay slower than a nonpigmented E. faecalis isolate in a solar simulator in simulated, clear seawater. This further supports the idea that the yellow carotenoid pigment in Enterococcus provides protection under sunlit conditions. The findings are in accordance with previous work with other carotenoid-containing nonphotosynthetic and photosynthetic bacteria that suggests that the carotenoid is able to quench reactive oxygen species capable of causing photoinactivation and photostress. The results suggest that using enterococcal species composition as a microbial source tracking tool may be hindered by the differential environmental persistence of pigmented and nonpigmented enterococci.

The quantitative polymerase chain reaction (qPCR) method provides rapid estimates of fecal indicator bacteria densities that have been indicated to be useful in the assessment of water quality. Primarily because this method provides faster results than standard culture-based methods, the U.S. Environmental Protection Agency is currently considering its use as a basis for revised ambient water quality criteria. In anticipation of this possibility, we sought to examine the relationship between qPCR-based and culture-based estimates of enterococci in surface waters. Using data from several research groups, we compared enterococci estimates by the two methods in water samples collected from 37 sites across the United States. A consistent linear pattern in the relationship between cell equivalents (CCE), based on the qPCR method, and colony-forming units (CFU), based on the traditional culturable method, was significant (P < 0.05) at most sites. A linearly decreasing variance of CCE with increasing CFU levels was significant (P < 0.05) or evident for all sites. Both marine and freshwater sites under continuous influence of point-source contamination tended to reveal a relatively constant proportion of CCE to CFU. The consistency in the mean and variance patterns of CCE versus CFU indicates that the relationship of results based on these two methods is more predictable at high CFU levels (e.g., log(10)CFU > 2.0/100 mL) while uncertainty increases at lower CFU values. It was further noted that the relative error in replicated qPCR estimates was generally higher than that in replicated culture counts even at relatively high target levels, suggesting a greater need for replicated analyses in the qPCR method to reduce relative error. Further studies evaluating the relationship between culture and qPCR should take into account analytical uncertainty as well as potential differences in results of these methods that may arise from sample variability, different sources of pollution, and environmental factors.

In the last decade, the use of culture-independent methods for detecting indicator organisms and pathogens in recreational waters has increased and has led to heightened interest in their use for routine water quality monitoring. However, a thorough understanding of the persistence of genetic markers in environmental waters is lacking. In the present study, we evaluate the persistence of enterococci, enterovirus, and human-specific Bacteroidales in seawater microcosms. Two microcosms consisted of seawater seeded with human sewage. Two additional seawater microcosms were seeded with naked Enterococcus faecium DNA and poliovirus RNA. One of each replicate microcosm was exposed to natural sunlight; the other was kept in complete darkness. In the sewage microcosms, concentrations of enterococci and enterovirus were measured using standard culture-dependent methods as well as QPCR and RT-QPCR respectively. Concentrations of human-specific Bacteroidales were determined with QPCR. In the naked-genome microcosms, enterococci and enterovirus markers were enumerated using QPCR and RT-QPCR, respectively. In the sewage microcosm exposed to sunlight, concentrations of culturable enterococci fell below the detection limit within 5 days, but the QPCR signal persisted until the end of the experiment (day 28). Culturable enterococci did not persist as long as infectious enteroviruses. The ability to culture enteroviruses and enterococci was lost before detection of the genetic markers was lost, but the human-specific Bacteroidales QPCR signal persisted for a similar duration as infectious enteroviruses in the sewage microcosm exposed to sunlight. In the naked-genome microcosms, DNA and RNA from enterococci and enterovirus, respectively, persisted for over 10d and did not vary between the light and dark treatments. These results indicate differential persistence of genetic markers and culturable organisms of public health relevance in an environmental matrix and have important management implications.

Enterococci are indicator bacteria used to assess the risk of acquiring enteric disease from swimming in marine waters. Previous work identified beach sands as reservoirs of enterococci which can be transported from the sand to the sea, where they may instigate beach advisories. The present study establishes that naturally occurring enterococci can replicate in beach sands under environmentally relevant conditions. In unseeded, nonsterile microcosm experiments, it was shown that intermittent wetting of sands by seawater, like that which would occur at the high tide line, stimulates the transient replication of enterococci at rates of 0.20 to 0.63 per day (equivalent to doubling times of 1.1 to 3.5 days). Replication was not observed in control microcosms that were not subjected to wetting. Enterococci were enumerated using both culture-dependent (membrane filtration and mEI media) and culture-independent (quantitative PCR [QPCR], 23S rRNA gene based) techniques, which allowed tracking of both culturable and total enterococcus populations. Inhibition of QPCR and DNA extraction efficiencies were accounted for in the interpretation of the QPCR results. The results provide evidence that enterococci may not be an appropriate indicator of enteric disease risk at recreational beaches subject to nonpoint sources of pollution.

Fecal indicator bacteria (FIB) concentrations in a single grab sample of water are used to notify the public about the safety of swimming in coastal waters. If concentrations are over a single-sample standard, waters are closed or placed under an advisory. Previous work has shown that notification errors occur often because FIB vary more quickly than monitoring results can be obtained (typically 24 h). Rapid detection technologies (such as quantitative polymerase chain reaction) that allow FIB quantification in hours have been suggested as a solution to notification errors. In the present study, I explore variability of enterococci (ENT) over time scales less than a day that might affect interpretation of FIB concentrations from a single grab sample, even if obtained rapidly. Five new data sets of ENT collected at 10 and 1 min periodicities for 24 and 1 h, respectively, are presented. Data sets are collected in diverse marine environments from a turbulent surf zone to a quiescent bay. ENT vary with solar and tidal cycles, as has been observed in previous studies. Over short time scales, ENT are extremely variable in each environment even the quiescent bay. Changes in ENT concentrations between consecutive samples (1 or 10 min apart) greater than the single-sample standard (104 most probable number per 100 mL) are not unusual. Variability, defined as the change in concentration between consecutive samples, is not distinct between environments. ENT change by 60% on average between consecutive samples, and by as much as 700%. Spectral analyses reveal no spectral peaks, but power-law decline of spectral density with frequency. Power-law exponents are close to 1 suggesting ENT time series share properties with 1/f noise and are fractal in nature. Since fractal time series have no characteristic time scale associated with them, it is not obvious how the fractal nature of ENT can be exploited for adaptive sampling or management. Policy makers, as well as scientists designing field campaigns for microbial source tracking and epidemiology studies, are cautioned that a single sample of water reveals little about the true water quality at a beach. Multiple samples must be taken to gain a snapshot into the patchy structure of microbial water quality and associated human health risk.

Beach health advisories are issued if enterococci (ENT) densities exceed the 30-d geometric mean or single-sample water quality criteria. Current ENT enumeration procedures require 1 day of incubation; therefore, beach managers make policy decisions using 1-day-old data. This is tantamount to using a model that assumes ENT density on day t is equal to ENT density on day t-1. Research has shown that ENT densities vary over time scales shorterthan a day, calling into question the usefulness of the current model for decision-making. We created Dynamic Partial Least Square Regression (DPLSR) models for ENT at water quality monitoring stations within two adjacent marine recreational sites, Huntington State Beach (HSB) and Huntington City (HCB) Beach, California, using publicly available environmental data and tested whether these models overcome the drawbacks of the current model. The DPLSR models provide a better prediction of ENT than the current models based on comparisons of root-mean-square errors of prediction and the numbers of type 1 and 2 errors. We compared outcomes in terms of predicted illness, swimmers deterred from entering the water, and net benefits to swimmers for hypothetical management scenarios where beach advisories were issued based on (a) the previously collected sample's ENT density in conjunction with the two water quality criteria, and (b) predictions from DPLSR models in conjunction with the single-sample standard. At both HSB and HCB the DPLSR scenario produced a more favorable balance between illness prevention and recreational access. The results call into question the current method of beach management and show that model-informed decision-making and elimination of the geometric mean standard will aid beach managers in achieving more favorable outcomes in terms of illness and access than are presently achieved using 1-day-old measurements, especially at beaches where water quality problems are chronic.

An analytical model of enterococci (ENT) concentrations in the surf zone of a long sandy beach is constructed considering the physical processes of dilution by rip currents and alongshore littoral drift, and the biological processes of inactivation and mortality by grazing. The solution is used to construct an expression for the length of shoreline adversely impacted by ENT from a point source. Two non-dimensional parameters are developed whose magnitude can be used to ascertain whether dilution, inactivation, or grazing is the dominant sink for ENT in the surf zone. The model is applied to beaches in southern California, USA. Model input parameters related to physical processes and inactivation are compiled from the literature. Laboratory experiments are conducted to determine grazing mortality rates of ENT (6.5 x 10(-6) s(-1)). Results indicate that at the field sites, between 1000 and 5000 m of shoreline are typically impacted by a continuous point source of ENT. Dilution is the primary cause of decline in ENT concentrations within the surf zone, with inactivation secondary and grazing tertiary. Results recommend strategic positioning of point sources and timing of effluent releases to take advantage of high dilution conditions. Our estimates for grazing mortality rates are within the same order of magnitude as some published inactivation rates, thus we cannot rule out the possibility that grazing is an important sink for ENT, especially in low dilution environments like enclosed bays.

Marine beach water quality is typically monitored in early morning once a week without respect to tidal condition. To assess the effect of tide on this public health warning system, we analyzed enterococci (ENT) data from 60 southern California marine beaches with differing geomorphology, orientation, and proximity to runoff sources. ENT concentrations during spring tides were significantly higher (p < 0.1) than those during neap tides at 50 of the beaches, and at the majority of these, water samples were also more than twice as likely to be out of compliance with the ENT single-sample standard during spring tides compared to neap tides. When tide range (spring/neap) and tide stage (ebb/flood) conditions were considered together, spring-ebb tides yielded the highest ENT concentrations and the greatest chance of exceeding the single-sample standard at the majority of beaches. The proximity to a terrestrial runoff source, the slope of the runoff source, the slope of the beach, and the orientation of the beach had minimal influence on the tidal modulation of ENT concentrations. The presence of spring and spring-ebb tide signals at such a great percentage of beaches suggests that tide should be considered in the design and interpretation of beach monitoring program data. It also suggests that ENT delivered by tidally forced sources other than terrestrial surficial runoff are widespread. Possibilities include ENT-laden groundwater (saline and fresh) from the beach aquifer as well as ENT-enriched sands, decaying wrack, and bird feces near the high water line.

Microbial source tracking and a mass balance approach were used to identify sources of fecal indicator bacteria (FIB) in the Hanalei River, Kaua'i, Hawai'i. Historically, concentrations enterococci and Clostridium perfringens were significantly higher during storm flows compared to non-storm flows in the Hanalei River, and correlated to total suspended solids in the river. During targeted dry weather studies, the Hanalei River bed sediments and streambank soils were documented to harbor E. coli, enterococci, and the human- and pig-specific fecal markers in Bacteroidales, suggesting that sediments and soils may be potential sources of these microorganisms to the Hanalei river. The human-specific marker in Bacteroidales was four times as likely to be detected in sediment and soil samples as in water samples. Furthermore, the occurrence of host-specific source tracking markers is indicative that a portion of FIB present in the Hanalei River are of fecal origin. A mass balance approach was used to explore causes of observed FIB loadings and losses along different reaches of the river. Resuspension or deposition of FIB-laden river sediments cannot account for changes in E. coli and enterococci concentrations along the river during dry weather. Additionally, losses due to bacterial inactivation were insignificant. Groundwater and ditches draining agricultural and urban lands were shown to provide sufficient FIB fluxes to account for the observed loads along some river reaches. The presence of the human-specific Bacteroidales marker in the river water, sediments and adjacent soils, as well as the presence of the human enterovirus marker in the water, suggests that there is widespread human fecal contamination in the Hanalei River that is likely a result of nearby wastewater disposal systems. [ABSTRACT FROM AUTHOR]

Stormwater biofilters are used to attenuate the flow and volume of runoff and reduce pollutant loading to aquatic systems. However, the capacity of biofilters to remove microbial contaminants remains inadequate. While biochar has demonstrated promise as an amendment to improve microbial removal in laboratory-scale biofilters, it is uncertain if the results are generalizable to the field. To assess biochar performance in a simulated field setting, sand and biochar-amended sand biofilters were periodically dosed with natural stormwater over a 61-week conditioning phase. Impact of media saturation was assessed by maintaining biofilters with and without a saturated zone. Biochar-amended biofilters demonstrated improved Escherichia coli removal over sand biofilters during the first 31 weeks of conditioning though media type did not impact E. coli removal during the last 30 weeks of conditioning. Presence of a saturated zone was not a significant factor influencing E. coli removal across the entire conditioning phase. Following conditioning, biofilters underwent challenge tests using stormwater spiked with wastewater to assess their capacity to remove wastewater-derived E. coli, enterococci, and male-specific (F+) coliphage. In challenge tests, biochar-amended biofilters demonstrated enhanced removal of all fecal indicators relative to sand biofilters. Additionally, saturated biofilters demonstrated greater removal of fecal indicators than unsaturated biofilters for both media types. Discrepant conclusions from the conditioning phase and challenge tests may be due to variable influent chemistry, dissimilar transport of E. coli indigenous to stormwater and those indigenous to wastewater, and differences in E. coli removal mechanisms between tests. Mobilization tests conducted following challenge tests showed minimal (<2.5%) observable mobilization of fecal indicators, regardless of media type and presence of a saturated zone. While our results emphasize the challenge of translating biochar’s performance from the laboratory to the field, findings of this study inform biofilter design to remove microbial contaminants from urban stormwater. [ABSTRACT FROM AUTHOR]

Recirculating seawater is an important component of submarine groundwater discharge, yet its role in transporting microbial contaminants from beach sand to coastal water is unknown. This study investigated the extent to which recirculating seawater carries fecal indicators, Enterococcus and bird-associated Catellicoccus, through the beach subsurface. Laboratory experiments and numerical modeling were performed to characterize the transport of fecal indicators suspended in seawater through medium-grained beach sand under transient and saturated flow conditions. Enterococcus was measured both by culture (cENT) and DNA assay (tENT), and Catellicoccus (CAT) by DNA assay. There were differences between transport of tENT and CAT compared to cENT through laboratory columns containing beach sands. Under transient flow conditions, first-order attachment rate coefficients (katt) of DNA markers were greater (~ 10 h[sup -1]) than katt of cENT ( ~1 h ), although under saturated conditions katt values were similar (~1 h[sup -1]). First-order detachment rate coefficients, kdet, of DNA markers were greater (~1 h[sup -1]) than kdet of cENT (~0.1h[sup -1]) under both types of flow conditions. Incorporating the rate coefficients into field-scale subsurface transport simulations showed that, in this sand type, the contribution of recirculating seawater to surf zone contamination is likely to be minimal unless bird feces are deposited close to the land-sea interface. [ABSTRACT FROM AUTHOR]

Most studies characterize microbial source tracking (MST) target performance using sensitivity and specificity metrics. However, it is important to also consider the temporal stability of MST targets in relation to regulated microbial pollutants. Differences among bacterial target stabilities may lead to erroneous conclusions about sources of contamination. The present study evaluates the relative stability of MST targets and fecal indicator organisms using the gull/pigeon-associated Catellicoccus marimammalium (CAT) marker and enterococci (ENT). The decay rates of CAT and ENT measured by culture (cENT) and QPCR (tENT) were compared in sand and seawater laboratory microcosms under environmentally relevant conditions (subject to tidal wetting versus no wetting in sand, and sunlit versus dark conditions in seawater). Bacterial targets were more persistent in beach sand than in seawater with decay rates on the order of 0.01–0.1 per day and 1 to 10 per day, respectively. Targets were more persistent in unwetted compared to wetted sand, and dark compared to sunlit seawater. During the first 8 days of the sand experiment, the decay rate k of CAT was greater than that of cENT. The decay rates of CAT, tENT, and cENT were similar in sand after day 8 and in dark seawater. In sunlit seawater, the decay rates were different between targets with k cENT > k CAT > k tENT . The decay rates presented here are useful for fate and transport models and also inform the use of MST marker concentrations to infer ENT sources in the environment. [ABSTRACT FROM AUTHOR]

Abstract: Microbial source tracking (MST) methods were evaluated in the Source Identification Protocol Project (SIPP), in which 27 laboratories compared methods to identify host sources of fecal pollution from blinded water samples containing either one or two different fecal types collected from California. This paper details lessons learned from the SIPP study and makes recommendations to further advance the field of MST. Overall, results from the SIPP study demonstrated that methods are available that can correctly identify whether particular host sources including humans, cows and birds have contributed to contamination in a body of water. However, differences between laboratory protocols and data processing affected results and complicated interpretation of MST method performance in some cases. This was an issue particularly for samples that tested positive (non-zero C t values) but below the limits of quantification or detection of a PCR assay. Although false positives were observed, such samples in the SIPP study often contained the fecal pollution source that was being targeted, i.e., the samples were true positives. Given these results, and the fact that MST often requires detection of targets present in low concentrations, we propose that such samples be reported and identified in a unique category to facilitate data analysis and method comparisons. Important data can be lost when such samples are simply reported as positive or negative. Actionable thresholds were not derived in the SIPP study due to limitations that included geographic scope, age of samples, and difficulties interpreting low concentrations of target in environmental samples. Nevertheless, the results of the study support the use of MST for water management, especially to prioritize impaired waters in need of remediation. Future integration of MST data into quantitative microbial risk assessments and other models could allow managers to more efficiently protect public health based on site conditions. [Copyright &y& Elsevier]

Abstract: The last decade has seen development of numerous new microbial source tracking (MST) methodologies, but many of these have been tested in just a few laboratories with a limited number of fecal samples. This method evaluation study examined the specificity and sensitivity of 41 MST methodologies by analyzing data generated in 27 laboratories. MST methodologies that targeted human, cow, ruminant, dog, gull, pig, horse, and sheep were tested against sewage, septage, human, cow, dog, deer, pig, chicken, pigeon, gull, horse, and goose fecal samples. Each laboratory received 64 blind samples containing a single source (singletons) or two sources (doubletons), as well as diluted singleton samples to assess method sensitivity. Laboratories utilized their own protocols when performing the methods and data were deposited in a central database before samples were unblinded. Between one and seven laboratories tested each method. The most sensitive and specific assays, based on an analysis of presence/absence of each marker in target and non-target fecal samples, were HF183 endpoint and HF183SYBR (human), CF193 and Rum2Bac (ruminant), CowM2 and CowM3 (cow), BacCan (dog), Gull2SYBR and LeeSeaGull (gull), PF163 and pigmtDNA (pig), HoF597 (horse), PhyloChip (pig, horse, chicken, deer), Universal 16S TRFLP (deer), and Bacteroidales 16S TRFLP (pig, horse, chicken, deer); all had sensitivity and specificity higher than 80% in all or the majority of laboratories. When the abundance of MST markers in target and non-target fecal samples was examined, some assays that performed well in the binary analysis were found to not be sensitive enough as median concentrations fell below a minimum abundance criterion (set at 50 copies per colony forming units of enterococci) in target fecal samples. Similarly, some assays that cross-reacted with non-target fecal sources in the binary analysis were found to perform well in a quantitative analysis because the cross-reaction occurred at very low levels. Based on a quantitative analysis, the best performing methods were HF183Taqman and BacH (human), Rum2Bac and BacR (ruminant), LeeSeaGull (gull), and Pig2Bac (pig); no cow or dog-specific assay met the quantitative specificity and sensitivity criteria. Some of the best performing assays in the study were run by just one laboratory so further testing of assay portability is needed. While this study evaluated the marker performance in defined samples, further field testing as well as development of frameworks for fecal source allocation and risk assessment are needed. [Copyright &y& Elsevier]

Sunlight influences microbial water quality of surface waters. Previous studies have investigated photoinactivation mechanisms and cellular photostress responses of fecal indicator bacteria (FIB), including Escherichia coli and enterococci, but further work is needed to characterize photostress responses of bacterial pathogens. Here we investigate the photoinactivation of Staphylococcus aureus (strain Newman), a pigmented, waterborne pathogen of emerging concern. We measured photodecay using standard culture-based assays and cellular membrane integrity and investigated photostress response by measuring the relative number of mRNA transcripts of select oxidative stress, DNA repair, and metabolism genes. Photoinactivation experiments were performed in both oxic and anoxic systems to further investigate the role of oxygen-mediated and non-oxygen-mediated photoinactivation mechanisms. S. aureus lost culturability much faster in oxic systems than in anoxic systems, indicating an important role for oxygen in photodecay mechanisms. S. aureus cell membranes were damaged by sunlight exposure in anoxic systems but not in oxic systems, as measured by cell membrane permeability to propidium iodide. After sunlight exposure, S. aureus increased expression of a gene coding for methionine sulfoxide reductase after 12 h of sunlight exposure in the oxic system and after 6 h of sunlight exposure in the anoxic system, suggesting that methionine sulfoxide reductase is an important enzyme for defense against both oxygen-dependent and oxygen-independent photostresses. This research highlights the importance of oxygen in bacterial photoinactivation in environmentally relevant systems and the complexity of the bacterial photostress response with respect to cell structure and transcriptional regulation. IMPORTANCE Staphylococcus aureus is a pathogenic bacterium that causes gastrointestinal, respiratory, and skin infections. In severe cases, S. aureus infection can lead to life-threatening diseases, including pneumonia and sepsis. Cases of communityacquired S. aureus infection have been increasing in recent years, pointing to the importance of considering S. aureus transmission pathways outside the hospital environment. Associations have been observed between recreational water contact and staphylococcal skin infections, suggesting that recreational waters may be an important environmental transmission pathway for S. aureus. However, prediction of human health risk in recreational waters is hindered by incomplete knowledge of pathogen sources, fate, and transport in this environment. This study is an in-depth investigation of the inactivation of a representative strain of S. aureus by sunlight exposure, one of the most important factors controlling the fate of microbial contaminants in clear waters, which will improve our ability to predict water quality changes and human health risk in recreational waters. [ABSTRACT FROM AUTHOR]

Field studies in fresh and marine waters consistently show diel fluctuations in concentrations of enterococci, indicators of water quality. We investigated sunlight inactivation of Enterococcus faecalis to gain insight into photoinactivation mechanisms and cellular responses to photostress. E. faecalis bacteria were exposed to natural sunlight in clear, filtered seawater under both oxic and anoxic conditions to test the relative importance of oxygen-mediated and non-oxygen-mediated photoinactivation mechanisms. Multiple methods were used to assess changes in bacterial concentration, including cultivation, quantitative PCR (qPCR), propidium monoazide (PMA)-qPCR, LIVE/DEAD staining using propidium iodide (PI), and cellular activity, including ATP concentrations and expression of the superoxide dismutase-encoding gene, sodA. Photoinactivation, based on numbers of cultivable cells, was faster in oxic than in anoxic microcosms exposed to sunlight, suggesting that oxygen-mediated photoinactivation dominated. There was little change in qPCR signal over the course of the experiment, demonstrating that the nucleic acid targets were not damaged to a significant extent. The PMA-qPCR signal was also fairly stable, consistent with the observation that the fraction of Pi-permeable cells was constant. Thus, damage to the membrane was minimal. Microbial ATP concentrations decreased in all microcosms, particularly the sunlit oxic microcosms. The increase in relative expression of the sodA gene in the sunlit oxic microcosms suggests that cells were actively responding to oxidative stress. Dark repair was not observed. This research furthers our understanding of photoinactivation mechanisms and the conditions under which diel fluctuations in enterococci can be expected in natural and engineered systems. [ABSTRACT FROM AUTHOR]

We present estimates of annual public health impacts, both illnesses and cost of illness, attributable to excess gastrointestinal illnesses caused by swimming in contaminated coastal waters at beaches in southern California. Beach-specific enterococci densities are used as inputs to two epidemiological dose response models to predict the risk of gastrointestinal illness at 28 beaches spanning 160 km of coastline in Los Angeles and Orange Counties. We use attendance data along with the health cost of gastrointestinal illness to estimate the number of illnesses among swimmers and their likely economic impact. We estimate that between 627,800 and 1,479,200 excess gastrointestinal illnesses occur at beaches in Los Angeles and Orange Counties each year. Using a conservative health cost of gastroenteritis, this corresponds to an annual economic loss of $21 or $51 million depending upon the underlying epidemiological model used (in year 2000 dollars). Results demonstrate that improving coastal water quality could result in e reduction of gastrointestinal illnesses locally and a concurrent savings in expenditures on related health care costs. [ABSTRACT FROM AUTHOR]

We simulate the influence of multiple sources of enterococci (ENT) as faecal indicator bacteria (FIB) in recreational water bodies on potential human health risk by considering waters impacted by human and animal sources, human and non-pathogenic sources, and animal and non-pathogenic sources. We illustrate that risks vary with the proportion of culturable ENT in water bodies derived from these sources and estimate corresponding ENT densities that yield the same level of health protection that the recreational water quality criteria in the United States seeks (benchmark risk). The benchmark risk is based on epidemiological studies conducted in water bodies predominantly impacted by human faecal sources. The key result is that the risks from mixed sources are driven predominantly by the proportion of the contamination source with the greatest ability to cause human infection (potency), not necessarily the greatest source(s) of FIB. Predicted risks from exposures to mixtures comprised of approximately 30% ENT from human sources were up to 50% lower than the risks expected from purely human sources when contamination is recent and ENT levels are at the current water quality criteria levels (35 CFU 100 mL -1 ). For human/non-pathogenic, human/gull, human/pig, and human/chicken faecal mixtures with relatively low human contribution, the predicted culturable enterococci densities that correspond to the benchmark risk are substantially greater than the current water quality criteria values. These findings are important because they highlight the potential applicability of site specific water quality criteria for waters that are predominantly un-impacted by human sources. [ABSTRACT FROM AUTHOR]

Highlights: [•] After 6h of sun E. faecalis concentrations decreased 6–7 orders of magnitude. [•] After 12h of sun E. faecalis transcribe RNA encoding for ribosomal and stress response proteins. [•] E. faecalis exposed to sunlight in oligotrophic seawater were not able to repair photo-damage. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Fecal indicator bacteria (FIB) are nearly ubiquitous in California (CA) beach sands. Sands were collected from 55 beaches along the CA coast. Ninety-one percent of the beaches had detectable enterococci (ENT) while 62% had detectable E. coli (EC) in their sands. The presence of a putative bacterial source (such as a river), the degree of wave shelter, and surrounding land use explained a significant (p < 0.05) fraction of the variation in both ENT and EC densities between beaches. Sand characteristics including moisture content, organic carbon, and percent fines, significantly (p < 0.05) influenced only EC densities in beach sand. We assayed 34 of 163 sand samples for salmonellae, but did not detect this bacterial pathogen. The potential for FIB to be transported from the sand to sea was investigated at a single wave-sheltered beach with high densities of ENT in beach sand: Lovers Point, CA (LP). We collected samples of exposed and submerged sands as well as water over a 24 h period in order to compare the disappearance or appearance of ENT in sand and the water column. Exposed sands had significantly higher densities of ENT than submerged sands with the highest densities located near the high tide line. Water column ENT densities began low, increased sharply during the first flood tide and slowly decreased over the remainder of the study. During the first flood tide, the number of ENT that entered the water column was nearly equivalent to the number of ENT lost from exposed sands when they were submerged by seawater. The decrease in nearshore ENT concentrations after the initial influx can be explained by ENT die-off and dilution with clean ocean water. While some ENT in the water and sand at LP might be of human origin because they were positive for the esp gene, others lacked the esp gene and were therefore equivocal with respect to their origin. Follow-up sampling at LP revealed the presence of the human specific Bacteroides marker in water and sand. [ABSTRACT FROM AUTHOR]