Development and evaluation of a colorimetric LAMP based-assay targeting the Bacteroides HF183 marker for tracking sewage pollution in environmental waters.

Surface waters are vulnerable to contamination by human and animal feces, posing risks to human health due to potential exposure to enteric pathogens. This research developed a colorimetric loop-mediated isothermal amplification (cLAMP) assay to detect sewage associated Bacteroides dorei HF183/BacR287 (HF183) marker in wastewater and environmental water samples. The host sensitivity and host specificity of the assay were evaluated, and their performance was compared to the Bacteroides HF183 qPCR assay using control materials (gBlocks), environmental water samples seeded with untreated sewage, and ambient environmental water samples. In serial dilutions of control materials, qPCR produced quantifiable data across all dilutions, while cLAMP detected the marker down to 0.001 pg/µL of control materials, which was two orders of magnitude less sensitive than qPCR. All untreated sewage samples (n = 12) tested positive for HF183 by both the qPCR and cLAMP assays, demonstrating a host sensitivity value of 1.00 (maximum value of 1.00). The host specificity by analysing 70 non-human fecal nucleic acid samples revealed cLAMP's specificity value of 0.81 compared to qPCR's 0.64. When testing sewage-seeded environmental water samples, both methods detected HF183 for the lowest amount of sewage, indicating similar detection sensitivity. The application of cLAMP for tracking sewage pollution in environmental waters showed promising results, with moderate agreement between cLAMP and qPCR (κ = 0.510). However, cLAMP occasionally missed detections compared to qPCR, particularly in low-concentration samples. Overall, the cLAMP HF183 assay demonstrated promising potential as a rapid and sensitive method for detecting sewage pollution, offering a viable alternative to qPCR in certain environmental monitoring scenarios.

Two risk assessments: Evaluating the use of indicator HF183 Bacteroides versus pathogen measurements for modelling recreational illness risks in an urban watershed.

The purpose of this study was to evaluate the performance of HF183 Bacteroides for estimating pathogen exposures during recreational water activities. We compared the use of Bacteroides-based exposure assessment to exposure assessment that relied on pathogen measurements. We considered two types of recreational water sites: those impacted by combined sewer overflows (CSOs) and those not impacted by CSOs. Samples from CSO-impacted and non-CSO-impacted urban creeks were analysed by quantitative polymerase chain reaction (qPCR) for HF183 Bacteroides and eight human gastrointestinal pathogens. Exposure assessment was conducted two ways for each type of site (CSO-impacted vs. non-CSO impacted): 1) by estimating pathogen concentrations from HF183 Bacteroides concentrations using published ratios of HF183 to pathogens in sewage and 2) by estimating pathogen concentrations from qPCR measurements. QMRA (quantitative microbial risk assessment) was then conducted for swimming, wading, and fishing exposures. Overall, mean risk estimates varied from 0.27 to 53 illnesses per 1,000 recreators depending on exposure assessment, site, activity, and norovirus dose-response model. HF183-based exposure assessment identified CSO-impacted sites as higher risk, and the recommended HF183 risk-based threshold of 525 genomic copies per 100 mL was generally protective of public health at the CSO-impacted sites but was not as protective at the non-CSO-impacted sites. In the context of our urban watershed, HF183-based exposure assessment over- and under-estimated risk relative to exposure assessment based on pathogen measurements, and the etiology of predicted pathogen-specific illnesses differed significantly. Across all sites, the HF183 model overestimated risk for norovirus, adenovirus, and Campylobacter jejuni, and it underestimated risk for E. coli and Cryptosporidium. To our knowledge, this study is the first to directly compare health risk estimates using HF183 and empirical pathogen measurements from the same waterways. Our work highlights the importance of site-specific hazard identification and exposure assessment to decide whether HF183 is applicable for monitoring risk.

Comparison of adsorption-extraction (AE) workflows for improved measurements of viral and bacterial nucleic acid in untreated wastewater.

The lack of standardized methods and large differences in virus concentration and extraction workflows have hampered Severe Acute Respiratory Syndrome (SARS-CoV-2) wastewater surveillance and data reporting practices. Numerous studies have shown that adsorption-extraction (AE) method holds promise, yet several uncertainties remain regarding the optimal AE workflow. Several procedural components may influence the recovered concentrations of target nucleic acid, including membrane types, homogenization instruments, speed and duration, and lysis buffer. In this study, 42 different AE workflows that varied these components were compared to determine the optimal workflow by quantifying endogenous SARS-CoV-2, human adenovirus 40/41 (HAdV 40/41), and a bacterial marker gene of fecal contamination (Bacteroides HF183). Our findings suggest that the workflow chosen had a significant impact on SARS-CoV-2 concentrations, whereas it had minimal impact on HF183 and no effect on HAdV 40/41 concentrations. When comparing individual components in a workflow, such as membrane type (MF-Millipore™ 0.45 µm MCE vs. Isopore™ 0.40 µm), we found that they had no impact on SARS-CoV-2, HAdV 40/41, and HF183 concentrations. This suggests that at least some consumables and equipment are interchangeable. Buffer PM1 + TRIzol-based workflows yielded higher concentrations of SARS-CoV-2 than other workflows. HF183 concentrations were higher in workflows without chloroform. Similarly, higher homogenization speeds (5000-10,000 rpm) led to increased concentrations of SARS-CoV-2 and HF183 but had no effect on HAdV 40/41. Our findings indicate that minor enhancements to the AE workflow can improve the recovery of viruses and bacteria from the wastewater, leading to improved outcomes from wastewater surveillance efforts.

Assessing the defecation practices of unsheltered individuals and their contributions to microbial water quality in an arid, urban watershed.

Outdoor defecation by people experiencing homelessness is frequently perceived as a potentially large source of human fecal pollution and a significant source of health risk in urban waterbodies with recreational contact. The goal of this study was to count the number of people experiencing homelessness and quantifies their sanitation habits in an urban river corridor setting, then use this information for estimating human fecal pollutant loading on a watershed scale. Two types of census counts were conducted including periodic point-in-time counts over six years and weekly counts of encampments. While the population census varied from count-to-count, the range of population estimates in the river corridor varied from 109 to 349 individuals during the six-year span, which mirrored the weekly counts of encampments. A face-to-face survey of people experiencing homelessness assessed the sanitation habits of the unsheltered population (N = 63), including outdoor defecation frequency and containment practices. Overall, 95 % of survey respondents reported defecating outdoors; 36 % practiced outdoor defecation between 4 and 7 days/week and 27 % practiced outdoor defecation <1 day/week. Of those that did practice outdoor defecation, 75 % contained their feces in a bucket or bag, thereby limiting fecal material contributions to the river; 6.7 % reported defecating on low ground near the river that could wash off when flood waters rise during a storm event. Only a single survey respondent reported defecating directly into the river. Based on literature values for average HF183 output for an adult human, and the average rainfall in the urban watershed, the total watershed contribution of HF183 averaged 1.2 × 1010 gene copies per storm event (95 % CI: 0.9 × 1010-1.6 × 1010) along the 41 km stretch of river in this study. This human fecal loading estimate is at least two orders of magnitude less than cumulative HF183 loading from all human sources measured at the bottom of the watershed.

Evaluation of four human-associated fecal biomarkers in wastewater in Southern Ontario.

Human fecal biomarkers (HFBs) have a longstanding history in the field of microbial source tracking (MST) serving as indicators of human fecal contamination in drinking and recreational water. Further, HFBs have aided in recent efforts to monitor human pathogen transmission within communities. The dilution of wastewater from various sources throughout the sewershed cannot be controlled and human fecal biomarkers (HFBs) can be used to normalize target human pathogen concentrations so that fluctuations in fecal matter in wastewater can be accounted for. In the current study, we monitored the prevalence of four HFBs - including two viruses, Pepper mild mottle virus (PMMoV), cross-assembly phage (crAssphage), as well as two human-associated Bacteroides markers, HF183 and BacHuman - in wastewater samples from ten Southern Ontario wastewater treatment plants and evaluated their temporal and spatial variation in context of environmental factors that may impact the ability of HFB to normalize pathogen concentrations in wastewater. Environmental variables including precipitation, wastewater flow rate, temperature, and concentrated mass were also analyzed for their potential correlation with HFB variation in wastewater. The four HFBs were detected at high concentrations across all 10 sampling locations. The median concentrations across all sampling sites were: PMMoV 3.6 Log gene copies (GC)/mL; crAssphage 5.0 Log GC/mL; HF183 6.8 Log GC/mL and BacHuman 6.9 Log GC/mL. All HFBs were found to be similarly stratified across all 10 sites, and the bacterial markers were consistently found at higher concentration compared to the viral HFBs at all sites. The coefficient of variation (CV) for each HFB was used to characterize the variability of each biomarker at each sewershed. BacHuman and crAssphage were found to have lower CV than PMMoV and HF183, indicating that BacHuman and crAssphage may perform better in reflecting the variations in abundance of human feces in wastewater or MST applications.

Assessment of multiple fecal contamination sources in surface waters using environmental mitochondrial DNA metabarcoding.

Waterborne diseases are transmitted to humans through the fecal contamination of water, where homeothermic species are the main reservoir. Fecal indicator bacteria (FIB) are often used to determine the occurrence of fecal contamination. However, FIB cannot provide the source of fecal contamination. Furthermore, as fecal inputs and contamination could originate from multiple sources (e.g., human, livestock, wildlife), multiple source tracking markers are required to identify fecal sources. From a previous study, we developed a mitochondrial DNA (mtDNA) metabarcoding approach to assess the presence of multiple homeotherms in four surface waters. Here, we have broadened our approach by sampling 86 surface water samples from the L'Assomption River and Ville-Marie watersheds (Province of Quebec, Canada). Fecal coliform levels were higher than the expected sanitary recommendations for recreational water (> 200 CFU/100 mL) in 73 % samples. The occurrence of mtDNA from human, livestock, domestic animals, wild mammals and wild birds was found in 40-88 % of the samples. Multivariate analyses showed significant covariations between homeothermic taxa and fecal coliforms, enterococci, ß-D-glucuronidase, conductivity, the human-specific Bacteroidales Hf183 genetic marker, and the human population, in the watersheds of L'Assomption River (p = 0.001) and Ville-Marie (p = 0.015) (Province of Quebec, Canada). Through the application of Bayes Theorem, it was determined that fecal coliforms co-occurred with the detection of bovine, beaver, robin and chicken mtDNA in 100 % of cases in the L'Assomption River watershed, and human mtDNA co-occurred with fecal coliforms in 93 % and 76 % of cases in L'Assomption River watershed and Ville-Marie sub-catchment, respectively. This study suggests that fecal contamination could be the result of multiple species, among which some wild animals may contribute to fecal inputs in surface waters, resulting in potential risk to human health. This reinforces the necessity of using the mtDNA metabarcoding method to monitor multi-animal species.

Widespread human waste pollution in surface waters observed throughout the urbanized, coastal communities of Lee County, Florida, USA.

The coastal communities of Lee County, Florida, USA have grown rapidly since the 1970s. In this county, drainage ditches, canals, creeks, and the Caloosahatchee River Estuary often have high concentrations of nutrients and bacteria limiting their designated uses. Septic systems have previously been identified as a major pollution source in some areas of Lee County; therefore, this study sought to identify the extent of this issue throughout the county. To accomplish this, surface water samples were collected at 25 ditch, creek, or canal sites suspected of human waste contamination from septic systems in various drainage basins throughout Lee County during January 2020-January 2021. Water samples were analyzed for nutrients, dual stable nitrate isotopes (δ15N-NO3-, δ18O-NO3-), fecal indicator bacteria (enterococci, Escherichia coli), a molecular tracer of human waste (HF183), and chemical tracers of human waste (the artificial sweetener sucralose, pharmaceuticals). Particulate organic matter (POM) and macrophytes were also collected and analyzed for stable carbon (δ13C) and nitrogen (δ15N) isotopes, as well as elemental composition (C:N:P). To broaden the assessment of stable isotope values and C:N:P, archived macrophyte samples from 2019 were also included in analyses. Ammonium concentrations were high (> 4.3 µM) in 55 % of samples. Fecal bacteria were high in 66 % of samples. HF183 was detected in 50 % of samples and positively correlated with enterococci (r = 0.32). Sucralose concentrations were high (> 380 ng/L) in 54 % of samples, while carbamazepine was detected in 40 % of samples. Human waste N sources were indicated by δ15N > 3.00 ‰ at 44 % of sites by δ15N-NO3-, 68 % of sites by POM, and at 100 % of sites where macrophyte samples were collected. This large-scale study provides evidence of widespread human waste pollution throughout Lee County and can help guide infrastructure improvements to promote sustainable development. These findings should be applicable to urbanized regions globally that are experiencing declines in water quality and harmful algal blooms due to development with inadequate infrastructure.

Risk of Gastroenteritis from Swimming at a Wastewater-Impacted Tropical Beach Varies across Localized Scales.

Population growth and changing climate are expected to increase human exposure to pathogens in tropical coastal waters. We examined microbiological water quality in three rivers within 2.3 km of each other that impact a Costa Rican beach and in the ocean outside their plumes during the rainy and dry seasons. We performed quantitative microbial risk assessment (QMRA) to predict the risk of gastroenteritis associated with swimming and the amount of pathogen reduction needed to achieve safe conditions. Recreational water quality criteria based on enterococci were exceeded in >90% of river samples but in only 13% of ocean samples. Multivariate analysis grouped microbial observations by subwatershed and season in river samples but only by subwatershed in the ocean. The modeled median risk from all pathogens in river samples was between 0.345 and 0.577, 10-fold above the U.S. Environmental Protection Agency (U.S. EPA) benchmark of 0.036 (36 illnesses/1,000 swimmers). Norovirus genogroup I (NoVGI) contributed most to risk, but adenoviruses raised risk above the threshold in the two most urban subwatersheds. The risk was greater in the dry compared to the rainy season, due largely to the greater frequency of NoVGI detection (100% versus 41%). Viral log10 reduction needed to ensure safe swimming conditions varied by subwatershed and season and was greatest in the dry season (3.8 to 4.1 dry; 2.7 to 3.2 rainy). QMRA that accounts for seasonal and local variability of water quality contributes to understanding the complex influences of hydrology, land use, and environment on human health risk in tropical coastal areas and can contribute to improved beach management. IMPORTANCE This holistic investigation of sanitary water quality at a Costa Rican beach assessed microbial source tracking (MST) marker genes, pathogens, and indicators of sewage. Such studies are still rare in tropical climates. Quantitative microbial risk assessment (QMRA) found that rivers impacting the beach consistently exceeded the U.S. EPA risk threshold for gastroenteritis of 36/1,000 swimmers. The study improves upon many QMRA studies by measuring specific pathogens, rather than relying on surrogates (indicator organisms or MST markers) or estimating pathogen concentrations from the literature. By analyzing microbial levels and estimating the risk of gastrointestinal illness in each river, we were able to discern differences in pathogen levels and human health risks even though all rivers were highly polluted by wastewater and were located less than 2.5 km from one another. This variability on a localized scale has not, to our knowledge, previously been demonstrated.

Influence of temperature and water quality on the persistence of human mitochondrial DNA, human Hf183 Bacteroidales, fecal coliforms and enterococci in surface water in human fecal source tracking context.

Mitochondrial DNA (mtDNA) is used as a genetic marker to track fecal contamination in surface water. Its potential to effectively discriminate between the nonpoint sources of fecal pollution (e.g. human, livestock) in water environments is relevant for water quality management. However, there is a lack of knowledge about the environmental persistence of mtDNA in relation to those of other microbial parameters, such as fecal indicator bacteria (FIB). In this study, mesocosms composed of water collected from four rivers and tap water were spiked with raw wastewater to mimic human fecal contamination. Mesocosms composed of raw wastewater were also studied. The mesocosms were incubated at 4 °C or at 22 °C for 189 days, from which the levels of human mtDNA (HumtDNA) and human Bacteroidales (Hf183) were measured by qPCR. The levels of FIB (fecal coliforms and enterococci) and heterotrophs were determined by culture methods along with the determination of physicochemical attributes. The decay rates of the genetic markers and FIB were determined with first-order decay rate models. The decay rates of HumtDNA (0.004-0.059 d-1), Hf183 (0.007-0.082 d-1), and the two FIBs (0.005-0.066 d-1) were similar at 4 °C, while the genetic markers both had higher decay rates (0.013-0.919 d-1) at 22 °C. Different HumtDNA decay rates were observed between the river mesocosms (0.043-0.919 d-1) and the wastewater and tap water mesocosms (0.004-0.095 d-1). Covariations of pH and conductivity among the HumtDNA, Hf183 and FIB decay rates were observed. HumtDNA and Hf183 had similar environmental persistence, whereas fecal coliforms and enterococci persisted longer at 22 °C. Finally, HumtDNA had the same trends of persistence in the four river mesocosms, suggesting a relative stability of this marker in different rivers. Our results suggest that HumtDNA could be more suitable for tracking the source of a recent fecal contamination in complement to FIB.

Highly variable removal of pathogens, antibiotic resistance genes, conventional fecal indicators and human-associated fecal source markers in a pilot-scale stormwater biofilter operated under realistic stormflow conditions.

Green stormwater infrastructure systems, such as biofilters, provide many water quality and other environmental benefits, but their ability to remove human pathogens and antibiotic resistance genes (ARGs) from stormwater runoff is not well documented. In this study, a field scale biofilter in Southern California (USA) was simultaneously evaluated for the breakthrough of a conservative tracer (bromide), conventional fecal indicators, bacterial and viral human-associated fecal source markers (HF183, crAssphage, and PMMoV), ARGs, and bacterial and viral pathogens. When challenged with a 50:50 mixture of untreated sewage and stormwater (to mimic highly contaminated storm flow) the biofilter significantly removed (p < 0.05) 14 of 17 microbial markers and ARGsin descending order of concentration reduction: ermB (2.5 log(base 10) reduction) > Salmonella (2.3) > adenovirus (1.9) > coliphage (1.5) > crAssphage (1.2) > E. coli (1.0) ∼ 16S rRNA genes (1.0) ∼ fecal coliform (1.0) ∼ intl1 (1.0) > Enterococcus (0.9) ∼ MRSA (0.9) ∼ sul1 (0.9) > PMMoV (0.7) > Entero1A (0.5). No significant removal was observed for GenBac3, Campylobacter, and HF183. From the bromide data, we infer that 0.5 log-units of attenuation can be attributed to the dilution of incoming stormwater with water stored in the biofilter; removal above this threshold is presumably associated with non-conservative processes, such as physicochemical filtration, die-off, and predation. Our study documents high variability (>100-fold) in the removal of different microbial contaminants and ARGs by a field-scale stormwater biofilter operated under transient flow and raises further questions about the utility of human-associated fecal source markers as surrogates for pathogen removal.

Genetic sequence data evidence that human faecal-associated HF183 sequences are on human skin and in urine.

AIMS: The DNA marker HF183 is a partial 16S rRNA gene sequence highly specific to human-associated Bacteroides including Bacteroides dorei. While HF183 is used to assess human faecal contamination in aquatic environments worldwide, little is known about the existence of HF183 and B. dorei in human microbiomes outside of the human gastrointestinal tract and faeces. METHODS AND RESULTS: Previously published human skin and urine microbiome data sets from five independent human body skin studies, the Human Microbiome Project (HMP) and three independent human urine studies were analysed. The HF183 gene sequence was detected in all skin data sets, with the ratios of positive samples ranging from 0.5% to 36.3%. Popliteal fossa (knee), volar forearm and inguinal (groin) creases were identified as hot spots. HF183 was detected in two of three urine data sets, with ratios of positive samples ranging from 0% to 37.5%. All HF183-containing sequences from these data sets were classified as associated with B. dorei. CONCLUSIONS: HF183 is widespread on human skin and present in urine. SIGNIFICANCE AND IMPACT OF STUDY: Skin and urine microbiomes could be sources of HF183 to environmental waters. Such non-faecal sources of HF183 might explain low concentrations of HF183 in recreational waters when swimmers are present.

An assessment of three methods for extracting bacterial DNA from beach sand.

AIMS: Beach water quality is regulated by faecal indicator bacteria levels, sand is not, despite known human health risk from exposure to beach sand. We compared the performance of three methods to extract bacterial DNA from beach sand as a step toward a standard method. METHODS AND RESULTS: The analytical sensitivity of quantitative polymerase chain reaction (qPCR) for Enterococcus was compared for the slurry (suspension, agitation, membrane filtration of supernatant), versus direct extraction using PowerSoil™ or PowerMax Soil™ kits. The slurry method had the lowest limit of detection at 20-80 gene copies g-1 , recovered significantly more DNA, and the only method that detected Enterococcus by qPCR in all samples; therefore, the only method used in subsequent experiments. The slurry method reflected the spatial variability of Enterococcus in individual transect samples. Mean recovery efficiency of the microbial source tracking marker HF183 from wastewater spiked marine and freshwater beach sand was 100.8% and 64.1%, respectively, but varied, indicating that the mixing protocol needs improvement. CONCLUSIONS: Among the three methods, the slurry method had the best analytical sensitivity and produced extracts that were useful for culture or molecular analysis. SIGNIFICANCE AND IMPACT OF STUDY: Standardization of methods for extraction of bacterial DNA from sand facilitates comparisons among studies, and ultimately contributes to the safety of recreational beaches.

Bather Shedding as a Source of Human Fecal Markers to a Recreational Beach.

Microbial source tracking (MST) can identify and locate surf zone fecal indicator bacteria (FIB) sources. However, DNA-based fecal marker results may raise new questions, since FIB and DNA marker sources can differ. Here, during 2 years of summertime (dry season) MST for a Goleta, California recreational beach, surf zone FIB were mainly from gulls, yet low level human-associated DNA-based fecal marker (HF183) was detected in 25 and 14% of surf zone water samples, respectively. Watershed sources were hypothesized because dry weather creek waters had elevated FIB, and runoff-generating rain events mobilized human (and dog) fecal markers and Salmonella spp. into creeks, with human marker HF183 detected in 40 and 50% of creek water samples, dog markers detected in 70 and 50% of samples, and Salmonella spp. in 40 and 33.3% of samples, respectively over 2 years. However, the dry weather estuary outlet was bermed in the first study year; simultaneously, creek fecal markers and pathogens were lower or similar to surf zone results. Although the berm breached in the second year, surf zone fecal markers stayed low. Watershed sediments, intertidal beach sands, and nearshore sediments were devoid of HF183 and dog-associated DNA markers. Based on dye tests and groundwater sampling, beach sanitary sewers were not leaking; groundwater was also devoid of HF183. Offshore sources appeared unlikely, since FIB and fecal markers decreased along a spatial gradient from the surf zone toward nearshore and offshore ocean waters. Further, like other regional beaches, surf zone HF183 corresponded significantly to bather counts, especially in the afternoons when there were more swimmers. However, morning detections of surf zone HF183 when there were few swimmers raised the possibility that the wastewater treatment plant (WWTP) offshore outfall discharged HF183 overnight which transported to the surf zone. These findings support that there may be lowest achievable limits of surf zone HF183 owing to several chronic and permanent, perhaps diurnal, low concentration sources.

Sources of Low Level Human Fecal Markers in Recreational Waters of Two Santa Barbara, CA Beaches: Roles of WWTP Outfalls and Swimmers.

Worldwide, fecal indicator bacteria (FIB) evidence coastal water contamination for which sources are unknown. Here, for two FIB-impacted Santa Barbara recreational beaches, hypothesized fecal sources were investigated over three dry seasons (summers) using nearly 2000 field samples of water (ocean, creek, groundwater), sand, sediments, effluent and fecal sources. In years 1 and 2, gull and dog feces were identified as the probable main FIB sources to surf zone waters, yet HF183 human fecal markers were consistently detected. Determining HF183 sources was therefore prioritized, via year 3 sub-studies. In lower watersheds, human and dog wastes were mobilized by small storms into creeks, but no storm drain outfalls or creeks discharged into surf zones. Beach area bathrooms, sewers, and a septic system were not sources: dye tracing discounted hydraulic connections, and shallow groundwater was uncontaminated. Sediments from coastal creeks and downstream scour ponds, nearshore marine sediments, and sands from inter- and supratidal zones contained neither HF183 nor pathogens. Two nearby wastewater treatment plant (WWTP) outfalls discharged HF183 into plumes that were either deep or distant with uncertain onshore transport. Regardless, local sources were evidenced, as surf zone HF183 detection rates mostly exceeded those offshore and nearshore (around boat anchorages). The presence of swimmers was associated with surf zone HF183, as swimmer counts (on weekdays, holidays, weekends, and during races) significantly correlated (p<0.05, n = 196) to HF183 detections. Besides comprehensively assessing all possible fecal sources, this study provides new explanations of chronic low-level human markers in recreational beach surf zones, suggesting likely lowest achievable HF183 thresholds.

crAssphage as a human molecular marker to evaluate temporal and spatial variability in faecal contamination of urban marine bathing waters.

Bathing water quality may be negatively impacted by diffuse pollution arising from urban and agricultural activities and wildlife, it is therefore important to be able to differentiate between biological and geographical sources of faecal pollution. crAssphage was recently described as a novel human-associated microbial source tracking marker. This study aimed to evaluate the performance of the crAssphage marker in designated bathing waters. The sensitivity and specificity of the crAss_2 marker was evaluated using faecal samples from herring gulls, dogs, sewage and a stream impacted by human pollution (n = 80), which showed that all human impacted samples tested positive for the marker while none of the animal samples did. The crAss_2 marker was field tested in an urban marine bathing water close to the discharge point of human impacted streams. In addition, the bathing water is affected by dog and gull fouling. Analysis of water samples taken at the compliance point every 30 min during a tidal cycle following a rain event showed that the crAss_2 and HF183 markers performed equally well (Spearman correlation ρ = 0.84). The levels of these marker and faecal indicators (Escherichia coli, intestinal enterococci, somatic coliphages) varied by up to 2.5 log10 during the day. Analysis of a high-tide transect perpendicular to the shoreline revealed high levels of localised faecal contamination 1 km offshore, with a concomitant spike in the gull marker. In contrast, both the crAss_2 and HF183 markers remained at a constant level, showing that human faecal contamination is homogenously distributed, while gull pollution is localised. Performance of the crAss_2 and HF183 assay was further evaluated in bimonthly compliance point samples over an 18-month period. The co-occurrence between the crAss_2 and HF183 markers in compliance sampling was 76%. A combination of both markers should be applied in low pollution impacted environments to obtain a high confidence level.

Identifying septic pollution exposure routes during a waterborne norovirus outbreak - A new application for human-associated microbial source tracking qPCR.

In June 2017, the Pennsylvania Department of Health (PADOH) was notified of multiple norovirus outbreaks associated with 179 ill individuals who attended separate events held at an outdoor venue and campground over a month period. Epidemiologic investigations were unable to identify a single exposure route and therefore unable to determine whether there was a persistent contamination source to target for exposure mitigation. Norovirus was detected in a fresh recreational water designated swimming area and a drinking water well. A hydrogeological site evaluation suggested a nearby septic leach field as a potential contamination source via ground water infiltration. Geological characterization revealed a steep dip of the bedrock beneath the septic leach field toward the well, providing a viral transport pathway in a geologic medium not previously documented as high risk for viral ground water contamination. The human-associated microbial source tracking (MST) genetic marker, HF183, was used as a microbial tracer to demonstrate the hydrogeological connection between the malfunctioning septic system, drinking water well, and recreational water area. Based on environmental investigation findings, venue management and local public health officials implemented a series of outbreak prevention strategies including discontinuing the use of the contaminated well, issuing a permit for a new drinking water well, increasing portable toilet and handwashing station availability, and promoting proper hand hygiene. Despite the outbreaks at the venue and evidence of ground water contamination impacting nearby recreational water and the drinking water well, no new norovirus cases were reported during a large event one week after implementing prevention practices. This investigation highlights a new application for human-associated MST methods to trace hydrological connections between multiple fecal pollutant exposure routes in an outbreak scenario. In turn, pollutant source information can be used to develop effective intervention practices to mitigate exposure and prevent future outbreaks associated with human fecal contaminated waters.

Relationships among microbial indicators of fecal pollution, microbial source tracking markers, and pathogens in Costa Rican coastal waters.

Tropical coastal waters are understudied, despite their ecological and economic importance. They also reflect projected climate change scenarios for other climate zones, e.g., increased rainfall and water temperatures. We conducted an exploratory microbial water quality study at a tropical beach influenced by sewage-contaminated rivers, and tested the hypothesis that fecal microorganisms (fecal coliforms, enterococci, Clostridium perfringens, somatic and male-specific coliphages, pepper mild mottle virus (PMMoV), Bacteroides HF183, norovirus genogroup I (NoVGI), Salmonella, Cryptosporidium and Giardia) would vary by season and tidal stage. Most microorganisms' concentrations were greater in the rainy season; however, NoVGI was only detected in the dry season and Cryptosporidium was the only pathogen most frequently detected in rainy season. Fecal indicator bacteria (FIB) levels exceeded recreational water quality criteria standards in >85% of river samples and in <50% of ocean samples, regardless of the FIB or regulatory criterion. Chronic sewage contamination was demonstrated by detection of HF183 and PMMoV in 100% of river samples, and in >89% of ocean samples. Giardia, Cryptosporidium, Salmonella, and NoVGI were frequently detected in rivers (39%, 39%, 26%, and 39% of samples, respectively), but infrequently in ocean water, particularly during the dry season. Multivariate analysis showed that C. perfringens, somatic coliphage, male-specific coliphage, and PMMoV were the subset of indicators that maximized the correlation with pathogens in the rivers. In the ocean, the best subset of indicators was enterococci, male-specific coliphage, and PMMoV. We also executed redudancy analyses on environmental parameters and microorganim concentrations, and found that rainfall best predicted microbial concentrations. The seasonal interplay of rainfall and pathogen prevalence undoubtedly influences beach users' health risks. Relationships are likely to be complex, with some risk factors increasing and others decreasing each season. Future use of multivariate approaches to better understand linkages among environmental conditions, microbial predictors (fecal indicators and MST markers), and pathogens will improve prediction of high-risk scenarios at recreational beaches.

Collection system investigation microbial source tracking (CSI-MST): Applying molecular markers to identify sewer infrastructure failures.

Collection System Investigation Microbial Source Tracking (CSI-MST) is a novel, sensitive approach for identifying sewer infrastructure deficiencies using molecular markers. This method requires both a detailed understanding of collection and conveyance system infrastructure and quickly turned around molecular data to advise an adaptive, targeted in-pipe approach to detect deficiencies. Here we explain the CSI-MST approach and provide several case study examples of how this approach can be adapted to different scale watersheds to identify potential sewer infrastructure issues. This approach has been used to locate and confirm the remediation of numerous needed infrastructure repairs in the southeastern Virginia region. The selected case studies presented here serve as a proof of concept-this methodology can be adopted by other utilities and municipalities to address necessary wastewater infrastructure repairs in different regions.

The challenges of microbial source tracking at urban beaches for Quantitative Microbial Risk Assessment (QMRA).

Urban beaches are frequently impacted from multiple sources of fecal contamination. This along with high beach usage underscores the importance of appropriate management that protects swimmer health. The USEPA has enabled the use of QMRA as a tool for quantifying swimmer health risk and setting site-specific water quality objectives. This study illustrates the challenges associated with human and non-human source identification and how these challenges influence the decision of whether QMRA at typical urban beaches for water quality management is appropriate. In this study, a similar and correlated spatial relationship with elevated Enterococcus and avian-specific markers was observed, suggesting shorebirds as a primary source of FIB. However, human-associated markers were also detected frequently but at low concentrations. Ultimately, a QMRA was not conducted because pathogen loading from potential human sources could not be confidently quantified, having consequences for health risk in receiving waters where recreational contact occurs.

Interlaboratory accuracy and precision among results of three sewage-associated marker genes in urban environmental estuarine waters and freshwater streams.

The application of quantitative polymerase chain reaction (qPCR) based microbial source tracking (MST) marker genes are increasingly being used to identify contaminating sources and inform management decisions. In this study, we assessed interlaboratory agreement on duplicate environmental water samples collected from estuarine and freshwater locations, by comparing results of qPCR based testing for Bacteroides HF183, crAssphage CPQ_056, and pepper mild mottle virus (PMMoV). The overall agreements (co-detection and non-co-detection) between CSIRO Land and Water (CLW) laboratory and Sydney Water (SW) laboratory for the HF183, crAssphage CPQ_056 and PMMoV marker genes for duplicate water samples were 74, 75 and 74%, respectively. Cohene's kappa (k) revealed fair to moderate agreements and acceptable relative percent difference (RPD) values of <15% for duplicate samples. The pooled mean abundances of HF183, CPQ_056, and PMMoV in measurable samples at the CLW laboratory were 5.19 ± 0.93, 5.12 ± 0.82, and 4.42 ± 0.65 log10 copies/L, respectively. However, the pooled mean abundances were significantly lower at the SW laboratory, HF183 (4.58 ± 0.84 log10 copies/L), crAssphage CPQ_056 (4.20 ± 0.63 log10 copies/L), and PMMoV (3.89 ± 0.41 log10 copies/L). At individual sample level, most of the paired samples had <1 log10 difference. Significant positive Spearman rank correlations were obtained between two laboratories for the HF183 (Rs = 0.65; p < 0.05), CPQ_056 (Rs = 0.79; p < 0.05), and PMMoV (Rs = 0.54; p < 0.05) marker genes. Several factors such as standards, qPCR platforms, PCR inhibitors, nucleic acid extraction efficiency and low levels of targets in some samples may have contributed to the observed discrepancies. Results presented in this study highlight the importance of standardized protocol, laboratory equipment (such as digital PCR), sample processing strategies and appropriate quality controls that may need implementation to further improve accuracy and precision of results between laboratories.