CrAssphage are recently-discovered DNA bacteriophages that are prevalent and abundant in human feces and sewage. We assessed the performance characteristics of a crAssphage quantitative PCR (qPCR) ...assay for quantifying sewage impacts in stormwater and surface water in subtropical Tampa, Florida. The mean concentrations of crAssphage in untreated sewage ranged from 9.08 to 9.98 log10 gene copies/L. Specificity was 0.927 against 83 non-human fecal reference samples and the sensitivity was 1.0. Cross-reactivity was observed in DNA extracted from soiled poultry litter but the concentrations were substantially lower than untreated sewage. The presence of the crAssphage marker was monitored in water samples from storm drain outfalls during dry and wet weather conditions in Tampa, Florida. In dry weather conditions, 41.6% of storm drain outfalls samples were positive for the crAssphage marker and the concentrations ranged from 3.60 to 4.65 log10 gene copies/L of water. After a significant rain event, 66.6% of stormwater outlet samples were positive for the crAssphage marker and the concentration ranged from 3.62 to 4.91 log10 gene copies/L of water. The presence of the most commonly used Bacteroides HF183 marker in storm drain outfalls was also tested along with the crAssphage. Thirteen samples (55%) were either positive (i.e., both markers were present) or negative (i.e., both markers were absent) for both the markers. Due to the observed cross-reactivity of this marker with DNA extracted from poultry litter samples, it is recommended that this marker should be used in conjunction with additional markers such as HF183. Our data indicate that the crAssphage marker is highly sensitive to sewage, is adequately specific, and will be a valuable addition to the MST toolbox.
•The crAssphage marker showed high host-sensitivity and -specificity values.•9.08–9.98 log10 gene copies/L of crAssphage was detected in untreated sewage.•CrAssphage marker was detected in storm drain outfall samples.•CrAssphage cross-reacted with poultry litter.
Sewage contamination of environmental waters is increasingly assessed by measuring DNA from sewage-associated microorganisms in microbial source tracking (MST) approaches. However, DNA can persist ...through wastewater treatment and reach surface waters when treated sewage/recycled water is discharged, which may falsely indicate pollution from untreated sewage. Recycled water discharged from an advanced wastewater treatment (AWT) facility into a Florida stream elevated the sewage-associated HF183 marker 1,000-fold, with a minimal increase in cultured Escherichia coli. The persistence of sewage-associated microorganisms was compared by qPCR in untreated sewage and recycled water from conventional wastewater treatment (CWT) and AWT facilities. E. coli (EC23S857) and sewage-associated markers HF183, H8, and viral crAssphage CPQ_056 were always detected in untreated sewage (6.5–8.7 log10 GC/100 mL). Multivariate analysis found a significantly greater reduction of microbial variables via AWT vs CWT. Bacterial markers decayed ~4–5 log10 through CWT, but CPQ_056 was ~100-fold more persistent. In AWT facilities, the log10 reduction of all variables was ~5. In recycled water, bacterial marker concentrations were significantly correlated (P ≤ 0.0136; tau ≥ 0.44); however, CPQ_056 was not correlated with any marker, suggesting varying drivers of decay. Concentrations of cultured E. coli carrying the H8 marker (EcH8) in untreated sewage were 5.24–6.02 log10 CFU/100 mL, while no E. coli was isolated from recycled water. HF183 and culturable EcH8 were also correlated in contaminated surface waters (odds ratio β1 = 1.701). Culturable EcH8 has a strong potential to differentiate positive MST marker signals arising from treated (e.g., recycled water) and untreated sewage discharged into environmental waters.IMPORTANCEGenes in sewage-associated microorganisms are widely accepted indicators of sewage pollution in environmental waters. However, DNA persists through wastewater treatment and can reach surface waters when recycled water is discharged, potentially causing false-positive indications of sewage contamination. Previous studies have found that bacterial and viral sewage-associated genes persist through wastewater treatment; however, these studies did not compare different facilities or identify a solution to distinguish sewage from recycled water. In this study, we demonstrated the persistence of bacterial marker genes and the greater persistence of a viral marker gene (CPQ_056 of crAssphage) through varying wastewater treatment facilities. We also aim to provide a tool to confirm sewage contamination in surface waters with recycled water inputs. This work showed that the level of wastewater treatment affects the removal of microorganisms, particularly viruses, and expands our ability to identify sewage in surface waters.
•Rainfall was the best predictor of microbial variables when river and ocean data were separated.•Sewage indicators HF183 and PMMoV were detected in >88% of samples.•Norovirus was detected only ...during dry season; Cryptosporidium was more prevalent in rainy season.•Multiple indicators, particularly viruses, correlated with pathogens; single indicators did not.•Multivariate analysis identified relationships of environmental and indicator variables with pathogens.
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.
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Estuarine water quality is declining worldwide due to increased tourism, coastal development, and a changing climate. Although well-established methods are in place to monitor water quality, ...municipalities struggle to use the data to prioritize infrastructure for monitoring and repair and to determine sources of contamination when they occur. The objective of this study was to assess water quality and prioritize sources of contamination within Town Creek Estuary (TCE), Beaufort, North Carolina, by combining culture, molecular, and geographic information systems (GIS) data into a novel contamination source ranking system. Water samples were collected from TCE at ten locations on eight sampling dates in Fall 2021 (n = 80). Microbiological water quality was assessed using US Environmental Protection Agency (U.S. EPA) approved culture-based methods for fecal indicator bacteria (FIB), including analysis of total coliforms (TC), Escherichia coli (EC), and Enterococcus spp. (ENT). The quantitative microbial source tracking (qMST) human-associated fecal marker, HF183, was quantified using droplet digital PCR (ddPCR). This information was combined with environmental data and GIS information detailing proximal sewer, septic, and stormwater infrastructure to determine potential sources of fecal contamination in the estuary. Results indicated FIB concentrations were significantly and positively correlated with precipitation and increased throughout the estuary following rainfall events (p < 0.01). Sampling sites with FIB concentrations above the U.S. EPA threshold also had the highest percentages of aged, less durable piping materials. Using a novel ranking system combining concentrations of FIB, HF183, and sewer infrastructure data at each site, we found that the two sites nearest the most aged sewage infrastructure and stormwater outflows were found to have the highest levels of measurable fecal contamination. This case study supports the inclusion of both traditional water quality measurements and local infrastructure data to support the current need for municipalities to identify, prioritize, and remediate failing infrastructure.
The use of microbial source tracking (MST) marker genes has grown in recent years due to the need to attribute point and non-point fecal contamination to specific sources. Quantitative microbial risk ...assessment (QMRA) is a modeling approach used to estimate health risks from exposure to feces-contaminated water and associated pathogens. A combination of these approaches quantitative MST (qMST) and QMRA can provide additional pathogen-related information for prioritizing and addressing health risks, compared to reliance on conventional fecal indicator bacteria (FIB). To inform expansion of this approach, a review of published qMST-QMRA studies was conducted to summarize the state of the science and to identify research needs. The reviewed studies primarily aimed to identify what levels of MST marker genes in hypothetical recreational waterbodies would exceed the United States Environmental Protection Agency (USEPA) risk benchmarks for primary contact recreators. The QMRA models calculated relationships between MST marker gene(s) and reference pathogens based on published data in the literature. The development of a robust, accurate relationship was identified as an urgent research gap for qMST-QMRA. This metric requires additional knowledge to quantify the relationship between MST marker genes and the degree of variability in decay of pathogens as a dynamic function of environmental conditions and combinations of fecal sources at multiple spatial and temporal scales. Improved characterization of host shedding rates of host-associated microorganisms (i.e., MST marker genes), as well as fate and transport of these microorganisms and their nucleic acids, would facilitate expansion of this approach to other exposure pathways. Incorporation of information regarding the recovery efficiency, and host-specificity of MST marker genes into QMRA model parameters, and the sensitivity analysis, would greatly improve risk management and site-specific water monitoring criteria.
•qMST and QMRA can provide information for prioritizing health risks.•Accurate relationship was identified as an urgent research gap for qMST-QMRA.•Recovery efficiency, host-specificity and sensitivity analysis need to be considered for QMRA model.
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.
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 log
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.
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.
Background: Gastrointestinal disease affects millions of people in the United States and places a substantial economic burden upon healthcare systems. Recreational waters polluted with fecal material ...are a main source for transmission of gastrointestinal disease. Georgia beaches are monitored for the presence of fecal indicator bacteria, but these bacteria are not well associated with enteric viruses. The United States Environmental Protection Agency (US EPA) has recently proposed coliphage (a virus of Escherichia coli) as an alternative indicator of fecal contamination in recreational waters. The present study compares fecal indicator bacteria and coliphage concentrations at two Georgia beaches with adjacent creeks that have a history of pollution. Methods: For one year, samples and environmental data were collected from four sites on Jekyll Island, GA, during the peak swimming season and the off-season. Samples were processed using US EPA-approved methods for membrane filtration and plaque formation. Statistical analyses were performed using t-tests and Spearman correlations. Results: The highest numbers of enterococci and significant differences with coliphage were found at Saint Andrews Creek during the swimming season and the off-season. The enterococci concentrations at Clam Creek sites did not exceed recommended recreational water criteria. During the off-season, concentrations of enterococci and coliphages were different at Clam Creek sites, indicating a potential risk for presence of enteric virus when enterococci could not be detected. Conclusions: The US EPA has proposed to adapt coliphage concentrations as an alternative indicator of water pollution for routine beach monitoring nationally. The present study provides a background for adoption of this method in Georgia. Measures of enterococci do not provide sufficient information about the associated human health risk. Inclusion of these viral indicators will improve decision making for beach closures and for protection of the health of swimmers. Keywords: coliphage, enterococci, enteric viruses, beach water quality, health risk, Georgia
Wastewater monitoring data can be used to estimate disease trends to inform public health responses. One commonly estimated metric is the rate of change in pathogen quantity, which typically ...correlates with clinical surveillance in retrospective analyses. However, the accuracy of rate of change estimation approaches has not previously been evaluated.
We assessed the performance of approaches for estimating rates of change in wastewater pathogen loads by generating synthetic wastewater time series data for which rates of change were known. Each approach was also evaluated on real-world data.
Smooth trends and their first derivatives were jointly sampled from Gaussian processes (GP) and independent errors were added to generate synthetic viral load measurements; the range hyperparameter and error variance were varied to produce nine simulation scenarios representing different potential disease patterns. The directions and magnitudes of the rate of change estimates from four estimation approaches (two established and two developed in this work) were compared to the GP first derivative to evaluate classification and quantitative accuracy. Each approach was also implemented for public SARS-CoV-2 wastewater monitoring data collected January 2021–May 2023 at 25 sites in North Carolina, USA.
All four approaches inconsistently identified the correct direction of the trend given by the sign of the GP first derivative. Across all nine simulated disease patterns, between a quarter and a half of all estimates indicated the wrong trend direction, regardless of estimation approach. The proportion of trends classified as plateaus (statistically indistinguishable from zero) for the North Carolina SARS-CoV-2 data varied considerably by estimation method but not by site.
Our results suggest that wastewater measurements alone might not provide sufficient data to reliably track disease trends in real-time. Instead, wastewater viral loads could be combined with additional public health surveillance data to improve predictions of other outcomes.
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•Wastewater surveillance is increasingly used to estimate population disease trends.•The accuracy of common trend estimation approaches has not been evaluated.•Synthetic wastewater time series were generated with known trend rates of change.•All approaches estimated the wrong trend direction on 25 % or more days.•Wastewater measurements alone may not reliably track disease trends in real time.
Fecal contamination at recreational beaches impacts the health of beachgoers, through the introduction of disease-causing microorganisms, and the well-being of communities dependent on income from ...recreational beach activities. Beach ecosystems are also impacted by sewage through the introduction of nutrients that can cause abnormal increases in autochthonous microorganisms which can impact the population of larger organisms in the ecosystem. Fecal contamination is introduced into sand via untreated sewage, direct deposition of human feces into sand, runoff, and deposition of animal feces into sand. The introduction of fecal contamination into sand exposes individuals to pathogens (disease causing microorganisms) which can result in gastrointestinal illness. While standard methods and regulatory guidelines exist for the monitoring of fecal contamination in water, none exist for sand despite the data that link recreational contact with sand to gastrointestinal illness. The detection of fecal-associated pathogens in the environment is difficult due to their diversity and low concentration, therefore contamination is monitored using fecal indicator bacteria (FIB). FIB are present in the gastrointestinal tract of animals and provide an indication of the presence of fecal contamination. Escherichia coli (freshwater) and enterococci (fresh and salt water) are commonly used as FIB globally and in the United States. In sand, FIB are present at higher concentrations than in water, as sand protects FIB from environmental stressors such as UV or predation and provides easier access to nutrients. FIB that survive long-term and replicate in sand are termed “naturalized.” Naturalized populations can complicate the identification of recent fecal contamination, as FIB monitoring techniques cannot differentiate between naturalized FIB and those recently introduced through sources of fecal contamination. The ubiquity of FIB among animals and humans means it is also difficult to determine the source of contamination. Some sources of fecal contamination (human as compared to cow or bird fecal contamination) contain a higher number of pathogens that are likely to infect humans (adenovirus in humans or Cryptosporidium spp. from cows). Therefore, microbial source tracking (MST) was developed to include a suite of host-associated genes (markers) of fecal microorganisms that are specific to different animals. These markers can differentiate between sources of fecal contamination, supplementing FIB monitoring and providing a more accurate depiction of the fecal contamination picture. Few studies have investigated the presence of MST markers in sand or determined relationships between MST markers and FIB in sand. The human health risk associated with exposure to fecal contamination can be assessed by epidemiological studies; however, these studies are typically expensive and require specialized teams that may not be available to organizations with limited resources. Quantitative microbial risk assessment (QMRA) is a mathematical modeling framework used to estimate human health risk (the likelihood someone becomes ill) from exposure to pathogens under different environmental scenarios. QMRA consists of four steps: Problem formulation, exposure assessment, dose-response modeling, and risk characterization. Problem formulation involves establishing the framework (reference pathogen identification, exposure pathway, sources of contamination) that helps target the risk management needs to be addressed. The exposure assessment then determines the concentration and frequency of exposure by individuals to the reference pathogen(s) and exposure pathway identified during the problem formulation step, also known as a dose. The dose is then compared to a dose-response model (typically sourced from the literature) to determine the probability that the estimated dose would lead to illness in an individual. Risk characterization then quantifies the level of risk based on the modeled data. Reverse QMRA, used in this experiment, estimates the probabilities of pathogen concentrations that correspond with a risk threshold that is defined by stakeholders. Few QMRAs have been conducted in sand, therefore, this dissertation is focused on understanding the human health risk from exposure to pathogens at a tropical beach impacted by sewage. Jacó beach is a tropical beach located on the Pacific coast of Costa Rica and is a popular tourist destination. A water quality study determined the fecal contamination was severely impacting the water quality of the beach and placing individuals at risk of illness from exposure to contaminated waters. Understanding the human health risk from exposure to pathogens in beach sand, in addition to the human health risk from water, ultimately helps to improve beach management decisions and public health. In chapter one, the analytical sensitivity of quantitative polymerase chain reaction (qPCR) for Enterococcus in sand was compared for the slurry (suspension, agitation, membrane filtration of supernatant), versus two direct extraction methods using PowerSoil™ or PowerMax Soil™ kits at a freshwater and saltwater beach in Tampa, Fl, USA. We found the slurry method had the lowest limit of detection at 20–80 gene copies g-1 (wet weight), recovered significantly more DNA, and was the only method that detected Enterococcus by qPCR in all samples; therefore, the slurry method was exclusively used in subsequent experiments. The slurry method reflected the spatial variability of Enterococcus in individual transect samples. Mean recovery efficiency of the human-associated microbial source tracking marker HF183 from marine and freshwater beach sand spiked with wastewater was 100.8% and 64.1%, respectively, but varied between dilutions, indicating that the mixing protocol needs improvement. The objective of chapter two was to determine the extent of the influence a contaminated waterbody has on the concentration of microbes in beach sand. Upstream, downstream and ocean samples were collected on a transect in sand at 0 m (origin), 2.5 m and 5.0 m from the riverbank or swash zone. Samples were also collected in sediment. Samples were analyzed for the presence of the MST marker HF183 and FIB Enterococcus using the slurry method followed by DNA extraction and qPCR. Median concentrations of Enterococcus decreased as distance from the river or ocean increased. Enterococcus ranged from 1.46 x 104 to 8.11 x 103 gene copies 100 g-1 to the same distance. HF183 and Enterococcus were positively correlated at the riverbank/swash zone, but not at other subsites due to frequent failure to detect HF183. Sediment samples did not differ in HF183 or Enterococcus concentrations along the river and HF183 was not detected in the ocean sediment. The frequency of detection for HF183 was significantly greater among samples along the riverbank than samples collected at the 2.5 m and 5 m subsites. Polluted waterbodies can influence microbe concentrations in sand but the extent of the influence of a waterbody on MST markers in sand requires further study.Chapter three employed a reverse QMRA in Copey River at Jacó Beach to estimate the varying probability that corresponds with meeting a risk target. The risk target was defined as 36 cases of gastrointestinal illness per 1000 people (36/1000), the health target set in the 2012 recreational water quality guidelines for the United States Environmental Protection Agency (USEPA). Data were used to calculate whether concentrations of Salmonella, Adenovirus, and Giardia exceeded or were below concentrations needed to meet a risk of 36/1000. Samples at two sites, one upstream and one downstream of where beachgoers tended to recreate at the riverbank were collected six times over three weeks. Samples were analyzed for the presence of enterococci (FIB), the human-host associated MST marker HF183 and sewage-associated MST marker PMMoV, and pathogens Salmonella, adenovirus, and Giardia. Sand samples were collected at the riverbank and analyzed for the fecal indicator bacteria enterococci, the human-host associated microbial source tracking marker genes HF183, sewage-associated marker pepper mild mottle virus (PMMoV), and pathogens Salmonella, Giardia, and human adenovirus. Enterococci and Salmonella were cultured using the slurry method followed by USEPA standard methods. HF183, PMMoV, and adenovirus were detected by (RT)-qPCR. Giardia was detected using microscopy following USEPA standard methodology. Enterococci were detected in all samples and had a geometric mean of 3.25 x 103 GC 100 g-1. Geometric means of the MST markers HF183 and PMMoV were 1.19 x 103 and 1.64 x103 GC 100 g-1 and were detected in 83% and 33% of samples, respectively. Salmonella was detected in 66.6% of samples and had a geometric mean of 2.45 x 102 CFU 100 g-1. Giardia and adenovirus were detected only once each at 45 oocysts 100 g-1 and 1.91 x 102 GC 100 g-1. QMRA analysis showed that adenovirus and Giardia concentrations exceeded the levels that coincide with the risk threshold of 36/1000 while Salmonella fell between the 50th and 75th % percentile probability of meeting the risk threshold. Exposure to sewage-borne pathogens in sand could contribute to the risk of illness for recreational users of Jacó Beach and should be analyzed in tandem with pathogens in water to implement the best strategy for the protection of public health. To implement a comprehensive beach monitoring strategy, it is important to understand all the contextual factors that affect public health at a beach. Recreation in sand has been linked to gastrointestinal illness, however there are no regulatory guidelines or standard methods for analysis of fecal microorganisms in sand. This research has shown that a comprehensive beach monitoring strategy should include sand monitoring strategies in tandem with water monitoring strategies.
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