Source Tracking Markers Research Articles

The probability of detecting host-specific microbial source tracking markers in surface waters was strongly associated with method and season.

Fecal contamination of surface waters presents significant human health and environmental risks. While many methods for understanding fecal contamination cannot differentiate between human and animal sources, microbial source tracking (MST) marker methods are used to identify fecal sources. To understand how to best employ MST marker data for managing fecal contamination risks, the present study compiled previously collected MST marker data (N = 12,878 samples) from across North America; each sample in the data set had been tested for fecal contamination from one or more of five sources (avian, canine, human, ruminant, swine). Using these data, this study aimed to characterize associations between non-methodological and methodological factors and detection of host-specific MST markers and determine how methodological differences may complicate the interpretation of these associations between studies. Variance partitioning analysis was used to quantify the variance in host-specific MST marker detection attributable to non-methodological and methodological factors. Conditional forest and regression analysis were utilized to assess the association between detection and select non-methodological and methodological factors. Between 10% (canine) and 38% (human) of total variance was uniquely attributable to non-methodological factors for any of the fecal sources considered, while between 50% (human) and 84% (canine) of variance could not be attributed to either methodological or non-methodological factors. This highlights the need for standardization of methods for MST marker detection across studies but also suggests that other factors, beyond those non-methodological variables considered here, influenced variation in the likelihood of MST marker detection.IMPORTANCEThis study underscores complications associated with comparing findings from studies that used different methodologies to detect the same fecal targets and highlights the difficulties associated with using non-comparable data to generalize findings and develop science-based risk management plans. These findings highlight the need for standardization of sampling and laboratory methods across microbial source tracking marker studies. Our findings build on previous research to suggest that one-size-fits-all approaches to managing fecal hazards in surface waterways may not be appropriate; instead, strategies tailored to specific water sources and conditions at the time of water use may be more effective.

The Identification of Predominant Faecal Contamination Sources in Water Using Host-Specific Genetic Markers in Water-Stressed Rural Communities of Vhembe District Municipality, South Africa

It is critical to attribute faecal contamination to its original source in order to assess public health risks and implement effective interventions to mitigate future contamination. This study aimed to identify the primary sources of faecal contamination in water using microbial source tracking markers in water-stressed rural communities. A total of 1128 water samples were collected sequentially from the main source (river/borehole) to the households. Six host-specific genetic markers were used to detect faecal contamination in the water samples (BacHum and HF183, BacCow, Pig-2-Bac, Cytb and BacCan). Of the 564 water samples tested during the wet season, 37.94% (n = 214) were positive for human and animal-specific Bacteroidales marker genes, while 31.73% (n = 179) of the 564 tested during the dry season were also positive. During the wet season, animal faecal contamination was more prevalent among the positive samples (Cytb: 20.56%, n = 44; BacCan: 19.16%, n = 41). By contrast, human-origin faecal contamination was dominant during the dry season (BacHum: 23.46%, n = 42; HF183: 21.23%, n = 38). Identifying the origin of faecal contamination will assist in implementing targeted intervention strategies for the effective prevention of pathogen transmission in water-stressed rural communities in order to protect public health.

Assessing human-source microbial contamination of stormwater in the U.S

Stormwater capture and use (SCU) projects have the potential to provide a significant portion of municipal water demand. However, uncertainty about the degree of microbial contamination in stormwater and the required treatment is a barrier for implementation of SCU projects. Stormwater runoff could become contaminated with human fecal matter in areas with deteriorating infrastructure where raw wastewater exfiltrates from sewer networks to stormwater collection networks, homeless encampments exist, or sanitary sewer overflows (SSOs) occur. Estimation of human fecal contamination can inform selection of stormwater treatment targets. This study investigates stormwater microbial contamination originating from human fecal matter using observed detections and concentrations of human microbial source tracking (MST) markers and potentially human-infectious pathogens (PHIPs). First, a systematic review complied measurements of human MST markers in wet and dry weather stormwater flows and influent wastewater. In addition, measurements of viral pathogens (e.g., adenoviruses, norovirus GI+GII, and enteroviruses) and protozoan pathogens (e.g., Giardia lamblia and Cryptosporidium parvum) in wet weather flows and influent wastewater were assessed. Human MST marker and PHIP data were statistically analyzed and applied to estimate a human fecal contamination analog (HFCA) which is an estimate of the amount of human fecal matter based on relative concentrations of microbial contaminants in stormwater compared to municipal wastewater. Human MST-based HFCAs in wet and dry weather flows ranged from <10−7.0 to 10−1.5 (median = 10−4.5) and 10−12 to 10−2.6 (median = 10−7.0), respectively. PHIP-based HFCAs in wet weather flows ranged from ∼10−8 to 10−0.14. Estimates of human MST-based HFCAs are more reliable than PHIP-based HFCAs because the current PHIP datasets are generally limited by the number of data points, percent detection, variability observed within the statistical distributions, and the geographical span of sampling locations. The use of human MST-based HFCAs is recommended to guide the selection of stormwater treatment process trains that are protective of public health based on the intended end use. Application of HFCA 10−1 (i.e., sewage dilution 10−1) remains a reasonable conservative estimate of human fecal contamination in stormwater to inform selection of pathogen log reduction targets based on the data presently available.

The use of E. coli phylogrouping and microbial source tracking (non-species specific, human-specific, bovine-specific bacteroidales markers) to elucidate hydro(geo)logical contamination mechanisms in southeastern Ontario, Canada

In Ontario, monitoring, maintenance, and treatment of private drinking systems (e.g. wells) are the responsibility of the well owner. Fecal contamination of drinking water threatens public health, particularly in rural communities which are often fully reliant on unregulated private groundwater as a primary drinking water source. Private well users face a higher risk of acute gastrointestinal illness compared to those served by municipally operated systems (Murphy et al., 2016). Accordingly, the current study sought to characterize the fecal indicator, E. coli, isolated from southeastern Ontario private groundwater wells, including phylogroups and host source. Results were examined in the context of antecedent climate and local hydrogeological setting to elucidate likely contaminant sources and pathways. A total of 737 E. coli isolates from 260 private wells were assigned to phylogroups using the Clermont PCR phylotyping method, with likely host source determined using host-specific Bacteroidales 16S rRNA RT qPCR assays. Multivariate models were developed for the main E. coli phylogroups (A, B1, B2, and D) and all microbial source tracking markers. Models were coupled for interpretation where possible, based on associations between phylogroups and MST markers. Preferential subsurface flow, and to a lesser degree, overland flow, were likely mechanisms of contamination across all models. Distinct temporal associations were found based on the fecal source. Multiple models were developed and will be discussed, in an attempt to elucidate source-specific contamination mechanisms, in support of risk assessment and appropriate protective actions.

Development of a novel crAss-like phage detection method with a broad spectrum for microbial source tracking

CrAssphage has been recognized as the most abundant and human-specific bacteriophage in the human gut. Consequently, crAssphage has been used as a microbial source tracking (MST) marker to monitor human fecal contamination. Many crAss-like phages (CLPs) have been recently discovered, expanding the classification into the new order Crassvirales. This study aims to assess CLP prevalence in South Korea and develop a detection system for MST applications. Thirteen CLPs were identified in six human fecal samples and categorized into seven genera via metagenomic analysis. The major head protein (MHP) displayed increased sequence similarity within each genus. Eight PCR primer candidates, designed from MHP sequences, were evaluated in animal and human feces. CLPs were absent in animal feces except for those from raccoons, which hosted genera VI, VIIa, and VIIb. CLPs were detected in 91.52% (54/59) of humans, with genus VI (38 out of 59) showing the highest prevalence, nearly double that of p-crAssphage in genus I (22 out of 59). This study highlights genus VI as a potent MST marker, broadening the detection range for CLPs. Human-specific and selectively targeted MST markers can significantly impact hygiene regulations, lowering public health costs through their application in screening liver, sewage, wastewater, and various environmental samples.

Benefits and Challenges of Applying Bacteriophage Biocontrol in the Consumer Water Cycle.

Bacteria (including disinfection- and antibiotic-resistant bacteria) are abundant in the consumer water cycle, where they may cause disease, and lead to biofouling and infrastructure damage in distributions systems, subsequently resulting in significant economic losses. Bacteriophages and their associated enzymes may then offer a biological control solution for application within the water sector. Lytic bacteriophages are of particular interest as biocontrol agents as their narrow host range can be exploited for the targeted removal of specific bacteria in a designated environment. Bacteriophages can also be used to improve processes such as wastewater treatment, while bacteriophage-derived enzymes can be applied to combat biofouling based on their effectiveness against preformed biofilms. However, the host range, environmental stability, bacteriophage resistance and biosafety risks are some of the factors that need to be considered prior to the large-scale application of these bacterial viruses. Characteristics of bacteriophages that highlight their potential as biocontrol agents are thus outlined in this review, as well as the potential application of bacteriophage biocontrol throughout the consumer water cycle. Additionally, the limitations of bacteriophage biocontrol and corresponding mitigation strategies are outlined, including the use of engineered bacteriophages for improved host ranges, environmental stability and the antimicrobial re-sensitisation of bacteria. Finally, the potential public and environmental risks associated with large-scale bacteriophage biocontrol application are considered, and alternative applications of bacteriophages to enhance the functioning of the consumer water cycle, including their use as water quality or treatment indicators and microbial source tracking markers, are discussed.

Scenario-based assessment of fecal pathogen sources affecting bathing water quality: novel treatment options to reduce norovirus and Campylobacter infection risks.

Wastewater discharge and runoff waters are significant sources of human and animal fecal microbes in surface waters. Human-derived fecal contamination of water is generally estimated to pose a greater risk to human health than animal fecal contamination, but animals may serve as reservoirs of zoonotic pathogens. In this study, quantitative microbial risk assessment (QMRA) tools were used to evaluate the hygienic impact of sewage effluents and runoff water from municipalities and animal farms on surface and bathing waters. The human-specific microbial source tracking (MST) marker HF183 was used to evaluate the dilution of fecal pathogens originating from the sewage effluent discharge to the downstream watershed. As novel risk management options, the efficiency of UV-LED disinfection and wetland treatment as well as biochar filtration was tested on-site for the contamination sources. According to the dilution pattern of the MST marker HF183, microbes from wastewater were diluted (2.3-3.7 log10) in the receiving waters. The scenario-based QMRA revealed, that the health risks posed by exposure to human-specific norovirus GII and zoonotic Campylobacter jejuni during the bathing events were evaluated. The risk for gastroenteritis was found to be elevated during wastewater contamination events, where especially norovirus GII infection risk increased (1-15 cases per day among 50 bathers) compared with the business as usual (BAU) situation (1 case per day). The noted C. jejuni infection risk was associated with animal farm contamination (1 case per day, versus 0.2-0.6 cases during BAU). Tertiary treatment of wastewater with wetland treatment and UV-LED disinfection effectively reduced the waterborne gastroenteritis risks associated with bathing. Based on the experiences from this study, a QMRA-based approach for health risk evaluations at bathing sites can be useful and is recommended for bathing site risk assessments in the future. In case of low pathogen numbers at the exposure sites, the MST marker HF183 could be used as a pathogen dilution coefficient for the watershed under evaluation. The full-scale implementation of novel tertiary treatment options at wastewater treatment plants (WWTPs) as well as on-site runoff water treatment options should be considered for infection risk management at locations where scenario-based QMRA implies elevated infection risks.

Is detection of enteropathogens and human or animal faecal markers in the environment associated with subsequent child enteric infections and growth: an individual participant data meta-analysis

Quantifying contributions of environmental faecal contamination to child diarrhoea and growth faltering can illuminate causal mechanisms behind modest health benefits in recent water, sanitation, and hygiene (WASH) trials. We aimed to assess associations between environmental detection of enteropathogens and human or animal microbial source tracking markers (MSTM) and subsequent child health outcomes. In this individual participant data meta-analysis we searched we searched PubMed, Embase, CAB Direct Global Health, Agricultural and Environmental Science Database, Web of Science, and Scopus for WASH intervention studies with a prospective design and concurrent control that measured enteropathogens or MSTM in environmental samples, or both, and subsequently measured enteric infections, diarrhoea, or height-for-age Z-scores (HAZ) in children younger than 5 years. We excluded studies that only measured faecal indicator bacteria. The initial search was done on Jan 19, 2021, and updated on March 22, 2023. One reviewer (AM) screened abstracts, and two independent reviewers (AM and RT) examined the full texts of short-listed articles. All included studies include at least one author that also contributed as an author to the present Article. Our primary outcomes were the 7-day prevalence of caregiver-reported diarrhoea and HAZ in children. For specific enteropathogens in the environment, primary outcomes also included subsequent child infection with the same pathogen ascertained by stool testing. We estimated associations using covariate-adjusted regressions and pooled estimates across studies. Data from nine published reports from five interventions studies, which included 8603 children (4302 girls and 4301 boys), were included in the meta-analysis. Environmental pathogen detection was associated with increased infection prevalence with the same pathogen and lower HAZ (ΔHAZ -0·09 [95% CI -0·17 to -0·01]) but not diarrhoea (prevalence ratio 1·22 [95% CI 0·95 to 1·58]), except during wet seasons. Detection of MSTM was not associated with diarrhoea (no pooled estimate) or HAZ (ΔHAZ -0·01 [-0·13 to 0·11] for human markers and ΔHAZ -0·02 [-0·24 to 0·21] for animal markers). Soil, children's hands, and stored drinking water were major transmission pathways. Our findings support a causal chain from pathogens in the environment to infection to growth faltering, indicating that the lack of WASH intervention effects on child growth might stem from insufficient reductions in environmental pathogen prevalence. Studies measuring enteropathogens in the environment should subsequently measure the same pathogens in stool to further examine theories of change between WASH, faecal contamination, and health. Given that environmental pathogen detection was predictive of infection, programmes targeting specific pathogens (eg, vaccinations and elimination efforts) can environmentally monitor the pathogens of interest for population-level surveillance instead of collecting individual biospecimens. The Bill & Melinda Gates Foundation and the UK Foreign and Commonwealth Development Office.

CrAss-Like Phages: From Discovery in Human Fecal Metagenome to Application as a Microbial Source Tracking Marker.

CrAss-like phages are a diverse group of bacteriophages genetically similar to the prototypical crAssphage (p-crAssphage), which was discovered in the human gut microbiome through a metagenomics approach. It was identified as a ubiquitous and highly abundant bacteriophage group in the gut microbiome. Initial co-occurrence analysis postulated Bacteroides spp. as the prospective bacterial host. Subsequent studies have confirmed multiple host species under Phylum Bacteroidetes and some Firmicutes. Detection of crAss-like phages in sewage-contaminated environmental water and robust correlation with enteric viruses and bacteria has culminated in their adoption as a microbial source tracking (MST) marker. Polymerase chain reaction (PCR) and real-time PCR assays have been developed utilizing the conserved genes in the p-crAssphage genome to detect human fecal contamination of different water sources, with high specificity. Numerous investigations have examined the implications of crAss-like phages in diverse disease conditions, including ulcerative colitis, obesity and metabolic syndrome, autism spectrum disorders, rheumatoid arthritis, atopic eczema, and other autoimmune disorders. These studies have unveiled associations between certain diseases and diminished abundance and diversity of crAss-like phages. This review offers insights into the diverse aspects of research on crAss-like phages, including their discovery, genomic characteristics, structure, taxonomy, isolation, molecular detection, application as an MST marker, and role as a gut microbiome modulator with consequential health implications.

β-D-glucuronidase activity triggered monitoring of fecal contamination using microbial and chemical source tracking markers at drinking water intakes

Intense rainfall and snowmelt events may affect the safety of drinking water, as large quantities of fecal material can be discharged from storm or sewage overflows or washed from the catchment into drinking water sources. This study used β-d-glucuronidase activity (GLUC) with microbial source tracking (MST) markers: human, bovine, porcine mitochondrial DNA markers (mtDNA) and human-associated Bacteroidales HF183 and chemical source tracking (CST) markers including caffeine, carbamazepine, theophylline and acetaminophen, pathogens (Giardia, Cryptosporidium, adenovirus, rotavirus and enterovirus), water quality indicators (Escherichia coli, turbidity) and hydrometeorological data (flowrate, precipitation) to assess the vulnerability of 3 drinking water intakes (DWIs) and identify sources of fecal contamination. Water samples were collected under baseline, snow and rain events conditions in urban and agricultural catchments (Québec, Canada). Dynamics of E. coli, HF183 and WWMPs were similar during contamination events, and concentrations generally varied over 1 order of magnitude during each event. Elevated human-associated marker levels during events demonstrated that urban DWIs were impacted by recent contamination from an upstream municipal water resource recovery facility (WRRF). In the agricultural catchment, mixed fecal pollution was observed with the occurrences and increases of enteric viruses, human bovine and porcine mtDNA during peak contaminating events. Bovine mtDNA qPCR concentrations were indicative of runoff of cattle-derived fecal pollutants to the DWI from diffuse sources following rain events. This study demonstrated that the suitability of a given MST or CST indicator depend on river and catchment characteristics. The sampling strategy using continuous online GLUC activity coupled with MST and CST markers analysis was a more reliable source indicator than turbidity to identify peak events at drinking water intakes.

Precipitation impacts the physicochemical water quality and abundance of microbial source tracking markers in urban Texas watersheds

This study investigated the sources of fecal pollution in surface and groundwaters collected from three urban watersheds in Texas during dry and wet weather and identified the impact of precipitation on water quality. Water samples (n = 316 and 107 for dry and wet events, respectively) were collected biweekly from eight sampling sites (6 sites from creeks and ponds, and 2 well sites) during two-year monitoring and analyzed for six physico-chemical parameters and ten qPCR-based assays targeting general (E. coli, Enterococcus, and universal Bacteroidales), human (BacHum and HF183), animal (Rum2Bac, BacCow, BacCan), and avian (Chicken/Duck-Bac and GFD) fecal markers. Elevated concentrations of NO3-N and NO2-N were observed in ponds and creeks sites during wet weather. Fecal markers analysis indicated higher concentrations of Rum2Bac, BacCow, and BacCan markers in most of pond and creek sites under wet weather, suggesting stormwater runoff contributed to non-point sources of fecal contamination by animal sources. Furthermore, sporadically higher concentrations of these markers were detected at groundwater sampling sites, demonstrating the significant human health risk. Multivariate statistical analysis such as cluster analysis (CA) and principal coordinate analysis (PCoA) was performed to identify relationship between sampling sites; while CA majorly classified ponds, creeks, and well sites separately, PCoA identified similarities in water quality characteristics between waters of wells with ponds and creeks. Overall, results indicate ruminant and dog fecal contamination is a major concern during storm events, consequently impacting surface and groundwater quality of the study.

Household environment and animal fecal contamination are critical modifiers of the gut microbiome and resistome in young children from rural Nicaragua

BackgroundEarly life plays a vital role in the development of the gut microbiome and subsequent health. While many factors that shape the gut microbiome have been described, including delivery mode, breastfeeding, and antibiotic use, the role of household environments is still unclear. Furthermore, the development of the gut antimicrobial resistome and its role in health and disease is not well characterized, particularly in settings with water insecurity and less sanitation infrastructure.ResultsThis study investigated the gut microbiome and resistome of infants and young children (ages 4 days-6 years) in rural Nicaragua using Oxford Nanopore Technology’s MinION long-read sequencing. Differences in gut microbiome diversity and antibiotic resistance gene (ARG) abundance were examined for associations with host factors (age, sex, height for age z-score, weight for height z-score, delivery mode, breastfeeding habits) and household environmental factors (animals inside the home, coliforms in drinking water, enteric pathogens in household floors, fecal microbial source tracking markers in household floors). We identified anticipated associations of higher gut microbiome diversity with participant age and vaginal delivery. However, novel to this study were the significant, positive associations between ruminant and dog fecal contamination of household floors and gut microbiome diversity. We also identified greater abundance of potential pathogens in the gut microbiomes of participants with higher fecal contamination on their household floors. Path analysis revealed that water quality and household floor contamination independently and significantly influenced gut microbiome diversity when controlling for age. These gut microbiome contained diverse resistome, dominated by multidrug, tetracycline, macrolide/lincosamide/streptogramin, and beta-lactam resistance. We found that the abundance of ARGs in the gut decreased with age. The bacterial hosts of ARGs were mainly from the family Enterobacteriaceae, particularly Escherichia coli.ConclusionsThis study identified the role of household environmental contamination in the developing gut microbiome and resistome of young children and infants with a One Health perspective. We found significant relationships between host age, gut microbiome diversity, and the resistome. Understanding the impact of the household environment on the development of the resistome and microbiome in early life is essential to optimize the relationship between environmental exposure and human health.92Jf2w8Y_xc1zDLBRpWCqWVideo Graphical

Occurrence and removal of fecal bacteria and microbial source tracking markers in a stormwater detention basin overlying the Edwards Aquifer recharge zone in Texas.

The Edwards Aquifer is the primary water resource for over 2 million people in Texas and faces challenges including fecal contamination of water recharging the aquifer, while effectiveness of best management practices (BMPs) such as detention basins in mitigating fecal pollution remains poorly understood. For this study, the inlet and outlet of a detention basin overlying the aquifer's recharge zone were sampled following storm events using automated samplers. Microbial source tracking and culture-based methods were used to determine the occurrence and removal of fecal genetic markers and fecal coliform bacteria in collected water samples. Markers included E. coli (EC23S857), Enterococcus (Entero1), human (HF183), canine (BacCan), and bird (GFD). Fecal coliforms, EC23S857, and Entero1 were detected following each storm event. GFD was the most frequent host-associated marker detected (91% of samples), followed by BacCan (46%), and HF183 (17%). Wilcoxon signed rank tests indicated significantly lower outlet concentrations for fecal coliforms, EC23S857, and Entero1, but not for HF183, GFD, and BacCan. Higher GFD and BacCan outlet concentrations may be due to factors independent of basin design, such as the non-point source nature of bird fecal contamination and domestic dog care practices in neighborhoods contributing to the basin. Mann-Whitney tests showed marker concentrations were not significantly higher during instances of fecal coliform water quality criterion exceedance, except for E. coli, and that fecal coliform concentrations were not significantly different based on marker detection. Overall, results suggest that the detention basin is effective in attenuating fecal contamination associated with fecal coliforms and the general markers, but not for host-associated markers. Consequently, management efforts should focus on mitigating dog and bird-associated fecal pollution in the study region.

Correlation of Antibiotic Resistance Gene and Microbial Source Tracking Marker Removal through Activated Sludge Wastewater Treatment

Correlation of Antibiotic Resistance Gene and Microbial Source Tracking Marker Removal through Activated Sludge Wastewater Treatment

High-Throughput Microfluidic Quantitative PCR Platform for the Simultaneous Quantification of Pathogens, Fecal Indicator Bacteria, and Microbial Source Tracking Markers.

Contamination of water with bacterial, viral, and protozoan pathogens can cause human diseases. Both humans and nonhumans can release these pathogens through their feces. To identify the sources of fecal contamination in the water environment, microbial source tracking (MST) approaches have been developed; however, the relationship between MST markers and pathogens is still not well understood most likely due to the lack of comprehensive datasets of pathogens and MST marker concentrations. In this study, we developed a novel microfluidic quantitative PCR (MFQPCR) platform for the simultaneous quantification of 37 previously validated MST markers, two fecal indicator bacteria (FIB), 22 bacterial, 11 viral, and five protozoan pathogens, and three internal amplification/process controls in many samples. The MFQPCR chip was applied to analyze pathogen removal rates during the wastewater treatment processes. In addition, multiple host-specific MST markers, FIB, and pathogens were successfully quantified in human and avian-impacted surface waters. While the genes for pathogens were relatively infrequently detected, positive correlations were observed between some potential pathogens such as Clostridium perfringens and Mycobacterium spp., and human MST markers. The MFQPCR chips developed in this study, therefore, can provide useful information to monitor and improve water quality.

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, β-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.

Validating Microbial Source Tracking Markers and Assessing the Efficacy of Culturable E. coli and Enterococcus Assays in Ozark Streams, USA

Fecal contamination threatens human health and contributes to the eutrophication of water resources. In Oklahoma, approximately 75% of assessed stream miles in the state are listed as impaired for fecal indicator bacteria (FIB). We tested the performance of seven microbial source tracking (MST) markers in six Northeast Oklahoma streams. All samples were tested with human (HF183), bovine (COWM2, COWM3), porcine (Pig-2-Bac), avian (Av4143), Escherichia coli, and Enterococcus markers using digital PCR (dPCR), as well as culturable assays for E. coli (Colisure) and Enterococcus (Enterolert). Rural and agricultural land uses were characterized by bovine sources of bacterial contamination. Human fecal contamination was found to be prominent in developed landscapes with several indicators for chronic human fecal pollution in an urban stream. All the streams met the criterion for Enterococcus impairment in 2019 and 2020; however, we found no relationships between culturable Enterococcus and the MST markers except in the urban stream, which had chronic human fecal pollution issues. The urban stream met the criterion for E. coli impairment, and E. coli was significantly correlated with the dominant MST markers in both rural and urban streams. We find that the culturable Enterococcus assay is not specific enough to be used for FIB water quality standards. We support the continued use of culturable E. coli assays to monitor for fecal contamination, and we recommend following-up with MST to verify fecal sources so informed mitigative actions can be taken to improve stream water quality.

Host-Associated Bacteroides 16S rDNA-Based Markers for Source Tracking of Fecal Pollution in Laguna Lake, Philippines.

Sources of fecal contamination in Laguna Lake, Philippines, were identified using a library-independent microbial source tracking method targeting host-associated Bacteroides 16S rDNA-based markers. Water samples from nine lake stations were assessed for the presence of the fecal markers HF183 (human), BoBac (cattle), Pig-2-Bac (swine), and DuckBac (duck) from August 2019 to January 2020. HF183 (average concentration = 1.91 log10 copies/mL) was the most frequently detected, while Pig-2-Bac (average concentration = 2.47 log10 copies/mL) was the most abundant. The detected marker concentrations in different stations corresponded to the land use patterns around the lake. Generally, all marker concentrations were higher during the wet season (August-October), suggesting the effect of rainfall-associated factors on the movement and retention of markers from sources. There was a significant association (ρ = 0.45; p < 0.001) between phosphate and the concentration of HF183, suggesting domestic sewage-derived pollution. The markers had acceptable sensitivity and specificity, i.e., HF183 (S = 0.88; R = 0.99), Pig-2-Bac (S = 1.00; R = 1.00), and DuckBac (S = 0.94; R = 1.00), and therefore may be used for the continuous monitoring of fecal pollution in the lake and in designing interventions to improve the quality of the lake water.

Effects of water, sanitation, and hygiene interventions on detection of enteropathogens and host-specific faecal markers in the environment: a systematic review and individual participant data meta-analysis

Water, sanitation, and hygiene (WASH) improvements are promoted to reduce diarrhoea in low-income countries. However, trials from the past 5 years have found mixed effects of household-level and community-level WASH interventions on child health. Measuring pathogens and host-specific faecal markers in the environment can help investigate causal pathways between WASH and health by quantifying whether and by how much interventions reduce environmental exposure to enteric pathogens and faecal contamination from human and different animal sources. We aimed to assess the effects of WASH interventions on enteropathogens and microbial source tracking (MST) markers in environmental samples. We did a systematic review and individual participant data meta-analysis, which included searches from Jan 1, 2000, to Jan 5, 2023, from PubMed, Embase, CAB Direct Global Health, Agricultural and Environmental Science Database, Web of Science, and Scopus, of prospective studies with water, sanitation, or hygiene interventions and concurrent control group that measured pathogens or MST markers in environmental samples and measured child anthropometry, diarrhoea, or pathogen-specific infections. We used covariate-adjusted regression models with robust standard errors to estimate study-specific intervention effects and pooled effect estimates across studies using random-effects models. Few trials have measured the effect of sanitation interventions on pathogens and MST markers in the environment and they mostly focused on onsite sanitation. We extracted individual participant data on nine environmental assessments from five eligible trials. Environmental sampling included drinking water, hand rinses, soil, and flies. Interventions were consistently associated with reduced pathogen detection in the environment but effect estimates in most individual studies could not be distinguished from chance. Pooled across studies, we found a small reduction in the prevalence of any pathogen in any sample type (pooled prevalence ratio [PR] 0·94 [95% CI 0·90-0·99]). Interventions had no effect on the prevalence of MST markers from humans (pooled PR 1·00 [95% CI 0·88-1·13]) or animals (pooled PR 1·00 [95% CI 0·97-1·03]). The small effect of these sanitation interventions on pathogen detection and absence of effects on human or animal faecal markers are consistent with the small or null health effects previously reported in these trials. Our findings suggest that the basic sanitation interventions implemented in these studies did not contain human waste and did not adequately reduce exposure to enteropathogens in the environment. Bill and Melinda Gates Foundation and the UK Foreign and Commonwealth Development Office.

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.