Microbial Source Tracking Markers Research Articles

Applying Microbial Source Tracking Techniques for Identification of Pathways of Faecal Pollution from Water Sources to Point of Use in Vhembe District, South Africa

A safe water supply is a necessity, but it remains one of the backlogs of services rendered in rural areas of developing countries. This leads to vulnerable communities using water from available sources that is unsafe as it is contaminated with faecal matter. Microbial source tracking (MST) methods are gold-standard techniques that detect the exact sources of faecal contamination. This study, therefore, tracked and identified the exact sources of faecal contamination from the catchment to the point of use in rural areas of Vhembe District Municipality. Collected water samples (n = 1048) were concentrated by membrane filtration for the enumeration and detection of E. coli, followed by DNA extraction. The extracted DNA was subjected to a quantitative polymerase chain reaction (qPCR) to track target host-specific Bacteroidales genetic markers from the water source to the point of use. Rivers and dams exhibited maximum E. coli counts of up to 90 CFU/100 mL during the wet season and up to 50 CFU/100 mL during the dry season. Due to the effective treatment of these water sources, no E. coli bacteria were detected in any of the sampled municipal drinking water treatment plants at the point of treatment, while this indicator bacterium was detected at the point of use (households), with a maximum of 4 CFU/100 mL recorded during both the wet and dry seasons. Overall, the most prevalent MST marker exhibited during the wet season was BacCan (dog-associated, 6.87%), followed by BacCow (cow-associated, 5.53%), while Pig-2-Bac (pig-associated, 2.48%) was the least prevalent. The most prevalent marker exhibited during the dry season was BacCan (5.34%), followed by BacCow, with Pig-2-Bac (1.72%) being the least prevalent. A positive correlation (r = 0.31, p = 0.001) was established between the presence of the MST markers and detected E. coli from water sources to the point of use. The knowledge of the faecal contamination attributes in both public and domestic domains will assist in developing prevention and control strategies.

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.

Quantitative microbial risk assessment for ingestion of antibiotic resistance genes from private wells contaminated by human and livestock fecal sources.

We used quantitative microbial risk assessment to estimate ingestion risk for intI1, erm(B), sul1, tet(A), tet(W), and tet(X) in private wells contaminated by human and/or livestock feces. Genes were quantified with five human-specific and six bovine-specific microbial source-tracking (MST) markers in 138 well-water samples from a rural Wisconsin county. Daily ingestion risk (probability of swallowing ≥1 gene) was based on daily water consumption and a Poisson exposure model. Calculations were stratified by MST source and soil depth over the aquifer where wells were drilled. Relative ingestion risk was estimated using wells with no MST detections and >6.1 m soil depth as a referent category. Daily ingestion risk varied from 0 to 8.8 × 10-1 by gene and fecal source (i.e., human or bovine). The estimated number of residents ingesting target genes from private wells varied from 910 (tet(A)) to 1,500 (intI1 and tet(X)) per day out of 12,000 total. Relative risk of tet(A) ingestion was significantly higher in wells with MST markers detected, including wells with ≤6.1 m soil depth contaminated by bovine markers (2.2 [90% CI: 1.1-4.7]), wells with >6.1 m soil depth contaminated by bovine markers (1.8 [1.002-3.9]), and wells with ≤6.1 m soil depth contaminated by bovine and human markers simultaneously (3.1 [1.7-6.5]). Antibiotic resistance genes (ARGs) were not necessarily present in viable microorganisms, and ingestion is not directly associated with infection. However, results illustrate relative contributions of human and livestock fecal sources to ARG exposure and highlight rural groundwater as a significant point of exposure.IMPORTANCEAntibiotic resistance is a global public health challenge with well-known environmental dimensions, but quantitative analyses of the roles played by various natural environments in transmission of antibiotic resistance are lacking, particularly for drinking water. This study assesses risk of ingestion for several antibiotic resistance genes (ARGs) and the class 1 integron gene (intI1) in drinking water from private wells in a rural area of northeast Wisconsin, United States. Results allow comparison of drinking water as an exposure route for antibiotic resistance relative to other routes like food and recreational water. They also enable a comparison of the importance of human versus livestock fecal sources in the study area. Our study demonstrates the previously unrecognized importance of untreated rural drinking water as an exposure route for antibiotic resistance and identifies bovine fecal material as an important exposure factor in the study setting.

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.

Assessing the Occurrence of Host-Specific Faecal Indicator Markers in Water Systems as a Function of Water, Sanitation and Hygiene Practices: A Case Study in Rural Communities of Vhembe District Municipality, South Africa.

In settings where humans and animals closely coexist, the introduction of faecal material into unprotected water sources significantly increases the risk of contracting diarrhoeal and zoonotic waterborne diseases. The data were gathered from a survey conducted through interviews at randomly sampled villages; additionally, water samples were collected in randomly selected households and their associated feeder catchments. Molecular techniques were used, specifically qPCR, to run host-specific Bacteroides microbial source tracking (MST) assays for human, cattle, pig, chicken and dog faecal contamination. Unexpectedly, the qPCR assays revealed dogs to be the most prevalent (40.65%) depositor of faecal matter in unprotected surface water, followed by humans (40.63%); this finding was contradictory to survey findings indicating cattle as the leading source. At the household level, dogs (16.67%) and chickens (15.28%) played prominent roles, as was expected. Reflecting on some of the basic daily practices in households, nearly 89.00% of the population was found to store water due to erratic supply, in contrast to 93.23% using an improved water source. Additionally, a significant association was found between water, sanitation and hygiene (WASH) variables and the occurrence of MST markers after performing a bivariate linear regression. However, the inconsistency between the MST results and household surveys suggests pervasive sanitation issues, even in households without domesticated animals.

Bacteroides: From a Fecal Pollutant to a Useful Tool in Solving Water Pollution

Bacteroides spp. are Gram-negative bacilli, non-endospore-forming and obligate anaerobes, belonging to Phylum Bacteroidetes, Class Bacteroidia, Order Bacteroidales, Family Bacteroidaceae, with 40 taxonomically recognized species. They are anaerobes, and hence, commonly found in warm-blooded animals’ guts and fecal microbiome, where they are the most dominant bacteria, having either mutually or commensally co-evolved with their host. However, they are also disease-causing and carriers of antibiotic resistance genes. Hence, release of Bacteroides-dominated feces by hosts causes fecal pollution, especially in water bodies. Despite being anaerobes, Bacteroides have a short lifespan. They leave their genetic materials behind as environmental DNA, which remain viable for PCR. DNA isolation can determine the feces’ source—a technique known as microbial source tracking (MST). There are two types of MST: library-dependent (LDM) and -independent (LIM). Bacteroides use falls under LIM, wherein animal associated Bacteroides markers determine the fecal contamination’s source. Due to extensive co-evolution between Bacteroides and hosts, most LIM-MST biomarkers are Bacteroides-based [PigBac (pigs), CowBac (cows), ChickenBac (chicken)] and human-based (HF183, HumBac), etc. MST also effectively determines fecal pollution extent from anthropogenic and agricultural sources. Therefore, MST can directly determine a definitive fecal pollutant source. The main limitation of LIM-MST is the lack of markers for other animals. Most available MST markers are directed toward domesticated animals (chickens, cows, pigs). Moreover, even existing markers are affected by geography and animal diet. These limitations warrant continued efforts to fill the gaps by designing more specific and sensitive MST animal markers. This review aims to initiate interest in the use of Bacteroides for efficient water quality monitoring.

Integrating microbial source tracking with quantitative microbial risk assessment to evaluate site specific risk based thresholds at two South Florida beaches.

Quantitative microbial risk assessment (QMRA) can be used to evaluate health risks associated with recreational beach use. This study developed a site-specific risk assessment using a novel approach that combined quantitative PCR-based measurement of microbial source tracking (MST) genetic markers (human, dog, and gull fecal bacteria) with a QMRA analysis of potential pathogen risk. Water samples (n = 24) from two recreational beaches were collected and analyzed for MST markers as part of a broader Beach Exposure And Child Health Study that examined child behavior interactions with the beach environment. We report here the measurements of fecal bacteria MST markers in the environmental DNA extracts of those samples and a QMRA analysis of potential health risks utilizing the results from the MST measurements in the water samples. Human-specific Bacteroides was enumerated by the HF183 Taqman qPCR assay, gull-specific Catellicoccus was enumerated by the Gull2 qPCR assay, and dog-specific Bacteroides was enumerated by the DogBact qPCR assay. Derived reference pathogen doses, calculated from the MST marker concentrations detected in recreational waters, were used to estimate the risk of gastrointestinal illness for both children and adults. Dose-response equations were used to estimate the probability of the risk of infection (Pinf) per a swimming exposure event. Based on the QMRA simulations presented in this study, the GI risk from swimming or playing in water containing a mixture of human and non-human fecal sources appear to be primarily driven by the human fecal source. However, the estimated median GI health risk for both beaches never exceeded the U.S. EPA risk threshold of 32 illnesses per 1,000 recreation events. Our research suggests that utilizing QMRA together with MST can further extend our understanding of potential recreational bather risk by identifying the source contributing the greatest risk in a particular location, therefore informing beach management responses and decision-making.

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.

The Tomato Brown Rugose Fruit Virus Movement Protein Gene Is a Novel Microbial Source Tracking Marker.

Microbial source tracking (MST) identifies sources of fecal contamination in the environment using host-associated fecal markers. While there are numerous bacterial MST markers that can be used herein, there are few such viral markers. Here, we designed and tested novel viral MST markers based on tomato brown rugose fruit virus (ToBRFV) genomes. We assembled eight nearly complete genomes of ToBRFV from wastewater and stool samples from the San Francisco Bay Area in the United States. Next, we developed two novel probe-based reverse transcription-PCR (RT-PCR) assays based on conserved regions of the ToBRFV genome and tested the markers' sensitivities and specificities using human and non-human animal stool as well as wastewater. The ToBRFV markers are sensitive and specific; in human stool and wastewater, they are more prevalent and abundant than a commonly used viral marker, the pepper mild mottle virus (PMMoV) coat protein (CP) gene. We used the assays to detect fecal contamination in urban stormwater samples and found that the ToBRFV markers matched cross-assembly phage (crAssphage), an established viral MST marker, in prevalence across samples. Taken together, these results indicate that ToBRFV is a promising viral human-associated MST marker. IMPORTANCE Human exposure to fecal contamination in the environment can cause transmission of infectious diseases. Microbial source tracking (MST) can identify sources of fecal contamination so that contamination can be remediated and human exposures can be reduced. MST requires the use of host-associated MST markers. Here, we designed and tested novel MST markers from genomes of tomato brown rugose fruit virus (ToBRFV). The markers are sensitive and specific to human stool and highly abundant in human stool and wastewater samples.

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.

Using watershed characteristics to enhance fecal source identification

Fecal pollution is one of the most prevalent forms of pollution affecting waterbodies worldwide, threatening public health and negatively impacting aquatic environments. Microbial source tracking (MST) applies polymerase chain reaction (PCR) technology to help identify the source of fecal pollution. In this study, we combine spatial data for two watersheds with general and host-associated MST markers to target human (HF183/BacR287), bovine (CowM2), and general ruminant (Rum2Bac) sources. Concentrations of MST markers in samples were determined with droplet digital PCR (ddPCR). The three MST markers were detected at all sites (n = 25), but bovine and general ruminant markers were significantly associated with watershed characteristics. MST results, combined with watershed characteristics, suggest that streams draining areas with low-infiltration soil groups and high agricultural land use are at an increased risk for fecal contamination. Microbial source tracking has been applied in numerous studies to aid in identifying the sources of fecal contamination, but these studies usually lack information on the involvement of watershed characteristics. Our study combined watershed characteristics with MST results to provide more comprehensive insight into the factors that influence fecal contamination in order to implement the most effective best management practices.

Microbial source tracking using molecular and cultivable methods in a tropical mixed-use drinking water source to support water safety plans

Microbial contamination deteriorates source water quality, posing a severe problem for drinking water suppliers worldwide and addressed by the Water Safety Plan framework to ensure high-quality and reliable drinking water. Microbial source tracking (MST) is used to examine different microbial pollution sources via host-specific intestinal markers for humans and different types of animals. However, the application of MST in tropical surface water catchments that provide raw water for drinking water supplies is limited. We analyzed a set of MST markers, namely, three cultivable bacteriophages and four molecular PCR and qPCR assays, together with 17 microbial and physicochemical parameters, to identify fecal pollution from general, human-, swine-, and cattle-specific sources. Seventy-two river water samples at six sampling sites were collected over 12 sampling events during wet and dry seasons. We found persistent fecal contamination via the general fecal marker GenBac3 (100 % detection; 2.10–5.42 log10 copies/100 mL), with humans (crAssphage; 74 % detection; 1.62–3.81 log10 copies/100 mL) and swine (Pig-2-Bac; 25 % detection; 1.92–2.91 log10 copies/100 mL). Higher contamination levels were observed during the wet season (p < 0.05). The conventional PCR screening used for the general and human markers showed 94.4 % and 69.8 % agreement with the respective qPCR results. Specifically, in the studied watershed, coliphage could be a screening parameter for the crAssphage marker (90.6 % and 73.7 % positive and negative predictive values; Spearman's rank correlation coefficient = 0.66; p < 0.001). The likelihood of detecting the crAssphage marker significantly increased when total and fecal coliforms exceeded 20,000 and 4000 MPN/100 mL, respectively, as Thailand Surface Water Quality Standards, with odds ratios and 95 % confidence intervals of 15.75 (4.43–55.98) and 5.65 (1.39–23.05). Our study confirms the potential benefits of incorporating MST monitoring into water safety plans, supporting the use of this approach to ensure high-quality drinking water supplies worldwide.

Occurrence and prevalence of antimicrobial resistance in urban karst groundwater systems based on targeted resistome analysis

Antimicrobial resistance (AMR) is a global crisis threatening human, animal, and environmental health. The natural environment, specifically water resources, has been recognized as a reservoir and dissemination pathway for AMR; however, urban karst aquifer systems have been overlooked. This is a concern as these aquifer systems provide drinking water to about 10 % of the global population; yet, the urban influence on the resistome in these vulnerable aquifers is sparingly explored. This study used high-throughput qPCR to determine the occurrence and relative abundance of antimicrobial resistant genes (ARG) in a developing urban karst groundwater system in Bowling Green, KY. Ten sites throughout the city were sampled weekly and analyzed for 85 ARGs, as well as seven microbial source tracking (MST) genes for human and animal sources, providing a spatiotemporal understanding of the resistome in urban karst groundwater. To further understand ARGs in this environment, potential drivers (landuse, karst feature type, season, source of fecal pollution) were considered in relation to the resistome relative abundance. The MST markers highlighted a prominent human influence to the resistome in this karst setting. The concentration of targeted genes varied between the sample weeks, but all targeted ARGs were prevalent throughout the aquifer regardless of karst feature type or season, with high concentrations captured for sulfonamide (sul1), quaternary ammonium compound (qacE), and aminoglycoside (strB) antimicrobial classes. Higher prevalence and relative abundance were detected during the summer and fall seasons, as well as at the spring features. Linear discriminant analysis suggested that karst feature type had higher influence on ARGs in the aquifer compared to season and the source of fecal pollution had the least influence. These findings can contribute to the development of effective management and mitigation strategies for AMR.