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.

Community intervention trial for estimating risk of acute gastrointestinal illness from groundwater-supplied non-disinfected drinking water.

By community intervention in 14 non-disinfecting municipal water systems, we quantified sporadic acute gastrointestinal illness (AGI) attributable to groundwater. Ultraviolet (UV) disinfection was installed on all supply wells of intervention communities. In control communities, residents continued to drink non-disinfected groundwater. Intervention and control communities switched treatments by moving UV disinfection units at the study midpoint (crossover design). Study participants (n = 1,659) completed weekly health diaries during four 12-week surveillance periods. Water supply wells were analyzed monthly for enteric pathogenic viruses. Using the crossover design, groundwater-borne AGI was not observed. However, virus types and quantity in supply wells changed through the study, suggesting that exposure was not constant. Alternatively, we compared AGI incidence between intervention and control communities within the same surveillance period. During Period 1, norovirus contaminated wells and AGI attributable risk from well water was 19% (95% CI, -4%, 36%) for children <5 years and 15% (95% CI, -9%, 33%) for adults. During Period 3, echovirus 11 contaminated wells and UV disinfection slightly reduced AGI in adults. Estimates of AGI attributable risks from drinking non-disinfected groundwater were highly variable, but appeared greatest during times when supply wells were contaminated with specific AGI-etiologic viruses.

Statewide Quantitative Microbial Risk Assessment for Waterborne Viruses, Bacteria, and Protozoa in Public Water Supply Wells in Minnesota.

Infection risk from waterborne pathogens can be estimated via quantitative microbial risk assessment (QMRA) and forms an important consideration in the management of public groundwater systems. However, few groundwater QMRAs use site-specific hazard identification and exposure assessment, so prevailing risks in these systems remain poorly defined. We estimated the infection risk for 9 waterborne pathogens based on a 2-year pathogen occurrence study in which 964 water samples were collected from 145 public wells throughout Minnesota, USA. Annual risk across all nine pathogens combined was 3.3 × 10-1 (95% CI: 2.3 × 10-1 to 4.2 × 10-1), 3.9 × 10-2 (2.3 × 10-2 to 5.4 × 10-2), and 1.2 × 10-1 (2.6 × 10-2 to 2.7 × 10-1) infections person-1 year-1 for noncommunity, nondisinfecting community, and disinfecting community wells, respectively. Risk estimates exceeded the U.S. benchmark of 10-4 infections person-1 year-1 in 59% of well-years, indicating that the risk was widespread. While the annual risk for all pathogens combined was relatively high, the average daily doses for individual pathogens were low, indicating that significant risk results from sporadic pathogen exposure. Cryptosporidium dominated annual risk, so improved identification of wells susceptible to Cryptosporidium contamination may be important for risk mitigation.

The Environmental Microbiology Minimum Information (EMMI) Guidelines: qPCR and dPCR Quality and Reporting for Environmental Microbiology.

Real-time quantitative polymerase chain reaction (qPCR) and digital PCR (dPCR) methods have revolutionized environmental microbiology, yielding quantitative organism-specific data of nucleic acid targets in the environment. Such data are essential for characterizing interactions and processes of microbial communities, assessing microbial contaminants in the environment (water, air, fomites), and developing interventions (water treatment, surface disinfection, air purification) to curb infectious disease transmission. However, our review of recent qPCR and dPCR literature in our field of health-related environmental microbiology showed that many researchers are not reporting necessary and sufficient controls and methods, which would serve to strengthen their study results and conclusions. Here, we describe the application, utility, and interpretation of the suite of controls needed to make high quality qPCR and dPCR measurements of microorganisms in the environment. Our presentation is organized by the discrete steps and operations typical of this measurement process. We propose systematic terminology to minimize ambiguity and aid comparisons among studies. Example schemes for batching and combining controls for efficient work flow are demonstrated. We describe critical reporting elements for enhancing data credibility, and we provide an element checklist in the Supporting Information. Additionally, we present several key principles in metrology as context for laboratories to devise their own quality assurance and quality control reporting framework. Following the EMMI guidelines will improve comparability and reproducibility among qPCR and dPCR studies in environmental microbiology, better inform engineering and public health actions for preventing disease transmission through environmental pathways, and for the most pressing issues in the discipline, focus the weight of evidence in the direction toward solutions.

Optical Properties of Water for Prediction of Wastewater Contamination, Human-Associated Bacteria, and Fecal Indicator Bacteria in Surface Water at Three Watershed Scales.

Relations between spectral absorbance and fluorescence properties of water and human-associated and fecal indicator bacteria were developed for facilitating field sensor applications to estimate wastewater contamination in waterways. Leaking wastewater conveyance infrastructure commonly contaminates receiving waters. Methods to quantify such contamination can be time consuming, expensive, and often nonspecific. Human-associated bacteria are wastewater specific but require discrete sampling and laboratory analyses, introducing latency. Human sewage has fluorescence and absorbance properties different than those of natural waters. To assist real-time field sensor development, this study investigated optical properties for use as surrogates for human-associated bacteria to estimate wastewater prevalence in environmental waters. Three spatial scales were studied: Eight watershed-scale sites, five subwatershed-scale sites, and 213 storm sewers and open channels within three small watersheds (small-scale sites) were sampled (996 total samples) for optical properties, human-associated bacteria, fecal indicator bacteria, and, for selected samples, human viruses. Regression analysis indicated that bacteria concentrations could be estimated by optical properties used in existing field sensors for watershed and subwatershed scales. Human virus occurrence increased with modeled human-associated bacteria concentration, providing confidence in these regressions as surrogates for wastewater contamination. Adequate regressions were not found for small-scale sites to reliably estimate bacteria concentrations likely due to inconsistent local sanitary sewer inputs.

Septic Systems and Rainfall Influence Human Fecal Marker and Indicator Organism Occurrence in Private Wells in Southeastern Pennsylvania.

In the United States, approximately 48 million people are served by private wells. Unlike public water systems, private well water quality is not monitored, and there are few studies on the extent and sources of contamination of private wells. We extensively investigated five private wells to understand the variability in microbial contamination, the role of septic systems as sources of contamination, and the effect of rainfall on well water quality. From 2016 to 2017, weekly or biweekly samples (n = 105) were collected from five private wells in rural Pennsylvania. Samples were tested for general water quality parameters, conventional and sewage-associated microbial indicators, and human pathogens. Total coliforms, human Bacteroides (HF183), and pepper mild mottle virus were detected at least once in all wells. Regression revealed significant relationships between HF183 and rainfall 8-14 days prior to sampling and between total coliforms and rainfall 8-14 or 0-14 days prior to sampling. Dye tracer studies at three wells confirmed the impact of household septic systems on well contamination. Microbiological measurements, chemical water quality data, and dye tracer tests provide evidence of human fecal contamination in the private wells studied, suggesting that household septic systems are the source of this contamination.

Cryptosporidium Incidence and Surface Water Influence of Groundwater Supplying Public Water Systems in Minnesota, USA.

Regulations for public water systems (PWS) in the U.S. consider Cryptosporidium a microbial contaminant of surface water supplies. Groundwater is assumed free of Cryptosporidium unless surface water is entering supply wells. We determined the incidence of Cryptosporidium in PWS wells varying in surface water influence. Community and noncommunity PWS wells ( n = 145) were sampled ( n = 964) and analyzed for Cryptosporidium by qPCR and immunofluorescence assay (IFA). Surface water influence was assessed by stable isotopes and the expert judgment of hydrogeologists using site-specific data. Fifty-eight wells (40%) and 107 samples (11%) were Cryptosporidium-positive by qPCR, and of these samples 67 were positive by IFA. Cryptosporidium concentrations measured by qPCR and IFA were significantly correlated ( p < 0.001). Cryptosporidium incidence was not associated with surface water influence as assessed by stable isotopes or expert judgment. We successfully sequenced 45 of the 107 positive samples to identify species, including C. parvum (41), C. andersoni (2), and C. hominis (2), and the predominant subtype was C. parvum IIa A17G2R1. Assuming USA regulations for surface water-supplied PWS were applicable to the study wells, wells positive for Cryptosporidium by IFA would likely be required to add treatment. Cryptosporidium is not uncommon in groundwater, even when surface water influence is absent.

Human-Associated Indicator Bacteria and Human-Specific Viruses in Surface Water: A Spatial Assessment with Implications on Fate and Transport.

Hydrologic, seasonal, and spatial variability of sewage contamination was studied at six locations within a watershed upstream from water reclamation facility (WRF) effluent to define relative loadings of sewage from different portions of the watershed. Fecal pollution from human sources was spatially quantified by measuring two human-associated indicator bacteria (HIB) and eight human-specific viruses (HSV) at six stream locations in the Menomonee River watershed in Milwaukee, Wisconsin from April 2009 to March 2011. A custom, automated water sampler, which included HSV filtration, was deployed at each location and provided unattended, flow-weighted, large-volume (30-913 L) sampling. In addition, wastewater influent samples were composited over discrete 7 day periods from the two Milwaukee WRFs. Of the 8 HSV, only 3 were detected, present in up to 38% of the 228 stream samples, while at least 1 HSV was detected in all WRF influent samples. HIB occurred more often with significantly higher concentrations than the HSV in stream and WRF influent samples ( p < 0.05). HSV yield calculations showed a loss from upstream to the most-downstream sub-watershed of the Menomonee River, and in contrast, a positive HIB yield from this same sub-watershed emphasizes the complexity in fate and transport properties between HSV and HIB. This study demonstrates the utility of analyzing multiple HSV and HIB to provide a weight-of-evidence approach for assessment of fecal contamination at the watershed level, provides an assessment of relative loadings for prioritizing areas within a watershed, and demonstrates how loadings of HSV and HIB can be inconsistent, inferring potential differences in fate and transport between the two indicators of human fecal presence.

Fate of Manure-Borne Pathogens during Anaerobic Digestion and Solids Separation.

Anaerobic digestion can inactivate zoonotic pathogens present in cattle manure, which reduces transmission of these pathogens from farms to humans through the environment. However, the variability of inactivation across farms and over time is unknown because most studies have examined pathogen inactivation under ideal laboratory conditions or have focused on only one or two full-scale digesters at a time. In contrast, we sampled seven full-scale digesters treating cattle manure in Wisconsin for 9 mo on a biweekly basis ( = 118 pairs of influent and effluent samples) and used real-time quantitative polymerase chain reaction to analyze these samples for 19 different microbial genetic markers. Overall, inactivation of pathogens and fecal indicators was highly variable. When aggregated across digester and season, log-removal values for several representative microorganisms-bovine , -like CowM3, and bovine polyomavirus-were 0.78 ± 0.34, 0.70 ± 0.50, and 0.53 ± 0.58, respectively (mean ± SD). These log-removal values were up to two times lower than expected based on the scientific literature. Thus, our study indicates that full-scale anaerobic digestion of cattle manure requires optimization with regard to pathogen inactivation. Future studies should focus on identifying the potential causes of this suboptimal performance (e.g., overloading, poor mixing, poor temperature control). Our study also examined the fate of pathogens during manure separation and found that the majority of microbes we detected ended up in the liquid fraction of separated manure. This finding has important implications for the transmission of zoonotic pathogens through the environment to humans.

Using Integrated Environmental Modeling to Assess Sources of Microbial Contamination in Mixed-Use Watersheds.

Microbial fate and transport in watersheds should include a microbial source apportionment analysis that estimates the importance of each source, relative to each other and in combination, by capturing their impacts spatially and temporally under various scenarios. A loosely configured software infrastructure was used in microbial source-to-receptor modeling by focusing on animal- and human-impacted mixed-use watersheds. Components include data collection software, a microbial source module that determines loading rates from different sources, a watershed model, an inverse model for calibrating flows and microbial densities, tabular and graphical viewers, software to convert output to different formats, and a model for calculating risk from pathogen exposure. The system automates, as much as possible, the manual process of accessing and retrieving data and completes input data files of the models. The workflow considers land-applied manure from domestic animals on undeveloped areas; direct shedding (excretion) on undeveloped lands by domestic animals and wildlife; pastureland, cropland, forest, and urban or engineered areas; sources that directly release to streams from leaking septic systems; and shedding by domestic animals directly to streams. The infrastructure also considers point sources from regulated discharges. An application is presented on a real-world watershed and helps answer questions such as: What are the major microbial sources? What practices contribute to contamination at the receptor location? What land-use types influence contamination at the receptor location? and Under what conditions do these sources manifest themselves? This research aims to improve our understanding of processes related to pathogen and indicator dynamics in mixed-use watershed systems.

Capturing Microbial Sources Distributed in a Mixed-use Watershed within an Integrated Environmental Modeling Workflow.

Many watershed models simulate overland and instream microbial fate and transport, but few provide loading rates on land surfaces and point sources to the waterbody network. This paper describes the underlying equations for microbial loading rates associated with 1) land-applied manure on undeveloped areas from domestic animals; 2) direct shedding (excretion) on undeveloped lands by domestic animals and wildlife; 3) urban or engineered areas; and 4) point sources that directly discharge to streams from septic systems and shedding by domestic animals. A microbial source module, which houses these formulations, is part of a workflow containing multiple models and databases that form a loosely configured modeling infrastructure which supports watershed-scale microbial source-to-receptor modeling by focusing on animal- and human-impacted catchments. A hypothetical application - accessing, retrieving, and using real-world data - demonstrates how the infrastructure can automate many of the manual steps associated with a standard watershed assessment, culminating in calibrated flow and microbial densities at the watershed's pour point.

Human virus and microbial indicator occurrence in public-supply groundwater systems: meta-analysis of 12 international studies.

Groundwater quality is often evaluated using microbial indicators. This study examines data from 12 international groundwater studies (conducted 1992-2013) of 718 public drinking-water systems located in a range of hydrogeological settings. Focus was on testing the value of indicator organisms for identifying virus-contaminated wells. One or more indicators and viruses were present in 37 and 15% of 2,273 samples and 44 and 27% of 746 wells, respectively. Escherichia coli (E. coli) and somatic coliphage are 7-9 times more likely to be associated with culturable virus-positive samples when the indicator is present versus when it is absent, while F-specific and somatic coliphages are 8-9 times more likely to be associated with culturable virus-positive wells. However, single indicators are only marginally associated with viruses detected by molecular methods, and all microbial indicators have low sensitivity and positive predictive values for virus occurrence, whether by culturable or molecular assays, i.e., indicators are often absent when viruses are present and the indicators have a high false-positive rate. Wells were divided into three susceptibility subsets based on presence of (1) total coliform bacteria or (2) multiple indicators, or (3) location of wells in karst, fractured bedrock, or gravel/cobble settings. Better associations of some indicators with viruses were observed for (1) and (3). Findings indicate the best indicators are E. coli or somatic coliphage, although both indicators may underestimate virus occurrence. Repeat sampling for indicators improves evaluation of the potential for viral contamination in a well.

Effects of Climate and Sewer Condition on Virus Transport to Groundwater.

Pathogen contamination from leaky sanitary sewers poses a threat to groundwater quality in urban areas, yet the spatial and temporal dimensions of this contamination are not well understood. In this study, 16 monitoring wells and six municipal wells were repeatedly sampled for human enteric viruses. Viruses were detected infrequently, in 17 of 455 samples, compared to previous sampling at these wells. Thirteen of the 22 wells sampled were virus-positive at least once. While the highest virus concentrations occurred in shallower wells, shallow and deep wells were virus-positive at similar rates. Virus presence in groundwater was temporally coincident, with 16 of 17 virus-positive samples collected in a six-month period. Detections were associated with precipitation and occurred infrequently during a prolonged drought. The study purposely included sites with sewers of differing age and material. The rates of virus detections in groundwater were similar at all study sites during this study. However, a relationship between sewer age and virus detections emerged when compared to data from an earlier study, conducted during high precipitation conditions. Taken together, these data indicate that sewer condition and climate affect urban groundwater contamination by human enteric viruses.

Human and Bovine Viruses and Bacteria at Three Great Lakes Beaches: Environmental Variable Associations and Health Risk.

Waterborne pathogens were measured at three beaches in Lake Michigan, environmental factors for predicting pathogen concentrations were identified, and the risk of swimmer infection and illness was estimated. Waterborne pathogens were detected in 96% of samples collected at three Lake Michigan beaches in summer, 2010. Samples were quantified for 22 pathogens in four microbial categories (human viruses, bovine viruses, protozoa, and pathogenic bacteria). All beaches had detections of human and bovine viruses and pathogenic bacteria indicating influence of multiple contamination sources at these beaches. Occurrence ranged from 40 to 87% for human viruses, 65-87% for pathogenic bacteria, and 13-35% for bovine viruses. Enterovirus, adenovirus A, Salmonella spp., Campylobacter jejuni, bovine polyomavirus, and bovine rotavirus A were present most frequently. Variables selected in multiple regression models used to explore environmental factors that influence pathogens included wave direction, cloud cover, currents, and water temperature. Quantitative Microbial Risk Assessment was done for C. jejuni, Salmonella spp., and enteroviruses to estimate risk of infection and illness. Median infection risks for one-time swimming events were approximately 2 × 10(-5), 8 × 10(-6), and 3 × 10(-7) [corrected] for C. jejuni, Salmonella spp., and enteroviruses, respectively. Results highlight the importance of investigating multiple pathogens within multiple categories to avoid underestimating the prevalence and risk of waterborne pathogens.

Improved HF183 quantitative real-time PCR assay for characterization of human fecal pollution in ambient surface water samples.

Quantitative real-time PCR (qPCR) assays that target the human-associated HF183 bacterial cluster within members of the genus Bacteroides are among the most widely used methods for the characterization of human fecal pollution in ambient surface waters. In this study, we show that a current TaqMan HF183 qPCR assay (HF183/BFDrev) routinely forms nonspecific amplification products and introduce a modified TaqMan assay (HF183/BacR287) that alleviates this problem. The performance of each qPCR assay was compared in head-to-head experiments investigating limits of detection, analytical precision, predicted hybridization to 16S rRNA gene sequences from a reference database, and relative marker concentrations in fecal and sewage samples. The performance of the modified HF183/BacR287 assay is equal to or improves upon that of the original HF183/BFDrev assay. In addition, a qPCR chemistry designed to combat amplification inhibition and a multiplexed internal amplification control are included. In light of the expanding use of PCR-based methods that rely on the detection of extremely low concentrations of DNA template, such as qPCR and digital PCR, the new TaqMan HF183/BacR287 assay should provide more accurate estimations of human-derived fecal contaminants in ambient surface waters.

Drinking water systems, hydrology, and childhood gastrointestinal illness in Central and Northern Wisconsin.

OBJECTIVES: This study investigated if the type of drinking water source (treated municipal, untreated municipal, and private well water) modifies the effect of hydrology on childhood (aged < 5 years) gastrointestinal illness. METHODS: We conducted a time series study to assess the relationship between hydrologic and weather conditions with childhood gastrointestinal illness from 1991 to 2010. The Central and Northern Wisconsin study area includes households using all 3 types of drinking water systems. Separate time series models were created for each system and half-year period (winter/spring, summer/fall). RESULTS: More precipitation (summer/fall) systematically increased childhood gastrointestinal illness in municipalities accessing untreated water. The relative risk of contracting gastrointestinal illness was 1.4 in weeks with 3 centimeters of precipitation and 2.4 in very wet weeks with 12 centimeters of precipitation. By contrast, gastrointestinal illness in private well and treated municipal areas was not influenced by hydrologic conditions, although warmer winter temperatures slightly increased incidence. CONCLUSIONS: Our study suggests that improved drinking water protection, treatment, and delivery infrastructure may improve public health by specifically identifying municipal water systems lacking water treatment that may transmit waterborne disease.

Accumulation and inactivation of avian influenza virus by the filter-feeding invertebrate Daphnia magna.

The principal mode of avian influenza A virus (AIV) transmission among wild birds is thought to occur via an indirect fecal-oral route, whereby individuals are exposed to virus from the environment through contact with virus-contaminated water. AIV can remain viable for an extended time in water; however, little is known regarding the influence of the biotic community (i.e., aquatic invertebrates) on virus persistence and infectivity in aquatic environments. We conducted laboratory experiments to investigate the ability of an aquatic filter-feeding invertebrate, Daphnia magna, to accumulate virus from AIV-dosed water under the hypothesis that they represent a potential vector of AIV to waterfowl hosts. We placed live daphnids in test tubes dosed with low-pathogenicity AIV (H3N8 subtype isolated from a wild duck) and sampled Daphnia tissue and the surrounding water using reverse transcription-quantitative PCR (RT-qPCR) at 3- to 120-min intervals for up to 960 min following dosing. Concentrations of viral RNA averaged 3 times higher in Daphnia tissue than the surrounding water shortly after viral exposure, but concentrations decreased exponentially through time for both. Extracts from Daphnia tissue were negative for AIV by cell culture, whereas AIV remained viable in water without Daphnia present. Our results suggest daphnids can accumulate AIV RNA and effectively remove virus particles from water. Although concentrations of viral RNA were consistently higher in Daphnia tissue than the water, additional research is needed on the time scale of AIV inactivation after Daphnia ingestion to fully elucidate Daphnia's role as a potential vector of AIV infection to aquatic birds.

Source and transport of human enteric viruses in deep municipal water supply wells.

Until recently, few water utilities or researchers were aware of possible virus presence in deep aquifers and wells. During 2008 and 2009 we collected a time series of virus samples from six deep municipal water-supply wells. The wells range in depth from approximately 220 to 300 m and draw water from a sandstone aquifer. Three of these wells draw water from beneath a regional aquitard, and three draw water from both above and below the aquitard. We also sampled a local lake and untreated sewage as potential virus sources. Viruses were detected up to 61% of the time in each well sampled, and many groundwater samples were positive for virus infectivity. Lake samples contained viruses over 75% of the time. Virus concentrations and serotypes observed varied markedly with time in all samples. Sewage samples were all extremely high in virus concentration. Virus serotypes detected in sewage and groundwater were temporally correlated, suggesting very rapid virus transport, on the order of weeks, from the source(s) to wells. Adenovirus and enterovirus levels in the wells were associated with precipitation events. The most likely source of the viruses in the wells was leakage of untreated sewage from sanitary sewer pipes.