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.

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.

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.

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.

Microbial source tracking and land use associations for antibiotic resistance genes in private wells influenced by human and livestock fecal sources.

Antimicrobial resistance is a growing public health problem that requires an integrated approach among human, agricultural, and environmental sectors. However, few studies address all three components simultaneously. We investigated the occurrence of five antibiotic resistance genes (ARGs) and the class 1 integron gene (intI1) in private wells drawing water from a vulnerable aquifer influenced by residential septic systems and land-applied dairy manure. Samples (n = 138) were collected across four seasons from a randomized sample of private wells in Kewaunee County, Wisconsin. Measurements of ARGs and intI1 were related to microbial source tracking (MST) markers specific to human and bovine feces; they were also related to 54 risk factors for contamination representing land use, rainfall, hydrogeology, and well construction. ARGs and intI1 occurred in 5%-40% of samples depending on target. Detection frequencies for ARGs and intI1 were lowest in the absence of human and bovine MST markers (1%-30%), highest when co-occurring with human and bovine markers together (11%-78%), and intermediate when co-occurring with just one type of MST marker (4%-46%). Gene targets were associated with septic system density more often than agricultural land, potentially because of the variable presence of manure on the landscape. Determining ARG prevalence in a rural setting with mixed land use allowed an assessment of the relative contribution of human and bovine fecal sources. Because fecal sources co-occurred with ARGs at similar rates, interventions intended to reduce ARG occurrence may be most effective if both sources are considered.

Risk of viral acute gastrointestinal illness from nondisinfected drinking water distribution systems.

Acute gastrointestinal illness (AGI) resulting from pathogens directly entering the piping of drinking water distribution systems is insufficiently understood. Here, we estimate AGI incidence from virus intrusions into the distribution systems of 14 nondisinfecting, groundwater-source, community water systems. Water samples for virus quantification were collected monthly at wells and households during four 12-week periods in 2006-2007. Ultraviolet (UV) disinfection was installed on the communities' wellheads during one study year; UV was absent the other year. UV was intended to eliminate virus contributions from the wells and without residual disinfectant present in these systems, any increase in virus concentration downstream at household taps represented virus contributions from the distribution system (Approach 1). During no-UV periods, distribution system viruses were estimated by the difference between well water and household tap virus concentrations (Approach 2). For both approaches, a Monte Carlo risk assessment framework was used to estimate AGI risk from distribution systems using study-specific exposure-response relationships. Depending on the exposure-response relationship selected, AGI risk from the distribution systems was 0.0180-0.0661 and 0.001-0.1047 episodes/person-year estimated by Approaches 1 and 2, respectively. These values represented 0.1-4.9% of AGI risk from all exposure routes, and 1.6-67.8% of risk related to drinking water exposure. Virus intrusions into nondisinfected drinking water distribution systems can contribute to sporadic AGI.

Fate and seasonality of antimicrobial resistance genes during full-scale anaerobic digestion of cattle manure across seven livestock production facilities.

Anaerobic digestion has been suggested as an intervention to attenuate antibiotic resistance genes (ARGs) in livestock manure but supporting data have typically been collected at laboratory scale. Few studies have quantified ARG fate during full-scale digestion of livestock manure. We sampled untreated manure and digestate from seven full-scale mesophilic dairy manure digesters to assess ARG fate through each system. Samples were collected biweekly from December through August (i.e., winter, spring, and summer; n = 235 total) and analyzed by quantitative polymerase chain reaction for intI1, erm(B), sul1, tet(A), and tet(W). Concentrations of intI1, sul1, and tet(A) decreased during anaerobic digestion, but their removal was less extensive than expected based on previous laboratory studies. Removal for intI1 during anaerobic digestion equaled 0.28 ± 0.03 log10 units (mean ± SE), equivalent to only 48% removal and notable given intI1's role in horizontal gene transfer and multiple resistance. Furthermore, tet(W) concentrations were unchanged during anaerobic digestion (p > 0.05), and erm(B) concentrations increased by 0.52 ± 0.03 log10 units (3.3-fold), which is important given erythromycin's status as a critically important antibiotic for human medicine. Seasonal log10 changes in intI1, sul1, and tet(A) concentrations were ≥50% of corresponding log10 removals by anaerobic digestion, and variation in ARG and intI1 concentrations among digesters was quantitatively comparable to anaerobic digestion effects. These results suggest that mesophilic anaerobic digestion may be limited as an intervention for ARGs in livestock manure and emphasize the need for multiple farm-level interventions to attenuate antibiotic resistance.

Virus contamination from operation and maintenance events in small drinking water distribution systems.

We tested the association of common events in drinking water distribution systems with contamination of household tap water with human enteric viruses. Viruses were enumerated by qPCR in the tap water of 14 municipal systems that use non-disinfected groundwater. Ultraviolet disinfection was installed at all active wellheads to reduce virus contributions from groundwater to the distribution systems. As no residual disinfectant was added to the water, any increase in virus levels measured downstream at household taps would be indicative of distribution system intrusions. Utility operators reported events through written questionnaires. Virus outcome measures were related to distribution system events using binomial and gamma regression. Virus concentrations were elevated in the wells, reduced or eliminated by ultraviolet disinfection, and elevated again in distribution systems, showing that viruses were, indeed, directly entering the systems. Pipe installation was significantly associated with higher virus levels, whereas hydrant flushing was significantly associated with lower virus levels. Weak positive associations were observed for water tower maintenance, valve exercising, and cutting open a water main. Coliform bacteria detections from routine monitoring were not associated with viruses. Understanding when distribution systems are most vulnerable to virus contamination, and taking precautionary measures, will ensure delivery of safe drinking water.

Quantitative Microbial Risk Assessment for Contaminated Private Wells in the Fractured Dolomite Aquifer of Kewaunee County, Wisconsin.

BACKGROUND: Private wells are an important source of drinking water in Kewaunee County, Wisconsin. Due to the region's fractured dolomite aquifer, these wells are vulnerable to contamination by human and zoonotic gastrointestinal pathogens originating from land-applied cattle manure and private septic systems. OBJECTIVE: We determined the magnitude of the health burden associated with contamination of private wells in Kewaunee County by feces-borne gastrointestinal pathogens. METHODS: This study used data from a year-long countywide pathogen occurrence study as inputs into a quantitative microbial risk assessment (QMRA) to predict the total cases of acute gastrointestinal illness (AGI) caused by private well contamination in the county. Microbial source tracking was used to associate predicted cases of illness with bovine, human, or unknown fecal sources. RESULTS: Results suggest that private well contamination could be responsible for as many as 301 AGI cases per year in Kewaunee County, and that 230 and 12 cases per year were associated with a bovine and human fecal source, respectively. Furthermore, Cryptosporidium parvum was predicted to cause 190 cases per year, the most out of all 8 pathogens included in the QMRA. DISCUSSION: This study has important implications for land use and water resource management in Kewaunee County and informs the public health impacts of consuming drinking water produced in other similarly vulnerable hydrogeological settings. https://doi.org/10.1289/EHP7815.

Sources and Risk Factors for Nitrate and Microbial Contamination of Private Household Wells in the Fractured Dolomite Aquifer of Northeastern Wisconsin.

BACKGROUND: Groundwater quality in the Silurian dolomite aquifer in northeastern Wisconsin, USA, has become contentious as dairy farms and exurban development expand. OBJECTIVES: We investigated private household wells in the region, determining the extent, sources, and risk factors of nitrate and microbial contamination. METHODS: Total coliforms, Escherichia coli, and nitrate were evaluated by synoptic sampling during groundwater recharge and no-recharge periods. Additional seasonal sampling measured genetic markers of human and bovine fecal-associated microbes and enteric zoonotic pathogens. We constructed multivariable regression models of detection probability (log-binomial) and concentration (gamma) for each contaminant to identify risk factors related to land use, precipitation, hydrogeology, and well construction. RESULTS: Total coliforms and nitrate were strongly associated with depth-to-bedrock at well sites and nearby agricultural land use, but not septic systems. Both human wastewater and cattle manure contributed to well contamination. Rotavirus group A, Cryptosporidium, and Salmonella were the most frequently detected pathogens. Wells positive for human fecal markers were associated with depth-to-groundwater and number of septic system drainfield within 229m. Manure-contaminated wells were associated with groundwater recharge and the area size of nearby agricultural land. Wells positive for any fecal-associated microbe, regardless of source, were associated with septic system density and manure storage proximity modified by bedrock depth. Well construction was generally not related to contamination, indicating land use, groundwater recharge, and bedrock depth were the most important risk factors. DISCUSSION: These findings may inform policies to minimize contamination of the Silurian dolomite aquifer, a major water supply for the U.S. and Canadian Great Lakes region. https://doi.org/10.1289/EHP7813.