Characterizing Differences in Sources of and Contributions to Fecal Contamination of Sediment and Surface Water with the Microbial FIT Framework.

Surface water monitoring and microbial source tracking (MST) are used to identify host sources of fecal pollution and protect public health. However, knowledge of the locations of spatial sources and their relative impacts on the environment is needed to effectively mitigate health risks. Additionally, sediment samples may offer time-integrated information compared to transient surface water. Thus, we implemented the newly developed microbial find, inform, and test framework to identify spatial sources and their impacts on human (HuBac) and bovine (BoBac) MST markers, quantified from both riverbed sediment and surface water in a bovine-dense region. Dairy feeding operations and low-intensity developed land-cover were associated with 99% (p-value < 0.05) and 108% (p-value < 0.05) increases, respectively, in the relative abundance of BoBac in sediment, and with 79% (p-value < 0.05) and 39% increases in surface water. Septic systems were associated with a 48% increase in the relative abundance of HuBac in sediment and a 56% increase in surface water. Stronger source signals were observed for sediment responses compared to water. By defining source locations, predicting river impacts, and estimating source influence ranges in a Great Lakes region, this work informs pollution mitigation strategies of local and global significance.

Identifying bioaugmentation candidates for bioremediation of polycyclic aromatic hydrocarbons in contaminated estuarine sediment of the Elizabeth River, VA, USA.

Estuarine sediments near former creosoting facilities along the Elizabeth River (Virginia, USA) are contaminated by polycyclic aromatic hydrocarbons (PAHs). In this study, we interrogated the bacterial community of the Elizabeth River with both culture-based and culture-independent methods to identify potential candidates for bioremediation of these contaminants. DNA-based stable isotope probing (SIP) experiments with phenanthrene and fluoranthene using sediment from the former Republic Creosoting site identified relevant PAH-degrading bacteria within the Azoarcus, Hydrogenophaga, and Croceicoccus genera. Targeted cultivation of PAH-degrading bacteria from the same site recovered 6 PAH-degrading strains, including one strain highly similar to Hydrogenophaga sequences detected in SIP experiments. Other isolates were most similar to organisms within the Novosphingobium, Sphingobium, Stenotrophomonas, and Alcaligenes genera. Lastly, we performed 16S rRNA gene amplicon microbiome analyses of sediment samples from four sites, including Republic Creosoting, with varying concentrations of PAHs. Analysis of these data showed a striking divergence of the microbial community at the highly contaminated Republic Creosoting site from less contaminated sites with the enrichment of several bacterial clades including those affiliated with the Pseudomonas genus. Sequences within the microbiome libraries similar to SIP-derived sequences were generally found at high relative abundance, while the Croceicoccus sequence was present at low to moderate relative abundance. These results suggest that Azoarcus and Hydrogenophaga strains might be good target candidates for biostimulation, while Croceicoccus spp. might be good targets for bioaugmentation in these sediments. Furthermore, this study demonstrates the value of culture-based and culture-independent methods in identifying promising bacterial candidates for use in a precision bioremediation scheme. KEY POINTS: • This study highlights the importance of using multiple strategies to identify promising bacterial candidates for use in a precision bioremediation scheme. • We used both selective cultivation techniques and DNA-based stable isotope probing to identify bacterial degraders of prominent PAHs at a historically contaminated site in the Elizabeth River, VA, USA. • Azoarcus and Hydrogenophaga strains might be good target candidates for biostimulation in Elizabeth River sediments, while Croceicoccus spp. might be good targets for bioaugmentation.

Transmission of Antimicrobial-Resistant Staphylococcus aureus Clonal Complex 9 between Pigs and Humans, United States.

Transmission of livestock-associated Staphylococcus aureus clonal complex 9 (LA-SA CC9) between pigs raised on industrial hog operations (IHOs) and humans in the United States is poorly understood. We analyzed whole-genome sequences from 32 international S. aureus CC9 isolates and 49 LA-SA CC9 isolates from IHO pigs and humans who work on or live near IHOs in 10 pig-producing counties in North Carolina, USA. Bioinformatic analysis of sequence data from the 81 isolates demonstrated 3 major LA-SA CC9 clades. North Carolina isolates all fell within a single clade (C3). High-resolution phylogenetic analysis of C3 revealed 2 subclades of intermingled IHO pig and human isolates differing by 0-34 single-nucleotide polymorphisms. Our findings suggest that LA-SA CC9 from pigs and humans share a common source and provide evidence of transmission of antimicrobial-resistant LA-SA CC9 between IHO pigs and humans who work on or live near IHOs in North Carolina.

Microbial Find, Inform, and Test Model for Identifying Spatially Distributed Contamination Sources: Framework Foundation and Demonstration of Ruminant Bacteroides Abundance in River Sediments.

Microbial pollution in rivers poses known ecological and health risks, yet causal and mechanistic linkages to sources remain difficult to establish. Host-associated microbial source tracking (MST) markers help to assess the microbial risks by linking hosts to contamination but do not identify the source locations. Land-use regression (LUR) models have been used to screen the source locations using spatial predictors but could be improved by characterizing transport (i.e., hauling, decay overland, and downstream). We introduce the microbial Find, Inform, and Test (FIT) framework, which expands previous LUR approaches and develops novel spatial predictor models to characterize the transported contributions. We applied FIT to characterize the sources of BoBac, a ruminant Bacteroides MST marker, quantified in riverbed sediment samples from Kewaunee County, Wisconsin. A 1 standard deviation increase in contributions from land-applied manure hauled from animal feeding operations (AFOs) was associated with a 77% (p-value <0.05) increase in the relative abundance of ruminant Bacteroides (BoBac-copies-per-16S-rRNA-copies) in the sediment. This is the first work finding an association between the upstream land-applied manure and the offsite bovine-associated fecal markers. These findings have implications for the sediment as a reservoir for microbial pollution associated with AFOs (e.g., pathogens and antibiotic-resistant bacteria). This framework and application advance statistical analysis in MST and water quality modeling more broadly.

Impacts of an Urban Sanitation Intervention on Fecal Indicators and the Prevalence of Human Fecal Contamination in Mozambique.

Fecal source tracking (FST) may be useful to assess pathways of fecal contamination in domestic environments and to estimate the impacts of water, sanitation, and hygiene (WASH) interventions in low-income settings. We measured two nonspecific and two human-associated fecal indicators in water, soil, and surfaces before and after a shared latrine intervention from low-income households in Maputo, Mozambique, participating in the Maputo Sanitation (MapSan) trial. Up to a quarter of households were impacted by human fecal contamination, but trends were unaffected by improvements to shared sanitation facilities. The intervention reduced Escherichia coli gene concentrations in soil but did not impact culturable E. coli or the prevalence of human FST markers in a difference-in-differences analysis. Using a novel Bayesian hierarchical modeling approach to account for human marker diagnostic sensitivity and specificity, we revealed a high amount of uncertainty associated with human FST measurements and intervention effect estimates. The field of microbial source tracking would benefit from adding measures of diagnostic accuracy to better interpret findings, particularly when FST analyses convey insufficient information for robust inference. With improved measures, FST could help identify dominant pathways of human and animal fecal contamination in communities and guide the implementation of effective interventions to safeguard health.

Microorganisms in the Placenta: Links to Early-Life Inflammation and Neurodevelopment in Children.

Prenatal exposure to various stressors can influence both early and later life childhood health. Microbial infection of the intrauterine environment, specifically within the placenta, has been associated with deleterious birth outcomes, such as preterm birth, as well as adverse neurological outcomes later in life. The relationships among microorganisms in the placenta, placental function, and fetal development are not well understood. Microorganisms have been associated with perinatal inflammatory responses that have the potential for disrupting fetal brain development. Microbial presence has also been associated with epigenetic modifications in the placenta, as well other tissues. Here we review research detailing the presence of microorganisms in the placenta and associations among such microorganisms, placental DNA methylation, perinatal inflammation, and neurodevelopmental outcomes.

Drinking water improvements and rates of urinary and gastrointestinal infections in Galápagos, Ecuador: Assessing household and community factors.

OBJECTIVES: The Galápagos provides an important setting to investigate the health impacts of a new drinking water treatment plant (DWTP) in a limited resource environment. We examine how household perceptions and practices affect the relationship between water quality and infections before and after DWTP. METHODS: Ethnographic data and self-reported infections were collected from 121 mothers and 168 children ages 2 to 10 from Isla San Cristóbal. Household tap water samples were tested for levels of fecal contamination. Community level infection rates were estimated using discharge records from the Ministry of Public Health. The effects of the new DWTP and fecal contamination levels on infections were tested using logistic and Poisson models. RESULTS: Perceptions of water quality and household practices influenced exposures to contaminated tap water. We found minimal change in drinking water sources with 85% of mothers sampled before the DWTP and 83% sampled after using bottled water, while >85% from the pooled sample used tap water for cooking and hygiene practices. The DWTP opening was associated with lower odds of fecal contamination in tap water, reported urinary infections, and community level rates of urinary and gastrointestinal infections. The household practice of recently washing the cistern contributed to higher contamination levels after the DWTP opened. CONCLUSIONS: To ensure access to clean water, public health works need to consider how household perceptions and practices influence tap water use and quality, in addition to infrastructure improvements. Exposures to contaminated tap water contribute to the burden of infectious disease in environments with inadequate water infrastructure.

Risk of Antibiotic-Resistant Staphylococcus aureus Dispersion from Hog Farms: A Critical Review.

The World Health Organization has declared antibiotic resistance "one of the biggest threats to global health." Mounting evidence suggests that antibiotic use in industrial-scale hog farming is contributing to the spread of antibiotic-resistant Staphylococcus aureus. To capture available evidence on these risks, we searched peer-reviewed studies published before June 2017 and conducted a meta-analysis of these studies' estimates of the prevalence of swine-associated, antibiotic-resistant S. aureus in animals, humans, and the environment. The 166 relevant studies revealed consistent evidence of livestock-associated methicillin-resistant S. aureus (MRSA) in hog herds (55.3%) raised with antibiotics. MRSA prevalence was also substantial in slaughterhouse pigs (30.4%), industrial hog operation workers (24.4%), and veterinarians (16.8%). The prevalence of swine-associated, multidrug-resistant S. aureus (MDRSA)-with resistance to three or more antibiotics-is not as well documented. Nonetheless, sufficient studies were available to estimate MDRSA pooled prevalence in conventional hog operation workers (15.0%), workers' household members (13.0%), and community members (5.37%). Evidence also suggests that antibiotic-resistant S. aureus can be present in air, soil, water, and household surface samples gathered in or near high-intensity hog operations. An important caveat is that prevalence estimates for humans reflect colonization, not active infection, and the health risks of colonization remain poorly understood. In addition, these pooled results may not represent risks in specific locations, due to wide geographic variation. Nonetheless, these results underscore the need for additional preventive action to stem the spread of antibiotic-resistant pathogens from livestock operations and a streamlined reporting system to track this risk.

Geostatistical Prediction of Microbial Water Quality Throughout a Stream Network Using Meteorology, Land Cover, and Spatiotemporal Autocorrelation.

Predictive modeling is promising as an inexpensive tool to assess water quality. We developed geostatistical predictive models of microbial water quality that empirically modeled spatiotemporal autocorrelation in measured fecal coliform (FC) bacteria concentrations to improve prediction. We compared five geostatistical models featuring different autocorrelation structures, fit to 676 observations from 19 locations in North Carolina's Jordan Lake watershed using meteorological and land cover predictor variables. Though stream distance metrics (with and without flow-weighting) failed to improve prediction over the Euclidean distance metric, incorporating temporal autocorrelation substantially improved prediction over the space-only models. We predicted FC throughout the stream network daily for one year, designating locations "impaired", "unimpaired", or "unassessed" if the probability of exceeding the state standard was ≥90%, ≤10%, or >10% but <90%, respectively. We could assign impairment status to more of the stream network on days any FC were measured, suggesting frequent sample-based monitoring remains necessary, though implementing spatiotemporal predictive models may reduce the number of concurrent sampling locations required to adequately assess water quality. Together, these results suggest that prioritizing sampling at different times and conditions using geographically sparse monitoring networks is adequate to build robust and informative geostatistical models of water quality impairment.

Exposure to Human-Associated Chemical Markers of Fecal Contamination and Self-Reported Illness among Swimmers at Recreational Beaches.

Anthropogenic chemicals have been proposed as potential markers of human fecal contamination in recreational water. However, to date, there are no published studies describing their relationships with illness risks. Using a cohort of swimmers at seven U.S. beaches, we examined potential associations between the presence of chemical markers of human fecal pollution and self-reported gastrointestinal (GI) illness, diarrhea, and respiratory illness. Swimmers were surveyed about their beach activities, water exposure, and baseline symptoms on the day of their beach visit, and about any illness experienced 10-12 days later. Risk differences were estimated using model-based standardization and adjusted for the swimmer's age, beach site, sand contact, rainfall, and water temperature. Sixty-two chemical markers were analyzed from daily water samples at freshwater and marine beaches. Of those, 20 were found consistently. With the possible exception of bisphenol A and cholesterol, no chemicals were consistently associated with increased risks of illness. These two chemicals were suggestively associated with 2% and 1% increased risks of GI illness and diarrhea in both freshwater and marine beaches. Additional research using the more sensitive analytic methods currently available for a wider suite of analytes is needed to support the use of chemical biomarkers to quantify illness risk and identify fecal pollution sources.

Rapid Detection of Escherichia coli in Water Using Sample Concentration and Optimized Enzymatic Hydrolysis of Chromogenic Substrates.

Methods for rapid detection of fecal indicator bacteria in water are important to ensure that water is safe for drinking, bathing, recreation, fishing and shellfish harvesting. In this study, we tested experimental conditions for bacterial hydrolysis of two promising enzymatic substrates, 5-Bromo-4-chloro-3-indolyl ß-D-glucuronide (X-Gluc) and Resorufin ß-D-glucuronide (REG), and optimized parameters such as temperature and pH to determine conditions for rapid reactions. We then innovated a membrane filter-based approach to facilitate more rapid enzyme-based detection of Escherichia coli in water based on the combination of an initial concentration step and optimized test conditions. For this approach, a water sample (10‒100 mL) is filtered through a 0.45-µm pore size filter with a diameter of 4 or 13 mm. After filtration, a newly designed rapid detection broth is added containing the enzymatic inducer Methyl-beta-D-Glucuronide sodium (MetGlu) and the substrate REG or X-Gluc. After a few (1‒7) hours of incubation at 35 °C, the filter shows pink color (for REG-containing broth) or green color (for X-Gluc containing broth) if E. coli is present. The study provides insights and approaches towards developing a simple, fast, and low-cost method to detect fecal indicator bacteria in water.

Exposure to human-associated fecal indicators and self-reported illness among swimmers at recreational beaches: a cohort study.

BACKGROUND: Fecal indicator bacteria used to assess illness risks in recreational waters (e.g., Escherichia coli, Enterococci) cannot discriminate among pollution sources. To address this limitation, human-associated Bacteroides markers have been proposed, but the risk of illness associated with the presence of these markers in recreational waters is unclear. Our objective was to estimate associations between human-associated Bacteroides markers in water and self-reported illness among swimmers at 6 U.S. beaches spanning 2003-2007. METHODS: We used data from a prospectively-enrolled cohort of 12,060 swimmers surveyed about beach activities and water exposure on the day of their beach visit. Ten to twelve days later, participants reported gastroinestinal, diarrheal, and respiratory illnesses experienced since the visit. Daily water samples were analyzed for the presence of human-associated Bacteroides genetic markers: HF183, BsteriF1, BuniF2, HumM2. We used model-based standardization to estimate risk differences (RD) and 95% confidence intervals (CI). We assessed whether the presence of Bacteroides markers were modifiers of the association between general Enterococcus and illness among swimmers using interaction contrast. RESULTS: Overall we observed inconsistent associations between the presence of Bacteroides markers and illness. There was a pattern of increased risks of gastrointestinal (RD = 1.9%; 95% CI: 0.1%, 3.7%), diarrheal (RD = 1.3%; 95% CI: -0.2%, 2.7%), and respiratory illnesses (RD = 1.1%; 95% CI: -0.2%, 2.5%) associated with BsteriF1. There was no evidence that Bacteroides markers acted as modifiers of Enterococcus and illness. Patterns were similar when stratified by water matrix. CONCLUSIONS: Quantitative measures of fecal pollution using Bacteroides, rather than presence-absence indicators, may be necessary to accurately assess human risk specific to the presence of human fecal pollution.

Decay of Coliphages in Sewage-Contaminated Freshwater: Uncertainty and Seasonal Effects.

Understanding the fate of enteric viruses in water is vital for protection of water quality. However, the decay of enteric viruses is not well characterized, and its uncertainty has not been examined yet. In this study, the decay of coliphages, an indicator for enteric viruses, was investigated in situ under both sunlit and shaded conditions as well as in summer and winter. The decay rates of coliphages and their uncertainties were analyzed using a Bayesian approach. The results from the summer experiments revealed that the decay rates of somatic coliphages were significantly higher in sunlight (1.29 ± 0.06 day-1) than in shade (0.96 ± 0.04 day-1), but the decay rates of male-specific (F+) coliphages were not significantly different between sunlight (1.09 ± 0.09 day-1) and shaded treatments (1.11 ± 0.08 day-1). The decay rates of both F+ coliphages (0.25 ± 0.02 day-1) and somatic coliphages (0.12 ± 0.01 day-1) in winter were considerably lower than those in summer. Temperature and chlorophyll a (chla) concentration varied significantly (p < 0.001) between the two seasons, suggesting that these parameters might be important contributors to the seasonal variation of coliphage decay. Additionally, the Bayesian approach provided full distributions of decay rates and reduced the uncertainty, offering useful information for comparing decay rates under different conditions.

A case control study of environmental and occupational exposures associated with methicillin resistant Staphylococcus aureus nasal carriage in patients admitted to a rural tertiary care hospital in a high density swine region.

BACKGROUND: Distinct strains of methicillin resistant Staphylococcus aureus (MRSA) have been identified on livestock and livestock workers. Industrial food animal production may be an important environmental reservoir for human carriage of these pathogenic bacteria. The objective of this study was to investigate environmental and occupational exposures associated with nasal carriage of MRSA in patients hospitalized at Vidant Medical Center, a tertiary hospital serving a region with intensive livestock production in eastern North Carolina. METHODS: MRSA nasal carriage was identified via nasal swabs collected within 24 hours of hospital admission. MRSA carriers (cases) were gender and age matched to non-carriers (controls). Participants were interviewed about recent environmental and occupational exposures. Home addresses were geocoded and publicly available data were used to estimate the density of swine in residential census block groups of residence. Conditional logistic regression models were used to derive odds ratio (OR) estimates and 95% confidence intervals (CI). Presence of the scn gene in MRSA isolates was assessed. In addition, multi locus sequence typing (MLST) of the MRSA isolates was performed, and the Diversilab® system was used to match the isolates to USA pulsed field gel electrophoresis types. RESULTS: From July - December 2011, 117 cases and 119 controls were enrolled. A higher proportion of controls than cases were current workforce members (41.2% vs. 31.6%) Cases had a higher odds of living in census block groups with medium densities of swine (OR: 4.76, 95% CI: 1.36-16.69) and of reporting the ability to smell odor from a farm with animals when they were home (OR: 1.51, 95% CI: 0.80-2.86). Of 49 culture positive MRSA isolates, all were scn positive. Twenty-two isolates belonged to clonal complex 5. CONCLUSIONS: Absence of livestock workers in this study precluded evaluation of occupational exposures. Higher odds of MRSA in medium swine density areas could reflect environmental exposure to swine or poultry.

Human viruses and viral indicators in marine water at two recreational beaches in Southern California, USA.

Waterborne enteric viruses may pose disease risks to bather health but occurrence of these viruses has been difficult to characterize at recreational beaches. The aim of this study was to evaluate water for human virus occurrence at two Southern California recreational beaches with a history of beach closures. Human enteric viruses (adenovirus and norovirus) and viral indicators (F+ and somatic coliphages) were measured in water samples over a 4-month period from Avalon Beach, Catalina Island (n = 324) and Doheny Beach, Orange County (n = 112). Human viruses were concentrated from 40 L samples and detected by nested reverse transcriptase polymerase chain reaction (PCR). Detection frequencies at Doheny Beach were 25.5% (adenovirus) and 22.3% (norovirus), and at Avalon Beach were 9.3% (adenovirus) and 0.7% (norovirus). Positive associations between adenoviruses and fecal coliforms were observed at Doheny (p = 0.02) and Avalon (p = 0.01) Beaches. Human viruses were present at both beaches at higher frequencies than previously detected in the region, suggesting that the virus detection methods presented here may better measure potential health risks to bathers. These virus recovery, concentration, and molecular detection methods are advancing practices so that analysis of enteric viruses can become more effective and routine for recreational water quality monitoring.

Survey of antibiotic-resistant bacteria isolated from bottlenose dolphins Tursiops truncatus in the southeastern USA.

Contamination of coastal waters can carry pathogens and contaminants that cause diseases in humans and wildlife, and these pathogens can be transported by water to areas where they are not indigenous. Marine mammals may be indicators of potential health effects from such pathogens and toxins. Here we isolated bacterial species of relevance to humans from wild bottlenose dolphins Tursiops truncatus and assayed isolated bacteria for antibiotic resistance. Samples were collected during capture-release dolphin health assessments at multiple coastal and estuarine sites along the US mid-Atlantic coast and the Gulf of Mexico. These samples were transported on ice and evaluated using commercial systems and aerobic culture techniques routinely employed in clinical laboratories. The most common bacteria identified were species belonging to the genus Vibrio, although Escherichia coli, Shewanella putrefaciens, and Pseudomonas fluorescens/putida were also common. Some of the bacterial species identified have been associated with human illness, including a strain of methicillin-resistant Staphylococcus aureus (MRSA) identified in 1 sample. Widespread antibiotic resistance was observed among all sites, although the percentage of resistant isolates varied across sites and across time. These data provide a baseline for future comparisons of the bacteria that colonize bottlenose dolphins in the southeastern USA.

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.

BACKGROUND: 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. METHODS: 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. FINDINGS: 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. INTERPRETATION: 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. FUNDING: The Bill & Melinda Gates Foundation and the UK Foreign and Commonwealth Development Office.

Probabilistic analysis showing that a combination of Bacteroides and Methanobrevibacter source tracking markers is effective for identifying waters contaminated by human fecal pollution.

Microbial source tracking assays to identify sources of waterborne contamination typically target genetic markers of host-specific microorganisms. However, no bacterial marker has been shown to be 100% host-specific, and cross-reactivity has been noted in studies evaluating known source samples. Using 485 challenge samples from 20 different human and animal fecal sources, this study evaluated microbial source tracking markers including the Bacteroides HF183 16S rRNA, M. smithii nifH, and Enterococcus esp gene targets that have been proposed as potential indicators of human fecal contamination. Bayes' Theorem was used to calculate the conditional probability that these markers or a combination of markers can correctly identify human sources of fecal pollution. All three human-associated markers were detected in 100% of the sewage samples analyzed. Bacteroides HF183 was the most effective marker for determining whether contamination was specifically from a human source, and greater than 98% certainty that contamination was from a human source was shown when both Bacteroides HF183 and M. smithii nifH markers were present. A high degree of certainty was attained even in cases where the prior probability of human fecal contamination was as low as 8.5%. The combination of Bacteroides HF183 and M. smithii nifH source tracking markers can help identify surface waters impacted by human fecal contamination, information useful for prioritizing restoration activities or assessing health risks from exposure to contaminated waters.

Relating Antimicrobial Resistance and Virulence in Surface-Water E. coli.

The role of the environment in the emergence and spread of antimicrobial resistance (AMR) is being increasingly recognized, raising questions about the public health risks associated with environmental AMR. Yet, little is known about pathogenicity among resistant bacteria in environmental systems. Existing studies on the association between AMR and virulence are contradictory, as fitness costs and genetic co-occurrence can be opposing influences. Using Escherichia coli isolated from surface waters in eastern North Carolina, we compared virulence gene prevalence between isolates resistant and susceptible to antibiotics. We also compared the prevalence of isolates from sub-watersheds with or without commercial hog operations (CHOs). Isolates that had previously been evaluated for phenotypic AMR were paired by matching isolates resistant to any tested antibiotic with fully susceptible isolates from the same sample date and site, forming 87 pairs. These 174 isolates were evaluated by conventional PCR for seven virulence genes (bfp, fimH, cnf-1, STa (estA), EAST-1 (astA), eae, and hlyA). One gene, fimH, was found in 93.1% of isolates. Excluding fimH, at least one virulence gene was detected in 24.7% of isolates. Significant negative associations were found between resistance to at least one antibiotic and presence of at least one virulence gene, tetracycline resistance and presence of a virulence gene, resistance and STa presence, and tetracycline resistance and STa presence. No significant associations were found between CHO presence and virulence, though some sub-significant associations merit further study. This work builds our understanding of factors controlling AMR dissemination through the environment and potential health risks.

Identifying sources of antibiotic resistance genes in the environment using the microbial Find, Inform, and Test framework.

Introduction: Antimicrobial resistance (AMR) is an increasing public health concern for humans, animals, and the environment. However, the contributions of spatially distributed sources of AMR in the environment are not well defined. Methods: To identify the sources of environmental AMR, the novel microbial Find, Inform, and Test (FIT) model was applied to a panel of five antibiotic resistance-associated genes (ARGs), namely, erm(B), tet(W), qnrA, sul1, and intI1, quantified from riverbed sediment and surface water from a mixed-use region. Results: A one standard deviation increase in the modeled contributions of elevated AMR from bovine sources or land-applied waste sources [land application of biosolids, sludge, and industrial wastewater (i.e., food processing) and domestic (i.e., municipal and septage)] was associated with 34-80% and 33-77% increases in the relative abundances of the ARGs in riverbed sediment and surface water, respectively. Sources influenced environmental AMR at overland distances of up to 13 km. Discussion: Our study corroborates previous evidence of offsite migration of microbial pollution from bovine sources and newly suggests offsite migration from land-applied waste. With FIT, we estimated the distance-based influence range overland and downstream around sources to model the impact these sources may have on AMR at unsampled sites. This modeling supports targeted monitoring of AMR from sources for future exposure and risk mitigation efforts.