Comparison of methods proposed for monitoring cefotaxime-resistant Escherichia coli in the water environment.

Escherichia coli is a promising subject for globally coordinated surveillance of antimicrobial resistance (AMR) in water environments due to its clinical relevance and widespread use as an indicator of fecal contamination. Cefotaxime-resistant E. coli was recently evaluated favorably for this purpose by the World Health Organization TriCycle Protocol, which specifies tryptone bile x-glucuronide (TBX) medium and incubation at 35°C. We assessed comparability with the U.S. Environmental Protection Agency-approved method for E. coli quantification, which uses membrane-thermotolerant E. coli (mTEC) agar and incubation at 44.5°C, in terms of recovery of E. coli and cefotaxime-resistant E. coli from wastewater influent and surface waters. Total E. coli concentrations in wastewater influent were 106-108 CFU/100 mL, while cefotaxime-resistant E. coli were ~100-fold lower. Total E. coli in surface waters were ~102 CFU/100 mL, and cefotaxime-resistant isolates were near the limit of detection (0.4 CFU/100 mL). Total and putative cefotaxime-resistant E. coli concentrations did not differ significantly between media or by incubation method; however, colonies isolated on mTEC were more frequently confirmed to species (97.1%) compared to those from TBX (92.5%). Incubation in a water bath at 44.5°C significantly decreased non-specific background growth and improved confirmation frequency on both media (97.4%) compared to incubation at 35°C (92.3%). This study helps to advance globally coordinated AMR in water environments and suggests that the TriCycle Protocol is adaptable to other standard methods that may be required in different locales, while also offering a means to improve specificity by decreasing the frequency of false-positive identification of cefotaxime-resistant E. coli by modifying incubation conditions.IMPORTANCEAs antibiotic-resistant bacteria in water environments are increasingly recognized as contributors to the global antibiotic resistance crisis, the need for a monitoring subject that captures antibiotic resistance trends on a global scale increases. The World Health Organization TriCycle Protocol proposes the use of cefotaxime-resistant Escherichia coli isolated on tryptone bile x-glucuronide agar. The U.S. Environmental Protection Agency (USEPA) criteria for safe recreational waters also use E. coli as an indicator but specify the use of mTEC agar at a higher incubation temperature (44.5°C vs 35°C). We assessed the comparability of these methods for isolating total and cefotaxime-resistant E. coli, finding overall good agreement and performance, but significantly higher specificity toward E. coli selection with the use of the USEPA incubation protocol and mTEC agar. This study is the first to directly compare these methods and provides evidence that the methods may be used interchangeably for global surveillance of antibiotic resistance in the environment.

Interventions can shift the thermal optimum for parasitic disease transmission.

Temperature constrains the transmission of many pathogens. Interventions that target temperature-sensitive life stages, such as vector control measures that kill intermediate hosts, could shift the thermal optimum of transmission, thereby altering seasonal disease dynamics and rendering interventions less effective at certain times of the year and with global climate change. To test these hypotheses, we integrated an epidemiological model of schistosomiasis with empirically determined temperature-dependent traits of the human parasite Schistosoma mansoni and its intermediate snail host (Biomphalaria spp.). We show that transmission risk peaks at 21.7 °C (Topt ), and simulated interventions targeting snails and free-living parasite larvae increased Topt by up to 1.3 °C because intervention-related mortality overrode thermal constraints on transmission. This Topt shift suggests that snail control is more effective at lower temperatures, and global climate change will increase schistosomiasis risk in regions that move closer to Topt Considering regional transmission phenologies and timing of interventions when local conditions approach Topt will maximize human health outcomes.

Risk of Gastroenteritis from Swimming at a Wastewater-Impacted Tropical Beach Varies across Localized Scales.

Population growth and changing climate are expected to increase human exposure to pathogens in tropical coastal waters. We examined microbiological water quality in three rivers within 2.3 km of each other that impact a Costa Rican beach and in the ocean outside their plumes during the rainy and dry seasons. We performed quantitative microbial risk assessment (QMRA) to predict the risk of gastroenteritis associated with swimming and the amount of pathogen reduction needed to achieve safe conditions. Recreational water quality criteria based on enterococci were exceeded in >90% of river samples but in only 13% of ocean samples. Multivariate analysis grouped microbial observations by subwatershed and season in river samples but only by subwatershed in the ocean. The modeled median risk from all pathogens in river samples was between 0.345 and 0.577, 10-fold above the U.S. Environmental Protection Agency (U.S. EPA) benchmark of 0.036 (36 illnesses/1,000 swimmers). Norovirus genogroup I (NoVGI) contributed most to risk, but adenoviruses raised risk above the threshold in the two most urban subwatersheds. The risk was greater in the dry compared to the rainy season, due largely to the greater frequency of NoVGI detection (100% versus 41%). Viral log10 reduction needed to ensure safe swimming conditions varied by subwatershed and season and was greatest in the dry season (3.8 to 4.1 dry; 2.7 to 3.2 rainy). QMRA that accounts for seasonal and local variability of water quality contributes to understanding the complex influences of hydrology, land use, and environment on human health risk in tropical coastal areas and can contribute to improved beach management. IMPORTANCE This holistic investigation of sanitary water quality at a Costa Rican beach assessed microbial source tracking (MST) marker genes, pathogens, and indicators of sewage. Such studies are still rare in tropical climates. Quantitative microbial risk assessment (QMRA) found that rivers impacting the beach consistently exceeded the U.S. EPA risk threshold for gastroenteritis of 36/1,000 swimmers. The study improves upon many QMRA studies by measuring specific pathogens, rather than relying on surrogates (indicator organisms or MST markers) or estimating pathogen concentrations from the literature. By analyzing microbial levels and estimating the risk of gastrointestinal illness in each river, we were able to discern differences in pathogen levels and human health risks even though all rivers were highly polluted by wastewater and were located less than 2.5 km from one another. This variability on a localized scale has not, to our knowledge, previously been demonstrated.

Antimicrobial Resistance Monitoring of Water Environments: A Framework for Standardized Methods and Quality Control.

Antimicrobial resistance (AMR) is a grand societal challenge with important dimensions in the water environment that contribute to its evolution and spread. Environmental monitoring could provide vital information for mitigating the spread of AMR; this includes assessing antibiotic resistance genes (ARGs) circulating among human populations, identifying key hotspots for evolution and dissemination of resistance, informing epidemiological and human health risk assessment models, and quantifying removal efficiencies by domestic wastewater infrastructure. However, standardized methods for monitoring AMR in the water environment will be vital to producing the comparable data sets needed to address such questions. Here we sought to establish scientific consensus on a framework for such standardization, evaluating the state of the science and practice of AMR monitoring of wastewater, recycled water, and surface water, through a literature review, survey, and workshop leveraging the expertise of academic, governmental, consulting, and water utility professionals.

An assessment of three methods for extracting bacterial DNA from beach sand.

AIMS: Beach water quality is regulated by faecal indicator bacteria levels, sand is not, despite known human health risk from exposure to beach sand. We compared the performance of three methods to extract bacterial DNA from beach sand as a step toward a standard method. METHODS AND RESULTS: The analytical sensitivity of quantitative polymerase chain reaction (qPCR) for Enterococcus was compared for the slurry (suspension, agitation, membrane filtration of supernatant), versus direct extraction using PowerSoil™ or PowerMax Soil™ kits. The slurry method had the lowest limit of detection at 20-80 gene copies g-1 , recovered significantly more DNA, and the only method that detected Enterococcus by qPCR in all samples; therefore, the only method used in subsequent experiments. The slurry method reflected the spatial variability of Enterococcus in individual transect samples. Mean recovery efficiency of the microbial source tracking marker HF183 from wastewater spiked marine and freshwater beach sand was 100.8% and 64.1%, respectively, but varied, indicating that the mixing protocol needs improvement. CONCLUSIONS: Among the three methods, the slurry method had the best analytical sensitivity and produced extracts that were useful for culture or molecular analysis. SIGNIFICANCE AND IMPACT OF STUDY: Standardization of methods for extraction of bacterial DNA from sand facilitates comparisons among studies, and ultimately contributes to the safety of recreational beaches.

Application of SourceTracker for Accurate Identification of Fecal Pollution in Recreational Freshwater: A Double-Blinded Study.

The efficacy of SourceTracker software to attribute contamination from a variety of fecal sources spiked into ambient freshwater samples was investigated. Double-blinded samples spiked with ≤5 different sources (0.025-10% vol/vol) were evaluated against fecal taxon libraries characterized by next-generation amplicon sequencing. Three libraries, including an initial library (17 nonlocal sources), a blinded source library (5 local sources), and a composite library (local and nonlocal sources), were used with SourceTracker. SourceTracker's predictions of fecal compositions in samples were made, in part, based on distributions of taxa within abundant genera identified as discriminatory by discriminant analyses but also using a large percentage of low abundance taxa. The initial library showed poor ability to characterize blinded samples, but, using local sources, SourceTracker showed 91% accuracy (31/34) at identifying the presence of source contamination, with two false positives for sewage and one for horse. Furthermore, sink predictions of source contamination were positively correlated (Spearman's ρ ≥ 0.88, P < 0.001) with spiked source volumes. Using the composite library did not significantly affect sink predictions ( P > 0.79) compared to those made using the local sources alone. Results of this study indicate that geographically associated fecal samples are required for SourceTracker to assign host sources accurately.

Ultrafiltration and Microarray for Detection of Microbial Source Tracking Marker and Pathogen Genes in Riverine and Marine Systems.

Pathogen identification and microbial source tracking (MST) to identify sources of fecal pollution improve evaluation of water quality. They contribute to improved assessment of human health risks and remediation of pollution sources. An MST microarray was used to simultaneously detect genes for multiple pathogens and indicators of fecal pollution in freshwater, marine water, sewage-contaminated freshwater and marine water, and treated wastewater. Dead-end ultrafiltration (DEUF) was used to concentrate organisms from water samples, yielding a recovery efficiency of >95% for Escherichia coli and human polyomavirus. Whole-genome amplification (WGA) increased gene copies from ultrafiltered samples and increased the sensitivity of the microarray. Viruses (adenovirus, bocavirus, hepatitis A virus, and human polyomaviruses) were detected in sewage-contaminated samples. Pathogens such as Legionella pneumophila, Shigella flexneri, and Campylobacter fetus were detected along with genes conferring resistance to aminoglycosides, beta-lactams, and tetracycline. Nonmetric dimensional analysis of MST marker genes grouped sewage-spiked freshwater and marine samples with sewage and apart from other fecal sources. The sensitivity (percent true positives) of the microarray probes for gene targets anticipated in sewage was 51 to 57% and was lower than the specificity (percent true negatives; 79 to 81%). A linear relationship between gene copies determined by quantitative PCR and microarray fluorescence was found, indicating the semiquantitative nature of the MST microarray. These results indicate that ultrafiltration coupled with WGA provides sufficient nucleic acids for detection of viruses, bacteria, protozoa, and antibiotic resistance genes by the microarray in applications ranging from beach monitoring to risk assessment.

Vancomycin-Resistant Enterococci and Bacterial Community Structure following a Sewage Spill into an Aquatic Environment.

UNLABELLED: Sewage spills can release antibiotic-resistant bacteria into surface waters, contributing to environmental reservoirs and potentially impacting human health. Vancomycin-resistant enterococci (VRE) are nosocomial pathogens that have been detected in environmental habitats, including soil, water, and beach sands, as well as wildlife feces. However, VRE harboring vanA genes that confer high-level resistance have infrequently been found outside clinical settings in the United States. This study found culturable Enterococcus faecium harboring the vanA gene in water and sediment for up to 3 days after a sewage spill, and the quantitative PCR (qPCR) signal for vanA persisted for an additional week. Culturable levels of enterococci in water exceeded recreational water guidelines for 2 weeks following the spill, declining about five orders of magnitude in sediments and two orders of magnitude in the water column over 6 weeks. Analysis of bacterial taxa via 16S rRNA gene sequencing showed changes in community structure through time following the sewage spill in sediment and water. The spread of opportunistic pathogens harboring high-level vancomycin resistance genes beyond hospitals and into the broader community and associated habitats is a potential threat to public health, requiring further studies that examine the persistence, occurrence, and survival of VRE in different environmental matrices. IMPORTANCE: Vancomycin-resistant enterococci (VRE) are harmful bacteria that are resistant to the powerful antibiotic vancomycin, which is used as a last resort against many infections. This study followed the release of VRE in a major sewage spill and their persistence over time. Such events can act as a means of spreading vancomycin-resistant bacteria in the environment, which can eventually impact human health.

Human-Associated Bacteroides spp. and Human Polyomaviruses as Microbial Source Tracking Markers in Hawaii.

Identification of sources of fecal contaminants is needed to (i) determine the health risk associated with recreational water use and (ii) implement appropriate management practices to mitigate this risk and protect the environment. This study evaluated human-associated Bacteroides spp. (HF183TaqMan) and human polyomavirus (HPyV) markers for host sensitivity and specificity using human and animal fecal samples collected in Hawaii. The decay rates of those markers and indicator bacteria were identified in marine and freshwater microcosms exposed and not exposed to sunlight, followed by field testing of the usability of the molecular markers. Both markers were strongly associated with sewage, although the cross-reactivity of the HF183TaqMan (also present in 82% of canine [n = 11], 30% of mongoose [n = 10], and 10% of feline [n = 10] samples) needs to be considered. Concentrations of HF183TaqMan in human fecal samples exceeded those in cross-reactive animals at least 1,000-fold. In the absence of sunlight, the decay rates of both markers were comparable to the die-off rates of enterococci in experimental freshwater and marine water microcosms. However, in sunlight, the decay rates of both markers were significantly lower than the decay rate of enterococci. While both markers have their individual limitations in terms of sensitivity and specificity, these limitations can be mitigated by using both markers simultaneously; ergo, this study supports the concurrent use of HF183TaqMan and HPyV markers for the detection of sewage contamination in coastal and inland waters in Hawaii. IMPORTANCE: This study represents an in-depth characterization of microbial source tracking (MST) markers in Hawaii. The distribution and concentrations of HF183TaqMan and HPyV markers in human and animal fecal samples and in wastewater, coupled with decay data obtained from sunlight-exposed and unexposed microcosms, support the concurrent application of HF183TaqMan and HPyV markers for sewage contamination detection in Hawaii waters. Both markers are more conservative and more specific markers of sewage than fecal indicator bacteria (enterococci and Escherichia coli). Analysis of HF183TaqMan (or newer derivatives) is recommended for inclusion in future epidemiological studies concerned with beach water quality, while better concentration techniques are needed for HPyV. Such epidemiological studies can be used to develop new recreational water quality criteria, which will provide direct information on the absence or presence of sewage contamination in water samples as well as reliable measurements of the risk of waterborne disease transmission to swimmers.

LA35 Poultry Fecal Marker Persistence Is Correlated with That of Indicators and Pathogens in Environmental Waters.

Disposal of fecally contaminated poultry litter by land application can deliver pathogens and fecal indicator bacteria (FIB) into receiving waters via runoff. While water quality is regulated by FIB enumeration, FIB testing provides inadequate information about contamination source and health risk. This microbial source tracking (MST) study compared the persistence of the Brevibacterium sp. strain LA35 16S rRNA gene (marker) for poultry litter with that of pathogens and FIB under outdoor, environmentally relevant conditions in freshwater, marine water, and sediments over 7 days. Salmonella enterica, Campylobacter jejuni, Campylobacter coli, Bacteroidales, and LA35 were enumerated by quantitative PCR (qPCR), and Enterococcus spp. and E. coli were quantified by culture and qPCR. Unlike the other bacteria, C. jejuni was not detectable after 48 h. Bacterial levels in the water column consistently declined over time and were highly correlated among species. Survival in sediments ranged from a slow decrease over time to growth, particularly in marine microcosms and for Bacteroidales. S. enterica also grew in marine sediments. Linear decay rates in water (k) ranged from -0.17 day(-1) for LA35 to -3.12 day(-1) for C. coli. LA35 levels correlated well with those of other bacteria in the water column but not in sediments. These observations suggest that, particularly in the water column, the fate of LA35 in aquatic environments is similar to that of FIB, C. coli, and Salmonella, supporting the hypothesis that the LA35 marker gene can be a useful tool for evaluating the impact of poultry litter on water quality and human health risk.

Sediment and vegetation as reservoirs of Vibrio vulnificus in the Tampa Bay Estuary and Gulf of Mexico.

The opportunistic pathogen Vibrio vulnificus occurs naturally in estuarine habitats and is readily cultured from water and oysters under warm conditions but infrequently at ambient conditions of <15°C. The presence of V. vulnificus in other habitats, such as sediments and aquatic vegetation, has been explored much less frequently. This study investigated the ecology of V. vulnificus in water by culture and quantitative PCR (qPCR) and in sediment, oysters, and aquatic vegetation by culture. V. vulnificus samples were taken from five sites around Tampa Bay, FL. Levels determined by qPCR and culture were significantly correlated (P = 0.0006; r = 0.352); however, V. vulnificus was detected significantly more frequently by qPCR (85% of all samples) compared to culture (43%). Culturable V. vulnificus bacteria were recovered most frequently from oyster samples (70%), followed by vegetation and sediment (∼50%) and water (43%). Water temperature, which ranged from 18.5 to 33.4°C, was positively correlated with V. vulnificus concentrations in all matrices but sediments. Salinity, which ranged from 1 to 35 ppt, was negatively correlated with V. vulnificus levels in water and sediments but not in other matrices. Significant interaction effects between matrix and temperature support the hypothesis that temperature affects V. vulnificus concentrations differently in different matrices and that sediment habitats may serve as seasonal reservoirs for V. vulnificus. V. vulnificus levels in vegetation have not been previously measured and reveal an additional habitat for this autochthonous estuarine bacterium.

Evidence for extraintestinal growth of bacteroidales originating from poultry litter.

Water quality monitoring techniques that target microorganisms in the order Bacteroidales are potential alternatives to conventional methods for detection of fecal indicator bacteria. Bacteroidales and members of the genus Bacteroides have been the focus of microbial source tracking (MST) investigations for discriminating sources of fecal pollution (e.g., human or cattle feces) in environmental waters. For accurate source apportionment to occur, one needs to understand both the abundance of Bacteroides in host feces and the survival of these host-associated microbial markers after deposition in the environment. Studies were undertaken to evaluate the abundance, persistence, and potential for growth of Bacteroidales originating from poultry litter under oxic and anoxic environmental conditions. Bacteroidales abundance, as determined by quantitative PCR (qPCR) with GenBac primers and probe, increased 2 to 5 log gene copies ml(-1) and 2 log gene copies g litter(-1) under most conditions during incubation of poultry litter in a variety of laboratory microcosm and field mesocosm studies. DNA sequencing of the Bacteroidales organisms in the litter identified taxa with sequences corresponding exactly to the GenBac primer and probe sequences and that were closely related to Bacteroides uniformis, B. ovatus, and B. vulgatus. These results suggest that MST studies using qPCR methods targeting Bacteroidales in watersheds that are affected by poultry litter should be interpreted cautiously. Growth of Bacteroidales originating from poultry litter in environmental waters may occur while Bacteroidales growth from other fecal sources declines, thus confounding the interpretation of MST results.

A synthesis of the effects of pesticides on microbial persistence in aquatic ecosystems.

Pesticides have a pervasive presence in aquatic ecosystems throughout the world. While pesticides are intended to control fungi, insects, and other pests, their mechanisms of action are often not specific enough to prevent unintended effects, such as on non-target microbial populations. Microorganisms, including algae and cyanobacteria, protozoa, aquatic fungi, and bacteria, form the basis of many food webs and are responsible for crucial aspects of biogeochemical cycling; therefore, the potential for pesticides to alter microbial community structures must be understood to preserve ecosystem services. This review examines studies that focused on direct population-level effects and indirect community-level effects of pesticides on microorganisms. Generally, insecticides, herbicides, and fungicides were found to have adverse direct effects on algal and fungal species. Insecticides and fungicides also had deleterious direct effects in the majority of studies examining protozoa species, although herbicides were found to have inconsistent direct effects on protozoans. Our synthesis revealed mixed or no direct effects on bacterial species among all pesticide categories, with results highly dependent on the target species, chemical, and concentration used in the study. Examination of community-level, indirect effects revealed that all pesticide categories had a tendency to reduce higher trophic levels, thereby diminishing top-down pressures and favoring lower trophic levels. Often, indirect effects exerted greater influence than direct effects. However, few studies have been conducted to specifically address community-level effects of pesticides on microorganisms, and further research is necessary to better understand and predict the net effects of pesticides on ecosystem health.

A novel microbial source tracking microarray for pathogen detection and fecal source identification in environmental systems.

Pathogen detection and the identification of fecal contamination sources are challenging in environmental waters. Factors including pathogen diversity and ubiquity of fecal indicator bacteria hamper risk assessment and remediation of contamination sources. A custom microarray targeting pathogens (viruses, bacteria, protozoa), microbial source tracking (MST) markers, and antibiotic resistance genes was tested against DNA obtained from whole genome amplification (WGA) of RNA and DNA from sewage and animal (avian, cattle, poultry, and swine) feces. Perfect and mismatch probes established the specificity of the microarray in sewage, and fluorescence decrease of positive probes over a 1:10 dilution series demonstrated semiquantitative measurement. Pathogens, including norovirus, Campylobacter fetus, Helicobacter pylori, Salmonella enterica, and Giardia lamblia were detected in sewage, as well as MST markers and resistance genes to aminoglycosides, beta-lactams, and tetracycline. Sensitivity (percentage true positives) of MST results in sewage and animal waste samples (21-33%) was lower than specificity (83-90%, percentage of true negatives). Next generation DNA sequencing revealed two dominant bacterial families that were common to all sample types: Ruminococcaceae and Lachnospiraceae. Five dominant phyla and 15 dominant families comprised 97% and 74%, respectively, of sequences from all fecal sources. Phyla and families not represented on the microarray are possible candidates for inclusion in subsequent array designs.

Agrochemicals indirectly increase survival of E. coli O157:H7 and indicator bacteria by reducing ecosystem services.

Storm water and agricultural runoff frequently contain agrochemicals, fecal indicator bacteria (FIB), and zoonotic pathogens. Entry of such contaminants into aquatic ecosystems may affect ecology and human health. This study tested the hypothesis that the herbicide atrazine and the fungicide chlorothalonil indirectly affect the survival of FIB (Escherichia coli and Enterococcus faecalis) and a pathogen (E. coli O157:H7) by altering densities of protozoan predators or by altering competition from autochthonous bacteria. Streptomycin-resistant E. coli, En. faecalis, and E. coli O157:H7 were added to microcosms composed of Florida river water containing natural protozoan and bacterial populations. FIB, pathogen, and protozoan densities were monitored over six days. Known metabolic inhibitors, cycloheximide and streptomycin, were used to inhibit autochthonous protozoa or bacteria, respectively. The inhibitors made it possible to isolate the effects of predation or competition on survival of allochthonous bacteria, and each treatment increased the survival of FIB and pathogens. Chlorothalonil's effect was similar to that of cycloheximide, significantly reducing protozoan densities and elevating densities of FIB and pathogens relative to the control. Atrazine treatment did not affect protozoan densities, but, through an effect on competition, resulted in significantly greater densities of En. faecalis and E. coli O157:H7. Hence, by reducing predaceous protozoa and bacterial competitors that facilitate purifying water bodies of FIBs and human pathogens, chlorothalonil and atrazine indirectly diminished an ecosystem service of fresh water.

Differential expression of a sodium-phosphate cotransporter among Vibrio vulnificus strains.

Vibrio vulnificus is an estuarine bacterium with pathogenic potential. Its three known biotypes differ in host distribution. We have found the nptA gene for a sodium-phosphate cotransporter, which is rare in bacteria, in each biotype. nptA transcript abundance differed significantly among biotypes, leading to the hypothesis that transcript levels differ under environmental conditions associated with estuarine and host environments. nptA transcript abundance was assessed in V. vulnificus biotypes 1 (C and E genotypes), 2 and 3 strains under varied salinity, phosphate concentration, and pH. Differences in transcript abundance separated strains into two groups. Type C and biotype 3 strains formed Group 1, while type E and biotype 2 strains formed Group 2. Group 2 strains had significantly greater nptA RNA transcript abundance than Group 1. Transcript abundance in the two groups also responded differently to pH and salinity, suggesting differential regulation of nptA in response to environmental conditions. Comparison of the deduced amino acid sequences of NptA among strains resulted in strain grouping similar to that based on transcript abundance. Variation in transcript abundance between groups may affect the ability of V. vulnificus strains to colonize hosts and/or to compete as free-living bacteria in various habitats.

Protozoan predation is differentially affected by motility of enteric pathogens in water vs. sediments.

Survival of enteric bacteria in aquatic habitats varies depending upon species, strain, and environmental pressures, but the mechanisms governing their fate are poorly understood. Although predation by protozoa is a known, top-down control mechanism on bacterial populations, its influence on the survival of fecal-derived pathogens has not been systematically studied. We hypothesized that motility, a variable trait among pathogens, can influence predation rates and bacterial survival. We compared the survival of two motile pathogens of fecal origin by culturing Escherichia coli O157 and Salmonella enterica Typhimurium. Each species had a motile and non-motile counterpart and was cultured in outdoor microcosms with protozoan predators (Tetrahymena pyriformis) present or absent. Motility had a significant, positive effect on S. enterica levels in water and sediment in the presence or absence of predators. In contrast, motility had a significant negative effect on E. coli O157 levels in sediment, but did not affect water column levels. The presence/absence of protozoa consistently accounted for a greater proportion of the variability in bacterial levels (>95 %) than in bacterial motility (<4 %) in the water column. In sediments, however, motility was more important than predation for both bacteria. Calculations of total CFU/microcosm showed decreasing bacterial concentrations over time under all conditions except for S. enterica in the absence of predation, which increased ∼0.5-1.0 log over 5 days. These findings underscore the complexity of predicting the survival of enteric microorganisms in aquatic habitats, which has implications for the accuracy of risk assessment and modeling of water quality.

Persistence of sewage-associated genetic markers in advanced and conventional treated recycled water: implications for microbial source tracking in surface waters.

Sewage contamination of environmental waters is increasingly assessed by measuring DNA from sewage-associated microorganisms in microbial source tracking (MST) approaches. However, DNA can persist through wastewater treatment and reach surface waters when treated sewage/recycled water is discharged, which may falsely indicate pollution from untreated sewage. Recycled water discharged from an advanced wastewater treatment (AWT) facility into a Florida stream elevated the sewage-associated HF183 marker 1,000-fold, with a minimal increase in cultured Escherichia coli. The persistence of sewage-associated microorganisms was compared by qPCR in untreated sewage and recycled water from conventional wastewater treatment (CWT) and AWT facilities. E. coli (EC23S857) and sewage-associated markers HF183, H8, and viral crAssphage CPQ_056 were always detected in untreated sewage (6.5-8.7 log10 GC/100 mL). Multivariate analysis found a significantly greater reduction of microbial variables via AWT vs CWT. Bacterial markers decayed ~4-5 log10 through CWT, but CPQ_056 was ~100-fold more persistent. In AWT facilities, the log10 reduction of all variables was ~5. In recycled water, bacterial marker concentrations were significantly correlated (P ≤ 0.0136; tau ≥ 0.44); however, CPQ_056 was not correlated with any marker, suggesting varying drivers of decay. Concentrations of cultured E. coli carrying the H8 marker (EcH8) in untreated sewage were 5.24-6.02 log10 CFU/100 mL, while no E. coli was isolated from recycled water. HF183 and culturable EcH8 were also correlated in contaminated surface waters (odds ratio ß1 = 1.701). Culturable EcH8 has a strong potential to differentiate positive MST marker signals arising from treated (e.g., recycled water) and untreated sewage discharged into environmental waters. IMPORTANCE: Genes in sewage-associated microorganisms are widely accepted indicators of sewage pollution in environmental waters. However, DNA persists through wastewater treatment and can reach surface waters when recycled water is discharged, potentially causing false-positive indications of sewage contamination. Previous studies have found that bacterial and viral sewage-associated genes persist through wastewater treatment; however, these studies did not compare different facilities or identify a solution to distinguish sewage from recycled water. In this study, we demonstrated the persistence of bacterial marker genes and the greater persistence of a viral marker gene (CPQ_056 of crAssphage) through varying wastewater treatment facilities. We also aim to provide a tool to confirm sewage contamination in surface waters with recycled water inputs. This work showed that the level of wastewater treatment affects the removal of microorganisms, particularly viruses, and expands our ability to identify sewage in surface waters.

To what extent do water reuse treatments reduce antibiotic resistance indicators? A comparison of two full-scale systems.

Water reuse is an essential strategy for reducing water demand from conventional sources, alleviating water stress, and promoting sustainability, but understanding the effectiveness of associated treatment processes as barriers to the spread of antibiotic resistance is an important consideration to protecting human health. We comprehensively evaluated the reduction of antibiotic resistance genes (ARGs) and antibiotic-resistant bacteria (ARB) in two field-operational water reuse systems with distinct treatment trains, one producing water for indirect potable reuse (ozone/biologically-active carbon/granular activated carbon) and the other for non-potable reuse (denitrification-filtration/chlorination) using metagenomic sequencing and culture. Relative abundances of total ARGs/clinically-relevant ARGs and cultured ARB were reduced by several logs during primary and secondary stages of wastewater treatment, but to a lesser extent during the tertiary water reuse treatments. In particular, ozonation tended to enrich multi-drug ARGs. The effect of chlorination was facility-dependent, increasing the relative abundance of ARGs when following biologically-active carbon filters, but generally providing a benefit in reduced bacterial numbers and ecological and human health resistome risk scores. Relative abundances of total ARGs and resistome risk scores were lowest in aquifer samples, although resistant Escherichia coli and Klebsiella pneumoniae were occasionally detected in the monitoring well 3-days downgradient from injection, but not 6-months downgradient. Resistant E. coli and Pseudomonas aeruginosa were occasionally detected in the nonpotable reuse distribution system, along with increased levels of multidrug, sulfonamide, phenicol, and aminoglycoside ARGs. This study illuminates specific vulnerabilities of water reuse systems to persistence, selection, and growth of ARGs and ARB and emphasizes the role of multiple treatment barriers, including aquifers and distribution systems.

The influence of predation and competition on the survival of commensal and pathogenic fecal bacteria in aquatic habitats.

The role of fecal indicator bacteria (FIB) in water quality assessment is to provide a warning of the increased risk of pathogen presence. An effective surrogate for waterborne pathogens would have similar survival characteristics in aquatic environments. Although the effect of abiotic factors such as sunlight and salinity on the survival of FIB and pathogens are becoming better understood, the effect of the indigenous microbiota is not well characterized. The influence of biotic factors on the survival of non-pathogenic Escherichia coli, Enterococcus faecalis, and E. coli O157:H7 were compared in fresh (river) water and sediments over 5 days. Treatments were (i) disinfection (filtration of water and baking of sediments) to remove indigenous protozoa (predators) and bacteria (competitors), and (ii) kanamycin treatment to reduce competition from indigenous bacteria. The disinfection treatment significantly increased survival of E. coli, E. coli O157:H7 and Ent. faecalis in the water column. In sediments, survival of FIB but not that of E. coli O157:H7 increased in disinfected treatments, indicating that the pathogen's survival was unaffected by the natural microbiota. Location (water or sediment) influenced bacterial survival more than species/type in the disinfection experiment. In the competition experiments where only the natural bacterial flora was manipulated, the addition of kanamycin did not affect the survival of Ent. faecalis, but resulted in greater survival of E. coli in water and sediment. Species/type influenced survival more than the level of competition in this experiment. This study demonstrates the complexity of interactions of FIB and pathogens with indigenous microbiota and location in aquatic habitats, and argues against over-generalizing conclusions derived from experiments restricted to a particular organism or habitat.