Wastewater surveillance for bacterial targets: current challenges and future goals.

Wastewater-based epidemiology (WBE) expanded rapidly in response to the COVID-19 pandemic. As the public health emergency has ended, researchers and practitioners are looking to shift the focus of existing wastewater surveillance programs to other targets, including bacteria. Bacterial targets may pose some unique challenges for WBE applications. To explore the current state of the field, the National Science Foundation-funded Research Coordination Network (RCN) on Wastewater Based Epidemiology for SARS-CoV-2 and Emerging Public Health Threats held a workshop in April 2023 to discuss the challenges and needs for wastewater bacterial surveillance. The targets and methods used in existing programs were diverse, with twelve different targets and nine different methods listed. Discussions during the workshop highlighted the challenges in adapting existing programs and identified research gaps in four key areas: choosing new targets, relating bacterial wastewater data to human disease incidence and prevalence, developing methods, and normalizing results. To help with these challenges and research gaps, the authors identified steps the larger community can take to improve bacteria wastewater surveillance. This includes developing data reporting standards and method optimization and validation for bacterial programs. Additionally, more work is needed to understand shedding patterns for potential bacterial targets to better relate wastewater data to human infections. Wastewater surveillance for bacteria can help provide insight into the underlying prevalence in communities, but much work is needed to establish these methods.IMPORTANCEWastewater surveillance was a useful tool to elucidate the burden and spread of SARS-CoV-2 during the pandemic. Public health officials and researchers are interested in expanding these surveillance programs to include bacterial targets, but many questions remain. The NSF-funded Research Coordination Network for Wastewater Surveillance of SARS-CoV-2 and Emerging Public Health Threats held a workshop to identify barriers and research gaps to implementing bacterial wastewater surveillance programs.

Correlation between wastewater and COVID-19 case incidence rates in major California sewersheds across three variant periods.

Monitoring for COVID-19 through wastewater has been used for adjunctive public health surveillance, with SARS-CoV-2 viral concentrations in wastewater correlating with incident cases in the same sewershed. However, the generalizability of these findings across sewersheds, laboratory methods, and time periods with changing variants and underlying population immunity has not been well described. The California Department of Public Health partnered with six wastewater treatment plants starting in January 2021 to monitor wastewater for SARS-CoV-2, with analyses performed at four laboratories. Using reported PCR-confirmed COVID-19 cases within each sewershed, the relationship between case incidence rates and wastewater concentrations collected over 14 months was evaluated using Spearman's correlation and linear regression. Strong correlations were observed when wastewater concentrations and incidence rates were averaged (10- and 7-day moving window for wastewater and cases, respectively, ρ = 0.73-0.98 for N1 gene target). Correlations remained strong across three time periods with distinct circulating variants and vaccination rates (winter 2020-2021/Alpha, summer 2021/Delta, and winter 2021-2022/Omicron). Linear regression revealed that slopes of associations varied by the dominant variant of concern, sewershed, and laboratory (ß = 0.45-1.94). These findings support wastewater surveillance as an adjunctive public health tool to monitor SARS-CoV-2 community trends.

Assessing cross-laboratory performance for quantifying coliphage using EPA Method 1642.

AIMS: Widespread adoption of the new U.S. Environmental Protection Agency (USEPA) Method 1642 for enumeration of coliphage in recreational water requires demonstration that laboratories consistently meet internal method performance goals and yield results that are consistent across laboratories. METHODS AND RESULTS: Here we assess the performance of six laboratories processing a series of blind wastewater- and coliphage-spiked samples along with laboratory blanks. All laboratories met the method-defined recovery requirements when performance was averaged across samples, with the few failures on individual samples mostly occurring for less-experienced laboratories on the initial samples processed. Failures that occurred on later samples were generally attributed to easily correctable activities. Failure rates were higher for somatic vs. F+ coliphage, attributable to the more stringent performance criteria associated with somatic coliphage. There was no difference in failure rate between samples prepared in a marine water matrix compared to that in phosphate-buffered saline. CONCLUSIONS: Variation among laboratories was similar to that previously reported for enterococci, the current bacterial indicator used for evaluating beach water quality for public health protection. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings suggest that laboratory performance is not an inhibitor to the adoption of coliphage as a new indicator for assessing recreational health risk.

RNA Viromics of Southern California Wastewater and Detection of SARS-CoV-2 Single-Nucleotide Variants.

Municipal wastewater provides an integrated sample of a diversity of human-associated microbes across a sewershed, including viruses. Wastewater-based epidemiology (WBE) is a promising strategy to detect pathogens and may serve as an early warning system for disease outbreaks. Notably, WBE has garnered substantial interest during the coronavirus disease 2019 (COVID-19) pandemic to track disease burden through analyses of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA. Throughout the COVID-19 outbreak, tracking SARS-CoV-2 in wastewater has been an important tool for understanding the spread of the virus. Unlike traditional sequencing of SARS-CoV-2 isolated from clinical samples, which adds testing burden to the health care system, in this study, metatranscriptomics was used to sequence virus directly from wastewater. Here, we present a study in which we explored RNA viral diversity through sequencing 94 wastewater influent samples across seven wastewater treatment plants (WTPs), collected from August 2020 to January 2021, representing approximately 16 million people in Southern California. Enriched viral libraries identified a wide diversity of RNA viruses that differed between WTPs and over time, with detected viruses including coronaviruses, influenza A, and noroviruses. Furthermore, single-nucleotide variants (SNVs) of SARS-CoV-2 were identified in wastewater, and we measured proportions of overall virus and SNVs across several months. We detected several SNVs that are markers for clinically important SARS-CoV-2 variants along with SNVs of unknown function, prevalence, or epidemiological consequence. Our study shows the potential of WBE to detect viruses in wastewater and to track the diversity and spread of viral variants in urban and suburban locations, which may aid public health efforts to monitor disease outbreaks. IMPORTANCE Wastewater-based epidemiology (WBE) can detect pathogens across sewersheds, which represents the collective waste of human populations. As there is a wide diversity of RNA viruses in wastewater, monitoring the presence of these viruses is useful for public health, industry, and ecological studies. Specific to public health, WBE has proven valuable during the coronavirus disease 2019 (COVID-19) pandemic to track the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) without adding burden to health care systems. In this study, we used metatranscriptomics and reverse transcription-droplet digital PCR (RT-ddPCR) to assay RNA viruses across Southern California wastewater from August 2020 to January 2021, representing approximately 16 million people from Los Angeles, Orange, and San Diego counties. We found that SARS-CoV-2 quantification in wastewater correlates well with county-wide COVID-19 case data, and that we can detect SARS-CoV-2 single-nucleotide variants through sequencing. Likewise, wastewater treatment plants (WTPs) harbored different viromes, and we detected other human pathogens, such as noroviruses and adenoviruses, furthering our understanding of wastewater viral ecology.

Acute Illness Among Surfers After Exposure to Seawater in Dry- and Wet-Weather Conditions.

Rainstorms increase levels of fecal indicator bacteria in urban coastal waters, but it is unknown whether exposure to seawater after rainstorms increases rates of acute illness. Our objective was to provide the first estimates of rates of acute illness after seawater exposure during both dry- and wet-weather periods and to determine the relationship between levels of indicator bacteria and illness among surfers, a population with a high potential for exposure after rain. We enrolled 654 surfers in San Diego, California, and followed them longitudinally during the 2013-2014 and 2014-2015 winters (33,377 days of observation, 10,081 surf sessions). We measured daily surf activities and illness symptoms (gastrointestinal illness, sinus infections, ear infections, infected wounds). Compared with no exposure, exposure to seawater during dry weather increased incidence rates of all outcomes (e.g., for earache or infection, adjusted incidence rate ratio (IRR) = 1.86, 95% confidence interval (CI): 1.27, 2.71; for infected wounds, IRR = 3.04, 95% CI: 1.54, 5.98); exposure during wet weather further increased rates (e.g., for earache or infection, IRR = 3.28, 95% CI: 1.95, 5.51; for infected wounds, IRR = 4.96, 95% CI: 2.18, 11.29). Fecal indicator bacteria measured in seawater (Enterococcus species, fecal coliforms, total coliforms) were strongly associated with incident illness only during wet weather. Urban coastal seawater exposure increases the incidence rates of many acute illnesses among surfers, with higher incidence rates after rainstorms.

Geochemical, metagenomic and metaproteomic insights into trace metal utilization by methane-oxidizing microbial consortia in sulphidic marine sediments.

Microbes have obligate requirements for trace metals in metalloenzymes that catalyse important biogeochemical reactions. In anoxic methane- and sulphide-rich environments, microbes may have unique adaptations for metal acquisition and utilization because of decreased bioavailability as a result of metal sulphide precipitation. However, micronutrient cycling is largely unexplored in cold (≤ 10°C) and sulphidic (> 1 mM ΣH(2)S) deep-sea methane seep ecosystems. We investigated trace metal geochemistry and microbial metal utilization in methane seeps offshore Oregon and California, USA, and report dissolved concentrations of nickel (0.5-270 nM), cobalt (0.5-6 nM), molybdenum (10-5600 nM) and tungsten (0.3-8 nM) in Hydrate Ridge sediment porewaters. Despite low levels of cobalt and tungsten, metagenomic and metaproteomic data suggest that microbial consortia catalysing anaerobic oxidation of methane (AOM) utilize both scarce micronutrients in addition to nickel and molybdenum. Genetic machinery for cobalt-containing vitamin B12 biosynthesis was present in both anaerobic methanotrophic archaea (ANME) and sulphate-reducing bacteria. Proteins affiliated with the tungsten-containing form of formylmethanofuran dehydrogenase were expressed in ANME from two seep ecosystems, the first evidence for expression of a tungstoenzyme in psychrophilic microorganisms. Overall, our data suggest that AOM consortia use specialized biochemical strategies to overcome the challenges of metal availability in sulphidic environments.

Longitudinal metatranscriptomic sequencing of Southern California wastewater representing 16 million people from August 2020-21 reveals widespread transcription of antibiotic resistance genes.

Municipal wastewater provides a representative sample of human fecal waste across a catchment area and contains a wide diversity of microbes. Sequencing wastewater samples provides information about human-associated and medically important microbial populations, and may be useful to assay disease prevalence and antimicrobial resistance (AMR). Here, we present a study in which we used untargeted metatranscriptomic sequencing on RNA extracted from 275 sewage influent samples obtained from eight wastewater treatment plants (WTPs) representing approximately 16 million people in Southern California between August 2020 - August 2021. We characterized bacterial and viral transcripts, assessed metabolic pathway activity, and identified over 2,000 AMR genes/variants across all samples. Because we did not deplete ribosomal RNA, we have a unique window into AMR carried as ribosomal mutants. We show that AMR diversity varied between WTPs (as measured through PERMANOVA, P < 0.001) and that the relative abundance of many individual AMR genes/variants increased over time (as measured with MaAsLin2, Padj < 0.05). Similarly, we detected transcripts mapping to human pathogenic bacteria and viruses suggesting RNA sequencing is a powerful tool for wastewater-based epidemiology and that there are geographical signatures to microbial transcription. We captured the transcription of gene pathways common to bacterial cell processes, including central carbon metabolism, nucleotide synthesis/salvage, and amino acid biosynthesis. We also posit that due to the ubiquity of many viruses and bacteria in wastewater, new biological targets for microbial water quality assessment can be developed. To the best of our knowledge, our study provides the most complete longitudinal metatranscriptomic analysis of a large population's wastewater to date and demonstrates our ability to monitor the presence and activity of microbes in complex samples. By sequencing RNA, we can track the relative abundance of expressed AMR genes/variants and metabolic pathways, increasing our understanding of AMR activity across large human populations and sewer sheds.

Relationship between coliphage and Enterococcus at southern California beaches and implications for beach water quality management.

Coliphage have been suggested as an alternative to fecal indicator bacteria for assessing recreational beach water quality, but it is unclear how frequently and at what types of beaches coliphage produces a different management outcome. Here we conducted side-by-side sampling of male-specific and somatic coliphage by the new EPA dead-end hollow fiber ultrafiltration (D-HFUF-SAL) method and Enterococcus at southern California beaches over two years. When samples were combined for all beach sites, somatic and male-specific coliphage both correlated with Enterococcus. When examined categorically, Enterococcus would have resulted in approximately two times the number of health advisories as somatic coliphage and four times that of male-specific coliphage,using recently proposed thresholds of 60 PFU/100 mL for somatic and 30 PFU/100 mL for male-specific coliphage. Overall, only 12% of total exceedances would have been for coliphage alone. Somatic coliphage exceedances that occurred in the absence of an Enterococcus exceedance were limited to a single site during south swell events, when this beach is known to be affected by nearby minimally treated sewage. Thus, somatic coliphage provided additional valuable health protection information, but may be more appropriate as a supplement to FIB measurements rather than as replacement because: (a) EPA-approved PCR methods for Enterococcus allow a more rapid response, (b) coliphage is more challenging owing to its greater sampling volume and laboratory time requirements, and (c) Enterococcus' long data history has yielded predictive management models that would need to be recreated for coliphage.

Longitudinal metatranscriptomic sequencing of Southern California wastewater representing 16 million people from August 2020-21 reveals widespread transcription of antibiotic resistance genes.

Municipal wastewater provides a representative sample of human fecal waste across a catchment area and contains a wide diversity of microbes. Sequencing wastewater samples provides information about human-associated and medically-important microbial populations, and may be useful to assay disease prevalence and antimicrobial resistance (AMR). Here, we present a study in which we used untargeted metatranscriptomic sequencing on RNA extracted from 275 sewage influent samples obtained from eight wastewater treatment plants (WTPs) representing approximately 16 million people in Southern California between August 2020 - August 2021. We characterized bacterial and viral transcripts, assessed metabolic pathway activity, and identified over 2,000 AMR genes/variants across all samples. Because we did not deplete ribosomal RNA, we have a unique window into AMR carried as ribosomal mutants. We show that AMR diversity varied between WTPs and that the relative abundance of many individual AMR genes/variants increased over time and may be connected to antibiotic use during the COVID-19 pandemic. Similarly, we detected transcripts mapping to human pathogenic bacteria and viruses suggesting RNA sequencing is a powerful tool for wastewater-based epidemiology and that there are geographical signatures to microbial transcription. We captured the transcription of gene pathways common to bacterial cell processes, including central carbon metabolism, nucleotide synthesis/salvage, and amino acid biosynthesis. We also posit that due to the ubiquity of many viruses and bacteria in wastewater, new biological targets for microbial water quality assessment can be developed. To the best of our knowledge, our study provides the most complete longitudinal metatranscriptomic analysis of a large population's wastewater to date and demonstrates our ability to monitor the presence and activity of microbes in complex samples. By sequencing RNA, we can track the relative abundance of expressed AMR genes/variants and metabolic pathways, increasing our understanding of AMR activity across large human populations and sewer sheds.

SARS-CoV-2 RNA is enriched by orders of magnitude in primary settled solids relative to liquid wastewater at publicly owned treatment works.

Wastewater-based epidemiology has gained attention throughout the world for detection of SARS-CoV-2 RNA in wastewater to supplement clinical testing. Raw wastewater consists of small particles, or solids, suspended in liquid. Methods have been developed to measure SARS-CoV-2 RNA in the liquid and the solid fraction of wastewater, with some studies reporting higher concentrations in the solid fraction. To investigate this relationship further, six laboratories collaborated to conduct a study across five publicly owned treatment works (POTWs) where both primary settled solids obtained from primary clarifiers and raw wastewater influent samples were collected and quantified for SARS-CoV-2 RNA. Settled solids and influent samples were processed by participating laboratories using their respective methods and retrospectively paired based on date of collection. SARS-CoV-2 RNA concentrations, on a mass equivalent basis, were higher in settled solids than in influent by approximately three orders of magnitude. Concentrations in matched settled solids and influent were positively and significantly correlated at all five POTWs. RNA concentrations in both settled solids and influent were correlated to COVID-19 incidence rates in the sewersheds and thus representative of disease occurrence; the settled solids methods appeared to produce a comparable relationship between SARS-CoV-2 RNA concentration measurements and incidence rates across all POTWs. Settled solids and influent methods showed comparable sensitivity, N gene detection frequency, and calculated empirical incidence rate lower limits. Analysis of settled solids for SARS-CoV-2 RNA has the advantage of using less sample volume to achieve similar sensitivity to influent methods.

A latitudinal diversity gradient in planktonic marine bacteria.

For two centuries, biologists have documented a gradient of animal and plant biodiversity from the tropics to the poles but have been unable to agree whether it is controlled primarily by productivity, temperature, or historical factors. Recent reports that find latitudinal diversity gradients to be reduced or absent in some unicellular organisms and attribute this to their high abundance and dispersal capabilities would suggest that bacteria, the smallest and most abundant organisms, should exhibit no latitudinal pattern of diversity. We used amplified ribosomal intergenic spacer analysis (ARISA) whole-assemblage genetic fingerprinting to quantify species richness in 103 near-surface samples of marine bacterial plankton, taken from tropical to polar in both hemispheres. We found a significant latitudinal gradient in richness. The data can help to evaluate hypotheses about the cause of the gradient. The correlations of richness with latitude and temperature were similarly strong, whereas correlations with parameters relating to productivity (chlorophyll, annual primary productivity, bacterial abundance) and other variables (salinity and distance to shore) were much weaker. Despite the high abundance and potentially high dispersal of bacteria, they exhibit geographic patterns of species diversity that are similar to those seen in other organisms. The latitudinal gradient in marine bacteria supports the hypothesis that the kinetics of metabolism, setting the pace for life, has strong influence on diversity.

Pathogenic Vibrio Species Are Associated with Distinct Environmental Niches and Planktonic Taxa in Southern California (USA) Aquatic Microbiomes.

Interactions between vibrio bacteria and the planktonic community impact marine ecology and human health. Many coastal Vibrio spp. can infect humans, representing a growing threat linked to increasing seawater temperatures. Interactions with eukaryotic organisms may provide attachment substrate and critical nutrients that facilitate the persistence, diversification, and spread of pathogenic Vibrio spp. However, vibrio interactions with planktonic organisms in an environmental context are poorly understood. We quantified the pathogenic Vibrio species V. cholerae, V. parahaemolyticus, and V. vulnificus monthly for 1 year at five sites and observed high abundances, particularly during summer months, with species-specific temperature and salinity distributions. Using metabarcoding, we established a detailed profile of both prokaryotic and eukaryotic coastal microbial communities. We found that pathogenic Vibrio species were frequently associated with distinct eukaryotic amplicon sequence variants (ASVs), including diatoms and copepods. Shared environmental conditions, such as high temperatures and low salinities, were associated with both high concentrations of pathogenic vibrios and potential environmental reservoirs, which may influence vibrio infection risks linked to climate change and should be incorporated into predictive ecological models and experimental laboratory systems. IMPORTANCE Many species of coastal vibrio bacteria can infect humans, representing a growing health threat linked to increasing seawater temperatures. However, their interactions with surrounding microbes in the environment, especially eukaryotic organisms that may provide nutrients and attachment substrate, are poorly understood. We quantified three pathogenic Vibrio species monthly for a duration of 1 year, finding that all three species were abundant and exhibited species-specific temperature and salinity distributions. Using metabarcoding, we investigated associations between these pathogenic species and prokaryotic and eukaryotic microbes, revealing genus and amplicon sequence variant (ASV)-specific relationships with potential functional implications. For example, pathogenic species were frequently associated with chitin-producing eukaryotes, such as diatoms in the genus Thalassiosira and copepods. These associations between high concentrations of pathogenic vibrios and potential environmental reservoirs should be considered when predicting infection risk and developing ecologically relevant model systems.

A Combined Digital PCR and Next Generation DNA-Sequencing Based Approach for Tracking Nearshore Pollutant Dynamics Along the Southwest United States/Mexico Border.

Ocean currents, multiple fecal bacteria input sources, and jurisdictional boundaries can complicate pollution source tracking and associated mitigation and management efforts within the nearshore coastal environment. In this study, multiple microbial source tracking tools were employed to characterize the impact and reach of an ocean wastewater treatment facility discharge in Mexico northward along the coast and across the Southwest United States- Mexico Border. Water samples were evaluated for fecal indicator bacteria (FIB), Enterococcus by culture-based methods, and human-associated genetic marker (HF183) and Enterococcus by droplet digital polymerase chain reaction (ddPCR). In addition, 16S rRNA gene sequence analysis was performed and the SourceTracker algorithm was used to characterize the bacterial community of the wastewater treatment plume and its contribution to beach waters. Sampling dates were chosen based on ocean conditions associated with northern currents. Evidence of a gradient in human fecal pollution that extended north from the wastewater discharge across the United States/Mexico border from the point source was observed using human-associated genetic markers and microbial community analysis. The spatial extent of fecal contamination observed was largely dependent on swell and ocean conditions. These findings demonstrate the utility of a combination of molecular tools for understanding and tracking specific pollutant sources in dynamic coastal water environments.

Standardizing data reporting in the research community to enhance the utility of open data for SARS-CoV-2 wastewater surveillance.

SARS-CoV-2 RNA detection in wastewater is being rapidly developed and adopted as a public health monitoring tool worldwide. With wastewater surveillance programs being implemented across many different scales and by many different stakeholders, it is critical that data collected and shared are accompanied by an appropriate minimal amount of metainformation to enable meaningful interpretation and use of this new information source and intercomparison across datasets. While some databases are being developed for specific surveillance programs locally, regionally, nationally, and internationally, common globally-adopted data standards have not yet been established within the research community. Establishing such standards will require national and international consensus on what metainformation should accompany SARS-CoV-2 wastewater measurements. To establish a recommendation on minimum information to accompany reporting of SARS-CoV-2 occurrence in wastewater for the research community, the United States National Science Foundation (NSF) Research Coordination Network on Wastewater Surveillance for SARS-CoV-2 hosted a workshop in February 2021 with participants from academia, government agencies, private companies, wastewater utilities, public health laboratories, and research institutes. This report presents the primary two outcomes of the workshop: (i) a recommendation on the set of minimum meta-information that is needed to confidently interpret wastewater SARS-CoV-2 data, and (ii) insights from workshop discussions on how to improve standardization of data reporting.

The challenges of microbial source tracking at urban beaches for Quantitative Microbial Risk Assessment (QMRA).

Urban beaches are frequently impacted from multiple sources of fecal contamination. This along with high beach usage underscores the importance of appropriate management that protects swimmer health. The USEPA has enabled the use of QMRA as a tool for quantifying swimmer health risk and setting site-specific water quality objectives. This study illustrates the challenges associated with human and non-human source identification and how these challenges influence the decision of whether QMRA at typical urban beaches for water quality management is appropriate. In this study, a similar and correlated spatial relationship with elevated Enterococcus and avian-specific markers was observed, suggesting shorebirds as a primary source of FIB. However, human-associated markers were also detected frequently but at low concentrations. Ultimately, a QMRA was not conducted because pathogen loading from potential human sources could not be confidently quantified, having consequences for health risk in receiving waters where recreational contact occurs.

Metagenomics of Wastewater Influent from Southern California Wastewater Treatment Facilities in the Era of COVID-19.

Sequencing wastewater may be useful for detecting pathogens and assaying microbial water quality. We concentrated, extracted, and sequenced nucleic acids from 17 composite influent wastewater samples spanning seven southern California wastewater treatment facilities in May 2020. Bacteria were the most proportionally abundant taxonomic group present, followed by viruses and archaea.

Quantification of pathogens and markers of fecal contamination during storm events along popular surfing beaches in San Diego, California.

Along southern California beaches, the concentrations of fecal indicator bacteria (FIB) used to quantify the potential presence of fecal contamination in coastal recreational waters have been previously documented to be higher during wet weather conditions (typically winter or spring) than those observed during summer dry weather conditions. FIB are used for management of recreational waters because measurement of the bacterial and viral pathogens that are the potential causes of illness in beachgoers exposed to stormwater can be expensive, time-consuming, and technically difficult. Here, we use droplet digital Polymerase Chain Reaction (digital PCR) and digital reverse transcriptase PCR (digital RT-PCR) assays for direct quantification of pathogenic viruses, pathogenic bacteria, and source-specific markers of fecal contamination in the stormwater discharges. We applied these assays across multiple storm events from two different watersheds that discharge to popular surfing beaches in San Diego, CA. Stormwater discharges had higher FIB concentrations as compared to proximal beaches, often by ten-fold or more during wet weather. Multiple lines of evidence indicated that the stormwater discharges contained human fecal contamination, despite the presence of separate storm sewer and sanitary sewer systems in both watersheds. Human fecal source markers (up to 100% of samples, 20-12440 HF183 copies per 100 ml) and human norovirus (up to 96% of samples, 25-495 NoV copies per 100 ml) were routinely detected in stormwater discharge samples. Potential bacterial pathogens were also detected and quantified: Campylobacter spp. (up to 100% of samples, 16-504 gene copies per 100 ml) and Salmonella (up to 25% of samples, 6-86 gene copies per 100 ml). Other viral human pathogens were also measured, but occurred at generally lower concentrations: adenovirus (detected in up to 22% of samples, 14-41 AdV copies per 100 ml); no enterovirus was detected in any stormwater discharge sample. Higher concentrations of avian source markers were noted in the stormwater discharge located immediately downstream of a large bird sanctuary along with increased Campylobacter concentrations and notably different Campylobacter species composition than the watershed that had no bird sanctuary. This study is one of the few to directly measure an array of important bacterial and viral pathogens in stormwater discharges to recreational beaches, and provides context for stormwater-based management of beaches during high risk wet-weather periods. Furthermore, the combination of culture-based and digital PCR-derived data is demonstrated to be valuable for assessing hydrographic relationships, considering delivery mechanisms, and providing foundational exposure information for risk assessment.

Local similarity analysis reveals unique associations among marine bacterioplankton species and environmental factors.

MOTIVATION: Characterizing the diversity of microbial communities and understanding the environmental factors that influence community diversity are central tenets of microbial ecology. The development and application of cultivation independent molecular tools has allowed for rapid surveying of microbial community composition at unprecedented resolutions and frequencies. There is a growing need to discern robust patterns and relationships within these datasets which provide insight into microbial ecology. Pearson correlation coefficient (PCC) analysis is commonly used for identifying the linear relationship between two species, or species and environmental factors. However, this approach may not be able to capture more complex interactions which occur in situ; thus, alternative analyses were explored. RESULTS: In this paper we introduced local similarity analysis (LSA), which is a technique that can identify more complex dependence associations among species as well as associations between species and environmental factors without requiring significant data reduction. To illustrate its capability of identifying relationships that may not otherwise be identified by PCC, we first applied LSA to simulated data. We then applied LSA to a marine microbial observatory dataset and identified unique, significant associations that were not detected by PCC analysis. LSA results, combined with results from PCC analysis were used to construct a theoretical ecological network which allows for easy visualization of the most significant associations. Biological implications of the significant associations detected by LSA were discussed. We also identified additional applications where LSA would be beneficial. AVAILABILITY: The algorithms are implemented in Splus/R and they are available upon request from the corresponding author.

Monodeuterated Methane, an Isotopic Tool To Assess Biological Methane Metabolism Rates.

Biological methane oxidation is a globally relevant process that mediates the flux of an important greenhouse gas through both aerobic and anaerobic metabolic pathways. However, measuring these metabolic rates presents many obstacles, from logistical barriers to regulatory hurdles and poor precision. Here we present a new approach for investigating microbial methane metabolism based on hydrogen atom dynamics, which is complementary to carbon-focused assessments of methanotrophy. The method uses monodeuterated methane (CH3D) as a metabolic substrate, quantifying the aqueous D/H ratio over time using off-axis integrated cavity output spectroscopy. This approach represents a nontoxic, comparatively rapid, and straightforward approach that supplements existing radiotopic and stable carbon isotopic methods; by probing hydrogen atoms, it offers an additional dimension for examining rates and pathways of methane metabolism. We provide direct comparisons between the CH3D procedure and the well-established 14CH4 radiotracer method for several methanotrophic systems, including type I and II aerobic methanotroph cultures and methane-seep sediment slurries and carbonate rocks under anoxic and oxic incubation conditions. In all applications tested, methane consumption values calculated via the CH3D method were directly and consistently proportional to 14C radiolabel-derived methane oxidation rates. We also employed this method in a nontraditional experimental setup, using flexible, gas-impermeable bags to investigate the role of pressure on seep sediment methane oxidation rates. Results revealed an 80% increase over atmospheric pressure in methanotrophic rates the equivalent of ~900-m water depth, highlighting the importance of this parameter on methane metabolism and exhibiting the flexibility of the newly described method. IMPORTANCE Microbial methane consumption is a critical component of the global carbon cycle, with wide-ranging implications for climate regulation and hydrocarbon exploitation. Nonetheless, quantifying methane metabolism typically involves logistically challenging methods and/or specialized equipment; these impediments have limited our understanding of methane fluxes and reservoirs in natural systems, making effective management difficult. Here, we offer an easily implementable, precise method using monodeuterated methane (CH3D) that advances three specific aims. First, it allows users to directly compare methane consumption rates between different experimental treatments of the same inoculum. Second, by empirically linking the CH3D procedure with the well-established 14C radiocarbon approach, we determine absolute scaling factors that facilitate rate measurements for several aerobic and anaerobic systems of interest. Third, CH3D represents a helpful tool in evaluating the relationship between methane activation and full oxidation in methanotrophic metabolisms. The procedural advantages, consistency, and novel research questions enabled by the CH3D method should prove useful in a wide range of culture-based and environmental microbial systems to further elucidate methane metabolism dynamics.

Incidence of gastrointestinal illness following wet weather recreational exposures: Harmonization of quantitative microbial risk assessment with an epidemiologic investigation of surfers.

We modeled the risk of gastrointestinal (GI) illness associated with recreational exposures to marine water following storm events in San Diego County, California. We estimated GI illness risks via quantitative microbial risk assessment (QMRA) techniques by consolidating site specific pathogen monitoring data of stormwater, site specific dilution estimates, literature-based water ingestion data, and literature based pathogen dose-response and morbidity information. Our water quality results indicated that human sources of contamination contribute viral and bacterial pathogens to streams draining an urban watershed during wet weather that then enter the ocean and affect nearshore water quality. We evaluated a series of approaches to account for uncertainty in the norovirus dose-response model selection and compared our model results to those from a concurrently conducted epidemiological study that provided empirical estimates for illness risk following ocean exposure. The preferred norovirus dose-response approach yielded median risk estimates for water recreation-associated illness (15 GI illnesses per 1000 recreation events) that closely matched the reported epidemiological results (12 excess GI illnesses per 1000 wet weather recreation events). The results are consistent with norovirus, or other pathogens associated with norovirus, as an important cause of gastrointestinal illness among surfers in this setting. This study demonstrates the applicability of QMRA for recreational water risk estimation, even under wet weather conditions and describes a process that might be useful in developing site-specific water quality criteria in this and other locations.