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Background: Wastewater monitoring data can be used to estimate disease trends to inform public health responses. One commonly estimated metric is the rate of change in pathogen quantity, which typically correlates with clinical surveillance in retrospective analyses. However, the accuracy of rate of change estimation approaches has not previously been evaluated., Objectives: We assessed the performance of approaches for estimating rates of change in wastewater pathogen loads by generating synthetic wastewater time series data for which rates of change were known. Each approach was also evaluated on real-world data., Methods: Smooth trends and their first derivatives were jointly sampled from Gaussian processes (GP) and independent errors were added to generate synthetic viral load measurements; the range hyperparameter and error variance were varied to produce nine simulation scenarios representing different potential disease patterns. The directions and magnitudes of the rate of change estimates from four estimation approaches (two established and two developed in this work) were compared to the GP first derivative to evaluate classification and quantitative accuracy. Each approach was also implemented for public SARS-CoV-2 wastewater monitoring data collected January 2021-May 2023 at 25 sites in North Carolina, USA., Results: All four approaches inconsistently identified the correct direction of the trend given by the sign of the GP first derivative. Across all nine simulated disease patterns, between a quarter and a half of all estimates indicated the wrong trend direction, regardless of estimation approach. The proportion of trends classified as plateaus (statistically indistinguishable from zero) for the North Carolina SARS-CoV-2 data varied considerably by estimation method but not by site., Discussion: Our results suggest that wastewater measurements alone might not provide sufficient data to reliably track disease trends in real-time. Instead, wastewater viral loads could be combined with additional public health surveillance data to improve predictions of other outcomes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Estuarine water quality is declining worldwide due to increased tourism, coastal development, and a changing climate. Although well-established methods are in place to monitor water quality, municipalities struggle to use the data to prioritize infrastructure for monitoring and repair and to determine sources of contamination when they occur. The objective of this study was to assess water quality and prioritize sources of contamination within Town Creek Estuary (TCE), Beaufort, North Carolina, by combining culture, molecular, and geographic information systems (GIS) data into a novel contamination source ranking system. Water samples were collected from TCE at ten locations on eight sampling dates in Fall 2021 (n = 80). Microbiological water quality was assessed using US Environmental Protection Agency (U.S. EPA) approved culture-based methods for fecal indicator bacteria (FIB), including analysis of total coliforms (TC), Escherichia coli (EC), and Enterococcus spp. (ENT). The quantitative microbial source tracking (qMST) human-associated fecal marker, HF183, was quantified using droplet digital PCR (ddPCR). This information was combined with environmental data and GIS information detailing proximal sewer, septic, and stormwater infrastructure to determine potential sources of fecal contamination in the estuary. Results indicated FIB concentrations were significantly and positively correlated with precipitation and increased throughout the estuary following rainfall events (p < 0.01). Sampling sites with FIB concentrations above the U.S. EPA threshold also had the highest percentages of aged, less durable piping materials. Using a novel ranking system combining concentrations of FIB, HF183, and sewer infrastructure data at each site, we found that the two sites nearest the most aged sewage infrastructure and stormwater outflows were found to have the highest levels of measurable fecal contamination. This case study supports the inclusion of both traditional water quality measurements and local infrastructure data to support the current need for municipalities to identify, prioritize, and remediate failing infrastructure., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Hynes et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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., Competing Interests: RUH is managing member of AquaVitas, LLC and founder of the ASU non-profit project OneWaterOneHealth operating in the same intellectual space. LPS is employed by Ceres Nanosciences.

Wastewater monitoring has provided health officials with early warnings for new COVID-19 outbreaks, but to date, no approach has been validated to distinguish signal (sustained surges) from noise (background variability) in wastewater data to alert officials to the need for heightened public health response. We analyzed 62 wk of data from 19 sites participating in the North Carolina Wastewater Monitoring Network to characterize wastewater metrics around the Delta and Omicron surges. We found that wastewater data identified outbreaks 4 to 5 d before case data (reported on the earlier of the symptom start date or test collection date), on average. At most sites, correlations between wastewater and case data were similar regardless of how wastewater concentrations were normalized and whether calculated with county-level or sewershed-level cases, suggesting that officials may not need to geospatially align case data with sewershed boundaries to gain insights into disease transmission. Although wastewater trend lines captured clear differences in the Delta versus Omicron surge trajectories, no single wastewater metric (detectability, percent change, or flow-population normalized viral concentrations) reliably signaled when these surges started. After iteratively examining different combinations of these three metrics, we developed the Covid-SURGE (Signaling Unprecedented Rises in Groupwide Exposure) algorithm, which identifies unprecedented signals in the wastewater data. With a true positive rate of 82%, a false positive rate of 7%, and strong performance during both surges and in small and large sites, our algorithm provides public health officials with an automated way to flag community-level COVID-19 surges in real time.

This study aimed to understand the performance and utility of US EPA-approved coliphage methods in comparison to fecal indicator bacteria (FIB) and molecular microbial source tracking (MST) methods in recreational waters. We used US EPA Method 1642 to quantify concentrations of coliphage along with culture- and molecular-based enumeration of E. coli and Enterococcus sp, and human fecal source marker HF183. We also conducted a feasibility assessment to determine the utility of US EPA Method 1642 for application to routine recreational water monitoring. Ten sampling events were conducted at three sampling sites over the duration of a year. Average concentrations of somatic (SC) (log10 1.48 PFU/100 mL) and male-specific (MSC) coliphages (log10 1.00 PFU/100 mL) at all sites were low with SC found across a broader range (0.3-3.1 log10 PFU/100 mL) of concentrations compared to MSC (non-detect-1.7 log10 PFU/100 mL). A feasibility assessment was conducted across US EPA Method 1642 for coliphage enumeration, culture-based FIB, defined substrate technology (DST) approaches Enterolert™ and Colilert®, and quantitative microbial source tracking (qMST) US EPA Method 1696. US EPA Method 1642 had the longest processing times, but also was moderate in cost, compared to the DST and qMST molecular methods. Given the poor correlations between MSC and SC with FIB and qMST markers in this study and the cumbersome nature of US EPA Method 1642, the method may not be the most applicable method for use in systems impacted predominantly by stormwater and other non-point source pollution. Findings from this study, however, provide guidance on the application of fecal indicator virus in ambient coastal surface waters., (© The Author(s) 2023. Published by Oxford University Press on behalf of Applied Microbiology International.)

Wastewater surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) may be useful for monitoring population-wide coronavirus disease 2019 (COVID-19) infections, especially given asymptomatic infections and limitations in diagnostic testing. We aimed to detect SARS-CoV-2 RNA in wastewater and compare viral concentrations to COVID-19 case numbers in the respective counties and sewersheds. Influent 24-hour composite wastewater samples were collected from July to December 2020 from two municipal wastewater treatment plants serving different population sizes in Orange and Chatham Counties in North Carolina. After a concentration step via HA filtration, SARS-CoV-2 RNA was detected and quantified by reverse transcription droplet digital polymerase chain reaction (RT-ddPCR) and quantitative PCR (RT-qPCR), targeting the N1 and N2 nucleocapsid genes. SARS-CoV-2 RNA was detected by RT-ddPCR in 100 % (24/24) and 79 % (19/24) of influent wastewater samples from the larger and smaller plants, respectively. In comparison, viral RNA was detected by RT-qPCR in 41.7 % (10/24) and 8.3 % (2/24) of samples from the larger and smaller plants, respectively. Positivity rates and method agreement further increased for the RT-qPCR assay when samples with positive signals below the limit of detection were counted as positive. The wastewater data from the larger plant generally correlated (⍴ ~0.5, p < 0.05) with, and even anticipated, the trends in reported COVID-19 cases, with a notable spike in measured viral RNA preceding a spike in cases when students returned to a college campus in the Orange County sewershed. Correlations were generally higher when using estimates of sewershed-level case data rather than county-level data. This work supports use of wastewater surveillance for tracking COVID-19 disease trends, especially in identifying spikes in cases. Wastewater-based epidemiology can be a valuable resource for tracking disease trends, allocating resources, and evaluating policy in the fight against current and future pandemics., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Wastewater based epidemiology (WBE) is useful for tracking and monitoring the level of disease prevalence in a community and has been used extensively to complement clinical testing during the current COVID-19 pandemic. Despite the numerous benefits, sources of variability in sample storage, handling, and processing methods can make WBE data difficult to generalize. We performed an experiment to determine sources of variability in WBE data including the impact of storage time, handling, and processing techniques on the concentration of SARS-CoV-2 in wastewater influent from three wastewater treatment plants (WWTP) in North Carolina over 19 days. The SARS-CoV-2 concentration in influent samples held at 4°C did not degrade significantly over the 19-day experiment. Heat pasteurization did not significantly impact the concentration of SARS-CoV-2 at two of the three WWTP but did reduce viral recovery at the WWTP with the smallest population size served. On each processing date, one filter from each sample was processed immediately while a replicate filter was frozen at -80°C. Once processed, filters previously frozen were found to contain slightly higher concentrations (<0.2 log copies/L) than their immediately processed counterparts, indicating freezing filters is a viable method for delayed quantification and may even improve recovery at WWTP with low viral concentrations. Investigation of factors contributing to variability during sample processing indicated that analyst experience level contributed significantly (p<0.001) to accepted droplet generation while extraction efficiency and reverse transcription efficiency contributed significantly (p<0.05) to day-to-day SARS-CoV-2 variability. This study provides valuable practical information for minimizing decay and/or loss of SARS CoV-2 in wastewater influent while adhering to safety procedures, promoting efficient laboratory workflows, and accounting for sources of variability., Competing Interests: The authors have declared that no competing interests exist.

The global spread of SARS-CoV-2 has continued to be a serious concern after WHO declared the virus to be the causative agent of the coronavirus disease 2019 (COVID-19) a global pandemic. Monitoring of wastewater is a useful tool for assessing community prevalence given that fecal shedding of SARS-CoV-2 occurs in high concentrations by infected individuals, regardless of whether they are asymptomatic or symptomatic. Using tools that are part of wastewater-based epidemiology (WBE) approach, combined with molecular analyses, wastewater monitoring becomes a key piece of information used to assess trends and quantify the scale and dynamics of COVID-19 infection in a specific community, municipality, or area of service. This study investigates a six-month long SARS-CoV-2 RNA quantification in influent wastewater from four municipal wastewater treatment plants (WWTP) serving the Charlotte region of North Carolina (NC) using both RT-qPCR and RT-ddPCR platforms. Influent wastewater was analyzed for the nucleocapsid (N) genes N1 and N2. Both RT-qPCR and RT-ddPCR performed well for detection and quantification of SARS-CoV-2 using the N1 target, while for the N2 target RT-ddPCR was more sensitive. SARS-CoV-2 concentration ranged from 10 3 to 10 5 copies/L for all four plants. Both RT-qPCR and RT-ddPCR showed a significant positive correlation between SARS-CoV-2 concentrations and the 7-day rolling average of clinically reported COVID-19 cases when lagging 5 to 12 days (ρ = 0.52-0.92, p < 0.001-0.02). A major finding of this study is that RT-qPCR and RT-ddPCR generated SARS-CoV-2 data that was positively correlated (ρ = 0.569, p < 0.0001) and can be successfully used to monitor SARS-CoV-2 signals across the WWTP of different sizes and metropolitan service functions without significant anomalies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier B.V.)

Wastewater surveillance for pathogens using reverse transcription-polymerase chain reaction (RT-PCR) is an effective and resource-efficient tool for gathering community-level public health information, including the incidence of coronavirus disease-19 (COVID-19). Surveillance of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) in wastewater can potentially provide an early warning signal of COVID-19 infections in a community. The capacity of the world's environmental microbiology and virology laboratories for SARS-CoV-2 RNA characterization in wastewater is increasing rapidly. However, there are no standardized protocols or harmonized quality assurance and quality control (QA/QC) procedures for SARS-CoV-2 wastewater surveillance. This paper is a technical review of factors that can cause false-positive and false-negative errors in the surveillance of SARS-CoV-2 RNA in wastewater, culminating in recommended strategies that can be implemented to identify and mitigate some of these errors. Recommendations include stringent QA/QC measures, representative sampling approaches, effective virus concentration and efficient RNA extraction, PCR inhibition assessment, inclusion of sample processing controls, and considerations for RT-PCR assay selection and data interpretation. Clear data interpretation guidelines (e.g., determination of positive and negative samples) are critical, particularly when the incidence of SARS-CoV-2 in wastewater is low. Corrective and confirmatory actions must be in place for inconclusive results or results diverging from current trends (e.g., initial onset or reemergence of COVID-19 in a community). It is also prudent to perform interlaboratory comparisons to ensure results' reliability and interpretability for prospective and retrospective analyses. The strategies that are recommended in this review aim to improve SARS-CoV-2 characterization and detection for wastewater surveillance applications. A silver lining of the COVID-19 pandemic is that the efficacy of wastewater surveillance continues to be demonstrated during this global crisis. In the future, wastewater should also play an important role in the surveillance of a range of other communicable diseases., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Crown Copyright © 2021. Published by Elsevier B.V. All rights reserved.)

Examinations of stormwater delivery in the context of tidal inundation are lacking. Along the coastal plains of the southeastern United States, tidal inundation is increasing in frequency and severity, often with dramatic adverse impacts on timely stormwater discharge, coastal flooding hazards, and even "sunny day flooding". Therefore, a comprehensive study was conducted to examine tidally-influenced stormwater outfalls discharging to Taylor's Creek, an estuary proximal to Beaufort, NC used regularly for recreation and tourism. Over a wide range of meteorological conditions, water samples were collected and analyzed for fecal indicator bacteria (FIB, used for water quality management) and previously published quantitative microbial source tracking (qMST) markers. Nineteen sampling events were conducted from July 2017-June 2018 with samples classified according to tidal state and defined as either inundated, receding, or transition. A first-of-its-kind multiple linear regression model was developed to predict concentrations of Enterococcus sp. by tidal cycle, salinity and antecedent rainfall. We demonstrated that the majority of variability associated with the concentration of Enterococcus sp. could be predicted by E. coli concentration and tidal phase. FIB concentrations were significantly (<0.05) influenced by tide with higher concentrations observed in samples collected during receding (low) tides (EC: log 3.12 MPN/100 mL; ENT: 2.67 MPN/100 mL) compared to those collected during inundated (high) (EC: log 2.62 MPN/100 mL; ENT: 2.11 MPN/100 mL) or transition (EC: log 2.74 MPN/100 mL; ENT: 2.53 MPN/100 mL) tidal periods. Salinity, was also found to significantly (<0.05) correlate with Enterococcus sp. concentrations during inundated (high) tidal conditions (sal: 17 ppt; ENT: 2.04 MPN/100 mL). Tide, not precipitation, was shown to be a significant driver in explaining the variability in Enterococcus sp. concentrations. Precipitation has previously been shown to be a driver of Enterococcus sp. concentrations, but our project demonstrates the need for tidal parameters to be included in the future development of water quality monitoring programs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021. Published by Elsevier B.V.)

Wastewater surveillance for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has garnered extensive public attention during the coronavirus disease pandemic as a proposed complement to existing disease surveillance systems. Over the past year, methods for detection and quantification of SARS-CoV-2 viral RNA in untreated sewage have advanced, and concentrations in wastewater have been shown to correlate with trends in reported cases. Despite the promise of wastewater surveillance, for these measurements to translate into useful public health tools, bridging the communication and knowledge gaps between researchers and public health responders is needed. We describe the key uses, barriers, and applicability of SARS-CoV-2 wastewater surveillance for supporting public health decisions and actions, including establishing ethics consideration for monitoring. Although wastewater surveillance to assess community infections is not a new idea, the coronavirus disease pandemic might be the initiating event to make this emerging public health tool a sustainable nationwide surveillance system, provided that these barriers are addressed.

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

Competing Interests: None declared.

Privately-owned drinking water wells serving fewer than 25 people (private wells) are prevalent and understudied across most of the US. Private wells primarily serve rural households located outside of municipal drinking water and sewerage service coverage areas. These wells are not regulated by United States Environmental Protection Agency (EPA) under the Safe Drinking Water Act, are not regularly monitored by any public agency or utility, and generally do not undergo disinfection treatment. Coliphages are a group of viruses that infect coliform bacteria and are useful viral surrogates for fecal contamination in water systems in much the same way that fecal indicator bacteria (FIB), such as E. coli and to a lesser extent total coliforms, are used to quantify fecal contamination. Coliphages are approved by the EPA for regulatory monitoring in groundwater wells in the USA, but are not routinely used for this purpose. The present study characterizes the occurrence of male-specific and somatic coliphages, along with FIB, in private wells ( n  = 122) across two different counties in North Carolina. While occurrences of E. coli were rare and frequency of total coliform was generally low (~20%), male-specific and somatic coliphages were detectable in 66% and 54% of samples, respectively. Concentrations of male-specific coliphages were higher than somatics at each county and on a monthly basis. Rainfall appears to be partly influencing higher coliphage concentrations in December, January and February. This research underscores the need for increased surveillance in private wells and consideration of using coliphages in order to better characterize occurrence of fecal contamination at the time of sampling, especially during rainier months., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 Published by Elsevier Ltd.)

Fecal contamination is observed downstream of municipal separate storm sewer systems in coastal North Carolina. While it is well accepted that wet weather contributes to this phenomenon, less is understood about the contribution of the complex hydrology in this low-lying coastal plain. A quantitative microbial assessment was conducted in Beaufort, North Carolina to identify trends and potential sources of fecal contamination in stormwater receiving waters. Fecal indicator concentrations were significantly higher in receiving water downstream of a tidally submerged outfall compared to an outfall that was permanently submerged (p < 0.001), though tidal height was not predictive of human-specific microbial source tracking (MST) marker concentrations at the tidally submerged site. Short-term rainfall (i.e. <12 h) was predictive of E. coli, Enterococcus spp., and human-specific MST marker concentrations (Fecal Bacteroides, BacHum, and HF183) in receiving waters. The strong correlation between 12-hr antecedent rainfall and Enterococcus spp. (r = 0.57, p < 0.001, n = 92) suggests a predictive model could be developed based on rainfall to communicate risk for bathers. Additional molecular marker data indicates that the delivery of fecal sources is complex and highly variable, likely due to the influence of tidal influx (saltwater intrusion from the estuary) into the low-lying stormwater pipes. In particular, elevated MST marker concentrations (up to 2.56 × 10 4 gene copies HF183/mL) were observed in standing water near surcharging street storm drain. These data are being used to establish a baseline for stormwater dynamics prior to dramatic rainfall in 2018 and to characterize the interaction between complex stormwater dynamics and water quality impairment in coastal NC., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Pollution - unwanted waste released to air, water, and land by human activity - is the largest environmental cause of disease in the world today. It is responsible for an estimated nine million premature deaths per year, enormous economic losses, erosion of human capital, and degradation of ecosystems. Ocean pollution is an important, but insufficiently recognized and inadequately controlled component of global pollution. It poses serious threats to human health and well-being. The nature and magnitude of these impacts are only beginning to be understood., Goals: (1) Broadly examine the known and potential impacts of ocean pollution on human health. (2) Inform policy makers, government leaders, international organizations, civil society, and the global public of these threats. (3) Propose priorities for interventions to control and prevent pollution of the seas and safeguard human health., Methods: Topic-focused reviews that examine the effects of ocean pollution on human health, identify gaps in knowledge, project future trends, and offer evidence-based guidance for effective intervention., Environmental Findings: Pollution of the oceans is widespread, worsening, and in most countries poorly controlled. It is a complex mixture of toxic metals, plastics, manufactured chemicals, petroleum, urban and industrial wastes, pesticides, fertilizers, pharmaceutical chemicals, agricultural runoff, and sewage. More than 80% arises from land-based sources. It reaches the oceans through rivers, runoff, atmospheric deposition and direct discharges. It is often heaviest near the coasts and most highly concentrated along the coasts of low- and middle-income countries. Plastic is a rapidly increasing and highly visible component of ocean pollution, and an estimated 10 million metric tons of plastic waste enter the seas each year. Mercury is the metal pollutant of greatest concern in the oceans; it is released from two main sources - coal combustion and small-scale gold mining. Global spread of industrialized agriculture with increasing use of chemical fertilizer leads to extension of Harmful Algal Blooms (HABs) to previously unaffected regions. Chemical pollutants are ubiquitous and contaminate seas and marine organisms from the high Arctic to the abyssal depths., Ecosystem Findings: Ocean pollution has multiple negative impacts on marine ecosystems, and these impacts are exacerbated by global climate change. Petroleum-based pollutants reduce photosynthesis in marine microorganisms that generate oxygen. Increasing absorption of carbon dioxide into the seas causes ocean acidification, which destroys coral reefs, impairs shellfish development, dissolves calcium-containing microorganisms at the base of the marine food web, and increases the toxicity of some pollutants. Plastic pollution threatens marine mammals, fish, and seabirds and accumulates in large mid-ocean gyres. It breaks down into microplastic and nanoplastic particles containing multiple manufactured chemicals that can enter the tissues of marine organisms, including species consumed by humans. Industrial releases, runoff, and sewage increase frequency and severity of HABs, bacterial pollution, and anti-microbial resistance. Pollution and sea surface warming are triggering poleward migration of dangerous pathogens such as the Vibrio species. Industrial discharges, pharmaceutical wastes, pesticides, and sewage contribute to global declines in fish stocks., Human Health Findings: Methylmercury and PCBs are the ocean pollutants whose human health effects are best understood. Exposures of infants in utero to these pollutants through maternal consumption of contaminated seafood can damage developing brains, reduce IQ and increase children's risks for autism, ADHD and learning disorders. Adult exposures to methylmercury increase risks for cardiovascular disease and dementia. Manufactured chemicals - phthalates, bisphenol A, flame retardants, and perfluorinated chemicals, many of them released into the seas from plastic waste - can disrupt endocrine signaling, reduce male fertility, damage the nervous system, and increase risk of cancer. HABs produce potent toxins that accumulate in fish and shellfish. When ingested, these toxins can cause severe neurological impairment and rapid death. HAB toxins can also become airborne and cause respiratory disease. Pathogenic marine bacteria cause gastrointestinal diseases and deep wound infections. With climate change and increasing pollution, risk is high that Vibrio infections, including cholera, will increase in frequency and extend to new areas. All of the health impacts of ocean pollution fall disproportionately on vulnerable populations in the Global South - environmental injustice on a planetary scale., Conclusions: Ocean pollution is a global problem. It arises from multiple sources and crosses national boundaries. It is the consequence of reckless, shortsighted, and unsustainable exploitation of the earth's resources. It endangers marine ecosystems. It impedes the production of atmospheric oxygen. Its threats to human health are great and growing, but still incompletely understood. Its economic costs are only beginning to be counted.Ocean pollution can be prevented. Like all forms of pollution, ocean pollution can be controlled by deploying data-driven strategies based on law, policy, technology, and enforcement that target priority pollution sources. Many countries have used these tools to control air and water pollution and are now applying them to ocean pollution. Successes achieved to date demonstrate that broader control is feasible. Heavily polluted harbors have been cleaned, estuaries rejuvenated, and coral reefs restored.Prevention of ocean pollution creates many benefits. It boosts economies, increases tourism, helps restore fisheries, and improves human health and well-being. It advances the Sustainable Development Goals (SDG). These benefits will last for centuries., Recommendations: World leaders who recognize the gravity of ocean pollution, acknowledge its growing dangers, engage civil society and the global public, and take bold, evidence-based action to stop pollution at source will be critical to preventing ocean pollution and safeguarding human health.Prevention of pollution from land-based sources is key. Eliminating coal combustion and banning all uses of mercury will reduce mercury pollution. Bans on single-use plastic and better management of plastic waste reduce plastic pollution. Bans on persistent organic pollutants (POPs) have reduced pollution by PCBs and DDT. Control of industrial discharges, treatment of sewage, and reduced applications of fertilizers have mitigated coastal pollution and are reducing frequency of HABs. National, regional and international marine pollution control programs that are adequately funded and backed by strong enforcement have been shown to be effective. Robust monitoring is essential to track progress.Further interventions that hold great promise include wide-scale transition to renewable fuels; transition to a circular economy that creates little waste and focuses on equity rather than on endless growth; embracing the principles of green chemistry; and building scientific capacity in all countries.Designation of Marine Protected Areas (MPAs) will safeguard critical ecosystems, protect vulnerable fish stocks, and enhance human health and well-being. Creation of MPAs is an important manifestation of national and international commitment to protecting the health of the seas., Competing Interests: All authors declare no Conflict of Interest in regard to the work presented in this paper with the following exceptions. – Author William H. Gaze declares no conflict of interest although he has received co-funding for PhD studentships from AstraZeneca.– Author Philippe Grandjean has provided paid expert assistance in legal cases involving populations exposed to PFAS.– Author Barbara Demeneix is an inventor of “Transgenic clawed frog embryos and used as detectors of endocrine disruption in the environment”, a French patent application filed in 2002 (n°FR0206669), that was extended through a PCT application filled in 2003. Applicants: Centre National de la Recherche Scientifique (CNRS) and Muséum National d’Histoire Naturelle (MNHN). Inventors: B. Demeneix and N. Turque. The patent has been extended worldwide: France (2007), Japan (2011), United States (2013), Canada (2013) and Europe (2015). There has been no financial compensation for the patent., (Copyright: © 2020 The Author(s).)

Vibrio parahaemolyticus is a ubiquitous and abundant member of native microbial assemblages in coastal waters and shellfish. Though V. parahaemolyticus is predominantly environmental, some strains have infected human hosts and caused outbreaks of seafood-related gastroenteritis. In order to understand differences among clinical and environmental V. parahaemolyticus strains, we used high quality DNA sequencing data to compare the genomes of V. parahaemolyticus isolates ( n = 43) from a variety of geographic locations and clinical and environmental sample matrices. We used phylogenetic trees inferred from multilocus sequence typing (MLST) and whole-genome (WG) alignments, as well as a novel classification and genome clustering approach that relies on protein motif fingerprints (MFs), to assess relationships between V. parahaemolyticus strains and identify novel molecular targets associated with virulence. Differences in strain clustering at more than one position were observed between the MLST and WG phylogenetic trees. The WG phylogeny had higher support values and strain resolution since isolates of the same sequence type could be differentiated. The MF analysis revealed groups of protein motifs that were associated with the pathogenic MLST type ST36 and a large group of clinical strains isolated from human stool. A subset of the stool and ST36-associated protein motifs were selected for further analysis and the motif sequences were found in genes with a variety of functions, including transposases, secretion system components and effectors, and hypothetical proteins. DNA sequences associated with these protein motifs are candidate targets for future molecular assays in order to improve surveys of pathogenic V. parahaemolyticus in the environment and seafood.

Of marine eubacteria, the genus Vibrio is intriguing because member species are relevant to both marine ecology and human health. Many studies have touted the relationships of Vibrio to environmental factors, especially temperature and salinity, to predict total Vibrio abundance but lacked the taxonomic resolution to identify the relationships among species and the key drivers of Vibrio dynamics. To improve next-generation sequencing (NGS) surveys of Vibrio , we have conducted both 16S small subunit rRNA and heat shock protein 60 ( hsp60 ) amplicon sequencing of water samples collected at two well-studied locations in the Neuse River Estuary, NC. Samples were collected between May and December 2016 with enhanced sampling efforts in response to two named storms. Using hsp60 sequences, 21 Vibrio species were identified, including the potential human pathogens V. cholerae , V. parahaemolyticus , and V. vulnificus Changes in the Vibrio community mirrored seasonal and storm-related changes in the water column, especially in response to an influx of nutrient-rich freshwater to the estuary after Hurricane Matthew, which initiated dramatic changes in the overall Vibrio community. Individual species dynamics were wide ranging, indicating that individual Vibrio taxa have unique ecologies and that total Vibrio abundance predictors are insufficient for risk assessments of potentially pathogenic species. Positive relationships between Vibrio , dinoflagellates, and Cyanobacteria were identified, as were intraspecies associations, which further illuminated the interactions of cooccurring Vibrio taxa along environmental gradients. IMPORTANCE The objectives of this research were to utilize a novel approach to improve sequence-based surveys of Vibrio communities and to demonstrate the usefulness of this approach by presenting an analysis of Vibrio dynamics in the context of environmental conditions, with a particular focus on species that cause disease in humans and on storm effects. The methods presented here enabled the analysis of Vibrio dynamics with excellent taxonomic resolution and could be incorporated into future ecological studies and risk prediction strategies for potentially pathogenic species. Next-generation sequencing of hsp60 and other innovative sequence-based approaches are valuable tools and show great promise for studying Vibrio ecology and associated public health risks., (Copyright © 2018 American Society for Microbiology.)

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., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Monitoring of Vibrio vulnificus and V. parahaemolyticus abundance is pertinent due to the ability of these species to cause disease in humans through aquatic vectors. Previously, we performed a multiyear investigation tracking Vibrio spp. levels in five sites along the southeastern North Carolina coast. From February 2013 to October 2015, total V. vulnificus and V. parahaemolyticus abundance was measured in water, oysters and clams. In the current study, pathogenic subpopulations were identified in these isolates using molecular markers, revealing that 5.3% of V. vulnificus isolates possessed the virulence-correlated gene (vcgC), and 1.9% of V. parahaemolyticus isolates harbored one or both of the virulence-associated hemolysin genes (tdh and trh). Total V. parahaemolyticus abundance was not sufficient to predict the abundance of pathogenic subpopulations. Specifically, pathogenic V. parahaemolyticus isolates were more often isolated in cooler waters and were sometimes isolated when no other V. parahaemolyticus strains were detectable. Vibrio vulnificus clinical (C-) genotypes correlated with total V. vulnificus; however, salinity, water depth and total suspended solids influenced C- and E-genotypes differently. Lastly, we documented individual oysters harboring significantly higher V. vulnificus levels for which there was no ecological explanation, a phenomenon that deserves closer attention due to the potentially elevated health hazard associated with these 'hot' shellfish., (© FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Filter feeding shellfish can concentrate pathogenic bacteria, including Vibrio vulnificus and Vibrio parahaemolyticus, as much as 100-fold from the overlying water. These shellfish, especially clams and oysters, are often consumed raw, providing a route of entry for concentrated doses of pathogenic bacteria into the human body. The numbers of foodborne infections with these microbes are increasing, and a better understanding of the conditions that might trigger elevated concentrations of these bacteria in seafood is needed. In addition, if bacterial concentrations in water are correlated with those in shellfish, then sampling regimens could be simplified, as water samples can be more rapidly and easily obtained. After sampling of oysters and clams, either simultaneously or separately, for over 2 years, it was concluded that while Vibrio concentrations in oysters and water were related, this was not the case for levels in clams and water. When clams and oysters were collected simultaneously from the same site, the clams were found to have lower Vibrio levels than the oysters. Furthermore, the environmental parameters that were correlated with levels of Vibrio spp. in oysters and water were found to be quite different from those that were correlated with levels of Vibrio spp. in clams., Importance: This study shows that clams are a potential source of infection in North Carolina, especially for V. parahaemolyticus These findings also highlight the need for clam-specific environmental research to develop accurate Vibrio abundance models and to broaden the ecological understanding of clam-Vibrio interactions. This is especially relevant as foodborne Vibrio infections from clams are being reported., (Copyright © 2016 American Society for Microbiology.)

The bacterial species, Vibrio parahaemolyticus and Vibrio vulnificus, are ubiquitous in estuaries and coastal waters throughout the world, but they also happen to be important human pathogens. They are concentrated by filter-feeding shellfish which are often consumed raw or undercooked, providing an important potential route of entry for an infective dose of these bacteria. Vibrio parahaemolyticus can cause abdominal cramping, nausea, diarrhoea, vomiting, chills and fever. Vibrio vulnificus can cause similar gastrointestinal-related symptoms, but can also spread to the bloodstream, resulting in primary septicaemia, and it can also cause disease via wound infections. The objective of this article is to summarize, for the first time, the incidence and importance of V. parahaemolyticus and V. vulnificus in South America, in environmental waters and seafood, especifically molluscan shellfish, as well as human infection cases and outbreaks. It appears that infections from V. parahaemolyticus have been more strongly related to shellfish ingestion and have been more frequently reported on the Pacific coast of South America. Conversely, V. vulnificus has been more frequently acquired by water contact with open wounds and its presence has been more heavily reported along the Atlantic coast of South America, and while documented to cause serious mortality, have been relatively few in number. The impacts of El Nino Southern Oscillation (ENSO) have been observed to cause an increase in V. parahaemolyticus outbreaks on the Pacific coast of South America. The implementation of a regulated monitoring approach, along with the use of faster, more accurate and virulence-specific detection approaches, such as PCR confirmation, should be considered to detect the presence of pathogenic Vibrio strains in environmental and seafood samples for protection of public health. Furthermore, improved clinical surveillance with suspected cases should be implemented. This review highlights the need for more research and monitoring of vibrios in South America, in water, shellfish and clinical samples., (© 2016 The Society for Applied Microbiology.)

Viruses are the most abundant biological entities in the world's oceans, and they play a crucial role in global biogeochemical cycles. In deep-sea ecosystems, archaea and bacteria drive major nutrient cycles, and viruses are largely responsible for their mortality, thereby exerting important controls on microbial dynamics. However, the relative impact of viruses on archaea compared to bacteria is unknown, limiting our understanding of the factors controlling the functioning of marine systems at a global scale. We evaluate the selectivity of viral infections by using several independent approaches, including an innovative molecular method based on the quantification of archaeal versus bacterial genes released by viral lysis. We provide evidence that, in all oceanic surface sediments (from 1000- to 10,000-m water depth), the impact of viral infection is higher on archaea than on bacteria. We also found that, within deep-sea benthic archaea, the impact of viruses was mainly directed at members of specific clades of Marine Group I Thaumarchaeota. Although archaea represent, on average, ~12% of the total cell abundance in the top 50 cm of sediment, virus-induced lysis of archaea accounts for up to one-third of the total microbial biomass killed, resulting in the release of ~0.3 to 0.5 gigatons of carbon per year globally. Our results indicate that viral infection represents a key mechanism controlling the turnover of archaea in surface deep-sea sediments. We conclude that interactions between archaea and their viruses might play a profound, previously underestimated role in the functioning of deep-sea ecosystems and in global biogeochemical cycles.

Viruses profoundly influence benthic marine ecosystems by infecting and subsequently killing their prokaryotic hosts, thereby impacting the cycling of carbon and nutrients. Previously conducted studies, based on different methodologies, have provided widely differing estimates of the relevance of viruses on benthic prokaryotes. There has been no attempt so far to compare these independent approaches, including contextual comparisons among different approaches for sample manipulation (i.e., dilution or not of the sediments during incubations), between methods based on epifluorescence microscopy (EFM) or radiotracers, and between the use of different radiotracers. Therefore, it has been difficult to identify the most suitable methodologies and protocols to be used as standard approaches for the quantification of viral infections of prokaryotes. Here, we compared for the first time different methods for determining viral and prokaryotic production rates in marine sediments collected at two benthic sites, differing in depth and environmental conditions. We used a highly replicated experimental design, testing the potential biases associated to the incubation of sediments as diluted or undiluted. In parallel, we also compared EFM counts with the 3 H-thymidine incubations for the determination of viral production rates, and the use of 3 H-thymidine versus 3 H-leucine radiotracers for the determination of prokaryotic production. We show here that, independent from sediment dilution, EFM-based values of viral production ranged from 1.4 to 4.6 × 10 7 viruses g -1 h -1 , and were similar but overall less variable compared to those obtained by the 3 H-thymidine method (0.3 to 9.0 × 10 7 viruses g -1 h -1 ). In addition, the prokaryotic production rates were not affected by sediment dilution, and the use of different radiotracers provided very consistent estimates (10.3-35.1 and 9.3-34.6 ngC g -1 h -1 using the 3 H-thymidine or 3 H-leucine method, respectively). These results indicated that viral lysis was responsible for the abatement of 55-81% of the prokaryotic heterotrophic production, corroborating previous findings of the major role of viruses in benthic deep-sea ecosystems. Moreover, our methodological comparison for the analysis of viral production in marine sediments suggests that microscopy-based approaches are simpler and more cost-effective than those based on radiotracers. These approaches also reduce time to results and overcome issues related to generation of radioactive waste.

Quantitative polymerase chain reaction (qPCR) has become a frequently used technique for quantifying enterococci in recreational surface waters, but there are several methodological options. Here we evaluated how three method permutations, type of mastermix, sample extract dilution and use of controls in results calculation, affect method reliability among multiple laboratories with respect to sample interference. Multiple samples from each of 22 sites representing an array of habitat types were analyzed using EPA Method 1611 and 1609 reagents with full strength and five-fold diluted extracts. The presence of interference was assessed three ways: using sample processing and PCR amplifications controls; consistency of results across extract dilutions; and relative recovery of target genes from spiked enterococci in water sample compared to control matrices with acceptable recovery defined as 50 to 200%. Method 1609, which is based on an environmental mastermix, was found to be superior to Method 1611, which is based on a universal mastermix. Method 1611 had over a 40% control assay failure rate with undiluted extracts and a 6% failure rate with diluted extracts. Method 1609 failed in only 11% and 3% of undiluted and diluted extracts analyses. Use of sample processing control assay results in the delta-delta Ct method for calculating relative target gene recoveries increased the number of acceptable recovery results. Delta-delta tended to bias recoveries from apparent partially inhibitory samples on the high side which could help in avoiding potential underestimates of enterococci--an important consideration in a public health context. Control assay and delta-delta recovery results were largely consistent across the range of habitats sampled, and among laboratories. The methodological option that best balanced acceptable estimated target gene recoveries with method sensitivity and avoidance of underestimated enterococci densities was Method 1609 without extract dilution and using the delta-delta calculation method. The applicability of this method can be extended by the analysis of diluted extracts to sites where interference is indicated but, particularly in these instances, should be confirmed by augmenting the control assays with analyses for target gene recoveries from spiked target organisms., (Published by Elsevier B.V.)

The human-pathogenic marine bacteria Vibrio vulnificus and V. parahaemolyticus are strongly correlated with water temperature, with concentrations increasing as waters warm seasonally. Both of these bacteria can be concentrated in filter-feeding shellfish, especially oysters. Because oysters are often consumed raw, this exposes people to large doses of potentially harmful bacteria. Various models are used to predict the abundance of these bacteria in oysters, which guide shellfish harvest policy meant to reduce human health risk. Vibrio abundance and behaviour varies from site to site, suggesting that location-specific studies are needed to establish targeted risk reduction strategies. Moreover, virulence potential, rather than simple abundance, should be also be included in future modeling efforts., (© 2016 The Author(s).)

Infectious marine diseases can decimate populations and are increasing among some taxa due to global change and our increasing reliance on marine environments. Marine diseases become emergencies when significant ecological, economic or social impacts occur. We can prepare for and manage these emergencies through improved surveillance, and the development and iterative refinement of approaches to mitigate disease and its impacts. Improving surveillance requires fast, accurate diagnoses, forecasting disease risk and real-time monitoring of disease-promoting environmental conditions. Diversifying impact mitigation involves increasing host resilience to disease, reducing pathogen abundance and managing environmental factors that facilitate disease. Disease surveillance and mitigation can be adaptive if informed by research advances and catalysed by communication among observers, researchers and decision-makers using information-sharing platforms. Recent increases in the awareness of the threats posed by marine diseases may lead to policy frameworks that facilitate the responses and management that marine disease emergencies require., (© 2016 The Author(s).)

Predation on bacteria and accompanying mortality are important mechanisms in controlling bacterial populations and recycling of nutrients through the microbial loop. The agents most investigated and seen as responsible for bacterial mortality are viruses and protists. However, a body of evidence suggests that predatory bacteria such as the Halobacteriovorax (formerly Bacteriovorax), a Bdellovibrio-like organism, contribute substantially to bacterial death. Until now, conclusive evidence has been lacking. The goal of this study was to better understand the contributors to bacterial mortality by addressing the poorly understood role of Halobacteriovorax and how their role compares with that of viruses. The results revealed that when a concentrated suspension of Vibrio parahaemolyticus was added into microcosms of estuarine waters, the native Halobacteriovorax were the predators that responded first and most rapidly. Their numbers increased by four orders of magnitude, whereas V. parahaemolyticus prey numbers decreased by three orders of magnitude. In contrast, the extant virus population showed little increase and produced little change in the prey density. An independent experiment with stable isotope probing confirmed that Halobacteriovorax were the predators primarily responsible for the mortality of the V. parahaemolyticus. The results show that Halobacteriovorax have the potential to be significant contributors to bacterial mortality, and in such cases, predation by Halobacteriovorax may be an important mechanism of nutrient recycling. These conclusions add another dimension to bacterial mortality and the recycling of nutrients.

The United States has federal regulations in place to reduce the risk of seafood-related infection caused by the estuarine bacteria Vibrio vulnificus and Vibrio parahaemolyticus. However, data to support the development of regulations have been generated in a very few specific regions of the nation. More regionally specific data are needed to further understand the dynamics of human infection relating to shellfish-harvesting conditions in other areas. In this study, oysters and water were collected from four oyster harvest sites in North Carolina over an 11-month period. Samples were analyzed for the abundances of total Vibrio spp., V. vulnificus, and V. parahaemolyticus; environmental parameters, including salinity, water temperature, wind velocity, and precipitation, were also measured simultaneously. By utilizing these data, preliminary predictive management tools for estimating the abundance of V. vulnificus bacteria in shellfish were developed. This work highlights the need for further research to elucidate the full suite of factors that drive V. parahaemolyticus abundance., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Vibrio vulnificus, a bacterium ubiquitous in oysters and coastal water, is capable of causing ailments ranging from gastroenteritis to grievous wound infections or septicemia. The uptake of these bacteria into oysters is often examined in vitro by placing oysters in seawater amended with V. vulnificus. Multiple teams have obtained similar results in studies where laboratory-grown bacteria were observed to be rapidly taken up by oysters but quickly eliminated. This technique, along with suggested modifications, is reviewed here. In contrast, the natural microflora within oysters is notoriously difficult to eliminate via depuration. The reason for the transiency of exogenous bacteria is that those bacteria are competitively excluded by the oyster's preexisting microflora. Evidence of this phenomenon is shown using in vitro oyster studies and a multiyear in situ case study. Depuration of the endogenous oyster bacteria occurs naturally and can also be artificially induced, but both of these events require extreme conditions, natural or otherwise, as explained here. Finally, the "viable but nonculturable" (VBNC) state of Vibrio is discussed. This bacterial torpor can easily be confused with a reduction in bacterial abundance, as bacteria in this state fail to grow on culture media. Thus, oysters collected from colder months may appear to be relatively free of Vibrio but in reality harbor VBNC cells that respond to exogenous bacteria and prevent colonization of oyster matrices. Bacterial-uptake experiments combined with studies involving cell-free spent media are detailed that demonstrate this occurrence, which could explain why the microbial community in oysters does not always mirror that of the surrounding water., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Monitoring concentrations of bacterial pathogens and indicators of fecal contamination in coastal and estuarine ecosystems is critical to reduce adverse effects to public health. During storm events, particularly hurricanes, floods, Nor'easters, and tropical cyclones, sampling of coastal and estuarine waters is not generally possible due to safety concerns. It is particularly important to monitor waters during these periods as it is at precisely these times that pathogenic bacteria such as Vibrio spp. and fecal indicator bacteria concentrations fluctuate, potentially posing significant risks to public health. Automated samplers, such as the Isco sampler, are commonly used to conduct remote sample collection. Remote sampling is employed during severe storm periods, thereby reducing risk to researchers. Water samples are then stored until conditions are safe enough to retrieve them, typically in less than 21h, to collect the samples. Concerns exist regarding potential "bottle effects", whereby containment of sample might result in altered results. While these effects are well documented in samples being held for 24h or more, there is little data on bottle effects occurring during the first 24h of containment, and less still on the specific effects related to this type of sampling regime. Estuarine water samples were collected in the fall of 2013, placed into an Isco autosampler and subsampled over time to determine the effects of storage within this type of autosampling device. Vibrio spp. and fecal indicator bacteria were quantified using replicated culture-based methods, including Enterolert™ and membrane filtration. The experiments demonstrated no significant impact of storage time when comparing concentrations of total Vibrio spp., Vibrio vulnificus, Vibrio parahaemolyticus, or Enterococcus spp. after storage compared to original concentrations. However, the findings also suggested that increased variability and growth can occur during the middle of the day. Therefore, if at all possible, analysis schedules should be modified to account for this variability, e.g. collection of samples after overnight storage should occur as early in the morning as practicable., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The most common cause of seafood-borne death in the United States is the bacterium Vibrio vulnificus which can be concentrated into high numbers in the tissues of oysters or other shellfish. The ability to quickly, accurately, and inexpensively isolate living strains of this organism from oyster tissues is crucial for effective research on this pathogen. In this report, we evaluate four methods for isolating and quantifying V. vulnificus from oyster tissues, the solid media CPC+ (a refined version of cellobiose-polymyxin B-colistin medium), CHROMagar Vibrio, VVX (Vibrio vulnificus X-gal), and a method termed "Triple plating". Up to 1225 presumptive isolates were detected by each method, and 335 were subjected to molecular typing. The selectivity and sensitivity of each method was examined and VVX was found to be the most accurate method, with each of the other methods being recommended for task-specific uses. CHROMagar Vibrio is recommended for ease of use and relative accuracy, CPC+ is best used to differentiate between clinically associated and environmental strains., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Recently, the United States Environmental Protection Agency (USEPA) revised their recreational water quality criteria, in which adjustments were made by approving enterococci (ENT) quantitative PCR (qPCR) as an alternative, rapid method and advocating the use of predictive models for water quality management. The implementation of qPCR-based methods and prediction models are meant to decrease the time between sample collection and public advisories and notifications. To date, few studies have compared qPCR-based models to culture-based prediction models and none of these studies have been conducted in coastal estuarine systems. In this study, we created prediction models using qPCR-based fecal indicator bacteria (FIB) data in dual-use recreational and shellfish harvesting waters and compared them to published ENT and Escherichia coli (EC) culture-based prediction models in eastern North Carolina estuaries. Furthermore, an empirical statistical model was created to predict qPCR inhibition levels so that proper remediation techniques can be applied when it is a problem. Predictor variable selection in both qPCR- and culture-based ENT models was very similar; both models included 14-day rain total, dissolved oxygen, and salinity/conductivity, with 89 and 90% of qPCR and culture data described, respectively. Using ENT management action thresholds, qPCR- and culture-based models showed high accuracy in management decisions. The qPCR model had 92 and 96% accuracy using the 110 and 1000 cell equivalents (CE)/100 ml thresholds, respectively, and the culture model had 90% accuracy in management decisions with the 110 MPN/100 ml threshold. EC models for qPCR- and culture-based concentrations used similar independent variables (14-day humidity, salinity/conductivity, a rain/storm variable, and a measure of air temperature), with each model explaining 26 and 55% of the data variation, respectively. When using different thresholds that were logs apart for management decisions, the two EC models accurately predicted management decisions; qPCR models correctly predicted management decisions 96 and 77% of the time (using 31 and 320 CE/100 ml, respectively) while culture models correctly predicted management decisions 96 and 88% percent of the time (with 31 and 320 MPN/100 ml, respectively). Equivalency between models was shown in our non-point source impacted estuaries, with ENT models performing slightly better than EC models. In addition, inhibition of the qPCR was a major issue that had to be addressed. An inhibition model was created with easily obtained meteorological data and accounted for a high level of data variability (adjusted R(2) = 0.82)., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Microbial source tracking (MST) methods were evaluated in the Source Identification Protocol Project (SIPP), in which 27 laboratories compared methods to identify host sources of fecal pollution from blinded water samples containing either one or two different fecal types collected from California. This paper details lessons learned from the SIPP study and makes recommendations to further advance the field of MST. Overall, results from the SIPP study demonstrated that methods are available that can correctly identify whether particular host sources including humans, cows and birds have contributed to contamination in a body of water. However, differences between laboratory protocols and data processing affected results and complicated interpretation of MST method performance in some cases. This was an issue particularly for samples that tested positive (non-zero Ct values) but below the limits of quantification or detection of a PCR assay. Although false positives were observed, such samples in the SIPP study often contained the fecal pollution source that was being targeted, i.e., the samples were true positives. Given these results, and the fact that MST often requires detection of targets present in low concentrations, we propose that such samples be reported and identified in a unique category to facilitate data analysis and method comparisons. Important data can be lost when such samples are simply reported as positive or negative. Actionable thresholds were not derived in the SIPP study due to limitations that included geographic scope, age of samples, and difficulties interpreting low concentrations of target in environmental samples. Nevertheless, the results of the study support the use of MST for water management, especially to prioritize impaired waters in need of remediation. Future integration of MST data into quantitative microbial risk assessments and other models could allow managers to more efficiently protect public health based on site conditions., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

An inter-laboratory study of the accuracy of microbial source tracking (MST) methods was conducted using challenge fecal and sewage samples that were spiked into artificial freshwater and provided as unknowns (blind test samples) to the laboratories. The results of the Source Identification Protocol Project (SIPP) are presented in a series of papers that cover 41 MST methods. This contribution details the results of the virus and bacteriophage methods targeting human fecal or sewage contamination. Human viruses used as source identifiers included adenoviruses (HAdV), enteroviruses (EV), norovirus Groups I and II (NoVI and NoVII), and polyomaviruses (HPyVs). Bacteriophages were also employed, including somatic coliphages and F-specific RNA bacteriophages (FRNAPH) as general indicators of fecal contamination. Bacteriophage methods targeting human fecal sources included genotyping of FRNAPH isolates and plaque formation on bacterial hosts Enterococcus faecium MB-55, Bacteroides HB-73 and Bacteroides GB-124. The use of small sample volumes (≤50 ml) resulted in relatively insensitive theoretical limits of detection (10-50 gene copies or plaques × 50 ml(-1)) which, coupled with low virus concentrations in samples, resulted in high false-negative rates, low sensitivity, and low negative predictive values. On the other hand, the specificity of the human virus methods was generally close to 100% and positive predictive values were ∼40-70% with the exception of NoVs, which were not detected. The bacteriophage methods were generally much less specific toward human sewage than virus methods, although FRNAPH II genotyping was relatively successful, with 18% sensitivity and 85% specificity. While the specificity of the human virus methods engenders great confidence in a positive result, better concentration methods and larger sample volumes must be utilized for greater accuracy of negative results, i.e. the prediction that a human contamination source is absent., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Quantitative polymerase chain reaction (qPCR) is increasingly being used for the quantitative detection of fecal indicator bacteria in beach water. QPCR allows for same-day health warnings, and its application is being considered as an option for recreational water quality testing in the United States (USEPA, 2011. EPA-OW-2011-0466, FRL-9609-3, Notice of Availability of Draft Recreational Water Quality Criteria and Request for Scientific Views). However, transition of qPCR from a research tool to routine water quality testing requires information on how various method variations affect target enumeration. Here we compared qPCR performance and enumeration of enterococci in spiked and environmental water samples using three qPCR platforms (Applied Biosystem StepOnePlus™, the BioRad iQ™5 and the Cepheid SmartCycler(®) II), two reference materials (lyophilized cells and frozen cells on filters) and two comparative CT quantification models (ΔCT and ΔΔCT). Reference materials exerted the biggest influence, consistently affecting results by approximately 0.5 log(10) unit. Platform had the smallest effect, generally exerting <0.1 log(10) unit difference in final results. Quantification model led to small differences (0.04-0.2 log(10) unit) in this study with relatively uninhibited samples, but has the potential to cause as much as 8-fold (0.9 log(10) unit) difference in potentially inhibitory samples. Our findings indicate the need for a certified and centralized source of reference materials and additional studies to assess applicability of the quantification models in analyses of PCR inhibitory samples., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Quantitative polymerase chain reaction (qPCR) offers a rapid, highly sensitive analytical alternative to the traditional culture-based techniques of microbial enumeration typically used in water quality monitoring. Before qPCR can be widely applied within surface water monitoring programs and stormwater assessment research, the relationships between microbial concentrations measured by qPCR and culture-based methods must be assessed across a range of water types. Previous studies investigating fecal indicator bacteria quantification using molecular and culture-based techniques have compared measures of total concentration, but have not examined particle-associated microorganisms, which may be more important from a transport perspective, particularly during the calibration of predictive water quality models for watershed management purposes. This study compared total, free-phase, and particle-associated Escherichia coli concentrations as determined by the Colilert defined substrate method and qPCR targeting the uidA gene in stream grab samples partitioned via a calibrated centrifugation technique. Free-phase concentrations detected through qPCR were significantly higher than those detected using Colilert although total concentrations were statistically equivalent, suggesting a source of analytical bias. Although a specimen processing complex was used to identify and correct for inhibition of the qPCR reaction, high particle concentrations may have resulted in underestimation of total cell counts, particularly at low concentrations. Regardless, qPCR-based techniques will likely have an important future role in stormwater assessment and management.