A novel microplate-based spectrophotometric method for the quantitative assessment of freshwater bacterial coaggregation kinetics.

Coaggregation, the specific recognition and adhesion of genetically distinct bacteria, is proposed to contribute to the development of freshwater biofilms. This work aimed to develop a microplate-based system to measure and model the kinetics of freshwater bacterial coaggregation. Blastomonas natatoria 2.1 and Micrococcus luteus 2.13 were evaluated for coaggregation ability using 24-well microplates containing novel dome shaped wells (DSWs) and standard flat-bottom wells. Results were compared to a tube-based visual aggregation assay. The DSWs facilitated the reproducible detection of coaggregation via spectrophotometry and the estimation of coaggregation kinetics using a linked mathematical model. Quantitative analysis using DSWs was more sensitive than the visual tube aggregation assay and subject to substantially less variation than flat-bottom wells. Collectively these results demonstrate the utility of the DSW-based method and improve upon the current toolkit for studying freshwater bacterial coaggregation.

Industrialization as a source of heavy metals and antibiotics which can enhance the antibiotic resistance in wastewater, sewage sludge and river water.

The spread of antibiotic resistance is closely related with selective pressure in the environment. Wastewater from industrialized regions is characterized by higher concentrations of these pollutants than sewage from less industrialized areas. The aim of this study was to compare the concentrations of contaminants such as antibiotics and heavy metals (HMs), and to evaluate their impact on the spread of genes encoding resistance to antimicrobial drugs in samples of wastewater, sewage sludge and river water in two regions with different levels of industrialization. The factors exerting selective pressure, which significantly contributed to the occurrence of the examined antibiotic resistance genes (ARGs), were identified. The concentrations of selected gene copy numbers conferring resistance to four groups of antibiotics as well as class 1 and 2 integron-integrase genes were determined in the analyzed samples. The concentrations of six HMs and antibiotics corresponding to genes mediated resistance from 3 classes were determined. Based on network analysis, only some of the analyzed antibiotics correlated with ARGs, while HM levels were correlated with ARG concentrations, which can confirm the important role of HMs in promoting drug resistance. The samples from a wastewater treatment plant (WWTP) located an industrialized region were characterized by higher HM contamination and a higher number of significant correlations between the analyzed variables than the samples collected from a WWTP located in a less industrialized region. These results indicated that treated wastewater released into the natural environment can pose a continuous threat to human health by transferring ARGs, antibiotics and HMs to the environment. These findings shed light on the impact of industrialization on antibiotic resistance dissemination.

Phospholipid fatty acid (PLFA) analysis as a tool to estimate absolute abundances from compositional 16S rRNA bacterial metabarcoding data.

Microbial biodiversity monitoring through the analysis of DNA extracted from environmental samples is increasingly popular because it is perceived as being rapid, cost-effective, and flexible concerning the sample types studied. DNA can be extracted from diverse media before high-throughput sequencing of the prokaryotic 16S rRNA gene is used to characterize the taxonomic diversity and composition of the sample (known as metabarcoding). While sources of bias in metabarcoding methodologies are widely acknowledged, previous studies have focused mainly on the effects of these biases within a single substrate type, and relatively little is known of how these vary across substrates. We investigated the effect of substrate type (water, microbial mats, lake sediments, stream sediments, soil and a mock microbial community) on the relative performance of DNA metabarcoding in parallel with phospholipid fatty acid (PLFA) analysis. Quantitative estimates of the biomass of different taxonomic groups in samples were made through the analysis of PLFAs, and these were compared to the relative abundances of microbial taxa estimated from metabarcoding. Furthermore, we used the PLFA-based quantitative estimates of the biomass to adjust relative abundances of microbial groups determined by metabarcoding to provide insight into how the biomass of microbial taxa from PLFA analysis can improve understanding of microbial communities from environmental DNA samples. We used two sets of PLFA biomarkers that differed in their number of PLFAs to evaluate how PLFA biomarker selection influences biomass estimates. Metabarcoding and PLFA analysis provided significantly different views of bacterial composition, and these differences varied among substrates. We observed the most notable differences for the Gram-negative bacteria, which were overrepresented by metabarcoding in comparison to PLFA analysis. In contrast, the relative biomass and relative sequence abundances aligned reasonably well for Cyanobacteria across the tested freshwater substrates. Adjusting relative abundances of microbial taxa estimated by metabarcoding with PLFA-based quantification estimates of the microbial biomass led to significant changes in the microbial community compositions in all substrates. We recommend including independent estimates of the biomass of microbial groups to increase comparability among metabarcoding libraries from environmental samples, especially when comparing communities associated with different substrates.

Assessment of cytotoxicity biomarkers on the microalga Chlamydomonas reinhardtii exposed to emerging and priority pollutants.

Contamination of aquatic ecosystems linked to anthropogenic activity is currently a major concern; therefore, ecotoxicological studies are needed to assess its effect on organisms. The main objective of this study was to investigate the effects of different pollutants on microalgae in search of sensitive biomarkers that can promote a common cytotoxic response regardless of the contaminant. Cultures of the freshwater microalga Chlamydomonas reinhardtii were exposed for 24 h to four chemicals, three emerging pollutants (benzophenone-3, bisphenol A and oxytetracycline) and one priority substance (atrazine). A cytometric panel was carried out to assess toxicity biomarkers including cellular growth, inherent cell properties, viability, vitality, cytoplasmic membrane potential and ROS levels. Lipid peroxidation, photosynthetic efficiency and transcriptional responses of photosynthesis- and oxidative stress-related genes using RT-qPCR were also studied. Some toxicity responses showed a similar pattern; a decrease in growth rate, vitality and photosynthetic efficiency and an increase in autofluorescence and in the number of cells with depolarised cytoplasmic membrane and were found for all chemicals tested. However, ATZ and OTC provoked a decrease in cell size, whereas BP-3 and BPA caused an increase in cell size, intracellular complexity and ROS levels and a decrease in cell viability. Assayed pollutants generally promoted an overexpression of genes related to cellular antioxidant defence system and a subexpression of photosynthesis-related genes. In addition to the traditional growth endpoint, cell vitality, autofluorescence and gene expression of catalase, glutathione peroxidase and Fe-superoxide dismutase were significantly affected for all chemicals tested, showing a common cytotoxic response. Among the tested substances, BP-3 provoked the strongest cytotoxic alterations on this microalga, pointing out that some emerging contaminants could be more harmful to organisms than priority pollutants.

Assessment of microbial risk during Australian industrial practices for Escherichia coli O157:H7 in fresh cut-cos lettuce: A stochastic quantitative approach.

Escherichia coli O157:H7 risk associated with the consumption of fresh cut-cos lettuce during Australian industrial practices was assessed. A probabilistic risk assessment model was developed and implemented in the @Risk software by using the Monte Carlo simulation technique with 1,000,000 iterations. Australian preharvest practices yielded predicted annual mean E. coli O157:H7 levels from 0.2 to -3.4 log CFU/g and prevalence values ranged from 2 to 6.4%. While exclusion of solar radiation from the baseline model yielded a significant increase in concentration of E. coli O157:H7 (-5.2 -log fold), drip irrigation usage, exclusion of manure amended soil and rainfall reduced E. coli O157:H7 levels by 7.4, 6.5, and 4.3-log fold, respectively. The microbial quality of irrigation water and irrigation type both had a significant effect on E. coli O157:H7 concentrations at harvest (p < 0.05). The probability of illness due to consumption of E. coli O157:H7 contaminated fresh cut-cos lettuce when water washing interventions were introduced into the processing module, was reduced by 1.4-2.7-log fold (p < 0.05). This study provides a robust basis for assessment of risk associated with E. coli O157:H7 contamination on fresh cut-cos lettuce for industrial practices and will assist the leafy green industry and food safety authorities in Australia to identify potential risk management strategies.

Experimental metatranscriptomics reveals the costs and benefits of dissolved organic matter photo-alteration for freshwater microbes.

Microbes and sunlight convert terrigenous dissolved organic matter (DOM) in surface waters to greenhouse gases. Prior studies show contrasting results about how biological and photochemical processes interact to contribute to the degradation of DOM. In this study, DOM leached from the organic layer of tundra soil was exposed to natural sunlight or kept in the dark, incubated in the dark with the natural microbial community, and analysed for gene expression and DOM chemical composition. Microbial gene expression (metatranscriptomics) in light and dark treatments diverged substantially after 4 h. Gene expression suggested that sunlight exposure of DOM initially stimulated microbial growth by (i) replacing the function of enzymes that degrade higher molecular weight DOM such as enzymes for aromatic carbon degradation, oxygenation, and decarboxylation, and (ii) releasing low molecular weight compounds and inorganic nutrients from DOM. However, growth stimulation following sunlight exposure of DOM came at a cost. Sunlight depleted the pool of aromatic compounds that supported microbial growth in the dark treatment, ultimately causing slower growth in the light treatment over 5 days. These first measurements of microbial metatranscriptomic responses to photo-alteration of DOM provide a mechanistic explanation for how sunlight exposure of terrigenous DOM alters microbial processing and respiration of DOM.

INT Toxicity over Natural Bacterial Assemblages from Surface Oligotrophic Waters: Implications for the Assessment of Respiratory Activity.

Plankton community respiration (R) is a major component of the carbon flux in aquatic ecosystems. However, current methods to measure actual respiration from oxygen consumption at relevant spatial scales are not sensitive enough in oligotrophic environments where respiration rates are very low. To overcome this drawback, more sensitive indirect enzymatic approaches are commonly used as R proxies. The in vivo electron transport system (ETSvivo) assay, which measures the reduction of (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl tetrazolium chloride salt, INT) to INT-formazan in the presence of natural substrate levels, was recently proposed as an indirect reliable estimation of R for natural plankton communities. However, under in vivo conditions, formazan salts could be toxic to the cells. Here, we test the toxicity of 0.2 mM of final INT concentration, widely used for ETSvivo assays, on natural bacterial assemblages collected in coastal and oceanic waters off Gran Canaria (Canary Islands, subtropical North Atlantic), in eight independent experiments. After 0.5 h of incubation, a significant but variable decline in cell viability (14-49%) was observed in all samples inoculated with INT. Moreover, INT also inhibited leucine uptake in less than 90 min of incubation. In the light of these results, we argue that enzymatic respiratory rates obtained with the ETSvivo method need to be interpreted with caution to derive R in oceanic regions where bacteria largely contribute to community respiration. Moreover, the variable toxicity on bacterial assemblages observed in our experiments questions the use of a single R/ETSvivo relationship as a universal proxy for regional studies.

Ecotoxicity assessment of microcystins from freshwater samples using a bioluminescent cyanobacterial bioassay.

The hepatotoxic cyanotoxins microcystins (MCs) are emerging contaminants naturally produced by cyanobacteria. Yet their ecological role remains unsolved, previous research suggests that MCs have allelopathic effects on competing photosynthetic microorganisms, even eliciting toxic effects on other freshwater cyanobacteria. In this context, the bioluminescent recombinant cyanobacterium Anabaena sp. PCC7120 CPB4337 (hereinafter Anabaena) was exposed to extracts of MCs. These were obtained from eight natural samples from freshwater reservoirs that contained MCs with a concentration range of 0.04-11.9 µg MCs L-1. MCs extracts included the three most common MCs variants (MC-LR, MC-RR, MC-YR) in different proportions (MC-LR: 100-0%; MC-RR: 100-0%; MC-YR: 14.2-0%). The Anabaena bioassay based on bioluminescence inhibition has been successfully used to test the toxicity of many emerging contaminants (e.g., pharmaceuticals) but never for cyanotoxins prior to this study. Exposure of Anabaena to MCs extracts induced a decrease in its bioluminescence with effective concentration decreasing bioluminescence by 50% ranging from 0.4 to 50.5 µg MC L-1 in the different samples. Bioluminescence responses suggested an interaction between MCs variants which was analyzed via the Additive Index method (AI), indicating an antagonistic effect (AI < 0) of MC-LR and MC-RR present in the samples. Additionally, MC extracts exposure triggered an increase of intracellular free Ca2+ in Anabaena. In short, this study supports the use of the Anabaena bioassay as a sensitive tool to assess the presence of MCs at environmentally relevant concentrations and opens interesting avenues regarding the interactions between MCs variants and the possible implication of Ca2+ in the mode of action of MCs towards cyanobacteria.

Is the Effect Assessment Approach for Fungicides as Laid Down in the European Food Safety Authority Aquatic Guidance Document Sufficiently Protective for Freshwater Ecosystems?

In Europe, the European Food Safety Authority aquatic guidance document describes the procedures for the derivation of regulatory acceptable concentrations (RACs) for pesticides in edge-of-field surface waters on the basis of tier-1 (standard test species), tier-2 (geometric mean and species sensitivity distributions [SSDs]), and tier-3 (model ecosystem studies) approaches. In the present study, the protectiveness of such a tiered approach was evaluated for fungicides. Acute and chronic RACs for tier-1 and tier-2B (SSDs) were calculated using toxicity data for standard and additional test species, respectively. Tier-3 RACs based on ecological thresholds (not considering recovery) could be derived for 18 fungicides. We show that tier-1 RACs, in the majority of cases, are more conservative than RACs calculated based on model ecosystem experiments. However, acute tier-2B RACs do not show a sufficient protection level compared with tier-3 RACs from cosm studies that tested a repeated pulsed exposure regime or when relatively persistent compounds were tested. Chronic tier-2B RACs showed a sufficient protection level, although they could only be evaluated for 6 compounds. Finally, we evaluated the suitability of the calculated RACs for 8 compounds with toxicity data for fungi. The comparison shows that the current RACs for individual fungicides, with a few exceptions (e.g., tebuconazole), show a sufficient protection level for structural and functional fungal endpoints. However, more data are needed to extend this comparison to other fungicides with different modes of action. Environ Toxicol Chem 2019;38:2279-2293. © 2019 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC.

The application of quantitative microbial risk assessment to natural recreational waters: A review.

This review examines the aims of and approaches to the Quantitative Microbial Risk Assessment (QMRA) of untreated recreational waters. The literature search was conducted on four databases and yielded 54 papers, which were analyzed on a quantitative (time-trend, geographical distribution, water type) and qualitative (aims, source of microbial data, pathogens and their measurement or estimation, ways to address variability and uncertainty, sensitivity analysis) basis. In addition, the parameters, implications, and limitations were discussed for each QMRA step. Since 2003, the number of papers has greatly increased, highlighting the importance of QMRA for the risk management of recreational waters. Nevertheless, QMRA still exhibits critical issues, above all regarding contamination data and dose-response relationships. To our knowledge, this is the first review to give a wide panoramic view on QMRA in relation to recreational exposure to untreated waters. This could be useful in identifying the current knowledge gaps and research needs.

Preliminary assessment of the suitability of commonly used antiseptics in the elimination of bacteria in bathing water.

The use of antiseptics for the removal of bacteria in water has become pertinent given that most hand dug wells, boreholes, and surface waters are already contaminated in their in situ condition before being used. But, the efficacy of these skin disinfectants (antiseptics) is usually not well spelt out for the aforementioned purpose, and usage may yield no good report. This study assessed the suitability of use of two widely used antiseptics in Nigeria (Dettol and Izal) on Bacillus spp., Escherichia coli and Klebsiella spp., in eliminating bacteria in bathing water. The water which were tested at four different concentrations (0.4 ml, 0.8 ml, 1.2 ml, and 1.6 ml) and at six different contact times (0, 5, 10, 15, 30, and 60 min, respectively) within which bathing is meant to take place after antiseptics were applied. Overall, One hundred and Forty-four (144) samples were analyzed, and based on our null hypothesis of no bacteria should be found in bathing water after disinfection, results showed that both antiseptics were not efficient in bacteria removal. However, the multivariable logistic regression model conducted revealed that both antiseptics were more active in destroying Klebsiella spp. than any other bacteria investigated with Izal showing more dominance (OR = 31.21; p < 0.05). The study further revealed that Izal is 3.6 times more likely to destroy bacteria than Dettol (p < 0.05), with more of the elimination occurring at contact time greater than 5 min (OR = 1.504; p = 0.043). Therefore, it is suggested that disinfectants and antiseptics of high motility and sufficient potency in a wide range of bacteria spectrum should be produced to meet the needs of consumers resulting in a better bathing water quality.

Assessment of environmental parameters with special emphasis on avifaunal breeding season in the coastal wetland of Point Calimere Wildlife Sanctuary, Southeast coast of India.

The present study focuses on the hydrographic parameters and the population level of microbial indicators in wetland ecosystems and their effects on bird breeding habitat in Point Calimere Wildlife Sanctuary. Water samples were collected during the bird breeding seasons at five different stations in the sanctuaries, and samples were analyzed by standard methods. Results were compared with CPCB and USEPA standards and clearly denoted that the water quality is not suitable for bird feeding and breeding habitat. One-way ANOVA showed a strong evidence (p < 0.01) of risk for birds breeding in this habitat. As a result of salt pan chemical industries, aquaculture continues to have a major effect on the homogenization and breeding habitat of avian species. Urgent action is needed to prohibit the unregulated economical activities and to regulate water quality monitoring to strictly follow the wildlife conservation rules and regulations. This effective action will help in maintaining species diversity and composition of historical monuments to provide suitable breeding sites in the sanctuary.

Illumina sequencing and assessment of new cost-efficient protocol for metagenomic-DNA extraction from environmental water samples.

In this study, the development and assessment of a modified, efficient, and cost-efficient protocol for mDNA (metagenomic DNA) extraction from contaminated water samples was attempted. The efficiency of the developed protocol was investigated in comparison to a well-established commercial kit (Epicentre, Metagenomic DNA Isolation Kit for Water). The comparison was in terms of degree of shearing, yield, purity, duration, suitability for polymerase chain reaction and next-generation sequencing in addition to the quality of next-generation sequencing data. The DNA yield obtained from the developed protocol was 2.6 folds higher than that of the commercial kit. No significant difference in the alpha (Observed species, Chao1, Simpson and PD whole tree) and beta diversity was found between the DNA samples extracted by the commercial kit and the developed protocol. The number of high-quality sequences of the samples extracted by the developed method was 20% higher than those obtained by the samples processed by the kit. The developed economic protocol successfully yielded high-quality pure mDNA compatible with complex molecular applications. Thus we propose the developed protocol as a gold standard for future metagenomic studies investigating a large number of samples.

Illumina sequencing and assessment of new cost-efficient protocol for metagenomic-DNA extraction from environmental water samples

Abstract In this study, the development and assessment of a modified, efficient, and cost-efficient protocol for mDNA (metagenomic DNA) extraction from contaminated water samples was attempted. The efficiency of the developed protocol was investigated in comparison to a well-established commercial kit (Epicentre, Metagenomic DNA Isolation Kit for Water). The comparison was in terms of degree of shearing, yield, purity, duration, suitability for polymerase chain reaction and next-generation sequencing in addition to the quality of next-generation sequencing data. The DNA yield obtained from the developed protocol was 2.6 folds higher than that of the commercial kit. No significant difference in the alpha (Observed species, Chao1, Simpson and PD whole tree) and beta diversity was found between the DNA samples extracted by the commercial kit and the developed protocol. The number of high-quality sequences of the samples extracted by the developed method was 20% higher than those obtained by the samples processed by the kit. The developed economic protocol successfully yielded high-quality pure mDNA compatible with complex molecular applications. Thus we propose the developed protocol as a gold standard for future metagenomic studies investigating a large number of samples.

Vibrio Pathogens: A Public Health Concern in Rural Water Resources in Sub-Saharan Africa.

Members of the Vibrio genus are autochthonous inhabitants of aquatic environments and play vital roles in sustaining the aquatic milieu. The genus comprises about 100 species, which are mostly of marine or freshwater origin, and their classification is frequently updated due to the continuous discovery of novel species. The main route of transmission of Vibrio pathogens to man is through drinking of contaminated water and consumption inadequately cooked aquatic food products. In sub-Saharan Africa and much of the developing world, some rural dwellers use freshwater resources such as rivers for domestic activities, bathing, and cultural and religious purposes. This review describes the impact of inadequately treated sewage effluents on the receiving freshwater resources and the associated risk to the rural dwellers that depends on the water. Vibrio infections remain a threat to public health. In the last decade, Vibrio disease outbreaks have created alertness on the personal, economic, and public health uncertainties associated with the impact of contaminated water in the aquatic environment of sub-Saharan Africa. In this review, we carried out an overview of Vibrio pathogens in rural water resources in Sub-Saharan Africa and the implication of Vibrio pathogens on public health. Continuous monitoring of Vibrio pathogens among environmental freshwater and treated effluents is expected to help reduce the risk associated with the early detection of sources of infection, and also aid our understanding of the natural ecology and evolution of Vibrio pathogens.

Quantitative contamination assessment of Escherichia coli in baby spinach primary production in Spain: Effects of weather conditions and agricultural practices.

A quantitative microbial contamination model of Escherichia coli during primary production of baby spinach was developed. The model included only systematic contamination routes (e.g. soil and irrigation water) and it was used to evaluate the potential impact of weather conditions, agricultural practices as well as bacterial fitness in soil on the E. coli levels present in the crop at harvest. The model can be used to estimate E. coli contamination of baby spinach via irrigation water, via soil splashing due to irrigation water or rain events, and also including the inactivation of E. coli on plants due to solar radiation during a variable time of culturing before harvest. Seasonality, solar radiation and rainfall were predicted to have an important impact on the E. coli contamination. Winter conditions increased E. coli prevalence and levels when compared to spring conditions. As regards agricultural practices, both water quality and irrigation system slightly influenced E. coli levels on baby spinach. The good microbiological quality of the irrigation water (average E. coli counts in positive water samples below 1 log/100mL) could have influenced the differences observed among the tested agricultural practices (water treatment and irrigation system). This quantitative microbial contamination model represents a preliminary framework that assesses the potential impact of different factors and intervention strategies affecting E. coli concentrations at field level. Taking into account that E. coli strains may serve as a surrogate organism for enteric bacterial pathogens, obtained results on E. coli levels on baby spinach may be indicative of the potential behaviour of these pathogens under defined conditions.

Application of a paper based device containing a new culture medium to detect Vibrio cholerae in water samples collected in Haiti.

Cholera is now considered to be endemic in Haiti, often with increased incidence during rainy seasons. The challenge of cholera surveillance is exacerbated by the cost of sample collection and laboratory analysis. A diagnostic tool is needed that is low cost, easy-to-use, and able to detect and quantify Vibrio cholerae accurately in water samples within 18-24h, and perform reliably in remote settings lacking laboratory infrastructure and skilled staff. The two main objectives of this study were to develop and evaluate a new culture medium embedded in a new diagnostic tool (PAD for paper based analytical device) for detecting Vibrio cholerae from water samples collected in Haiti. The intent is to provide guidance for corrective action, such as chlorination, for water positive for V. cholerae epidemic strains. For detecting Vibrio cholerae, a new chromogenic medium was designed and evaluated as an alternative to thiosulfate citrate bile salts sucrose (TCBS) agar for testing raw water samples. Sensitivity and specificity of the medium were assessed using both raw and spiked water samples. The Vibrio cholerae chromogenic medium was proved to be highly selective against most of the cultivable bacteria in the water samples, without loss of sensitivity in detection of V. cholerae. Thus, reliability of this new culture medium for detection of V. cholerae in the presence of other Vibrio species in water samples offers a significant advantage. A new paper based device containing the new chromogenic medium previously evaluated was compared with reference methods for detecting V. cholerae from spiked water sample. The microbiological PAD specifications were evaluated in Haiti. More precisely, a total of 185 water samples were collected at five sites in Haiti, June 2014 and again in June 2015. With this new tool, three V. cholerae O1 and 17 V. cholerae non-O1/O139 strains were isolated. The presence of virulence-associated and regulatory genes, including ctxA, zot, ace, and toxR, was confirmed using multiplex PCR. The three V. cholerae O1 isolates were positive for three of the four virulence-associated and regulatory genes. Twelve of the V. cholerae non-O1/O139 isolates were found to carry toxR, but none were ctxA+, zot+, or ace+. However, six of the V. cholerae non-O1/O139 isolates were resistant to penicillin, ampicillin, trimethoprim/sulfamethoxazole, nalidixic acid, and ciprofloxacin. The paper based analytical device (PAD) provides advantages in that standard culture methods employing agar plates are not required. Also, intermediary isolation steps were not required, including transfer to selective growth media, hence these steps being omitted reduced time to results. Furthermore, experienced technical skills also were not required. Thus, PAD is well suited for resource-limited settings.

Estimated Costs of Sporadic Gastrointestinal Illness Associated with Surface Water Recreation: A Combined Analysis of Data from NEEAR and CHEERS Studies.

BACKGROUND: The burden of illness can be described by addressing both incidence and illness severity attributable to water recreation. Monetized as cost, attributable disease burden estimates can be useful for environmental management decisions. OBJECTIVES: We characterize the disease burden attributable to water recreation using data from two cohort studies using a cost of illness (COI) approach and estimate the largest drivers of the disease burden of water recreation. METHODS: Data from the NEEAR study, which evaluated swimming and wading in marine and freshwater beaches in six U.S. states, and CHEERS, which evaluated illness after incidental-contact recreation (boating, canoeing, fishing, kayaking, and rowing) on waterways in the Chicago area, were used to estimate the cost per case of gastrointestinal illness and costs attributable to water recreation. Data on health care and medication utilization and missed days of work or leisure were collected and combined with cost data to construct measures of COI. RESULTS: Depending on different assumptions, the cost of gastrointestinal symptoms attributable to water recreation are estimated to be $1,220 for incidental-contact recreation (range $338-$1,681) and $1,676 for swimming/wading (range $425-2,743) per 1,000 recreators. Lost productivity is a major driver of the estimated COI, accounting for up to 90% of total costs. CONCLUSIONS: Our estimates suggest gastrointestinal illness attributed to surface water recreation at urban waterways, lakes, and coastal marine beaches is responsible for costs that should be accounted for when considering the monetary impact of efforts to improve water quality. The COI provides more information than the frequency of illness, as it takes into account disease incidence, health care utilization, and lost productivity. Use of monetized disease severity information should be included in future studies of water quality and health. Citation: DeFlorio-Barker S, Wade TJ, Jones RM, Friedman LS, Wing C, Dorevitch S. 2017. Estimated costs of sporadic gastrointestinal illness associated with surface water recreation: a combined analysis of data from NEEAR and CHEERS Studies. Environ Health Perspect 125:215-222; http://dx.doi.org/10.1289/EHP130.

Evaluation of Techniques for Measuring Microbial Hazards in Bathing Waters: A Comparative Study.

Recreational water quality is commonly monitored by means of culture based faecal indicator organism (FIOs) assays. However, these methods are costly and time-consuming; a serious disadvantage when combined with issues such as non-specificity and user bias. New culture and molecular methods have been developed to counter these drawbacks. This study compared industry-standard IDEXX methods (Colilert and Enterolert) with three alternative approaches: 1) TECTA™ system for E. coli and enterococci; 2) US EPA's 1611 method (qPCR based enterococci enumeration); and 3) Next Generation Sequencing (NGS). Water samples (233) were collected from riverine, estuarine and marine environments over the 2014-2015 summer period and analysed by the four methods. The results demonstrated that E. coli and coliform densities, inferred by the IDEXX system, correlated strongly with the TECTA™ system. The TECTA™ system had further advantages in faster turnaround times (~12 hrs from sample receipt to result compared to 24 hrs); no staff time required for interpretation and less user bias (results are automatically calculated, compared to subjective colorimetric decisions). The US EPA Method 1611 qPCR method also showed significant correlation with the IDEXX enterococci method; but had significant disadvantages such as highly technical analysis and higher operational costs (330% of IDEXX). The NGS method demonstrated statistically significant correlations between IDEXX and the proportions of sequences belonging to FIOs, Enterobacteriaceae, and Enterococcaceae. While costs (3,000% of IDEXX) and analysis time (300% of IDEXX) were found to be significant drawbacks of NGS, rapid technological advances in this field will soon see it widely adopted.

Epidemiologic evaluation of multiple alternate microbial water quality monitoring indicators at three California beaches.

INTRODUCTION: Advances in molecular methods provide new opportunities for directly measuring pathogens or host-associated markers of fecal pollution instead of relying on fecal indicator bacteria (FIB) alone for beach water quality monitoring. Adoption of new indicators depends on identifying relationships between either the presence or concentration of the indicators and illness among swimmers. Here we present results from three epidemiologic studies in which a broad range of bacterial and viral indicators of fecal contamination were measured simultaneously by either culture or molecular methods along with Enterococcus to assess whether they provide better health risk prediction than current microbial indicators of recreational water quality. METHODS: We conducted prospective cohort studies at three California beaches -- Avalon Bay (Avalon), Doheny State Beach (Doheny), Surfrider State Beach (Malibu) -- during the summers of 2007, 2008 and 2009. The studies enrolled 10,785 swimmers across the beaches and recorded each swimmer's water exposure. Water and sand samples were collected several times per day at multiple locations at each beach and analyzed for up to 41 target indicators using 67 different methodologies. Interviewers contacted participants by phone 10-14 days later and recorded symptoms of gastrointestinal illness occurring after their beach visit. Regression models were used to evaluate the association between water quality indicators and gastrointestinal illness among swimmers at each beach. RESULTS: F+ coliphage (measured using EPA Method 1602) exhibited a stronger association with GI illness than did EPA Method 1600 at the two beaches where it was measured, while a molecular method, F+ RNA Coliphage Genotype II, was the only indicator significantly associated with GI illness at Malibu. MRSA, a known pathogen, had the strongest association with GI illness of any microbe measured at Avalon. There were two methods targeting human-associated fecal anaerobic bacteria that were more strongly associated with GI illness than EPA Method 1600, but only at Avalon. No indicator combinations consistently had a higher odds ratio than EPA Method 1600, but one composite indicator, based on the number of pathogens detected at a beach, was significantly associated with gastrointestinal illness at both Avalon and Doheny when freshwater flow was high. DISCUSSION: While EPA Method1600 performed adequately at two beaches based on its consistency of association with gastrointestinal illness and the precision of its estimated associations, F+ coliphage measured by EPA Method 1602 had a stronger association with GI illness under high risk conditions at the two beaches where it was measured. One indicator, F+ Coliphage Genotype II was the only indicator significantly associated with GI illness at Malibu. Several indicators, particularly those targeting human associated bacteria, exhibited relationships with GI illness that were equal to or greater than that of EPA Method 1600 at Avalon, which has a focused human fecal source. Our results suggest that site-specific conditions at each beach determine which indicator or indicators best predict GI illness.