Quantification of Protozoa and Viruses from Small Water Volumes.

Large sample volumes are traditionally required for the analysis of waterborne pathogens. The need for large volumes greatly limits the number of samples that can be processed. The aims of this study were to compare extraction and detection procedures for quantifying protozoan parasites and viruses from small volumes of marine water. The intent was to evaluate a logistically simpler method of sample collection and processing that would facilitate direct pathogen measures as part of routine monitoring programs. Samples were collected simultaneously using a bilayer device with protozoa capture by size (top filter) and viruses capture by charge (bottom filter). Protozoan detection technologies utilized for recovery of Cryptosporidium spp. and Giardia spp. were qPCR and the more traditional immunomagnetic separation-IFA-microscopy, while virus (poliovirus) detection was based upon qPCR versus plaque assay. Filters were eluted using reagents consistent with the downstream detection technologies. Results showed higher mean recoveries using traditional detection methods over qPCR for Cryptosporidium (91% vs. 45%) and poliovirus (67% vs. 55%) whereas for Giardia the qPCR-based methods were characterized by higher mean recoveries (41% vs. 28%). Overall mean recoveries are considered high for all detection technologies. Results suggest that simultaneous filtration may be suitable for isolating different classes of pathogens from small marine water volumes. More research is needed to evaluate the suitability of this method for detecting pathogens at low ambient concentration levels.

An alternative approach to water regulations for public health protection at bathing beaches.

New approaches should be considered as the US Environmental Protection Agency (EPA) moves rapidly to develop new beach monitoring guidelines by the end of 2012, as these guidelines serve as the basis by which states and territories with coasts along the oceans and Great Lakes can then develop and implement monitoring programs for recreational waters. We describe and illustrate one possible approach to beach regulation termed as the "Comprehensive Toolbox within an Approval Process (CTBAP)." The CTBAP consists of three components. The first is a "toolbox" consisting of an inventory of guidelines on monitoring targets, a series of measurement techniques, and guidance to improve water quality through source identification and prevention methods. The second two components are principles of implementation. These include first, "flexibility" to encourage and develop an individualized beach management plan tailored to local conditions and second, "consistency" of this management plan to ensure a consistent national level of public health protection. The results of this approach are illustrated through a case study at a well-studied South Florida recreational marine beach. This case study explores different monitoring targets based on two different health endpoints (skin versus gastrointestinal illness) and recommends a beach regulation program for the study beach that focuses predominately on source prevention.

Spatial and temporal variation in indicator microbe sampling is influential in beach management decisions.

Fecal indicator microbes, such as enterococci, are often used to assess potential health risks caused by pathogens at recreational beaches. Microbe levels often vary based on collection time and sampling location. The primary goal of this study was to assess how spatial and temporal variations in sample collection, which are driven by environmental parameters, impact enterococci measurements and beach management decisions. A secondary goal was to assess whether enterococci levels can be predictive of the presence of Staphylococcus aureus, a skin pathogen. Over a ten-day period, hydrometeorologic data, hydrodynamic data, bather densities, enterococci levels, and S. aureus levels including methicillin-resistant S. aureus (MRSA) were measured in both water and sand. Samples were collected hourly for both water and sediment at knee-depth, and every 6 h for water at waist-depth, supratidal sand, intertidal sand, and waterline sand. Results showed that solar radiation, tides, and rainfall events were major environmental factors that impacted enterococci levels. S. aureus levels were associated with bathing load, but did not correlate with enterococci levels or any other measured parameters. The results imply that frequencies of advisories depend heavily upon sample collection policies due to spatial and temporal variation of enterococci levels in response to environmental parameters. Thus, sampling at different times of the day and at different depths can significantly impact beach management decisions. Additionally, the lack of correlation between S. aureus and enterococci suggests that use of fecal indicators may not accurately assess risk for some pathogens.

Daily measures of microbes and human health at a non-point source marine beach.

Studies evaluating the relationship between microbes and human health at non-point source beaches are necessary for establishing criteria which would protect public health while minimizing economic burdens. The objective of this study was to evaluate water quality and daily cumulative health effects (gastrointestinal, skin, and respiratory illnesses) for bathers at a non-point source subtropical marine recreational beach in order to better understand the inter-relationships between these factors and hence improve monitoring and pollution prevention techniques. Daily composite samples were collected, during the Oceans and Human Health Beach Exposure Assessment and Characterization Health Epidemiologic Study conducted in Miami (Florida, USA) at a non-point source beach, and analyzed for several pathogens, microbial source tracking markers, indicator microbes, and environmental parameters. Analysis demonstrated that rainfall and tide were more influential, when compared to other environmental factors and source tracking markers, in determining the presence of both indicator microbes and pathogens. Antecedent rainfall and F+ coliphage detection in water should be further assessed to confirm their possible association with skin and gastrointestinal (GI) illness outcomes, respectively. The results of this research illustrate the potential complexity of beach systems characterized by non-point sources, and how more novel and comprehensive approaches are needed to assess beach water quality for the purpose of protecting bather health.

Evaluation of conventional and alternative monitoring methods for a recreational marine beach with nonpoint source of fecal contamination.

The objectives of this work were to compare enterococci (ENT) measurements based on the membrane filter, ENT(MF) with alternatives that can provide faster results including alternative enterococci methods (e.g., chromogenic substrate (CS), and quantitative polymerase chain reaction (qPCR)), and results from regression models based upon environmental parameters that can be measured in real-time. ENT(MF) were also compared to source tracking markers (Staphylococcus aureus, Bacteroidales human and dog markers, and Catellicoccus gull marker) in an effort to interpret the variability of the signal. Results showed that concentrations of enterococci based upon MF (<2 to 3320 CFU/100 mL) were significantly different from the CS and qPCR methods (p < 0.01). The correlations between MF and CS (r = 0.58, p < 0.01) were stronger than between MF and qPCR (r ≤ 0.36, p < 0.01). Enterococci levels by MF, CS, and qPCR methods were positively correlated with turbidity and tidal height. Enterococci by MF and CS were also inversely correlated with solar radiation but enterococci by qPCR was not. The regression model based on environmental variables provided fair qualitative predictions of enterococci by MF in real-time, for daily geometric mean levels, but not for individual samples. Overall, ENT(MF) was not significantly correlated with source tracking markers with the exception of samples collected during one storm event. The inability of the regression model to predict ENT(MF) levels for individual samples is likely due to the different sources of ENT impacting the beach at any given time, making it particularly difficult to to predict short-term variability of ENT(MF) for environmental parameters.

Traditional and molecular analyses for fecal indicator bacteria in non-point source subtropical recreational marine waters.

The use of enterococci as the primary fecal indicator bacteria (FIB) for the determination of recreational water safety has been questioned, particularly in sub/tropical marine waters without known point sources of sewage. Alternative FIB (such as the Bacteroidales group) and alternative measurement methods (such as rapid molecular testing) have been proposed to supplement or replace current marine water quality testing methods which require culturing enterococci. Moreover, environmental parameters have also been proposed to supplement current monitoring programs. The objective of this study was to evaluate the health risks to humans from exposure to subtropical recreational marine waters with no known point source. The study reported symptoms between one set of human subjects randomly assigned to marine water exposure with intensive environmental monitoring compared with other subjects who did not have exposure. In addition, illness outcomes among the exposed bathers were compared to levels of traditional and alternative FIB (as measured by culture-based and molecular-based methods), and compared to easily measured environmental parameters. Results demonstrated an increase in self-reported gastrointestinal, respiratory and skin illnesses among bathers vs. non-bathers. Among the bathers, a dose-response relationship by logistic regression modeling was observed for skin illness, where illness was positively related to enterococci enumeration by membrane filtration (odds ratio = 1.46 [95% confidence interval = 0.97-2.21] per increasing log10 unit of enterococci exposure) and positively related to 24 h antecedent rain fall (1.04 [1.01-1.07] per increasing millimeters of rain). Acute febrile respiratory illness was inversely related to water temperature (0.74 [0.56-0.98] per increasing degree of water temperature). There were no significant dose-response relationships between report of human illness and any of the other FIB or environmental measures. Therefore, for non-point source subtropical recreational marine waters, this study suggests that humans may be at increased risk of reported illness, and that the currently recommended and investigational FIB may not track gastrointestinal illness under these conditions; the relationship between other human illness and environmental measures is less clear.

The BEACHES Study: health effects and exposures from non-point source microbial contaminants in subtropical recreational marine waters.

BACKGROUND: Microbial water-quality indicators, in high concentrations in sewage, are used to determine whether water is safe for recreational purposes. Recently, the use of these indicators to regulate recreational water bodies, particularly in sub/tropical recreational marine waters without known sources of sewage, has been questioned. The objectives of this study were to evaluate the risk to humans from exposure to subtropical recreational marine waters with no known point source, and the possible relationship between microbe densities and reported symptoms in human subjects with random-exposure assignment and intensive individual microbial monitoring in this environment. METHODS: A total of 1303 adult regular bathers were randomly assigned to bather and non-bather groups, with subsequent follow-up for reported illness, in conjunction with extensive environmental sampling of indicator organisms (enterococci). RESULTS: Bathers were 1.76 times more likely to report gastrointestinal illness [95% confidence interval (CI) 0.94-3.30; P = 0.07]; 4.46 times more likely to report acute febrile respiratory illness (95% CI 0.99-20.90; P = 0.051) and 5.91 times more likely to report a skin illness (95% CI 2.76-12.63; P < 0.0001) relative to non-bathers. Evidence of a dose-response relationship was found between skin illnesses and increasing enterococci exposure among bathers [1.46 times (95% CI 0.97-2.21; P = 0.07) per increasing log(10) unit of enterococci exposure], but not for gastrointestinal or respiratory illnesses. CONCLUSIONS: This study indicated that bathers may be at increased risk of several illnesses relative to non-bathers, even in the absence of any known source of domestic sewage impacting the recreational marine waters. There was no dose-response relationship between gastroenteritis and increasing exposure to enterococci, even though many current water-monitoring standards use gastroenteritis as the major outcome illness.

Estimation of enterococci input from bathers and animals on a recreational beach using camera images.

Enterococci, are used nationwide as a water quality indicator of marine recreational beaches. Prior research has demonstrated that enterococci inputs to the study beach site (located in Miami, FL) are dominated by non-point sources (including humans and animals). We have estimated their respective source functions by developing a counting methodology for individuals to better understand their non-point source load impacts. The method utilizes camera images of the beach taken at regular time intervals to determine the number of people and animal visitors. The developed method translates raw image counts for week days and weekend days into daily and monthly visitation rates. Enterococci source functions were computed from the observed number of unique individuals for average days of each month of the year, and from average load contributions for humans and for animals. Results indicate that dogs represent the larger source of enterococci relative to humans and birds.

Presence of pathogens and indicator microbes at a non-point source subtropical recreational marine beach.

Swimming in ocean water, including ocean water at beaches not impacted by known point sources of pollution, is an increasing health concern. This study was an initial evaluation of the presence of indicator microbes and pathogens and the association among the indicator microbes, pathogens, and environmental conditions at a subtropical, recreational marine beach in south Florida impacted by non-point sources of pollution. Twelve water and eight sand samples were collected during four sampling events at high or low tide under elevated or reduced solar insolation conditions. The analyses performed included analyses of fecal indicator bacteria (FIB) (fecal coliforms, Escherichia coli, enterococci, and Clostridium perfringens), human-associated microbial source tracking (MST) markers (human polyomaviruses [HPyVs] and Enterococcus faecium esp gene), and pathogens (Vibrio vulnificus, Staphylococcus aureus, enterovirus, norovirus, hepatitis A virus, Cryptosporidium spp., and Giardia spp.). The enterococcus concentrations in water and sand determined by quantitative PCR were greater than the concentrations determined by membrane filtration measurement. The FIB concentrations in water were below the recreational water quality standards for three of the four sampling events, when pathogens and MST markers were also generally undetectable. The FIB levels exceeded regulatory guidelines during one event, and this was accompanied by detection of HPyVs and pathogens, including detection of the autochthonous bacterium V. vulnificus in sand and water, detection of the allochthonous protozoans Giardia spp. in water, and detection of Cryptosporidium spp. in sand samples. The elevated microbial levels were detected at high tide and under low-solar-insolation conditions. Additional sampling should be conducted to further explore the relationships between tidal and solar insolation conditions and between indicator microbes and pathogens in subtropical recreational marine waters impacted by non-point source pollution.

Correlations between microbial indicators, pathogens, and environmental factors in a subtropical estuary.

The objective of this study was to evaluate whether indicator microbes and physical-chemical parameters were correlated with pathogens within a tidally influenced Estuary. Measurements included the analysis of physical-chemical parameters (pH, salinity, temperature, and turbidity), measurements of bacterial indicators (enterococci, fecal coliform, Escherichia coli, and total coliform), viral indicators (somatic and MS2 coliphage), viral pathogens (enterovirus by culture), and protozoan pathogens (Cryptosporidium and Giardia). All pathogen results were negative with the exception of one sample which tested positive for culturable reovirus (8.5MPN/100L). Notable physical-chemical parameters for this sample included low salinity (<1ppt) and high water temperature (31 degrees C). Indicator bacteria and indicator virus levels for this sample were within average values typically measured within the study site and were low in comparison with levels observed in other freshwater environments. Overall results suggest that high levels of bacterial and viral indicators were associated with low salinity sites.

Quantitative evaluation of bacteria released by bathers in a marine water.

Enterococci, a common fecal indicator, and Staphylococcus aureus, a common skin pathogen, can be shed by bathers affecting the quality of recreational waters and resulting in possible human health impacts. Due to limited information available concerning human shedding of these microbes, this study focused on estimating the amounts of enterococci and S. aureus shed by bathers directly off their skin and indirectly via sand adhered to skin. Two sets of experiments were conducted at a marine beach located in Miami-Dade County, Florida. The first study, referred to as the "large pool" study, involved 10 volunteers who immersed their bodies in 4700L during four 15min cycles with exposure to beach sand in cycles 3 and 4. The "small pool" study involved 10 volunteers who were exposed to beach sand for 30min before they individually entered a small tub. After each individual was rinsed with off-shore marine water, sand and rinse water were collected and analyzed for enterococci. Results from the "large pool" study showed that bathers shed concentrations of enterococci and S. aureus on the order of 6x10(5) and 6x10(6) colony forming units (CFU) per person in the first 15min exposure period, respectively. Significant reductions in the bacteria shed per bather (50% reductions for S. aureus and 40% for enterococci) were observed in the subsequent bathing cycles. The "small pool" study results indicated that the enterococci contribution from sand adhered to skin was small (about 2% of the total) in comparison with the amount shed directly from the bodies of the volunteers. Results indicated that bathers transport significant amounts of enterococci and S. aureus to the water column, and thus human microbial bathing load should be considered as a non-point source when designing recreational water quality models.