Effect of beach management policies on recreational water quality.

When beach water monitoring programs identify poor water quality, the causes are frequently unknown. We hypothesize that management policies play an important role in the frequency of fecal indicator bacteria (FIB) exceedances (enterococci and fecal coliform) at recreational beaches. To test this hypothesis we implemented an innovative approach utilizing large amounts of monitoring data (n > 150,000 measurements per FIB) to determine associations between the frequency of contaminant exceedances and beach management practices. The large FIB database was augmented with results from a survey designed to assess management policies for 316 beaches throughout the state of Florida. The FIB and survey data were analyzed using t-tests, ANOVA, factor analysis, and linear regression. Results show that beach geomorphology (beach type) was highly associated with exceedance of regulatory standards. Low enterococci exceedances were associated with open coast beaches (n = 211) that have sparse human densities, no homeless populations, low densities of dogs and birds, bird management policies, low densities of seaweed, beach renourishment, charge access fees, employ lifeguards, without nearby marinas, and those that manage storm water. Factor analysis and a linear regression confirmed beach type as the predominant factor with secondary influences from grooming activities (including seaweed densities and beach renourishment) and beach access (including charging fees, employing lifeguards, and without nearby marinas). Our results were observable primarily because of the very large public FIB database available for analyses; similar approaches can be adopted at other beaches. The findings of this research have important policy implications because the selected beach management practices that were associated with low levels of FIB can be implemented in other parts of the US and around the world to improve recreational beach water quality.

Differential Impacts of Land-Based Sources of Pollution on the Microbiota of Southeast Florida Coral Reefs.

Coral reefs are dynamic ecosystems known for decades to be endangered due, in large part, to anthropogenic impacts from land-based sources of pollution (LBSP). In this study, we utilized an Illumina-based next-generation sequencing approach to characterize prokaryotic and fungal communities from samples collected off the southeast coast of Florida. Water samples from coastal inlet discharges, oceanic outfalls of municipal wastewater treatment plants, treated wastewater effluent before discharge, open ocean samples, and coral tissue samples (mucus and polyps) were characterized to determine the relationships between microbial communities in these matrices and those in reef water and coral tissues. Significant differences in microbial communities were noted among all sample types but varied between sampling areas. Contamination from outfalls was found to be the greatest potential source of LBSP influencing native microbial community structure among all reef samples, although pollution from inlets was also noted. Notably, reef water and coral tissue communities were found to be more greatly impacted by LBSP at southern reefs, which also experienced the most degradation during the course of the study. The results of this study provide new insights into how microbial communities from LBSP can impact coral reefs in southeast Florida and suggest that wastewater outfalls may have a greater influence on the microbial diversity and structure of these reef communities than do contaminants carried in runoff, although the influences of runoff and coastal inlet discharge on coral reefs are still substantial.IMPORTANCE Coral reefs are known to be endangered due to sewage discharge and to runoff of nutrients, pesticides, and other substances associated with anthropogenic activity. Here, we used next-generation sequencing to characterize the microbial communities of potential contaminant sources in order to determine how environmental discharges of microbiota and their genetic material may influence the microbiomes of coral reef communities and coastal receiving waters. Runoff delivered through inlet discharges impacted coral microbial communities, but impacts from oceanic outfalls carrying treated wastewater were greater. Geographic differences in the degree of impact suggest that coral microbiomes may be influenced by the microbiological quality of treated wastewater.

Use of quantitative real-time PCR for direct detection of serratia marcescens in marine and other aquatic environments.

Serratia marcescens is the etiological agent of acroporid serratiosis, a distinct form of white pox disease in the threatened coral Acropora palmata. The pathogen is commonly found in untreated human waste in the Florida Keys, which may contaminate both nearshore and offshore waters. Currently there is no direct method for detection of this bacterium in the aquatic or reef environment, and culture-based techniques may underestimate its abundance in marine waters. A quantitative real-time PCR assay was developed to detect S. marcescens directly from environmental samples, including marine water, coral mucus, sponge tissue, and wastewater. The assay targeted the luxS gene and was able to distinguish S. marcescens from other Serratia species with a reliable quantitative limit of detection of 10 cell equivalents (CE) per reaction. The method could routinely discern the presence of S. marcescens for as few as 3 CE per reaction, but it could not be reliably quantified at this level. The assay detected environmental S. marcescens in complex sewage influent samples at up to 761 CE ml(-1) and in septic system-impacted residential canals in the Florida Keys at up to 4.1 CE ml(-1). This detection assay provided rapid quantitative abilities and good sensitivity and specificity, which should offer an important tool for monitoring this ubiquitous pathogen that can potentially impact both human health and coral health.

Clonally related methicillin-resistant Staphylococcus aureus isolated from short-finned pilot whales (Globicephala macrorhynchus), human volunteers, and a bayfront cetacean rehabilitation facility.

In May of 2011, a live mass stranding of 26 short-finned pilot whales (Globicephala macrorhynchus) occurred in the lower Florida Keys. Five surviving whales were transferred from the original stranding site to a nearby marine mammal rehabilitation facility where they were constantly attended to by a team of volunteers. Bacteria cultured during the routine clinical care of the whales and necropsy of a deceased whale included methicillin-sensitive and methicillin-resistant Staphylococcus aureus (MSSA and MRSA). In order to investigate potential sources or reservoirs of MSSA and MRSA, samples were obtained from human volunteers, whales, seawater, and sand from multiple sites at the facility, nearby recreational beaches, and a canal. Samples were collected on 3 days. The second collection day was 2 weeks after the first, and the third collection day was 2 months after the last animal was removed from the facility. MRSA and MSSA were isolated on each day from the facility when animals and volunteers were present. MSSA was found at an adjacent beach on all three collection days. Isolates were characterized by utilizing a combination of quantitative real-time PCR to determine the presence of mecA and genes associated with virulence, staphylococcal protein A typing, staphylococcal cassette chromosome mec typing, multilocus sequence typing, and pulsed field gel electrophoresis (PFGE). Using these methods, clonally related MRSA were isolated from multiple environmental locations as well as from humans and animals. Non-identical but genetically similar MSSA and MRSA were also identified from distinct sources within this sample pool. PFGE indicated that the majority of MRSA isolates were clonally related to the prototype human strain USA300. These studies support the notion that S. aureus may be shed into an environment by humans or pilot whales and subsequently colonize or infect exposed new hosts.

Human-associated methicillin-resistant Staphylococcus aureus from a subtropical recreational marine beach.

Reports of Staphylococcus aureus including methicillin-resistant S. aureus (MRSA) detected in marine environments have occurred since the early 1990 s. This investigation sought to isolate and characterize S. aureus from marine waters and sand at a subtropical recreational beach, with and without bathers present, in order to investigate possible sources and to identify the risks to bathers of exposure to these organisms. During 40 days over 17 months, 1,001 water and 36 intertidal sand samples were collected by either bathers or investigators at a subtropical recreational beach. Methicillin-sensitive S. aureus (MSSA) and MRSA were isolated and identified using selective growth media and an organism-specific molecular marker. Antimicrobial susceptibility, staphylococcal cassette chromosome mec (SCCmec) type, pulsed-field gel electrophoresis (PFGE) pattern, multi-locus sequence type (MLST), and staphylococcal protein A (spa) type were characterized for all MRSA. S. aureus was isolated from 248 (37 %) bather nearby water samples at a concentration range of <2-780 colony forming units per ml, 102 (31 %) ambient water samples at a concentration range of <2-260 colony forming units per ml, and 9 (25 %) sand samples. Within the sand environment, S. aureus was isolated more often from above the intertidal zone than from intermittently wet or inundated sand. A total of 1334 MSSA were isolated from 37 sampling days and 22 MRSA were isolated from ten sampling days. Seventeen of the 22 MRSA were identified by PFGE as the community-associated MRSA USA300. MRSA isolates were all SCCmec type IVa, encompassed five spa types (t008, t064, t622, t688, and t723), two MLST types (ST8 and ST5), and 21 of 22 isolates carried the genes for Panton-Valentine leukocidin. There was a correlation (r = 0.45; p = 0.05) between the daily average number of bathers and S. aureus in the water; however, no association between exposure to S. aureus in these waters and reported illness was found. This report supports the concept that humans are a potential direct source for S. aureus in marine waters.

Integrating microbial source tracking with quantitative microbial risk assessment to evaluate site specific risk based thresholds at two South Florida beaches.

Quantitative microbial risk assessment (QMRA) can be used to evaluate health risks associated with recreational beach use. This study developed a site-specific risk assessment using a novel approach that combined quantitative PCR-based measurement of microbial source tracking (MST) genetic markers (human, dog, and gull fecal bacteria) with a QMRA analysis of potential pathogen risk. Water samples (n = 24) from two recreational beaches were collected and analyzed for MST markers as part of a broader Beach Exposure And Child Health Study that examined child behavior interactions with the beach environment. We report here the measurements of fecal bacteria MST markers in the environmental DNA extracts of those samples and a QMRA analysis of potential health risks utilizing the results from the MST measurements in the water samples. Human-specific Bacteroides was enumerated by the HF183 Taqman qPCR assay, gull-specific Catellicoccus was enumerated by the Gull2 qPCR assay, and dog-specific Bacteroides was enumerated by the DogBact qPCR assay. Derived reference pathogen doses, calculated from the MST marker concentrations detected in recreational waters, were used to estimate the risk of gastrointestinal illness for both children and adults. Dose-response equations were used to estimate the probability of the risk of infection (Pinf) per a swimming exposure event. Based on the QMRA simulations presented in this study, the GI risk from swimming or playing in water containing a mixture of human and non-human fecal sources appear to be primarily driven by the human fecal source. However, the estimated median GI health risk for both beaches never exceeded the U.S. EPA risk threshold of 32 illnesses per 1,000 recreation events. Our research suggests that utilizing QMRA together with MST can further extend our understanding of potential recreational bather risk by identifying the source contributing the greatest risk in a particular location, therefore informing beach management responses and decision-making.

Microbial communities in the water surface microlayer and associations with microbes in aerosols, beach sand, and bulk water.

The water surface microlayer (SML) serves as a boundary through which microbes can be exchanged. To evaluate exchanges of microbes, this study compared microbial communities within different reservoirs, with an emphasis on the water SML and aerosols. Additionally, the microbial communities during a sewage spill and perigean tides were evaluated and the results were compared to times without these events. Results show that during perigean tides and during the sewage spill, levels of culturable bacteria were highest and showed an increase via sequencing in potential pathogenic bacteria (Corynebacterium and Vibrio, which increased from 3.5%-1800% depending on sample type). In the aerosol samples, Corynebacterium (average of 2.0%), Vibrio (1.6%), and Staphylococcus (10%), were the most abundant genera. Aerosolization factors, which were used to examine the transfer of the microbes, were high for these three genera. Measurements of general marine bacteria (GMB) by culture showed a weak but significant correlation between culturable GMB in aerosol samples versus in water and in the SML. More research is needed to evaluate the exchange of pathogens between the SML and air, given the increase in potentially pathogenic microbes within the SML during rare events and evidence that suggests that microbes maintain viability during transfers across reservoirs.

Interlaboratory comparison of real-time PCR protocols for quantification of general fecal indicator bacteria.

The application of quantitative real-time PCR (qPCR) technologies for the rapid identification of fecal bacteria in environmental waters is being considered for use as a national water quality metric in the United States. The transition from research tool to a standardized protocol requires information on the reproducibility and sources of variation associated with qPCR methodology across laboratories. This study examines interlaboratory variability in the measurement of enterococci and Bacteroidales concentrations from standardized, spiked, and environmental sources of DNA using the Entero1a and GenBac3 qPCR methods, respectively. Comparisons are based on data generated from eight different research facilities. Special attention was placed on the influence of the DNA isolation step and effect of simplex and multiplex amplification approaches on interlaboratory variability. Results suggest that a crude lysate is sufficient for DNA isolation unless environmental samples contain substances that can inhibit qPCR amplification. No appreciable difference was observed between simplex and multiplex amplification approaches. Overall, interlaboratory variability levels remained low (<10% coefficient of variation) regardless of qPCR protocol.

Fecal indicator bacteria levels at a marine beach before, during, and after the COVID-19 shutdown period and associations with decomposing seaweed and human presence.

Studies are limited that evaluate seaweed as a source of bacteria to beach waters. The objective of the current study was to evaluate whether seaweed, along with humans and other animals, could be the cause of beach advisories due to elevated levels of enterococci. The monitoring period occurred a year prior to and through the COVID-19 beach shutdown period, which provided a unique opportunity to evaluate bacteria levels during prolonged periods without recreational activity. Samples of water, sediment, and seaweed were measured for enterococci by culture and qPCR, in addition to microbial source tracking by qPCR of fecal bacteria markers from humans, dogs, and birds. During periods of elevated enterococci levels in water, these analyses were supplemented by chemical source tracking of human-associated excretion markers (caffeine, sucralose, acetaminophen, ibuprofen, and naproxen). Results show that enterococci with elevated levels of human fecal markers persist in the seaweed and sediment and are the likely contributor to elevated levels of bacteria to the nearshore waters. During the shutdown period the elevated levels of enterococci in the sediment were isolated to the seaweed stranding areas. During periods when the beaches were open, enterococci were distributed more uniformly in sediment across the supratidal and intertidal zones. It is hypothesized from this study that human foot traffic may be responsible for the spread of enterococci throughout these areas. Overall, this study found high levels of enterococci in decomposing seaweed supporting the hypothesis that decomposing seaweed provides an additional substrate for enterococci to grow.

Shedding of Staphylococcus aureus and methicillin-resistant Staphylococcus aureus from adult and pediatric bathers in marine waters.

BACKGROUND: Staphylococcus aureus including methicillin resistant S. aureus, MRSA, are human colonizing bacteria that commonly cause opportunistic infections primarily involving the skin in otherwise healthy individuals. These infections have been linked to close contact and sharing of common facilities such as locker rooms, schools and prisons Waterborne exposure and transmission routes have not been traditionally associated with S. aureus infections. Coastal marine waters and beaches used for recreation are potential locations for the combination of high numbers of people with close contact and therefore could contribute to the exposure to and infection by these organisms. The primary aim of this study was to evaluate the amount and characteristics of the shedding of methicillin sensitive S. aureus, MSSA and MRSA by human bathers in marine waters. RESULTS: Nasal cultures were collected from bathers, and water samples were collected from two sets of pools designed to isolate and quantify MSSA and MRSA shed by adults and toddlers during exposure to marine water. A combination of selective growth media and biochemical and polymerase chain reaction analysis was used to identify and perform limited characterization of the S. aureus isolated from the water and the participants. Twelve of 15 MRSA isolates collected from the water had identical genetic characteristics as the organisms isolated from the participants exposed to that water while the remaining 3 MRSA were without matching nasal isolates from participants. The amount of S. aureus shed per person corresponded to 105 to 106 CFU per person per 15-minute bathing period, with 15 to 20% of this quantity testing positive for MRSA. CONCLUSIONS: This is the first report of a comparison of human colonizing organisms with bacteria from human exposed marine water attempting to confirm that participants shed their own colonizing MSSA and MRSA into their bathing milieu. These findings clearly demonstrate that adults and toddlers shed their colonizing organisms into marine waters and therefore can be sources of potentially pathogenic S. aureus and MRSA in recreational marine waters. Additional research is needed to evaluate recreational beaches and marine waters as potential exposure and transmission pathways for MRSA.

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.

Trends in regional enterococci levels at marine beaches and correlations with environmental, global oceanic changes, community populations, and wastewater infrastructure.

An increase in the number of advisories issued for recreational beaches across south Florida (due to the fecal indicator bacteria, enterococci) has been observed in recent years. To evaluate the possible reasons for this increase, we reviewed weekly monitoring data for 18 beaches in Miami-Dade County, Florida, for the years 2000-2019. Our objective was to evaluate this dataset for trends in enterococci levels and correlations with various factors that might have influenced enterococci levels at these beaches. For statistical analyses, we divided the 20-year period of record into 5-year increments (2000-2004, 2005-2009, 2010-2014, and 2015-2019). The Wilcoxon rank sum test was used to identify statistically significant differences between the geometric mean of different periods. When all 18 beaches were collectively considered, a significant increase (p = 0.03) in enterococci was observed during 2015-2019, compared to the prior 15-year period of record. To better understand the potential causes for this increase, correlations were evaluated with environmental parameters (rainfall, air temperature, and water temperature), global oceanic changes (sea level and Sargassum), community populations (county population estimates and beach visitation numbers), and wastewater infrastructure (sewage effluent flow rates to ocean outfalls and deep well injection). In relation to the enterococci geometric mean, the correlation with Sargassum was statistically significant at a 95% confidence interval (p = 0.035). Population (p = 0.078), air temperature (p = 0.092), and sea level (p = 0.098) were statistically significant at 90% confidence intervals. Rainfall, water temperature, beach visitation numbers, and sewage effluent flow rates via deep well injection had positive correlations but were not significant factors. Sewage effluent flow rates to ocean outfalls had a negative correlation.

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.

Microbial Source Tracking of Fecal Indicating Bacteria in Coral Reef Waters, Recreational Waters, and Groundwater of Saipan by Real-Time Quantitative PCR.

The Commonwealth of the Northern Mariana Islands (CNMI) recently identified the need to improve its capacity for detecting and tracking land-based sources of pollution (LBSP) in coastal waters, particularly microbial contaminants like fecal indicator bacteria (FIB). Reported here is a baseline study of a suite of host-specific FIB microbial source tracking (MST) markers in the coastal shoreline and reef waters around the island of Saipan. Three sampling campaigns were conducted in September 2017, March 2018, and August 2018. Samples were collected from the nearshore surface waters of Saipan, the reef waters of Saipan Lagoon, and groundwater from beaches along the Saipan Lagoon shoreline. Measurements of submarine groundwater discharge (SGD) into nearshore waters and isotopic source tracking of nitrogen inputs were conducted concurrently with MST. Environmental DNA was extracted from the samples and analyzed by quantitative polymerase chain reaction (qPCR) for MST gene markers of fecal Bacteroidales specifically associated with humans, dogs, cows, and pigs, and for an MST gene marker of Catellicoccus associated with seabirds. MST assessments were combined with local knowledge, assessments of sanitary infrastructure, and routine watershed surveys. This study identified hotspots of human FIB along the western Saipan Lagoon shoreline in both surface waters and groundwater, plus another hotspot of human FIB at a popular tourist bathing area known as the Grotto. FIB hotspots on the Lagoon shoreline coincided with areas of high SGD and nitrogen isotopic data indicating sewage-derived N inputs. It appears that faulty sanitary infrastructure may be contributing to inputs to Saipan Lagoon, while bather shedding is likely a primary input for the Grotto area. Moderate levels of dog fecal contamination were common and widespread across the island. High levels of seabird fecal contamination were more random, both spatially and temporally, and mostly concentrated along the less developed northeast region of Saipan. No significant levels of cow or pig fecal marker were detected in coastal water samples. This study provides demonstration and establishment of analytical capacity to resource management in CNMI for MST technology to aid in trouble-shooting water quality issues involving land-based sources of microbial contaminants to CNMI coastal waters.

Pepper mild mottle virus as an indicator of fecal pollution.

Accurate indicators of fecal pollution are needed in order to minimize public health risks associated with wastewater contamination in recreational waters. However, the bacterial indicators currently used for monitoring water quality do not correlate with the presence of pathogens. Here we demonstrate that the plant pathogen Pepper mild mottle virus (PMMoV) is widespread and abundant in wastewater from the United States, suggesting the utility of this virus as an indicator of human fecal pollution. Quantitative PCR was used to determine the abundance of PMMoV in raw sewage, treated wastewater, seawater exposed to wastewater, and fecal samples and/or intestinal homogenates from a wide variety of animals. PMMoV was present in all wastewater samples at concentrations greater than 1 million copies per milliliter of raw sewage. Despite the ubiquity of PMMoV in human feces, this virus was not detected in the majority of animal fecal samples tested, with the exception of chicken and seagull samples. PMMoV was detected in four out of six seawater samples collected near point sources of secondary treated wastewater off southeastern Florida, where it co-occurred with several other pathogens and indicators of fecal pollution. Since PMMoV was not found in nonpolluted seawater samples and could be detected in surface seawater for approximately 1 week after its initial introduction, the presence of PMMoV in the marine environment reflects a recent contamination event. Together, these data demonstrate that PMMoV is a promising new indicator of fecal pollution in coastal environments.

Viable cell sorting of dinoflagellates by multiparametric flow cytometry.

Electronic cell sorting for isolation and culture of dinoflagellates and other marine eukaryotic phytoplankton was compared to the traditional method of manually picking cells using a micropipette. Trauma to electronically sorted cells was not a limiting factor, as fragile dinoflagellates, such as Karenia brevis (Dinophyceae), survived electronic cell sorting to yield viable cells. The rate of successful isolation of large-scale (> 4 litres) cultures was higher for manual picking than for electronic cell sorting (2% vs 0.5%, respectively). However, manual picking of cells is more labor intensive and time consuming. Most manually isolated cells required repicking, as the cultures were determined not to be unialgal after a single round of isolation; whereas, no cultures obtained in this study from electronic single-cell sorting required resorting. A broad flow cytometric gating logic was employed to enhance species diversity. The percentages of unique genotypes produced by manual picking or electronic cell sorting were similar (57% vs 54%, respectively), and each approach produced a variety of dinoflagellate or raphidophyte genera. Alternatively, a highly restrictive gating logic was successfully used to target K. brevis from a natural bloom sample. Direct electronic single-cell sorting was more successful than utilizing a pre-enrichment sort followed by electronic single-cell sorting. The appropriate recovery medium may enhance the rate of successful isolations. Seventy percent of isolated cells were recovered in a new medium (RE) reported here, which was optimized for axenic dinoflagellate cultures. The greatest limiting factor to the throughput of electronic cell sorting is the need for manual postsort culture maintenance and assessment of the large number of isolated cells. However, when combined with newly developed automated methods for growth screening, electronic single-cell sorting has the potential to accelerate the discovery of new algal strains.

Oceanographic habitat and the coral microbiomes of urban-impacted reefs.

Coral reefs are in decline worldwide. In response to this habitat loss, there are efforts to grow, outplant, and restore corals in many regions. The physical oceanographic habitat of corals-such as sea temperature, waves, ocean currents, and available light-is spatially heterogeneous. We therefore hypothesize that outplant location may affect microbiomes, and ultimately, coral health and restoration success. We evaluated the influence of the physical oceanographic habitat on microbes in wild Porites astreoides and Siderastrea siderea. Tissue samples were collected at four Florida reefs in March, June, and September of 2015. We estimated oceanographic conditions from moored instruments, diver observations, remote sensing data, and numerical models. We analyzed microbiomes using amplicon 16S rRNA high-throughput sequencing data. We found microbial alpha-diversity negatively correlated with in situ sea temperature (which represented both the annual cycle and upwelling), as well as modeled alongshore currents, in situ sea-level, and modeled tide. Microbial beta-diversity correlated positively with significant wave height and alongshore currents from models, remotely-sensed relative turbidity, and in situ temperature. We found that archaea from the order Marine Group II decrease with increases in significant wave height, suggesting that this taxon may be influenced by waves. Also, during times of high wave activity, the relative abundance of bacteria from the order Flavobacteriales increases, which may be due to resuspension and cross-shelf transport of sediments. We also found that bacteria from the order SAR86 increase in relative abundance with increased temperature, which suggests that this taxon may play a role in the coral microbiome during periods of higher temperature. Overall, we find that physical oceanographic variability correlates with the structure of these coral microbiomes in ways that could be significant to coral health.

The coastal environment and human health: microbial indicators, pathogens, sentinels and reservoirs.

Innovative research relating oceans and human health is advancing our understanding of disease-causing organisms in coastal ecosystems. Novel techniques are elucidating the loading, transport and fate of pathogens in coastal ecosystems, and identifying sources of contamination. This research is facilitating improved risk assessments for seafood consumers and those who use the oceans for recreation. A number of challenges still remain and define future directions of research and public policy. Sample processing and molecular detection techniques need to be advanced to allow rapid and specific identification of microbes of public health concern from complex environmental samples. Water quality standards need to be updated to more accurately reflect health risks and to provide managers with improved tools for decision-making. Greater discrimination of virulent versus harmless microbes is needed to identify environmental reservoirs of pathogens and factors leading to human infections. Investigations must include examination of microbial community dynamics that may be important from a human health perspective. Further research is needed to evaluate the ecology of non-enteric water-transmitted diseases. Sentinels should also be established and monitored, providing early warning of dangers to ecosystem health. Taken together, this effort will provide more reliable information about public health risks associated with beaches and seafood consumption, and how human activities can affect their exposure to disease-causing organisms from the oceans.

Multi-laboratory survey of qPCR enterococci analysis method performance in U.S. coastal and inland surface waters.

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.

Localization of polyketide synthase encoding genes to the toxic dinoflagellate Karenia brevis.

Karenia brevis is a toxic marine dinoflagellate endemic to the Gulf of Mexico. Blooms of this harmful alga cause fish kills, marine mammal mortalities and neurotoxic shellfish poisonings. These harmful effects are attributed to a suite of polyketide secondary metabolites known as the brevetoxins. The carbon framework of all polyketides is assembled by a polyketide synthase (PKS). Previously, PKS encoding genes were amplified from K. brevis culture and their similarity to a PKS gene from the closely related protist, Cryptosporidium parvum, suggested that these genes originate from the dinoflagellate. However, K. brevis has not been grown axenically. The associated bacteria might be the source of the toxins or the PKS genes. Herein we report the localization of PKS encoding genes by a combination of flow cytometry/PCR and fluorescence in situ hybridization (FISH). Two genes localized exclusively to K. brevis cells while a third localized to both K. brevis and associated bacteria. While these genes have not yet been linked to toxin production, the work describes the first definitive evidence of resident PKS genes in any dinoflagellate.