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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., (© The Author(s) 2023. Published by Oxford University Press on behalf of FEMS.)

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.

The contribution of fecal pollution from dogs in urbanized areas can be significant and is an often underestimated problem. Microbial source tracking methods (MST) utilizing quantitative PCR of dog-associated gene sequences encoding 16S rRNA of Bacteroidales are a useful tool to estimate these contributions. However, data about the performance of available assays are scarce. The results of a multi-laboratory study testing two assays for the determination of dog-associated Bacteroidales (DogBact and BacCan-UCD) on 64 single and mixed fecal source samples created from pooled fecal samples collected in California are presented here. Standardization of qPCR data treatment lowered inter-laboratory variability of sensitivity and specificity results. Both assays exhibited 100% sensitivity. Normalization methods are presented that eliminated random and confirmed non-target responses. The combination of standardized qPCR data treatment, use of normalization via a non-target specific Bacteroidales assay (GenBac3), and application of threshold criteria improved the calculated specificity significantly for both assays. Such measures would reasonably improve MST data interpretation not only for canine-associated assays, but for all qPCR assays used in identifying and monitoring fecal pollution in the environment., (Copyright © 2013. Published by Elsevier Ltd.)

Here we report results from a multi-laboratory (n = 11) evaluation of four different PCR methods targeting the 16S rRNA gene of Catellicoccus marimammalium originally developed to detect gull fecal contamination in coastal environments. The methods included a conventional end-point PCR method, a SYBR(®) Green qPCR method, and two TaqMan(®) qPCR methods. Different techniques for data normalization and analysis were tested. Data analysis methods had a pronounced impact on assay sensitivity and specificity calculations. Across-laboratory standardization of metrics including the lower limit of quantification (LLOQ), target detected but not quantifiable (DNQ), and target not detected (ND) significantly improved results compared to results submitted by individual laboratories prior to definition standardization. The unit of measure used for data normalization also had a pronounced effect on measured assay performance. Data normalization to DNA mass improved quantitative method performance as compared to enterococcus normalization. The MST methods tested here were originally designed for gulls but were found in this study to also detect feces from other birds, particularly feces composited from pigeons. Sequencing efforts showed that some pigeon feces from California contained sequences similar to C. marimammalium found in gull feces. These data suggest that the prevalence, geographic scope, and ecology of C. marimammalium in host birds other than gulls require further investigation. This study represents an important first step in the multi-laboratory assessment of these methods and highlights the need to broaden and standardize additional evaluations, including environmentally relevant target concentrations in ambient waters from diverse geographic regions., (Published by Elsevier Ltd.)

Many PCR-based methods for microbial source tracking (MST) have been developed and validated within individual research laboratories. Inter-laboratory validation of these methods, however, has been minimal, and the effects of protocol standardization regimes have not been thoroughly evaluated. Knowledge of factors influencing PCR in different laboratories is vital to future technology transfer for use of MST methods as a tool for water quality management. In this study, a blinded set of 64 filters (containing 32 duplicate samples generated from 12 composite fecal sources) were analyzed by three to five core laboratories with a suite of PCR-based methods utilizing standardized reagents and protocols. Repeatability (intra-laboratory variability) and reproducibility (inter-laboratory variability) of observed results were assessed. When standardized methodologies were used, intra- and inter-laboratory %CVs were generally low (median %CV 0.1-3.3% and 1.9-7.1%, respectively) and comparable to those observed in similar inter-laboratory validation studies performed on other methods of quantifying fecal indicator bacteria (FIB) in environmental samples. ANOVA of %CV values found three human-associated methods (BsteriF1, BacHum, and HF183Taqman) to be similarly reproducible (p > 0.05) and significantly more reproducible (p < 0.05) than HumM2. This was attributed to the increased variability associated with low target concentrations detected by HumM2 (approximately 1-2 log10copies/filter lower) compared to other human-associated methods. Cow-associated methods (BacCow and CowM2) were similarly reproducible (p > 0.05). When using standardized protocols, variance component analysis indicated sample type (fecal source and concentration) to be the major contributor to total variability with that from replicate filters and inter-laboratory analysis to be within the same order of magnitude but larger than inherent intra-laboratory variability. However, when reagents and protocols were not standardized, inter-laboratory %CV generally increased with a corresponding decline in reproducibility. Overall, these findings verify the repeatability and reproducibility of these MST methods and highlight the need for standardization of protocols and consumables prior to implementation of larger scale MST studies involving multiple laboratories., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

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.

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.

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., (Published by Elsevier Ltd.)

Traditionally, the use of enterococci has been recommended as the fecal indicator bacteria of choice for testing marine recreational water quality, and prior studies have shown that bathers shed large numbers of enterococci into the water. The current study expands upon prior research by evaluating shedding from both toddlers and adults, and by the expansion of measurements to include enterococci shedding via three different methods (membrane filter (MF), chromogenic substrate (CS), and quantitative polymerase chain reaction (qPCR)) and shedding of alternative fecal indicator bacteria (Bacteroidales human markers UCD and HF8 via qPCR). Two sets of experiments were conducted. The first experiment consisted of two groups of 10 adults who bathed together in a large pool. The second study consisted of 14 toddlers who bathed individually in a small pool which allowed for sand recovery. Sand recovery was used to estimate the amount of sand transported on the bodies of toddlers and to estimate the number of fecal indicator bacteria released from this sand. The numbers of estimated enterococci shed per adult ranged from 1.8 x 10(4) to 2.8 x 10(6) CFU, from 1.9 x 10(3) to 4.5 x 10(6) MPN, and from 3.8 x 10(5) to 5.5 x 10(6) GEU based on the MF, CS, and qPCR methods, respectively. The estimated numbers of Bacteroidales human markers ranged from 1.8 x 10(4) to 1.3 x 10(6) for UCD, and ranged from the below detection limit to 1.6 x 10(5) for HF8. The estimated amount of sand transported per toddler (n=14) into the water column after sand exposure was 8+/-6g on average. When normalizing the numbers of enterococci shed from toddlers via sand by the 3.9 body surface area ratio, the differences between toddlers and adults were insignificant. Contributions of sands to the total enterococci (MF) shed per toddler was 3.7+/-4.4% on average. Although shedding via beach sand may contribute a small fraction of the microbial load during initial bathing, it may have a significant role if bathers go to water repetitively after sand exposure.

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.