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 ( = 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.

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.

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.

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.

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.

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.

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.

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.

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.

Dynamics of marine bacterial community diversity of the coastal waters of the reefs, inlets, and wastewater outfalls of southeast Florida.

Coastal waters adjacent to populated southeast Florida possess different habitats (reefs, oceanic inlets, sewage outfalls) that may affect the composition of their inherent microbiomes. To determine variation according to site, season, and depth, over the course of 1 year, we characterized the bacterioplankton communities within 38 nearshore seawater samples derived from the Florida Area Coastal Environment (FACE) water quality survey. Six distinct coastal locales were profiled - the Port Everglades and Hillsboro Inlets, Hollywood and Broward wastewater outfalls, and associated reef sites using culture-independent, high-throughput pyrosequencing of the 16S rRNA V4 region.

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.

Performance evaluation of canine-associated Bacteroidales assays in a multi-laboratory comparison study.

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.

Multi-laboratory evaluations of the performance of Catellicoccus marimammalium PCR assays developed to target gull fecal sources.

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.

Evaluation of the repeatability and reproducibility of a suite of qPCR-based microbial source tracking methods.

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.

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.

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).

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.

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.

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.

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.

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 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.

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.

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.