Assessment of Microbial Indicators for Monitoring Recreational Water Quality in Marine Sub/Tropical Environments (Recreational Microbes)

Principal Investigators: Helena Solo-Gabriele Ph.D. and John Wang Ph.D.
Collaborating Principal Investigators: Kelly Goodwin PhD (NOAA-AOML), Jay M. Fleisher, PhD (Nova Southeastern University)
Co-Investigators: Lorraine Backer, Samir Elmir, Jack Fell, Lora Fleming, Peter Minnett, Carol Palmer, Eugene Rankey
Participating Facilities Cores: Remote Sensing and Genomics

Abstract

Microbial water quality indicators are found in high concentrations in sewage, and are thus used to determine whether or not a water body is safe for recreational purposes. Recently, concerns have been raised about the appropriate use of microbial indicators to regulate recreational uses of water bodies. This is particularly true for water bodies located in tropical and subtropical environments, due to the potential for microbial regrowth in these latitudes, and the fact that studies used to set national guidelines were not conducted in tropical and subtropical environments. The primary goal of this study is to evaluate the relationship between human health and the physical and microbial characteristics of a coastal body of water and its shoreline. This goal is addressed by measuring human health effects and microbial water quality at a recreational beach site within a subtropical climate, and by developing a predictive, coupled model of hydrodynamic characteristics and microbial fate that can be used to distinguish impacts from sewage sources versus re-growth of microbes. Each of these sources could possibly result in different health effects as observed at the beach site. Substantial pilot data have been collected for the subtropical study site chosen for this research. These pilot data, as well as the literature, indicate that exposure to contaminated recreational marine waters may result in human health effects, and that the shoreline sediments are one likely source of indicator microbes. Human health is evaluated in the project through an epidemiologic study that randomly assigns exposure to water or beach with coordinated individual environmental sampling and repeated follow-up of reported human health effects. Water quality is evaluated through two sets of environmental measurements. The first phase of the environmental assessment focuses on identifying the distribution and sources of enterococci (the current, federally-recommended indicator microbe) within the study site. Enterococci is monitored through an intensive effort aimed at identifying sources, in particular shoreline sources, and correlations with suspended sediment concentrations. The data gathered from this intensive sampling effort will be used to develop source functions for an enterococci fate model to be coupled with a hydrodynamic oceanographic model developed for the area. The coupled enterococci model will be used in a predictive fashion to determine when and where within the study beach the epidemiologic study should take place to evaluate baseline levels of the indicator due to re­growth. These baseline levels will be compared with measured concentrations in the water column to identify time periods that may be impacted by sewage. The second phase of environmental measurements will focus on the analysis of multiple microbes coincident with times that participants participate in the epidemiologic study. Microbe measurements will include traditional (enterococci, Escherchia coli , fecal coliform) and non-traditional ( Clostridium perfringens , coliphage) microbial indicators, as well as direct measurement of microbial pathogens ( Staphylococcus aureus , enterovirus, Norwalk virus, hepatitis A, Cryptosporidium parvum , and Giardia lamblia ). Analysis of the viral and protozoan pathogens will include traditional PCR and Rresults from microbe analysis will be cross­checked against new Luminex technology. A primary goal of this research is to develop a rapid (same day), accurate high throughput and sensitive molecular test for the identification of pathogenic microorganisms in marine and freshwater environments. The Remote Sensing Core and the Genomics Core will participate in this interdisciplinary collaborative research study.

The results from the coupled hydrodynamic and microbial fate model will be compared with the pathogen and human health data for the purposes of predicting beach closures due to health hazards. Ultimately, the human, environmental and oceanographic data will be used to develop a predictive model with broad applicability to beaches in the tropics and subtropics with appropriate modifications for local conditions. The final result will be the improvement of recreational water quality monitoring in tropical/subtropical marine environments, and an increase in public confidence in the results from microbial water quality monitoring and modeling.

Specific Aims and Hypotheses.

The primary goal of this study is to evaluate the relationship between human health, and the physical and microbial characteristics of a coastal water body and its shoreline. This relationship will be evaluated by comparing reported human illnesses with microbial water quality within a subtropical environment and by evaluating whether re-growth of the currently recommended indicator microbe (enterococci) is responsible for a significant proportion of the microbe burden within a beach and its associated water body. The relationship between environmental and sewage sources of contamination will be evaluated through a coupled hydrodynamic/water quality model that includes a fate component to simulate microbe re-growth and die-off. The secondary goals of this project include testing a new innovative technology referred to herein as Luminex for rapid, less expensive detection of indicator microbes and pathogens, and evaluating the inter-relationship between enterococci re-growth and beach sand. Such goals will be addressed through interdisciplinary research that includes physical oceanographers, environmental scientists, and biomedical experts.

This research project is innovative in that it evaluates the relationships between marine processes (water circulation and contaminant transport) and public health (reported illness at a beach site) in a subtropical area. It is an interdisciplinary study involving public health professionals (from the University of Miami Medical School, University of Florida College of Veterinary Medicine, Miami-Dade Department of Health, and Centers for Disease Control and Prevention [CDC]) and researchers (from the University of Miami Marine School and College of Engineering) that focus on environmental monitoring and ocean process modeling. The epidemiologic study will address a number of shortcomings characteristic of previous studies, including assessing individual exposure to seawater or beach sand, creating a coupled hydrodynamic/microbial water quality model to evaluate sources of microorganisms to the beach site, and assessing the applicability of current microbial indicators to predict human health risks specifically in subtropical recreational waters. The environmental monitoring component of this study will include analysis of both indicator microbes and pathogens, which due to its expense, are not commonly evaluated. It is also rare for studies to evaluate both point and non-point sources of microbes. This study will also evaluate the suitability of a new innovative technology, Luminex, for evaluating concentrations of indicator microbes and pathogens in water. If this technology is found to perform well, it can revolutionize the way in which water quality is monitored at beach sites due to much more rapid results and lower costs than traditional analytical methods.

These goals will be attained through the following six research modules.

Coupled Hydrodynamic/Microbial Water Quality Model (Wang)

Intensive Environmental Monitoring of Enterococci (Solo-Gabriele/Elmir)

Sediment Study (Rankey/Solo-Gabriele/Elmir)

Epidemiologic Study (Backer/Fleming/Elmir)

Microbe Monitoring Using Traditional Methods (Palmer/Solo-Gabriele/Elmir)

Monitoring Using innovative Luminex Technology (Fell/Palmer)

Publications & reports

Solo-Gabriele, H.M., Durbin, M.E., Abdelzaher, A.M., Heybeck, N.F., Elmir, S., Goodwin, K.D., and Sinigalliano, C., 2005.  Factors that Influence Fecal Indicator Microbe Levels.  Proceedings of the Annual Meeting of the Florida Branch of the American Society for Microbiology.

Abstract

Fecal indicator microbes have been traditionally used for regulatory purposes to assess the quality of recreational beach waters. The detection of elevated levels of fecal indicator microbes are usually followed by beach closures or advisories due to presumed health risks. However, recreational water quality standards have been developed from studies at sites characterized by point sources of sewage contamination, a source known to contain human pathogens and an abundance of fecal indicator microbes. Increasing evidence suggests that sewage is not the only source of fecal indicator microbes and that this group of microbes is capable of regrowth within the environment, specifically within soils. Many researchers have indicated that local climate plays a role in the regrowth phenomena. Regrowth is most prevalent in freshwater systems that are characterized by limited flushing; however, regrowth has been observed in marine systems. The predominant characteristics of the sediments that appear to facilitate regrowth are sediments characterized by elevated organic content and by relatively shallow embankments that are periodically wetted and dried such as during storm events and varying tidal cycles. Shallow embankments provide for a large change in surface area in contact with the water relative to a small change in tidal height. Regrowth thus represents an additional non-point source of fecal indicator microbes to the environment in addition to the conventional sewage and animal sources. Of interest is to determine whether elevated levels of fecal indicator microbes from regrowth are correlated with human health. During this presentation, data will be presented to support the observations above. Furthermore, future plans will be described for a study focused on evaluating human health effects from a beach site characterized by non-point sources of fecal indicator microbes. (PDF)

M. E. Durbin, A. M. Zaher, N. F. Heybeck, H. M. Solo-Gabriele, S. Elmir, K. D. Goodwin, C. Sinigalliano, 2005. The Inter-Tidal Zone is the Source of Enterococci to a Subtropical Recreational Beach.  Proceedings of the Annual National Conference of the American Society for Microbiology.

 

Proceedings of the 2004 National Beaches Conference http://www.epa.gov/OST/beaches/meetings/2004/index.htm

 J. Fleisher, PhD-Nova Southeastern University- Risk Perception Bias and Self Reported Symptoms, 2004 National Beaches Conference pp. 301 & 302. (PDF ABSTRACT)

K. Goodwin, PhD-NOAA/AOML- Immobilized DNA Probes to Rapidly Detect Toxic Dinoflagellates and Sewage- Indicating Bacteria, 2004 National Beaches Conference pp. 420 (PDF ABSTRACT)

K. Goodwin, PhD-NOAA/AOML- Hybridization Based Detection of Fecal Bacteria, 2004 National Beaches Conference pp. 420 & 421 (PDF ABSTRACT)

K. Goodwin, PhD-NOAA/AOML- Assay and Remote Sensor Development for Molecular Biological Water Quality Monitoring, 2004 National Beaches Conference pp. 268-272 (PDF ABSTRACT)

 

Fleming LE, Solo Gabriele H, Elmir S, Shibata T, Squicciarini D, Quirino W, Arguello M, Van De Bogart G. A Pilot Study of Microbial Contamination of Subtropical Recreational Waters. Fl J Env Health March 2004;29-33. (PDF)

Abstract
Microbial water quality indicators are used to determine whether a water body is safe for recreational purposes. There have been concerns raised about the appropriate use of microbial indicators to regulate recreational uses of water bodies, in particular those located in tropical and sub-tropical environments.

This prospective cohort pilot study evaluated the relationship between microbial water quality indicators and public health within two public beaches without known sewage discharge, but with historically high microbial levels for one beach, in subtropical Miami-Dade County (Florida). Monitoring was conducted in three phases: daily water monitoring, beach sand sampling, and spatially intense water sampling. An epidemiological questionnaire from a Los Angeles recreational beach-goer study was used to assess the self-reported swimming-related symptoms and exposures. There was no significant association between the number nor the type of reported symptoms and the different sampling months or beach sites, although persons who returned repeatedly to the beach were more likely to report symptoms. The number of indicator organisms correlated negatively with the frequency of symptoms reported by recreational beach goers. Results of the daily monitoring indicated that different indicators provided conflicting results concerning beach water quality.

Larger epidemiologic studies with individual exposure monitoring are recommended to further evaluate these potentially important associations in subtropical recreational waters.

Shibata T, Solo Gabriele HM, Fleming LE, Elmir S. Monitoring Marine Recreational Water Quality Using Multiple Microbial Indicators in an Urban Tropical Environment. Water Research 2004;38:3119-3131. (PDF)

 

ASLO Conference Abstract

DETECTION OF FECAL INDICATORS IN RECREATIONAL WATERS USING THE LUMINEX 100 SYSTEM
Environmental research and monitoring require fast microbial detection. High throughput identifications of hundreds of species can be accomplished with a suspension array technique: Luminex xMAP. That technology measures multiple analytes simultaneously in a single reaction vessel with probe bound, color coded, microspheres that are laser identified with microfluidics. Target DNA is amplified and labeled with biotin. Upon hybridization, microspheres bearing target amplicons are classified by their spectral addresses using a laser. Detection of the amplicon is based on streptavidin coupled phycoerythrin fluorescence. Fecal indicators are amplified with 5 primer pairs in two multiplex PCR reactions. We designed probes targeting the bacterial species and groups: Escherichia coli, Bacteroides distasonis, Enterococci faecalis, Enterococci faecium, the Bacteroides fragilis group and the total coliform group. Probes are hybridized in a multiplex format to the amplicons. The probe array is sensitive and specific to the targets in an assay of a mixed known cultured bacterial DNA. Using this probe array, fecal indicators where detected in environmental seawater samples. Traditional culturing techniques are used to verify the presence of identified bacteria in the environmental samples. This system enables rapid, reliable detection of microbial contaminants in environmental samples.

Baums, I. B., Kiesling T., Goodwin K.D., and Fell J.W. 2005.  Detection of fecal indicators in recreational waters using the Luminex 100 system. American Society for Limnology and Oceanography (ASLO) 2005 Aquatic Sciences Meeting 20-25 Feb. Salt Lake City, Utah (Abstract)

Presentations

“Freshwater and Coastal Bathing Areas and Indicator Microbes”.  January 2005.  Promoting Public Health Through Safe Water, sponsored by the Florida Department of Health and the Florida Department of Environmental Protection.  Orlando, FL.  Presented by Helena Solo-Gabriele.

“Recreational Microbes.”  January 2005.  First Annual Oceans & Human Health Center Science Symposium, Rosenstiel School of Marine & Atmospheric Sciences (RSMAS).  Fairchild Botanical Gardens, Miami, FL. Presented by Helena Solo-Gabriele and John Wang.

“Development of Molecular Biological Tools for Monitoring Coastal Water Quality Monitoring.”  March 2005.  Sea Tech, Dania, FL.  Presented by Kelly Goodwin.

“Factors that Influence Fecal Indicator Microbe Levels.”  April 2005.  Annual Meeting of the Florida Branch of the American Society for Microbiology.  Ft. Lauderdale, FL.  Presented by Helena Solo-Gabriele

“The Source of Enterococci to a Subtropical Recreational Beach is the Inter-Tidal Zone.”   April 2005.  Annual Meeting of the Florida Branch of the American Society for Microbiology.  Ft. Lauderdale, FL.  Presented by Mary Durbin

“The Inter-Tidal Zone is the Source of Enterococci to a Subtropical Recreational Beach.”  May 2005.  105th General Meeting of the American Society for Microbiology, Atlanta, GA.  Poster presented by Mary Durbin.

“Impact of Environmental Sources of Fecal Indicators on Water Quality in Sub-Tropical Climates of Florida.”  November 2005.  Sustainable Beaches Conference, Clean Beaches Council, St. Petersburg, FL.  Presentation by Helena Solo-Gabriele.  Abstract authored by:  H. M. Solo-Gabriele, M. E. Durbin, A. M. Abdelzaher, N. F. Heybeck, J.D. Wang, S. Elmir, K. D. Goodwin, C. Sinigalliano

Abstract
Fecal indicator microbes have been traditionally used for regulatory purposes to assess the quality of recreational beach waters.  The detection of elevated levels of fecal indicator microbes are usually followed by beach closures or advisories due to presumed health risks.  However, recreational water quality standards have been developed from studies at sites characterized by point sources of sewage contamination, a source known to contain human pathogens and an abundance of fecal indicator microbes.  Increasing evidence suggests that sewage is not the only source of fecal indicator microbes and that this group of microbes is capable of regrowth within the environment, specifically within soils.  Many researchers have indicated that local climate plays a role in the regrowth phenomena.  Regrowth is most prevalent in freshwater systems that are characterized by limited flushing; however, regrowth has been observed in marine systems.  The predominant characteristics of the sediments that appear to facilitate regrowth are sediments characterized by elevated organic content and by relatively shallow embankments that are periodically wetted and dried such as during storm events and varying tidal cycles.  Shallow embankments provide for a large change in surface area in contact with the water relative to a small change in tidal height.  Regrowth thus represents an additional non-point source of fecal indicator microbes to the environment in addition to the conventional sewage and animal sources.  Of interest is to determine whether elevated levels of fecal indicator microbes from regrowth are correlated with human health.  During this presentation, data will be presented to support the observations above.  Furthermore, future plans will be described for a study focused on evaluating human health effects from a beach site characterized by non-point sources of fecal indicator microbes. 

 

“Beach Microbial Indicators and Pathogens in a Subtropical Non-Point Source Environment.”  November 2005.  Sustainable Beaches Conference, Clean Beaches Council, St. Petersburg, FL.  Presentation prepared by J. Wang and presented by H. Solo-Gabriele.  Abstract authored by: J D Wang, H Solo-Gabriele, M Durbin, A Zaher, L Fleming, I Baums, S Elmir, J Fleisher, K Goodwin

Abstract
Waters used for swimming and other recreational purposes where primary contact takes place must be of suitable quality to insure that the user is reasonably free from risk of contracting disease resulting from such contact.  The timely closure of beaches when it is likely that bathers would be exposed to a health hazard is based on regular monitoring of beach water for indicator organisms.  Several studies have shown that the presently used indicator organisms: enterococci and Fecal Coliforms may often lead to unnecessary closures or may not provide the early warning required. Under worst case scenarios serious economic consequences or unexpected health hazards could result.  The presence of pathogenic organisms in recreational waters is thought largely to result from the introduction of human and/or animal sewage into the waters.  Assessment of the risk of a person becoming ill when swimming in waters containing sewage is an extremely complex task that depends on the survival rates of various pathogens in the receiving waters and the route of exposure.  For convenience, an ”indicator organism,” that is always present in sewage, is often used to signal the presence of sewage contamination and thus the possibility of pathogens being present.  Limits of indicator organisms have been established based on studies that related the number of indicator organisms present at a particular location to risk of disease in people bathing at that location.  Recent research has, however, questioned the use of indicator organisms as a health indicator where no known point source of sewage pollution can be identified, particularly in sub/tropical marine recreational waters. This research has questioned the appropriateness of the use of indicator organisms as a measure of health risk. Developing a better warning system, a management problem for sustainable beaches, could have substantial human health and economic benefits.  One of the relatively heavily used public beaches close to the City of Miami has no point sources of sewage close enough to impact it on a regular basis.  However, water samples collected by the Health Department frequently contain relatively high numbers of indicator bacteria and at times exceed EPA standards.  The Oceans and Human Health Center of the University of Miami is pursuing research at this beach to clarify the usefulness of accepted indicator organisms as a warning tool in view of evidence that these organisms may survive and multiply in the sands of subtropical beaches.  In one component of this research project supported by NIEHS and NSF we are developing molecular probes to rapidly and economically determine the presence of indicators and pathogens, and we are adapting the Luminex XMapTM system to provide high-throughput, multi-plexed analysis. Another component of our research seeks to understand the environmental factors that support the regrowth of indicator organisms in the beach sand and cause the ultimate transport of organisms into the water column. Yet another component aims at determining the source of the indicator organisms using source tracking techniques as well as video monitoring of beach usage to establish loads from humans and animals, such as dogs and birds.  Finally an epidemiological study is being planned to determine the risk to bathers as a function of indicator organism concentrations and levels of proven pathogens in the water. Integrating this information with a suite of environmental data on temperature, wind, rain, radiation, tides, water currents and turbulence, etc. into a predictive model will not only improve our understanding of possible health risk of non-point sources of pollution but will enable more reliable assessment of the day-to-day health risk to bathers.

“The Impact of Bathing load and Beach Sand Transported by Bathers on the Microbial Water Quality of Recreational Waters.” November 2005.  Sustainable Beaches Conference, Clean Beaches Council, St. Petersburg, FL.  Presentation prepared by S. Elmir. 

Abstract
Limited data exists concerning the concentrations of enterococci and Staph. aureus contributed to the water column in recreational water bodies by bathers, a non-point source. USEPA recommends the use of enterococci to measure the microbial water quality in marine waters. Staphylococcus aureus is a pathogen found on people’s skin, nose, and mouth and can be transmitted through open wounds and skin abrasions. The purpose of this study is to review the design, implementation, and results of two field experiments to estimate 1- the concentrations of enterococci and Staph. aureus shed by bathers and 2- the amount of beach sand and the corresponding concentration of enterococci that can be transported by bathers into the water column. The first and second experiments are called the “Big” and “Small” pool experiments respectively.  The study site is “Dog” Beach located in Miami Dade County, Florida, a subtropical environment and located within the southern portion of Biscayne Bay. There are no fecal point sources present at Dog beach. The big pool experiment involved 10 volunteers who immersed their bodies in the inflatable pool for 4, 15 minute cycles with volunteers exposed to beach sand in cycles 3 and 4.  Volunteers immersed their heads 3 times during each cycle.  The pool was sanitized and filled with off-shore ocean water after each cycle. The off-shore ocean water was characterized by microbe levels below the detection limit.  For the small pool experiment 5 volunteers were exposed to beach sand for 30 minutes before they individually entered a small tub. Each individual was rinsed with clean off-shore ocean water while in the tub. After each rinse, sand and rinsed water were collected and analyzed for enterococci.  Results showed that: 1-bathers shed considerable concentrations of enterococci and Staph. aureus, 2- concentrations of Staph. aureus shed were significantly higher than enterococci, 3- significant decrease in shedding of both Staph. aureus and enterococci between cycles in all four cycles, and 4- significant amount of sand can be transported by bathers into the water column. In conclusion, bathing load as a non-point source contributes significant concentrations of enterococci and Staph. aureus to the water column, thus bathing load should be considered as one of the main non-point sources when designing recreational water quality models, and future studies are needed to validate and refine such models.

“Adaptation of the Luminex 100 System to Use in Recreational Waters.” November 2005.  Sustainable Beaches Conference, Clean Beaches Council, St. Petersburg, FL.  Presentation prepared by K. Goodwin.  Abstract authored by: I. B. Baums, T. Kiesling, K.D. Goodwin, J.W. Fell. 

Abstract
The Luminex 100 System is a suspension array technique that allows rapid, high-throughput, multiplexed detection of targets, which holds promise for detection of microbial contaminants in recreational waters.  We have designed a set of primers and probes for the Luminex system to allow simultaneous detection of a variety of fecal-indicating bacteria. The probe array is sensitive and specific to targets present in a mixture of known bacterial DNA and targets have been detected in environmental samples.  Luminex results are compared to culture techniques and DNA sequencing, with particular emphasis on Bacteroides.

“Recreational Microbes and Environmental Monitoring Results.”  January 2006.  Second Annual Research Symposium of the University of Miami Oceans and Human Health Center, Key Biscayne, FL.  Presented by Helena Solo-Gabriele and John Wang.

 

"Efforts to Process Coastal Water Samples for Downstream Molecular Analysis" K.D. Goodwin, C. Garcia, J.A. Bonilla, T.D. Bonilla, D. Wanless, A. Abdelzaher, M.J LaGier, H. Solo-Gabriele. (Poster)

ABSTRACT
Molecular techniques offer a powerful tool for detecting microbial contaminants. However, sample concentration is necessary to achieve detection of fecal indicators and human pathogens in coastal water samples, despite the sensitivity offered by molecular methods. Bacterial targets of interest are rare in comparison to indigenous populations; therefore, methods of concentration based on size exclusion produce samples containing large amounts of nontarget organisms. Concentrationof PCR inhibitors results in diminishing returns, resulting in a concentration conundrum – increasing the level of concentration often reduces the likelihood of achieving detection. Recovery efficiencies tend to be low for concentration protocols and the nucleic acid extraction and purification steps that typically follow. Low recoveries decrease overall detection sensitivity and increase chances that organisms of public health concern could go undetected. Appropriate extraction controls are needed in order to correct for losses during sample processing. The desire to detect multiple types of organisms (e.g., protists, bacteria, and viruses) further complicates sample processing. Furthermore, concentration creates a process bottle-neck, interfering with attempts to achieve rapid detection of microbial contaminants. Strategies attempted to process coastal water samples include membrane filtration, tangential flow filtration (TFF), virus sorption, inclusion of extraction controls, and attempts to streamline sample processing by eliminating the DNA extraction step via filter PCR or PCR of crude lysate. TFF failed to provide adequate sensitivity for both viral and bacterial targets. However, attempts to eliminate the DNA extraction step showed promise in cases in which PCR inhibition could be overcome. Extraction controls showed variable recoveries ranging from 6-35%.

 

 

 

SPECIAL LINKS

2005 NRDC Report concerning microbes at beaches