1

FINAL REPORT

THE PILOT EPIDEMIOLOGIC ASSESSMENT OF MICROBIAL INDICATORS 

FOR 

MONITORING RECREATIONAL WATER QUALITY 

IN 

MARINE SUB/TROPICAL ENVIRONMENTS

Principal Investigators:

Lora E Fleming MD PhD MPH MSc, (University of Miami)

Helena Solo Gabriele PhD (University of Miami)

Jay M.Fleisher PhD (Nova Southeastern University)

Samir Elmir PhD PE (Miami Dade County Public Health Department)

Chris Sinigalliano PhD (NOAA Southeast Miami)

Lisa Plano MD PhD (University of Miami)

John Wang PhD (University of Miami)

Collaborators

University of Miami: Mr Jonathan Kish MPH, Tomoyuki Shibata PhD, Amir Abdelzaher MS, Guoqing He PhD, 

Mary Wright MS, Julie Hollenbeck BA

NOAA Southeast Miami Lab: Kelly Goodwin PhD, Maribeth Gidley DO MPH, David Wanless MS 

Miami-Dade Dept. of Health: Gary Miller

Centers for Disease Control (CDC): Lorrie Backer PhD

University of Florida: Carol Palmer PhD, Fred Bonilla PhD, Tonya Bonilla PhD

BCS Laboratories: Troy Scott PhD, George Lukasik PhD

University of South Florida: Valerie Harwood PhD, Shannon McQuaig MS

NOAA Charleston: Jill Stewart PhD, Jerold Dickerson PhD

WHOI: Rebecca Gast PhD

University of Missouri: Charles (Andy) Carson PhD, Helen Yampara-Iquise PhD
2

ACKNOWLEDGEMENTS

The researchers would like to dedicate this research to the 

memory of Ms Seana Campbell, a very talented, hardworking 

and creative young researcher who enriched all people whose 

lives she touched, and who died too young.

The researchers would also like to thank the following University of

Miami and FIU Students, other student researchers, and other

researchers for their assistance in the performance of this study: 

Cristina Ortega (NSF REU), Kelly Withum, Anna Christy Garza, 

Hasan Abdel Fattah, Karen Kajder, Matthew Straney, Michael Lara, 

Xiaofang Zhu, Reshma Ramoutar, Michael Schoor (NSF REU), Erik 

Kumetz, Katie Herleman, Yasiel Hernandez, Rafael Hernandez

(Howard Hughes Scholar), Maria Del Carmen Vazquez MD, Evelyn Davila, John Clark PhD (NIEHS F30), 

Paulo Pinheiro MD, Zachary Lipschultz, Sharde Chambers, Derek Schesser, Jakub Bartowiak, Gloria 

Scorzetti; Florida Dept of Health: Erin O’Connell MPH, Pedro (Paco) Castellones MPH, Gary Miller, Racquel 

Mohammed, Michael Rybolowik, Gerard Philizaire, Erin May; EPA Intern: Lauren Crowder; NOAA Hollings 

Scholar: Rene M. Boiteau

The researchers would also like to thank the following individuals and organizations for their support without 

which this study as well as our ongoing research would not have been possible: Dr Sharon Smith, Dr Peter 

Minette, and the NSF NIEHS Oceans and Human Health Center; SeaAquarium Staff and Facility; American 

Express; the University of Miami Rosenstiel School of Marine and Atmospheric Sciences (RSMAS) facilities, 

RSMAS security, RSMAS Marine Biology and Fisheries (MBF Division), and the Schmale and Fell laboratories, 

the RSMAS Business Office, Aida Diaz and Sidney Hartley; Miami Dade Parks & Recreations; Miami Dade 

County Dept of Health (Environmental Health, Microbiology); Dr David Kay and his European collaborators;

Florida Department of Health, and the Grants Manager for this project, David Polk; University of Miami -

College of Engineering; University of Miami – Miller School of Medicine; the NOAA AOML Laboratory; Mote 

Marine Laboratory: Kate Nierenberg MS.

This Pilot Epidemiology Proposal was funded in part from the following sources: the National Center for 

Environmental Health (NCEH), Centers for Disease Control and Prevention (CDC); Florida Dept of Health (FL 

DOH) through monies from the Florida Dept of Environmental Protection (FL DEP); the Environmental 

Protection Agency (EPA) Internship Program; the National Science Foundation (NSF) and the National 

Institute of Environmental Health Sciences (NIEHS) Oceans and Human Health Center at the University of 

Miami Rosenstiel School (NSF 0CE0432368; NIEHS 1 P50 ES12736) and NSF REU in Oceans and Human 

Health, and the NSF SGER (NSF SGER 0743987) in Oceans and Human Health. We would also like to thank 

IDEXX for their support of our project through the provision of supplies needed for the chromogenic substrate 

analysis of enterococci.
3
4

ABSTRACT

Microbial water quality indicators are found in high concentrations in sewage, and are used to determine 

whether or not water 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

(although not exclusively) true for water bodies located in sub/tropical environments with no known point 

sources, due to the potential for microbial regrowth in sub/tropics, and the fact that studies used to set national 

guidelines were not conducted in sub/tropical environments nor with no known point sources. The primary 

goal of this study was to evaluate the relationship between human health, and the physical and microbial 

characteristics of non point source sub/tropical recreational marine waters and its shoreline. This goal was 

addressed by measuring human health effects and microbial water quality at a recreational beach site within a 

sub/tropical climate in conjunction with the ongoing development of a predictive, coupled hydrodynamic and 

microbial fate model that hopefully in the future can be used to distinguish impacts from sewage sources 

versus regrowth of microbes, each of these sources possibly resulting in different health effects as observed at 

the beach site. 

Substantial environmental pilot data had already been collected for the sub/tropical Study Site chosen for this 

research. These pilot data, as well as the literature, indicated that exposure to contaminated recreational 

marine waters may result in human health effects, and that the shoreline sediments were one likely source of 

indicator microbes. In the Pilot Project, human health was evaluated in the through an epidemiologic study 

which randomly assigned volunteer adult participants (residents of south Florida who regularly bathed in 

recreational marine waters) to bather and non-bather groups, with subsequent follow up for reported illness. 

This epidemiologic effort was conducted in conjunction with an ongoing extensive sampling of indicator 

organisms and pathogens as well as hydrodynamic modeling. The environmental assessment was conducted 

such that measures of indicator organism exposures were assigned directly to each individual bather, as well 

as additional environmental measures. Microbe measurements included traditional (enterococci, fecal coliform, 

E.coli) and non-traditional (C. perfringens, Bacteroides) microbial indicators, as well as direct measurement of 

microbial pathogens (S. aureus, enterovirus, C. parvum, and G. lamblia). Individual samples were assayed for 

enterococci and Staphylococcus aureus. Daily composite samples made from individual samples collected on 

each day were assayed for multiple fecal indicator microbes (enterococci, C. perfringens, fecal coliform, E. coli)

and waterborne pathogens (enterovirus, Cryptosporidium, and Giardia), and S. aureus as a skin infection 

agent. In addition to these organisms, daily “special” samples were also assayed for enteric viruses, 

Cryptosporidium, and Giardia. Environmental parameters were also measured throughout the study.

The study results indicated that there was an increased risk of reporting gastrointestinal symptoms with bathing 

exposure at the non point source subtropical recreational marine water. Furthermore, there appeared to be an 

association between enterococcus (by membrane filtration method) levels and report of bather gastrointestinal 

symptoms at or above 40 CFU/100 mL of enterococcus based on an individual sample. The Study Site, which 

is a non-point source of fecal contamination, showed intermittent high levels of indicator microbes in this study. 

Nevertheless, these intermittent high levels of indicator microbes did not coincide consistently with the 

presence of these pathogens. Future studies should also focus on developing fate and transport models, 

epidemiologic studies, and quantitative microbial risk assessments which incorporate human, microbiological 

and oceanographic data. 

INTRODUCTION

The description of the methods and results below is focused on the Pilot Epidemiologic Project of the ongoing 

NSF NIEHS Oceans and Human Health Center “Recreational Microbes” Study; the extensive environmental 

monitoring and hydrodynamic modeling which occurred concomitantly with the epidemiologic study are 

currently funded by an ongoing Research Study of the NSF NIEHS Oceans and Human Health Center of the 

University of Miami with collaborators from NOAA, the CDC, the Miami Dade County Dept of Health, University 

of Florida, University of South Florida, and other collaborators. This Pilot Epidemiologic Study provided for the 

collection of human epidemiologic data in conjunction with the ongoing already-funded environmental 

monitoring and hydrodynamic modeling, allowing for “leverage” of funding and resources. Ultimately, the 
5

human, environmental and oceanographic data will be used to develop a predictive microbial fate and transport 

model with broad applicability to beaches and recreational marine waters in the sub/tropics with appropriate 

modifications for local conditions. Model results will complement recreational water quality monitoring in the 

sub/tropical marine environment without a known point source thereby improving the likelihood of reliable risk 

assessment.

STATEMENT OF PROBLEM

Based on risk assessments from the World Health Organization (WHO) and academic research sources,

Shuval (2003) estimated that globally each year there are in excess of 120 million cases of gastrointestinal 

disease and in excess of 50 million cases of more severe respiratory diseases caused by swimming and 

bathing in wastewater-polluted coastal waters. Dwight et al (2005) estimated that the economic burden per 

gastrointestinal illness (GI) was $36.58 while it was $77.76 per acute respiratory disease, $37.86 per ear 

ailment, and $27.31 per eye ailment (2001 values in Orange County, California). The authors then estimated 

the cumulative public health burden from the excess illnesses associated with coastal water pollution to be 3.3 

million dollars/year from just 2 recreational marine beaches in California. Finally, Given et al (2006) used 

epidemiological dose-response models to predict the risk of gastrointestinal illness at 28 beaches spanning 

160 km of coastline in Los Angeles and Orange Counties (California). They estimated between 627,800 to

1,479,200 excess cases of gastrointestinal illnesses occurred at these beaches each year, corresponding to an

annual economic loss of 21 to 51 million dollars (in year 2000 dollars). 

Recently, concerns have been raised about the appropriate use of microbial indicators to prevent illness and to 

regulate recreational uses of recreational marine water bodies, particularly true for water bodies located in 

sub/tropical environments and without known point sources, due to the potential for microbial regrowth in 

sub/tropics, and the fact that studies used to set national guidelines were not conducted in sub/tropical 

environments, focused on locations with known point sources, lacked individual exposure information, and had 

epidemiologic methodologic weaknesses (Haile 1999, Wade 2003, Dwight 2004, Colford 2007, US EPA 2007, 

Yamahara 2007, Boehm in press). 

SPECIFIC AIMS AND HYPOTHESES

The primary goal of the Pilot Epidemiologic Study was to conduct a prospective randomized epidemiologic 

study to assess the human health risks associated with swimming in sub/tropical seawater contaminated with 

infectious microorganisms (while minimizing the significant bias seen in prior epidemiological studies) in 

collaboration with ongoing intensive environmental monitoring and hydrodynamic modeling. The Pilot 

Epidemiological Study was designed to address 3 major methodological weaknesses inherent in previously 

published epidemiological studies assessing the health effects of bathing in waters contaminated with domestic 

sewage. These methodological weaknesses were: 1) Failure to control for the substantial amount of temporal and 

spatial variation in indicator organism density shown to occur within just a few hours and within several meters at 

marine bathing locations; 2) Failure to relate indicator organism density directly to the individual bather; and 3) 

Failure to rigorously control for non-water-related risk factors for the outcome illness under study. The design of 

the Pilot Epidemiological Study addressed these weaknesses by controlling bather exposure, by individual study 

participants monitoring of their own environmental exposures (indicator organism densities), by pre- and postexposure interviews with each study participant, and by randomization of exposure among only bather participants 

(Cabelli 1982, Dufour 1984, Calderon 1991, Haile 1999, Wade 2003, Kay 2004, Dwight 2004, Wade 2006, 

Boehm 2007, Colford 2007, US EPA 2007, Yamahara 2007, Boehm in press). Furthermore, the Study was 

designed to evaluate the exposures of and health risks to bathers in sub/tropical recreational marine waters 

with no known point source of microbial pollution.

Specific Aims: The objectives of this Pilot Epidemiologic Study were to begin to evaluate the 

relationship between microbe densities and reported symptoms in human subjects with random 

assignment to recreational marine water exposure and intensive individual microbial monitoring in 

subtropical recreational marine waters with no known point source. 
6

The hypotheses tested for the epidemiologic study were: 

H1: People exposed to recreational waters contaminated with infectious microorganisms would be 

significantly more likely to report symptoms than unexposed similar people who go to the 

beach but do not go into the water.

H2: The organism densities sampled at the time of exposure in the marine waters would be associated

with reported human symptoms.

Ultimately, the human, environmental and oceanographic data will be used to develop a predictive model with 

broad applicability to beaches and recreational marine waters in the sub/tropics with appropriate modifications 

for local conditions. 

BACKGROUND

The primary significance of this Pilot Study was that it began to address an important data gap. The use of 

indicator microbes for water quality monitoring in sub/tropical (tropical and sub-tropical) environments has been 

questioned, in particular for sub/tropical coastal zones where there are no known point sources of pollution 

(Haile 1999, Wade 2003, Colford 2007, US EPA 2007, Yamahara 2007, Boehm in press). The premise is that 

indicator microbes within these regions regrow due to climate conditions conducive for their regrowth, and are 

thus not representative of sewage sources of pathogens (Yamahara 2007). The few epidemiologic studies 

conducted previously in sub/tropical environments (Fujioka 1994, Kueh 1995) showed no statistically 

significant relationship between human health and enterococcus, the currently recommended indicator microbe 

for evaluating marine water quality in the US (Haile 1999, Colford 2007). Although these studies challenged 

the use of current methods for sub/tropical environments with non point sources, they did not establish a 

substantial case for a new method or new set of indicators. Furthermore, these prior studies did not include an 

oceanographic component aimed at simulating the distribution and number of microbes in water during 

different environmental conditions. 

The Pilot Epidemiologic Study discussed in this Report differed in that it was conducted in conjunction with 

coordinated ongoing interdisciplinary efforts, including intensive individual microbial exposure monitoring and 

an oceanographic component to evaluate the distribution and inter-relation of microbes, human illness and 

other environmental factors; furthermore, this study used a different epidemiologic methodology in which 

bathers were randomly assigned to exposure, and then collected their own environmental water exposure 

samples. The researchers in this Pilot Epidemiologic Project consider that this combination of interdisciplinary 

efforts will ultimately build a unique and stronger case concerning which microbe or monitoring methodology is 

the most effective for establishing human related illness in sub/tropical marine environments and non point 

sources in sub/tropical recreational marine waters. 

Human Health Data: Several epidemiologic studies have found that, compared with bathing in 

uncontaminated waters, bathing in temperate recreational waters contaminated with domestic sewage or storm 

drain runoff (ie. known point sources) has been associated with an increased risk for transmission of infectious 

diseases, including gastroenteritis, febrile respiratory illness, eye and ear infections, and skin infections and 

rashes. However, there were weaknesses in the designs of many of these studies, including the absence of 

data capable of assigning individual estimates of exposure to each bather resulting in exposure 

misclassification, a failure to account for other risk factors for the illnesses of interest (including the possibly

inappropriate comparison of cohorts of bathers to cohorts of non-bathers), the presence of a known point

source (rather than the more common scenario of no known point source), and purely temperate not 

sub/tropical marine environments (Pruss 1998, Wade 2003, Craun 2006, US EPA 2007, Boehm in press).

Pruss (1998) and Wade (2003) reviewed all the significant existing epidemiologic studies on the health effects 

from exposure to recreational water. They found that most studies reported a dose-related increase in 

swimming-related illnesses associated with increases in recreational water indicator bacteria counts. The 

indicator organisms that reportedly associated best with the health outcomes were enterococci/fecal 

streptococci for marine and freshwater, and E. coli for freshwater. Of note, the majority of these studies were 
7

conducted in the northern US and northern Europe, with few studies performed in sub/tropical marine 

recreational waters and/or without known point sources.

In a series of studies of randomly assigned bathers vs non-bathers in the United Kingdom, Fleisher, et al. 

(1991, 1993, 1996, 1998) attempted to address some of the methodologic issues of prior studies. A summary 

of findings is presented in Table 1. These results, coupled with the evidence that infectious microorganisms 

can occur in sub/tropical waters in the absence of specific contamination events and the dirth of information 

describing the impact of these illnesses (i.e., the severity of the illness or need for medical care), indicated that 

the actual public health risk associated with bathing in sub/tropical waters containing microorganisms, including 

those from EPA’s list of microbial indicators, is unknown.

Table 1: Summary of results from the work of Fleisher et al. (1991, 1993, 1996, 1998) 

Number of Study 

Subjects

Indicator

(Concentration)

Reporting Symptoms

(Percentage) 

Statistical Test 

Significance

Fleisher et al (1993)

Fecal streptococcus* GI Chi Square(trend)

278 Non-bathers 7-14

78 <35 7-18

128 >35 20-24 P< 0.01- 0.11

Fleisher et al (1996)

Fecal streptococcus*

(quartiles)

Acute febrile Respiratory 

illness

Chi Square (trend)

665 Non-bathers 3

124 0-14 4

138 15-27 5.8

125 28*50 1.6

158 51-158 7.6 P<0.04

Fecal Coliforms* Ear ailments

636 Non-bathers 2.8

120 0-40 6.7

113 41-79 6.2

152 80-133 5.9

134 134-661 14.2 P< 0.001

Fleisher et al (1998)

Not specified GI illness Attributable Proportion

605 Non-bathers 9.7

546 Bathers 14.8 34%

Acute Febrile Resp. Illness

665 Non-bathers 3.0

546 Bathers 5.0 40%

Ear Ailments

636 Non-bathers 2.8

525 Bathers 8.2 66%

Eye Ailments

658 Non-bathers 2.1

538 Bathers 4.5 53%

*Number of organisms per 100 ml

Finally, as noted above, beaches may also be impacted by non-point sources. These are sources that can 

include storm water runoff, animal inputs, and/or human bather shedding (Gerba 2000, Elmir 2007, US EPA 
8

2007, Boehm in press). In non-point source beaches, the limited number of epidemiologic studies conducted 

to date have not indicated an association between enterococci (and other microbial indicators) and illness,

even though some of these studies did find an association between bathing exposure and health risk (Haile 

1999, Fleming 2004, Colford 2007). Therefore, the association between illness and a specific microbial 

indicator has not been well established at beaches dominated by non-point sources.

PRELIMINARY DATA

The researchers (Solo Gabriele 2002, Fleming 2004, Shibata 2004, Elmir 2007, Abdelzaher 2008, Wright 

2008, Solo-Gabriele 2008) have conducted a series of studies to characterize the Study Site, including 

extensive environmental monitoring, source tracking, experimental simulation, and small epidemiologic studies 

(including bather shedding studies).

The researchers conducted a pilot epidemiological and water quality study at two sub/tropical public beaches, 

including the current Study Site, located in the southern part of Biscayne Bay, Miami (FL) next to both the 

University of Miami Rosenstiel School and the NOAA Southeast Center. This very small pilot study was funded 

by the NIEHS Marine and Fresh Water Biomedical Sciences Center and the Miami Dade Dept of Health. The 

Study Site for original pilot and for the current Pilot Epidemiologic Study is approximately one mile long, 

relatively shallow, and characterized by poor water circulation. The beach has a history of poor water quality: 

during 2000, the beach exceeded the EPA Poor Water Quality Guideline (PWQG) for enterococci 29.2% of the 

time. The results showed that there was no significant relationship between density of microbes and health 

effects although the sample size was very small (208 subjects); the water quality at comparison beach was 

better than the Study Site regardless of the season (wet vs. dry); there was no fecal pollution point source 

identified in the sanitary survey; intensive spatial water quality monitoring indicated that the shoreline at the 
9

Study Site appeared to be the source of microbes; and the concentrations of indicator microbes were usually 

lower during low tide as compared to high tide. The detection of those indicators at the shoreline without a 

known point source of fecal pollution again raised issues concerning the suitability of those indicators for 

measuring the recreational marine water quality in sub/tropical climates. 

Over the past 4.5 years of NSF NIEHS Oceans and Human Health Center and collaborative NOAA and NSF 

funding, the Recreational Microbes Research Group has made significant progress. The group developed and 

implemented several sediment and water sampling and testing efforts within the Study Site. The overall results 

suggest that the inter-tidal zone (sand that is periodically wet and dry between low and high tide) may be the 

source of indicator microbes at this beach. The release of indicators from this source is a function of tidal 

height, antecedent rainfall, wind direction and intensity, and other factors which influence microbial transport 

from the sediments. The sampling efforts supporting these observations have included: 1) intensive sediment 

sampling effort, 2) hourly sampling continuously for 48 hours, 3) sampling along transects at set tidal heights, 

4) direct sampling of runoff, and 5) evaluation of the microbial contribution from runoff, human swimming and 

animal feces (Elmir 2006; Elmir 2007). Last summer, the research team conducted a baseline assessment of 

the Study Site for the presence of pathogens with funding from the Florida Dept of Health. The results from the 

4 sampling efforts showed that pathogens were detected when indicator microbes were elevated. Elevated 

indicator levels and pathogens were noted during a high tide sampling event characterized by low solar 

radiation. As a result of these prior activities at the Study Site, many important logistical, administrative and 

training activities were completed including: developing and implementing safety, sampling training and 

analyses protocols, meetings with the epidemiological, sediment and modeling groups, governmental 

agencies, and working with a variety of universities and public and private local laboratories. Those activities 

were essential for the success of the Pilot Epidemiologic Study discussed below.

STUDY METHODS

The objectives of this Pilot Epidemiologic Study were to begin to evaluate the relationship between microbe 

densities and reported symptoms in human subjects with random assignment to recreational marine water 

exposure and intensive individual microbial monitoring in subtropical recreational marine waters with no known 

point source. 

Epidemiologic Methods

The design of the Pilot Epidemiologic Study was based on information collected by the Investigators during 

past studies at the Study Site described above (Shibata 2004, Fleming 2004, Elmir 2007, Wright 2008) and on 

the work of Fleisher et al (1991, 1993, 1996, 1998) (see Table 1 above). The investigators conducted a 

prospective randomized study with study participant exposure randomly assigned to either potentially 

contaminated marine recreational water or beach-only exposure. 

Local adult residents (at least 18 years old) who regularly reported bathing in recreational marine waters were 

recruited to participate in the study. After recruitment (see below), the participants were screened for basic 

demographics, current illnesses and symptoms, alternative risk factors, and a brief health history; they were 

then given an appointment for the study beach day. At the study beach day, participants read and signed an 

IRB approved Consent Form (by Florida Dept of Health and University of Miami Human Subjects Committees; 

see Appendix) and a photography release. Participants were again interviewed briefly about current illnesses 

and symptoms, food consumption, and beach exposure since the baseline survey. Study participants were 

then randomized to “water exposure” or “no water exposure” (ie. bather vs non-bather, respectively). Study 

participants were instructed to: 1) Spend 15 minutes in knee-deep water in a demarcated study environmental 

sampling area and dunk their heads completely under water 3 times (“bather”), or 2) Spend time on the beach 

at a demarcated study environmental sampling site away from the water (“non-bather”). After participating in 

the beach day protocol, participants were given a $50 American Express gift card for their cooperation in the 

study. They were also given an appointment to complete an extensive follow-up questionnaire seven (7) day 

after the beach visit. After completing the follow-up questionnaire, study participants were sent an additional 

$25 American Express gift card to compensate them for any costs associated with participating in the study. 
10

Of note, all study materials were available in Spanish and English (see Appendix), and many of the study staff 

were bilingual. The questionnaires (as well as the study design) were adapted from those provided by Dr 

David Kay and Dr Jay Fleisher from the prospective randomized intervention studies in Europe (see Appendix). 

Adaptations included some language changes to “American” English, as well as logistical changes due to lack 

of resources (instead of follow up at 7 days and 3 weeks, due to resources and logistics, these follow ups were 

combined into a single questionnaire and event at 7 days post exposure).

Study Populations: Only local adult residents (not tourists) were recruited to participate in the study due to 

logistics of the 7 day follow up, as well as the possibility that tourists might represent an unusually susceptible 

subpopulation. An individual met the study inclusion criteria if: 1) at least 18 years old; 2) regularly bathed in 

recreational marine waters; 3) lived in South Florida at least 6 months or more per year; 4) did not have any 

chronic gastrointestinal (GI), respiratory disease, skin, or eye disease; 5) was willing to be exposed to bathing 

waters at the Study Site; 6) was willing to be randomized; and 7) understood and spoke either English or 

Spanish.

Disease Endpoint Criteria: The disease endpoints of interest in this study were the self-reported symptoms of 

gastrointestinal illness, acute febrile respiratory illness (as specified by ICD 9 461-466, 480), eye or ear 

infections, and skin infections/irritation within seven days of going to the beach. These health endpoints were

based on information collected during past studies by the Investigators at the Study Site and on the work of 

Fleisher et al. (1991, 1993, 1996, 1998) (see Table 1). Of note, in this Report, only the results for 

gastrointestinal symptoms are reported.
11

Recruitment Methods: Volunteers were recruited by a variety of methods: word of mouth, newspaper articles, 

flyers, emails, as well as on-site. Team members staffed booths at Miami-Dade Community College, University of 

Miami Coral Gables Campus, Florida International University, and the Study Site to actively recruit individuals at 

these locales and to answer questions from potential participants. During recruitment, OHH employees fluent in 

both English and Spanish were present in order to remove potential selection bias. Potential participants were 

explained the qualifications for becoming a participant, instruction on study activities (including obtaining informed 

consent, completion of health data questionnaires, as well as the beach day activities), and contact information for 

registration purposes; these materials were also available in Spanish and English. Interested participants were 

allowed to select one day of the 15 possible on which to participate; subjects were only allowed to participate 

once. 

Pre-exposure Interview: Prior to arriving at the beach, participants verbally consented to complete a baseline 

health questionnaire document over the phone in Spanish and English. This was completed no more than 7 

days prior to the sample collection day. Participants who had registered to participate on a specific sample 

collection day were telephoned each day during the week leading up to the event in attempt to complete the 

baseline health questionnaire. An individual who had not completed the questionnaire prior to the sample 

collection day was not allowed to participate in the beach activities. 

Beach Study Day Data Collection: The epidemiologic study data collection activities were conducted over a 

period of 15 days in coordination with the environmental research group beginning on December 15, 2007 and 

ending on June 21, 2008. Each sampling day, 56-125 participants were randomly assigned to spend a period 

of 15 minutes in demarcated swim zones or 15 minutes in a demarcated non-exposure zone(approximately 

671 people/group overall). Overall study participation took between 30 minutes to 1.5 hours depending on the 

volume of participants, and was performed between 8 am and noon on a range of week and weekend days.

On the actual study beach day, an IRB-approved Consent Form was explained and signed, and then a second 

pre-exposure day interview was administered to each study participant. This interview was administered by a 

trained interviewer, focusing on any non-water-related behavior that could either cause or confound any 

association found between bathing and the outcome illnesses under study, any current illnesses, recent food 

consumption, as well as any recent exposure to bathing waters. Participants reporting significant illness on the 

study day were excluded from the study.

Study subjects were randomly assigned to either a bather group or a non-bather group. Individual bathers were 

then assigned to the bathing station where staff members supervised the exposure activity of the bather, including 

the time, location, unusual activities, and duration each individual bather spent in the water. Bathers were

required to spend 15 minutes in the water at knee-deep water, and to immerse their head three times completely 

under water. Using ropes, a 20-meter stretch of beach was subdivided into 5 meter intervals forming six bathing 

exposure zones. Exposure of any individual bather was therefore restricted to a single 5 -meter-wide swim zone. 

Each subject was instructed on the technique to take their own water sample near the surface during their head 

immersions as well as provided with an appropriate water sampling container. Staff members instructed 

participants to thoroughly rinse the collection container before filling completely with the marine water. Staff 

members observed the subjects at all times to insure that the subjects performed the water sampling appropriately 

as well as followed the 3 head immersion protocol and remained only 15 minutes in the water. When the subjects 

left the water, they gave their individual water samples to the Study Staff for microbial analysis processing (as 

described below). 

No bather was allowed to enter the water more than once during the actual study exposure. The non-bather 

group was restricted to a roped-off area distant from the water and sand. Staff members were assigned to ensure 

that non-bathers did not enter the water. For logistical and safety reasons, the ratio of staff members to study 

subjects was approximately 1:9.

After all participants had completed their 15 minutes of “exposure,” they were thanked, given a hard copy of their 

Consent Form, an appointment for a telephone follow up interview in 7 days, and a American Express Card Gift 

Certificate for $50.
12

Follow up interview: This short telephone follow-up interview was used to assess the occurrence of illnesses 

(including questions about symptoms occurring since the beach visit, whether they sought medical care for 

their symptoms, and whether the symptoms caused them to miss work or school), as well as alternative risk 

factors for the study outcomes (including subsequent marine water exposures and food consumption) and 

subsequent beach visits. If participants were not able to be reached during the initial follow-up call time, they 

were called back subsequent times between seven (7) and twenty-one (21) days following the beach 

exposure. After completing the follow-up questionnaire, study participants were sent an additional $25 

American Express gift card through US Mail to compensate them for any costs associated with participating in 

the study. 

Environmental Sampling

The Study Site, (described above in Preliminary Data), has been well characterized in terms of the microbial 

content of water and sediments including an identification of the sources of microbes to water and sediments. 

This characterization has been facilitated through a hydrodynamics model developed from a prior pilot study 

and the ongoing research of the NSF NIEHS Oceans and Human Health Center at the University of Miami, as 

well as NOAA, Miami Dade County Public Health Department and CDC. Of note, the environmental monitoring 

and assessment are ongoing and funded by the NSF NIEHS Oceans and Human Health Center Recreational 

Microbes Research Project and collaborators. The environmental assessment described below focused on the 

analysis of several microbes in water including pathogenic viruses, bacteria, and protozoa and the traditional 

indicator microbes. Funds were provided by the FL DOH and CDC with the NSF NIEHS Oceans and 

Human Health Recreational Microbes Research Project, NSF SGER and NSF REU, NOAA, and many 

collaborators for microbial analyses described below. 

Water Quality Monitoring at Study Site: While each study participant was in the water, each subject collected 

their own environmental water sample from the demarcated study sites as described above. All water samples 

were placed in a cooler containing ice packs and transported back to the Microbe Laboratory at the University of 

Miami Rosentiel School of Marine and Atmospheric Sciences (RSMAS)(<5 minutes by car and 1⁄4 mile) for 

processing. 

Three different sets of samples were collected: 1) individual; 2) composite; and, 3) special samples. All 

samples were collected in the morning, approximately from 8 am to 12 pm on each sampling day. Individual 

water samples were collected at knee depth by epidemiological study participants using 5-L sterilized plastic 

containers after 15 minutes of bathing. Sampling points from the shoreline ranged from 10 to 40 m depending 

on tidal stage corresponding with the knee height. From 30 to 61 individual samples were collected per 

sampling day. Daily composite samples were prepared by pooling 1 L of each individual sample collected on 

the day into a 50-L sterilized carboy. For daily special samples, 10 liters of marine water were collected every 

15 to 20 minutes from the time the first bather entered the water and pooled into two 250-L sterilized plastic 

drums. In order to minimize the impacts from epidemiological study participants, the sampling point for the 

“special” samples was approximately 5 to 10 m away from bathers. 

Individual water samples were place in a cooler containing icepacks and after the collection of a batch of six, 

samples were transported to the microbial analysis laboratories at the University of Miami Rosentiel School 

(RSMAS) and National Oceanographic Atmospheric Administration Atlantic Oceanographic and Meteorological 

laboratory (NOAA/AOML), which are located in within a few minutes from the Study Site (<5 minutes) for 

processing. The composite and special samples were kept outside but in the shade under tents during 

individual sampling, and then delivered to the RSMAS Lab shortly after the end of sampling efforts (around 

noon to 1 pm). 

Other Environmental Measurements: Water temperature, pH, and salinity were measured for individual, 

composite, and special samples in the field using a YSI probe (600R series sonde,YSI Inc., Yellow Springs, 

OH). Turbidity was also measured for these samples using a HACH turbidimeter. Additionally, other 

environmental conditions (e.g. tidal stage, rainfall, wind, and solar radiation) were obtained from National 

Ocean and Atmospheric Administration (NOAA) and the University of Miami, RSMAS (NSF NIEHS OHH 

Center Remote Sensing Facilities Core). 
13

Investigators also surveyed the bather density and animal populations in the study area during participant 

exposure, as well as evaluated data from a 24 hour 365 day per year digital camera focused on the Study Site 

and installed by the NSF NIEHS OHH Center Recreational Microbes Research Project with permission of 

Miami Dade County and assistance from the Miami Dade County Dept of Health and Miami SeaAquarium

(Wright 2008). These data are being enumerated and evaluated by Dr John Wang and his research group as 

part of the ongoing development of an oceanographic fate and transport model for the Study Site.

Microbial Assays: Individual samples were assayed for enterococci and Staphylococcus aureus. Daily 

composite samples made from individual samples collected on each day were assayed for multiple fecal 

indicator microbes (enterococci, C. perfringens, fecal coliform, E. coli, Bacteroides) waterborne pathogens 

(enterovirus, Cryptosporidium, and Giardia), and S. aureus as a skin infection agent. In addition to these 

organisms, daily special samples were also assayed for enteric viruses, Cryptosporidium, and Giardia. 

Fecal indicator microbes 

Enterococci: Enterococci, the microbial indicator organism currently recommended by the US EPA for marine 

waters, was enumerated using a traditional membrane filter method as recommended by the US EPA (USEPA 

1997, 2000) and a chromogenic substrate method as proposed by IDEXX known by the tradename 

EnterolertTM (Westbrook, Maine) (APHA 1995). The membrane filter method involved placing the membrane 

filter containing the bacteria on a selective medium (mEI agar) and incubating the sample at 41°C for 24 hours. 

Colonies with a blue halo were then counted as enterococci. The chromogenic substrate method in simple 
14

term utilizes enzymes specific to particular microbial groups. These enzymes were attached to dyes which 

were then released when the target microbe was present in the sample. Enumeration of the microbe 

population was based upon the use of a tray (Quanti-Tray/2000) which separates the sample into a set of test 

wells. The number of test wells that showed the characteristic fluorescence under UV light were then counted 

and used in conjunction with a standardized table to provide the concentration in terms of the most probable 

number (MPN). 

C. perfringens: There is scientific evidence that indicates that C. perfringens behave similar to enteric viruses 

in marine and fresh waters, and that this bacteria does not multiply outside the human gastro-intestinal tract 

under aerobic conditions (Fujioka 1985; Fujioka 1997). Therefore, using C. perfringens as part of a monitoring 

program for the microbiological recreational water quality can be a valuable alternative, particularly in 

sub/tropical marine environments, where the regrowth of the other indicator microbes is suspected to occur. 

The state of Hawaii has elected to use C. perfringens in their water quality monitoring, and the USEPA has 

recently approved a membrane filtration method to detect and enumerate C. perfringens spores in water and 

sediments. C. perfringens was analyzed using the membrane filter method (USEPA 1995) requiring the use of 

mCP agar. Samples were incubated anaerobically at 44.5± 0.2°C for 24 hours. Plates were then exposed to 

ammonium hydroxide fumes after the incubation and dark pink to magenta colonies were counted as C. 

perfringens. 

Fecal coliforms: Fecal coliforms were originally recommended by the US EPA for recreational water monitoring 

(U.S.EPA 1976), and still used by the Florida Department of Health (FL DOH) and Florida Department of 

Environmental Protection (FL DEP). Enumeration of fecal coliforms was based upon the MF method (APHA 

1995; Method 9222 D). Filters were placed on modified mFC agar (Becton Dickinson, Sparks, MD) and were 

incubated at 44.5 ± 0.5oC for 24 hours; colonies that were various shades of blue were counted as fecal 

coliforms.

E. coli: The use of E. coli is recommended for fresh water monitoring by the US EPA (US EPA 1986). The MF 

method was used for enumerating using modified mTEC agar (US EPA 2002, Method 1603). Samples were 

incubated at 35 ± 0.5°C for 2 hours, and then incubated at 44.5 ± 0.2°C for 22 hours. Target red or magenta 

colonies were counted and reported as E. coli. 

Bacteroides: Bacteroides are found in the intestine of humans and other warm blood animals; thus, detection 

of bacteroides in water implies that the water has been contaminated with human or other animal feces. For B. 

thetaiotaomicron analysis, 400 mL of water from each sample was filtered through a 0.20 μm polycarbonate 

filter (Millipore Corp., Billerica, MA). The polycarbonate filter was placed into a 15mL conical tube. The tube 

was vortexed for 2 minutes and the filter removed. Conical tubes were first concentrated by centrifugation at 

2052 g for 15 minutes at 4oC. The supernatant was discarded, except for 200 μl, which was used to 

resuspend the pellet. DNA was extracted with the POWERSOIL DNA Extraction Kit (MOBIO Laboratories, 

INC.). The extracted DNA was analyzed for presence of B. thetaiotaomicron by PCR (Eppendorf Master Mix 

2.5, Brinkman Instruments, Inc, Westbury, NY) with primers (B.thetaF and B.thetaR). 

Pathogenic microorganisms

Due to presumption of low concentrations of pathogens in the recreational marine water from nonpoint sources 

of fecal contamination, daily composite and special samples were concentrated by two different methods. The 

first method of sample processing used traditional large volume filtrations. EPA method 1623 

(Cryptosporidium and Giardia in water by filtration/IMS/FA) for protozoan pathogens and EPA’s Manual for 

Virology (Chapter 14, Concentration and Processing of Waterborne Viruses by Positive Charge 1 MDS 

Cartridge Filters and Organic Flocculation) were followed with the exception of: a) type of filter used, as 

positively charged filters are adequate for freshwater only; b) the addition of acid and salt consistent with the 

needs of a negatively charged filter; and. c) the option to add acid and salt using an equilibration basin or fluid 

proportioner system. Negatively charged filters were utilized in accordance with those specified through 

Standard Methods for the Analysis of Water and Wastewater, Section 9510 C – Virus Concentration from 

Large Sample Volumes by Adsorption to and Elution from Microporous Filters, 21st Edition, 2005. The 

preparation of feed solutions for electronegative filters followed methods as outlined in Standard Methods, 
15

Section 9510 C. The elution and virus culture procedure followed EPA’s ICR Microbial Laboratory Manual, 

Section VIII – Virus Monitoring Protocol for the ICR, 1996. The method of acid and salt addition used an 

equilibration basin as described in EPA Method's 5 and 6 from the Manual for Virology). The second method 

of sample processing, called the bilayer method, used 10 L of composite sample (Abdelzaher 2008). The top 

and bottom filters were eluted separately for protozoan (Cryptosporidium and Giardia) and viruses (norovirus, 

enterovirus, and Hepatitis A virus). 

Water concentration for viruses and protozoa was conducted at the NSF NIEHS OHH Lab, RSMAS, University 

of Miami. Once concentrated onto filters, these samples were sent to universities or government laboratories 

for elution and analysis. 

Pathogenic Viruses: The presence of enteric viruses in coastal waters pose a public health risk since these 

viruses are typically transmitted via the fecal-oral route, and because for many viruses, only a small number of 

virus particles can initiate an infection (Haas et al. 1999). Because both the Florida Keys and the Tampa Bay 

region have been investigated for the presence of viruses in coastal waters and the presence of enteric viruses 

has been confirmed (Griffin 2003), it is likely that investigating the Miami region of Florida would also reveal the 

presence of viral pathogens in the coastal environment. After concentration of the special samples using 

Filterite filters (with pH and salt adjustment), the samples were sent to BCS laboratories in Gainesville, FL for 

elution and analysis for enteric virus using traditional virus culture methods. Concentrated composite samples 

were assayed for enterovirus based on qPCR. 

Pathogenic Protozoa: For the special samples, the filters were eluted, the pellet was concentrated, and the 

Cryptosporidium and Giardia were purified using immuno-magnetic separation. The purified pellet was stained 

with a fluorescent antibody and the numbers of oocysts/cysts were counted using a microscope. Protozoan in 

the composite samples were assayed based on qPCR. 

Staphylococcus aureus: S. aureus has been commonly associated with skin infections in marine waters 

(Charoenca 1993, Charoenca 1995). S. aureus was assayed based culture method using Baird-Parker agar 

base with Egg Yolk Tellurite Enrichment followed by a series of confirmation tests using Mannitol Salt agar, 

Latex agglutination, and PCR. Furthermore, the presence of Methicillin-resistant S. aureus (MRSA) was

identified base on PCR.

Epidemiologic Study Sample Size Calculations: The sample size for the epidemiologic study was calculated 

based on information collected during past studies by the Investigators at the Study Site and on the work of 

Fleisher et al., (1991, 1993, 1996, 1998) (see Table I). Using GI symptoms as reported by Fleisher et al (1993) 

and assuming 95% confidence and 80% power, approximately 1300 study participants (650 bathers and 650 

non-bathers) were needed to show an increase in reported GI symptoms of 5% in bathers vs. Non-bathers. 

Randomization, Epidemiologic Study Database & Statistical Analysis: Block randomization was used to 

maximize the probability of achieving equal number of study participants in the 2 groups under study. Block size 

consisted of a random ordering of blocks of two, four and six study participants per individual block. It should be 

noted that the random ordering of block size eliminates any chance of clinical personnel discovering the blocking 

pattern. Block sizes of two, four and six were chosen to maximize the probability of equal numbers of participants 

in each study group. All participants were not told their randomized status until completing the exposure day 

interview on the beach.

The Study Database was created in ACCESS. The data were analyzed using the statistical software package, 

SAS, for the purposes of the epidemiologic analyses. All univariate analyses were performed using the SAS 

package of statistical programs. Simple stratified analyses were conducted using EPINFO, while all logistic 

modeling was computed using SAS.

Initial analyses evaluated the effects of randomization for all demographic variables to insure equal distribution 

of demographic risk factors among exposed (ie. bathers) and unexposed (ie. non-bathers) populations. The 

basic analyses were geared to answer two questions: (1) "Is there an excess of the illnesses under study among 
16

bathers vs. non-bathers?"; and (2) "Is there a dose-response relationship between indicator organism density and 

the incidence of the outcome illnesses studied among bathers?" 

1) Bather vs. Non-Bather Comparisons: Success of randomization was assessed by comparing the distribution of 

non-water-related risk factors for the illness under study among bathers vs. non-bathers using univariate chisquare analysis. Where expected cell frequencies were less than 5.0, Fishers Exact Test (2-tailed) was used in 

lieu of the Pearson X2

The results of the environmental monitoring are presented first, followed by the descriptive results of the 

epidemiologic study and by the results of the hypotheses which combine both the epidemiologic and 

environmental data. Of note, since data collection was completed at the end of June 2008, there continue to 

be ongoing microbiologic and statistical analyses. Thus, limited data results are presented below with the 

expectations that additional results will be published subsequently in peer reviewed publications. In particular, 

only gastrointestinal symptoms are reported for the following reasons: a) these were the most numerous 

symptoms reported and thus yield the most statistically robust analyses; b) these are the symptoms most 

commonly associated with exposure to recreational marine waters in the literature; and c) these are the 

symptoms upon which the EPA regulations are based for the monitoring of these waters using enterococcus as 

statistic. Assuming randomization was successful, the crude rates of illness for bathers and 

non-bathers were adjusted for any of these extraneous risk factors. If randomization was not successful for any 

non-water-related risk factor, the effect of this failure was assessed for any confounding of the association found 

between illnesses among non-bathers vs. bathers. 

A second chi-square analysis was conducted for each of the illnesses under study to assess at what indicator 

organism density rates of illness among bathers were significantly greater than rates of illness among non-bathers. 

In the context of public health planning, specifically the setting of recreational water quality criteria, this analysis 

provided insight into possible thresholds of effect (i.e., an indicator organism density below which bathers 

experience no excess illness relative to non-bathers). It must be emphasized that this analysis was not meant to 

definitively define such a threshold, but rather to generate hypotheses regarding the possible existence of such 

thresholds to be tested by future epidemiological/statistical research, particularly for sub/tropical locations without 

known point sources.

2) Analyses Restricted To Bathers Only: In order to assess the relationship between indicator organism density 

and each illness under study, while identifying and controlling for non-water-related risk factors for such illness, 

analyses were restricted to bathers only. Exposure (i.e., indicator organism density) was related to each bather 

using the time an individual exited the water, the swim zone used, and the indicator organism density recorded. . 

Chi-square analyses were then used to identify any statistically significant trends among the observed incidence of 

a particular outcome illness among non-bathers vs. bathers at increasing levels of indicator organism exposure. 

Bathers were divided into different distributions of indicator organism exposure (e.g quartiles), and trends then 

assessed for each indicator organism assayed. These series of analyses included the rates of illness observed 

among non-bathers. When a particular outcome illness did not show a statistically significant trend for any of the 

indicator organisms assayed, rates of illness among bathers in the highest quartile of indicator organism exposure 

were compared to rates of illness among bathers in the lowest quartile using chi-square analysis.

Multiple logistic regression modeling was used to evaluate relationships between indicator organism density and 

illness among bathers while controlling for extraneous non-water-related risk factors identified via a prior univariate 

chi square analysis of postulated non-water-related risk factors or confounders among ill vs. non-ill bathers. Initial 

criteria for entering non-water-related risk factors or possible confounders into the logistic regression model were

at p < 0.15, although retention in the model was at p<0.05. A backward step-wise elimination strategy was

employed in logistic regression modeling used to control for non-water-related risk factors or confounders for the 

illness under analysis. For convenience, based on the workings of the statistical package used for logistic 

regression modeling, a forward step-wise procedure was used to assess the magnitude of any confounding of any 

observed indicator organism exposure-disease outcome relationships by non-water-related risk factors.

RESULTS
17

the recommended indicator organism. The possible association between gastrointestinal symptoms and the 

indicator organism, enterococcus, was evaluated using enterococcus as analyzed by the membrane filtration 

method. In addition, although some data are presented for the pathogen analyses, no statistical analyses were 

performed of these particular data. Again, in the future, the Research Group plans to expand on the results of 

this Report by publishing in the peer-reviewed scientific literature.

Overview of Environmental Results

Microbial Concentrations

The following microbial analyses results are provisional and pending final review; subsequent papers in the 

peer reviewed literature will contain results which will supercede the microbial results outlined below.

Individual Samples: A total of 668 water samples collected by individual epidemiologic participants were 

assayed for enterococci and Staphylococcus aureus over 15 individual study days from December 2007 

through June 2008 (Table 2). Although some of these waters samples were not used in the epidemiologic 

analyses due to loss to follow up (see above), the data presented below are for all 668 samples.

Table 2: Summary of individual enterococci and S. aureus levels for each monitoring day. 

Date N

Enterococci CFU/100ml 

(MF) Enterococci MPN/100ml (CS) S. aureus CFU/100ml

Min Max Ave GM Min Max Ave GM Min Max Ave GM

12/15/07 30 1 83 6±15 2 10 120 26±29 17 1 60 6±12 2

01/12/08 34 1 123 27±34 11 10 201 44±48 27 1 760 40±135 3

01/26/08 42 1 1580 65±242 12 5 152 53±39 38 1 100 12±22 3

02/16/08 48 1 559 27±85 4 5 316 39±51 23 1 400 30±84 3

02/23/08 53 1 271 28±41 15 5 137 28±25 20 1 220 17±41 3

03/08/08 38 19 3320 350±817 98 20 2844 310±710 88 1 200 11±35 2

03/22/08 38 2 757 149±184 58 5 644 123±133 67 1 20 3±4 2

03/29/08 55 1 219 20±34 8 5 73 13±13 9 1 532 14±72 2

04/12/08 49 1 159 33±39 13 5 120 19±22 12 1 440 18±66 3

05/10/08 53 1 1760 66±244 13 5 163 43±37 28 1 200 10±33 2

05/18/08 52 20 618 100±91 81 5 888 108±122 81 1 2 1±0.2 1

06/01/08 61 1 820 75±114 34 5 311 63±65 35 1 240 22±44 5

06/07/08 53 1 1140 34±159 5 5 74 23±19 16 1 40 4±7 2

06/13/08 31 1 621 66±146 10 5 453 47±87 21 1 780 41±141 5

06/21/08 30 10 849 73±152 40 5 693 94±122 63 1 560 40±104 7

Ave 44 4 859 75±160 27 7 479 69±101 36 1 304 18± 53 3

N is the number of individual samples collected on each sampling date; Min and Max are lowest and highest 

microbial levels on the day; Ave is arithmetic mean with standard deviation; GM is the geometric mean. 

Enterococci were measured based on the membrane filter (MF) and chromogenic substrate (CS) methods

Enterococci: The concentration of enterococci in individual samples ranged from the detection limit of 2 to 3320 

CFU/100mL with an approximate average of 75 (standard deviation, ±160) CFU/100mL based on the MF 

method. Those samples with below detection limit values were set at half the detection limit value (1 

CFU/100mL) for the statistical analyses. The enterococci concentrations based on the CS method ranged 

from below 10 to 2840 MPN/100mL with an approximate average of 69±101 MPN/100mL. Overall, enterococci 

levels based on the MF and CS (n = 668) were statistically not different. The two methods were strongly 

correlated (r = 0.87) for higher values (when MF values were larger than 35 CFU/100mL). The correlation 

between the methods was weak (r = 0.19) for MF values equal to or below 35 CFU/100mL. 

Staphylococcus aureus: The concentrations of S. aureus ranged from below 2 to 780 CFU/100mL with an 

approximate average of 18±53 CFU/100mL based on the cultured method followed by PCR confirmation. 

Methicillin Resistant S. aureus (MRSA) was detected from 1% (7/668) of individual samples. The 
18

concentrations of MRSA ranged from below 2 to 68 with an approximate average of 1±3 CFU/100mL. The 

levels of S. aureus and enterococci were not correlated (r < 0.01). 

Daily Samples: Results of microbial assays from 15 composite and 15 special samples were summarized in 

the Tables 3 and 4. 

Table 3: Multiple fecal indicator microbes in daily “composite” samples

Date

Enterococci Clostridium 

perfringens

Fecal

coliform

E. coli Bacteroides 

thetaiotaomicron

MF CS MF MF MF PCR

CFU

/100ml

MPN

/100ml

CFU

/100ml

CFU

/100ml

CFU

/100ml per 400ml

12/15/07 1 55 1 3 1 -

01/12/08 15 52 <0.7 104 1 +

01/26/08 4 61 7 27 31 -

02/16/08 15 18 <0.7 10 3 +

02/23/08 13 13 1295 20 7 +

03/08/08 99 69 1653 196 83 -

03/22/08 109 100 402 61 27 -

03/29/08 1 23 13 66 3 +

04/12/08 3 13 4 1 1 -

05/10/08 29 124 5 5 3 -

05/18/08 50 43 34 9 23 -

06/01/08 13 15 24 127 25 -

06/07/08 <0.7 11 6 7 <0.7 -

06/13/08 14 48 159 159 30 -

06/21/08 1 68 16 147 17 -

Ave 25 47 241 63 17

MF and CS stand for membrane filter and chromogenic substrate methods, respectively; 

Ave is the arithmetic mean of 15 daily samples; One half the detection limit was used for 

samples measuring below detection limit values. 

Fecal Indicators

Enterococci: The concentrations of enterococci in daily composite samples ranged from below the detection 

limit of 2 to 109 CFU/100mL with an approximate average of 25±36 CFU/100mL based on the MF method. 

The enterococci concentrations based on the CS method ranged from 11 to 124 MPN/100mL with an average 

of 47±34 MPN/100mL. Enterococci levels based on the CS method showed slightly higher results than the 

MF, but statistically not different (p = 0.08). 

C. perfringens: The concentrations of fecal coliform in the composite samples ranged from below 2 to 1650 

CFU/100mL, with an average of 241±547 CFU/100mL based on the MF method. 

Fecal coliforms: The concentrations of fecal coliforms in the composite samples ranged from 2 to 196 

CFU/100mL, with an average of 63±67 CFU/100mL based on the MF method. 

E. coli: The concentrations of E. coli in the composite samples ranged from below 0.7 to 83 CFU/100mL with 

an approximate average of 17±22 CFU/100mL based on the MF method.

Bacteroides thetaiotaomicron was detected in four composite samples (per 400 mL) based on PCR. 

GI Pathogens 

Enteric virus: The concentration of enteric virus in the daily “special” samples ranged from below detection limit 

of 1 to 2.2 MPN/100L. The concentrations of Pan Specific Enterovirus in the daily “composite” samples were 

below detection limits (0.5 – 1.1/100mL) for all daily composite samples. 
19

Cryptosporidium: Cryptosporidium was detected from only one daily “special” sample (1.6 oocysts/100L or 

0.002 oocysts/100mL), while other samples were below the detection limit of 1 oocyst/100 L or 0.001 

oocyst/100mL. The concentrations of Cryptosporidium in the daily “composite” samples were all below 

detection limits ranging from 0.1 to 0.3 oocysts/100mL. 

Giardia: Giardia were detected in two “special” samples (1.1 and 2.1 cysts/100L or 0.001 and 0.002 

cystes/100mL), whereas the remaining samples were below the detection limit of 1 cyst/100L or 0.001 

cyste/100mL. The concentrations of Giardia in the daily “composite” samples were all below detection limits, 

ranging from 0.1 to 0.3 cysts/100mL. 

Table 4: Pathogens in daily samples (composite and *special) 

Date

*Enteric

virus

Enterovirus Cryptosporidium Giardia

Staphylococc

us

aureus

*Culture qPCR *IMS-FA qPCR *IMS-FA qPCR MF

/100ml /100ml /100ml /100ml /100ml /100ml /100ml

12/15/07 0.0016 <0.5 0.0016 <0.1 0.0011 <0.1 8

01/12/08 <0.0005 <0.5 <0.0001 <0.1 <0.0001 <0.1 9

01/26/08 <0.0005 <0.5 <0.0001 <0.1 <0.0001 <0.1 24

02/16/08 <0.0005 <0.5 <0.0001 <0.1 <0.0001 <0.1 6

02/23/08 <0.0005 <0.5 <0.0001 <0.1 <0.0001 <0.1 2

03/08/08 <0.0006 <1.1 <0.0001 <0.3 <0.0001 <0.3 <0.7

03/22/08 <0.0006 <0.7 <0.0001 <0.2 0.0021 <0.2 5

03/29/08 <0.0005 <0.5 <0.0001 <0.1 <0.0001 <0.1 <0.7

04/12/08 <0.0005 <0.5 <0.0001 <0.1 <0.0001 <0.1 11

05/10/08 0.0022 <0.5 0.0009 <0.1 0.0023 <0.1 <0.7

05/18/08 0.0005 <0.6 <0.0001 <0.1 <0.0001 <0.1 <0.7

06/01/08 <0.0006 <0.7 0.0010 <0.2 <0.0001 <0.2 14

06/07/08 <0.0005 <0.5 <0.0001 <0.1 0.0015 <0.1 12

06/13/08 0.0005 <0.5 <0.0001 <0.1 <0.0001 <0.1 2

06/21/08 0.0005 <0.5 0.0005 <0.1 0.0010 <0.1 13

IMS-FA is immunomagnetic separation and immunofluorescent assay

Skin Pathogens

S. aureus: The concentrations of S. aureus ranged from less than 2 to 24 CFU/100mL with an approximate 

average of 10±6 CFU/100mL. 

Physical, Chemical, and Hydro-meteorologic Parameters

Time: The Study Site water quality was evaluated for 15 days from December 15, 2007 to June 21, 2008. The 

individual samples were collected between 7:44 to 11: 52 AM with an average time of 9:52 AM. 

pH: The pH values of individual samples ranged from 6.05 to 8.82 with an average of 7.97± 0.30. The average 

pH of daily individual (sample mean, μ = 7.95 ±0.26), composite (μ = 8.02±0.28), and special samples (μ = 

7.94±0.35) were statistically the same. 

Salinity: The salinity values of individual samples ranged from 32.12 to 38.58‰ with an average of 

35.73±1.18‰. The average salinity of daily individual (μ = 35.65±1.32‰), composite (μ = 35.64±1.43‰), and 

special samples (μ = 35.77±1.28‰) were statistically the same. 

Temperature: The temperatures of individual samples ranged from 20.6 to 31.3 oC, with an average of 

26.0±2.3oC. The average temperatures of daily individual (μ = 25.3± 2.6oC), composite (μ = 26.4±3.0oC), and 

special samples (μ =25.8±3.0oC) were statistically the same. 
20

Turbidity: The turbidity values ranged from 0.4 to 117 NTU, with an average of 11.8±13.5 NTU for the 

individual samples. The average turbidities of daily individual (μ = 12.4±13.2 NTU), composite (μ = 

14.5±14.7NTU), and special samples (μ = 12.1±9.1 NTU) were statistically the same. 

Tide: The tidal stage ranged from -0.1 to 0.8 m with an average of 0.2±0.2 m during individual sampling. 

Rainfall: Only 3 rainfall events were observed during monitoring efforts. Cumulative rainfalls in the previous six 

hours and during sampling were 12, 0.51, and 0.77 mm on March 8, May 18, and June 1, 2008, respectively. 

Wind: Wind direction ranged from 10 to 310o, with an average of 149±79o. The wind speed ranged from 2.4 to 

11.3 m/s, with an average of 5.8±2.4 m/s during individual sampling. 

Solar Radiation: The solar radiation ranged from -13.9 to 35.5 W/m2

, with an average of -0.5±3.4 W/m2 during 

field sampling. 

Epidemiologic

Demographics of Study Population: There were 1341 subjects who participated in the 15 beach study days

from December 2007 through June 2008; 38 (2.9 % were lost to follow up) resulting in a total cohort of 1303. 

After randomization, there were 652 bathers and 651 non-bathers (Table 5). Based on these results (as well 

as data not shown), the randomization of subjects into bather and non-bather exposure for the purposes of the 

study was successful in terms of equal distribution of demographics.

Table 5. Demographics of Study Population

H1: People exposed to recreational waters contaminated with infectious microorganisms will be 

significantly more likely to report symptoms than unexposed similar people who go to the 

beach but do not go into the water.

Gastrointestinal (GI) Symptoms

For the purposes of these analyses, of the 1341 participants enrolled in the study, 38 (2.91%) were lost to 

follow-up. Of the remaining 1303 study participants, 64 (4.8%), reported having a chronic condition that 

predisposed to symptoms of gastroenteritis (e.g., colitis, irritable bowel syndrome, etc.), or reported symptoms 

of gastroenteritis on the exposure day, and were excluded from the study analysis for gastrointestinal 

symptoms. Therefore the final sample size for the gastrointestinal symptom analyses was 1239.

Prior to analysis, gastrointestinal symptoms were categorized as either: all cases of vomiting or diarrhea, or all 

reported cases of indigestion or nausea accompanied by a fever. “Diarrhea” was defined as having three or 

Variable All Participants Bather Non-bather p

N (%) Bather Status 1303 (100) 652 (50.04) 651 (49.96)

Mean Age (+/- SE) 32.39 +/- 12.97 22.98+/- 253.68 29.01+/- 57.92 0.52

N (%) Female 751 (57.64) 370 (49.27) 381 (50.73) 0.54

N (%) Hispanic ethnicity 499 (38.30) 243 (48.70) 256 (51.30) 0.40

N (%) Occupation

-Office

-Catering/leisure

-Agriculture

-Student/School/University

-Factory

-Caring for Others

-Building/Construction

-Other

1299 (100)

295 (22.71)

14 (1.08)

10 (0.77)

572 (44.03)

8 (0.62)

62 (4.77)

37 (2.85)

301 (23.17)

141 (47.80)

9 (64.29)

5 (50)

285 (49.83)

7 (87.50)

26 (41.94)

23 (62.16)

154 (51.16)

154 (52.20)

5 (35.71)

5 (50)

287 (50.17)

1 (12.50)

36 (58.06)

14 (37.84)

147 (48.84)

0.12

SE= Standard Error
21

more runny stools within a 24-hour period. This categorization of symptoms of gastroenteritis was, therefore, 

less likely to be affected by inter-individual variation in perception of specific symptoms, and thus provided a 

more reliable estimate of the occurrence of gastrointestinal symptoms among study subjects. Furthermore, 

this categorization of symptoms was less likely to be subject to any recall bias among bathers or non-bathers, 

given the nature of the symptom and the relatively short period between exposure and follow-up. In addition, 

this categorization of symptoms was quite similar to those used other published large prospective study of 

recreational waterborne illness, and thus provides the opportunity for comparison of results Fleisher et al. 

(1991, 1993, 1996, 1998).

Overall, analysis of the crude rates of gastrointestinal symptoms showed bathers to be 1.76 times more likely 

to suffer gastrointestinal symptoms relative to Non-bathers (odds ratio=1.76; 95% Confidence interval 

(CI)=0.98–3.19; p=0.057). A variety of confounding factors were considered (only statistically significant 

comparisons shown), including: age, gender, number of people in household, medications and illnesses in the 

weeks prior to the study exposure, the foods consumed from 3 days prior to throughout the 7 day follow up 

period including any “take out” food (bathers vs non-bathers: 54.2% vs 45.8%; p=0.02), illness in the 

household throughout the 7 day follow up period (69.6% vs 34.4%; p=0.0002), and additional bathing during 

the 7 day follow up period. 

The crude rates shown above did not take into account the possible confounding effect due to non-bathingwater-related risk factors for gastroenteritis. Adjustment for possible confounders/extraneous risk factors 

(Table 6) still demonstrated that Bathers were at the same elevated risk relative to non-bathers (1.76 (0.94-

3.30); p=0.07). These analyses confirmed that the observed differences in the overall rate of gastrointestinal

symptoms among bathers vs. non-bathers were not confounded by the non-water-related risk factors 

evaluated in this study.

Table 6. Results of Unconditional Multiple Logistic Regression Analysis: Bathers vs Non-bathers

Risk factor Chi 

square

P 

value

Odds Ratio (95% Confidence 

Interval)

GI Symptoms 3.17 0.07 1.76 (0.94-3.30)

Excessive tiredness 3 week prior

to initial interview lasting <24 hours 

7.21 0.007 4.65 (1.51–14.27)

Eat salad after Trial day 4.54 0.03 0.75 (0.57–0.98)

Anybody else in household ill since 

exposure day

7.91 0.005 0.49 (0.30-0.81)

Eat Ice-Cream since trial day 3.50 0.06 0.79 (0.62-1.01)

H2: The organism densities sampled at the time of exposure in the marine waters will be associated

with reported human symptoms

Gastrointestinal (GI) Symptoms & Enterococcus (MF)

The crude rates of GI symptoms are shown in Table 7 among non-bathers compared to bathers at quartiles of 

Enterococci exposure (personal samples by the membrane filtration method). There was a statistically 

significant trend between increasing Enterococci counts and increasing risk of gastrointestinal symptoms 

(p=0.04). Using Enterococci as the measure of exposure, there was a statistically significantly increased risk 

of 2.89 times for gastrointestinal symptoms for Bathers compared to non-bathers at highest quartile of 

exposure (2.89; 1.23–6.76; p=0.006). 

The crude rates of GI symptoms among non-bathers compared to bathers in increasing 20 unit intervals of 

Enterococci exposure are shown in Table 7. There was a statistically significant trend between increasing 

Enterococci counts and increasing risk of GI symptoms (p=0.01). There was an increased risk of 2.47 times for 

GI symptoms for Bathers compared to non-bathers at highest quartile of exposure (0.96-6.10; p=0.052), 

although not quite reaching statistical significance. Bathers exposed to greater than 40 Enterococci at higher 

risk relative to bathers exposed to less than 40 Enterococci /100 mL of individual sample. In addition, bathers 

exposed to less than 40 enterococci/100 ml had rates of illness similar to the non-bathers.
22

The crude rates shown above did not take into account the possible confounding effect due to non-bathingwater-related risk factors for gastro-enteritis. Subsequent analyses, therefore, assessed the effect of 

enterococcus on the reported illness among bathers, to assess the effect of confounding by non-water-related 

risk factors for gastroenteritis, including diarrhea lasting 24 hours within 3 weeks of the study (Ill Bather vs non 

ill bather: 25.0% vs 75.0%; p=0.06), and taking any medicines in the 4 weeks prior to exposure (8.1% vs 

91.9%; p=0.008). 

Table 7. Crude Rates of gastrointestinal symptoms among non-bathers vs. bathers by Enterococcus levels in 

personal samples

Exposure Status N Rate/100 P value (Trend)

Enterococcus Quartiles

Non-bathers 620 2.90 0.04

Bathers (0-4) 171 5.85

Bathers (5-20) 145 2.07

Bathers (21-61) 149 4.70

Bathers (61-3320) 99 4.14

Enterococcus counts by 20

Non-bathers 620 2.90 0.01

Bathers (0-20) 306 4.25

Bathers (21-40) 102 2.94

Bathers (41-60) 53 7.55*

Bathers (61-80) 40 7.50*

Bathers (81+) 118 6.78*

Enterococcus measurements by membrane filtration method; *p < 0.10

Unconditional multiple logistic regression analysis was used to model the relationship between Enterococci 

exposures less than vs. greater than or equal to 40 CFU/100 mL and GI illness among bathers while 

controlling for non-water-related risk factors/confounders. Thus, bathers were at 2.05 greater risk of being ill 

when exposed to enterococcus levels greater than or equal to 40 CFU/100 mL than bathers exposed to 

enterococcus levels less than 40 CFU/100 mL (2.05 (0.97-4.30); p=0.059) (Table 8).

Table 8. Multiple logistic regression analyses among Ill vs Non Ill bathers and enterococcus exposure less than 

40 CFU/100 mL vs. greater than 40 CFU/100 mL while controlling for possible non-water related risk 

factors/confounders.

Risk factor Chi square P value Odds Ratio (95% Confidence Interval)

Enterococcus * 3.58 0.059 2.05 (0.97–4.30)

Diarrhea 3 or more times

lasting 24 hours during

the 3 weeks prior to exposure 

4.21 0.04 0.17 (0.03-0.92)

Taking Tablets/ Medicines 

taken 4 weeks prior to exposure

5.91 0.01 0.39 (0.18 – 0.83)

*Enterococci modeled less than 40 vs. greater than or equal to 40 enterococci/100 ml.

DISCUSSION

The objectives of this Pilot Epidemiologic Study were to begin to evaluate the relationship between microbe 

densities and reported symptoms in human subjects with random assignment to recreational marine water 

exposure and intensive individual microbial monitoring in subtropical recreational marine waters with no known 

point source. Our study results indicated that there was an increased risk of reporting symptoms with bathing 

exposure at the non point source subtropical recreational marine water. Furthermore, there appeared to be an 
23

association between enterococcus (by membrane filtration method) levels and report of bather symptoms at or 

above 40 CFU/100 mL of enterococcus based on an individual sample. The Study Site, which is a non-point 

source of fecal contamination, showed high levels of indicator microbes intermittently during this study. 

Nevertheless, these intermittent high levels of indicator microbes did not correlate on a one-to-one basis with 

the presence or absence of these pathogens; sometimes pathogens were detected when enterococci levels 

were elevated, and sometimes pathogens were not detected during periods of elevated enterococci (see 

Tables 2-4).

Environmental Sampling

Water samples collected by individual epidemiologic study participants contain elevated levels of enterococci. 

The percentage of the total individual samples that exceeded the single sample limit of enterococci 

(104/100mL) as recommended in the US EPA guideline were 13% based on the MF and CS methods. This 

result was consisted with the monitoring data as collected by the Florida Department of Health (FL DOH 2008)

which also monitored the same Study Site during the previous year. During four days (March 8th, March 22nd, 

May 18th, and June 21st, 2008) during the 15- day monitoring period, the geometric means of individuals on 

each sampling day exceeded 35/100mL (US EPA 1986) based on the MF and CS methods. Additionally, the 

geometric mean of individual samples on January 26th, 2008 also exceeded 35/100 mL based on the CS 

method. S. aureus were detected from most individual samples, but there is no guideline for dermatitis illness. 

S. aureus levels were neither correlated with enterococci nor environmental parameters. The source of S. 

aureus is believed to be the bathers themselves. 

Viruses and protozoans were not detected from the composite samples unlike the special samples. This 

result was not likely due to differences in the samples, but rather to the different sample concentration methods 

used to process each type of sample. The special samples were processed based on the conventional large 

filtration method while the composite samples were processed based on the alternative Bilayer method, which 

is applicable for small water sample volumes with high concentrations of microbes. Since the water at the 

Study Site contained very low levels of viruses and protozoan, they were not detected from the composite 

samples which were based on the analysis of smaller sample volumes. 

 

Limitations and Strengths (Pruss 1998, Wade 2003, Fleisher 2006, Craun 2006, EPA 2007, Boehm in press)

There are a number of limitations to this Pilot Epidemiologic study, in part due to limited resources. First, there 

was no objective measurement of symptoms or illness, but rather self report; ideally, objective ascertainment 

by healthcare providers at 7 and also if possible 21 days after exposure would be important. Of note, future

data analyses will be performed to evaluate the possibility that increased risk perception may lead to increased 

self report of disease by the participants (Fleisher 2006). 

The lack of children in this study was also a limitation since children have been identified as the potential high 

risk group from exposure to microbial pollution of recreational marine waters (Wade 2008; EPA 2007). In 

addition to the Pilot Epidemiologic Study, the investigators have performed some pilot studies of bather 

shedding in toddlers wearing diapers and plan to pursue additional studies in children in the future. By using 

only residents, not tourists, and in particular residents that were willing to go bathing at the Study Site, there 

was some indication from the Hawaii studies (Fujioka 1994) that we may have selected for persons less 

susceptible to illnesses caused by exposure to microbial pollution in recreational marine waters due to self 

selection and the exclusion of persons with obvious immunologic susceptibility

Nevertheless we believe that our study had a number of important strengths (Fleisher 1991, Fleisher 1993, 

Fleisher 1996, Fleisher 1998, Pruss 1998, Wade 2003, Fleisher 2006, Craun 2006, EPA 2007, Boehm in 

. Nevertheless, as Fleisher et al 

found increased report of illness among their resident participants in the United Kingdom, we also found an 

increased risk of reported illness even among our potentially less susceptible resident bather participants in the 

exposed bather group for report of gastrointestinal symptoms.

As noted above, individual monitoring was only able to evaluate the indicator organism (enterococcus) and the 

pathogen, S. aureus; due to expense and logistics, additional individual pathogen monitoring was not possible, 

only daily for the 15 study days. 
24

press). To our knowledge, this was the first study performed in the US as a randomized design, and the first 

study in the world as a randomized design in non point source sub/tropical recreational marine waters. As

noted above, we believe that the randomized design is very important since it minimizes the biases contained 

in previous non-randomized studies as explained above. We were also able to perform extensive individual 

environmental sampling in a Study Site that had been extremely well characterized as a non point source. 

Finally, we were able to capture a wide variability in the indicator organism measurements as well as perform 

the study with a large sample size over a 7 month period with variable environmental conditions.

CONCLUSIONS & RECOMMENDATIONS

The results of this study demonstrated that even non point source recreational marine waters, at least in 

sub/tropical areas, should be evaluated for possible exposures and health risks to human bathers. 

Furthermore, although our results indicated some association between the currently used indicator organism, 

enterococcus, and reported health risks in human bathers, the apparent lack of direct association between the 

enterococcus association and measurement of pathogens needs to be evaluated further. Future studies 

should also focus on developing fate and transport models, quantitative microbial risk assessments, and 

epidemiologic studies which incorporate human, microbiological and oceanographic data. 
25

REFERENCES

Abdelzaher AM, Solo-Gabriele HM, Wright ME, Palmer CJ. Sequential concentration of bacteria and viruses 

from marine waters using a dual membrane system. J Environ Qual 2008;23;37(4):1648-55. 

American Public Health Association (APHA). Standard Methods for the Examination of Water and Wastewater, 

19th ed. American Public Health Association, Inc., Washington D.C, 1995.

Atmar RL, Neill FH, Romalde JL, et al. Detection of Norwalk virus and hepatitis A virus in shellfish tissues with 

the PCR. Appl Environ Microbiol 1995;61:3014-3018

Boehm AB, Ashbolt NJ, Colford JM, Dunbar LE, Fleming LE, Gold MA, Hansel J, Hunter PR, Ichida AM, 

McGee CD, Soller JA, Weisberg SB. A sea change ahead for recreational water quality criteria, Journal of 

Water and Health in press.

Boehm AB. Enterococci concentrations in diverse coastal environments exhibit extreme variability. Environ Sci 

Technol 2007;15;41(24):8227-32.

Bofill-Mas S, Pina S, Girones R. Documenting the epidemiologic patterns of polyomaviruses in human 

populations by studying their presence in urban sewage. Appl Environ Microbiol 2000;66:238-45.

Burkhardt III W, Calci KR, Watkins WD, et al. Inactivation of Indicator Microorganisms in Estuarine Waters. 

Water Research 2000;34(8):2207-2214

Cabelli VJ, Dufour AP, McCabe LJ, Levin MA. Swimming associated gastroenteritis and water quality. Am J 

Epidemiol 1982;115:606–616.

Calderon RL, Mood EW, Dufour AP. Health effects of swimmers and nonpoint sources of contaminated water. 

International Journal of Environmental Health Research 1991;1(1):21– 31.

Charoenca N, Fujioka RS. Assessment of Staphylococcus Bacteria in Hawaii's Marine Recreational Waters. 

Water Science and Technology 1993;27(3/4): 283-289.

Charoenca N, Fujioka RS. 1995. Association of staphylococcal skin infections and swimming. Water Science 

and Technology 1995;31(5-6):11-17.

Colford JM Jr, Wade TJ, Schiff KC, Wright CC, Griffith JF, Sandhu SK, Burns S, Sobsey M, Lovelace G, 

Weisberg SB. Water quality indicators and the risk of illness at beaches with nonpoint sources of fecal 

contamination. Epidemiology 2007;18(1):27-35.

Craun GF, Calderon RL, Wade TJ. Assessing waterborne risks: an introduction. J Water Health 2006;4 Suppl 

2:3-18.

Current WL, Garcia L.S. Cryptosporidiosis. Clin Lab Med 1992;11:873–897.

Dufour AP. Health Effects Criteria for Fresh Recreational Waters. U.S. Environmental Protection Agency. EPA600/1-84-004, 1984. Cincinnati, OH. http://www.epa.gov/microbes/frc.pdf

Dwight RH, Fernandez LM, Baker DB, Semenza JC, Olson BH. Estimating the economic burden from illnesses 

associated with recreational coastal water pollution--a case study in Orange County, California. J Environ 

Management 2005;76(2):95-103. 

Dwight RH, Baker DB, Semenza JC, Olson BH. Health effects associated with recreational coastal water use: 

urban versus rural California. Am J Public Health 2004;94(4):565-7.
26

Elmir, SM, 2006. Development Of A Water Quality Model Which Incorporates Non-Point Microbial Sources. 

Ph.D. Thesis, University of Miami, Coral Gables, FL

Elmir SM, Wright ME, Solo-Gabriele HM, Abdelzaher A, Fleming LE, Miller G, Rybolowik M, Shih M-T P, Pillai

SP, Cooper JA, Quaye EA. Quantitative Evaluation of Bacteria Released by Bathers in a Marine Water. Water 

Research 2007;41:3-10 (with Supplement available on line)

Fleisher JM, Jones F, Stanwell-Smith R, et al. 1993. Water and non-water risk factors for gastroenteritis 

among bathers exposed to sewage-contaminated marine waters. International Journal of Epidemiology

1993;22:698-708.

Fleisher JM. A Re-Analysis of the Data Supporting U.S. Federal Bacteriological Water Quality Criteria Governing 

Marine Recreational Waters. Water Pollution Control Federation Journal 1991;63:259-265.

Fleisher JM, Kay D, Wyer MD, et al. Estimates of the severity of illnesses associated with bathing in marine 

recreational waters contaminated with domestic sewage. International Journal of Epidemiology 1998;27:722-

726.

Fleisher JM, Kay D, Jones F, et al. 1996. Marine Waters Contaminated with Domestic Sewage: Nonenteric Illness 

Associated with Bather Exposure in the United United Kingdom. American Journal of Public Health 

1996;86(9):1228-1234. 

Fleisher JM, Kay D. Risk perception bias, self-reporting of illness, and the validity of reported results in an 

epidemiologic study of recreational water associated illnesses. Mar Pollut Bull 2006;52(3):264-8. 

Fleming LE, Solo Gabriele H, Elmir S, Shibata T, et al. A Pilot Study of Microbial Contamination of Subtropical 

Recreational Waters. Fl J Env Health 2004;March:29-33.

Fowler TL, Fujioka RS, Tice AD, Ishikawa AK, Yoneyanna BS. Modification of CHROMagar staph. aureus to 

enumerate staphylococcus aureus from marine recreational waters in Hawaii. ASM 104th General Meeting; 

May, 2004. 

Fujioka RS, Roll K, Morens D. A Pilot Epidemiological Study of Health Risks Associated with Swimming at 

Kuhio Beach. Hawaii Water Resources Research Center, 1994.

Fujioka RS, Roll B, Byappanahilli M. Appropriate recreational water quality standards for Hawaii and other 

tropical regions based on concentrations of Clostridium perfringens. Proceedings of the Water Environment 

Federation, 70th Annual Conference and Exposition 1997;4: 406-411.

Fujioka, R.S., Shizumura, L.K. 1985. Clostridium perfringens, a reliable indicator of stream water quality. 

Journal Water Pollution Control Federation, 57(10): 986-992.

Gerba, C.P. 2000. Assessment of enteric pathogen shedding by bathers during recreational activity and its 

impact on water quality. Quantitative Microbiology 2(1), 55-68.

Given S, Pendleton LH, Boehm AB. Regional public health cost estimates of contaminated coastal waters: a 

case study of gastroenteritis at southern California beaches. Environ Sci Technol 2006;40(16):4851-8.

Griffin, D.W., K.A. Donaldson, J.H. Paul, and J.B. Rose. 2003. Pathogenic human viruses in coastal waters. 

Clin. Miicrobiol. Rev. 16:129-143.

Haas, C. N., J. B. Rose, and C. P. Gerba. 1999. Microbial agents and their transmission, p. 35-50. In C. N. 

Haas, J. B. Rose, and C. P. Gerba (ed.), Quantitative microbial risk assessment. J. Wiley and Sons, Inc., New 

York, N.Y.
27

Haile RW, Witte JS, Gold M, Cressey R, McGee C, Millikan RC, Glasser A, Harawa N, Ervin C, Harmon P, 

Harper J, Dermand J, Alamillo J, Barrett K, Nides M, Wang G. The health effects of swimming in ocean water 

contaminated by storm drain runoff. Epidemiology1999;10(4):355-63. 

Kay D, Bartram J, Prüss A, Ashbolt N, Wyer MD, Fleisher JM, Fewtrell L, Rogers A, Rees G. Derivation of 

numerical values for the World Health Organization guidelines for recreational waters. Water Res 

2004;38(5):1296-304.

Kueh CS, Tam TY, Lee T et al. Epidemiological study of swimming associated illnesses relating to bathing 

beach water quality. Wat SciTech 1995;31(5):1-4.

McQuaig SM, Scott TM, Harwood VJ, Farrah SR, Lukasik JO. Detection of Human-Derived Fecal Pollution in 

Environmental Waters by Use of a PCR-Based Human Polyomavirus Assay. Applied and Environmental 

Microbiology 2006;72(12):7567-7574.

Pruss A. Review of epidemiological studies on health effects from exposure to recreational water. International 

Journal of Epidemiology 1998;27(1):1-9.

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.

Shuval H. Estimating the global burden of thalassogenic diseases: human infectious diseases caused by 

wastewater pollution of the marine environment. J Water Health 2003;1(2):53-64. 

Solo-Gabriele HM, Fleming L, Elmir S. A Pilot Study Evaluation and Sanitary Survey of Microbial Recreational 

Water Quality Indicators in the Subtropical Marine Environment. Prepared for the National Institute of 

Environmental Health Sciences (NIEHS) Marine and Freshwater Biomedical Sciences Center, Rosenteil 

School of Marine and Atmospheric Sciences and the Miami-Dade County Health Department, 2002.

Solo-Gabriele, HM, et al., 2008. Pathogen Monitoring of a South Florida Beach Which Has Been 

Characterized by Elevated Microbe Levels. Final Report Submitted to the Florida Department of Health, 

Tallahasses, FL.

US Environmental Protection Agency (US EPA). Method for Detection and Enumeration of Clostridium 

perfringens in Water and Sediments by Membrane Filtration, EPA-600-R-95-030. U.S. Environmental 

Protection Agency, Edison Laboratory, Environmental Services Division, Region 2 and Environmental 

Monitoring Systems Laboratory - Cincinnati, OH, 1995.

United States Environmental Protection Agency (USEPA). Report of the Experts Scientific Workshop on 

Critical Research Needs for the Development of New or Revised Recreational Water Quality Criteria.

Washington, DC. EPA 823-R-07-006, http://www.epa.gov/waterscience/criteria/recreation/, 2007.

US Environmental Protection Agency (US EPA). Membrane Filter Test Method for Enterococci in Water, EPA821-R-97-004. U.S. Environmental Protection Agency, Office of Water, Washington D.C, 1997.

US Environmental Protection Agency (US EPA). Improved Enumeration Methods for the Recreational Water 

Quality Indicators: Enterococci and Escherichia coli. U.S. Environmental Protection Agency, Office of Science 

and Technology, Washington DC, 2000.

US Environmental Protection Agency (US EPA). US EPA Method 1623, Cryptosporidium and Giardia in water 

by filtration/IMS/FA, EPA-821-R-01-025, April 2001, Office of Water, Washington D.C. 
28

Wade TJ, Calderon RL, Brenner KP, Sams E, Beach M, Haugland R, Wymer L, Dufour AP. High sensitivity of 

children to swimming-associated gastrointestinal illness: results using a rapid assay of recreational water 

quality. Epidemiology 2008;19(3):375-83.

Wade TJ, Calderon RL, Sams E, Beach M, Brenner KP, Williams AH, Dufour AP. Rapidly measured indicators 

of recreational water quality are predictive of swimming-associated gastrointestinal illness. Environ Health 

Perspect 2006;114(1):24-8.

Wade TJ, Pai N, Eisenberg JN, Colford JM Jr. Do U.S. Environmental Protection Agency water quality 

guidelines for recreational waters prevent gastrointestinal illness? A systematic review and meta-analysis.

Environ Health Perspect 2003;111(8):1102-9. 

Wright ME. Evaluation of Enterococci, an Indicator Microbe, and the Sources that Impact the Water Quality at 

a Subtropical Non-Point Source Recreational Beach. Masters Thesis in Engineering. Coral Gables (FL): 

University of Miami, 2008.

Yamahara KM, Layton BA, Santoro AE, Boehm AB.Beach sands along the California coast are diffuse sources 

of fecal bacteria to coastal waters. Environ Sci Technol. 2007;41(13):4515-21.
29

APPENDICES

Human Subjects (IRB) Approval

• UM

• FL DOH

Study Consent Form (English & Spanish)

Study Questionnaires (English & Spanish)

- Of note, in this study, we combined the 2 follow up questionnaires due to limited resources: we 

used the first page of the 7 day follow up and then the entire Postal Questionnaire; both 

questionnaires were administered over the phone in English and Spanish at day 7 after the Study 

exposure.

- The original Questionnaires were provided by Dr David Kay (dave@crehkay.demon.co.uk) and 

have been used in similarly designed studies in Europe