Preliminary evidence for human fecal contamination in corals of
the Florida Keys, USA
Erin K. Lipp
, Jennifer L. Jarrell
, Dale W. Griﬃn
, Jerzy Lukasik
, Joan B. Rose
College of Marine Science, University of South Florida, 140, 7th Ave. S, St. Petersburg, FL 33701, USA
Biosecure, 4641 W 6th St. Suite A, Gainesville, FL 32609, USA
Corals and reef environments are under increased stress from anthropogenic activities, particularly those in the vicinity of heavily
populated areas such as the Florida Keys. The potential adverse impacts of wastewater can aﬀect both the environment and human
health; however, because of the high decay rate of bacterial indicators in coral reef waters it has been diﬃcult to document the
presence of microbial contaminants and to assign risks in these environments. Here we show initial evidence that microorganisms
associated with human feces are concentrated along the surface of coral heads relative to the overlying water column in the Florida
Keys. Bacterial indicators (fecal coliform bacteria, enterococci or Clostridium perfringens) were detected in 66.7% of the coral surface
microlayer (CSM) samples at levels between ﬁve and 1000 CFU/100 ml, but were found infrequently and at low numbers in the
overlying water column ( 62.5 CFU/100 ml). Similarly, enterovirus nucleic acid sequences, an indicator of human-speciﬁc waste,
were detected in 93.3% of the CSM samples and only once in the water column by cell culture. Results show that coral mucus may
accumulate enteric microorganisms in reef environments, and may indicate a risk to public and environmental health despite low
indicator levels in the surrounding water. Ó2002 Elsevier Science Ltd. All rights reserved.
Keywords: Pollution; Corals; Enteroviruses; Indicator bacteria; Florida Keys; Coral mucus; RT-PCR
Corals and reef environments are under increased
stress from anthropogenic activities, particularly those
in the vicinity of heavily populated areas such as the
Florida Keys. Researchers have noted a loss of diversity
in corals and an increase in diseased or damaged corals
throughout the Florida Keys National Marine Sanc-
tuary (Dustan and Halas, 1987; Harvell et al., 1999;
Porter et al., 1999). In a recent review of coral disease by
Green and Bruckner (2000), the authors noted that 97%
of coral disease in the Caribbean region was docu-
mented in reefs moderately to highly impacted by
human activities. Risks to public health may also be an
issue in these areas. Recently, human waste has been
found to contribute to a high prevalence of enteric vi-
ruses in nearshore waters and canals of the Florida
Keys, and a risk to swimmers in Key West has been
documented (Griﬃn et al., 1999; Nobles et al., 2000).
Despite this, conclusive evidence that wastewater is
reaching and adversely impacting Keys’ reef environ-
ments and corals is lacking and the topic remains con-
troversial. The potential adverse impacts of wastewater
aﬀect both the environment and human health; however,
because of the high decay rate of bacterial indicators in
coral reef waters it has been diﬃcult to document the
presence of microbial contaminants and to assign risks.
Previous studies have shown that fecal bacteria
and viruses from human waste do not survive long in
saline, warm and highly transparent waters generally
found in reef and other marine environments (Solic and
Krstuvolic, 1992). Yet in marine sediment, enteric
Corresponding author. Present address: Department of Environ-
mental Health Science, University of Georgia, 206 Environmental
Health Science Building, Athens, GA 30602, USA. Tel.: +1 706 542
2454; fax: +1 706 542 7472.
E-mail address: email@example.com (E.K. Lipp).
Present address: North Inlet––Winyah Bay National Estuarine
Research Reserve, Georgetown, SC 29442, USA.
Present address: United States Geological Survey, Center for
Coastal Geology and Regional Marine Studies, St. Petersburg, FL
0025-326X/02/$ - see front matter Ó2002 Elsevier Science Ltd. All rights reserved.
PII: S0 0 2 5 - 3 2 6 X ( 0 1 ) 0 0 3 3 2 - 0
Marine Pollution Bulletin 44 (2002) 666–670
microorganisms have been detected at signiﬁcantly ele-
vated levels relative to the overlying water column (Lipp
et al., 2001a). A similar accumulation and enhanced
survival may also occur at the coral surface, where
overlying water column conditions may promote rapid
die-oﬀ of bacteria and viruses from wastewater. Indeed,
high levels of native bacteria, relative to the overlying
water column, have been noted (Paul et al., 1986), and
bacteria may be chemotactically attracted to mucus
(Ducklow and Mitchell, 1979). Thus, we hypothesized
that the surface microlayer of coral heads (mucus) might
also accumulate microbial indicators of waste and
human viruses, and could thereby provide more direct
evidence for human impacts on reef environments. To
our knowledge this is the ﬁrst report on the detection of
fecal indicators and human enteric viruses concentrated
in coral surface microlayers (CSM).
2. Materials and methods
One-liter grab samples of surface water were collected
from four sites, between Long Key and Marathon, in
the Florida Keys National Marine Sanctuary in March
2000 (Fig. 1). At each site, a large volume of surface
water (100 l) was also concentrated by adsorption/elu-
tion for cell culture analysis of enteroviruses (Lipp et al.,
2001b; USEPA, 1994). In addition, surface microlayers
(mucus) from 15 corals were sampled by gentle aspira-
tion with a sterile 60 ml syringe. Corals included Mon-
tastraea annularis complex, Siderastraea radians,S.
sideraea and Solenastraea bournouni. Physical and
chemical conditions observed at each site are listed in
Indicator bacteria were enumerated by membrane
ﬁltration and growth on selective media. Duplicate
aliquots of water and mucus were vortexed and ﬁltered
onto 47 mm, 0.45 lm pore size, ﬁlters and placed on
appropriate selective media. For Clostridium perfringens,
ﬁlters were incubated on mCP medium (AcuMedia,
Baltimore, MD) at 45 °C for 24 h. Yellow colonies that
turned pink upon exposure to ammonium hydroxide
fumes (30 s) were counted (Bisson and Cabelli, 1979).
Filters were incubated on mEI medium at 41 °C for 24 h
and colonies with a blue halo were counted as entero-
cocci (USEPA, 1997). Fecal coliform bacteria were
enumerated on mFC medium; blue colonies were coun-
ted after incubating at 44.5 °C for 24 h (APHA, 1992).
Coliphage were detected and enumerated using the agar
overlay method of Adams (1959), with an Escherichia
coli host (ATCC strain 15597). Five replicate plates of 2
ml aliquots were assayed for each sample and plaques
were counted after 18–24 h incubation at 37 °C.
To assay for infectious enteroviruses in the water
column, concentrated samples were inoculated onto
BGM (Buﬀalo Green Monkey kidney) cells and exam-
ined for cytopathogenic eﬀects for six weeks (repre-
senting three passages). CSM samples were tested for
enteroviruses using RT-PCR followed by internal probe
hybridization. Viral RNA was extracted using the
RNeasy Kit from Qiagen (Valencia, CA) and 10 llof
puriﬁed RNA was used in RT-PCR according to the
conditions described by De Leon et al. (1990). The tar-
get 197 bp amplicon was conﬁrmed by dot-blot hy-
bridization with a biotin-labeled probe internal to this
region, and detected by chemiluminescence (Griﬃn
et al., 1999, 2000; Table 1). Poliovirus and echovirus
were used as positive controls.
Fig. 1. Map of sampling stations in the Florida Keys. The dotted line box in the inset map of Florida shows the boundary of the Florida Keys
National Marine Sanctuary. The arrow shows the direction of the Florida Current as it passes to the south and east of the Florida Keys. Legend: Key
22––Channel 2; Key 23––100 m oﬀ Long Key, Florida Keys Marine Lab; Key 24––Marathon Government Center Boat Basin; Key 25––Long
Key, beach at Florida Keys Marine Lab.
E.K. Lipp et al. / Marine Pollution Bulletin 44 (2002) 666–670 667
Fecal coliform bacteria and enterococci were detected
only once in the water column (at Key 24) at 0.5 and 2.5
CFU/100 ml, respectively, while C. perfringens were
never found (Table 2). In contrast, all of the bacterial
indicators were detected at least once from CSM sam-
ples collected from three of the four sites (Key 22, 24,
25) (Table 3). Furthermore, nine of the 15 coral heads
sampled were positive for one or more of the fecal in-
dicators (Table 3). The highest indicator levels were
found in CSM samples from Key 24 with an average of
74 CFU fecal coliform bacteria, 252 CFU enterococci
and 33 CFU C. perfringens per 100 ml. Coliphage were
never detected in water or CSM samples.
Infectious enteroviruses were detected in the water
column only at Key 24 (Table 2), but enterovirus se-
quences were found in CSM samples from all stations
and 93.3% of the mucus samples (14/15) (Table 3, Fig.
2). In 40% of the cases, a strong signal was observed
(Fig. 2). Most positive samples were collected from M.
annularis complex, at Key 22, where the average depth
Primer and probe sequences for the speciﬁc detection of enteroviruses by RT-PCR (De Leon et al., 1990; Griﬃn et al., 1999, 2000)
Target Sequence Product size (bp)
50untranslated region (highly conserved) Primer 50-CCTCCGGCCCCTGAATG-30197
Physical and chemical conditions, and enteric microorganism in the water column overlying selected patch reefs
Site °C Salinity pH Tidal
Key 22 (Channel 2) 23.9 32 8.1 Flood <0.5 <0.5 <0.5 <10 <1
Key 23 (100 m oﬀ Long
Key Marine Lab)
24.7 35 8.0 Flood <0.5 <0.5 <0.5 <10 <1
Key 24 (Marathon Gov’t
28.0 35 7.7 Flood 0.5 2.5 <0.5 <10 1.13
Key 25 (beach at Long
Key Marine Lab)
26.0 35 7.9 Flood <0.5 <0.5 <0.5 <10 <1
Samples were collected between 11:00 a.m. and 3:15 p.m., over a period of two days.
Tide data were obtained from Tides and Currents Pro software v2.5b (Nautical Software, Beaverton, OR).
Enteric microorganisms in CSM samples (CFU or PFU/100 ml)
Site ID Species Fecal coliform
Coliphage Ampliﬁed enterovi-
Key 22 M1 Montastraea annularis complex <5<5<5<10 þþþ
M2 Montastraea annularis complex <5<530 <10 þþ
M3 Montastraea annularis complex <5<55 <10 þþþ
X<5<5 11.7 <10
Key 23 M4 Siderastraea sideria <5<5<5<10 þ
M5 Solenastraea bournouni <5<5<5<10 þþ
Key 24 M6 Siderastraea radians 60 80 10 <10 þ
M7 Siderastraea radians 70 55 15 <10 þ
M8 Siderastraea radians 115 1000 20 <10 þ
M9 Siderastraea radians 50 50 90 <10 þ
M10 Siderastraea radians 75 75 30 <10 þþ
X 74 252 33 <10
Key 25 M11 Siderastraea radians <5<5<5<10 þ
M12 Siderastraea radians <5<5<5<10 þ
M13 Siderastraea radians <5<5<5<10
M14 Siderastraea radians <5<525 <10 þ
M15 Siderastraea radians <565<5<10 þþ
X¼mean concentration in CSM per site).
668 E.K. Lipp et al. / Marine Pollution Bulletin 44 (2002) 666–670
of sampled coral heads was 1.5 m. Strong positive
samples were also found at Key 23 from S. bournouni
(collected at 1 m). Only one strong positive was noted
from Siderea radians samples collected at Key 24, where
the average depth was 0.25 m.
Communities in the Florida Keys rely almost exclu-
sively on on-site wastewater disposal (Paul et al., 2000).
There are at least 24,000 septic systems and 5000–10,000
illegal cesspools, which are underlain by a porous
limestone substrate (Paul et al., 2000; Shinn et al., 1994).
Previous work has traced the migration of wastewater in
septic tanks and shallow injection wells to nearshore
environments throughout the Florida Keys (Griﬃn
et al., 1999; La Pointe et al., 1990; Paul et al., 1995, 1997,
2000). In particular, areas sampled at Long Key (Key 23
and 25) and Marathon (Key 24) have been shown to
receive wastewater from septic systems on those res-
pective islands, based on viral tracers studies (Paul et al.,
1997, 2000). Despite tracer evidence for wastewater
migration, it has been diﬃcult to routinely document
contamination because indicators rapidly die oﬀ in these
warm, saline and highly transparent waters (Solic and
Krstuvolic, 1992). Here we have shown that relative to
the overlying water column, coral mucus can accumu-
late enteric bacteria and viruses in near shore patch reefs
of the Florida Keys that are inﬂuenced by poor waste-
water treatment and disposal practices (e.g., septic sys-
tems and shallow injection wells).
While bacterial indicators, in general, were found at
higher levels in the CSM, we observed diﬀerences in the
frequency of detection of certain indicators. Clostridium
perfringens was the most prevalent fecal indicator in
CSM samples but was never recovered from the water
column. We speculate that this disparity could have
been due to low oxygen conditions on some of the coral
heads, which might select for this anaerobic bacterium;
however D.O. was not measured. The ability of the
species to form spores might also have provided for
better survival. The absence of coliphage in all samples
was most likely related to a low tolerance for warm
saline waters; MS2 coliphage survives <36 h at 32‰and
30 °C (McLaughlin, 2000). Coliphage were also isolated
infrequently in previous studies in the Florida Keys
despite a high prevalence of enteric viruses (Griﬃn et al.,
Enteroviruses, which speciﬁcally indicate contami-
nation with human waste, were detected frequently
throughout the CSM samples, while only one water
column sample was positive. There was no correlation
between the presence of enteroviruses and indicator
bacteria; however, indicator bacteria are known to be
inadequate proxies for enteric viruses (e.g., Bitton
et al., 1983; Griﬃn et al., 2001; Havelaar et al., 1993;
Lipp et al., 2001a). Patterns of enterovirus detection in
coral mucus noted here suggest that there might be an
association between viral accumulation and speciﬁc
coral species and/or sample location and depth. Strong
virus signal was most common in samples from M.
annularis complex and/or those collected at greater
depths. Rohwer et al. (2001) suggest that speciﬁc bac-
teria-coral associations exist and, thus, it is possible
that the native microbial community between species
could diﬀerentially inﬂuence the stability of enteric vi-
ruses that might be accumulated in coral mucus. Fur-
thermore, diﬀerences in mucus composition could have
also aﬀected RT-PCR inhibition between sites and/or
species. Alternatively, samples collected at greater
depths tended to produce greater hybridization signal
intensity. Therefore, light attenuation could have
played a role in maintaining enteric viruses, and viral
nucleic acids, in CSM where they could be further
protected from photodamage (Johnson et al., 1997;
Lyons et al., 1998).
In this exploratory study we have shown that micro-
bial contaminants associated with human wastewater
can be isolated more frequently and at higher concen-
trations in coral mucus than in the overlying water
column in nearshore areas of the Florida Keys. The
CSM might oﬀer a better record of fecal contamination
in reef areas where fecal indicators and pathogens are
otherwise diﬃcult to detect. Evidence of human entero-
viruses also suggests a public health risk in these envi-
ronments, in the absence of detectable levels of fecal
indicators in the water column. Ongoing and future
work will explore changes in enteric bacterial and viral
levels over time and space, and will assess potential
wastewater impacts in oﬀshore reef areas in both rec-
reational and protected areas. Should similar observa-
tions be made along oﬀshore reefs, sampling of coral
mucus for enteric bacteria and viruses may oﬀer a means
of early and rapid detection of wastewater pollution
and, eventually, might be used to determine risk to coral
and reef health, as well as human health.
Fig. 2. Dot-blot hybridization for RT-PCR ampliﬁed enterovirus se-
quences detected by chemiluminescence. Row A, columns 1–12: M1–
M12. Row B, columns 1–3: M13–M15; columns 4, 5 and 6: poliovirus
positive control, echovirus positive control, negative control, respec-
E.K. Lipp et al. / Marine Pollution Bulletin 44 (2002) 666–670 669
Corals were sampled in the Florida Keys National
Marine Sanctuary under permit # FKNMS-2000-010.
We would like to thank Walter Jaap and Jennifer
Wheaton of the Florida Marine Research Institute,
Florida Wildlife Conservation Commission (FWCC) for
logistical support. We are also grateful to Dave Eaken
and John Dotten (FWCC) for sampling support and to
Eugene Shinn (USGS) for careful review of this manu-
Adams, M.H., 1959. Bacteriophages. Interscience Publications, New
American Public Health Association, 1992. Standard Methods for the
Evaluation of Water and Wastewater, 18th ed. Washington, DC.
Bisson, J.W., Cabelli, V.J., 1979. Membrane ﬁltration enumeration
method for Clostridium perfringens. Appl. Env. Microbiol. 37,
Bitton, G., Farrah, S.R., Ruskin, R.H., Butner, J., Chou, Y.J., 1983.
Survival of pathogenic and indicator microorganisms in ground
water. Ground Water 21, 405–410.
De Leon, R., Sheih, Y.-S.C., Baric, R.S., Sobsey, M.D., 1990.
Detection of enteroviruses and hepatitis A virus in environmental
samples by gene probes and polymerase chain reaction. In: Proceed-
ings of the Water Quality Conference San Diego, CA. American
Water Works Association, vol. 18, pp. 833–853.
Ducklow, H.W., Mitchell, R., 1979. Bacterial populations and
adaptations in the mucus layer on living corals. Limn. Ocean. 24,
Dustan, P., Halas, J.C., 1987. Changes in the reef-coral community of
Carysfort Reef, Key Largo, Florida: 1974–1982. Coral Reefs 6,
Green, E.P., Bruckner, A.W., 2000. The signiﬁcance of coral disease
epizootiology for coral reef conservation. Biol. Conserv. 96,
Griﬃn, D.W., Gibson, C.J., Lipp, E.K., Riley, K., Paul, J.H.,
Rose, J.B., 1999. Detection of viral pathogens by reverse transcrip-
tase PCR and of microbial indicators by standard methods in
the canals of the Florida Keys. Appl. Env. Microbiol. 65, 4118–
Griﬃn, D.W., Gibson, C.J., Lipp, E.K., Riley, K., Paul, J.H., Rose,
J.B., 2000. Detection of viral pathogens by reverse transcrip-
tase PCR and of microbial indicators by standard methods in the
canals of the Florida Keys (Erratum). Appl. Env. Microbiol. 66,
Griﬃn, D.W., Lipp, E.K., McLaughlin, M.R., Rose, J.B., 2001.
Marine recreation and public health microbiology: quest for the
ideal indicator. Bioscience 51, 817–825.
Harvell, C.D., Kim, K., Burkholder, J.M., Colwell, R.R., Epstein,
P.R., Grimes, J., Hofman, E.E., Lipp, E.K., Osterhaus, A.D.M.E.,
Overstreet, R., Porter, J.W., Smith, G.W., Vasta, G., 1999.
Diseases in the ocean: Emerging pathogens, climate links, and
anthropogenic factors. Science 285, 1505–1510.
Havelaar, A.H., Olphen, M.V., Drost, Y.C., 1993. F-speciﬁc RNA
bacteriophages are adequate model organisms for enteric viruses in
fresh water. Appl. Env. Microbiol. 59, 2956–2962.
Johnson, D.C., Enriquez, C.E., Pepper, I.L., Davis, T.L., Gerba, C.P.,
Rose, J.B., 1997. Survival of Giardia,Cryptosporidium,poliovirus
and Salmonella in marine waters. Wat. Sci. Tech. 35, 261–268.
La Pointe, B.F., O’Connell, J.D., Garrett, G.S., 1990. Nutrient
couplings between on-site waste disposal systems, groundwaters,
and nearshore surface waters of the Florida Keys. Biogeochemistry
Lipp, E.K., Kurz, R., Vincent, R., Rodriguez-Palacios, C., Farrah,
S.R., Rose, J.B., 2001a. The eﬀects of seasonal variability and
weather on microbial fecal pollution and enteric pathogens in a
subtropical estuary. Estuaries 24, 266–276.
Lipp, E.K., Lukasik, J., Rose, J.B., 2001b. Human enteric viruses and
parasites in the marine environment. In: Paul, J.H. (Ed.), Methods
in Microbiol., vol. 30. Academic Press, London, pp. 559–588.
Lyons, M.M., Aas, P., Pakulski, J.D., Van Waasbergen, L., Miller,
R.V., Mitchell, D.L., Jeﬀrey, W.H., 1998. DNA damage induced
by ultraviolet radiation in coral reef microbial communities. Mar.
Biol. 130, 537–543.
McLaughlin, M.R., 2000. Evaluation of the Bacteriodes fragilis phage
assay as an alternative indicator of sewage pollution. M.S. Thesis,
Department of Marine Science, University of South Florida, p. 72.
Nobles, R.E., Brown, P., Rose, J.B., Lipp, E.K., 2000. The investi-
gation and analysis of swimming-associated illness using the fecal
indicator enterococcus in southern Florida’s marine waters. Flor-
ida J. Env. Health 169, 15–19.
Paul, J.H., DeFlaun, M., Jeﬀery, W.H., 1986. Elevated levels of
microbial activity in the coral surface microlayer. Mar. Ecol. Prog.
Ser. 33, 29–40.
Paul, J.H., Rose, J.B., Brown, J., Shinn, E., Miller, S., Farrah, S.,
1995. Viral tracer studies indicate contamination of marine waters
by sewage disposal practices in Key Largo, Florida. Appl. Env.
Microbiol. 61, 2230–2234.
Paul, J.H., Rose, J.B., Jiang, S., Zhou, X., Cochran, P., Kellogg, C.,
Kang, J.B., Griﬃn, D., Farrah, S., Lukasik, J., 1997. Evidence for
groundwater and surface marine water contamination by waste
disposal wells in the Florida Keys. Wat. Res. 31, 1448–1454.
Paul, J.H., McLaughlin, M.R., Griﬃn, D.W., Lipp, E.K., Stokes, R.,
Rose, J.B., 2000. Rapid movement of wastewater from onsite
disposal systems into surface waters in the Lower Florida Keys.
Estuaries 23, 662–668.
Porter, J.W., Lewis, S.K., Porter, K.G., 1999. The eﬀects of multiple
stressors on the Florida Keys coral reef ecosystem: a landscape
hypothesis and a physiological test. Limn. Ocean 44, 941–949.
Rohwer, F., Breitbart, M., Jara, J., Azam, F., Knowlton, N., 2001.
Diversity of bacteria associated with the Caribbean coral Mon-
tastraea franksi. Coral Reefs 20, 85–91.
Shinn, E.A., Rees, R.S., Reich C.D., 1994. Fate and pathways of
injection-well eﬄuent in the Florida Keys. USGS Report 94–276,
Solic, M., Krstuvolic, N., 1992. Separate and combined eﬀects of solar
radiation, temperature, salinity and pH on the survival of fecal
coliforms in seawater. Mar. Poll. Bull. 24, 411–416.
US Environmental Protection Agency, 1994. Monitoring requirements
for public drinking water supplies: proposed rule. Federal Register
US Environmental Protection Agency, 1997. Method 1600: Membrane
Filter Test Method for Enterococci in Water. EPA-821-R-97-004.
670 E.K. Lipp et al. / Marine Pollution Bulletin 44 (2002) 666–670