Detection of spatial fluctuations of non-point source fecal pollution in coral reef surrounding waters in southwestern Puerto Rico using PCR-based assays M. Bonkosky a , E.A. Hernández-Delgado b, * , B. Sandoz a , I.E. Robledo c , J. Norat-Ramírez a , H. Mattei a a Department of Environmental Health, University of Puerto Rico, Medical Sciences Campus, Graduate School of Public Health, P.O. Box 365067, San Juan, 00936-5067, Puerto Rico b UPR, Department of Biology, Center for Applied Tropical Ecology and Conservation, Coral Reef Research Group, P.O. Box 23360, San Juan 00931-3360, Puerto Rico c UPR–MSC, Microbiology and Medical Zoology Department, P.O. Box 365067, San Juan 00936-5067, Puerto Rico article info Keywords: Bacteroides Clostridium coccoides Coral reefs Microbial source tracking Non-point source coastal fecal pollution PCR abstract Human fecal contamination of coral reefs is a major cause of concern. Conventional methods used to monitor microbial water quality cannot be used to discriminate between different fecal pollution sources. Fecal coliforms, enterococci, and human-specific Bacteroides (HF183, HF134), general Bacteroides-Prevo- tella (GB32), and Clostridium coccoides group (CP) 16S rDNA PCR assays were used to test for the presence of non-point source fecal contamination across the southwestern Puerto Rico shelf. Inshore waters were highly turbid, consistently receiving fecal pollution from variable sources, and showing the highest fre- quency of positive molecular marker signals. Signals were also detected at offshore waters in compliance with existing microbiological quality regulations. Phylogenetic analysis showed that most isolates were of human fecal origin. The geographic extent of non-point source fecal pollution was large and impacted extensive coral reef systems. This could have deleterious long-term impacts on public health, local fish- eries and in tourism potential if not adequately addressed. Ó 2008 Published by Elsevier Ltd. 1. Introduction Fecal contamination of coastal waters is a paramount concern in tropical developing countries (Byamukama et al., 2005). Wastewa- ter pollution can negatively affect both the marine environment and human health (Fong and Lipp, 2005). In this context, it is important to determine sources of fecal contamination in order to prevent diseases and assure water quality (Scott et al., 2002), since many of these areas are used for fishing and recreational pur- poses. Non-point source fecal pollution is a major threat to coral reefs, often resulting in an increased stress to benthic communities (Pastorok and Bilyard, 1985), an increased frequency of coral mor- tality (Frías-López et al., 2002; Kaczmarsky et al., 2005), and in long-term phase shifts in benthic community structure (Done, 1992). Chronic sewage pollution often prevents natural coral reef recovery following natural disturbance (Jokiel et al., 1993). How- ever, long-term fecal pollution impacts in coral reefs have been poorly documented. This could often be the result of lack of fund- ing, trained personnel or laboratory facilities, or the inadequacy of standard microbial indicators to detect potential impacts. The United States Environmental Protection Agency (USEPA) recommended the use of Escherichia coli, a member of the fecal coliform group, as well as enterococci, as microbiological indica- tors of fecal pollution for marine waters (USEPA, 2000). However, it has been suggested that E. coli may not be a reliable indicator in tropical and subtropical environments, because of its ability to replicate in polluted soils (Desmarais et al., 2002; Solo-Gabriele et al., 2000), as well as in tropical freshwater and coastal environ- ments (Hazen and Toxanzos, 1990; Hernández-Delgado, 1991). Members of Bacteroides spp. have been suggested as alternate fecal pollution indicators (Allsop and Stickler, 1985; Fiksdal et al., 1985; Dick et al., 2005) as it constitutes one of the most numerous mem- bers of the human colonic flora (Finegold et al., 1983; Sghir et al., 2000). Bacteroides spp. is Gram-negative, anaerobic bacilli or coc- co-bacilli, each species is morphologically distinct, and most are encapsulated, and are capable of outcompeting other members of the gastrointestinal flora. They can also be opportunistic patho- gens, causing a variety of infections throughout the body (Sheehan and Harding, 1989). However, their use as indicator organisms using standard plate culturing techniques has been very limited due to their complicated growth requirements. Recently, a series of novel culture-independent microbial source tracking (MST) methods have been developed to determine the source of fecal contamination (Field et al., 2003; Simpson et al., 2004; Shanks et al., 2006). One of these methods consists in detect- ing host-specific molecular markers using the 16S rRNA gene of Bacteroides (Bernhard and Field, 2000a,b). These molecular markers can amplify DNA sequences from this potential microbial fecal indicator and identify its source (whether it is human or animal) 0025-326X/$ - see front matter Ó 2008 Published by Elsevier Ltd. doi:10.1016/j.marpolbul.2008.09.008 * Corresponding author. Tel.: +787 764 0000x2009; fax: +787 764 2610. E-mail address: coral_giac@yahoo.com (E.A. Hernández-Delgado). Marine Pollution Bulletin 58 (2009) 45–54 Contents lists available at ScienceDirect Marine Pollution Bulletin journal homepage: www.elsevier.com/locate/marpolbul