Ernesto Otero

Improving fluorometry as a source tracking method to detect human fecal contamination

In a continuing effort to develop inexpensive source tracking methods to detect human fecal contamination in environmental waters, targeted sampling was combined with fluorometry. Targeted sampling works by identifying hot spots of fecal contamination through multiple samplings over ever-decreasing distances. Fluorometry identifies human fecal contamination by detecting optical brighteners, primarily from laundry detergents. Because organic matter fluoresces and interferes with fluorometry, two locations were chosen for sampling: waters relatively low in organic matter at Mayagüez Bay, Puerto Rico, and waters relatively high in organic matter at St. Simons Island, Georgia. In Puerto Rico, targeted sampling and fluorometry quickly and easily identified two hot spots of human fecal contamination in the Yagüez River, which flows through the city of Mayagüez. Another source tracking method, detection of theesp gene, confirmed their human origin. On St. Simous Island, targeted sampling and fiuorometry identified two hot spots of potential human fecal contamination. Detection of theesp gene confirmed the human origin of one site but not the other, most likely because background organic matter fluorescence interfered with fhiorometry. A separate experiment showed that adding a 436-um emission filter to the fluorometer reduced this background fluorescence by > 50%. With the 436-nm Filter in place, another sampling was conducted on St. Simons Island, and the second hot spot was identified as fecal contamination from birds. As long as the fluorometer was equipped with a 436-nm filter and organic matter concentrations were considered, targeted sampling combined with fluorometry was a relatively inexpensive method for identifying human fecal contamination in water.

Assessment of non-point sources of fecal pollution in coastal waters of Puerto Rico and Trinidad.

Traditional and molecular methods (PCR) were used to detect, quantify and identify the source of fecal pollution in coastal sites of Puerto Rico and Trinidad. Enterococci and Escherichia coli standard plate counts were used as a general indicator of fecal contamination while the PCR detection of Bifidobacteria adolescentis and human or bovine specific Bacteroidales were used to examine potential sources. Seven of 14 sites in Trinidad including Maracas Bay which is a major public beach contained significant fecal contamination based on enterococci numbers counts exceeding established thresholds for areas of direct contact. Forty six percent of the 27 stations in Puerto Rico were over the established thresholds for enterococci and 49% according to E. coli counts. About 31% of the stations examined in Puerto Rico had evidence of human derived fecal contamination. Human fecal pollution was detected in only one station from Trinidad. Bovine derived contamination was detected only once.

Detection of Helicobacter pylori in the coastal waters of Georgia, Puerto Rico and Trinidad

Fecal pollution in the coastal marine environments was assessed at eleven sampling locations along the Georgia coast and Trinidad, and nine sites from Puerto-Rico. Membrane filtration (EPA method 1604 and method 1600) was utilized for Escherichia coli and enterococci enumeration at each location. Quantitative polymerase chain reaction (qPCR) amplification of the 16S ribosomal RNA gene was used to determine the presence of the Helicobacter pylori in marine samples. There was no significant correlation between the levels of E. coli, enterococci and H. pylori in these water samples. H. pylori was detected at four of the 31 locations sampled; Oak Grove Island and Village Creek Landing in Georgia, Maracas river in Trinidad, and Ceiba Creek in Puerto Rico. The study confirms the potential public health risk to humans due to the widespread distribution of H. pylori in subtropical and tropical costal marine waters.

Spatial and temporal patterns of water quality indicators in reef systems of southwestern Puerto Rico.

Abstract. Baseline information on the water quality status of coral reefs and associated habitats of La Parguera, southwestern PR, is presented. Measurements of temperature, salinity, chlorophyll a, turbidity, fluorescence of dissolved organic matter, bacterial abundance and productivity, bacteriophytoplankton, sedimentation and stable isotopes of particulate organic matter were made at various stations within the area from 2003 through 2005. Significant daily fluctuations were observed at all stations and seasonal changes indicate inputs of materials from terrestrial sources even at the shelf edge. The &dgr;13C data indicates low inputs from terrestrial particles (min = -18 ‰ vs. PDB) while variations of dissolved organic matter fluorescence suggest periodic inputs of terrestrially derived matter and nutrients throughout the shelf, though most notable towards the coast. Chlorophyll a (mostly > 0.5µg.l-1), bacteria (ca. 1 ×106 cells.ml-1) and bacteriophytoplankton (72-122 ×103 cell.ml-1) indicate the presence of significant nutrient inputs while particle &dgr;15N shows minimum to moderate enrichment from anthropogenic sources (max = 3.5 ‰ vs. air). Sedimentation rates were mostly <100 g.m-2.d-1, being higher during stormy conditions. Significant co-variation between turbidity and sedimentation was observed to be useful for modeling sedimentation patterns. Higher levels of the different water quality indictors were observed closer to the coast.

Detection of human-derived fecal contamination in Puerto Rico using carbamazepine, HF183 Bacteroides, and fecal indicator bacteria.

The level of fecal pollution in 17 sites in Puerto Rico was determined by Escherichia coli (E.coli) enumeration using an enzyme substrate medium and Quanti-Tray®/2000. Human fecal pollution was identified using an enzyme-linked immunosorbent assay for the detection of carbamazepine (CBZ) and quantitative polymerase chain reaction (qPCR) detection of the human Bacteroides marker, HF183. Carbamazepine was detected in 16 out of 17 sites, including Condado Lagoon, a popular recreational area. Elevated E.coli levels (>410 CFU 100 mL(-1)) were detected in 13 sites. Average CBZ concentrations ranged from 0.005 μg L(-1) to 0.482 μg L(-1) and 7 sites were positive for HF183. Higher CBZ concentrations were associated with the detection of HF183 (Mann-Whitney test; U=42.0; df=7; 1-tailed P value=0.013). This was the second study to determine surface water concentrations of CBZ in the Caribbean and the first in Puerto Rico.

Molecular detection of atrazine catabolism gene atzA in coastal waters of Georgia, Puerto Rico and Trinidad

In this study, quantitative polymerase chain reaction targeting the atrazine catabolism gene, atzA, was used to detect the presence of atrazine degrading bacteria as an indicator of atrazine contamination in 11 sites in Georgia, nine coastal sites in Puerto Rico and 11 coastal sites in Trinidad. The atzA gene was detected in five stations in Georgia (Oak Grove Island entrance, Blythe Island Recreation Park, Jekyll Island., Village Creek Landing and Dunbar Creek Sea Island Rd Bridge). In Puerto Rico gene was detected in five sites (Boquilla, Oro Creek, Fishers Association, Ceiba Creek and Sabalos Creek) while seven sites in Trinidad (Carli Bay, Las Cuevas Bay, Quinam Bay, Salybia River, Salybia Bay, Maracas River and Maracas Bay) showed the presence of atzA.

Detection of verotoxin producing Escherichia coli in marine environments of the Caribbean.

The goal of this study was to determine the potential for Enterohemorrhagic Escherichia coli O157:H7 (EHEC) contamination in tropical marine waters. Samples were collected from urban, suburban, and rural sites around the islands of Puerto Rico and The Republic of Trinidad and Tobago. Quantification of E. coli and EHEC was evaluated using MI plates and qPCR. EHEC was detected in six sites in Puerto Rico: West of La Parguera Town, Boquilla, Oro Creek, Fishers Association, Joyuda Lagoon, and Boqueron Wetland Creek and in two rural sites in Trinidad: Balandra Bay and Quinam Bay. Plate count enumeration of E. coli was not a reliable indicator for the presence of EHEC. The sites where EHEC was detected on both islands are used for recreational bathing, water sports and recreational/commercial fisheries and therefore pose a public potential health risk.