Mitochondrial DNA marker: A PCR approach for tracking rat (Rattus rattus and Rattus norvegicus) fecal pollution in surface water systems.

Rats act as reservoirs for a wide range of zoonotic pathogens and can negatively impact human health. In this study, we developed a novel dye base mitochondrial DNA (mtDNA) PCR-assay (RatMt) specifically targeting a 180 bp fragment of the NADH dehydrogenase subunit 2 gene for detecting fecal pollution from two species of rats (Rattus rattus and Rattus norvegicus) in environmental samples. Estimation of Escherichia coli concentrations in Rattus norvegicus fecal pellets suggested that there were approximately 2.24 × 10 4 ± 4.86 × 103 MPN/g of fecal pellet. The RatMt PCR assay was robust, had a detection limit of rat feces in water of 0.274 ± 0.14 mg/100 mL and was 100 % specific for detecting Rattus rattus and Rattus norvegicus fecal mtDNA. Fecal Indicator Bacteria (FIB) along an urbanized gradient in Pensacola-Bay was assessed by the IDEXX Colilert™ - 18 and indicated that the majority of the fifteen sampling sites in the Pensacola-Bay area had E. coli concentrations >410 MPN/100 mL. Rattus rattus and Rattus norvegicus mtDNA were detected in all the urban marine sites, three of the urban freshwater sampling areas, and three of the forested sampling sites. The RatMt PCR assay is a useful tool for rapidly detecting Rattus rattus and Rattus norvegicus fecal pollution in environmental samples.

Identifying the primary sources of fecal contamination along the beaches and rivers of Trinidad.

The aim of this study was to identify the main sources of fecal pollution at popular beaches and rivers in the island of Trinidad. Escherichia coli enumeration and microbial source tracking (MST) were used to identify the primary sources of fecal bacteria contamination at the sites. Nineteen sites exceeded USEPA water quality standards for safe recreational use. Highest levels of fecal contamination were recorded on the central and west coasts of the island and included Brickfield River (4,839 MPN 100 ml-1), Orange Valley Bay (2,406.6 MPN 100 ml-1) and Chaguaramas Bay (1,921.2 MPN 100 ml-1). MST detected human (HF183) fecal pollution at ∼63%, birds at ∼67%, chicken at ∼36% and cattle (BacCow) at ∼34% of the sites. MST is a useful and rapid method for identifying major sources of fecal pollution in rivers and beaches. In Trinidad water bodies, the main sources of fecal pollution were humans and birds. The large number of sites with elevated levels of fecal pollution detected is particularly alarming and represents a serious public health risk.

Detection of Brucella suis, Campylobacter jejuni, and Escherichia coli Strains in Feral Pig (Sus scrofa) Communities of Georgia.

Feral pigs (Sus scrofa) are an environmentally destructive invasive species that act as a reservoir for zoonotic pathogens. The aim of this study was to determine the presence of Brucella suis, Campylobacter jejuni, and of Escherichia coli in feces of feral pigs from Georgia. Fecal samples were collected from 87 feral pigs from forested and agricultural regions of Georgia. DNA was extracted from the fecal samples and quantitative PCR (qPCR) was used to screen for each of the four pathogens. The qPCR assays indicated that B. suis and eaeA-containing strains of E. coli was present in about 22% and 28% of the samples, respectively. C. jejuni was undetected in any of the feral pig fecal samples. The incidence of B. suis was higher in the pigs from forested region, whereas E. coli strains possessing eaeA gene incidence was higher in the pigs from agricultural regions. In Georgia, feral pigs harbor infectious agents and are a growing threat to the transmission of pathogens to native wildlife, humans, and food crops.

Evaluation of physical, chemical and microbiological parameters of water quality in the Harris Neck estuarine marshes along the Georgia coast.

Analysis of the physical, chemical and biological parameters assessing water quality in Harris Neck estuary indicated that the average dissolved oxygen level was 8.6 mg/L, it maintained moderate levels of total dissolved nitrogen (2.7-4.6 mg/L) and total dissolved phosphorous (<0.05 mg/L), chlorophyll a was above 5.0 µg/L and it is contaminated with low levels of fecal bacteria. Bifidobacterium adolescentis, a putative marker of human fecal pollution, was detected once at stations 3 and 5. Overall the Harris Neck water quality analyses indicated a relatively pristine and a healthy functioning marine environment.

Comparison of four polymerase chain reaction methods for the rapid detection of human fecal pollution in marine and inland waters.

We compared the effectiveness of three PCR protocols for the detection of Bifidobacterium adolescentis and one PCR protocol for detecting Bacteroidales as indicators of human fecal pollution in environmental samples. Quantitative PCR indicated that a higher concentration of B. adolescentis DNA was recovered from sewage samples on the 0.2 mum filters compared to the 0.45 mum filters, and there was no evidence of qPCR inhibitors in the DNA extracts. With the Matsuki method (1999), B. adolescentis was detected only in undiluted sewage samples. The King method (2007) performed well and detected B. adolescentis in all of the sewage dilutions (from undiluted to 10(-4)). In contrast, the Bonjoch approach (2004) was effective at detecting B. adolescentis at lower dilutions (10(-3)) of sewage samples and it gave false positive results with some (3/8) pig fecal samples. Human-specific Bacteroidales (HuBacs) were detected in the lower diluents of sewage samples but was positive in pig (6/8) and cattle fecal samples. PCR detection of B. adolescentis in marine samples from Puerto Rico and freshwater samples from Georgia indicated that the PCR method of King et al. (2007) and the modified Layton method for HuBac were in agreement in detecting human fecal pollution in most sites.

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.

Quantification of enterococci and bifidobacteria in Georgia estuaries using conventional and molecular methods.

Fecal pollution is a serious threat to the estuarine environment along the Georgia coast. Culture-dependant and molecular methodologies were utilized to compare and evaluate the abundance of fecal indicator bacteria in four Georgia estuaries (Darien River, Frederica River, Gulley Hole Creek, and St. Marys River). The functionality of enterococci and bifidobacteria as indicator organisms in marine environments was assessed, as well as Bifidobacterium adolescentis densities. At each study site, enterococci were enumerated as colony forming units (CFU) on mEI agar. For quantitative polymerase chain reaction (qPCR), genus- and species-specific primer sets were used to quantify bifidobacteria and B. adolescentis as 16S rRNA gene copies and enterococci as tuf gene copies. A high correlation (r=0.925) was observed between CFU and qPCR enumeration of enterococci. Enterococci densities in the estuarine rivers ranged from 3-449CFU/100ml on mEI plates and 4.58-5.39Log(10) gene copies/100ml by qPCR. Bifidobacteria densities ranged from 3.62-4.14Log(10) gene copies/100ml and suggested the Frederica River as least affected by fecal bacteria and the Darien River as most affected by fecal pollution. A correlation of 0.46 was observed among qPCR densities of enterococci and bifidobacteria at all sample sites. Quantitative polymerase chain reaction detection of B. adolescentis was a rapid (i.e., less than 2h) indicator of presumptive human fecal pollution and suggested that Gulley Hole Creek, the Darien River, and the St. Marys River were affected by fecal bacteria derived from a human source. Gulley Hole Creek and the Darien River had the highest levels of fecal pollution detected in the studied estuaries. Molecular quantification of bifidobacteria may be a more accurate method of determining immediate health risks associated with fecal pollution in estuarine water than traditional and contemporary assessments of enterococci.

Rapid detection of human fecal contamination in estuarine environments by PCR targeting of Bifidobacterium adolescentis.

Detection of Bifidobacterium adolescentis was used as an effective genetic marker of human fecal contamination in Georgia estuaries. Enterococci enumerations on mEI media indicated that a tributary to the Little Satilla River with 516 CFU/100 ml was the most polluted of all the rivers tested. Extracted DNA from eight river water samples was subjected to a two-step nested PCR protocol using genus and species specific primers for Bifidobacterium spp. and B. adolescentis. B. adolescentis was detected from extracted DNA in Dunbar River, Black Bank Creek, and in a Little Satilla River tributary which demonstrates the presence of human fecal contamination in these three rivers. In the five other estuaries tested including West Point-Federica River and the Altamaha River, which both had less than 16 CFU/100 ml of enterococci, B. adolescentis was not detected.