Identification of fecal pollution sources using rDNA marker for the detection of specific fecal Bacteroides in recreational waters of Arenales River (Salta, Argentina).

HÉCTOR A. CRISTÓBAL; RAMIRO POMA; VERÓNICA RAJAL

Florianopolis

Congreso; 17th International Symposium on Health-Related Water Microbiology (WaterMicro 2013); 2013

In Argentina, the existing legislation establishes to control microbial contamination of water (and food) analysis of bacterial standards (total and fecal coliforms) during routine monitoring. However, these bacterial indicators not accurately predict the risk of other pathogens such as protozoa, bacteria uncultivable and enteric viruses. It constitutes a risk to health, as the relationship between human pathogen and the indicators used can vary among populations. The use of contaminated water for human consumption is causing diseases such as diarrhea, dysentery, hepatitis, typhoid fever or respiratory infections. Bacteroides species are promising as novel fecal indicators for various reasons; is the normal flora of warm-blooded organisms, high concentration in feces and potential host specificity, which allowing discriminate among livestock and human fecal contamination in water. The proposed method called Microbial Source Tracking is based on the detection of genetic marker (16S ribosomal DNA) in Bacteroides that identify the sources of fecal contamination in water. In previous study, the validation of the systems to detect Bacteroides in our region, informed us that the AllBac system has a specificity of 88% for detecting Bacteroides universal. To detect in water, Bacteroides of human origin, the BacHum system presents 87% specificity and 95% sensitivity; and the detection Bacteroides of bovine origin, the BacRum and BacB2 systems were the most promising. Aims The study aimed evaluates the use of Bacteroides as new indicators in type-specific identification of the origin of fecal pollution in environmental waters of the province of Salta. Establish a correlation of the presence of bacterial indicators Bacteroidales with current standards established by legislation (fecal and total coliforms). Methods To carry out diagnostic studies, which allow us to evaluate recreational water quality in different places from Salta, water samples collected of four month were analyzed from five sites (P5, P6, P7, P10 and P11) of Arenales river. These points were selected in previous studies as critical and highly contaminated sites, determined high concentrations of fecal and total coliform, Entorococus, viruses and parasites. Bacteroides concentrations present in water samples can be small. Therefore, and to reach the detection limit of the method used, it is necessary to perform a step of concentration of microorganisms in the water samples. Was collected 20 liters of water from each point, and concentration was performed (about 400 times) by ultrafiltration system to a final volume of approximately 50 ml. DNA extraction was performed from the concentrates of the samples using the kit PureLinkTM Viral RNA-DNA Mini (Invitrogen). Real-time PCR assays were performed to detect 16S rDNA in Bacteroides species and differentiate among universal (AllBc), human (BacHum) and cow (BacB2). All system were performed by duplicate with the equal PCR condition, and used to estimate the fecal contamination of five points of water collected. Results It is important remark that the monitoring sectioned was conducted in the dry season and during the winter months (April, May, June and July). From a total of 20 samples, 19 samples were positive for AllBac assays to detect of universal Bacteroides. For detection of human Bacteroides, 17 samples were positives with BacHum system assays. For detection of bovines Bacteroides, only 2 samples were positives with BacB2 system assays. It stands that was observed in the majority of the tested points a high concentration in April and May compared to other months. The points selected of Arenales river correspond to wastewater discharges (P5), Sauces park (P6), channel stormwater Esteco (P7) and nearby to the treatment plant (P10, P11) of city Salta. However, the P5 and P11 points showed maximum values ​​with respects to the other points. Conclusions The dumping of domestic and industrial effluents pollute untreated the state of the river Arias-Arenales. The specificity of data obtained in this study confirms the high bacterial fecal contamination of human found in the water tested. We conclude that the Arenales River in the stretch that crosses the city of Salta, represents a high risk from the point of view of health. The river in any of the sections not should be used for recreational purposes that involve direct contact with the water.