INIQUI   05448
INSTITUTO DE INVESTIGACIONES PARA LA INDUSTRIA QUIMICA
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Identification of fecal pollution using 16S rDNA marker for the detection of specific fecal Bacteroides in recreational waters of Arenales river from Salta, Argentina
Autor/es:
HÉCTOR A. CRISTÓBAL; HUGO R. POMA; VERÓNICA B. RAJAL
Lugar:
Florianópolis
Reunión:
Simposio; 17th International Symposium on Health Related Water Microbiology - Watermicro 2013; 2013
Resumen:
In Argentina, the legislation establishes the detection of bacterial indicators (total and fecal coliforms) to control microbial contamination in water and food (during routine monitoring). However, they do not predict accurately the presence of other pathogens such as protozoa, uncultivable bacteria, and viruses, which also constitute a health risk since the relationship human pathogens - indicators vary among populations. Another disadvantage is that fecal indicator criteria do not allow the identification of the contamination source either human or animal. The use of contaminated water for human consumption and recreational activity are the cause of multiple diseases such as diarrhea, dysentery, hepatitis, typhoid fever or respiratory infections. On the other hand, Bacteroides species are promising as novel fecal indicators for various reasons: they are part of the normal flora in warm-blooded organisms, are in high concentration in feces, and have potential host specificity which allows discriminating the origin of fecal contamination source in waters. The method called Microbial Source Tracking (MST) is based on the detection of a genetic marker (16S ribosomal DNA) in Bacteroides that identify the sources of fecal contamination in water. The validation of the systems to detect Bacteroides in our region was performed in a previous study. This research work also showed that AllBac system had 88% of sensitivity for the detection of universal Bacteroides, while BacHum system presented 87% of specificity and a sensitivity of 95% for detection of Bacteroides from humans, and the BacRum and BacB2 systems presented 96 and 98% of specificity, respectively; meanwhile the sensitivity was 50 and 95% , respectively. Aims The objectives of this study were: to use Bacteroides as new indicators in type-specific identification of the origin of fecal pollution in recreational waters of the province of Salta and to evaluate the correlation between Bacteroidales with current bacterial indicators established by legislation. Methods In the 2009, a year monthly monitoring was carried out to screen the concentration of viral contamination in eleven sampling sites across Arenales river. Briefly, 20 liters of water samples were previously concentrated 400 times using hallow fiber ultrafiltration method. Bacteroides assays were performed in 20 samples corresponding to five sites during four months. In previous studies, these sites have already been determinate as critical and highly contaminated, because of high concentrations of fecal and total coliform, Entorococus, viruses and parasites were detected. Selected sites were: a wastewater discharge, a recreational site, a stormwater channel and upstream and downstream wastewater treatment plant of the Salta city. Nucleic acids (RNA/DNA) extractions were performed from the concentrated samples using the PureLinkTM Viral RNA-DNA Mini kit (Invitrogen). Concentrations of DNA were calculated by fluorometry using Qubit 2.0 (Invitrogen). Real-time PCR (qPCR) assays were performed to detect 16S ribosomal DNA in Bacteroides species and discriminate between universal (AllBc), human (BacHum) and cow (BacB2) fecal sources. All systems were performed by duplicate under the qPCR standard amplification, the conditions consisted: 50°C for 2 min; 95°C for 10 min; 40 cycles of 95° for 15 seg and 60°C for 1 min used to quantify the fecal contamination Results To detect universal Bacteroides were positive in 19 of the 20 waters samples. For the detection of human Bacteroides assays, 17 waters samples were positives; meanwhile for the detection of bovines Bacteroides assays, only 2 waters samples were positives. It is important to highlight, that the selected monitoring sites belong to the dry season (April, July winter months). In this study, a high concentration of Bacteroides were observed in the majority of the selected sites, especially in April and May compared to other months. In this season the pollution in the river is increased due to illegal effluent discharges and rainfall scarcity. Conclusions Results obtained in the present study confirm the water contamination assayed by a precise detection of fecal bacteria (Bacteroides). According to MST study mostly of the fecal pollution had a human origin. This demonstrates the potential of MST to detect illegal discharges of domestic and untreated industrial effluents which pollute continuously the river. We can conclude that the Arenales river in the section that crosses Salta city, represents a high public health risk. For this reason, the river should not be employed for recreational purposes specially those which involve a direct contact of the users.