Use of Viability-Based Methods for Improved Detection of Recent Fecal Contamination in a Microbial Source Tracking Study Near Tijuana, Mexico

Current microbial source tracking methods heavily rely on the use of quantitative PCR (qPCR) assays to differentiate human and non-human sources of fecal contamination. However, traditional qPCR measures DNA from viable, viable but not culturable (VBNC), and dead cells, which may confound the use of this technique for detecting recent fecal contamination from waters receiving treated sewage effluent. In this study, fecal indicator bacteria (FIB), six host-associated markers, and two viability-based methods for rapid detection and assessment of fecal contamination were used in a microbial source tracking study to identify sources impairing water quality and sediments within the San Antonio de los Buenos watershed in Tijuana, Mexico. Horse- and gull-associated markers were detected in 4 and 8% of samples tested, respectively. The human- and dog-associated markers were positive in 74 and 63% of watershed samples and 92 and 75% of storm drain samples, respectively. Propidium monoazide (PMA) successfully inhibited amplification of DNA from dead cells in environmental creek waters that receive large volumes of treated wastewater effluent. Accordingly, PMA-qPCR measurements were more comparable to measurements made by culture-based methods (IDEXX). The covalently linked immunomagnetic separation/adenosine triphosphate (Cov-IMS/ATP) method showed a strong linear relationship to culture methods when compared to measurements made by the qPCR Entero1a assay. Both the PMA-qPCR and the Cov-IMS/ATP methods show promise for improved assessment of water quality and recent fecal contamination in sewage impacted waters, including areas receiving discharge from wastewater treatment plants, where measurement by qPCR does not effectively differentiate between DNA from live and dead cells. This study serves as an important positive control for non-point source pollution studies.