Q-255. Rapid Quantitative PCR Method for Quantification of Bacteroides as an Alternative Indicator of Fecal Contamination

A. D. Blackwood, R. T. Noble;
Univ. of North Carolina, Morehead City, NC.

Traditional methods for measuring fecal indicator bacteria (FIB), such as fecal coliforms (E. coli), and Enterococcus, have been used for decades due to their association with swimming-related illness. Recent studies have found that FIB are capable of persisting, and even reproducing in sediment, sand, and aquatic environments, making them unreliable and inaccurate. Several Bacteroides spp. have been suggested as potential alternative indicators since they are obligate anaerobes and therefore not likely to persist in aquatic environments. We have developed a rapid, culture-independent method for analyzing Bacteroides spp. using quantitative PCR (QPCR) targeting the 16S rRNA gene. QPCR is a desirable and essential tool in the field of environmental microbiology due to improved rapidity, sensitivity, specificity, and reproducibility of assay results when compared with traditional culture-based methods. The assay is specifically designed to quantify the most common human Bacteroides spp., B. fragilis, B. thetaiotaomicron, B. distasonis, and B. uniformis. Wastewater treatment plant influent averaged 3 x 109 cells per 100 ml, and septic system waste averaged 1 x 108 per 100 ml, with concentrations for human fecal contamination several fold greater than that observed for domestic animal scat. The average PCR amplification efficiency of the assay is 100%, and with no exhibited cross reactivity with other undesired targets present in fecal contamination (e.g. Enterococcus spp., E. coli). We present the details of the method, including analyses of a wide range of fecal samples from different organisms. We have analyzed both point and non-point sources of contaminated runoff to demonstrate the relationships to other well established FIB and Bacteroides spp. markers. Quantification of this target is being used in a large stormwater runoff assessment project, to prioritize stormwater outfalls for application of best management practices. The assay is a promising tool for early and rapid detection of human fecal contamination, such as sewage spills, and has the potential to be used as an alternate indicator for protection of public health.