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Evaluation of a real-time quantitative PCR method with propidium monazide treatment for analyses of viable fecal indicator bacteria in wastewater samples
Citation:
VARMA, M., R. FIELDS, M. K. STINSON, B. Rukovets, E. C. CHERN, L. J. WYMER, AND R. A. HAUGLAND. Evaluation of a real-time quantitative PCR method with propidium monazide treatment for analyses of viable fecal indicator bacteria in wastewater samples. Presented at 2008 American Society for Microbiology General Meeting, Boston, MA, June 01, 2008 - June 05, 2208.
Impact/Purpose:
1. Determine effects of storage by freezing of NEEAR study samples on QPCR measurements of enterococci. 2. Identify, from an expanded pool of candidates, the general fecal indicator microorganism(s) (i.e. different taxonomic or phylogenetically-related groups from all animal sources) whose densities in NEEAR study water samples, as measured by QPCR analysis, best correlate with illness rates monitored in the NEEAR study. 3. Determine whether illnesses rates in the NEEAR studies are better correlated with QPCR measurements of fecal indicator organisms from human as opposed to general sources (subject to the availability or development of suitable QPCR assays for human fecal indicators).
Description:
The U.S. EPA is currently evaluating rapid, real-time quantitative PCR (qPCR) methods for determining recreational water quality based on measurements of fecal indicator bacteria DNA sequences. In order to potentially use qPCR for other Clean Water Act needs, such as updating criteria for disinfected POTW effluents, the ability to distinguish live organisms would be desirable. Propidium monoazide (PMA) has been shown to be selective in penetrating the cellular membranes of dead microorganisms where, upon exposure to light, it renders the DNA sequences in these cells unavailable for PCR detection. Therefore only sequences from live organisms should be measured by qPCR following PMA treatment. In this study a qPCR method incorporating pretreatment of samples with PMA was evaluated using pure cultures and wastewater samples collected from 3 different POTWs at 4 treatment stages including the chlorine-disinfected effluents. Treatment of heat-killed, cultured Enterococcus faecalis and Bacteroides thetaiotaomicron cells with PMA resulted in ~3-4 log reductions in QPCR-detectable target sequences compared with levels detected from live cells exposed to PMA or killed cells that were not exposed to PMA. Similar results were seen with cell concentrates from 1 ml buffer and wastewater samples spiked with E. faecalis, however, an inhibitory effect on PMA activity was indicated when 10ml of the wastewater samples were processed. Analyses of wastewater samples collected from the POTWs during normal dry weather operation showed that culturable Enterococcus and fecal coliform bacteria counts were more closely associated with QPCR-determined levels of target sequences from Enterococcus and Bacteroidales in PMA treated samples than with those in samples without PMA treatment. This trend was not as evident in samples collected from the same plants during wet weather operation where blending of primary and secondary-treated samples occurred prior to disinfection. Further studies are needed to determine the efficacy of PMA treatment for distinguishing viable organisms in different wastewater and surface water matrices by qPCR analysis.