Q-251. Evaluation of a Real-Time Quantitative PCR Method with Propidium Monoazide Treatment for Analyses of Viable Fecal Indicator Bacteria in Wastewater Samples
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.
Notice: Although this work was reviewed by EPA and approved for publication, it
may not necessarily reflect official Agency policy.