Comparison of Propidium Monoazide Real-time PCR and a Conventional Culture-based Method (EPA Method 1603) for Detection of Viable Escherichia coli in Water

Introduction: Escherichia coli is a fecal indicator bacterium that is monitored to assess water quality by the U.S. Environmental Protection Agency (EPA).  Real-time PCR (qPCR) assays enable rapid and sensitive detection of E. coli.  However, qPCR may overestimate target numbers because it quantifies DNA from both viable and dead cells.

Purpose: The objective of this study was to compare numbers of viable E. coli in water as measured by the EPA culture-based method with those measured by propidium monoazide (PMA)-qPCR.  PMA is a dye that can penetrate dead cells and bind to cellular DNA, preventing its amplification via a subsequent PCR.

Methods: E. coli ATCC 25922 was serially diluted to generate cell suspensions ranging from 10 to 10⁷ CFU ml^-1.  Dead cells were obtained by heating the suspensions at 85°C for 15 min.  Suspensions were inoculated into sterile distilled water.  Numbers of E. coli in water samples were measured by EPA Method 1603.  Water samples were treated with PMA and DNA was extracted and amplified by TaqMan® real-time PCR targeting the uidA gene to detect only viable E. coli cells.

Results: PMA-qPCR could detect as low as 10³ CFU ml^-1 viable E. coli in distilled water samples, while completely preventing false-positive PCR results generated by 10⁴ CFU ml^-1 of dead E. coli cells.  Numbers of viable E. coli measured using PMA-qPCR were significantly correlated with those measured using EPA Method 1603.  PMA-qPCR could detect viable E. coli in water within 3 h whereas EPA Method 1603 required more than 24 h.  Studies detecting viable E. coli in environmental water samples are ongoing.

Significance: In conclusion, good qualitative agreement was found between EPA Method 1630 and the PMA-qPCR assay in terms of detecting E. coli in distilled water. The PMA-qPCR assay could be a promising alternative to detect only viable E. coli in waters.