Detection of Viable Escherichia coli in Environmental Water Using a Combined Propidium Monoazide Staining-Real-time PCR

Introduction: Escherichia coli, as a principal fecal indicator bacterium, is used to monitor water quality worldwide. With the detection of E. coli by real-time PCR (qPCR), a rapid and sensitive reflection of water quality and safety can be achieved. 

Purpose: The objectives of this study were to specifically detect viable E. coli by targeting its ycjM gene in a propidium monoazide (PMA)-qPCR assay, and to investigate the specificity, efficiency, and accuracy of the assay for environmental water.

Methods: Four strains of E. coli isolated from animal feces, were freshly grown, combined and serially diluted for inoculating into water samples. Spiked tap water and other environmental water samples, including water from Lake of the Ozarks, Missouri River and Mississippi River, were filtered or centrifuged for cell collection. Samples were then treated with PMA, followed by DNA isolation and TaqMan® qPCR detection.

Results: For pure cultures, 5 µM PMA with a 10-min light exposure was efficient at inhibiting the amplification of DNA from 10⁵ CFU /ml dead E. coli cells, with a detection limit of 10² CFU/100 ml. For tap and environmental waters collected in the winter, a higher PMA concentration of 10 µM was required and as low as 10³ CFU/100 ml viable cells could be detected in the presence of 10⁵ CFU/100 ml dead cells. For water samples collected during the summer, 10² CFU/10 ml viable cells could be detected in the presence of 10⁴CFU/10 ml dead cells, after a 20 µM PMA treatment. Significant and strong correlations were found between the PMA-qPCR and EPA Method 1603.

Significance: With proper optimization steps to remove suspended solids in environmental water samples, the PMA-qPCR could effectively and accurately differentiate between viable and dead E. coli cells by suppressing the amplification of DNA from dead cells.