Detection of viable Escherichia coli in environmental water using combined propidium monoazide staining and quantitative PCR

Abstract

The objectives of this study were to specifically detect viable Escherichia coli in environmental waters by targeting the ycjM gene in a propidium monoazide (PMA)-qPCR assay. PMA is a viability dye that can inhibit the amplification of DNA from dead cells, thus allowing for the detection and quantification of only viable cells. The ycjM primers were used to target E. coli that directly originated from the feces of warm blooded animals, and avoid false positive detection caused by "naturalized" E. coli that can exist in the environment. In this study, tap water and environmental waters were inoculated with E. coli isolated from animal feces. Following cell collection, samples were treated with PMA, followed by DNA isolation and qPCR detection. 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 viable cells. For tap and environmental waters collected in the winter, a 10 μM PMA 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 10⁴ CFU/10 mL dead cells, after a 20 μM PMA treatment. No significant differences were found among the PMA-qPCR assay and two other standard culture-based methods for detection of viable E. coli in environmental water. In conclusion, with proper pretreatment of environmental water samples, this PMA-qPCR assay that targets the ycjM gene could quantify viable E. coli cells that directly come from the feces of warm-blooded animals, and therefore effectively and accurately indicate the quality of environmental water.

Keywords: E. coli; Environmental waters; PMA; Viable cells; qPCR; ycjM.