Detection of Live Shiga Toxin-Producing Escherichia coli with Long-Read Sequencing

A requirement of any foodborne pathogen testing method is that it only detects live bacteria. Ethidium monoazide (EMA) and propidium monoazide (PMA) are dyes that penetrate the membranes of dead cells and form cross-linkages in the DNA, which prevents its amplification in PCR. This study investigated whether treatment with EMA or PMA would inhibit the sequencing of DNA from dead Escherichia coli. Range finding experiments with qPCR were conducted to determine the optimal concentrations of EMA and PMA needed to inhibit the amplification of DNA from dead cells while not influencing live cells. An EMA concentration that differentiated between live and dead cells could not be established. However, a PMA concentration of 25 µM effectively prevented qPCR amplification of DNA from dead E. coli while not impacting the amplification of live E. coli DNA. Sequencing experiments were conducted with PMA-treated live, untreated live, PMA-treated dead, and untreated dead E. coli. There were no significant differences in the detection of virulence genes of interest between the PMA-treated live, untreated live, and untreated dead E. coli. However, no DNA sequencing data were obtained from the PMA-treated dead E. coli. These results suggest that PMA could be incorporated into sample preparation methods prior to sequencing to selectively detect live cells of foodborne pathogens.