Detection of live Escherichia coli with long-read sequencing

Authors: Counihan, Katrina; Tilman, Shannon; Chen, Chin Yi; He, Yiping

Submitted to: International Journal of Molecular Sciences
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/28/2025
Publication Date: 3/1/2025
Citation: Counihan, K.L., Tilman, S.M., Chen, C., He, Y. 2025. Detection of live Escherichia coli with long-read sequencing. International Journal of Molecular Sciences. https://doi.org/10.3390/ijms26052228.
DOI: https://doi.org/10.3390/ijms26052228

Interpretive Summary: Tests for bacteria in food that can infect people and make them sick must detect only live bacteria. Two chemicals, ethidium monoazide (EMA) and propidium monoazide (PMA), are able to attach to the DNA of dead bacteria, which prevents their detection in certain tests. This study investigated whether treatment with EMA or PMA would inhibit sequencing of DNA from dead Escherichia coli. Experiments were conducted to find the optimal concentration of EMA and PMA to use. It was determined that EMA did not work well. However, PMA did work well at a concentration of 20 µM. Sequencing experiments were conducted with PMA-treated live, untreated live, PMA-treated dead, and untreated dead E. coli. The PMA did not prevent the sequencing of the PMA-treated live, untreated live, and untreated dead E. coli. However, no DNA was sequenced from the PMA-treated dead E. coli. These results suggest that PMA could be used with sample preparation methods prior to sequencing to detect only live bacteria.

Technical Abstract: 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 sequencing of DNA from dead Escherichia coli. Range finding experiments with qPCR were conducted to determine the optimal concentrations of EMA and PMA. 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 dead E. coli while not influencing live E. coli. 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 was obtained from the PMA-treated dead E. coli. These results suggest that PMA could be incorporated into sample preparation methods prior to sequencing to detect only live foodborne pathogens.