Dead or alive? A novel flow cytometric method to determine microbial viability in Mycoplasma genitalium and Neisseria gonorrhoeae

Neisseria gonorrhoeae and Mycoplasma genitalium infect more than 150 million people each year and are rapidly acquiring antimicrobial resistance (AMR), to all available treatments. PCR tools used for diagnosis and test-of-cure, amplify nucleic acids regardless of microbial viability, leading to false positives and subsequent overuse of antibiotics. Assays that accurately assess microbial viability and replication after exposure to antibiotics and other stimuli are needed to improve diagnostics. In this study, we present a flow cytometric method to quantitatively assess N. gonorrhoeae and M. genitalium viability and replication. We utilised a membrane exclusion dye (Fixable Viability Stain 700) to differentiate live and dead cells and a cell trace dye (CellTrace Violet) to track cell replication. The flow cytometry assay was assessed with and without nutrient starvation and inactivation (permeabilisation or UV exposure) to validate the assays' ability to resolve replicating, non-replicating, and non-viable bacterial populations. The outcomes of flow cytometry were compared to qPCR assays and culture-based approaches. Whereas qPCR overestimated viable cell counts (two-to-four-fold overestimation) and could not distinguish between viable and non-viable cells (p > 0.05), flow cytometry could reliably distinguish these populations, as well as replicating and non-replicating subpopulations. This novel flow cytometry method can be used to improve upon existing techniques for in vitro experimentation such as antimicrobial sensitivity testing and could be further utilised for cell sorting, enabling downstream analyses at the single-cell and population level, providing a basis for the discovery of viability targets for diagnostic tools.