Practical application of PMA-qPCR assay for determination of viable cells of inter-species biofilm of Candida albicans-Staphylococcus aureus.

Determining the number of viable cells by calculating colony-forming units is time-consuming. The evaluation of mixed biofilms consisting of different species is particularly problematic. Therefore, the aim of this study was to optimize a molecular method-propidium monoazide quantitative polymerase chain reaction (PMA-qPCR)-for accurate and consistent differentiation between living and dead cells. In the practical experimental example, the number of genome copies representing living cells was determined in a mixed biofilm of Candida albicans-Staphylococcus aureus inhibited by photodynamic inactivation. Optimal conditions such as PMA concentration and the duration of light exposure, the optimization of DNA isolation from the mixed biofilm and standardization of PMA-qPCR parameters were tested prior to the main experiment. The genome copy number was calculated based on the known amount of genomic DNA in the qPCR and the genome size of the respective microorganism. The results showed that photodynamic inactivation in the presence of 1 mM methylene blue decreased the total genome copy number from 1.65 × 10⁸ to 3.19 × 10⁷, and from 4.39 × 10⁷ to 1.91 × 10⁷ for S. aureus and C. albicans (P < 0.01), respectively. The main disadvantage is the overestimation of the number of living cells represented by genome copy numbers. Such cells are unable to reproduce and grow (no vitality) and are continuously dying. On the other hand, PMA-qPCR determines the copy numbers of all microbial species, including a mix of eukaryotic yeasts and prokaryotic bacteria in a biofilm in one step, which is a great advantage.