PHOTODYNAMIC INACTIVATION IN THE CONTROL OF BACTERIAL PERSISTENCE: PHENOTYPIC REVERSAL UNDER STRESS CONDITIONS

Persistent bacterial infections represent a significant clinical challenge, particularly in chronic and recurrent infections. Subpopulations in a persistence state — characterized by reversible, non-hereditary metabolic dormancy — survive even when exposed to high antibiotic concentrations, contributing to therapeutic failures. Staphylococcus aureus exposed to Oxacillin exhibits this behavior, forming biphasic killing curves characteristic of persistent populations, and retaining susceptibility after antibiotic removal, which reinforces its non-genetic nature. Photodynamic Inactivation (PDI), based on the generation of reactive oxygen species (ROS) by light-activated photosensitizers (PSs), represents a promising alternative or combination to traditional antimicrobial treatments. However, its unmodulated application may induce transient tolerance, as the oxidative stress induced by PDI can also lead to a dormancy state in the surviving population. With this condition in mind, PDI treatment was optimized by introducing a metabolic recovery period to overcome the transient tolerance state previously observed. Other stress factors may also lead to similar results, turning the persistent formation as a stress response into a topic of interest when studying combining antibacterial therapies. As such, in this research, for the time-dependent killing curve (time-kill assays) and photodynamic inactivation assays, a population of S. aureus was exposed to different stressors for 3h incubation periods: 1) Thermal stress: incubation at 40 °C; 2) Acidic pH: incubation in BHI medium adjusted to pH 4 by adding hydrochloric acid (HCl) and acetic acid (CH₃COOH); 3) Hyperosmotic stress: incubation in medium containing 0.5 M, 1 M, 1.5 M, and 2 M NaCl. By incorporating environmental stress conditions into the analysis of persistent populations, it was possible to observe distinct behaviors associated with the chosen stressors. High incubation temperatures did not produce significant increases in the persister fraction assessed by time-kill assays, when compared to stressors such as hyperosmotic medium and acidic medium, which caused a significant increase in the observed antibiotic tolerance. The introduction of stress conditions also proved to be a differentiating factor when analyzed in the context of PDI treatment, reducing its bactericidal effect and resulting in greater bacterial survival — possibly related to decreased metabolic activity in stressed cells and increased difficulty in photosensitizer internalization.