Inactivation of Gram (−) bacteria Salmonella enterica by chlorophyllin-based photosensitization: Mechanism of action and new strategies to enhance the inactivation efficiency

Data confirmed that photosensitization (Chl 1.5 × 10− 5 M, for 1-120 min, 405 nm, 0-46.1 J/cm2) reduced S. enterica population, just by 2.05 log (CFU/ml). Fluorimetric measurements indicated that just minor part of Chl was bound to Salmonella in suspension. Addition of sodium azide (NaN3) (10 mM) protected bacteria from killing, what means that 1O2 took place in photochemical reactions. Gene expression data confirmed that Chl-based photosensitization induced oxidative stress in bacteria cells, since mostly genes responsible for detoxification of ROS (OxyR, AhpC, GrxA) have been expressed in Salmonella. Moreover, the expression of genes, responsible for the inhibition of oxidative respiration (AtpC), cell division and down-regulation of metabolism (SulA) have been detected. In addition, Chl-based photosensitization induced significant release of intracellular components (absorbing at λ260 nm and λ280 nm) in bacteria that indicated increased membrane permeability. Thus, the combination of two antimicrobials (Chl-based photosensitization and chitosan (CHS)) with the same target (cellular membrane) in the presence of light drastically reduced viable Salmonella population (by 7.28 log). Combined treatment of photosensitization and high power pulsed UV light (HPPL) was also very effective, since reduced viable Salmonella by 7.5 log. Bacterial regrowth experiments clearly indicated that after both combined treatments Salmonella lost its ability to proliferate, and SEM images confirmed that after both treatments no viable bacteria have been found at all.