Pseudomonas psychrophila Biofilm Formation Inhibition by Thymol Adaptation

This study investigated the effects of thymol (TH) adaptation on biofilm formation and the metabolic profile of the multiresistant slaughterhouse isolate Pseudomonas psychrophila M33T02.2. After exposure to increasing sublethal concentrations of thymol, the adapted P. psychrophila M33T02.2 showed decreased biofilm-forming capacity, reduced swarming motility, and lower rhamnolipid production compared to the wild-type strain. Confocal microscopy further showed that the biofilms developed by the adapted strain were less homogeneous, confirming their inability to develop well-structured biofilms. To further understand these changes at the metabolic level, high-performance liquid chromatography (UHPLC-Orbitrap-MS/MS) identified redox metabolism intermediates and energy balance-related metabolites as most important variables. 20 metabolites were underexpressed for the TH-adapted strain, including glutathione disulfide, guanosine diphosphate, and flavin adenine mono- and di-nucleotide, among others. Therefore, we conclude that repeated exposure to TH prevents the emergence of resistance mechanisms associated with biofilm formation, acting at the level of redox state and energy imbalance.