<i>Pseudomonas psychrophila</i> 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 <i>Pseudomonas psychrophila</i> M33T02.2. After exposure to increasing sublethal concentrations of thymol, the adapted <i>P. psychrophila</i> 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.