Modulatory Effects of Satureja montana L. Essential Oil on Biofilm Formation and Virulence Factors of Pseudomonas aeruginosa

<b>Background</b>: Antimicrobial resistance is a major global health threat, particularly from pathogens such as <i>Pseudomonas aeruginosa</i>, known for forming biofilms and producing virulence factors that cause persistent infections. Essential oils (EOs) offer promising alternatives to conventional antimicrobial therapy due to their antimicrobial and antibiofilm properties. This study aimed to investigate the modulatory effects of a thymol-rich EO from <i>Satureja montana</i> L. on planktonic growth, biofilm formation, swarming motility, proteolytic activity and pyocyanin production of <i>P. aeruginosa</i> PAO1. <b>Methods</b>: The essential oil, isolated by hydrodistillation from <i>S. montana</i> aerial parts, was analysed by GC-MS. The minimum inhibitory concentration (MIC) of the EO and thymol was determined using the broth microdilution method. Sub-MICs were tested for planktonic growth and biofilm formation. Virulence was assessed by testing swarming motility, proteolytic activity and pyocyanin production. <b>Results</b>: The EO was characterised by a very high content of monoterpenes, with thymol dominating (56.47%). MIC for both EO and thymol was 4 mg/mL. They showed a biphasic effect: higher concentrations significantly inhibited planktonic growth (36-58% reduction; <i>p</i> < 0.05), while lower concentrations promoted it (10-17% increase; <i>p</i> < 0.05). Biofilm biomass varied, but the biofilm index indicated promotion at higher concentrations (0.125-0.5 mg/mL; <i>p</i> < 0.05). Both inhibited swarming at 0.5 mg/mL (thymol was more effective). Thymol decreased proteolytic activity, while EO increased pyocyanin production. <b>Conclusions</b>: <i>S. montana</i> essential oil and thymol show concentration-dependent modulation of <i>P. aeruginosa</i> growth, biofilms and virulence, suggesting their potential as anti-virulence agents, although the biphasic responses require careful dosing.