Transcriptomic analysis reveals the multifaceted inhibitory mechanisms of thymol on the survival and virulence of Vibrio alginolyticus.

Vibrio alginolyticus is a widespread marine pathogen responsible for major disease outbreaks in aquaculture and zoonotic infections in humans. Its pathogenicity is tightly regulated by quorum sensing and driven by biofilm formation, motility, and multiple virulence factors. The organism's high adaptability and virulence complicate traditional antibiotic treatments, especially amid rising antibiotic resistance. Although thymol, a natural monoterpenoid, is known for its broad-spectrum bactericidal activity, its effects on the survival and virulence of V. alginolyticus have not been previously investigated. In this study, we evaluated the inhibitory potential of thymol against V. alginolyticus. Experimental results demonstrated that thymol significantly reduced the survival of both planktonic and mature biofilm cells, induced reactive oxygen species generation, and disrupted membrane integrity. Furthermore, thymol at sub-inhibitory concentrations markedly impaired quorum sensing regulated biofilm formation and motility. Global transcriptomic analysis revealed that thymol exerts a multifaceted inhibitory mechanism by downregulating key genes involved in antioxidant defense, membrane biosynthesis, energy metabolism, ABC transporter function, quorum sensing, biofilm formation, and motility. In addition, computational studies revealed strong binding affinities between thymol and the upstream key quorum sensing regulators LuxS and LuxU, with hydrogen bonds formed at key active site residues, further supporting its anti-virulence potential. To our knowledge, this is the first study demonstrating that thymol acts as a potent inhibitor of V. alginolyticus survival and virulence. These findings highlight thymol as a promising natural agent for managing V. alginolyticus associated infections in aquaculture systems.