Antibacterial mechanisms of terpinen-4-ol against Aeromonas hydrophila

Aeromonas hydrophila (A. hydrophila) is a highly harmful pathogenic bacterium in aquaculture, and its increasingly severe drug resistance poses a threat to global aquaculture and food safety. It is an urgent need to develop new antibacterial agents. Terpinen-4-ol is a natural plant monomer with broad-spectrum antibacterial activity, but its effect and mechanism against A. hydrophila remain unclear. In this study, the minimum inhibitory concentration (MIC) of terpinen-4-ol against A. hydrophila was 7.56 mM (1.17 mg/mL), at which it effectively inhibited bacterial growth. Scanning electron microscopy (SEM) revealed morphological changes in treated A. hydrophila, including cell shrinkage, deformation, and flagellar detachment or loss. Detection of nucleic acid, protein, alkaline phosphatase (ALP), and β-galactosidase leakage, together with propidium iodide (PI) staining, showed that terpinen-4-ol caused intracellular content leakage and increased PI fluorescence intensity. Measurements of succinate dehydrogenase (SDH) activity and reactive oxygen species (ROS) levels revealed that terpinen-4-ol at 1 MIC reduced SDH activity by 55.7% and increased ROS levels by 5-fold. Crystal violet staining of biofilms further demonstrated that terpinen-4-ol at 1/2 MIC inhibited biofilm formation by 78.3%. In conclusion, terpinen-4-ol exerted its antibacterial effect on A. hydrophila by disrupting the integrity of the cell membrane and wall to cause the leakage of intracellular contents, interfering with bacterial metabolism, and inhibiting biofilm formation. This study provides potential strategies for controlling A. hydrophila infections in aquaculture and lays the foundation for future research into the antibacterial mechanism of terpinen-4-ol.