Mechanistic Analysis for the Inhibition of Staphylococcus aureus by Terpinen-4-ol

Objective To analyze the chemical composition of tea tree oil (TTO) and elucidate the antibacterial mechanisms of its major component, terpinen-4-ol (T-4-O), against Staphylococcus aureus . Methods The chemical constituents of TTO were determined using GC MS. The antibacterial mechanism of T-4-O was investigated from multiple perspectives, including its in vitro antibacterial activity, effects on bacterial growth, morphology, cell membrane and cell wall integrity, as well as leakage of cell lysates and proteins, cell cycle progression, and transcriptional responses. Results GC-MS analysis revealed that TTO contained 15 compounds, primarily alcohols, alkenes, alkanes, and esters, among which T-4-O was the most abundant component, accounting for 31.09%. T-4-O exhibited strong antibacterial activity against S. aureus , with a minimum inhibitory concentration (MIC) of 2.12 mg/mL and a minimum bactericidal concentration (MBC) of 8.47 mg/mL. At 2 × MIC, T-4-O effectively suppressed bacterial growth. Furthermore, T-4-O disrupted metabolic pathways associated with biofilm formation, leading to approximately 50% inhibition of S. aureus biofilm development. It also induced structural disorganization of the cell membrane and impaired cell wall synthesis, resulting in leakage of cellular lysates, proteins, and other intracellular components. As a consequence, normal cell division and growth were inhibited, and the bacterial cell cycle was arrested at the R phase. Ultimately, these cumulative effects caused cell membrane rupture and bacterial death. Conclusion T-4-O exerts significant antibacterial effects against S. aureus primarily by disrupting cell membrane integrity, inhibiting biofilm formation, and inducing cell cycle arrest, collectively leading to bacterial death.