Linalool potentiates tetracycline against multidrug-resistant Staphylococcus aureus via biofilm disruption

The rapid global spread of multidrug-resistant (MDR) bacteria is undermining the effectiveness of existing antibiotics and motivating the search for adjunct strategies. Here, we evaluated the in vitro antibacterial activity of linalool and explored its associated cellular effects against multidrug-resistant Staphylococcus aureus (MDRSA). Checkerboard assays and fractional inhibitory concentration index (FICI) analysis were used to assess the inhibitory effect of linalool in combination with tetracycline antibiotics on MDRSA. Targeted metabolomics was employed to investigate the impact of linalool on MDRSA. Bacterial morphology was observed using transmission and scanning electron microscopy. Bacterial content leakage and changes in oxidative stress-related parameters were measured separately. In combination with tetracycline antibiotics, linalool showed synergistic or additive interactions based on FICI analysis and was associated with reduced bacterial growth and biofilm formation. Electron microscopy observations revealed that linalool caused indentations and ruptures on bacterial surfaces, leading to an increase in the leakage of bacterial contents (DNA, and RNA). Metabolomics revealed that linalool regulates various microbial metabolic processes, including microbial metabolism in different environments, lysine degradation, two-component systems, ABC transporters, and arginine biosynthesis, further supporting its antibacterial activity. Linalool treatment was associated with altered SOD- and H2O2-related readouts in MDRSA, consistent with perturbations in redox homeostasis and cellular metabolism; these changes were more pronounced under combination treatment. The combination of linalool with tetracycline-class antibiotics was associated with broader physiological perturbations. These findings provide a rationale for further mechanistic investigations and in vivo evaluation of linalool as a potential adjuvant to tetracycline antibiotics.