Minimum Inhibitory Concentration (MIC) Determination

The minimal inhibitory concentration (MIC) was defined as the lowest concentration of a compound for which no growth was observed. The MIC determination assays were performed as established by the Clinical and Laboratory Standards Institute [CLSI] (2005) guidelines. The twofold standard micro broth dilution method was performed in 96-well plates. Briefly, bacterial strains were incubated overnight in 3 mL of MH broth at 37°C with shaking at 220 rpm. The bacterial cultures were then diluted in phosphate-buffered solution (PBS) to McFarland 0.5 (1 × 10⁸ CFU/mL). To each well, 188 μL of MH broth, 10 μL of the EO, extract or compound (7.81–1,000 μg/mL), and 2 μL of a bacterial suspension were added to McFarland 0.5 to achieve a final volume of 200 μL. In addition, some wells were used as solvent and sterility controls. The plates were incubated at 37°C for 24 h, and the optical density was measured at 600 nm (OD₆₀₀) in an Elisa lector (Thermo Scientific Multiskan FC Model, Waltham, MA, United States). The MICs of EtBr, a universal substrate of expulsion pumps, and carbonyl cyanide m-chlorophenyl hydrazone (CCCP) were used as the controls for EPIs and determined by the same method. All results are expressed as μg/mL, and experiments were performed in triplicate in three individual experiments.