2.5.2. Determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC)

The MIC and MBC of GIE against VREF were evaluated according to methods described in the CLSI 2021 guidelines [24]. Briefly, GIE was serially diluted in a 96-well microtiter plate to a final concentration of 250 to 0.98 mg/mL in MHB. A suspension of E. faecium (5 × 10⁵ CFU/mL) was added to each well, and plates were incubated at 37 °C for 18 h. Tetracycline (TE) and deionized water (DI) were the positive and negative controls, respectively. To determine MIC values, 0.05% resazurin (Thermo Fisher Scientific, Lancashire, UK) was added. The MIC was defined as the lowest concentration that completely inhibited bacterial growth, indicated by a blue color [24]. MBC values were determined by streaking cultures from wells with significant MIC results onto TSA plates to assess bacterial viability.