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

The MICs for the peptidomimetics and conventional antibiotics (vancomycin, ciprofloxacin, gentamicin, rifampicin, linezolid, azithromycin and nisin) were determined by using the two-fold broth microdilution assay. Overnight cultures of the enterococcal isolates were diluted in physiological saline (0.9% NaCl) to reach a turbidity of 0.5 McFarland units (Sensititre^® nephelometer and the Sensititre^® McFarland Standard). Bacterial suspensions were adjusted to 5 × 10⁵ CFU/mL in MHB II or BHI broth containing two-fold dilutions of peptidomimetics or conventional antibiotics in a final volume of 100 µL. The plates were incubated for 24 h at 37 °C without shaking. The MIC was defined as the concentration of the agent that completely prevented visible growth. All experiments were performed in three biological replicates.

Since BHI better supports the growth of enterococci as compared to MHB II, which is traditionally used for susceptibility studies, we also assessed the MICs of compounds 2, 5 and 10 against E. faecium (MV388) and E. faecalis (MV269) when propagated in BHI. The MICs of the three compounds against both isolates were identical in BHI and MHB II. Hence, the following experiments were conducted in BHI medium.

After the MIC determination of compound 2, 5 and 10, aliquots of 50 μL from all the tubes that showed no visible bacterial growth were plated on BHI agar plates and incubated for 24 h at 37 °C. The minimum bactericidal concentration (MBC) is defined as the lowest concentration of the peptidomimetics that kills 99.9% of the bacterial population. All experiments were performed in three biological replicates.