3.9. Determination of the MICs and MBCs of Compounds and Control Drugs against Clinically Important Gram-Positive and Gram-Negative Bacteria

The minimum inhibitory concentrations (MICs) of the tested compounds and control drugs, linezolid, vancomycin, and gentamicin, were determined using the broth microdilution method according to the guidelines outlined by the Clinical and Laboratory Standards Institute (CLSI) [48] against clinically relevant bacterial (Staphylococcus aureus, MRSA, Escherichia coli, Enterococcus faecalis and Enterococcus faecium strains. S. aureus, MRSA, E. coli, Enterococcus faecalis and Enterococcus faecium were grown aerobically overnight on tryptone soy agar (TSA) plates at 37° C. Afterwards, a bacterial solution equivalent to 0.5 McFarland standard was prepared and diluted in cation-adjusted Mueller–Hinton broth (CAMHB) (for S. aureus, MRSA, and E. coli) to achieve a bacterial concentration of about 5 × 10⁵ CFU/mL. Enterococcus faecalis and Enterococcus faecium 0.5 McFarland standard solution was diluted in tryptone soy broth (TSB) to achieve a bacterial concentration of about 5 × 10⁵ CFU/mL. Compounds and control drugs were added in the first row of 96-well plates and serially diluted with the corresponding media containing bacteria. Plates were then incubated as previously described. MICs reported in Table 2 are the minimum concentration of the compounds and control drugs that could completely inhibit the visual growth of bacteria. The minimum bactericidal concentration (MBC) was tested by spotting 4 µL from wells with no growth onto TSA plates. Plates were incubated at 37 ° C for at least 18 h before recording the MBC. The MBC was categorized as the lowest concentration that reduced bacterial growth by 99.9% [45,46,47].