4.4. Evaluation of In Vitro Antibacterial Activity

The MIC values of Z33 against MRSA ATCC 43300 and S. aureus clinical isolates were measured according to the broth microdilution guidelines provided by the Clinical and Laboratory Standards Institute (CLSI) [18]. S. aureus ATCC 29213 was used as the quality control strain, and the quality control range was referred to the CLSI. The MIC value is defined as the lowest concentration of the compounds with no visible growth of bacteria. The MIC assay was independently repeated three times.

The MBC values of antibacterial agents were measured after incubation at 37 °C for 24 h. Briefly, 100 µL of bacterial solution from the three wells behind the corresponding well of MIC was absorbed and spread on the Mueller–Hinton Agar (MHA). The MBC refers to the lowest concentration corresponding to the amount of colony-forming unit (CFU) ≤ 5 on the medium after 18~24 h of incubation at 37 °C.

The time–kill kinetics curve of Z33 was determined according to previous reports [17]. MRSA ATCC 43300 strains were grown in Mueller–Hinton Broth (MHB) at the starting inoculum of 10⁶ CFU/mL. Z33 and tiamulin were diluted with MHB and added to the bacterial suspension to reach final concentrations from 1 × MIC to 32 × MIC, respectively. MHB without antibiotics was used as a control. At various time points (0, 3, 6, 9, and 24 h), 100 µL of samples were diluted with 0.9% saline and plated on MHA medium. CFU were recorded after 24 h of incubation at 37 °C.

The PAE was measured using the method described by Jin et al. [19]. MRSA ATCC 43300 cells were collected in the logarithmic phase and resuspended in MHB. The bacterial suspension (10⁶ CFU/mL) was inoculated in MHB containing Z33 (final concentration is 2 × or 4 × MIC) and incubated for 1 or 2 h at 37 °C with shaking (210 rpm). The group without the drug served as a control. After treatment with various concentrations of Z33, samples were subjected to one-thousand-fold dilution with MHB to remove the drug and incubated at 37 °C. In total, 100 µL of the samples was serially diluted in 0.9% saline and plated onto MHA at various time points (0, 1, 2, 4, 8 h). MHA plates were incubated at 37 °C for 18~24 h to calculate CFU. The growth kinetic curves of MRSA treated with different drug concentrations were established by plotting log₁₀ CFU/mL of bacterial counts versus time. PAE was presented in hours and calculated according to the following equation: PAE = T_A − T_C, where T_A and T_C are the time required for MRSA to rise by 1 log₁₀ CFU/mL after drug removal in the treated and untreated cultures, respectively.