Minimum inhibitory concentration (MIC) of HY-133 against S. aureus strain ATCC 29213 was determined in sterile 96-well microplates (Greiner Bio One International, Kremsmünster, Austria) following the Clinical and Laboratory Standards Institute (CLSI) guidelines, as previously described [12 (link),17 ,18 ]. Briefly, two-fold dilution series of HY-133 were prepared in cation-adjusted Mueller-Hinton broth (CAMHB, Becton Dickinson, Franklin Lakes, NJ, USA), with final concentrations ranging from 0.016–8 µg/mL. S. aureus ATCC 29213 was inoculated via direct colony suspension from stationary phase cultures grown overnight on Columbia blood agar (Becton Dickinson). A final concentration of 5 × 105 CFU/mL was used for inoculation of the 96-well microplates.
For determination of the minimum bactericidal concentration (MBC), 10 µL aliquots of culture from each microwell displaying the MIC and concentrations above were inoculated onto tryptic soy agar (TSA, Becton Dickinson) plates and incubated at 37 °C overnight. The MBC was defined as the concentration of HY-133 resulting in <500 CFU/mL (killing rate of 99.9%) [18 ]. Tests were performed in triplicate, and median values were taken for analysis.
For determination of the minimum bactericidal concentration (MBC), 10 µL aliquots of culture from each microwell displaying the MIC and concentrations above were inoculated onto tryptic soy agar (TSA, Becton Dickinson) plates and incubated at 37 °C overnight. The MBC was defined as the concentration of HY-133 resulting in <500 CFU/mL (killing rate of 99.9%) [18 ]. Tests were performed in triplicate, and median values were taken for analysis.
Free full text: Click here
