The antibacterial activity was assessed by determination of the MIC of the compounds using the microdilution method, in a 96-well plate, according to the Clinical and Laboratory Standard Institute (CLSI) guidelines [72 ]. The media used was MHB II. The concentrations tested ranged from 64 µg/mL to 4 µg/mL, which were later converted to µM. The MIC was determined by visual inspection. DMSO, in subinhibitory concentrations (1% v/v), was used as a solvent for the compounds.
The combined effect of the compounds and clinically relevant antimicrobial drugs was evaluated by determining the antibiotic MIC in the presence of each compound. Briefly, the MIC of cefotaxime (CTX) (Duchefa Biochemie, Haarlem, The Netherlands) was determined in the presence of the highest concentration of each compound tested that did not affect bacterial growth of E. coli SA/2 when the compound was used alone. The antibiotic tested was serially diluted, whereas the concentration of each compound was kept fixed at 64 µg/mL, which were later converted to µM. Antibiotic MICs were determined as described above.
The combined effect of the compounds and clinically relevant antimicrobial drugs was evaluated by determining the antibiotic MIC in the presence of each compound. Briefly, the MIC of cefotaxime (CTX) (Duchefa Biochemie, Haarlem, The Netherlands) was determined in the presence of the highest concentration of each compound tested that did not affect bacterial growth of E. coli SA/2 when the compound was used alone. The antibiotic tested was serially diluted, whereas the concentration of each compound was kept fixed at 64 µg/mL, which were later converted to µM. Antibiotic MICs were determined as described above.