Determination of antibacterial synergy

MICs of 135C in combination with a range of antibiotics with varying mechanisms of action were determined against S. aureus NCTC 6571 to investigate whether synergy or antagonism occurred. The antibiotics tested were ciprofloxacin, rifampicin, vancomycin, erythromycin, gentamicin and oxacillin. The checkerboard broth microdilution method was used as described previously²¹. Briefly, each antibiotic was serially diluted two-fold in 50 μL volumes in a 96-well microtitre tray (Falcon, Becton Dickinson, USA), after which 50 μL of 135C at varying concentrations (ranging from 0.06–4 μg/ml) was added to each dilution series. The remainder of the assay was performed as described above. For each well of the microtitre plate that corresponded to an MIC, the fractional inhibitory concentration index (∑FIC) was determined using the equation: ∑FIC = FIC_A + FIC_B = (MIC of drug A in combination/MIC of drug A alone) + (MIC of drug B in combination/MIC of drug B alone). The ∑FIC_min and ∑FIC_max were determined as the lowest and the highest ∑FIC, respectively. The interaction was deemed synergistic if the ∑FIC was ≤ 0.5, additive if the ∑FIC was between 0.5 and 1.0, indifferent if the ∑FIC was between 1.0 and 2.0 and antagonistic if the ∑FIC was > 2.0^22,23.This assay was performed in triplicate per antibacterial agent/135C combination.