Time-kill synergy testing.

Antimicrobial stocks were diluted in 9.5 ml of RPMI 1640 in 25- by 150-mm glass round-bottom tubes to the appropriate starting concentrations. Antimicrobial concentrations for the time-kill study were based on the checkerboard array results. For a given drug combination and strain, we identified the well in the checkerboard array with the lowest concentration of MIN or RIF among wells with an FIC_I of ≤0.5 and noted the concentration of both drugs in that well. Time-kill drug concentrations were then chosen as follows. For AMB plus RIF, RIF at the concentration in this well was tested with AMB at the concentration in the well and AMB at twice this concentration. For AMB plus MIN, the same formula was used, except that MIN was tested at twice the concentration in the well because there was no effect at lower concentrations in any strain tested. For CAS plus MIN, MIN at the concentration in the well and twice that concentration was tested in combination with CAS at the concentration in the well. Negative (sterility) control and positive growth control tubes containing no antimicrobials were also prepared. A 1.0 McFarland suspension of yeast cells from an overnight plate was prepared in 0.9% sodium chloride, and 0.5 ml of this suspension was added to each tube for a final starting concentration of 1 × 10⁵ to 5 × 10⁵ CFU/ml (39). Cultures were incubated with shaking in ambient air at 35°C for 48 h. At 0, 3, 6, 24, and 48 h, aliquots were removed from the culture tube and a 10-fold dilution series was prepared in 0.9% sodium chloride. A 10-μl drop from each dilution was plated onto Sabouraud dextrose agar and incubated overnight. The colonies within each drop were then counted and cell density was calculated (40). Only dilutions containing 3 to 40 colonies per drop were counted in order to prevent quantification distortion associated with very low numbers of colonies while ensuring that individual colonies could be distinguished by eye (41). If more than one dilution for a given sample was usable, the cell densities of the two drops were averaged. If no drops were usable, the densities for consecutive drops above and below the usable range were averaged. The lower limit of detection with this method is 300 CFU/ml. A combination was considered synergistic if it resulted in a ≥2-log₁₀ reduction in CFU/ml compared to the most active agent alone and fungicidal if it resulted in a ≥3-log₁₀ CFU/ml reduction compared to the starting inoculum. Synergy and fungicidal activity were evaluated at 24 and 48 h.