Checkerboard titration assay for the detection of synergy in vitro.

The checkerboard titration method was used to evaluate synergy between TXA707 and cefdinir against MRSA, VISA, VRSA, LRSA, and MSSA isolates. In this assay (performed with 96-well microtiter plates), TXA707 was serially diluted 2-fold in CAMH broth along the rows of the microtiter plate, while cefdinir was diluted along the columns. The final volume in each well was 0.1 ml. After the compounds were added, each well was inoculated with 5 × 10⁵ CFU of log-phase bacteria. The microtiter plates were incubated aerobically for 16 to 24 h at 37°C, at which point bacterial growth was visualized. Fractional inhibitory concentrations (FICs) of the agents in each well that did not exhibit visible growth compared to agent-free control wells were calculated with the following two relationships: (i) FIC_TXA707 = MIC of TXA707 in combination with cefdinir/MIC of TXA707 alone; (ii) FIC_cefdinir = MIC of cefdinir in combination with TXA707/MIC of cefdinir alone. These FICs allow the determination of the FIC index (FICI) with the following relationship: FICI = FIC_TXA707 + FIC_cefdinir. An FICI of ≤0.5 indicates synergy between the two agents, while an FICI between 0.5 and 2.0 indicates additivity.