Fractional Inhibitory Concentration Index

To determine the fractional inhibitory concentration index (FICI), ultrapure water was used to dissolve the ceftriaxone sodium and PPIX-MED so as to give stock concentrations of 4, 2, 0.5, and 0.25 MIC. Using the checkerboard design method, 50 μl of each drug was added at different concentrations in the horizontal and vertical columns of a 96-well plate, respectively. Then, 100 μl of bacterial (10⁶ CFU/ml) suspension was added and mixed by slight shaking. The plates were kept in the dark for 30 min at 37°C and then exposed to light for 30 min. After light exposure, the samples were incubated in the dark at 37°C for 16–20 h. The MIC of each antimicrobial in the combination was read and interpreted using previously described methods. For each antimicrobial combination, we calculated the FICI by computing the ratio of the MIC of the combination divided by the MIC of the antimicrobial alone for each agent and then adding those two ratios together (see Equation 2). The FICI data were interpreted using the following criteria: synergy, FICI ≤ 0.5; indifference, FICI 0.5–4.0; and antagonism, FICI > 4.0 (Barbee et al., 2014).