We evaluated whether Ω76 and colistin have any synergistic, additive, or antagonistic effects on A. baumannii (P1270) using the checkerboard assay. This was performed by calculating the fractional inhibitory concentration (∑FIC; Eq. 2) for a given combination of antibiotics.FIC=FICcolistin+FICΩ76FICcolistin=min(MBCcolistin value on checkerboard)MBCcolistinFICΩ76=min(MBCΩ76 value on checkerboard)MBCΩ76(2)

Here, ∑FIC ≤ 0.5 indicates synergy, 0.5 < ∑ FIC ≤ 4 indicates an additive effect, and ∑FIC > 4 indicates antagonism (53).

A checkerboard assay was performed on a 96-well polypropylene plate. Each well contained 200 μl of Mueller-Hinton medium, differing concentrations of antibacterial agents, and an inoculum of 5 × 104 CFU per well of A. baumannii (P1270), as depicted in fig. S2A. The 96-well checkerboard assay was incubated at 37°C for 12 hours. Thirty microliters of a 0.02% (w/v) aqueous resazurin solution was then pipetted into each well. Further incubation was performed at 37°C for 12 hours. Growth was estimated on the basis of fluorescence measurements (excitation, 530 nm; emission, 590 nm). The ∑FIC value for the chosen antibiotic combination was then estimated on the basis of bacterial growth.

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