Determination of Minimum Inhibitory Concentration

Linezolid minimum inhibitory concentration (MIC) was identified using 2 methods. The first method was broth macrodilution. Turbidity of an exponential-phase Mtb culture was adjusted to a bacterial density of 1.5 × 10⁵ CFU/mL, and then treated with linezolid concentrations in Middlebrook 7H9 broth of 0, 0.075, 0.15, 0.30, 0.6, 1.25, 2.5, and 5.0 mg/L in conical tubes in triplicate. On day 7, cultures were washed twice with normal saline to prevent drug carryover, serially diluted, and cultured on Middlebrook 7H10 agar supplemented with 10% OADC (herein termed “agar”). Cultures were incubated at 37°C and colonies counted 21 days later. MIC was defined as the lowest concentration that prevented at least 99% of the growth observed in the absence of linezolid. The second method was an Epsilometer test (Etest). Two hundred microliters of Mtb in exponential phase growth with turbidity adjusted to 1.5 × 10⁸ CFU/mL were used to streak agar. Cultures were incubated at 37°C with 5% CO₂ for a period of 48 hours, after which the Etest strip was applied and the cultures incubated for another 7 days, at which point the result was read. The MIC was defined as the concentration at which the ellipse or zone of inhibition intersects the MIC reading scale.