MIC measurements.

MICs of Leu₁₀-teixobactin were determined for all isolates using broth microdilution in Mueller-Hinton broth (MHB; Oxoid, England) supplemented with 0.1% Tween 80 to avoid the adhesion of the compound to the 96-well microtiter plates (19). Stock solutions of Leu₁₀-teixobactin were prepared in dimethyl sulfoxide (Sigma-Aldrich) immediately before each experiment. The solution was then serially diluted in MHB medium to create a range of solutions from 0.125 to 128 μg/ml, which were then added to 96-well microtiter plates (Techno Plas). All tested strains were exposed to a final concentration of DMSO (2.5%, vol/vol) that had no inhibitory effect on their growth. The subculture-isolated colonies were added to 0.9% saline using a sterile swab and then measured using a McFarland meter (bioMérieux) to reach a 0.5 McFarland turbidity standard. The culture was then diluted 100-fold to give an approximate starting inoculum of 10⁶ CFU/ml in each well of the 96-well microtiter plates. The plates were then incubated at 37°C for 18 to 24 h. MICs were determined as the lowest concentrations that inhibited the visible growth of the bacteria.