Minimum inhibitory concentration (MIC).

MIC was determined by broth micro-dilution according to CLSI guidelines. The test medium formost species was cation-adjusted Mueller-Hinton broth (MHB). The same test medium was supplemented with 3% lysed horse blood (Cleveland Scientific, Bath, OH) for growing Streptococci. Haemophilus Test Medium was used for H. influenzae (Teknova, Hollister, CA), Middlebrook 7H9 broth (Difco) was used for mycobacteria, Schaedleranaerobe broth (Oxoid) was used for C. difficile, and fetal bovine serum (ATCC) was added to MHB (1:10) to test the effect of serum. All test media were supplemented with 0.002% polysorbate 80 to prevent drug binding to plastic surfaces³⁹, and cell concentration was adjusted to approximately 5 × 10⁵ cells per ml. After 20 h of incubation at 37 °C (2 days for M. smegmatis, and 7 days for M. tuberculosis), the MIC was defined as the lowest concentration of antibiotic with no visible growth. Expanded panel antibacterial spectrum of teixobactin was tested at Micromyx, Kalamazoo, MI, in broth assays. Experiments were performed with biological replicates.