MIC determination by broth microdilution.

Assays were performed according to CLSI M07-A11 guidelines (9) in triplicate, with the exception of the indicated procedural variations and use of nonstandard AST media. The experimental parameters were varied one at a time during testing, and results were compared with those obtained using the standard reference BMD method (CAMHB). Falcon 96-well polystyrene plates (Corning; catalog number 351177) were used in all assays, except for indicated analyses using Falcon 96-well polypropylene plates (Corning; catalog number 351190). Assay plates were incubated for 18 h at 35 ± 2°C in ambient air, and MICs were determined visually unless otherwise indicated. To investigate the impact of freeze-thawing on exebacase activity in dilution panels (in indicated media), plates were transferred to a −80°C freezer within 2 h of preparation; after 24 h (unless otherwise indicated), the plates were removed and thawed for 1 h in ambient air prior to the addition of inocula (final cell density = 5 × 10⁵ CFU/ml) and overnight incubation at 35°C ± 2°C to determine MICs.