*2.3. MIC Measurement*

Colistin MIC was determined using the broth microdilution method established by the Clinical and Laboratory Standards Institute (CLSI) [24]. Whole-population MIC was determined for each KP population replicate at the end of every 3 days of serial passage and prior to transfer into the subsequent treatment concentration. The range of antibiotic selection began at a baseline level of <sup>1</sup> <sup>2</sup>MIC or 0.5 μg/mL for the ancestor KP clone on days 1–3 and was subsequently increased to a level of 1024 × MIC or 1024 μg/mL for the final days 33–36. Cation-adjusted Mueller–Hinton Broth 2 (MHB2) (Sigma-Aldrich, MO, USA) was used to inoculate each planktonic KP population for overnight incubation at 37 ◦C prior to MIC testing to increase bacterial cell count. KP biofilm populations were isolated by transferring a single colonized polystyrene bead into a 15 mL conical tube containing 2 mL of phosphate-buffered saline for ultrasonic homogenization (DPS-20 model, PRO Scientific Inc., Oxford, CT, USA). Prior to MIC testing, planktonic and homogenized biofilm populations were cultured in MHB2 medium for 24 h at 37 ◦C in a shaking incubator (Corning LSE 71L model, Corning, NY, USA). MIC testing was

performed using sterile 96-well, microplates (Greiner, Frickenhausen, Germany). Bacterial concentration was adjusted to approximately 5 × 105 CFU/mL with PBS prior to plating. MIC values were determined by measuring turbidity via optical density readings at 570 nm wavelength using a Gen 5 Microplate Reader and Imaging software (BioTek Instruments, Winooski, VT, USA, Version 3.04). Samples selected for MIC testing were preserved in 8% dimethyl sulfoxide for further genomic analysis.
