**2. Materials and Methods**

#### *2.1. Bacteria Strain and Culture Media*

*Klebsiella pneumoniae* American Type Culture Collection (ATCC) strain 43816 was first grown on cation-adjusted Mueller–Hinton Broth 2 (MHB2) agar medium (Sigma-Aldrich, St. Louis, MO, USA) overnight, and a single colony was transferred into liquid M9 medium supplemented with <sup>1</sup> <sup>2</sup>MIC level of colistin sulfate treatment (Research Products International, Mount Prospect, IL, USA). M9 minimal growth medium was designed using M9 salts (6 g/L Na2HPO4, 3 g/L KH2PO4, 0.5 g/L NaCl, and 1.0 g/L NH4Cl) supplemented with 0.1 mM CaCl2, 2 mM L-Glutamine, 2 mM MgSO4, and 4% glucose to reflect the essential components required by mammalian cells and biofilm formation to prevent additional nutrient selection effects [19,20].

#### *2.2. Bacterial Transfer and Resistance Selection*

To investigate the timing and relevance of resistance genes concerning MIC increase, we performed bacterial experimental evolution studies using a serial passage of KP planktonic culture and a single colonized biofilm bead under colistin selection pressure (Supplementary Figure S1). Three replicate populations were subjected to colistin selection according to bacteria lifestyle. For planktonic populations, 1/100 or approximately 2.5 × <sup>10</sup><sup>6</sup> colony forming units (CFU)/mL of total overnight culture was transferred into fresh M9 media supplemented with colistin. For biofilm populations, a bead transfer-based biofilm evolution system was used according to a method described previously [21–23]. Briefly, a single colonized 7 mm polystyrene bead, approximately 2.5 × 105 CFU/mL, was transferred into M9 medium supplemented with colistin and two sterile polystyrene beads. The sterile beads facilitate the processes of bacteria attachment, biofilm formation, and dispersal. The concentration of colistin treatment was doubled every three days to steadily enhance bacteria resistance selection. Evolution under antibiotic selection continued for 36 days without complete inhibition of growth after each doubling of treatment concentration (Figure 2A).

**Figure 2.** Colistin selection and population MIC result by bacterial lifestyle. (**A**) Colistin treatment for KP planktonic and biofilm populations started at <sup>1</sup> <sup>2</sup> MIC; the selection concentrations were doubled every three days for 36 days. (**B**) Colistin population MIC results averaged among three replicate colistin-treated KP populations by bacteria lifestyle. Rapid resistance to colistin was observed for (**C**) planktonic and (**D**) biofilm individually evolved populations after 36 days of colistin selection pressure. By day 36, there was a 1024–2048-fold increase in colistin MIC compared to baseline level. The resistance is distinguished in gray background when MIC is above the CLSI-determined clinical breakpoint at 4 μg/mL for colistin.
