*3.1. Rapid MIC Increase in Colistin-Treated KP Populations*

The baseline MIC for the colistin-susceptible ancestor KP clone (ATCC 43816) was determined to be 1 μg/mL. Rapid increases in MIC were observed for individually evolved KP populations through experimental evolution under colistin selection with increasing concentrations (Figure 2A) independent of bacteria lifestyle (Figure 2B). However, there was a slower rate of MIC increase within the first 3 days and for the extent of evolution for biofilm- compared to planktonic-evolved KP. For planktonic-evolved KP populations, the most substantial jumps in MIC occurred during the first 3 days of colistin selection. More gradual and consistent twofold increases occurred every 3 days afterward and for the remaining days of evolution. Compared to the planktonic lifestyle, biofilm populations showed modest (twofold) increases from baseline MIC after 24 h of incubation at <sup>1</sup> <sup>2</sup>MIC level treatment. After 24 h of antibiotic selection, colistin MIC increased over 32-fold for planktonic populations and twofold for biofilm populations (Figures 1D and 2C). By day 3, planktonic populations 1–3 showed 64-, 512-, and 64-fold increases in MIC compared to 128-, 16-, and 64-fold increases for biofilm populations 1–3. Similar to planktonic populations, after the first 3 days of selection, biofilm populations showed consistent 2–4-fold increases in MIC for the remainder of the 36 day evolution experiment. The CLSI breakpoint for colistin of 4 μg/mL was surpassed after only 24 and 48 h evolution at <sup>1</sup> <sup>2</sup>MIC level of selection for planktonic and biofilm lifestyles, respectively. After day 27, KP showed further twofold increases in MIC with no hindrance in growth under selection pressure for both lifestyles. Following 36 days of selection, 2048-fold increases in colistin MIC were observed for all three planktonic populations (Figure 2C) as well as 1024-, 2048-, and 2048-fold increases in biofilm populations 1–3, respectively (Figure 2D).
