*3.4. Timing of Colistin Resistance Mutations by Functional Roles*

Planktonic and biofilm-evolved KP in this study shared many COL-R mutations with various functions in capsule production, cell membrane integrity, energy metabolism, and modification of LPS structure and biosynthesis (Tables 3 and 4). Defense against reactive oxygen species (ROS) by *bcp* was a mutation only observed in planktonic-evolved KP, while mutations in genes regulating fatty-acid biosynthesis (*fadR* and *acpP*), biofilm formation (*qseC*), and peptide transport (*sbmA*) were only observed in biofilm-evolved KP (Tables 3 and 4). A mutation in DUF3413 domain-containing protein was observed in both planktonic and biofilm KP, but remains functionally uncharacterized. However, the generation time of this mutation appears to align with other mutations on genes responsible for the modification of LPS (*lpxC* and *lpxD*) (Figure 4). Next, we assessed the timing of mutations according to their functional roles observed for both planktonic- and biofilm-evolved KP to better understand their influence on the COL-R mechanism.
