3.4.1. Capsule Production

Hypervirulent KP has been characterized on the basis of several features, including the virulence gene *rmpA*, a regulator of the mucoid phenotype A, which is an activator for capsular polysaccharide synthesis [30,31]. Low *rmpA* expression has been shown to be correlated with a hypervirulence-negative phenotype in KP [30]. However, less is understood regarding the impact of *rmpA* on COL-R and the potential evolutionary tradeoff between hypervirulence and COL-R. An insertion in *rmpA* was acquired by day 1 of selection in both bacteria lifestyles and was nearly fixed after 24 h. The timing of this insertion aligns with 64-, 32-, and 32-fold increases in MIC for planktonic populations 1–3 (Table 3) and twofold increases in MIC for biofilm populations (Table 4). Notably, these MIC increases were similarly observed for *mscL* and *nadA* mutations, which have functions in cell membrane integrity and energy metabolism, respectively. Our results suggest that capsule production is one of the earliest functional groups modified by colistin selection (Figure 4).

#### 3.4.2. Cell Membrane Integrity

Genes with roles in cell membrane integrity that were altered following colistin selection include *mscL* and *baeS*, which were generated by day 1 and day 36 in planktonic and biofilm populations, respectively (Tables 3 and 4, Figure 4). SNPs in *baeS* were observed in planktonic population 3 and biofilm population 2 on the final day of selection at 75% and 100% frequency, respectively (Tables 3 and 4).

**Table 3.** Functional roles of COL-R mutations in relation to timing, population frequency (percentage), and colistin MIC (μg/mL) for planktonic-evolved KP. Resistant MICs are shown in red, and mutations independent to planktonic lifestyle are shown in blue.


Abbreviations: mutation frequency—Freq; hypermucoviscous—HMV; nicotinamide adenine dinucleotide—NAD; lipopolysaccharide—LPS; reactive oxygen species—ROS.

#### 3.4.3. Energy Metabolism

Quinolinate synthase NadA, XylR family transcriptional regulator, and pyrroloquinoline– quinone synthase PqqC are associated with energy metabolism and were modified through colistin selection. SNPs in *nadA* were generated by day 1 and persisted throughout selection. Through carbon catabolite repression, bacteria may activate transcription factor XylR to regulate the metabolism of L-arabinose and D-xylose in place of glucose [32]. Mutations in XylR family transcriptional regulator were only acquired in planktonic populations 2 and 3 on day 36 (Table 3). *PqqC* expression is required for the biosynthesis of pyrroloquinoline quinone, a vitamin and redox cofactor of bacterial dehydrogenases, important for cell growth and metabolic reactions [33]. A mutation in *pqqC* was generated only in biofilm population 3 on day 24 but persisted until the end of selection at 100% frequency (Table 4). While mutations in *nadA* were fixed early in selection, mutations in *xylR* and *pqqC* were required at later timepoints for planktonic and biofilm populations, respectively (Figure 4). It is likely that alterations in bacterial metabolism are direct responses to the environmental stress posed by increasing colistin selection pressure.

**Table 4.** Functional roles of COL-R mutations in relation to timing, population frequency (percentage), and colistin MIC (μg/mL) for biofilm-evolved KP. Resistant MICs are shown in red, sensitive MICs are shown in green, and mutations independent to biofilm lifestyle are shown in green.


Abbreviations: mutation frequency—Freq; hypermucoviscous—HMV; nicotinamide adenine dinucleotide—NAD; lipopolysaccharide—LPS; antimicrobial peptide—AMP.
