*3.2. Temporal Regulation of COL-R Mutations in Planktonic KP*

After 24 h of <sup>1</sup> <sup>2</sup>MIC level colistin selection, all three replicate planktonic KP populations showed an insertion in mucoid phenotype A regulator RmpA, as well as single-nucleotide polymorphisms (SNPs) in quinolinate synthase NadA and large-conductance mechanosensitive channel protein MscL, at nearly 100% frequency (Table 1, Figure 3A–C). These mutations coincided with a substantial rise in colistin MIC equivalent to 64-, 32-, and 32-fold increases compared to baseline MIC level (1 μg/mL) for populations 1–3, respectively. By day 6 of selection, KP population 1 showed an SNP in two-component system sensor histidine kinase PhoQ at 93% frequency, which aligned with a 128-fold increase in colistin MIC compared to baseline. In addition, this mutation increased to 100% frequency in population 1 in accordance with a 512-fold increase in colistin MIC. By day 36, a SNP at a new position in *phoQ* was acquired at 100% frequency as well as an SNP in two-component system sensor histidine kinase PmrB at high frequency, in line with a 2048-fold increase in colistin MIC compared to baseline (Figure 3A). Despite detection at different positions, mutations in *phoQ* were persistently observed from day 6 onward until day 36, which may contribute to the continued enhanced colistin resistance (Table 1, Figure 3A).

For planktonic population 2, two different SNPs in *phoQ* acquired by day 3 of selection corresponded with a 512-fold increase in colistin MIC. This dramatic rise in MIC during the first 3 days of selection was prominent in population 2, compared to populations 1 and 3, which showed more gradual increases over selection time (Figure 3A–C). Population 2 showed a similar pattern of *phoQ* SNPs generated at different positions through the course of evolution to that of population 1 (Table 1). Colistin MIC level remained consistent from days 3 to 27, in alignment with the duration of a unique SNP in *phoQ* and an SNP in thioredoxin-dependent thiol peroxidase BCP, observed on days 15–27. The final jump in MIC (2048-fold) was linked to mutations at unique positions of UDP-3O-(3-hydroxymyristoyl)glucosamine N-acyltransferase LpxD, *phoQ*, and *bcp*, all at 100% frequency. Interestingly, no *prmB* mutations were observed for population 2 for the extent of colistin resistance evolution (Table 1, Figure 3B).

**Table 1.** Timepoint and MIC of acquired COL-R mutations in planktonic evolved KP populations. COL-R mutations are represented for each individually evolved KP population with colistin MIC (μg/mL) and population frequency. Mutations with shared gene positions between populations are shaded gray. Shared mutations in genes that are acquired in independently evolved populations are color-coded for comparison. Mutations unique to individual populations are in white background. The shading is darker for mutations with higher population frequency.


Abbreviations: single-nucleotide polymorphism—SNP; insertion—INS; deletion—DEL.

Planktonic population 3 showed a unique pattern of SNPs in *phoQ* and *pmrB* with enhanced selection. On day 6, a SNP in *pmrB* was acquired and increased to 86.3% frequency by day 15. On day 15, a substantial rise in MIC was seen (512-fold) along with an additionally acquired SNP in two-component system response regulator PhoP that exists in the population until day 27 (Table 1, Figure 3C). On day 27, three unique mutations in *phoQ* (two positions) and *lpxD* were observed for which the MIC was maintained at 512-fold compared to baseline. The *phoQ* mutation seen at the same position as population 2 occurred at 73% frequency in population 3 on day 36, consistent with a 2048-fold increase in colistin MIC. The shifts in *phoQ* SNP positions with increased selection pressure likely contributed to the enhanced evolution of colistin resistance. The temporal regulation of COL-R mutations can be seen via the generation of key mutations sequentially, in alignment with considerable increases in MIC, with many alterations rising in population frequency or becoming fixed with enhanced selection pressure (Table 1, Figure 3A–C).

**Figure 3.** Temporal regulation of colistin-resistant mutations in planktonic and biofilm KP populations. Timelines of acquired mutations having mutation frequencies greater than 10% in known and theorized COL-R genes are represented by horizontal bars throughout a 36 day evolution. The duration of evolution in which the COL-R mutations exist within each planktonic (**A**–**C**) and biofilm (**D**–**F**) population is compared against colistin MIC. For both lifestyles, mutations in genes *rmpA*, *nadA*, and *mscL* were generated after 24 h at <sup>1</sup> <sup>2</sup> MIC colistin selection and were subsequently fixed in each population. Planktonic populations 1 (**A**) and 2 (**B**) acquired a mutation in *bcp* around midpoint of evolution followed by mutations in *lpxD*. Biofilm populations 1 (**D**) and 3 (**F**) acquired mutations in *lpxC* around midpoint of evolution. Mutations in two component system sensor regulator genes *phoQ*/*phoP* and histidine kinase genes *pmrB*/*pmrA* appeared sporadically, in relation to notable (at least twofold) increases in colistin MIC.

#### *3.3. Temporal Regulation of COL-R Mutations in Biofilm KP*

Similar to the "first wave" of colistin resistance acquired in planktonic KP, after 24 h selection, all three replicate KP biofilm populations showed mutations in *rmpA*, *nadA*, and *mscL* at 100% frequency (Table 2, Figure 3D–F). Insertions in *rmpA*, and SNPs in *nadA* and *mscL* coincided with 128-, 16-, and 64-fold increases in colistin MIC by day 3 of selection for biofilm populations 1–3, respectively. After 6 days of selection, biofilm population 1 acquired three SNPs in *pmrB* at unique positions, as well as an SNP in *phoQ*, consistent with a 128-fold increase in MIC. These mutations generated by day 6 were lost. Subsequent SNPs in *phoQ* and UDP-3-O-acyl-N-acetylglucosamine deacetylase LpxC were later acquired by day 24, which likely contributed to additional MIC increase (Figure 3D). In addition, a SNP in *lpxC* by day 15 aligned with another twofold increase in MIC, equivalent to a 256-fold increase from baseline. The final day of selection showed a 1024-fold increase in MIC, which overlapped with the timing of a deletion in PhoP/PhoQ regulator and DNA-binding transcriptional repressor MgrB–KdgR and a SNP in two-component system response regulator PmrA (Table 2, Figure 3D).

**Table 2.** Timepoint and MIC of acquired COL-R mutations in biofilm evolved KP populations. COL-R mutations are represented for each individually evolved KP population with colistin MIC (μg/mL) and population frequency. Mutations with shared gene positions between populations are shaded gray. Shared mutations in genes that are acquired in independently evolved populations are colorcoded for comparison. Mutations unique to individual populations are in white background. The shading is darker for mutations with higher population frequency.


Abbreviations: single-nucleotide polymorphism—SNP; insertion—INS; deletion—DEL.

For biofilm population 2, a similar timeframe for generating two-component system sensor histidine kinase mutations was seen to that of population 1 (Table 1, Figure 3E). By day 6, an SNP of 75% frequency was acquired in *phoQ*, consistent with a 64-fold increase in MIC from baseline. Two unique SNPs in *pmrB* and *phoQ* were generated by day 15 (Figure 3E), with a slight twofold increase in MIC. A 512-fold increase in MIC from baseline occurred by day 24, with the generation of an SNP in *phoQ* and deletion in two-component system sensor histidine kinase QseC, which remain fixed in the population for the extent of the selection. By day 36 of selection, a newly appeared mutation in two-component system sensor histidine kinase BaeS aligned with a 2048-fold increase in colistin MIC from baseline (Table 2, Figure 3E).

For biofilm KP population 3, a delay in the generation of two-component system sensor histidine kinase mutations was observed until day 15 of selection (Table 2, Figure 3F). By day 15, a mutation in two-component system sensor histidine kinase EnvZ was generated, consistent with a 128-fold increase in MIC from baseline. Another twofold increase in MIC was seen by day 24, for which mutations in *lpxC* and *pmrB* were acquired and fixed for the extent of selection. By day 36 of selection, two unique mutations in *envZ* and *phoP* were generated, which aligned with a 2048-fold increase in MIC from baseline (Table 2, Figure 3F). Through monitoring the MIC change via increasing concentrations of colistin selection, key mutations in two-component system sensor and regulators, as well as SNPs in *lpxC*, appear to drive COL-R in biofilm populations. The modification in mutation position for these critical genes and the increase in population frequency over time are also likely contributing factors to enhanced colistin resistance, independent of bacterial lifestyle. Next, we aimed to further characterize COL-R in KP by studying the timing of these mutations in relation to gene function.
