*3.7. Loss of Hypermucoviscous Phenotype with Colistin Selection*

Our results indicated that rapid COL-R was initially dependent on mutations in mucoid phenotype A regulator gene *rmpA*, leading to changes in capsular polysaccharide synthesis and the hypermucoviscous (HMV) phenotype, independent of bacterial lifestyle. To demonstrate the *rmpA* mutations and alterations in HMV, we conducted the string test for hypervirulence on 1 day evolved (under <sup>1</sup> <sup>2</sup> MIC colistin treatment) planktonic population clones compared to the evolutionary ancestor clone. We found a significant decrease in the percentage of colonies that passed string tests following 1 day of colistin treatment for all three planktonic populations (Supplementary Figure S2). These results suggest that there may be an evolutionary tradeoff between hypervirulence and progressive resistance to colistin.

### **4. Discussion**

Colistin resistance has been extensively studied with meaningful mutations and their roles in the resistance mechanism identified. Modifications in lipid A moiety of LPS, overexpression of two-component regulatory systems PhoPQ and PmrAB, plasmid-mediated transfer of mobilized colistin resistance genes *mcr-1* to *mcr-8*, and the inactivation of the PhoQ/PhoP signaling regulator MgrB are some of the most understood mechanisms of colistin resistance in Gram-negative bacteria [14,37,44–50]. In this study, we narrow the knowledge gap in understanding the importance of mutation timing in the progression of COL-R by gene function for both planktonic and biofilm KP lifestyles. We show a similar pattern of resistance mutation timing through colistin selection between bacteria lifestyles (Figure 4). The "first wave" of early resistance was consistent between lifestyles, with the generation of mutations relevant to capsule production, cell membrane integrity, and energy metabolism. Mutations in *mscL* and *nadA*, regulating osmotic stress and energy metabolism, respectively, were likely immediate responses to environmental stress posed by colistin treatment. NadA is a catalyst in the biosynthesis of nicotinamide adenine

dinucleotide (NAD+), an essential cofactor, signaling molecule, and coenzyme for redox reactions of energy metabolism [51,52]. MscL is a pore-forming membrane protein that protects the cell from osmotic downshock, by promoting the efflux of various molecules such as potassium, glutamate, and proline, from the cytosol [53]. MscL is considered a potential drug target by acting as an emergency release valve, allowing for the uptake of extracellular molecules, including antibiotics [54]. These mutations may promote the development of further resistance mutations with roles in LPS modification and synthesis.

Following the "first wave", sporadic mutations affecting LPS structure were observed after day 2 in planktonic populations and occurred later on, by day 6, in biofilm populations. It appears that the positioning of the two-component system sensor and response regulator mutations responsible for LPS modifications may impact COL-R progression. For instance, for planktonic and biofilm populations, mutations at unique gene positions were generated in *phoQ* and *pmrB*. It is possible that mutation positions facilitating optimized COL-R became fixed in each population through enhanced selection pressure. Interestingly, mutations in LPS biosynthesis, likely leading to LPS loss, were generated following modifications in lipid A. For both lifestyles, lipid A modifications preceded mutations in *lpxC* and *lpxD* genes responsible for LPS biosynthesis. This suggests that LPS production may have been altered through colistin selection. Complete loss of LPS is a known mechanism of COL-R in *A. baumannii*, through modifications in *lpxA*, *lpxC*, and *lpxD*, the primary genes involved in lipid A synthesis, leading to a dramatic increase in colistin MIC of greater than 256 μg/mL [49]. Loss of LPS through deletions in *lpx* genes associated with COL-R has also been observed in *Escherichia coli* [55]. However, less is understood regarding the potential loss of LPS in KP, leading to rapid COL-R. Decreased LPS and modifications in LPS are likely the dominant mechanisms of COL-R observed in both planktonic and biofilm evolutions in our study. In addition, an uncharacterized gene mutation (*IT767\_0158*) was detected in both lifestyles following mutations *lpxC* and/or *lpxD*, suggesting that this gene may have roles in regulating LPS. Further investigation is necessary to understand the role of these gene mutations and whether their function is related to LPS biosynthesis.

We also found that bacterial evolution to resistance is likely specialized according to bacterial lifestyle in terms of mutation timing and MIC increase. The biofilm environment itself is designed to protect and respond in defense to environmental stressors, shown by a lag time in COL-R development compared with planktonic-evolved populations. In addition, biofilm development requires more time and metabolic demand for cell attachment, colonization, and maturation, compared to free-living cell propagation. Several mutations were theoretically specialized for biofilm-evolved COL-R, including mutations with roles in peptide transport, fatty-acid biosynthesis, and biofilm formation (*sbmA*, *fadR*, *acpP*, and *qseC*). The sensory kinase QseC is part of the two-component-based quorum sensing system (QseBC), which responds to environmental stress, including changes in osmotic pressure, heat shock, and oxidative stress [56]. Further investigation is required to understand the role of the quorum sensing system QseBC in COL-R development. Mutations generated at later timepoints (on or past day 27) had roles in regulating LPS modifications (*mgrB*, *phoP*, and *arnC*), as well as the envelope stress response and efflux pump expression (*baeS*). Mutations in *baeS*, generated by day 36 for both lifestyles, were likely attributed to bacterial defense against interactions of colistin at the bacterial cell membrane. The membrane-bound sensor histidine kinase BaeS is part of a two-component system involved in envelope stress response that responds to environmental stressors and regulates the expression of different efflux pumps [57]. The pattern of COL-R mutation timing between bacteria lifestyles appears to be both similar and specialized due to growing metabolic demands and likely differences in bacteria susceptibility to colistin treatment.

Through our long-term 36 day experimental evolution, we were able to monitor changes in mutation position and frequency and addition of newly acquired mutations, with the intent to better understand how KP continues to adapt to increasing colistin pressure. These methods allowed us to assess the temporal regulation and interactions of COL-R mutations for over 374 generations (36 days × ~10.2 generations per day). Additionally, we were able to study the timing of various impacted COL-R genes and their functions by bacterial lifestyle. By performing the experimental evolutions using both planktonic and biofilm growth, we were able to account for the effects and demands of bacteria lifestyle as a part of the COL-R mechanism and compare/contrast the most beneficial mutations that led to substantial colistin MIC increase with enhanced selection pressure.

A limitation of in vitro experimental evolution studies is the inability to capture the resistance mechanisms associated with plasmid-mediated transfer in addition to somatic gene mutations. As expected, we did not observe any horizontal transfer of plasmid-borne COL-R genes such as the *mcr* variants, which have been rising in prevalence in nature and human patients [15]. Nonetheless, the system of in vitro experimental evolutions under a controlled environment allows us to unmistakably identify the most beneficial chromosomal alterations leading to progressively enhanced colistin resistance.

Considering the rapid resistance to colistin detected for KP in both lifestyles, it is crucial to determine if the COL-R isolates that could exist in clinical settings will remain susceptible to the newly approved β-lactam/β-lactamase dual-inhibitor antibiotics. CAZ/AVI has been approved for administration in the United States since 2015 to combat ESBL and carbapenemase-producing infections. Avibactam prevents ceftazidime hydrolysis by carbapenemases (KPCs) and ESBLs, while the third-generation cephalosporin ceftazidime exhibits bactericidal activity by inhibiting cell-wall synthesis [58]. Our results demonstrate the diversity in bacterial resistance mechanisms to various antimicrobials, including AMPs and conventional dual-inhibitor antibiotic treatments designed to prevent resistance development. These results support that CAZ/AVI remains a critical treatment option for colistin-resistant infections, which is increasingly vital with the rise in high mortality and hypervirulent MDR bacterial infections in the clinical setting.

In summary, this study presents a clearer understanding of the timing and significance of COL-R mutations in KP to ultimately aid in the development of new clinical treatments that successfully eradicate and/or prevent CR-KP infections in patients. Experimental approaches using in vitro and in vivo models of antibiotic selection similar to laboratory systems described here and isolates sampled from patients before and after treatment [59,60] would facilitate the identification of potentially critical resistance mutations. In addition, we can study the influence of epigenetic factors such as DNA methylation of bacteria, which regulate the expression of genes [61]. With further gain- or loss-of-function studies to investigate the impact of individual and combinations of mutations on antimicrobial resistance [62–64], these data are useful for the clinical situation by elucidating the critical targets that are utilized by bacteria for adaptive resistance to antimicrobials. Along with understanding the influence of timing of critical mutations and timing of affected genes by their functional roles for cell survival through adaptive evolution to resistance, studies like this will be important in enhancing drug development to prevent or lessen the rapid resistance to antimicrobials.

**Supplementary Materials:** The following supporting information can be downloaded at https: //www.mdpi.com/article/10.3390/pharmaceutics15010270/s1: Figure S1. Schematic of planktonic and biofilm experimental evolution workflow; Figure S2. Evolved KP isolates quickly lost their hypervirulence.

**Author Contributions:** Conceptualization, J.M.K. and Y.P.D.; formal analysis, J.M.K. and Y.P.D.; investigation, J.M.K. and Y.P.D.; funding acquisition Y.P.D.; resources, Y.P.D.; supervision, Y.P.D.; writing—original draft, J.M.K.; writing—review and editing, Y.P.D. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the National Institutes of Health (R01 AI-133351 to Y.P.D.). The funding agencies had no role in the study design, data collection, and analysis, the decision to publish, or the preparation of the manuscript.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data are contained within the article or Supplementary Materials. The sequencing data have been deposited with links to BioProject accession number PRJNA922161 in the NCBI BioProject database (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA922161).

**Acknowledgments:** The authors thank Jaydep Halder and Pranav Kumar Kaliperumal for their assistance in carrying out string test experiments.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.
