*3.5. Theoretical Pathways of Colistin Resistance in K. pneumoniae*

On the basis of the similarities in mutations observed with increasing colistin MIC by bacterial lifestyle, we theorize a pathway of colistin resistance, including the timing of affected gene functions and their corresponding mutations (Figure 5). We predict that an initial mutation in *rmpA* leads to loss of capsule polysaccharide (cps) synthesis, followed by regulation in osmotic pressure and efflux (mutations in *mscL* and *baeS*). Next, one could observe changes in energy metabolism through a modification in *nadA*. Additionally, mutations in *mgrB*, *phoQ*/*phoP*, and *pmrB* led to the addition of 4-amino-4-deoxy-L-arabinose and/or the transfer of phosphoethanolamine (pEth) by enzyme phosphoethanolamine transferase. Lastly, mutations in *lpxA* and *lpxD* critical for lipid A synthesis contribute to LPS loss. These mutations allow for the development and progression of colistin resistance in hypervirulent *K. pneumoniae*.

**Figure 5.** Theoretical pathways of colistin resistance in hypervirulent-*K. pneumoniae*. Colistin selection led to changes in five major gene functional groups, possibly in a sequential manner in both bacterial lifestyles: capsule production, cell membrane integrity, energy metabolism, modifications of LPS, and LPS biosynthesis loss. We posit that the mutations related to these functional groups facilitate and allow for enhanced colistin resistance in *K. pneumoniae*.
