*2.6. Mutation Selection Criteria*

A series of criteria were distinguished to select relevant genes and their corresponding mutations of interest. Mutation prediction using breseq and mutation comparison tables were designed and analyzed to identify alterations in genes that were observed in independently evolved populations at greater than 20% frequency. Most of these mutations increased in frequency with enhanced selection pressure or became fixed at 100% frequency in these populations. We studied the function of genes for which these mutations were observed to speculate their potential role in and importance for the resistance mechanism. Using a thorough review of the current literature, we noted mutations that have been previously identified as critical for colistin-resistance development. In addition, we included mutations that have not been studied extensively in *K. pneumoniae* to theorize how they may be influential in the resistance pathway according to their timing and associated gene functions.

### *2.7. String Test for Hypervirulence*

The string test was performed to assess changes in hypervirulence following <sup>1</sup> 2MIC colistin treatment for planktonic KP populations. Tryptic soy agar plates containing 40 mg/L Congo Red (Sigma, MO, USA) and 20 mg/L Coomassie Brilliant Blue (Bio-Rad, Watford, UK) dyes were prepared and used to plate various bacterial dilutions to achieve comparable single-colony counts between each treated population and the evolution ancestor clone. A sterile inoculation loop was used to touch the surface of each single colony and measure the length of mucoid string produced. A positive test was determined for a mucoid string of 5 mm or greater in length, a feature observed clinically to define the

hypervirulent phenotype [29]. Statistical comparisons between treatment populations and the original clone were made using one-way ANOVA. A *p*-value < 0.05 was considered to be statistically significant.

### **3. Results**
