*2.4. Whole-Genome DNA Sequencing*

KP (whole population or single clone) genomic DNA was extracted using a DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany). Biofilm attached to beads was dissociated by sonication in sterile PBS before DNA extraction. Planktonic cultures were centrifuged and pelleted before DNA extraction. Whole-genome DNA sequencing was performed using an Illumina NextSeq 2000 platform with a paired-end mode of 2 × 150 base pairs and sequencing depth coverage of 200 Mbp for individual clones and 650 Mbp for wholepopulation bacterial genomes (SeqCenter, Pittsburgh, PA, USA).

#### *2.5. Comprehensive Mutation Analysis*

Data preprocessing for raw sequencing data included gentle quality trimming using trimmomatic (version 0.38, parameter: PE -phred33 LEADING:20 TRAILING:20 SLIDING-WINDOW:4:20 MINLEN:70) to filter out low-quality and unpaired reads before computational mutation analysis [25]. Bowtie2 (version 2.4.1), as part of the breseq workflow, was used to build an index of the reference genome and align reads to the index reference genome *K. pneumoniae* strain ATCC 43816 (serotype O1:K2) [26,27]. The reference genome was downloaded from NCBI GenBank with accession number SRR13008124. Breseq was used to detect mutations relative to the reference genome in consensus mode and polymorphism mode for clonal and mixed-population samples, respectively [28]. Both mixed-population and clonal samples were analyzed to identify and confirm the most integral COL-R mutations generated with increased selection. Breseq gdtools COMPARE subcommand was used to create side-by-side mutation comparison tables for individual populations to show how genetic variants and their frequencies change over time.
