Genomic Insights into Chromosomal Colistin Resistance and Virulence–Resistance Convergence in MDR/XDR Klebsiella pneumoniae from Tertiary Hospitals in Peshawar, Pakistan

Background: Klebsiella pneumoniae is a World Health Organization-listed critical priority pathogen and a major cause of healthcare-associated infections, driven by the global emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) lineages and their alarming convergence with hypervirulence. Methods: In this study, 152 clinical specimens, including urine, blood, pus, wound swabs, and respiratory samples, were collected from tertiary care hospitals in Peshawar, Pakistan. Standard microbiological and biochemical methods identified 55 K. pneumoniae isolates. Antimicrobial susceptibility testing (AST) was performed using the Kirby–Bauer disk diffusion and broth microdilution methods, with results interpreted according to Clinical and Laboratory Standards Institute (CLSI) guidelines. MDR and XDR phenotypes were defined based on European Centre for Disease Prevention and Control (ECDC) criteria. Whole-genome sequencing (WGS) was conducted on 16 phenotypically confirmed MDR/XDR isolates, followed by comprehensive bioinformatic analyses to characterize sequence types (STs), acquired antimicrobial resistance genes, resistance-associated chromosomal mutations, virulence determinants, plasmid replicons, and phylogenetic relationships. Results: Among 55 confirmed K. pneumoniae isolates, 19 (34.5%) were classified as MDR and 10 (18.2%) as XDR. WGS revealed substantial genomic heterogeneity, identifying 11 distinct STs, with ST39 being the most prevalent. Resistance to multiple antibiotic classes was mediated by the combined presence of plasmid-borne carbapenemases and extended-spectrum β-lactamases, alongside chromosomal mutations affecting outer membrane porins (OmpK35/OmpK36), fluoroquinolone targets (gyrA/parC), efflux regulation (ramR, marR), and lipid A modification pathways associated with colistin resistance (mgrB, pmrA/pmrB, arnC, crrB). IncF-family plasmids predominated and frequently co-occurred with additional resistance-associated replicons. Notably, one isolate exhibited an expanded virulence gene repertoire, including multiple siderophore systems and a complete type II secretion system, consistent with a hypervirulence-associated genomic profile. Phylogenetic analyses demonstrated close relatedness to international lineages from Asia, the Middle East, and Europe, indicating regional and transnational dissemination. Conclusions: This study highlights the complex interplay between plasmid-mediated gene acquisition and chromosomal adaptive mutations driving MDR and XDR phenotypes in K. pneumoniae circulating in Peshawar, Pakistan. The identification of hypervirulence-associated genetic features within an MDR background underscores the growing threat posed by convergent lineages and emphasizes the need for sustained WGS-based surveillance to inform infection control and antimicrobial stewardship strategies.