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.

Multidrug-resistant (MDR) Klebsiella pneumoniae represents a major clinical and public health challenge worldwide, particularly in regions with limited genomic surveillance. This study investigated the clinical, phenotypic, and genomic characteristics of clinical K. pneumoniae isolates recovered from a tertiary-care hospital in Peshawar, Pakistan. A total of 2400 non-duplicate clinical specimens were processed, and antimicrobial susceptibility testing was performed according to CLSI guidelines. Whole-genome sequencing (WGS) was conducted on a purposively selected subset of 18 isolates representing diverse resistance and phenotypic profiles. Genomic analyses included multilocus sequence typing, resistome and virulome profiling, identification of resistance-associated chromosomal mutations, plasmid replicon typing, and phylogenomic comparison with publicly available international genomes. K. pneumoniae was identified in 256/2400 (10.7%) specimens, predominantly from urine samples. MDR and extensively drug-resistant (XDR) phenotypes were detected in 83.2% and 13.3% of isolates, respectively. WGS revealed substantial genomic heterogeneity, with ST147 identified as the most frequent lineage among sequenced isolates. Extended-spectrum β-lactamase genes, particularly bla_CTX-M-15, together with carbapenemase genes including bla_OXA-48-like and bla_NDM-5, were identified in multiple isolates alongside resistance-associated chromosomal alterations in gyrA, parC, ompK36, mgrB, pmrB, and ramR. Yersiniabactin-associated loci were detected in all sequenced isolates, whereas canonical hypervirulence-associated determinants (rmpA, iuc, iro) were absent. These findings highlight the complex genomic landscape of MDR K. pneumoniae in Pakistan and underscore the need for continued genomic surveillance and antimicrobial stewardship.