Abstract
Multidrug resistant K. pneumoniae is a public health treat in Pakistan, yet longitudinal genomic data on the evolution of resistance and virulence remain limited. This study integrates phenotypic and whole-genome analyses to explore the evolution of resistance and virulence in clinical isolates. In this study, 39 clinical K. pneumoniae isolates from a single center in Lahore, Pakistan were characterized. Comparative genomic analysis of six representative isolates was performed against 45 historical Pakistani isolates (2010 vs. 2019). Pan-genome analysis, resistance and virulence gene profiling, plasmid replicon identification, genotype-phenotype concordance, and phylogenetic reconstruction was performed. Alarmingly, 90% of isolates were MDR while 13% were extensively drug-resistant. Carbapenem resistance exceeded 60%, mainly due to bla(NDM−1) and bla(NDM−5) in high-risk sequence types ST-15, ST-397 and ST-870. Genotype-phenotype concordance analyses demonstrated strong predictive accuracy for beta-lactams but also revealed discordances against other classes indicative of complex resistance mechanisms. Pan-genome analysis of 51 isolates revealed high levels of genome plasticity, with just 18.7% of genes forming the core genome, and significant increases of accessory genome with a preponderance of virulence-linked factors. Representative isolates produced variable biofilm, with strong producers carrying both adhesion genes and plasmid-borne resistance. Collectively, these findings underscore evolving MDR K. pneumoniae population from Pakistan. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s42770-026-01906-y.