Comparative genomic insights into multidrug resistance in classical and hypervirulent K. pneumoniae clinical isolates

Abstract Klebsiella pneumoniae is a highly adaptable opportunistic pathogen responsible for various infections, particularly in immunocompromised individuals. The recent genetic evolution of K. pneumoniae has led to the emergence of strains exhibiting both hypervirulence and multidrug resistance (MDR). In this study, we compared the genomic characteristics of MDR in classical and hypervirulent K. pneumoniae clinical isolates. Sixty-four isolates were collected from two healthcare institutions in Khyber Pakhtunkhwa, Pakistan, and identified using standard microbiological techniques. Whole-genome sequencing (WGS) was performed on 30 selected isolates using an Illumina platform. The sequencing data were analysed with the Kleborate tool. WGS analysis identified sequence types ST147 (n = 7, 23%) and ST2629 (n = 5, 16.6%) as the most prevalent lineages. Additionally, 13 other ST were detected, including ST147-1LV (n = 1, 3.3%) and ST859 (n = 1, 3.3%), which exhibited hypervirulent MDR traits. Among K-loci-associated virulence determinants, KL64 was the most predominant (n = 7, 23%), while the O-serotype O1/O2v1 was found in 22 isolates. A diverse range of antimicrobial resistance (AMR) determinants was observed across isolates. Plasmid-mediated quinolone resistance (PMQR) genes ( qnr S1, qnr B1, qnr B4, qep A2) and aminoglycoside-modifying enzymes ( aac (3)-IIa, aac (6′)-Ib-cr, strA , strB ) were detected in both classical and hypervirulent strains. β-lactamase genes included bla- SHV variants ( bla - SHV-1 , bla - SHV-11 , bla - SHV-25 , bla - SHV-187 ), bla- CTX - M-15 , bla - VEB-5 , bla - OXA-1 , bla- OXA -48 , and bla - OXA - 181 . Additional resistance genes conferred resistance to macrolides ( ermB , mphB ), phenicols ( catB4, catA1, floR ), sulfonamides ( sul1, sul2 ), tetracyclines ( tetA , tetB , tetD ), and trimethoprim ( dfrA1, dfrA12, dfrA14 ). Acquired AmpC and other β-lactamases ( DHA-1 , TEM-1D , CMH-1 , CMY-6 , LAP-2 , Amp C1) were also present. The most prevalent plasmid replicons were Col(pHAD28), IncFIB(K), and IncR, with the hypervirulent ST147-1LV isolate carrying the highest number. These findings underscore the significant public health threat posed by hypervirulent and MDR K. pneumoniae strains, highlighting the extensive burden of virulence and resistance genes that complicate treatment strategies.