Klebsiella pneumoniae is a Gram-negative bacterium that has emerged as a significant opportunistic pathogen responsible for a wide range of infections, from mild urinary tract infections to severe pneumonia, particularly in immunocompromised patients or those with underlying medical conditions (Dong et al., 2022). A growing concern within the medical community is the emergence of multidrug-resistant (MDR) and hypervirulent (hv) strains of K. pneumoniae (Heng et al., 2024a, Lan et al., 2021). These clones are resistant to last-resort medications, such as carbapenems, which are essential for treating serious infections, thereby posing a significant threat to clinical therapy (Yang et al., 2024). Carbapenem-resistant (CR) K. pneumoniae isolates harbour various resistance genes, primarily located on plasmids, including those encoding carbapenemases such as KPC, NDM, and OXA (Ma et al., 2023). The hv K. pneumoniae are known to harbor a ∼200 kb virulence plasmid, which includes mucoid phenotype regulator genes (rmpA and rmpA2), aerobactin synthesis (iuc gene cluster), and salmochelin synthesis (iro gene clusters) that help cause the tissue-invasive infection compared to the classic K. pneumoniae (Bolourchi et al., 2022, Russo et al., 2018, Yang et al., 2023, Yang et al., 2019). A serious clinical consequence of this is the co-occurrence of carbapenem resistance and hypervirulence in a single strain (Arcari and Carattoli, 2023), known as carbapenem-resistant hypervirulent (CRhv) K. pneumoniae. Studies have documented the presence of CRhv K. pneumoniae in various geographical locations, including Asia, Europe, North America, and South America, highlighting its widespread dissemination (Cejas et al., 2014, Heng et al., 2024a, Hussain et al., 2023, Yang et al., 2019, Yao et al., 2015). The incidence of CR K. pneumoniae, hv K. pneumoniae, and CRhv K. pneumoniae worldwide remains a serious issue (Arcari and Carattoli, 2023), underscoring the urgent need for thorough surveillance and effective intervention strategies.

Plasmids are extrachromosomal DNA molecules capable of autonomous replication within bacterial cells and play an essential role in this process (Actis et al., 1999). They serve as mobile genetic elements that can readily transfer resistance genes and virulence determinants between bacterial isolates (Actis et al., 1999, Robertson and Nash, 2018, Smalla et al., 2015). Profiling the K. pneumoniae plasmidome can serve as an effective surveillance tool by allowing us to trace the transmission of specific plasmids related to outbreaks and detect newly emerging resistance mechanisms. Traditional methods for plasmid analysis involve labor-intensive steps such as plasmid isolation and PCR amplification of specific resistance genes, which only provide a snapshot of the plasmid content without capturing the full plasmidome population structure (Smalla et al., 2015). Long-read sequencing has the potential to recover complete plasmids from genomes, but small plasmids (<20 kb) are often filtered out during assembly (Wick et al., 2021). Next-generation sequencing (NGS) technologies, which enable high-throughput sequencing of entire bacterial genomes, have generated substantial genomic data globally over time. However, assembling complete plasmid sequences from complex genomic datasets can be computationally demanding due to spliced and short contigs, as well as shared segments between plasmids and chromosomes (Robertson and Nash, 2018, Tang et al., 2023, Wick et al., 2021). Consequently, our understanding of plasmid dynamics within K. pneumoniae populations remains incomplete, limiting our ability to predict, characterize, and control the spread and convergence of CR K. pneumoniae, hv K. pneumoniae, and CRhv K. pneumoniae

In this study, we present a novel approach for the comprehensive characterization and profiling of the K. pneumoniae plasmidome on a global scale. By integrating NGS technologies and bioinformatics tools, we aim to profile the dynamics of the K. pneumoniae plasmidome. Our research provides a high-resolution view of the plasmid content, offering insights into the origins and trends of resistance and virulence gene transfer among these pathogens across different plasmids. The findings from this study will not only enhance our understanding of the role of the plasmidome in K. pneumoniae transmission and evolution but also aid in the development of targeted interventions to prevent the spread of CR K. pneumoniae, hv K. pneumoniae, and CRhv K. pneumoniae strains.