Klebsiella pneumoniae is a significant causative agent of hospital- and community-acquired infections, presenting with various clinical manifestations. Notably, the rapid spread of carbapenem-resistant K. pneumoniae (CRKP) has become a global threat, leading to high morbidity and mortality rates. In this context, researchers have employed high-throughput sequencing to investigate drug resistance mechanisms in this pathogen, particularly focusing on plasmids, which carry crucial resistance genes. Understanding these mechanisms is essential to develop effective countermeasures.


Whole Plasmid Sequencing for Drug Resistance Gene Analysis

To analyze drug resistance mechanisms in K. pneumoniae, researchers have adopted whole plasmid sequencing, a powerful method to obtain the complete genetic information of plasmids. By predicting and annotating all genes in the genome, they aim to identify genes associated with various phenotypes, including drug resistance, heavy metal resistance, and environmental adaptability.


Importance of Validating Drug Resistance Genes

While drug resistance genes identified through plasmid sequencing may show resistance in heterologous hosts, it is imperative to ascertain their resistance in their natural reservoirs. Additionally, drug-resistant genes can be transferred horizontally between bacteria in the form of plasmids, leading to the dissemination of drug resistance. Thus, validating “candidate” drug resistance genes becomes crucial.


Role of Whole Plasmid Sequencing in Identifying and Annotating Drug Resistance Genes

Whole Plasmid Sequencing technology enables the screening and annotation of “candidate” drug resistance genes. By identifying these genes, researchers can initiate drug resistance functional verification experiments, leading to a deeper understanding of the resistance mechanisms. This comprehensive approach accelerates the discovery of drug resistance mechanisms and facilitates the development of appropriate countermeasures.


Plasmid Whole Genome Sequencing for Analyzing Drug Resistance Mechanisms in CRKP

Researchers have utilized plasmid whole genome sequencing to investigate the drug resistance mechanisms in CRKP strains isolated from pediatric patients in Shanghai. One such strain was identified as ST3936 and KL30. The isolate harbored a 5508387 bp ring chromosome and two plasmids: pNDM-IMP-1 (347317 bp) and pNDM-IMP-2 (144371 bp). The strain carried multiple resistance genes, including those conferring resistance to aminoglycosides, fluoroquinolones, carbapenems, and other β-lactam drugs. Of particular significance were the carbapenemase encoding genes blaNDM-1 (2 copies) and blaIMP-4, co-located on the plasmid pNDM-IMP-1. This plasmid, with a size of 347.3 kb, was classified as unclassifiable and contained most of the resistance genes, except oqxAB and blaLEN17. Comparative genomics analysis further revealed that pNDM – IMP – 1 shared similarities with pKP1814 – 1 in terms of plasmid skeletons and essential functional genes.


Implications and Preventive Measures

This study represented the first report on the co-existence of IMP-4 and NDM-1 carbapenemases in highly virulent Klebsiella pneumoniae. The presence of a multidrug-resistant hybrid plasmid, pNDM-IMP-1, suggested its potential origin from Klebsiella pneumoniae, with the ISCR1 element contributing to the aggregation of blaNDM-1 and blaIMP-4 genes. To effectively prevent outbreaks and transmission, continuous monitoring of these strains is crucial.


Whole Genome Sequencing for Identifying Superplasmids in HvKP

The emergence of carbapenem-resistant HvKP (CR HvKP) and ESBL-producing HvKP strains poses significant challenges due to their simultaneous acquisition of high virulence and multidrug-resistant genes, often mediated by plasmids. Recent reports have indicated the presence of “super plasmids” harboring both virulence and MDR genes, facilitating the rapid dissemination of these traits.


Characteristics of Superplasmids

Superplasmids are defined by several key characteristics: (1) they contain hypervirulence and MDR genes on a single plasmid, (2) they carry complete conjugative elements that enable self-transmissibility, (3) they demonstrate stability and conservation, and (4) they impose no fitness cost to the host strain. The simultaneous carriage of high virulence and MDR genes poses significant challenges to clinical prevention, control, and treatment of infections caused by such strains. Vigilant surveillance is essential to prevent the dissemination of these efficient resistance/virulence plasmids in healthcare settings.



Whole plasmid sequencing has emerged as a valuable tool in the analysis of drug resistance genes in Klebsiella pneumoniae. Understanding the underlying resistance mechanisms through this approach is pivotal in formulating personalized treatment.