Genomic evolution and dissemination of non-conjugative virulence plasmid of ST65 carbapenem-resistant and hypervirulent Klebsiella pneumoniae strains in a Chinese hospital

Abstract

Background: The global rise in infections caused by hypervirulent and carbapenem-resistant K. pneumoniae (CR-hvKp) represents a growing public health threat. This study investigates ST65 CR-hvKp strains, with a focus on their genomic attributes and the mechanisms underlying the transmission of non-conjugative virulence plasmids.

Methods: Two clinical K2-ST65 CR-hvKp isolates (P6 and P10) were identified. Plasmid conjugation experiments were performed to assess the dissemination of the virulence plasmid. Antimicrobial susceptibility testing and virulence assays, including serum resistance, siderophore production, and G. mellonella larvae infection models, were used to characterize resistance and virulence phenotypes. Comprehensive bioinformatic analyses were performed to explore genetic evolution.

Results: Genomic analyses showed that both P6 and P10 carry a non-conjugative virulence plasmid, a conjugative untyped KPC plasmid, and a conjugative IncM2 plasmid. These isolates displayed broad-spectrum anti-microbial resistance and multiple virulence phenotypes, although they failed to sustain both hypervirulence and carbapenem resistance over time. The IncM2 plasmid was shown to be essential for the transfer of non-conjugative virulence plasmid. Mechanistic studies highlighted IS26-mediated plasmid fusion and the role of IncM2 plasmids in mobilizing non-conjugative virulence plasmids. The resulting transconjugants exhibited multidrug resistance, enhanced capsule production, and increased siderophore production.

Conclusions: This study provides new insights into the genomic dynamics of ST65-CR-hvKp strains and uncovers key mechanisms, such as IS26-mediated plasmid fusion and IncM2-mediated mobilization, which facilitate the dissemination of non-conjugative virulence plasmids. Understanding these mechanisms is crucial for developing effective strategies to manage and prevent the spread of these clinically challenging strains.

Keywords: Klebsiella pneumoniae; ST65; carbapenem resistance; hypervirulence; plasmid transmission.

Figure 1

Identification of ST65 hypervirulent and carbapenem-resistant K. pneumoniae. Phylogenetic analysis and basic characteristics of ST65 K. pneumoniae strains. The phylogenetic tree was constructed based on the core genome sequences of P6 and P10 strains (isolated in this study), and nine ST65 K. pneumoniae strains retrieved from the GenBank database.

Figure 2

Plasmid profiles of P6, P10 and their transconjugants. S1-PFGE analysis was performed on three TP6 transconjugants (TP6-1, TP6-2, and TP6-3) and three TP10 transconjugants (TP10-1, TP10-2, and TP10-3) that were randomly selected to perform. * indicate virulence plasmids (pVir), Δ indicate IncM2 plasmids (pInc), # indicate KPC plasmids (pP3).

Figure 3

Comparative genome analysis of hybrid plasmid (TP10-pVir) and proposed mechanism of plasmid fusion. (A-C) Comparative genome analysis of hybrid plasmid identified in TP10-1, TP10-2, and TP10-3, respectively. (D) Proposed model of IS26-mediated plasmid fusion using TP10-2-p1 as an example. Red arrows represent IS26 elements, and orange bold string represent the 8 bp target site duplication (GACTAAAC).

Figure 4

Transmission mechanisms and mobilization of non-conjugative virulence plasmids. (A) Elimination of pP3 and pInc plasmids from P6. The P6-P3cured and P6-IncMcured strains were successfully constructed. Lane 1: pVir gene; lane 2: pP3 gene; lane 3: pInc gene. (B) Conjugation frequencies of pVir plasmids of P6-P3cured and P6-IncMcured strains. ****P< 0.0001, ns, not significant. (C) Growth curves of P6 and P6-P3cured strains. (D) Alignment of OriT sequences from pP6Vir, pK2044, and pP6Inc. Multiple sequence alignment was performed using MUSCLE sequence alignment tool. The nic site is highlighted with a red box.

Figure 5

Virulence phenotypes of P6, P10, and their transconjugants. (A) Growth curves of P6, P10, and their transconjugants. (B) Viscosity measurements. (C) CPS production assays. (D) Siderophore production assessed using CAS agar. (E) Quantitative measurement of siderophore production. (F) Serum killing assay results. (G) Survival curves of Galleria mellonella larvae infected with the indicated strains. ****P< 0.0001, ***P< 0.001, **P< 0.01, *P< 0.05, ns, not significant.