Genetic diversity and evolution of the virulence plasmids encoding aerobactin and salmochelin in Klebsiella pneumoniae

Abstract

Virulence plasmids of hypervirulent Klebsiella pneumoniae (hvKp) have the potential to transfer to drug-resistant strains or integrate with other plasmids, facilitating the genome evolution of threatening pathogens. We conducted an in-depth analysis of the publicly available 156 complete genome sequences of hvKp together with a multi-region clinical cohort of 171 hvKp strains from China to provide evidence for the virulence plasmid evolution. Virulence plasmids were frequently detected in the ST23 and ST11 K. pneumoniae strains. Multidrug-resistant hvKp (MDR-hvKp) occupied a large proportion of hvKp, and the coexistence of virulence and resistance plasmids may be the major cause. Virulence plasmids commonly possessed multiple replicons, of which IncFIB_K was the most prevalent (84.6%). We identified 49 IncFIB_K alleles among 583 IncFIB_K plasmids, and they could be divided into Clades I, II, and III. We further observed that conjugative and non-conjugative virulence plasmids could be distinguished by IncFIB_K genetic diversity, and IncFIB_K subtyping could also indirectly indicate a chimeric preference of conjugative virulence plasmids. On this basis, we developed an open-access web tool called KpVR for IncFIB_K subtyping. In conclusion, the genetic diversity of IncFIB_K virulence plasmids could be used for tracking the evolution of virulence plasmids, and further preventing the emergence of MDR-hvKp strains.

Keywords: Hypervirulent Klebsiella pneumoniae; conjugative transfer; epidemiology; genome evolution; virulence plasmid.

Figure 1.

The distribution of hvKp strains and virulence plasmids. MLST of the 119 completely sequenced hvKp strains available in GenBank (a) and 171 clinical hvKp isolates (b). The distribution of iucA and iroB in GenBank (c) and clinical strains (d). (e) The replicons of the virulence plasmids. The dot matrix shows different multi-replicon profiles and the histograms on the top show the numbers of plasmids with the corresponding profiles. Histograms on the left represent numbers of corresponding replicons. (f) The occurrences of distinct virulence plasmids in various ST clones. The tree was constructed by using the maximum-likelihood method with 1,000 bootstrap replicates in MEGA7. The replicons are represented by different shapes and colors. The same type of virulence plasmids is framed in red

Figure 2.

Descriptions of IncFIB_K alleles. (a) Phylogenetic tree, occurrences, and other replicons of IncFIB_K plasmids. On the left is the phylogenetic tree of all IncFIB_K alleles. The tree was constructed by using the maximum-likelihood method with 1,000 bootstrap replicates in MEGA7. Colors indicate Clade I, II, and III from up to bottom. The next bar plots are the occurrences of the corresponding plasmids. Colors indicate virulence and non-virulence plasmids. Other co-occurring replicons were shown on the right. (b) IncFIB_K alleles in IncFIB_K virulence plasmids available in GenBank. (c) IncFIB_K alleles of virulence plasmids in clinical hvKP isolates

Figure 3.

IncFIB_K virulence plasmids in distinct genetic groups. (a) The differences of AMR genes and other replicons of IncFIB_K virulence plasmids in Clade I and Clade III. (b) The iuc and iro genes of IncFIB_K virulence plasmids in Clade I and Clade III. Different colors indicate different lineages of iuc and iro genes

Figure 4.

Comparative analysis of conjugative virulence plasmid with other similar plasmids. (a) Comparative analysis of conjugative virulence plasmid p205880-1 in Clade I. The plasmid p205880-1 (accession: CP030303.1) was used as the reference plasmid to perform genome alignment with classical virulence plasmid pK2044 (accession: AP006726.1) and IncFII_K P3 plasmid (accession: CP059380.1). (b) Comparative analysis of conjugative plasmid pAP855 in Clade III. The plasmid pAP855 (accession: CP035384.1) was used as the reference plasmid to perform genome alignment with classical virulence plasmid pK2044 (accession: AP006726.1) and IncFII_K plasmid pKPM501 (accession: CP031735.1)