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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 IncFIBK was the most prevalent (84.6%). We identified 49 IncFIBK alleles among 583 IncFIBK 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 IncFIBK genetic diversity, and IncFIBK 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 IncFIBK subtyping. In conclusion, the genetic diversity of IncFIBK virulence plasmids could be used for tracking the evolution of virulence plasmids, and further preventing the emergence of MDR-hvKp strains.

ObjectiveTo investigate the epidemiology of carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-HvKP) and hypervirulent carbapenem-resistant Klebsiella pneumoniae (Hv-CRKP).MethodsTotally 436 K. pneumoniae strains were collected from 7 hospitals in mainland China between 2017.01 and 2018.02. Sequence types, serotypes, antimicrobial-resistance and virulence genes were analyzed. Additionally, string test, capsule stain, Periodic Acid Schiff stain, fitness analysis, quantitative real-time PCR and mouse lethality test were also performed. Molecular combinations were used to screen putative blaKPC(+)-HvKP and Hv-blaKPC(+)-KP, followed by the confirmation of mouse lethality test.ResultsDiverse detection rates were found for the virulence genes, ranging from c-rmpA (0.0%) to entB (100.0%). According to the molecular criteria, 127, 186, 9 and 26 strains were putatively denoted as HvKP, blaKPC(+)-KP, blaKPC(+)-HvKP and Hv-blaKPC(+)-KP. Mouse lethality test confirmed 2 blaKPC(+)-HvKP strains (JS184 and TZ20) and no Hv-blaKPC(+)-KP. JS184 showed K2 serotype, thin capsule, positive exopolysaccharid and string test. TZ20 presented K20 serotype, thin capsule, negative exopolysaccharide and string test. Compared with the positive control NTUH-K2044, equal galF expression and growth curves were confirmed for JS184 and TZ20.ConclusionsMolecular determination of CR-HvKP and Hv-CRKP brings remarkable bias compared with mouse lethality test. The exact prevalence of CR-HvKP is less than 1.0%, which of Hv-CRKP is much lower.

ObjectiveTo investigate the overall distributions of key virulence genes in Klebsiella pneumoniae, especially the hypervirulent blaKPC-positive K. pneumoniae (Hv-blaKPC(+)-KP).MethodsA total of 521 complete genomes of K. pneumoniae from GenBank were collected and analyzed. Multilocus sequence typing, molecular serotyping, antibiotic-resistance, virulence genes and plasmid replicon typing were investigated.ResultsPositive rates of virulence genes highly varied, ranging from 2.9 (c-rmpA/A2) to 99.6% (entB). Totally 207 strains presented positive fimH, mrkD, entB and wzi and 190 showed positive fimH, mrkD, entB, irp2 and wzi, which were the two primary modes. A total of 94, 165 and 29 strains were denoted as hypervirulent K. pneumoniae (HvKP), blaKPC(+)-KP and Hv-blaKPC(+)-KP. ST11 accounted for 17 among the 29 Hv-blaKPC(+)-KP strains; Genes iucA, p-rmpA2 and p-rmpA were positive in 28, 26 and 18 Hv-blaKPC(+)-KP strains respectively. Among the 29 Hv-blaKPC(+)-KP strains exhibiting four super clusters from GenBank, IncHI1B plasmids carrying virulence genes and IncFII ones with blaKPC were responsible for both 23 strains respectively.ConclusionsPositive rates of virulence genes vary remarkably in K. pneumoniae. Genes iucA, p-rmpA2 and p-rmpA were primary ones inducing Hv-blaKPC(+)-KP. IncHI1B plasmids carrying virulence genes and IncFII ones with blaKPC constitute the primary combination responsible for Hv-blaKPC(+)-KP. The making of Hv-blaKPC(+)-KP is mostly via blaKPC(+)-KP acquiring another plasmid harboring virulence genes.