IntroductionThe rapidly increased isolation rate of CR-HvKP worldwide has brought great difficulties in controlling clinical infection. Moreover, it has been demonstrated that the transmission of drug-resistant genes among bacteria can be mediated by outer membrane vesicles (OMVs), which is a new way of horizontal gene transfer (HGT). The transmission of virulence genes among bacteria has also been well studied; however, it remains unclear whether virulence and drug-resistant genes can be co-transmitted simultaneously. Co-transmission of virulence and drug-resistant genes is essential for the formation and prevalence of CR-HvKP.MethodsFirst, we isolated OMVs from CR-HvKP by cushioned-density gradient ultracentrifugation (C-DGUC). TEM and DLS were used to examine the morphology and size of bacterial OMVs. OMV-mediated gene transfer in liquid cultures and the acquisition of the carbapenem gene and virulence gene was confirmed using colony-PCR. Antimicrobial susceptibility testing, mCIM and eCIM were conducted for the resistance of transformant. Serum killing assay, assessment of the anti-biofilm effect and galleria mellonella infection model, mucoviscosity assay, extraction and quantification of capsules were verified the virulence of transformant. Pulsed-field gel electrophoresis (PFGE), S1 nuclease-pulsed-field gel electrophoresis (S1-PFGE), Southern blotting hybridization confirmed the plasmid of transformant.ResultsFirstly, OMVs were isolated from CR-HvKP NUHL30457 (K2, ST86). TEM and DLS analyses revealed the spherical morphology of the vesicles. Secondly, our study demonstrated that CR-HvKP delivered genetic material, incorporated DNA within the OMVs, and protected it from degradation by extracellular exonucleases. Thirdly, the vesicular lumen DNA was delivered to the recipient cells after determining the presence of virulence and carbapenem-resistant genes in the CR-HvKP OMVs. Importantly, S1-PFGE and Southern hybridization analysis of the 700603 transformant strain showed that the transformant contained both drug-resistant and virulence plasmids.DiscussionIn the present study, we aimed to clarify the role of CRHvKP-OMVs in transmitting CR-HvKP among K. pneumoniae. Collectively, our findings provided valuable insights into the evolution of CR-HvKP.

Hypervirulent variants of Klebsiella pnuemoniae (hvKP), which causes life-threatening infections, is a global priority pathogen and frequently harbours virulence plasmids. The virulence plasmids have emerged as the predominant vehicles carrying the major pathogenic determinants of hypermucoviscosity and hypervirulence phenotypes. In the present study, we characterized a novel virulence plasmid in AP8555, an ST23 hvKP strain, which induced a metastatic infection and fatal septic shock in a critically ill patient. The serum killing assay, the quantitative biofilm formation assay, the G.mellonella infection model, and the mouse lethality assay demonstrated that AP8555 was almost as virulent as the hvKP strain NUTH-K2044. The plasmid pAP855 could be conjugated to Klebsiella quasipneumoniae ATCC700603 and E. coli J53 at a frequency of 7.2× 10−5 and 8.7× 10−7, respectively. Whole-genome sequencing and bioinformatics analysis confirmed that the plasmid was novel, clustered to the incompatibility type of IncHI1B/IncFIB/IncFII and presented high similarity to the pK2044 plasmid. In contrast, a 130-kb large-fragment insertion was observed on the plasmid, which introduced a genetic hybrid zone with multiple conjugation-related genes of type IV secretion systems (T4SS) and CcdAB toxin-antitoxin systems (TAS) to the plasmid. In the transconjugants, the presence of pAP855 had a negative impact on bacterial fitness, but enhancing the virulence-associated phenotypes. In vitro evolution experiments showed that pAP855 in the transconjugants could not be stably inherited after 10 days of passage. Our study not only reports a novel hybrid plasmid but also highlights the putative pathway of conjugative virulence plasmid formation and evolution by means of genetic rearrangement through sequence insertion. These findings indicate that structural versatility could contribute to the dissemination of cointegrate virulence plasmid, although the plasmid incurred a fitness cost. Therefore, continuous monitoring the acquisition of conjugative virulence plasmids may have critical value for plasmid research and increase awareness of hvKP.