Horizontal gene transfer via OMVs co-carrying virulence and antimicrobial-resistant genes is a novel way for the dissemination of carbapenem-resistant hypervirulent Klebsiella pneumoniae

Abstract

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.

Sporadic clone Escherichia coli ST615 as a vector and reservoir for dissemination of crucial antimicrobial resistance genes

“…Today, three canonical mechanisms of HGT are recognized, including transformation, transduction, and conjugation ( Dubnau and Blokesch, 2019 ). Also, there are other non-canonical mechanisms including vesiduction, which involves secretion and uptake of EV ( Li P. et al., 2022 ; Marinacci et al., 2023 ). In stressing habitats such as the nosocomial habitat, genome evolution is driven by antibiotic selection.…”

“…Of the DNA that is transferred, there are genes associated with AMR, which has huge clinical implications since the potential of propagation of a gene depends to a great extent on the competence to transmit ( Chatterjee et al., 2017 ; Wagner et al., 2018 ). Earlier studies have reported the transfer by EV of a penicillin resistance gene in Neisseria gonorrhoeae ( Dorward et al., 1989 ); the bla OXA-24 ( Rumbo et al., 2011 ) and the bla NDM-1 carbapenemase genes in Acinetobacter baumannii ( Chatterjee et al., 2017 ); the bla CTX-M-15 and bla TEM-1 genes in E. coli O104:H4 ( Bielaszewska et al., 2020 ); and finally, the bla NDM , bla KPC , bla SHV , bla CTX-M-9 , and aac(6′)-Ib genes in K. pneumoniae strains ( Li P. et al., 2022 ). In the present study, we wonder about the ability of the E. coli M19736 clinical strain belonging to the sporadic clone ST615, which was one of the first isolates harboring the mcr-1 gene in Latin America ( Tijet et al., 2017 ), to adapt to the XDR phenotype ( Magiorakos et al., 2012 ) by conjugation and/or transformation assays.…”

Whole-Genome Sequencing Revealed the Fusion Plasmids Capable of Transmission and Acquisition of Both Antimicrobial Resistance and Hypervirulence Determinants in Multidrug-Resistant Klebsiella pneumoniae Isolates

“…The formation of K. pneumoniae genetic lineages exhibiting both high virulence and carbapenem resistance can occur by several pathways associated with horizontal gene transfer [ 43 , 48 ], and the conjugation was considered its most significant mechanism [ 49 ]. Hypervirulent isolates can acquire MDR plasmids, and MDR isolates can also acquire plasmids carrying virulence factors.…”

Analysis of the hypovirulent Klebsiella pneumoniae with the NDM-5 gene on IncN plasmids

“…The CR-hvKP NUHL 30457, KP4089, the transformant, and EC600 were transferred to the Bioscreen C-Pro fully automated growth curve analyzer for continuous measurement of strain OD, as previously described ( 13 ).…”