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Acquisition of the Conjugative Virulence Plasmid From a CG23 Hypervirulent Klebsiella pneumoniae Strain Enhances Bacterial Virulence.

ABSTRACT: The emergence of hypervirulent and carbapenem-resistant Klebsiella pneumoniae (hv-CRKP) has become a hot topic and confounding problem for clinicians and researchers alike. Conjugative virulence plasmids have the potential to cause more threatening dissemination of hv-CRKP strains. We previously identified K2606, a CG23 clinical hypervirulent strain of Klebsiella pneumoniae harboring a conjugative virulence plasmid designated pK2606. In this study we examined hypervirulence levels using assays of biofilm formation, serum resistance, and wax larvae and mouse in vivo infection models. Moreover, to define the transfer ability of pK2606 and whether this confers hypervirulence to other strains we performed plasmid transconjugation experiments between K2606 and the ST11 CRKP strain HS11286 along with E. coli J53. We found that although biofilm formation and serum resistance were not significantly increased, the transconjugants acquired the ability of produce high level of siderophores and also caused high mortality of wax larvae and mice. Furthermore, we identified pK2606-like conjugative virulence plasmids in GenBank, providing evidence that such plasmids may have begun to spread throughout China. These findings provide an evidence base for the possible mechanisms of the emergence of hv-CRKP strains and highlight the potential of pK2606-like conjugative virulence plasmids to spread worldwide.

SUBMITTER: Tian D

PROVIDER: S-EPMC8485780 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundKlebsiella pneumoniae, as a global priority pathogen, is well known for its capability of acquiring mobile genetic elements that carry resistance and/or virulence genes. Its virulence plasmid, previously deemed nonconjugative and restricted within hypervirulent K. pneumoniae (hvKP), has disseminated into classic K. pneumoniae (cKP), particularly carbapenem-resistant K. pneumoniae (CRKP), which poses alarming challenges to public health. However, the mechanism underlying its transfer from hvKP to CRKP is unclear.MethodsA total of 28 sequence type (ST) 11 bloodstream infection-causing CRKP strains were collected from Ruijin Hospital in Shanghai, China, and used as recipients in conjugation assays. Transconjugants obtained from conjugation assays were confirmed by XbaI and S1 nuclease pulsed-field gel electrophoresis, PCR detection and/or whole-genome sequencing. The plasmid stability of the transconjugants was evaluated by serial culture. Genetically modified strains and constructed mimic virulence plasmids were employed to investigate the mechanisms underlying mobilization. The level of extracellular polysaccharides was measured by mucoviscosity assays and uronic acid quantification. An in silico analysis of 2608 plasmids derived from 814 completely sequenced K. pneumoniae strains available in GenBank was performed to investigate the distribution of putative helper plasmids and mobilizable virulence plasmids.ResultsA nonconjugative virulence plasmid was mobilized by the conjugative plasmid belonging to incompatibility group F (IncF) from the hvKP strain into ST11 CRKP strains under low extracellular polysaccharide-producing conditions or by employing intermediate E. coli strains. The virulence plasmid was mobilized via four modes: transfer alone, cotransfer with the conjugative IncF plasmid, hybrid plasmid formation due to two rounds of single-strand exchanges at specific 28-bp fusion sites or homologous recombination. According to the in silico analysis, 31.8% (242) of the putative helper plasmids and 98.8% (84/85) of the virulence plasmids carry the 28-bp fusion site. All virulence plasmids carry the origin of the transfer site.ConclusionsThe nonconjugative virulence plasmid in ST11 CRKP strains is putatively mobilized from hvKP or E. coli intermediates with the help of conjugative IncF plasmids. Our findings emphasize the importance of raising public awareness of the rapid dissemination of virulence plasmids and the consistent emergence of hypervirulent carbapenem-resistant K. pneumoniae (hv-CRKP) strains.

Project description:Carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) has received considerable attention. Typically, the genetic elements that confer virulence are harbored by nonconjugative plasmids. In this study, we report a CR-hvKP strain, CY814036, of high-risk sequence type 25 (ST25) and the K2 serotype, which is uncommon among K. pneumoniae isolates but caused serious lung infection in a tertiary teaching hospital in China. Whole-genome sequencing (WGS) revealed a rare conjugative plasmid, pCY814036-iucA, carrying a virulence-associated iuc operon (iucABCD-iutA) coding for aerobactin and determinants of multidrug resistance (MDR), coexisting with a conjugative blaKPC-2-bearing plasmid, pCY814036-KPC2, in the same strain. A conjugation assay showed that pCY814036-iucA and pCY814036-KPC2 could be efficiently cotransmitted from CY814036 to Escherichia coli EC600. Further phenotypic investigation, including antimicrobial susceptibility tests, serum resistance assays, and mouse infection models, confirmed that pCY814036-iucA was capable of cotransferring multidrug resistance and hypervirulence features to the recipient. pCY814036-KPC2 also conferred resistance to antibiotics, including β-lactams and aminoglycosides. Overall, the rare coexistence of a conjugative MDR-virulence plasmid and a blaKPC-2-bearing plasmid in a K. pneumoniae isolate offers a possible mechanism for the formation of CR-hvKP strains and the potential to significantly accelerate the propagation of high-risk phenotypes. IMPORTANCE The increased reporting of carbapenem-resistant hypervirulent K. pneumoniae is considered a worrisome concern to human health care and has restricted the choice of effective antibiotics for clinical treatment. Moreover, virulence plasmids with complete conjugation modules have been identified, which evolved via homologous recombination. Here, we characterize an ST25 CR-hvKP strain, CY814036, harboring both a conjugative MDR-virulence plasmid and a blaKPC-2-bearing plasmid in China. This study highlights that the cotransmission of drug resistance and virulence plasmids increases therapeutic difficulties and worsens clinical prognoses. Also, active surveillance of the conjugative MDR-virulence plasmid is necessary.

Project description:Hypervirulent Klebsiella pneumoniae causes liver abscess and potentially devastating metastatic complications. The majority of Klebsiella-induced liver abscess are caused by the CG23-I sublineage of hypervirulent Klebsiella pneumoniae. This and some other lineages possess a >200-kb virulence plasmid. We discovered a novel protein IroP nestled in the virulence plasmid-encoded salmochelin operon that cross-regulates and suppresses the promoter activity of chromosomal type 3 fimbriae (T3F) gene transcription. IroP is itself repressed by iron through the ferric uptake regulator. Iron-rich conditions increase T3F and suppress capsule mucoviscosity, leading to biofilm formation and cell adhesion. Conversely, iron-poor conditions cause a transcriptional switch to hypermucoid capsule production and T3F repression. The likely acquisition of iroP on mobile genetic elements and successful adaptive integration into the genetic circuitry of a major lineage of hypervirulent K. pneumoniae reveal a powerful example of plasmid chromosomal cross talk that confers an evolutionary advantage. Our discovery also addresses the conundrum of how the hypermucoid capsule that impedes adhesion could be regulated to facilitate biofilm formation and colonization. The acquired ability of the bacteria to alternate between a state favoring dissemination and one that favors colonization in response to iron availability through transcriptional regulation offers novel insights into the evolutionary success of this pathogen. IMPORTANCE Hypervirulent Klebsiella pneumoniae contributes to the majority of monomicrobial-induced liver abscess infections that can lead to several other metastatic complications. The large virulence plasmid is highly stable in major lineages, suggesting that it provides survival benefits. We discovered a protein IroP encoded on the virulence plasmid that suppresses expression of the type 3 fimbriae. IroP itself is regulated by iron, and we showed that iron regulates hypermucoid capsule production while inversely regulating type 3 fimbriae expression through IroP. The acquisition and integration of this inverse transcriptional switch between fimbriae and capsule mucoviscosity shows an evolved sophisticated plasmid-chromosomal cross talk that changes the behavior of hypervirulent K. pneumoniae in response to a key nutrient that could contribute to the evolutionary success of this pathogen.

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Acquisition of the Conjugative Virulence Plasmid From a CG23 Hypervirulent Klebsiella pneumoniae Strain Enhances Bacterial Virulence.

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