K1/K2 hvKP strains acquire carbapenem-resistance plasmids, known as CR-hvKp, and carbapenem-resistant Klebsiella pneumoniae (CRKP) strains obtain virulence plasmids, recognized as hv-CRKP. The two different evolution patterns of hypervirulent combined carbapenem-resistant Klebsiella pneumoniae may lead to their different prevalence in hospitals. Our study aimed to investigate the prevalence of hv-CRKP and CR-hvKp strains and to analyze factors influencing their evolution and prevalence. We collected 890 K. pneumoniae genomes from GenBank and 530 clinical K. pneumoniae isolates from nine hospitals. Our study found that hv-CRKP strains were more prevalent than CR-hvKp strains and both were dominated by bla_KPC-2 gene. The bla_KPC-2-carrying plasmids could mobilize non-conjugative virulence plasmids from hvKp strains to CRKP strains. The conserved oriT of virulence plasmids and the widespread of conjugative helper plasmids were potential factors for the mobilization of non-conjugative virulence plasmids. HvKp strains with KPC plasmid could hardly simultaneously exhibit hypervirulence and carbapenem resistance as CRKP strains with virulence plasmid, and we found that rfaH mutation reduced capsular synthesis and increased carbapenem resistance of the CR-hvKp strain. In summary, this study revealed that hv-CRKP strains were more suitable for survival in hospital settings than CR-hvKp strains and the widespread conjugative KPC-producing plasmids contributed to the emergence and prevalence of hv-CRKP strains.

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.

Fecal colonization with carbapenem-resistant Enterobacteriaceae (CRE) is a risk factor for bacterial translocation resulting in subsequent endogenous infections. The purpose of this study is to investigate the prevalence of CRE strains colonization in stool samples of outpatient in a tertiary pediatric hospital of Shanghai, China. In a retrospective study, fecal samples were consecutively obtained from patients in 2016 and screening test for CRE was conducted by using home-made MacConkey agar. Antimicrobial susceptibility was determined by the broth microdilution method and β-lactamases were characterized by polymerase chain reaction (PCR) assays and DNA sequencing. Multilocus sequence typing (MLST) was performed for the genetic relationships of the isolates. A total of 880 fecal samples were included for this screening test and 32 CRE strains were identified in 32 non-duplicate fecal samples from 32 children (1.3 ± 1.5 years), with a carriage rate of 3.6%. These strains mainly distributed in Klebsiella pnuemoniae (37.5%) and Escherichia coli (37.5%). All CRE strains showed high resistance to most of the routinely used antibiotics (> 90%) except for polymyxin B and tigecycline. The blaNDM gene was the major carbapenemase gene harbored by gastrointestinal CRE strains, followed by blaKPC-2, blaIMP-26, and blaIMP-4. Other β-Lactamase genes including blaCTX-M, blaSHV, blaTEM-1, and blaDHA-1 were also detected. MLST analysis revealed that various sequence types (STs) were detected in these strains, with ST11 and ST37 being more prevalent in K.pneumoniae and ST101 in E.coli. This study revealed the prevalence of CRE fecal carriage in children from outpatient and urgent implementation of infection control measure should be conducted to limit the spread of CRE strains.

Hypervirulent and carbapenem-resistant Klebsiella pneumoniae poses a severe threat to public health for its high pathogenicity, transmissibility, and drug resistance. This study aims to explore the evolutionary path and the role of Tn3 transposon in the virulence and carbapenem resistance transmission of K. pneumoniae . Bioinformatics analysis and some experimental tests such as plasmid conjugation experiments, antimicrobial susceptibility testing, and virulence-associated tests were performed to explore the virulence and drug resistance transmission. The complete genome sequencing, S1 nuclease pulsed-field gel electrophoresis, and bioinformatics analysis were used to investigate their transmission mechanism mediated by Tn3 transposons. Three hypervirulent and carbapenem-resistant K. pneumoniae isolates, which were obtained from different patients at different time points in the same ward, harbored a virulence plasmid and a carbapenem resistance plasmid. They were confirmed to have first evolved from hypervirulent isolates and then acquired a bla _KPC -positive plasmid (CR-hvKp evolutionary pattern). The non-conjugative virulence plasmid pVir could be transferred to other bacterial strains via mobilization by the conjugative IncN/U-type plasmid pKPC, as well as by fusing with the conjugative pKPC plasmid (mediated by Tn3-based homologous recombination) to be self-transmissible, thus transferring drug resistance and virulence. The cointegration, pVir/KPC fusion plasmid, was further resolved into pVir and pKPC between the duplicated copies of the Tn3 transposon resolution site mediated by site-specific recombination. Therefore, Tn3 transposons can mediate hypervirulent and carbapenem-resistant K. pneumoniae strains transferring drug resistance and virulence to other bacteria. We must be vigilant to emerging transposon-mediated hypervirulent and carbapenem-resistant pathogens. IMPORTANCE: Carbapenem-resistant Klebsiella pneumoniae (CRKP) is resistant to most common antibiotics, becoming the most important and prevalent nosocomial opportunity pathogen. Besides, K. pneumoniae can also cause severe community-acquired infections, such as primary liver abscess and endophthalmitis. These pathogens are commonly referred to as hvKp. CRKP and hvKp have evolved separately, each occupying its own clonal lineage and exhibiting a variety of properties. Our study provides important insights into the evolutionary events related to the arousal of virulence and drug resistance in K. pneumoniae through plasmid transmission, mediated by Tn3 transposon. Our study also provides evidence that multiple mechanisms contribute to the successful transfer of non-conjugative virulence plasmid, and the involvement of transposons enhances the efficiency. A good knowledge of its transmission mechanisms is fundamental to finding effective strategies to combat these threatening pathogens. Transposons are widely present in bacteria, spreading resistance and virulence genes between the environment and humans. Therefore, emerging transposon-mediated hypervirulent and carbapenem-resistant pathogens should be highly valued.

Background: Carbapenem-resistant E. coli (CREco) pose a significant public health threat due to their multidrug resistance. Colistin is often a last-resort treatment against CREco; however, the emergence of colistin resistance gene mcr-1 complicates treatment options. Methods: Two E. coli strains (ECO20 and ECO21), recovered from hospitalized patients in distinct wards, exhibited resistance to carbapenems and colistin. Whole-genome sequencing and phenotypic characterization were employed to study resistance patterns, plasmid profiles, transferability of resistance and virulence genes, and siderophore production capabilities. Comparative genome analysis was used to investigate the genetic environment of mcr-1, bla_NDM−7, and virulence clusters. Results: Both E. coli strains exhibited thr presence of both mcr-1 and bla_NDM−7 genes, showing high resistance to multiple antibiotics. Genomic analysis revealed the clonal transmission of these strains, possessing identical plasmid profiles (pMCR, pNDM, and pVir) associated with colistin resistance, carbapenem resistance, and virulence factors. Conjugation experiments confirmed the transferability of these plasmids, indicating their potential to disseminate resistance and virulence traits to other strains. Comparative genomic analyses unveiled the distribution of mcr-1 (IncX4-type) and bla_NDM (IncX3-type) plasmids across diverse bacterial species, emphasizing their adaptability and threat. The novelty of pVir indicates its potential role in driving the evolution of highly adaptable and pathogenic strains. Conclusions: Our findings underscore the co-occurrence of mcr-1, bla_NDM−7, and siderophore-producing plasmids in E. coli, which poses a significant concern for global health. This research is crucial to unravel the complex mechanisms governing plasmid transfer and recombination and to devise robust strategies to control their spread in healthcare settings.

Background: The global rise in infections caused by hypervirulent and carbapenem-resistant K. pneumoniae (CR-hvKp) represents a growing public health threat. This study investigates ST65 CR-hvKp strains, with a focus on their genomic attributes and the mechanisms underlying the transmission of non-conjugative virulence plasmids.Methods: Two clinical K2-ST65 CR-hvKp isolates (P6 and P10) were identified. Plasmid conjugation experiments were performed to assess the dissemination of the virulence plasmid. Antimicrobial susceptibility testing and virulence assays, including serum resistance, siderophore production, and G. mellonella larvae infection models, were used to characterize resistance and virulence phenotypes. Comprehensive bioinformatic analyses were performed to explore genetic evolution.Results: Genomic analyses showed that both P6 and P10 carry a non-conjugative virulence plasmid, a conjugative untyped KPC plasmid, and a conjugative IncM2 plasmid. These isolates displayed broad-spectrum anti-microbial resistance and multiple virulence phenotypes, although they failed to sustain both hypervirulence and carbapenem resistance over time. The IncM2 plasmid was shown to be essential for the transfer of non-conjugative virulence plasmid. Mechanistic studies highlighted IS26-mediated plasmid fusion and the role of IncM2 plasmids in mobilizing non-conjugative virulence plasmids. The resulting transconjugants exhibited multidrug resistance, enhanced capsule production, and increased siderophore production.Conclusions: This study provides new insights into the genomic dynamics of ST65-CR-hvKp strains and uncovers key mechanisms, such as IS26-mediated plasmid fusion and IncM2-mediated mobilization, which facilitate the dissemination of non-conjugative virulence plasmids. Understanding these mechanisms is crucial for developing effective strategies to manage and prevent the spread of these clinically challenging strains.