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Abstract Background Carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) co-producing bla KPC and bla NDM poses a serious threat to public health. This study aimed to investigate the mechanisms underlying the resistance and virulence of CR-hvKP isolates collected from a Chinese hospital, with a focus on bla KPC and bla NDM dual-positive hvKP strains. Methods Five CR-hvKP strains were isolated from a teaching hospital in China. Antimicrobial susceptibility and plasmid stability testing, plasmid conjugation, pulsed-field gel electrophoresis, and whole-genome sequencing (WGS) were performed to examine the mechanisms of resistance and virulence. The virulence of CR-hvKP was evaluated through serum-killing assay and Galleria mellonella lethality experiments. Phylogenetic analysis based on 16 highly homologous carbapenem-resistant K. pneumoniae (CRKP) producing KPC-2 isolates from the same hospital was conducted to elucidate the potential evolutionary pathway of CRKP co-producing NDM and KPC. Results WGS revealed that five isolates individually carried three unique plasmids: an IncFIB/IncHI1B-type virulence plasmid, IncFII/IncR-type plasmid harboring KPC-2 and IncC-type plasmid harboring NDM-1. The conjugation test results indicated that the transference of KPC-2 harboring IncFII/IncR-type plasmid was unsuccessful on their own, but could be transferred by forming a hybrid plasmid with the IncC plasmid harboring NDM. Further genetic analysis confirmed that the pJNKPN26-KPC plasmid was entirely integrated into the IncC-type plasmid via the copy-in route, which was mediated by TnAs1 and IS26. Conclusion KPC-NDM-CR-hvKP likely evolved from a KPC-2-CRKP ancestor and later acquired a highly transferable bla NDM-1 plasmid. ST11-KL64 CRKP exhibited enhanced plasticity. The identification of KPC-2-NDM-1-CR-hvKP highlights the urgent need for effective preventive strategies against aggravated accumulation of resistance genes.

Background: Carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) co-producing blaKPC and blaNDM poses a serious threat to public health. This study aimed to investigate the mechanisms underlying the resistance and virulence of CR-hvKP isolates collected from a Chinese hospital, with a focus on blaKPC and blaNDM dual-positive hvKP strains. Methods: Five CR-hvKP strains were isolated from a teaching hospital in China. Antimicrobial susceptibility and plasmid stability testing, plasmid conjugation, pulsed-field gel electrophoresis, and whole-genome sequencing (WGS) were performed to examine the mechanisms of resistance and virulence. The virulence of CR-hvKP was evaluated through serum-killing assay and Galleria mellonella lethality experiments. Phylogenetic analysis based on 16 highly homologous carbapenem-resistant K. pneumoniae (CRKP) producing KPC-2 isolates from the same hospital was conducted to elucidate the potential evolutionary pathway of CRKP co-producing NDM and KPC. Results: WGS revealed that five isolates individually carried three unique plasmids: an IncFIB/IncHI1B-type virulence plasmid, IncFII/IncR-type plasmid harboring KPC-2 and IncC-type plasmid harboring NDM-1. The conjugation test results indicated that the transference of KPC-2 harboring IncFII/IncR-type plasmid was unsuccessful on their own, but could be transferred by forming a hybrid plasmid with the IncC plasmid harboring NDM. Further genetic analysis confirmed that the pJNKPN26-KPC plasmid was entirely integrated into the IncC-type plasmid via the copy-in route, which was mediated by TnAs1 and IS26. Conclusion: KPC-NDM-CR-hvKP likely evolved from a KPC-2-CRKP ancestor and later acquired a highly transferable blaNDM-1 plasmid. ST11-KL64 CRKP exhibited enhanced plasticity. The identification of KPC-2-NDM-1-CR-hvKP highlights the urgent need for effective preventive strategies against aggravated accumulation of resistance genes. [ABSTRACT FROM AUTHOR]

Introduction: Carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-HvKP) strains combining virulence and multidrug resistance (MDR) features pose a great public health concern. The aimof this study is to explore the evolutionary characteristics of virulence in CR-HvKP by investigating the genetic features of resistance and virulence hybrid plasmids. Methods: The resistance and virulence phenotypes were determined by using antimicrobial susceptibility testing and the mouse bacteremia infection model, respectively. Plasmid profiles were investigated by S1 nuclease pulsed-field gel electrophoresis (S1-PFGE) and Southern blotting, conjugation assay, and whole genome sequencing (WGS). Bioinformatics tools were used to uncover the genetic features of the resistance and virulence hybrid plasmids. Results: Two ST11-KL64 CRKP clinical isolates (KP18-3-8 and KP18-2079), which exhibited enhanced virulence compared with the classic CRKP, were detected positive for blaKPC-2 and rmpA2. The virulence level of the hypermucoviscous strain KP18-3-8 was higher than that of KP18-2079. S1-PFGE, Southern hybridization and WGS analysis identified two novel hybrid virulence plasmids in KP18-3-8 (pKP1838-KPC-vir, 228,158 bp) and KP18-2079 (pKP1838-KPC-vir, 182,326 bp), respectively. The IncHI1B/repB-type plasmid pKP1838-KPC-vir co-harboring blaKPC-2 and virulence genes (rmpA2, iucABCD and iutA) but lacking type IV secretion system could transfer into non-hypervirulent ST11 K. pneumoniae with the assistance of a helper plasmid in conjugation. The IncFII/IncR-type virulence plasmid pKP18-2079-vir may have been generated as a result of recombination between a typical pLVPK-like virulence plasmid and an MDR plasmid. Conclusion: Our studies further highlight co-evolution of the virulence and resistance plasmids in ST11-CRKP isolates. Close surveillance of such hybrid virulence plasmids in clinical K. pneumoniae should be performed. [ABSTRACT FROM AUTHOR]

ABSTRACT The emergence of nosocomial infections caused by hypervirulent and carbapenem-resistant K. pneumoniae (hv-CRKP) has become a significant public health challenge. The genetic traits of virulence and resistance plasmids in hv-CRKP have been extensively studied; however, research on the adaptive evolution strategies of clinical strains inside the host was scarce. This study aimed to understand the effects of antibiotic treatment on the phenotype and genotype characteristics of hv-CRKP. We investigated the evolution of hv-CRKP strains isolated from the same patient to elucidate the transition between hospital invasion and colonization. A comparative genomics analysis was performed to identify single nucleotide polymorphisms in the rmpA promoter. Subsequent validation through RNA-seq and gene deletion confirmed that distinct rmpA promoter sequences exert control over the mucoid phenotype. Additionally, biofilm experiments, cell adhesion assays, and animal infection models were conducted to illuminate the influence of rmpA promoter diversity on virulence changes. We demonstrated that the P12T and P11T promoters of rmpA possess strong activity, which leads to the evolution of CRKP into infectious and virulent strains. Meanwhile, the specific sequence of polyT motifs in the rmpA promoter led to a decrease in the lethality of hv-CRKP and enhanced cell adhesion and colonization. To summarize, the rmpA promoter of hv-CRKP is utilized to control capsule production, thereby modifying pathogenicity to better suit the host’s ecological environment.IMPORTANCEThe prevalence of hospital-acquired illness caused by hypervirulent carbapenem-resistant Klebsiella pneumoniae (hv-CRKP) is significant, leading to prolonged antibiotic treatment. However, there are few reports on the phenotypic changes of hv-CRKP in patients undergoing antibiotic treatment. We performed a comprehensive examination of the genetic evolutionary traits of hv-CRKP obtained from the same patient and observed variations in the promoter sequences of the virulence factor rmpA. The strong activity of the promoter sequences P11T and P12T enhances the consistent production of capsule polysaccharides, resulting in an invasive strain. Conversely, weak promoter activity of P9T and P10T is advantageous for exposing pili, hence improving bacterial cell attachment ability and facilitating bacterial colonization. This finding also explains the confusion of some clinical strains carrying wild-type rmpA but exhibiting a low mucoid phenotype. This adaptive alteration facilitates the dissemination of K. pneumoniae within the hospital setting.

ABSTRACT The combination of hypervirulent Klebsiella pneumoniae (hvKP) infection with carbapenem and tigecycline resistance leads to significant challenges to clinical treatment, with limited available antibiotics and poor patient prognoses. The hvKP12 isolate was obtained from a blood sample of a 74-year-old female in a Chinese teaching hospital. Whole-genome sequencing and microbial characterization were performed to understand the evolutionary mechanism of its resistance. The patient infected with hvKP12 died due to pyemia after a 17-day tigecycline treatment. The antimicrobial susceptibility test identified that hvKP12 was resistant to tigecycline and carbapenems. Variants of tet(A) and the overexpression of efflux pumps related to tigecycline resistance were detected in hvKP12. Conjugation experiments with blaNDM and blaKPC plasmids failed in the laboratory environment. Additionally, phylogenetic analysis suggested that hvKP12 was a clinical high-risk clone of ST11-KL64. We found that the blaKPC-2 gene segment was formed by IS26-mediated gene cluster translocation. Interestingly, the evolutionary pathway of hvKP12 suggested that the KPC-2-producing carbapenem-resistant K. pneumoniae (KPC-2–CRKP) strain evolved into a KPC-NDM-CRKP strain by acquiring the NDM plasmid. To our knowledge, this is the first report of tigecycline-resistant ST11-KL64 carbapenem-resistant hvKP (CR-hvKP) bacteria coproducing blaKPC and blaNDM, causing a fatal blood infection. IMPORTANCE Infections with CRKP coproducing KPC and NDM currently have limited clinical antibacterial options, and tigecycline is used as the last line of defense for therapy. However, this study found that CR-hvKP infection with tigecycline resistance, which may lead to many bacteria being resistant to most commonly used antibiotics, brought significant challenges to clinical treatment. The clonal propagation of ST11-KL64 CRKP should receive sufficient attention.

The emergence of nosocomial infections caused by hypervirulent and carbapenem-resistant K. pneumoniae (hv-CRKP) has become a significant public health challenge. The genetic traits of virulence and resistance plasmids in hv-CRKP have been extensively studied; however, research on the adaptive evolution strategies of clinical strains inside the host was scarce. This study aimed to understand the effects of antibiotic treatment on the phenotype and genotype characteristics of hv-CRKP. We investigated the evolution of hv-CRKP strains isolated from the same patient to elucidate the transition between hospital invasion and colonization. A comparative genomics analysis was performed to identify single nucleotide polymorphisms in the rmpA promoter. Subsequent validation through RNA-seq and gene deletion confirmed that distinct rmpA promoter sequences exert control over the mucoid phenotype. Additionally, biofilm experiments, cell adhesion assays, and animal infection models were conducted to illuminate the influence of rmpA promoter diversity on virulence changes. We demonstrated that the P12T and P11T promoters of rmpA possess strong activity, which leads to the evolution of CRKP into infectious and virulent strains. Meanwhile, the specific sequence of polyT motifs in the rmpA promoter led to a decrease in the lethality of hv-CRKP and enhanced cell adhesion and colonization. To summarize, the rmpA promoter of hv-CRKP is utilized to control capsule production, thereby modifying pathogenicity to better suit the host's ecological environment.IMPORTANCEThe prevalence of hospital-acquired illness caused by hypervirulent carbapenem-resistant Klebsiella pneumoniae (hv-CRKP) is significant, leading to prolonged antibiotic treatment. However, there are few reports on the phenotypic changes of hv-CRKP in patients undergoing antibiotic treatment. We performed a comprehensive examination of the genetic evolutionary traits of hv-CRKP obtained from the same patient and observed variations in the promoter sequences of the virulence factor rmpA . The strong activity of the promoter sequences P11T and P12T enhances the consistent production of capsule polysaccharides, resulting in an invasive strain. Conversely, weak promoter activity of P9T and P10T is advantageous for exposing pili, hence improving bacterial cell attachment ability and facilitating bacterial colonization. This finding also explains the confusion of some clinical strains carrying wild-type rmpA but exhibiting a low mucoid phenotype. This adaptive alteration facilitates the dissemination of K. pneumoniae within the hospital setting., Competing Interests: The authors declare no conflict of interest.

Background: Carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) co-producing bla KPC and bla NDM poses a serious threat to public health. This study aimed to investigate the mechanisms underlying the resistance and virulence of CR-hvKP isolates collected from a Chinese hospital, with a focus on bla KPC and bla NDM dual-positive hvKP strains., Methods: Five CR-hvKP strains were isolated from a teaching hospital in China. Antimicrobial susceptibility and plasmid stability testing, plasmid conjugation, pulsed-field gel electrophoresis, and whole-genome sequencing (WGS) were performed to examine the mechanisms of resistance and virulence. The virulence of CR-hvKP was evaluated through serum-killing assay and Galleria mellonella lethality experiments. Phylogenetic analysis based on 16 highly homologous carbapenem-resistant K. pneumoniae (CRKP) producing KPC-2 isolates from the same hospital was conducted to elucidate the potential evolutionary pathway of CRKP co-producing NDM and KPC., Results: WGS revealed that five isolates individually carried three unique plasmids: an IncFIB/IncHI1B-type virulence plasmid, IncFII/IncR-type plasmid harboring KPC-2 and IncC-type plasmid harboring NDM-1. The conjugation test results indicated that the transference of KPC-2 harboring IncFII/IncR-type plasmid was unsuccessful on their own, but could be transferred by forming a hybrid plasmid with the IncC plasmid harboring NDM. Further genetic analysis confirmed that the pJNKPN26-KPC plasmid was entirely integrated into the IncC-type plasmid via the copy-in route, which was mediated by TnAs1 and IS26., Conclusion: KPC-NDM-CR-hvKP likely evolved from a KPC-2-CRKP ancestor and later acquired a highly transferable bla NDM-1 plasmid. ST11-KL64 CRKP exhibited enhanced plasticity. The identification of KPC-2-NDM-1-CR-hvKP highlights the urgent need for effective preventive strategies against aggravated accumulation of resistance genes., (© 2024. The Author(s).)

Introduction: Carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-HvKP) strains combining virulence and multidrug resistance (MDR) features pose a great public health concern. The aim of this study is to explore the evolutionary characteristics of virulence in CR-HvKP by investigating the genetic features of resistance and virulence hybrid plasmids., Methods: The resistance and virulence phenotypes were determined by using antimicrobial susceptibility testing and the mouse bacteremia infection model, respectively. Plasmid profiles were investigated by S1 nuclease pulsed-field gel electrophoresis (S1-PFGE) and Southern blotting, conjugation assay, and whole genome sequencing (WGS). Bioinformatics tools were used to uncover the genetic features of the resistance and virulence hybrid plasmids., Results: Two ST11-KL64 CRKP clinical isolates (KP18-3-8 and KP18-2079), which exhibited enhanced virulence compared with the classic CRKP, were detected positive for bla KPC-2 and rmpA2 . The virulence level of the hypermucoviscous strain KP18-3-8 was higher than that of KP18-2079. S1-PFGE, Southern hybridization and WGS analysis identified two novel hybrid virulence plasmids in KP18-3-8 (pKP1838-KPC-vir, 228,158 bp) and KP18-2079 (pKP1838-KPC-vir, 182,326 bp), respectively. The IncHI1B/repB-type plasmid pKP1838-KPC-vir co-harboring bla KPC-2 and virulence genes ( rmpA2, iucABCD and iut A) but lacking type IV secretion system could transfer into non-hypervirulent ST11 K. pneumoniae with the assistance of a helper plasmid in conjugation. The IncFII/IncR-type virulence plasmid pKP18-2079-vir may have been generated as a result of recombination between a typical pLVPK-like virulence plasmid and an MDR plasmid., Conclusion: Our studies further highlight co-evolution of the virulence and resistance plasmids in ST11-CRKP isolates. Close surveillance of such hybrid virulence plasmids in clinical K. pneumoniae should be performed., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Zhang, Li, Zhao, Dong, Zhao, Zhao, Xia, Chi, Wang, Li, Qin and Fu.)

To investigate the in vivo evolution of the mucoid-phenotype of ST11-KL64 carbapenem-resistant Klebsiella pneumoniae (CRKP) isolated from the same patients and gain insights into diverse evolution and biology of these strains. Whole genome sequencing and bioinformatic analysis were used to determine the mutation involved in the mucoid phenotype of ST11-KL64 CRKP. Gene knockout, bacterial morphology and capsular polysaccharides (CPS) extraction were used to verify the role of wzc and wcaJ in the mucoid phenotypes. Antimicrobial susceptibility, growth assay, biofilm formation, host cell adhesion and virulence assay were used to investigate the pleiotropic role of CPS changes in ST11-KL64 CRKP strains. Mutation of wzc S682N led to hypermucoid phenotype, which had negative impact on bacterial fitness and resulted in reduced biofilm formation and epithelial cell adhesion; while enhanced resistance to macrophage phagocytosis and virulence. Mutations of wcaJ gene led to non-mucoid phenotype with increased biofilm formation and epithelial cell adhesion, but reduced resistance of macrophage phagocytosis and virulence. Using virulence gene knockout, we demonstrated that CPS, rather than the pLVPK-like virulence plasmid, has a greater effect on mucoid phenotypic changes. CPS could be used as a surrogate marker of virulence in ST11-KL64 CRKP strains. ST11-KL64 CRKP strains sacrifice certain advantages to develop pathogenicity by changing CPS with two opposite in vivo evolution strategies. [ABSTRACT FROM AUTHOR]

The combination of hypervirulent Klebsiella pneumoniae (hvKP) infection with carbapenem and tigecycline resistance leads to significant challenges to clinical treatment, with limited available antibiotics and poor patient prognoses. The hvKP12 isolate was obtained from a blood sample of a 74-year-old female in a Chinese teaching hospital. Whole-genome sequencing and microbial characterization were performed to understand the evolutionary mechanism of its resistance. The patient infected with hvKP12 died due to pyemia after a 17-day tigecycline treatment. The antimicrobial susceptibility test identified that hvKP12 was resistant to tigecycline and carbapenems. Variants of tet (A) and the overexpression of efflux pumps related to tigecycline resistance were detected in hvKP12. Conjugation experiments with bla NDM plasmids failed in the laboratory environment. Additionally, phylogenetic analysis suggested that hvKP12 was a clinical high-risk clone of ST11-KL64. We found that the bla KPC -mediated gene cluster translocation. Interestingly, the evolutionary pathway of hvKP12 suggested that the KPC-2-producing carbapenem-resistant K. pneumoniae (KPC-2-CRKP) strain evolved into a KPC-NDM-CRKP strain by acquiring the NDM plasmid. To our knowledge, this is the first report of tigecycline-resistant ST11-KL64 carbapenem-resistant hvKP (CR-hvKP) bacteria coproducing bla KPC-2 gene segment was formed by IS 26 Infections with CRKP coproducing KPC and NDM currently have limited clinical antibacterial options, and tigecycline is used as the last line of defense for therapy. However, this study found that CR-hvKP infection with tigecycline resistance, which may lead to many bacteria being resistant to most commonly used antibiotics, brought significant challenges to clinical treatment. The clonal propagation of ST11-KL64 CRKP should receive sufficient attention.bla KPC and bla NDM , causing a fatal blood infection. IMPORTANCE Infections with CRKP coproducing KPC and NDM currently have limited clinical antibacterial options, and tigecycline is used as the last line of defense for therapy. However, this study found that CR-hvKP infection with tigecycline resistance, which may lead to many bacteria being resistant to most commonly used antibiotics, brought significant challenges to clinical treatment. The clonal propagation of ST11-KL64 CRKP should receive sufficient attention.