Search

ABSTRACT Multidrug-resistant hypervirulent Klebsiella pneumoniae (MDR-hvKp) has been emerging worldwide. However, the clinical, microbiological, and genomic characteristics of newly emerged MDR sequence type 65 (ST65) hvKp are unclear. We conducted active longitudinal genomic surveillance of K. pneumoniae in the hospital starting in 2017. Clinical characteristics, including demographic data, infection type, and outcomes, were collected. Whole-genome sequencing was performed to clarify phylogenetic and plasmid features, and phenotype determined by growth curves, plasmid transferability and stability, hypermucoviscosity, biofilm formation, and serum survival were analyzed to microbiologically characterize ST65 in depth. Ten ST65 (1.4%, 10/720) isolates were detected from 720 K. pneumoniae isolates in total. Nine patients (90%, 9/10) were older than 60 years and had multiple underlying diseases. All ST65 K. pneumoniae isolates harbored iucA, rmpA, rmpA2, iroB, and peg344 and were identified as hvKp. Surprisingly, two MDR-hvKp isolates that grew slowly were observed. Isolate PEKP4222 harbored a pLVPK-like plasmid and a conjunctive MDR plasmid. Isolate P1 harbored blaKPC-3 in a new plasmid, pP1-54, resulting in an extensively drug-resistant (XDR) phenotype; this isolate, which might have evolved from a strain harboring blaKPC-2, resulted in fatal infection. The pP1-54 plasmid could not be transferred to Escherichia coli by conjugation but could be stably inherited vertically. Interestingly, P1 also carried the pLVPK-like plasmid and acquired various antimicrobial resistance genes, and blaCTX-M-3 was detected in the IncB/O/K/Z plasmid. The convergence of XDR and hypervirulence within classical ST65 hvKp is emerging, highlighting the need for enhanced genomic surveillance. IMPORTANCE XDR-hvKp poses a great challenge to public health. ST65, a classical hvKp subtype, mostly presented with hypermucoviscosity, which restricts antimicrobial resistance acquisition. However, few studies have demonstrated the clinical, microbiological, and genomic characteristics of ST65, especially MDR-ST65 hvKp. Here, we first reported that ST65 hvKp acquired blaKPC-3 and then conferred the XDR-hvKp phenotype. Genomic context analysis concluded that the blaKPC-3 gene might have evolved from blaKPC-2. Additionally, the pLVPK-like plasmid seemed to acquire more resistance genes, and blaCTX-M-3 located in the IncB/O/K/Z plasmid was observed. The XDR-hvKp phenotype could be stably inherited vertically, indicating that strains harboring blaKPC-3 and pLVPK-like plasmids could persistently exist in hospital settings. These data suggest that genomic adaptation is rapid and that enhanced surveillance is essential.

Objectives: In this study, we report the complete genome sequence of a ST65 hypervirulent Klebsiella pneumoniae isolate carrying mcr-8 from China. The aim was to investigate its molecular characteristics and resistant mechanism., Methods: A colistin-resistant hvKP was isolated from an inpatient in China. The whole genome was sequenced on Illumina NovaSeq 6000 and long-read ONT platforms. de novo assembly was conducted using SPAdes and Unicycler. S1 nuclease pulsed-field gel electrophoresis, Southern-blot, and antimicrobial susceptibility testing were performed. Sequence type, antimicrobial resistance and virulence-related genes were predicted from the sequence. The circular maps of multiple plasmids comparisons were drawn by the BLAST Ring Image., Results: The complete genome sequence of K. pneumoniae ACESH00926 consists of one chromosome and two plasmids. ACESH00926 belongs to K2 ST65 according to the MLST scheme. ACESH00926 showed high resistance to colistin (MIC > 8 µg/mL). Several ARGs were identified, including mobile colistin-resistant gene mcr-8 which was located in an IncFIl(K)/IncFIA(HI1) type plasmid. The bigger plasmid was a pK244-like virulence plasmid. It carrying a series of virulence genes, such as the regulator of the mucoid phenotype (rmpA and rmpA2), salmochelin siderophore biosynthesis (iroB), ABC transporter (iroC), ferric aerobactin receptor (iutA), aerobactin siderophore biosynthesis protein (iucC), and aerobactin synthetase (iucA) encoded genes. And another plasmid carrying mcr-8 with a conserved genetic context (dgkA-sasA-copR-mcr-8-ccdA-ccdB-xerD-repE-parM-umuC-lexA-klcA)., Conclusions: It is necessary to emphasize the necessity of monitoring a combination of colistin-resistant and hypervirulent Klebsiella pneumoniae strains in the future., Competing Interests: Declaration of competing interest None declared., (Copyright © 2024. Published by Elsevier Ltd.)

Klebsiella pneumoniae is a Gram-negative bacterium implicated as the causative pathogen in several medical health issues with different strains causing different pathologies including pneumonia, bloodstream infections, meningitis and infections from wounds or surgery. In this study, four captive African marmosets housed in Thailand were found dead. Necropsy and histology revealed congestion of hearts, kidneys and adrenal glands. Twenty-four bacterial isolates were obtained from these four animals with all isolates yielding identical phenotypes indicative of K. pneumoniae based on classical identification schema. All the isolates show the susceptibility to amikacin, cephalexin, doxycycline, gentamicin, and enrofloxacin with intermediate susceptibility to amoxycillin/clavulanic acid. One isolate (20P167W) was chosen for genome analysis and determined to belong to sequence type 65 (ST65). The genome of 20P167W possessed multiple virulence genes including mrk gene cluster and iro and iuc gene cluster (salmochelin and aerobactin, respectively) as well as multiple antibiotic resistance genes including blaSHV−67, blaSHV−11, oqxA, oqxB, and fosA genes resembling those found in human isolates; this isolate has a close genetic relationship with isolates from humans in Ireland, but not from Thailand and California sea lions. Phylogenetic studies using SNP show that there was no relation between genetic and geographic distributions of all known strains typing ST65, suggesting that ST65 strains may spread worldwide through multiple international transmission events rather than by local expansions in humans and/or animals. We also predict that K. pneumoniae ST65 has an ability to acquire genetic mobile element from other bacteria, which would allow Klebsiella to become an even greater public health concern.

The emergence of New Delhi Metallo-β-lactamase (NDM)-producing Klebsiella pneumoniae has aroused critical concern worldwide. Herein, we reported the first emergence of NDM-5-producing K. pneumoniae isolates in a 68-year-old lung transplant recipient, who died of septic shock 13 days after surgery. The K. pneumoniae strain KP22937 isolated from the bloodstream of the patient was analyzed for phenotypes and genotypes. KP22937 belonged to sequence type (ST) 65 and capsule serotype K2, contained iucABCDiutA and iroBCDN virulence clusters, showed high virulence to mice, and was therefore considered a hypervirulent K. pneumoniae. The blaNDM-5 gene was located on a genomic island region of the IncX3-type plasmid pNDM22937, which was successfully transferred to Escherichia coli EC600 with insignificant fitness costs. The transconjugant demonstrated similar antimicrobial susceptibility and growth kinetics to the recipient E. coli EC600. The plasmid pNDM22937 was almost identical to the blaNDM-5-carrying IncX3 plasmids previously reported in K. pneumoniae strains with different ST types and in other species. Our findings raise concerns about the horizontal spread of blaNDM-5 gene mediated by IncX3 plasmid, where hypervirulent K. pneumoniae strains are also involved. Stricter control measures are needed to prevent the dissemination of the novel clone in hospital settings.

Yiqi Fu,1,* Min Xu,2,* Yanchao Liu,2 Ang Li,3 Jianying Zhou1 1Department of Respiratory Diseases, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, China; 2Key Laboratory of Clinical In Vitro Diagnostic Techniques of Zhejiang Province, Center of Clinical Laboratory, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, China; 3Department of Precision Medicine Center, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China *These authors contributed equally to this work Background: Capsular serotype K2 Klebsiella pneumoniae of sequence type (ST) 65 has been recognized as a hypervirulent clone. Simultaneous presence of different blaCTX-M genes has never been reported in this clone. In the present study, the genetic characteristics and virulence phenotype of a CTX-M-3 and CTX-M-14 coproducing ST65 K. pneumoniae human isolate, KP_06, that caused an intracranial infection, are evaluated. Methods: The potential virulence of KP_06 was assayed by in vitro and in vivo methods. The molecular biology and whole-genome sequencing technology were used to analyze the genomic features associated with the virulence of this strain. Results: The KP_06 exhibited typical features of hypervirulent K. pneumoniae (hvKP), showing hypermucoviscosity phenotype and belonging to K2 and ST65. Apart from virulence genes linked to hvKP, including rmpA, rmpA2, and clb cluster and genes encoding siderophores, it was found to harbor a ~170 kb pLVPK-like virulence plasmid. In contrast to most hvKP, KP_06 was resistant to cephalosporins and the coexistence of blaCTX-M-3 and blaCTX-M-14 was detected. Further experiments demonstrated that this strain was classified as a nonbiofilm producer and serum sensitivity (grade 1) and killed only 30% of Galleria mellonella inoculated with 1×106 colony-forming unit of the specimen within 48 hours, suggesting relatively low virulence. Comparative genomic analysis of KP_06 with five K2 hypermucoviscous K. pneumoniae (HMKP) revealed seven unique orthologies with varied function in this strain. Intriguingly, the virulence genes identified in KP-06 were unexpectedly more diverse than those observed in five other K2 HMKP strains. Conclusion: Our data support the notion that neither virulence-associated genes (clusters) nor the pLVPK-like virulence plasmid is sufficient for the hypervirulence of K. pneumoniae. Future studies aiming to explore the virulence of K. pneumoniae should take genome-based profile together with experimental work. The detailed mechanism involving in the impaired virulence of KP_06 remains to be further explored. Keywords: Klebsiella pneumoniae, virulence factor, serotype K2, ST65, blaCTX-M, comparative genome

The rise of a new hypervirulent variant of Klebsiella pneumoniae (hvKp) was recently reported, mainly linked to the ST23 lineage. The hvKp variants can cause severe infections, including hepatic abscesses, bacteremia, and meningitis, with a particularly disconcerting propensity to cause community-acquired, life-threatening infection among young and otherwise healthy individuals. The present study aimed to report the clinical characteristics of a hypermucoviscous K. pneumoniae strain isolated in Italy and sustaining recurrent meningitis in a patient of Peruvian origin. A further objective was to retrospectively investigate, by means of whole-genome sequencing (WGS) analysis, the genomic features of such an isolate. The hypermucoviscosity phenotype of the strain (sk205y205t) was determined using the string test. Genomic information was obtained by WGS (Illumina) and bioinformatic analysis. Strain sk205y205t was susceptible to most antibiotics, despite the presence of some resistance genes, including blaSHV-11, blaSHV-67, fosA, and acrR. The isolate belonged to ST65 and serotype K2, and exhibited several virulence factors related to the hvKp variant. Among these, were the siderophore genes entB, irp2, iroN, iroB, and iucA; the capsule-regulating genes rmpA and rmpA2; and the type 1 and 3 fimbriae fimH27 and mrkD, respectively. A further operon, encoding the genotoxin colibactin (clbA-Q), was also identified. The virulence plasmids pK2044, pRJA166b, and pNDM. MAR were also detected. Phylogenetic investigation showed that this Italian strain is highly similar to a Chinese isolate, suggesting a hidden circulation of this hvKp ST65 K2 lineage.

Background: Capsular serotype K2 Klebsiella pneumoniae of sequence type (ST) 65 has been recognized as a hypervirulent clone. Simultaneous presence of different blaCTX-M genes has never been reported in this clone. In the present study, the genetic characteristics and virulence phenotype of a CTX-M-3 and CTX-M-14 coproducing ST65 K. pneumoniae human isolate, KP_06, that caused an intracranial infection, are evaluated. Methods: The potential virulence of KP_06 was assayed by in vitro and in vivo methods. The molecular biology and whole-genome sequencing technology were used to analyze the genomic features associated with the virulence of this strain. Results: The KP_06 exhibited typical features of hypervirulent K. pneumoniae (hvKP), showing hypermucoviscosity phenotype and belonging to K2 and ST65. Apart from virulence genes linked to hvKP, including rmpA, rmpA2, and clb cluster and genes encoding siderophores, it was found to harbor a ~170 kb pLVPK-like virulence plasmid. In contrast to most hvKP, KP_06 was resistant to cephalosporins and the coexistence of blaCTX-M-3 and blaCTX-M-14 was detected. Further experiments demonstrated that this strain was classified as a nonbiofilm producer and serum sensitivity (grade 1) and killed only 30% of Galleria mellonella inoculated with 1×106 colony-forming unit of the specimen within 48 hours, suggesting relatively low virulence. Comparative genomic analysis of KP_06 with five K2 hypermucoviscous K. pneumoniae (HMKP) revealed seven unique orthologies with varied function in this strain. Intriguingly, the virulence genes identified in KP-06 were unexpectedly more diverse than those observed in five other K2 HMKP strains. Conclusion: Our data support the notion that neither virulence-associated genes (clusters) nor the pLVPK-like virulence plasmid is sufficient for the hypervirulence of K. pneumoniae. Future studies aiming to explore the virulence of K. pneumoniae should take genome-based profile together with experimental work. The detailed mechanism involving in the impaired virulence of KP_06 remains to be further explored. [ABSTRACT FROM AUTHOR]

Multidrug-resistant hypervirulent Klebsiella pneumoniae (MDR-hvKp) has been emerging worldwide. However, the clinical, microbiological, and genomic characteristics of newly emerged MDR sequence type 65 (ST65) hvKp are unclear. We conducted active longitudinal genomic surveillance of K. pneumoniae in the hospital starting in 2017. Clinical characteristics, including demographic data, infection type, and outcomes, were collected. Whole-genome sequencing was performed to clarify phylogenetic and plasmid features, and phenotype determined by growth curves, plasmid transferability and stability, hypermucoviscosity, biofilm formation, and serum survival were analyzed to microbiologically characterize ST65 in depth. Ten ST65 (1.4%, 10/720) isolates were detected from 720 K. pneumoniae isolates in total. Nine patients (90%, 9/10) were older than 60 years and had multiple underlying diseases. All ST65 K. pneumoniae isolates harbored iucA , rmpA , rmpA2 , iroB , and peg344 and were identified as hvKp. Surprisingly, two MDR-hvKp isolates that grew slowly were observed. Isolate PEKP4222 harbored a pLVPK-like plasmid and a conjunctive MDR plasmid. Isolate P1 harbored bla KPC-3 in a new plasmid, pP1-54, resulting in an extensively drug-resistant (XDR) phenotype; this isolate, which might have evolved from a strain harboring bla was detected in the IncB/O/K/Z plasmid. The convergence of XDR and hypervirulence within classical ST65 hvKp is emerging, highlighting the need for enhanced genomic surveillance. KPC-2 XDR-hvKp poses a great challenge to public health. ST65, a classical hvKp subtype, mostly presented with hypermucoviscosity, which restricts antimicrobial resistance acquisition. However, few studies have demonstrated the clinical, microbiological, and genomic characteristics of ST65, especially MDR-ST65 hvKp. Here, we first reported that ST65 hvKp acquired bla CTX-M-3 was detected in the IncB/O/K/Z plasmid. The convergence of XDR and hypervirulence within classical ST65 hvKp is emerging, highlighting the need for enhanced genomic surveillance. IMPORTANCE XDR-hvKp poses a great challenge to public health. ST65, a classical hvKp subtype, mostly presented with hypermucoviscosity, which restricts antimicrobial resistance acquisition. However, few studies have demonstrated the clinical, microbiological, and genomic characteristics of ST65, especially MDR-ST65 hvKp. Here, we first reported that ST65 hvKp acquired bla KPC-3 and then conferred the XDR-hvKp phenotype. Genomic context analysis concluded that the bla KPC-3 gene might have evolved from bla KPC-2 . Additionally, the pLVPK-like plasmid seemed to acquire more resistance genes, and bla CTX-M-3 located in the IncB/O/K/Z plasmid was observed. The XDR-hvKp phenotype could be stably inherited vertically, indicating that strains harboring bla KPC-3 and pLVPK-like plasmids could persistently exist in hospital settings. These data suggest that genomic adaptation is rapid and that enhanced surveillance is essential.

Klebsiella pneumoniae is a Gram-negative bacterium implicated as the causative pathogen in several medical health issues with different strains causing different pathologies including pneumonia, bloodstream infections, meningitis and infections from wounds or surgery. In this study, four captive African marmosets housed in Thailand were found dead. Necropsy and histology revealed congestion of hearts, kidneys and adrenal glands. Twenty-four bacterial isolates were obtained from these four animals with all isolates yielding identical phenotypes indicative of K. pneumoniae based on classical identification schema. All the isolates show the susceptibility to amikacin, cephalexin, doxycycline, gentamicin, and enrofloxacin with intermediate susceptibility to amoxycillin/clavulanic acid. One isolate (20P167W) was chosen for genome analysis and determined to belong to sequence type 65 (ST65). The genome of 20P167W possessed multiple virulence genes including mrk gene cluster and iro and iuc gene cluster (salmochelin and aerobactin, respectively) as well as multiple antibiotic resistance genes including bla SHV -67 , bla SHV -11 genes resembling those found in human isolates; this isolate has a close genetic relationship with isolates from humans in Ireland, but not from Thailand and California sea lions. Phylogenetic studies using SNP show that there was no relation between genetic and geographic distributions of all known strains typing ST65, suggesting that ST65 strains may spread worldwide through multiple international transmission events rather than by local expansions in humans and/or animals. We also predict that oqxA, oqxB ST65 has an ability to acquire genetic mobile element from other bacteria, which would allow Klebsiella to become an even greater public health concern.fosA genes resembling those found in human isolates; this isolate has a close genetic relationship with isolates from humans in Ireland, but not from Thailand and California sea lions. Phylogenetic studies using SNP show that there was no relation between genetic and geographic distributions of all known strains typing ST65, suggesting that ST65 strains may spread worldwide through multiple international transmission events rather than by local expansions in humans and/or animals. We also predict that K. pneumoniae ST65 has an ability to acquire genetic mobile element from other bacteria, which would allow Klebsiella to become an even greater public health concern., 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., (Copyright © 2022 Pinpimai, Banlunara, Roe, Dittmer, Biggs, Tantilertcharoen, Chankow, Bunpapong, Boonkam and Pirarat.)

The rise of a new hypervirulent variant of Klebsiella pneumoniae (hvKp) was recently reported, mainly linked to the ST23 lineage. The hvKp variants can cause severe infections, including hepatic abscesses, bacteremia, and meningitis, with a particularly disconcerting propensity to cause community-acquired, life-threatening infection among young and otherwise healthy individuals. The present study aimed to report the clinical characteristics of a hypermucoviscous K. pneumoniae strain isolated in Italy and sustaining recurrent meningitis in a patient of Peruvian origin. A further objective was to retrospectively investigate, by means of whole-genome sequencing (WGS) analysis, the genomic features of such an isolate. The hypermucoviscosity phenotype of the strain (sk205y205t) was determined using the string test. Genomic information was obtained by WGS (Illumina) and bioinformatic analysis. Strain sk205y205t was susceptible to most antibiotics, despite the presence of some resistance genes, including blaSHV-11, blaSHV-67, fosA, and acrR. The isolate belonged to ST65 and serotype K2, and exhibited several virulence factors related to the hvKp variant. Among these, were the siderophore genes entB, irp2, iroN, iroB, and iucA; the capsule-regulating genes rmpA and rmpA2; and the type 1 and 3 fimbriae fimH27 and mrkD, respectively. A further operon, encoding the genotoxin colibactin (clbA-Q), was also identified. The virulence plasmids pK2044, pRJA166b, and pNDM. MAR were also detected. Phylogenetic investigation showed that this Italian strain is highly similar to a Chinese isolate, suggesting a hidden circulation of this hvKp ST65 K2 lineage. [ABSTRACT FROM AUTHOR]

Background: Capsular serotype K2 Klebsiella pneumoniae of sequence type (ST) 65 has been recognized as a hypervirulent clone. Simultaneous presence of different bla genes has never been reported in this clone. In the present study, the genetic characteristics and virulence phenotype of a CTX-M-3 and CTX-M-14 coproducing ST65 CTX-M genes has never been reported in this clone. In the present study, the genetic characteristics and virulence phenotype of a CTX-M-3 and CTX-M-14 coproducing ST65 K. pneumoniae human isolate, KP_06, that caused an intracranial infection, are evaluated., Methods: The potential virulence of KP_06 was assayed by in vitro and in vivo methods. The molecular biology and whole-genome sequencing technology were used to analyze the genomic features associated with the virulence of this strain., Results: The KP_06 exhibited typical features of hypervirulent K. pneumoniae (hvKP), showing hypermucoviscosity phenotype and belonging to K2 and ST65. Apart from virulence genes linked to hvKP, including rmpA , rmpA2 , and clb cluster and genes encoding siderophores, it was found to harbor a ~170 kb pLVPK-like virulence plasmid. In contrast to most hvKP, KP_06 was resistant to cephalosporins and the coexistence of bla CTX-M-3 and bla CTX-M-14 was detected. Further experiments demonstrated that this strain was classified as a nonbiofilm producer and serum sensitivity (grade 1) and killed only 30% of Galleria mellonella inoculated with 1×10 6 colony-forming unit of the specimen within 48 hours, suggesting relatively low virulence. Comparative genomic analysis of KP_06 with five K2 hypermucoviscous K. pneumoniae (HMKP) revealed seven unique orthologies with varied function in this strain. Intriguingly, the virulence genes identified in KP-06 were unexpectedly more diverse than those observed in five other K2 HMKP strains., Conclusion: Our data support the notion that neither virulence-associated genes (clusters) nor the pLVPK-like virulence plasmid is sufficient for the hypervirulence of K. pneumoniae . Future studies aiming to explore the virulence of K. pneumoniae should take genome-based profile together with experimental work. The detailed mechanism involving in the impaired virulence of KP_06 remains to be further explored., Competing Interests: Disclosure The authors report no conflicts of interest in this work.