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Outbreak of KPC-2 Carbapenem-resistant Klebsiella pneumoniae ST76 and Carbapenem-resistant K2 Hypervirulent Klebsiella pneumoniae ST375 strains in Northeast China: molecular and virulent characteristics.

ABSTRACT: BACKGROUND:Carbapenem-resistant hypervirulent Klebsiella pneumoniae strains have recently come into existence worldwide; however, researchers in northeast China are not aware of their clinical features and molecular characteristics. METHODS:Here, the molecular and virulent characteristics of 44 carbapenem-resistant K. pneumoniae (CRKP) isolates collected from January 2015 to December 2017 were studied. Multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) were carried out to define the clonal relatedness among the isolates. PCR and capsular serotyping of the virulence-associated genes, as well as biofilm formation and serum complement-mediated killing assays, were employed to determine the virulent potential. The genomic features and associated mobile genetic elements of JmsCRE57 were detected by whole genome sequencing. RESULTS:The only positive isolate was JmsCRE57, which belonged to the ST375 serotype K2 that expressed uge, mrkD, fimH, kpn, aerobactin and rmpA virulence-associated genes and showed strong biofilm formation and serum sensitivity. Sequencing results showed that the JmsCRE57 genome mainly consisted of a circular chromosome, three antimicrobial resistant plasmids and a virulent plasmid. The antimicrobial resistant plasmid expressing blaKPC-2, blaCTX-M-15, aph(3″)-Ib, aph(6)-Id, qnrB1, aac(3)-IIa, aac(6')-Ib-cr, blaOXA-1, blaTEM-1B, catB4, sul2, dfrA14 and blaSHV-99. The virulent plasmid belonged to the IncHI1B group, which is mainly composed of mucoid phenotype genes and siderophore-associated genes. The remaining CRKP strains that expressed uge, fimH, mrkD and kpn virulence-associated genes were not successfully typed. CONCLUSION:Our results provide new insights on the epidemiology of carbapenem-resistant K2 hypervirulent K. pneumoniae ST375 and CRKP ST76 strains in northeast China, which may help control their future outbreaks.

SUBMITTER: Su S

PROVIDER: S-EPMC7331116 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) has been increasingly reported worldwide. Here, we report an outbreak caused by sequence type 859-K19 (ST859-K19) CR-hvKP isolates in a teaching hospital in China. Interestingly, K. pneumoniae ST859 was a single-locus variant of ST11 but has never been reported before. A total of 11 nonrepetitive ST859 CR-hvKP isolates were collected from 11 patients, 3 of which died of severe CR-hvKP infection. Antimicrobial susceptibility assay results showed that all the 11 CR-hvKP isolates exhibited high-level resistance to commonly used antibiotics, only remaining susceptible to colistin, tigecycline, and ceftazidime/avibactam. Whole-genome sequencing (WGS) was performed using the Illumina platform for the 11 CR-hvKP isolates, and RJ9299 was further sequenced using the PacBio platform. A phylogram tree using WGS data revealed that all the 11 CR-hvKP isolates were clustered in 1 clade, which probably indicated clone transmission. Determinants of resistance and virulence gene analysis using WGS data confirmed the 11 isolates had almost identical resistance gene profiles (blaKPC-2, blaTEM-1B, blaSHV-187, rmtB, fosA6) and virulence gene (rmpA, rmpA2, iucABCDiutA) profiles, which hint at clone spread again. The complete genome size of RJ9299 was 5,875 kb, including a 5,445-kb chromosome, a 215-kb virulence plasmid (pVir-CR-hvKP-RJ9299), a 109-kb blaKPC-2-harboring plasmid (pKPC-2-RJ9299), and three circular plasmids. Comparative genomics showed pVir-RJ9299 (IncHI1B type) and pKPC-2-RJ9299 (IncFII-IncR) possessed over 99% similarity to pLVPK and pKPC-CR-hvKP-C789, respectively. Serum resistance assays and a Galleria mellonella infection model showed the 11 isolates exhibited different levels of virulence. This is the first report of an outbreak caused by ST859 CR-hvKP isolates. IMPORTANCE The emergence of carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) in China has posed a great threat to public health, especially in the highly transmissible ST11 clone. With the transmission of virulence and resistance determinants, CR-hvKP isolates have been reported in an increasing number of sequence types (STs), including ST23, ST65, ST1797, ST43, ST231, ST147, ST15, ST383, ST268, ST595, ST375, ST48, and ST307. Here, we report an outbreak caused by ST859-K19 CR-hvKP isolates in a teaching hospital in China. ST859 is a single-locus variant of ST11. There is no literature on ST859 K. pneumoniae in public databases, let alone ST859 CR-hvKP isolates. To our knowledge, this is the first report to depict the molecular and genomic characteristics of ST859 CR-hvKP isolates. Active surveillance approaches should be implemented to promptly find the spread of CR-hvKP isolates in health care settings.

Project description:ObjectiveThis study aimed to comprehensively investigate hypervirulent carbapenem-resistant Klebsiella pneumoniae (CR-hvKP) in the Ningbo region. Importantly, we sought to elucidate its molecular characteristics and pathogenic mechanisms. This information will provide evidence-based insights for preventing and controlling nosocomial infections and facilitate improved clinical diagnosis and treatment in this region.Methods96 carbapenem-resistant Klebsiella pneumoniae strains were collected from the Ningbo region between January 2021 and December 2022. Whole genome sequencing and bioinformatic methods were employed to identify and characterize CR-hvKP strains at the molecular level. The minimum inhibitory concentrations (MICs) of common clinical antibiotics were determined using the VITEK-2 Compact automatic microbiological analyzer. Plasmid conjugation experiments evaluated the transferability of resistance plasmids. Finally, mouse virulence assays were conducted to explore the pathogenic mechanisms.ResultsAmong the 96 strains, a single CR-hvKP strain, designated CR-hvKP57, was identified, with an isolation frequency of 1.04%. Whole-genome sequencing revealed the strain to be ST23 serotype with a K1 capsule. This strain harbored three plasmids. Plasmid 1, a pLVPK-like virulence plasmid, carried multiple virulence genes, including rmpA, rmpA2, iroB, iucA, and terB. Plasmid 2 contained transposable element sequences such as IS15 and IS26. Plasmid 3, classified as a resistance plasmid, harbored the bla KPC-3 carbapenem resistance gene. Mouse virulence assays demonstrated a high mortality rate associated with CR-hvKP57 infection. Additionally, there was a significant increase in IL-1β, IL-6, and TNF-α levels in response to CR-hvKP57 infection, indicating varying degrees of inflammatory response. Western blot experiments further suggested that the pathogenic mechanism involves activation of the NF-κB signaling pathway.ConclusionThis study confirms the emergence of hypervirulent CR-hvKP in the Ningbo region, which likely resulted from the acquisition of a pLVPK-like virulence plasmid and a bla KPC-3 resistance plasmid by the ST23-K1 type Klebsiella pneumoniae. Our findings highlight the urgent need for more judicious use of antibiotics to limit the emergence of resistance. Additionally, strengthening infection prevention and control measures is crucial to minimize the spread of virulence and resistance plasmids.

Project description:Carbapenem-resistant Enterobacteriaceae (CRE) are resistant to most antibiotics, making CRE infections extremely difficult to treat with available agents. Klebsiella pneumoniae carbapenemases (KPC-2 and KPC-3) are predominant carbapenemases in CRE in the United States. Nacubactam is a bridged diazabicyclooctane (DBO) β-lactamase inhibitor that inactivates class A and C β-lactamases and exhibits intrinsic antibiotic and β-lactam "enhancer" activity against Enterobacteriaceae In this study, we examined a collection of meropenem-resistant K. pneumoniae isolates carrying blaKPC-2 or blaKPC-3; meropenem-nacubactam restored susceptibility. Upon testing isogenic Escherichia coli strains producing KPC-2 variants with single-residue substitutions at important Ambler class A positions (K73, S130, R164, E166, N170, D179, K234, E276, etc.), the K234R variant increased the meropenem-nacubactam MIC compared to that for the strain producing KPC-2, without increasing the meropenem MIC. Correspondingly, nacubactam inhibited KPC-2 (apparent Ki [Ki app] = 31 ± 3 μM) more efficiently than the K234R variant (Ki app = 270 ± 27 μM) and displayed a faster acylation rate (k2/K), which was 5,815 ± 582 M-1 s-1 for KPC-2 versus 247 ± 25 M-1 s-1 for the K234R variant. Unlike avibactam, timed mass spectrometry revealed an intact sulfate on nacubactam and a novel peak (+337 Da) with the K234R variant. Molecular modeling of the K234R variant showed significant catalytic residue (i.e., S70, K73, and S130) rearrangements that likely interfere with nacubactam binding and acylation. Nacubactam's aminoethoxy tail formed unproductive interactions with the K234R variant's active site. Molecular modeling and docking observations were consistent with the results of biochemical analyses. Overall, the meropenem-nacubactam combination is effective against carbapenem-resistant K. pneumoniae Moreover, our data suggest that β-lactamase inhibition by nacubactam proceeds through an alternative mechanism compared to that for avibactam.

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Outbreak of KPC-2 Carbapenem-resistant Klebsiella pneumoniae ST76 and Carbapenem-resistant K2 Hypervirulent Klebsiella pneumoniae ST375 strains in Northeast China: molecular and virulent characteristics.

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