Characterization of a Novel Hybrid Plasmid Coharboring bla KPC-2 and qnrVC4 in a Clinical Citrobacter freundii Strain.
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ABSTRACT:
SUBMITTER: Zhu Y
PROVIDER: S-EPMC7577163 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:We report two KPC-producing Citrobacter freundii isolates from unrelated patients. In one case, bla KPC-2 was harbored on a novel variant of a Tn4401 transposon of an IncN plasmid conjugated together with a coresident IncA plasmid, whereas in the other one, bla KPC-3 was on a Tn4401a transposon located on an IncX3-IncA self-conjugative plasmid fusion. The interplay among plasmids carrying bla KPC and the coresident IncA plasmids offers new information on plasmids coresident within clinically relevant enterobacteria.
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Project description:The emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates in Egyptian hospitals has been reported. However, the genetic basis and analysis of the plasmids associated with carbapenem-resistant hypervirulent K. pneumoniae (CR-HvKP) in Egypt have not been presented. Therefore, we attempted to decipher the plasmid sequences that are responsible for transferring the determinants of carbapenem resistance, particularly bla NDM-1 and bla KPC-2 Out of 34 K. pneumoniae isolates collected from two tertiary hospitals in Egypt, 31 were CRKP. Whole-genome sequencing revealed that our isolates were related to 13 different sequence types (STs). The most prevalent ST was ST101, followed by ST383 and ST11. Among the CRKP isolates, one isolate named EBSI036 has been reassessed by Nanopore sequencing. Genetic environment analysis showed that EBSI036 carried 20 antibiotic resistance genes and was identified as a CR-HvKP strain: it harbored four plasmids, namely, pEBSI036-1-NDM-VIR, pEBSI036-2-KPC, pEBSI036-3, and pEBSI036-4. The two carbapenemase genes bla NDM-1 and bla KPC-2 were located on plasmids pEBSI036-1-NDM-VIR and pEBSI036-2-KPC, respectively. The IncFIB:IncHI1B hybrid plasmid pEBSI036-1-NDM-VIR also carried some virulence factors, including the regulator of the mucoid phenotype (rmpA), the regulator of mucoid phenotype 2 (rmpA2), and aerobactin (iucABCD and iutA). Thus, we set out in this study to analyze in depth the genetic basis of the pEBSI036-1-NDM-VIR and pEBSI036-2-KPC plasmids. We report a high-risk clone ST11 KL47 serotype of a CR-HvKP strain isolated from the blood of a 60-year-old hospitalized female patient from the intensive care unit (ICU) in a tertiary care hospital in Egypt, which showed the cohabitation of a novel hybrid plasmid coharboring the bla NDM-1 and virulence genes and a bla KPC-2-carrying plasmid.IMPORTANCE CRKP has been registered in the critical priority tier by the World Health Organization and has become a significant menace to public health. The emergence of CR-HvKP is of great concern in terms of both disease and treatment. In-depth analysis of the carbapenemase-encoding and virulence plasmids may provide insight into ongoing recombination and evolution of virulence and multidrug resistance in K. pneumoniae Thus, this study serves to alert contagious disease clinicians to the presence of hypervirulence in CRKP isolates in Egyptian hospitals.
| S-EPMC8265623 | biostudies-literature
Project description:To date, blaNDM and blaKPC genes have been found predominantly in clinical settings around the world. In contrast, bacteria harbouring these two genes from natural environments are relatively less well studied compared to those found in clinical settings. In this study, a carbapenem-resistant Raoultella ornithinolytica strain, WLK218, was isolated from urban river sediment in Zhengzhou City, Henan Province, China. This isolate was subjected to PCR and antimicrobial susceptibility testing. PCR results showed that this isolate was positive for both the blaNDM-1 and blaKPC-2 genes. The antimicrobial susceptibility testing results showed that this isolate exhibited resistance or intermediate resistance to all the antibiotics tested except for streptomycin (susceptible) and cefepime (susceptible-dose dependent). The complete genome sequence of the WLK218 isolate was then determined by using a combination of the PacBio and Illumina sequencing technologies. The de novo assembly of the genome generated one chromosome and six plasmids. Among the six plasmids, the blaNDM-1 gene was carried on the IncX3 plasmid pWLK-NDM, while the blaKPC-2 gene was located on the untypeable plasmid pWLK-KPC. This is the first report of an environmental Raoultella ornithinolytica isolate co-harbouring the blaNDM-1 and blaKPC-2 genes.
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Project description:The complete and annotated nucleotide sequence of a 54,036-bp plasmid harboring a blaKPC-2 gene that is clonally present in Citrobacter isolates from different species is presented. The plasmid belongs to incompatibility group N (IncN) and harbors the class A carbapenemase KPC-2 in a unique genetic environment.
| S-EPMC4241661 | biostudies-literature
Project description:Carbapenemase-producing Klebsiella pneumoniae emerged as a nosocomial pathogen causing morbidity and mortality in patients. For infection prevention it is important to track the spread of K. pneumoniae and its plasmids between patients. Therefore, the major aim was to recapitulate the contents and diversity of the plasmids of genetically related K. pneumoniae strains harboring the beta-lactamase gene blaKPC-2 or blaKPC-3 to determine their dissemination in the Netherlands and the former Dutch Caribbean islands from 2014 to 2019. Next-generation sequencing was combined with long-read third-generation sequencing to reconstruct 22 plasmids. wgMLST revealed five genetic clusters comprised of K. pneumoniae blaKPC-2 isolates and four clusters consisted of blaKPC-3 isolates. KpnCluster-019 blaKPC-2 isolates were found both in the Netherlands and the Caribbean islands, while blaKPC-3 cluster isolates only in the Netherlands. Each K. pneumoniae blaKPC-2 or blaKPC-3 cluster was characterized by a distinct resistome and plasmidome. However, the large and medium plasmids contained a variety of antibiotic resistance genes, conjugation machinery, cation transport systems, transposons, toxin/antitoxins, insertion sequences and prophage-related elements. The small plasmids carried genes implicated in virulence. Thus, implementing long-read plasmid sequencing analysis for K. pneumoniae surveillance provided important insights in the transmission of a KpnCluster-019 blaKPC-2 strain between the Netherlands and the Caribbean.
| S-EPMC7546619 | biostudies-literature
Project description:A bla KPC-2-carrying Citrobacter freundii isolate developed ceftazidime-avibactam resistance during treatment with this agent. The initial and follow-up isolates exhibited ceftazidime-avibactam MICs of 4 and 64?µg/ml, respectively. Overexpression of AcrAB-TolC and porin alterations were detected in both isolates, but no other resistance mechanism was observed. After passaging the initial clinical isolate in ceftazidime-avibactam at a fixed concentration of 4?µg/ml and a 4:1 ratio, resistance to all ?-lactams was noted, and a percentage of the bla KPC-2 sequencing reads had mutations leading to the alterations D176Y (bla KPC-2-D176Y [78%]) or R164S plus P147L (bla KPC-2-R164S + P147L [82%]). Further investigation of the follow-up isolate showed that 11% of the bla KPC-2 reads had mutations leading to D179Y substitution (bla KPC-2-D179Y). In the absence of selective pressure, ceftazidime-avibactam MICs of the passaged and follow-up isolates revealed that 7 or 8 out of 20 screened colonies reverted to susceptible and possessed bla KPC-2 wild-type sequences. Recombinant plasmids carrying the bla KPC-2 alterations observed were transformed in Escherichia coli, and MIC values for ceftazidime ± avibactam were elevated. Lower MICs for ceftriaxone, cefepime, aztreonam, meropenem, and imipenem for the mutated KPC-2-producing isolates were observed compared to those of the isolates producing a wild-type KPC-2. Avibactam at a fixed concentration of 4?µg/ml restored the activity of all ?-lactams tested for the recombinant strains. The heterogenous population of wild-type and mutated bla KPC-2 and the reversibility of the genotypes observed suggest a significant challenge for managing KPC-producing isolates that develop ceftazidime-avibactam resistance during therapy.IMPORTANCE The development of ceftazidime-avibactam resistance among KPC-producing isolates during treatment with this agent has been reported. Usually isolates that become resistant have a mutated bla KPC gene that confers resistance to ceftazidime-avibactam and susceptibility to meropenem. We report a Citrobacter freundii isolate that developed ceftazidime-avibactam resistance due to mutations within the coding region of the bla KPC-2 ?-loop previously reported; however, in this case, only 11% of the whole-genome sequencing reads had mutations, making this alteration difficult to detect and the treatment of these isolates more challenging. In addition to bla KPC, the initial and the follow-up patient isolates displayed hyperexpression of the AcrAB-TolC efflux system and disruption of the outer membrane protein (OMP) OmpF, which contribute to carbapenem resistance. Experiments performed to confirm our findings included generating mutant isolates from the initial patient isolate, passaging the isolates for purity in drug-free medium, resulting in a reversible phenotype, and cloning the mutations to demonstrate the resistance conferred.
| S-EPMC6158515 | biostudies-literature
Project description:Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae have become widespread in hospitals and the environment. Here, we describe a blaKPC-2-carrying plasmid called pCRE3-KPC, which was recovered from a clinical multidrug-resistant Citrobacter braakii CRE3 strain in China. The complete nucleotide sequence of pCRE3-KPC was determined by combining MiSeq and MinION sequencing and then compared with those of three related plasmids. Plasmid conjugal transfer and electroporation tests, modified carbapenem inactivation method, and bacterial antimicrobial susceptibility test were carried out. We compared this plasmid with three related plasmids to verify that the backbone of pCRE3-KPC was composed of the backbones of the IncR plasmid and IncP6 plasmid. Further bioinformatics analysis showed that pCRE3-KPC carried two resistance-related regions (the blaKPC-2 gene cluster and the aacC2-tmrB-related region). The aacC2-tmrB-related region included two novel insertion sequences (ISCfr28 and ISCfr16).IMPORTANCE Reports of human-pathogenic C. braakii strains, especially of strains showing resistance to carbapenems, are rare. To the best of our knowledge, our results represent the first detection of carbapenemase gene blaKPC-2 in C. braakii strains. In addition, we have studied detailed genetic characteristics of the novel IncR/IncP6 hybrid plasmid pCRE3-KPC, which was isolated from a clinical multidrug-resistant Citrobacter braakii CRE3 strain. Our results may provide further insight into the horizontal transfer of multidrug resistance genes in bacteria and into the genomic diversity and molecular evolution of plasmids.
| S-EPMC7096625 | biostudies-literature
Project description:While conducting genomic surveillance of carbapenemase-producing Enterobacteriaceae (CPE) from patient colonisation and clinical infections at Birmingham's Queen Elizabeth Hospital (QE), we identified an N-type plasmid lineage, pQEB1, carrying several antibiotic resistance genes, including the carbapenemase gene bla KPC-2. The pQEB1 lineage is concerning due to its conferral of multidrug resistance, its host range and apparent transmissibility, and its potential for acquiring further resistance genes. Representatives of pQEB1 were found in three sequence types (STs) of Citrobacter freundii, two STs of Enterobacter cloacae, and three species of Klebsiella. Hosts of pQEB1 were isolated from 11 different patients who stayed in various wards throughout the hospital complex over a 13 month period from January 2023 to February 2024. At present, the only representatives of the pQEB1 lineage in GenBank were carried by an Enterobacter hormaechei isolated from a blood sample at the QE in 2016 and a Klebsiella pneumoniae isolated from a urine sample at University Hospitals Coventry and Warwickshire (UHCW) in May 2023. The UHCW patient had been treated at the QE. Long-read whole-genome sequencing was performed on Oxford Nanopore R10.4.1 flow cells, facilitating comparison of complete plasmid sequences. We identified structural variants of pQEB1 and defined the molecular events responsible for them. These have included IS26-mediated inversions and acquisitions of multiple insertion sequences and transposons, including carriers of mercury or arsenic resistance genes. We found that a particular inversion variant of pQEB1 was strongly associated with the QE Liver speciality after appearing in November 2023, but was found in different specialities and wards in January/February 2024. That variant has so far been seen in five different bacterial hosts from six patients, consistent with recent and ongoing inter-host and inter-patient transmission of pQEB1 in this hospital setting.
| S-EPMC11368168 | biostudies-literature
Project description:The imipenem and meropenem-resistant strains Citrobacter freundii HS70 and Escherichia coli HS510 were isolated from patients in Shanghai, China. By isoelectric focusing, PCR amplification and sequencing, these strains were each found to produce four β-lactamases: TEM-1, KPC-3, SHV-7 and CTX-M-14. A conjugation experiment and plasmid restriction digestion revealed that the bla (KPC-3) gene was located on the same plasmid in both isolates. Bidirectional primer walking sequencing showed that the nucleotide sequence surrounding the 3.8 kb bla(KPC-3) contained a 671-bp insertion similar to that previously characterized in China. The insertion was located between the promoter and the coding region of the bla(KPC-3) gene. Susceptibility testing performed on recombinant strains carrying the bla(KPC-3) gene with or without the insertion revealed that minimum inhibitory concentrations of imipenem, meropenem, cefepime, and cefotaxime for E. coli EMU-KPC3 (without insertion) were four times higher than that of E. coli EKPC3 (with insertion). The 671 bp insertion reduced bla(KPC-3) expression significantly. Taken together, these results suggest that KPC-3-producing C. freundii and E. coli have begun to emerge in our hospital.
| S-EPMC3052496 | biostudies-literature
Project description:The ability of a bacterial population to survive in different niches, as well as in stressful and rapidly changing environmental conditions, depends greatly on its genetic content. To survive such fluctuating conditions, bacteria have evolved different mechanisms to modulate phenotypic variations and related strategies to produce high levels of genetic diversity. Laboratories working in microbiological diagnosis have shown that Citrobacter freundii is very versatile in its colony morphology, as well as in its biochemical, antigenic and pathogenic behaviours. This phenotypic versatility has made C. freundii difficult to identify and it is frequently confused with both Salmonella enterica and Escherichia coli. In order to determine the genomic events and to explain the mechanisms involved in this plasticity, six C. freundii isolates were selected from a phenotypic variation study. An I-CeuI genomic cleavage map was created and eight housekeeping genes, including 16S rRNA, were sequenced. In general, the results showed a range of both phenotypes and genotypes among the isolates with some revealing a greater similarity to C. freundii and some to S. enterica, while others were identified as phenotypic and genotypic intermediary states between the two species. The occurrence of these events in natural populations may have important implications for genomic diversification in bacterial evolution, especially when considering bacterial species boundaries. In addition, such events may have a profound impact on medical science in terms of treatment, course and outcomes of infectious diseases, evading the immune response, and understanding host-pathogen interactions.
| S-EPMC3771896 | biostudies-literature