Skip to main content
PMC home page
Genome Announcements logoLink to Genome Announcements
. 2014 Nov 13;2(6):e01157-14. doi: 10.1128/genomeA.01157-14

Complete Nucleotide Sequence of a Citrobacter freundii Plasmid Carrying KPC-2 in a Unique Genetic Environment

Yancheng Yao a, Can Imirzalioglu a, Torsten Hain a, Martin Kaase b, Soeren Gatermann b, Martin Exner c, Martin Mielke d, Anja Hauri e, Yolanta Dragneva f, Rita Bill f, Constanze Wendt g, Angela Wirtz h, Eugen Domann a,✉, Trinad Chakraborty a
PMCID: PMC4241661  PMID: 25395635

Abstract

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.

GENOME ANNOUNCEMENT

Carbapenems remain the most effective antibiotics for the treatment of serious infections caused by multi-resistant Gram-negative bacteria producing extended-spectrum β-lactamases (ESBL). The rise of carbapenem-resistant Gram-negative bacteria is increasingly being reported and is now a matter of great clinical concern. Carbapenem resistance in Enterobacteriaceae is mainly due to the production of carbapenemases, the most common of which is the K. pneumoniae carbapenemase (KPC) family of enzymes (1–3).

Recently, isolation of a cluster of carbapenem-resistant Citrobacter species was reported from a single hospital environment in southern Hesse, Germany (4). Most isolates were typed as C. freundii but several isolates of carbapenem-resistant C. amalonaticus, C. braakii, and C. koseri were also detected. Citrobacter species are environmental pathogens that can colonize the intestinal tract of humans and animals. They are generally considered low-grade pathogens that rarely cause infections. However, these bacteria have been associated with a wide spectrum of infections involving the central nervous system and the gastrointestinal, urinary, and respiratory tracts (5).

Preliminary characterization revealed that all isolates harbored a nonconjugable blaKPC-2 gene. In order to determine the genetic localization of the KPC-2 we determined the genome sequence of 11 representative strains (8 C. freundii, 1 C. amalonaticus, 1 C. braakii, 1 C. koseri). DNA sequencing libraries were prepared using the Nextera XT kit (Illumina, San Diego) according to the manufacturer’s instructions. Individually tagged libraries were sequenced as a part of a flowcell as 2×300 base paired-end reads using the Illumina MiSeq platform (Illumina, San Diego). A total of 12,447,167,642 sequences were produced and the sequences from each isolate were separately assembled using CLC Genomics Workbench version 7.0.4. We identified contigs harboring blaKPC-2 by using ResFinder (http://cge.cbs.dtu.dk/services/ResFinder/) and assembled the flanking sequences to generate a closed contig comprised of 54,036 bp with 82 coding sequences (CDS) (6). Open reading frame (ORF) finding and gene annotation was done by using RAST (http://rast.nmpdr.org/) and a genetic map of the resulting contigs was generated with MAUVE (7, 8) and with the plasmid reference nucleotide sequence of pKPC_FCF/3SP (accession no. CP004367.2). Further analysis revealed that blaKPC-2 is located on an IncN plasmid and inserted in a region between the traI and traG genes (9–11). The blaKPC-2 gene is part of a 9,571-bp insertion with a unique genetic environment comprising at one end of a Tn4401 element with the ISKpn6 and Kpc-2 genes and an adjacent Tn3-like segment (12–14), harboring a blaTEM1b, ISCfr1, and aac3-IId genes flanked by a 137 bp direct repeat. All sequenced strains harbor genetically identical plasmids, suggesting its horizontal spread among the different Citrobacter species. The plasmid, derived from C. freundii isolate Cfr08698 encoding blaKPC-2, was designated pCfr-08698KPC-2.

Nucleotide sequence accession number.

The nucleotide sequence of the C. freundii plasmid carrying blaKPC-2 has been deposited in the EMBL database under accession no. LN610760.

ACKNOWLEDGMENTS

We thank Christina Gerstmann and Alexandra Amend for excellent technical assistance and Katrin Gentil for reading the manuscript.

This work was supported by grants from the German Centre of Infection Research (DZIF) to C.I. and T.C. (FKZ: 80 00 701-2 HZI).

Footnotes

Citation Yao Y, Imirzalioglu C, Hain T, Kaase M, Gatermann S, Exner M, Mielke M, Hauri A, Dragneva Y, Bill R, Wendt C, Wirtz A, Domann E, Chakraborty T. 2014. Complete nucleotide sequence of a Citrobacter freundii plasmid carrying KPC-2 in a unique genetic environment. Genome Announc. 2(6):e01157-14. doi:10.1128/genomeA.01157-14.

REFERENCES

  • 1. Munoz-Price LS, Poirel L, Bonomo RA, Schwaber MJ, Daikos GL, Cormican M, Cornaglia G, Garau J, Gniadkowski M, Hayden MK, Kumarasamy K, Livermore DM, Maya JJ, Nordmann P, Patel JB, Paterson DL, Pitout J, Villegas MV, Wang H, Woodford N, Quinn JP. 2013. Clinical epidemiology of the global expansion of Klebsiella pneumoniae carbapenemases. Lancet Infect. Dis. 13:785–796. 10.1016/S1473-3099(13)70190-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2. Nordmann P, Dortet L, Poirel L. 2012. Carbapenem resistance in Enterobacteriaceae: here is the storm!. Trends Mol. Med. 18:263–272. 10.1016/j.molmed.2012.03.003. [DOI] [PubMed] [Google Scholar]
  • 3. Tzouvelekis LS, Markogiannakis A, Psichogiou M, Tassios PT, Daikos GL. 2012. Carbapenemases in Klebsiella pneumoniae and other Enterobacteriaceae: an evolving crisis of global dimensions. Clin. Microbiol. Rev. 25:682–707. 10.1128/CMR.05035-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4. Robert-Koch-Institut 23 September 2014. Häufung von KPC-2 produzierenden Stämmen verschiedener Enterobacteriaceae-Spezies in Hessen. Epidemiolog. Bull. 10.1016/j.khinf.2014.08.009. [DOI] [Google Scholar]
  • 5. Abbott SL. 2007. Klebsiella, Enterobacter, Citrobacter, Serratia, Plesiomonas, and other Enterobacteriaceae, p 698–715 In Murray PR, Baron EJ, Jorgensen JH, Landry ML, Pfaller MA. (ed), Manual of clinical microbiology, 9th ed. ASM Press, Washington, D.C. [Google Scholar]
  • 6. Zankari E, Hasman H, Cosentino S, Vestergaard M, Rasmussen S, Lund O, Aarestrup FM, Larsen MV. 2012. Identification of acquired antimicrobial resistance genes. J. Antimicrob. Chemother. 67:2640–2644. 10.1093/jac/dks261. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7. Aziz RK, Bartels D, Best AA, DeJongh M, Disz T, Edwards RA, Formsma K, Gerdes S, Glass EM, Kubal M, Meyer F, Olsen GJ, Olson R, Osterman AL, Overbeek RA, McNeil LK, Paarmann D, Paczian T, Parrello B, Pusch GD, Reich C, Stevens R, Vassieva O, Vonstein V, Wilke A, Zagnitko O. 2008. The RAST server: Rapid Annotations using Subsystems Technology. BMC Genomics 9:75. 10.1186/1471-2164-9-75. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8. Darling AE, Mau B, Perna NT. 2010. progressiveMauve: multiple genome alignment with gene gain, loss, and rearrangement. PLoS One 5:e11147. 10.1371/journal.pone.0011147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9. Andrade LN, Curiao T, Ferreira JC, Longo JM, Clímaco EC, Martinez R, Bellissimo-Rodriguez F, Basile-Filho A, Evaristo MA, Del Peloso PF, Ribeiro VB, Barth AL, Paula MC, Baquero F, Cantón R, da Costa Darini AL, Coque TM. 2011. Dissemination of blaKPC-2 by the spread of Klebsiella pneumoniae clonal complex 258 clones (ST258, ST11, ST437) and plasmids (IncFII, IncN, IncL/M) among Enterobacteriaceae species in Brazil. Antimicrob. Agents Chemother. 55:3579–3583. 10.1128/AAC.01783-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10. Chmelnitsky I, Shklyar M, Leavitt A, Sadovsky E, Navon-Venezia S, Ben Dalak M, Edgar R, Carmeli Y. 2014. Mix and match of KPC-2 encoding plasmids in Enterobacteriaceae-comparative genomics. Diagn. Microbiol. Infect. Dis. 79:255–260. 10.1016/j.diagmicrobio.2014.03.008. [DOI] [PubMed] [Google Scholar]
  • 11. Eikmeyer F, Hadiati A, Szczepanowski R, Wibberg D, Schneiker-Bekel S, Rogers LM, Brown CJ, Top EM, Pühler A, Schlüter A. 2012. The complete genome sequences of four new IncN plasmids from wastewater treatment plant effluent provide new insights into IncN plasmid diversity and evolution. Plasmid 68:13–24. 10.1016/j.plasmid.2012.01.011. [DOI] [PubMed] [Google Scholar]
  • 12. Cain AK, Hall RM. 2012. Evolution of IncHI2 plasmids via acquisition of transposons carrying antibiotic resistance determinants. J. Antimicrob. Chemother. 67:1121–1127. 10.1093/jac/dks004. [DOI] [PubMed] [Google Scholar]
  • 13. Cuzon G, Naas T, Nordmann P. 2011. Functional characterization of Tn4401, a Tn3-based transposon involved in blaKPC gene mobilization. Antimicrob. Agents Chemother. 55:5370–5373. 10.1128/AAC.05202-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14. Pérez-Chaparro PJ, Cerdeira LT, Queiroz MG, de Lima CP, Levy CE, Pavez M, Lincopan N, Gonçalves EC, Mamizuka EM, Sampaio JL, Nunes MR, McCulloch JA. 2014. Complete nucleotide sequences of two blaKPC-2-bearing IncN Plasmids isolated from sequence type 442 Klebsiella pneumoniae clinical strains four years apart. Antimicrob. Agents Chemother. 58:2958–2960. 10.1128/AAC.02341-13. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Genome Announcements are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES