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

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Protocol publication

[…] 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 ().Recently, isolation of a cluster of carbapenem-resistant Citrobacter species was reported from a single hospital environment in southern Hesse, Germany (). 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 ().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) (). 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 (, ) 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 (). 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 (), 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. […]

Pipeline specifications

Software tools CLC Genomics Workbench, RAST, Mauve
Databases NMPDR
Application Nucleotide sequence alignment
Organisms Citrobacter freundii