title: Complete sequencing of an IncHI1 plasmid encoding the carbapenemase NDM-1, the ArmA 16S RNA methylase and a resistance-nodulation-cell division/multidrug efflux pump.
abstract: OBJECTIVE
  To characterize the pNDM-CIT plasmid identified in Citrobacter freundii carrying genes encoding the metallo-β-lactamase NDM-1 and the 16S RNA methylase ArmA.
  
  
  METHODS
  The complete DNA sequence of pNDM-CIT was obtained by using the 454-Genome Sequencer FLX procedure on a library obtained using plasmid DNA purified from the pNDM-CIT Escherichia coli J53 transconjugant. Contig assembly and predicted gaps were confirmed and filled by PCR-based gap closure. Comparative analysis with IncHI1 incompatibility group plasmids was performed using BLASTN and BLASTP algorithms.
  
  
  RESULTS
  Plasmid pNDM-CIT was 288::920 bp and revealed an IncHI1 plasmid scaffold, showing novel resistance and potential virulence determinants. The bla(NDM-1) gene was identified within a novel genetic context, flanked by a duplication of the class 1 integron on both sides. The replicase gene repAciN, originating from Acinetobacter spp. plasmids, was identified in a close association with the Tn1548::armA transposon and the macrolide resistance mel-mph2 cluster. The same structure was identified in silico from a series of enterobacterial plasmids carrying the armA gene. The repAciN gene probably represents a remnant sign of the original occurrence of the armA gene in Acinetobacter plasmids. A CP4-like prophage sequence was identified in pNDM-CIT, containing a resistance-nodulation-cell division/multidrug resistance (RND/MDR) efflux pump cluster surrounded by two IS1-like elements. This resistance determinant, associated with such a prophage sequence, has never been reported on plasmids.
  
  
  CONCLUSIONS
  Plasmid pNDM-CIT differed significantly from all known bla(NDM-1)-carrying plasmids identified in Enterobacteriaceae, since it combines the metallo-β-lactamase NDM-1, the 16S RNA methylase ArmA and a cryptic prophage carrying the RND/MDR efflux pump.
authors.affiliation: Department of Infectious,  Parasitic and Immuno-Mediated Diseases, Istituto Superiore di Sanità, Rome,  Italy.
authors.forename: Monika
authors.initials: M
authors.lastname: Dolejska
authors.forename: Laura
authors.initials: L
authors.lastname: Villa
authors.forename: Laurent
authors.initials: L
authors.lastname: Poirel
authors.forename: Patrice
authors.initials: P
authors.lastname: Nordmann
authors.forename: Alessandra
authors.initials: A
authors.lastname: Carattoli
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification; D002455:Cell Division / Q000235:genetics*; D016971:Citrobacter freundii / Q000201:enzymology / Q000235:genetics* / Q000472:pathogenicity; D024901:Drug Resistance, Multiple, Bacterial / Q000235:genetics*; D006801:Humans; D010957:Plasmids / Q000235:genetics* / Q000302:isolation & purification; D012336:RNA, Ribosomal, 16S / Q000235:genetics* / Q000302:isolation & purification; D017422:Sequence Analysis, DNA / Q000379:methods; D001618:beta-Lactamases / Q000235:genetics* / Q000302:isolation & purification; D012359:tRNA Methyltransferases / Q000235:genetics / Q000302:isolation & purification
chemical_list: D001426:Bacterial Proteins; D012336:RNA, Ribosomal, 16S; D012359:tRNA Methyltransferases; D001618:beta-Lactamases; C552864:beta-lactamase NDM-1