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

Objectives: 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.