Complete sequence of a novel 178-kilobase plasmid carrying bla(NDM-1) in a Providencia stuartii strain isolated in Afghanistan

Abstract

In response to global concerns over the spread of the New Delhi metallo-β-lactamase gene 1, bla(NDM-1), a monthly surveillance program was initiated in September 2010. All carbapenem-resistant Gram-negative strains forwarded to our facility are screened for this gene. To date, 321 carbapenem-resistant isolates, encompassing 11 bacterial species, have been tested. In February 2011, two strains of Providencia stuartii, submitted from a military hospital in Afghanistan, tested positive for bla(NDM-1). Both strains were identical by pulsed-field gel electrophoresis (PFGE). bla(NDM-1) was carried on a large plasmid, pMR0211, which was sequenced by emulsion PCR and pyrosequencing. pMR0211 is 178,277 bp in size and belongs to incompatibility group A/C. The plasmid consists of a backbone with considerable homology to pAR060302 from Escherichia coli, and it retains many of the antibiotic resistance genes associated with it. The plasmid also shares common elements with the pNDM-HK plasmid, including bla(NDM-1), armA, and sul1. However, gene orientation is reversed, and a 3-kb fragment from this region is absent from pMR0211. pMR0211 also contains additional genes, including the aminoglycoside-modifying enzyme loci aadA and aac(6'), the quinolone resistance gene qnrA, a gene with highest homology to a U32 family peptidase from Shewanella amazonensis, and the bla(OXA-10) gene. The finding of this gene in an intrinsically colistin-resistant species such as Providencia stuartii is especially worrisome, as it renders the organism resistant to nearly every available antibiotic. The presence of multiple insertion sequences and transposons flanking the region containing the bla(NDM-1) gene further highlights the potential mobility associated with this gene.

Fig 1

Structure of pMR0211. (A) Alignment of pAR060302 (4) and pNDM-HK (12) with pMR0211 showing areas of homology between all three plasmids. White shading (on pAR060302 and pNDM-HK only) indicates areas with no homology to pMR0211. Red shading indicates regions with homology to pAR060302. Green shading indicates regions with homology to pNDM-HK. Blue shading (on pMR0211 only) indicates regions with no orthologs in pAR060302 and pNDM-HK. Solid lines connecting the plasmids indicate insertion and deletion events in pMR0211 compared to the other plasmids. The sizes of the respective plasmids are indicated at the 3′ terminus. (B) Circular representation of plasmid pMR0211. Colored shading represents genes and regions with homology to pAR060302, pNDM-Hk, and pMR0211 as described above. The repA gene and putative antibiotic resistance genes with their respective orientations are indicated in the outermost region by block arrows with the same color scheme. White block arrows indicate the orientation and position of putative insertion sequence elements and transposons.

Fig 2

Structure of the bla_NDM-1 gene and surrounding regions in pMR0211 and pNDM-HK. Gene orientation is reversed in both plasmids, as indicated. A 3,036-bp deletion in pMR0211 is indicated by the dotted line on pMR0211 and corresponds to the genes shaded in black on pNDM-HK. Putative gene annotations were assigned using xBASE (www.xbase.ac.uk) (6).