Complete sequencing of an IncFII NDM-1 plasmid in Klebsiella pneumoniae shows structural features shared with other multidrug resistance plasmids
2013
Sir, Since the first report of New Delhi metallo-b-lactamase 1 (NDM-1) in 2009, it has spread worldwide in Europe, America, Africa, Asia andAustralia. ThefirstcasewasaSwedishpatientwhohadpreviously been treated in India. Retrospective analysis of carbapenemaseproducing Enterobacteriaceaetraced the blaNDM-1 geneto Escherichia coli collected from an Indian hospital in 2006. Recently, blaNDM-1producing bacteria were identified from the environment in New Delhi and Vietnam. Also, the blaNDM-1 gene has been found on a mobile plasmid in an Acinetobacter lwoffii isolate from chicken meat. We reported the first case of NDM-1-producing E. coli in Japan from a patient who had been treated in India in 2009. Subsequently, we determined the complete sequence of the IncA/C blaNDM-1positive plasmid (pNDM-1_Dok01), which suggested a possible origin from plant pathogens such as Pseudoxanthomonas and Xanthomonas spp. Here, we report another NDM-1 plasmid, in Klebsiella pneumoniae isolated from a second Japanese patient, who acquired the blaNDM-1-producing organism locally in Japan. From 15 September 2010 to 28 December 2010 the Ministry of Health, Labour and Welfare of the Japanese government conducted nationwide surveillance of multidrug-resistant Enterobacteriaceae that showed resistance to three classes of antibiotics: carbapenems, fluoroquinolones and aminoglycosides. A woman aged over 90 years, who resided in a nursing home and had not travelled overseas, was admitted to a local hospital due to aspiration pneumonia. Multidrug-resistant K. pneumoniae NDM-1-Saitama, which is susceptible only to aztreonam, was isolated from her urine, and subsequent PCR analysis revealed the presence of the blaNDM-1 gene. Multilocus sequence typing (http://www.pasteur.fr/ recherche/genotype/PF8/mlst/Kpneumoniae.html) indicated that NDM-1-Saitama belongs to sequence type ST42, which had not been reported previously. Complete sequencing of the plasmid from the clinical isolate K. pneumoniae NDM-1-Saitama by Illumina Hiseq (San Diego, USA) and gap closing by PCR demonstrated a 49441 bp IncFII plasmid (DDBJ/EMBL/GenBank accession number AB759690) (Figure 1 and Table S1, available as Supplementary data at JAC Online) that bears 53 genes predicted by EMBOSS GetORF software (http://emboss.sourceforge.net/). This plasmid did not possess any transfer operon and was not transferable by conjugation. Replicon typing showed IncFII (K1:A-:B-). The repA and repB genes were located from nucleotide 7776 to nucleotide 13111, which showed 98% identity with the IncFII (K1:A-:B-) pKPN4 plasmid in K. pneumoniae MGH 78578 (accession number CP000649). The plasmid pNDM-1-Saitama consists of a 22 kb backbone and a 27 kb multidrug resistance region. Half of the backbone (nucleotides 1–10815) showed a 100% perfect match with the sequence of K. pneumoniae plasmid pKP048 that contains blaKPC-2. 8 The second half of the backbone (nucleotides 10816–22344) showed only partial similarity to the plasmids containing blaNDM-1 (pNDM-OM, pNDM-HK, pNDM-CIT, pKOX_R1 and pMR0211) and blaCTX-M-3 (pCTX-M3) (Figure S1, available as Supplementary data at JAC Online). The multidrug resistance region of pNDM-1-Saitama (nucleotides 22345–49441) contains 13 drug resistance elements, including blaNDM-1, bleMBL, DblaDHA-1, sul1, armA, mel and mph2 (Figure 1). The complete sequence of an ISAba125 insertion sequence upstream of the blaNDM-1 gene suggests horizontal transfer of the blaNDM-1 gene between Enterobacteriaceae and Acinetobacter. The 235 sequences and 210 sequences also provide a promoter for NDM-1 expression. Interestingly, a part of the multidrug resistance region (nucleotides 30585–49333) was bracketed by insertion sequences ISAba125 and IS26 at both ends. This region is 100% identical to IncL/M plasmids pNMD-HK (accession number HQ451074) and pNDM-OM (accession number JX988621), except that the ISAba125 and IS26 insertion sequences are complete with inverted repeats in pNDM-1-Saitama. This composite transposon structure suggests that recombination events took place between two different transposons for the acquisition of these multidrug resistance genes. The blaNDM-1 gene followed by the bleomycin resistance protein gene bleMBL was acquired from transposon Tn125 with the ISAba125 insertion sequence, while truncated class C b-lactamase DblaDHA-1, dihydropteroate synthase sul1, 16S rRNA methylase armA, macrolide efflux protein mel and macrolide 2′-phophotransferase mph2 were provided by another transposon, Tn1548, with the IS26 insertion sequence. The blaNDM-1-bleMBL region is also found in another IncFII plasmid, pGUE-NDM, (accession number JQ364967) and IncN2 plasmids p271A (accession number JF785549) and pTR3 (accession number JQ349086). The DblaDHA-1-sul1-armA-mel-mph2 region was provided by the Tn1548 transposon found in KPC-bearing
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