Complete nucleotide sequence of the IncN plasmid encoding IMP-6 and CTX-M-2 from emerging carbapenem-resistant Enterobacteriaceae in Japan

Abstract

We have determined the DNA sequence of Klebsiella pneumoniae multidrug resistance plasmid pKPI-6, which is a self-transmissible IncN-type plasmid. pKPI-6 harboring blaIMP-6 and blaCTX-M-2 confers a stealth-type carbapenem resistance phenotype on members of the family Enterobacteriaceae that is not detectable with imipenem. pKPI-6 is already epidemic in Japan, favoring the dissemination of IMP-6 and CTX-M-2 in members of the family Enterobacteriaceae.

FIG 1

Structural comparison of pKPI-6, pKP96, and pN3. The nucleotide sequence of pKPI-6 plasmid DNA purified from a transconjugant was determined by the random shotgun sequencing method as described previously (15). Collected sequences were assembled by using Phrap software (version 1.080730) (16). Gaps were closed by direct sequencing of PCR products amplified with oligonucleotide primers designed to anneal each end to the neighboring contigs. Sequence reads were processed by using MetaGeneAnnotator (17), the InSilico molecular cloning software package, genomics edition (InSilico Biology Inc., Yokohama, Japan), the program BLASTP (18), and GenomeMatcher software (19). Color shadings between plasmids indicate homologous regions. Sequences of the terminal direct repeats of the acquired region are shown. ORFs are represented by pentagons, and annotated genes are colored on the basis of predicted gene function as follows: antimicrobial resistance genes, red; conjugation genes, green; transposons, yellow; integrons, orange; plasmid maintenance genes, violet; hypothetical genes, blue. Two regions, segments of Tn1721 and bla_CTX-M-2-ISEcp1, are visually extended at the bottom to show the difference between pKPI-6 and pKP96. Boxed nucleotide sequences represent direct duplications. Comparative analysis and generation of the image shown were performed with GenomeMatcher software (19). IRL, left inverted repeat; IRR, right inverted repeat.

FIG 2

Schematic representation of the genetic content of the bla_IMP-6 integron (In722) lacking ISKpn22. The attI1 and attC elements are shown as triangles. 5′CS and 3′CS represent the 5′ and 3′ conserved segments, respectively.

FIG 3

Summary of the PCR scanning analysis of IMP-6-positive plasmids from 13 K. pneumoniae, 1 K. oxytoca, and 6 E. coli clinical strains from the Kinki region. The plasmid structures in clinical isolates were examined by PCR scanning with 16 sets of primers (numbered 1 to 16; see Table S2 in the supplemental material) designed to cover all of plasmid pKPI-6. PCR amplification for the scan of pKPI-6 was performed with Quick Taq HS DyeMix (Toyobo, Tokyo, Japan) with 30 cycles of denaturing at 96°C for 20 s, annealing at 50°C for 30 s, and polymerization at 68°C for 5 min. By comparing the length of each amplified fragment to pKPI-6, the regional heterogeneity was determined. The gene arrangement of pKPI-6 is presented at the top. PCR products covering the entire plasmid sequence are depicted as numbered gray squares. The numbers represent the primer sets used for PCR scanning (see Table S2 in the supplemental material). Solid lines represent the regions amplified. Where the length of the amplified fragment is different from that of pKPI-6, the estimated size of the fragment is shown in parentheses. If no PCR product was obtained when a prolonged extension time was used, no estimated size is shown.