LAUSR

OBJECTIVES To analyse plasmids carrying blaIMP-6 in isolates of Klebsiella pneumoniae obtained from multicentre carbapenem-resistant Enterobacteriaceae surveillance. METHODS We characterised plasmids harbouring blaIMP-6 by the whole-genome sequencing of four K. pneumoniae isolates carrying blaIMP-6, and compared them with the pKPI-6 plasmid widespread in western Japan through PFGE, Southern blotting, bacterial conjugation, and qPCR. RESULTS Whole-genome sequencing analysis revealed that three of the four isolates carried approximately 50 kbp plasmids similar to the pKPI-6 plasmid, however, one isolate carried a 250 kbp plasmid harbouring blaIMP-6 (pE196_IMP6). So far, all of the reported plasmids carrying blaIMP-6 were similar to the pKPI-6 plasmid, and this plasmid was a novel blaIMP6-carrier. The size and transferability of this plasmid was confirmed by Southern hybridisation and conjugation experiments. We demonstrated that the generation of plasmid pE196_IMP6 was due to an intramolecular transposition mediated by IS26, and a homologous recombination between plasmids pKPI-6 and pE013 that was obtained from another carbapenem-resistant Enterobacteriaceae isolate in this analysis. As a result of co-integration with pE013, pE196_IMP6 acquired six additional pairs of type II toxin-antitoxin systems that pKPI-6 does not carry. Transcription of all of the toxin-antitoxin systems were confirmed in an isolate carrying pE196_IMP6 by qPCR. CONCLUSIONS We detected a novel plasmid carrying blaIMP-6, and revealed the origin of this plasmid. Toxin-antitoxin system acquisition could enable pE196_IMP6 maintenance persistently through successions, even without selection pressure by the clinical usage of antimicrobials, generating broad dissemination and longer carbapenem-resistant Enterobacteriaceae colonisation duration in patients.