Plasmid And Chromosomal Integration Of Four Novel Bla_IMP-carrying Transposons

Objectives:This study intends to identify the genetic characterization of four novel bla_IMP-carrying transposons from Pseudomonas aeruginosa,Klebsiella pneumoniae,and Enterobacter spp.,and to explore the diversity of plasmid and chromosomal integration of transposons and the regularity and complexity of evolutionary processes.Methods:Four clinically isolated multi-drug resistant?MDR?strains,namely P.aeruginosa 60512,K.pneumoniae 447,P.aeruginosa 12939 and Enterobacter spp.A1137,which produced carbapenemase IMP,were selected for further analysis.The species of the strains were distinguished using 16S rRNA gene sequencing,the identification of species specific markers and the phylogenetic tree.Activities of class A/B/D carbapenemases in bacterial cell extracts were determined via a modified Carba NP test.The major carbapenemase genes were screened for by PCR amplification.Genomic DNA was isolated from each of the 60512,12939 and A1137isolates using a Qiagen blood&cell culture DNA maxi kit and plasmid DNA was isolated from the 447-IMP-EC600 transconjugant?corresponding to the 447 isolate?using a Qiagen Large Construct Kit.The genomic DNA of strain 60512 and the plasmid DNA of strain 447 were sequenced from a mate-pair library using a MiSeq sequencer.For 12939 and A1137,genome sequencing was performed with a sheared DNA library on a PacBio RSII sequencer.Conjugal transfer or electroporation experiments were carried out with the rifampin-resistant Escherichia coli EC600.Bacterial antimicrobial susceptibilities were tested by BioMérieux VITEK 2 and interpreted as per Clinical and Laboratory Standards Institute?CLSI?guidelines^[1].Sequence annotation and comparison were performed using bioinformatics of each of plasmids and the drug-resistance regions of the chromosomes.Results:P.aeruginosa 60512,K.pneumoniae 447,P.aeruginosa 12939 and Enterobacter spp.A1137,which produced carbapenemase IMP,were subjected to genome sequencing.The complete nucleotide sequences of the plasmids p60512-IMP?GenBank accession number MF344578?from the 60512 isolate and p447-IMP?GenBank accession number KY978631?from the 447 isolate and those of the chromosomes of strains 12939?GenBank accession number CP024477?and A1137?GenBank accession number CP021851?were determined,and four novel bla_IMP-carrying transposons Tn6394,Tn6375,Tn6411 and Tn6397 with related sequences were performed.Tn6394 and Tn6375 were located in p60512-IMP and p447-IMP,respectively,while Tn6411 and Tn6397 were integrated into the chromosomes of 12939 and A1137,respectively.Tn6394 was an ISPa17-based transposition unit that harbored bla_IMP-1-carrying In992.Tn6375 and Tn6411,which belonged to the Tn3-and Tn7-family unit transposons,harbored In73(carrying bla_IMP-8)and In992,respectively.Tn6397 was a large integrative and conjugative element carrying Tn6378.Tn6378,the same as Tn6375,also carried class 1 integron In73.Plasmid p447-IMP and Tn6397 could be transferred into strain EC600 through conjugation,generatingE.colitransconjugants447-IMP-EC600and A1137-IMP-EC600,respectively,while p60512-IMP could be transferred into strain EC600 by electroporation,yielding the electroporant 60512-IMP-EC600.ALL the wild-type strains and the related transconjugants or electroporant produced carbapenemae IMP,and were resistant to ampicillin,ceftazidime and meropenem.Conclusions:Complex transposition and homologue recombination events have occurred during original formation and further plasmid and chromosomal integration of these four transposons,which were the important vectors carrying multiple resistance elements,and will promote accumulation and spread of antimicrobial resistance strains in the world.