Genome and Plasmid Analysis of blaIMP-4-Carrying Citrobacter freundii B38

Sequencing of the blaIMP-4-carrying C. freundii B38 using the PacBio SMRT technique revealed that the genome contained a chromosome of 5,134,500 bp and three plasmids, pOZ172 (127,005 bp), pOZ181 (277,592 bp), and pOZ182 (18,467 bp). Plasmid pOZ172 was identified as IncFIIY, like pP10164-NDM and pNDM-EcGN174. It carries a class 1 integron with four cassettes (bla(IMP-4)-qacG2-aacA4-aphA15) and a complete hybrid tni module (tniR-tniQ-tniB-tniA). The recombination of tniR from Tn402 (identical) with tniQBA from Tn5053 (99%) occurred within the res site of Tn402/5053. The Tn402/5053-like integron, named Tn6017, was inserted into Tn1722 at the res II site. The replication, partitioning, and transfer systems of pOZ181 were similar to those of IncHI2 plasmids (e.g., R478) and contained a sul1-type class 1 integron with the cassette array orf-dfrA1-orfgcu37-aadA5 linked to an upstream Tn1696 tnpA-tnpR and to a downstream 3' conserved sequence (3'-CS) and ISCR1. A Tn2 transposon encoding a bla(TEM-1) beta-lactamase was identified on pOZ182. Other interesting resistance determinants encoded on the B38 chromosome included multidrug resistance (MDR) efflux pumps, an AmpC beta-lactamase, and resistances to Cu, Ag, As, and Zn. This is the first report of a complete tni module linked to a bla(IMP-4)-carrying class 1 integron, which, together with other recently reported non-sul1 integrons, represents the emergence of a distinct evolutionary lineage of class 1 integrons lacking a 3-CS (qacE Delta 1-sul1). The unique cassette array, complete tni module of Tn6017, and incompatibility group of pOZ172 suggest a blaIMP- 4 evolutionary pathway in C. freundii B38 different from that for other blaIMP- 4 genes found in Gram-negative bacteria in the Western Pacific region.