Genetic characterization of a novel blaDIM-2-carrying megaplasmid p12969-DIM from clinical Pseudomonas putida

Abstract

Objectives: To characterize a blaDIM-2-carrying 409 kb megaplasmid p12969-DIM of Pseudomonas putida 12969 from a patient with pneumonia in China. Methods: The complete nucleotide sequence of p12969-DIM was determined with a paired-end library and a mate-pair library using next-generation sequencing technology. Results: BlaDIM-2, a close blaDIM-1 variant, was identified in p12969-DIM. DIM-2 differs from DIM-1 by two amino acid substitutions Ser119Leu and Ser209Pro. The p12969-DIM backbone is highly similar to pOZ176, but the IncP-2-type stability/replication/conjugal transfer system in the pOZ176 backbone is absent from p12969-DIM. The p12969-DIM accessory regions, a 45.7 kb MDR and a novel insertion sequence, ISPpu23, are almost entirely distinct from pOZ176. The MDR region contains a novel Tn21-subgroup transposon Tn6286 inserted with two class 1 integrons, In1225 and In1226; a Tn5503-family transposon-like element inserted with a strAB locus; and a novel Tn21-subgroup transposon-like element inserted with a class 1 integron, In1224. The three integrons carry blaDIM-2 as well as a number of additional genes conferring resistance to quinolones, aminoglycosides, chloramphenicol, florfenicol, trimethoprim, streptomycin, quaternary ammonium compounds and sulphonamides. p12969-DIM has two distinct replication/stability systems, repA/parAB-parB2 of an unknown incompatibility group in the backbone and repABC/mazFE of the IncQ2 group in the MDR region. Conclusions: The MDR region of p12969-DIM harbours many resistance genes as well as a second replication/stability system. This article is the first report of a fully sequenced blaDIM-carrying plasmid.