The carbapenem resistance gene blaOXA-23 is disseminated by a conjugative plasmid containing the novel transposon Tn6681 in Acinetobacter johnsonii M19

Abstract

Background: Carbapenem resistant Acinetobacter species have caused great difficulties in clinical therapy in the worldwide. Here we describe an Acinetobacter johnsonii M19 with a novel bla_OXA-23 containing transposon Tn6681 on the conjugative plasmid pFM-M19 and the ability to transferand carbapenem resistance.

Methods: A. johnsonii M19 was isolated under selection with 8 mg/L meropenem from hospital sewage, and the minimum inhibitory concentrations (MICs) for the representative carbapenems imipenem, meropenem and ertapenem were determined. The genome of A. johnsonii M19 was sequenced by PacBio RS II and Illumina HiSeq 4000 platforms. A homologous model of OXA-23 was generated, and molecular docking models with imipenem, meropenem and ertapenem were constructed by Discovery Studio 2.0. Type IV secretion system and conjugation elements were identified by the Pathosystems Resource Integration Center (PATRIC) server and the oriTfinder. Mating experiments were performed to evaluate transfer of OXA-23 to Escherichia coli 25DN.

Results: MICs of A. johnsonii M19 for imipenem, meropenem and ertapenem were 128 mg/L, 48 mg/L and 24 mg/L, respectively. Genome sequencing identified plasmid pFM-M19, which harbours the carbapenem resistance gene bla_OXA-23 within the novel transposon Tn6681. Molecular docking analysis indicated that the elongated hydrophobic tunnel of OXA-23 provides a hydrophobic environment and that Lys-216, Thr-217, Met-221 and Arg-259 were the conserved amino acids bound to imipenem, meropenem and ertapenem. Furthermore, pFM-M19 could transfer bla_OXA-23 to E. coli 25DN by conjugation, resulting in carbapenem-resistant transconjugants.

Conclusions: Our investigation showed that A. johnsonii M19 is a source and disseminator of bla_OXA-23 and carbapenem resistance. The ability to transfer bla_OXA-23 to other species by the conjugative plasmid pFM-M19 raises the risk of spread of carbapenem resistance. The carbapenem resistance gene bla_OXA-23 is disseminated by a conjugative plasmid containing the novel transposon Tn6681 in Acinetobacter johnsonii M19.

Keywords: Acinetobacter johnsonii; Carbapenem resistance; Conjugative plasmid; Novel transposon Tn6681; blaOXA-23.

Fig. 1

Neighbor-joining tree generated on the basis of 16S rDNA gene sequences and showing the relationship of M19 to other Acinetobacter species. Bootstrap values are shown as percentages of 1000 replicates when those values were greater than 50%. The scale bar represents 0.5% substitution per nucleotide position

Fig. 2

Tertiary structure modelling of A. johnsonii M19 OXA-23 with carbapenems. a Molecular docking models showing embedding of carbapenems in the OXA-23 cavity. b Hydrophobicity of OXA-23 surface and hydrophobic tunnel. Meropenem, imipenem and ertapenem are represented, respectively, by red, blue and green stick models

Fig. 3

Linear comparison of structures harbouring bla_OXA-23. IRR, right inverted repeat; IRL, left inverted repeat. All block arrows indicate length and orientation. The numbers in each block arrow or box represent the nucleotide sequence identity compared with Tn6681. The distance between ISAba1 or ΔISAba1 and the bla_OXA-23 start codon is indicated by the number of base pairs below a curved arrow. Direct repeat sequences are indicated

Fig. 4

Sequence comparisons of the ISAba1 junction upstream of bla_OXA-23. a Sequence alignment of left inverted repeat (IRL) of ISAba14_L and right inverted repeat (IRR) of ISAba14_R. b Sequence comparisons of the ISAba1 junctions upstream of bla_OXA-23 genes. The start codon of the bla_OXA-23 gene is underlined. The − 35 and extended − 10 regions of the promoter are marked

Fig. 5

Schematic representation of plasmid pFM-M19 and amplification of bla_OXA-23 and specific region of pFM–M19 from six transconjugant. a The asterisk on ISAba1 indicates a truncated gene. b, c M, DNA marker. The following templates were used in PCR: Lanes 1, plasmid fragments extracted from M19; Lanes 2–7, plasmid fragments extracted from the six transconjugants MAT-1 to MAT-6; Lanes 8, genome of 25DN

Fig. 6

Proposed pathway for transfer of the bla_OXA-23 gene and dissemination of carbapenem resistance from A. johnsonii M19