Contribution of a plasmid-borne blaOXA-58 gene with its hybrid promoter provided by IS1006 and an ISAba3-like element to beta-lactam resistance in acinetobacter genomic species 13TU

Abstract

The contribution of the blaOXA-58 gene and its promoter to beta-lactam resistance has not been validated in Acinetobacter spp. other than Acinetobacter baumannii. We identified a multidrug-resistant (including carbapenem resistance) Acinetobacter genomic species 13TU in which blaOXA-58 was the only detected carbapenemase gene. The blaOXA-58 gene was plasmid located, flanked by ISAba3 (downstream) and an ISAba3-like element (upstream). An IS1006 element was inserted into ISAba3-like (IS1006-DeltaISAba3-like) to generate a hybrid promoter for blaOXA-58, with a -35 promoter located in IS1006 and a -10 promoter in ISAba3-like. The reference strain of Acinetobacter genomic species 13TU, ATCC 17903, revealed higher MICs of amoxicillin, ticarcillin, and piperacillin and heteroresistance to imipenem and meropenem when it was transformed with a shuttle vector containing a fragment encompassing DeltaISAba3-like-blaOXA-58, compared to the same host containing only blaOXA-58. When the fragment was changed from DeltaISAba3-like-blaOXA-58 to IS1006-DeltaISAba3-like-blaOXA-58, the ATCC 17903 transformant revealed a markedly higher level of blaOXA-58 transcription (12-fold), increased cefuroxime and piperacillin-tazobactam MICs, and homoresistance to imipenem and meropenem. Different roles of the insertion elements preceding the blaOXA-58 gene in Acinetobacter genomic species 13TU are demonstrated. The ISAba3-like--blaOXA-58 construct can mediate resistance to penicillin derivatives but only heteroresistance to carbapenems. The insertion of IS1006 into ISAba3-like, generating a hybrid promoter, could further enhance the transcription of blaOXA-58 and mediate homoresistance to carbapenems and also enhanced resistance to piperacillin-tazobactam.

FIG. 1.

Identification of the plasmid localization of bla_OXA-58 by I-CeuI mapping. (A) Pulsed-field gel electrophoresis of I-CeuI-restricted TVICU14 fragments. Hybridization of the fragments with a 16S rRNA probe (B) or bla_OXA-58 probe (C). Lanes 1, lambda ladder PFGE marker; lanes 2, DNA molecular weight marker II (DIG-labeled); lanes 3, I-CeuI-restricted fragments of TVICU14.

FIG. 2.

Schematic map of the partial genetic organization of pTVICU14. (A) Linear map of partial pTVICU14 with relevant features. The arrows indicate the predicted ORFs and their transcriptional directions. The names of various features are indicated below or above the map. The locations of the HpaI and EcoRV digestion sites are marked. The thick vertical lines indicate the previously described Re27 recombination point (28). Sequences in the fragments marked in Roman numerals were almost identical to those found in previously sequenced A. baumannii plasmids. (B) Regions of high similarity with other sequences reported in the GenBank database are indicated by a continuous line. (C) The details of predicted proteins. The figure is not to scale.

FIG. 3.

Transcript start site and promoter region of the bla_OXA-58 gene predicted based on results of 5′ RACE PCR and sequence analysis. The asterisk denotes the initiation site of the transcript. The boxed characters are potential promoters. The −10 promoter is provided by the tnpA of the ΔISAba3-like element, but the −35 promoter is provided by the right inverted repeat of IS1006. An 8-bp direct repeat generated during the IS1006 insertion is in boldface. Gray blocked characters are genes coding for the transposase tnpA of IS1006, the ΔISAba3-like element, and bla_OXA-58.

FIG. 4.

Imipenem Etest results of the transformants of ATCC 17903 bearing different recombinant plasmids: ATCC 17903(pOXA-58-5) that harbors IS1006-ΔISAba3-like-bla_OXA-58 (A), ATCC 17903(pOXA-58-6) that harbors ΔISAba3-like-bla_OXA-58 (B), and ATCC 17903(pOXA-58-7) that contains only bla_OXA-58 (C).