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Antimicrobial Agents and Chemotherapy, November 2007, p. 3844-3852, Vol. 51, No. 11
0066-4804/07/$08.00+0     doi:10.1128/AAC.01512-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

An OXA-66/OXA-51-Like Carbapenemase and Possibly an Efflux Pump Are Associated with Resistance to Imipenem in Acinetobacter baumannii ^,

Department of Biotechnology and Laboratory Science in Medicine, School of Biomedical Science and Engineering,¹ Institute of Tropical Medicine, and Department of Parasitology, School of Medicine, National Yang-Ming University,² Division of Infectious Diseases, Department of Medicine, Mackay Memorial Hospital, Taipei, Taiwan³

Received 1 December 2006/ Returned for modification 8 January 2007/ Accepted 15 August 2007

We investigated the mechanisms involved in imipenem resistance in 23 clinical strains of Acinetobacter baumannii. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed the presence of a 30-kDa protein in imipenem-intermediate A. baumannii (IIAB) and imipenem-resistant A. baumannii (IRAB) strains; this protein was almost undetectable in imipenem-susceptible A. baumannii (ISAB) strains. The 30-kDa protein was identified as an OXA-51-like carbapenemase using two-dimensional gel electrophoresis and mass spectrometry. Similar to other recent findings, bla_OXA-51-like genes were found to exist in all 23 clinical strains; however, the transcript levels of bla_OXA-51-like in the IIAB and IRAB were higher than in the ISAB strains using reverse transcriptase PCR (RT-PCR) and real-time RT-PCR assays. This change was due to the presence of an insertion sequence, ISAba1, upstream of bla_OXA-51-like in the IIAB and IRAB strains that was not present in the ISAB strains. The introduction of bla_OXA-66 (a bla_OXA-51-like gene), identified in ISAB ab1254 and IRAB ab1266, into Escherichia coli TOP10 cells resulted in 3.95-fold and 7.90-fold elevations in resistance to imipenem, respectively. Furthermore, when ISAB ab8 and ISAB ab1254 and their in vitro-selected imipenem-resistant mutants ISAB ab8(r) and ISAB ab1254(r) were compared, the results showed no change in the bla_OXA-66/bla_OXA-51-like gene sequences, in expression of the gene, and in the outer membrane protein profiles. However, there was a four- to eightfold reduction in imipenem resistance upon adding carbonyl cyanide m-chlorophenylhydrazone. Taken together, these results suggest that the OXA-66/OXA-51-like carbapenemase contributes to intrinsic resistance to imipenem; however, drug export by an efflux pump may be more important and/or occur more frequently in imipenem-resistant A. baumannii. Furthermore, this is the first report of a Taiwanese strain of an OXA-66/OXA-51-like carbapenemase that confers imipenem resistance in A. baumannii.

Published ahead of print on 27 August 2007.

Supplemental material for this article may be found at http://aac.asm.org/.

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