title: An OXA-66/OXA-51-like carbapenemase and possibly an efflux pump are associated with resistance to imipenem in Acinetobacter baumannii.
abstract: 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.
authors.affiliation: Department of Biotechnology and Laboratory Science in Medicine, School of Biomedical Science and Engineering, National Yang-Ming University, 155 Li-Nong St., Sec. 2, Peitou, Taipei 112, Taiwan. huws@ym.edu.tw
authors.forename: Wensi S
authors.initials: WS
authors.lastname: Hu
authors.forename: Shu-Man
authors.initials: SM
authors.lastname: Yao
authors.forename: Chang-Phone
authors.initials: CP
authors.lastname: Fung
authors.forename: Yi-Ping
authors.initials: YP
authors.lastname: Hsieh
authors.forename: Chang-Pan
authors.initials: CP
authors.lastname: Liu
authors.forename: Jing-Fang
authors.initials: JF
authors.lastname: Lin
mesh_terms: D000151:Acinetobacter Infections / Q000382:microbiology; D040981:Acinetobacter baumannii / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D024881:Drug Resistance, Bacterial / Q000235:genetics; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D006801:Humans; D015378:Imipenem / Q000494:pharmacology*; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D009154:Mutation; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012689:Sequence Homology, Nucleic Acid; D001618:beta-Lactamases / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D012333:RNA, Messenger; D015378:Imipenem; D001618:beta-Lactamases; C063912:carbapenemase