Dataset Information

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.

SUBMITTER: Hu WS

PROVIDER: S-EPMC2151406 | biostudies-literature | 2007 Nov

REPOSITORIES: biostudies-literature

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An OXA-66/OXA-51-like carbapenemase and possibly an efflux pump are associated with resistance to imipenem in Acinetobacter baumannii.

Hu Wensi S WS Yao Shu-Man SM Fung Chang-Phone CP Hsieh Yi-Ping YP Liu Chang-Pan CP Lin Jing-Fang JF

Antimicrobial agents and chemotherapy 20070827 11

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-dimen ...[more]

PMID: 17724156

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Project description:Purpose:A novel variant of OXA-23, named OXA-423, was identified in an Acinetobacter baumannii clinical isolate. The aim of this study was to analyse the resistance phenotype of OXA-423. Methods:The A. baumannii strain WY-0713 was isolated from an intensive care unit patient. PCR was used to detect the bla OXA-23-like genes. Amplifying, cloning and sequencing were performed for the complete bla OXA-23-like. The novel bla OXA-423 and its ancestor bla OXA-23 were cloned into the expression vector pET-28b(+), and transformed into E. coli Rosetta (DE3) for antibiotic susceptibility testing. SDS-PAGE, modified Hodge test and CarbaNP test were used for detecting the expression of OXA-423 and OXA-23. Results:PCR screening of A. baumannii WY-0713 was positive for bla OXA-23-like genes. Sequencing of the PCR product identi?ed a novel bla OXA-23-like, named bla OXA-423 which encoding OXA-423. OXA-423 differed from OXA-23 by a crucial amino acid substitution (Val128Ala). The V128A substitution was located at the conserved active-site motifs SAV of OXA-23. Antibiotic susceptibility testing performed using isogenic E. coli showed that the MICs of E. coli Rosetta (pET-OXA-423) for penicillins and carbapenems were lower (reduced MICs 4-fold to 16-fold) than that of E. coli Rosetta (pET-OXA-23). The MICs of cefotaxime, ceftazidime and aztreonam for both transformants remained the same as the acceptor strain. Moreover, OXA-423 was slightly inhibited by sulbactam, clavulanic acid and tazobactam. SDS-PAGE analysis showed that OXA-423 and OXA-23 were conspicuously expressed. Modified Hodge test and CarbaNP test were positive demonstrated both of them were functional. Conclusion:OXA-423, the first report of an amino acid substitution located at conserved active-site motifs of OXA-23, conferred lower MIC values of penicillins and carbapenems as compared with OXA-23, while without affecting the resistance pro?les of expanded-spectrum cephalosporins and aztreonam.

Project description:IntroductionMultidrug-resistant Acinetobacter baumannii (A. baumannii) has become one of the greatest threats worldwide to the therapeutic management of infections. Our previous research confirmed an in vitro synergistic effect of amlodipine and imipenem against A. baumannii, and this study is designed to understand its mechanism.MethodsSixty-four non-duplicate A. baumannii isolates were collected and tested for antimicrobial susceptibility by the disk diffusion method. PCR amplification and sequencing were used to identify the presence of the adeB, adeE, adeH, adeJ, abeM and abeS efflux pump genes. The minimal inhibitory concentrations of imipenem, imipenem+amlodipine and imipenem+carbonyl cyanide m-chlorophenyl-hydrazone against these isolates were also determined by the broth microdilution method before and after siRNA silencing of the expression of the adeABC efflux pump.ResultsIn this study, the combination of amlodipine with imipenem showed synergistic antimicrobial activity against sixty-four A. baumannii isolates when compared with the activity of imipenem alone (p<0.025). In the multidrug-resistant group, AML was more effective than carbonyl cyanide m-chlorophenyl-hydrazone (p<0.001). The efflux pump genes adeB, adeE, adeH, adeJ, abeM and abeS were detected in 100% (4/64), 75% (48/64), 0% (0/64), 100% (64/64), 96.9% (62/64) and 96.9% (62/64) of the sixty-four A. baumannii isolates, respectively. The expression of the adeABC efflux pump genes in the multidrug-resistant group (5.05±19.25) is clearly higher than in the non-multidrug-resistant group (0.17±0.20), (p = 0.01). A gene silencing test verified that the mRNA expression levels of adeABC were decreased at 12 h and increased at 24 h, while the reversal of imipenem resistance by amlodipine disappeared at 12 h and reappeared at 24 h.ConclusionsThe combination of amlodipine with imipenem exhibits an in vitro synergistic antimicrobial effect on multidrug-resistant A. baumannii, which may be due to the inhibition of the AdeABC efflux pump.

Dataset Information

An OXA-66/OXA-51-like carbapenemase and possibly an efflux pump are associated with resistance to imipenem in Acinetobacter baumannii.

Publications

An OXA-66/OXA-51-like carbapenemase and possibly an efflux pump are associated with resistance to imipenem in Acinetobacter baumannii.

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