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Mechanisms of Resistance

Mechanisms of Azole Resistance in Petite Mutants of Candida glabrata

Sophie Brun, Thierry Bergès, Pascal Poupard, Carole Vauzelle-Moreau, Gilles Renier, Dominique Chabasse, Jean-Philippe Bouchara
Sophie Brun
1Groupe d'Etude des Interactions Hôte-Parasite, UPRES-EA 3142, Laboratoire de Parasitologie-Mycologie
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Thierry Bergès
2Laboratoire de Génétique de la Levure, CNRS UMR 6161, Faculté des Sciences, 86022 Poitiers Cedex
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Pascal Poupard
3UMR Pathologie Végétale 77, Faculté des Sciences, 49045 Angers Cedex, France
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Carole Vauzelle-Moreau
2Laboratoire de Génétique de la Levure, CNRS UMR 6161, Faculté des Sciences, 86022 Poitiers Cedex
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Gilles Renier
4Laboratoire d'Immunologie, Centre Hospitalier Universitaire, 49033 Angers Cedex
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Dominique Chabasse
1Groupe d'Etude des Interactions Hôte-Parasite, UPRES-EA 3142, Laboratoire de Parasitologie-Mycologie
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Jean-Philippe Bouchara
1Groupe d'Etude des Interactions Hôte-Parasite, UPRES-EA 3142, Laboratoire de Parasitologie-Mycologie
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  • For correspondence: jean-philippe.bouchara@univ-angers.fr
DOI: 10.1128/AAC.48.5.1788-1796.2004
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  • FIG. 1.
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    FIG. 1.

    Flow cytometric analysis of rhodamine 123-stained cells of C. glabrata parent isolates and their fluconazole-resistant or ETB-induced mutants. Yeasts were incubated (grey area) or not (black thick line) with 1 mM sodium azide before rhodamine 123 staining. The fluorescence of cells incubated without the fluorochrome (black area) is presented as a control.

  • FIG. 2.
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    FIG. 2.

    Oxygen consumption by the C. glabrata parent (solid line) and fluconazole-resistant (dashed line) or ETB-induced (dotted line) mutant strains. A marked decrease in oxygen consumption was found for the mutant cells.

  • FIG. 3.
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    FIG. 3.

    Transmission electron micrographs of C. glabrata parent isolate 90.1085 (A) and its fluconazole-resistant (B) and ETB-induced (C) mutants. Note the absence of mitochondria in the respiration-deficient cells compared to the numerous mitochondrial sections (arrowheads) with obvious cristae in the parent cell. N, nucleus. Bars, 1 μm.

  • FIG. 4.
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    FIG. 4.

    Electrophoretic patterns of mtDNA of C. glabrata parent isolate 90.1085 (lane 1) and its fluconazole-resistant (lane 2) and ETB-induced (lane 3) mutants. mtDNA was analyzed by agarose gel electrophoresis after digestion with EcoRV. Lane M, molecular size markers (Marker III; Roche Molecular Biochemicals, Meylan, France). Sizes are shown in base pairs.

  • FIG. 5.
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    FIG. 5.

    Flow cytometric analysis of rhodamine 6G uptake and efflux for C. glabrata parent isolates and their derived petite mutants. Histograms are given for cells incubated with rhodamine 6G (black area) and after removal of the free dye and an additional 15-min incubation (black line). Unlabeled controls are represented in grey.

  • FIG. 6.
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    FIG. 6.

    Expression of azole resistance genes in C. glabrata parent isolates and their derived petite mutants. (A) Northern blot analysis. Total RNAs were extracted, separated, and blotted as indicated in Materials and Methods. Hybridizations were carried out with 32P-labeled antisense RNA probes specific for either CgCDR1, CgCDR2, CgERG11, or CgACT1. (B) Relative mRNA levels in parent isolates and their derived fluconazole-resistant and ETB-induced mutants. The intensities of the autoradiographic signals were quantified and are shown as the ratio of CgCDR1, CgCDR2, or CgERG11 to CgACT1.

  • FIG. 7.
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    FIG. 7.

    Analysis of the sterol composition of C. glabrata parent isolates and their fluconazole-resistant and ETB-induced mutants. Sterols were extracted from approximately 50 mg of lyophilized cells. The different sterol species were separated by GC and identified by their retention times relative to cholesterol. Data are representative of the results obtained from two independent cultures. SQU, squalene; LAN, lanosterol; ZYM, zymosterol; OBI, other biosynthesis intermediates; ERG, ergosterol.

Tables

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  • TABLE 1.

    Oligonucleotides used for CgERG11 sequencing

    PrimerNucleotide sequence (5′-3′)Nucleotide coordinatesa
    1 senseCTACAATCGAGTGAGCTTG+17 to +35
    1 antisenseGTAGAACACAAGTGGTGG+729 to +746
    2 senseCCATCACATGGCAATTGC+688 to +705
    2 antisenseGGTCATCTTAGTACCATCC+1445 to +1463
    3 senseGGTCGTTGAACTATTGGAG+584 to +602
    3 antisenseGGACCCAAGTAGACAGTC+863 to +880
    4 senseCGTGAGAAGAACGATATCC+1380 to +1398
    4 antisenseCACCTTCAGTTGGGTAAC+2047 to +2064
    5 senseCGCTTACTGTCAATTGGG+1991 to +2008
    5 antisenseGTCATATGCTTGCACTGC+2397 to +2414
    • ↵ a Nucleotide coordinates refer to the CgERG11 GenBank sequence (accession no. L40389 ).

  • TABLE 2.

    Susceptibility to polyenes and azoles of C. glabrata parent isolates and their derived petite mutantsa

    Antifungal agentDiam (mm) of growth inhibition zone
    Isolate 90.1085Isolate 94.5579
    ParentResistant mutantETB-induced mutantParentResistant mutantETB-induced mutant
    Amphotericin B284040263030
    Nystatin284040263532
    Tioconazole403232404033
    Miconazole24 (M)TR1125 (M)1715
    Isoconazole24 (M)TRTR24 (M)1111
    Ketoconazole28 (M)11TR30 (M)1212
    Fluconazole26 (M)TRTR28 (M)TRTR
    • ↵ a In vitro susceptibility testing was performed by the disk diffusion method on Casitone agar plates with Neosensitab tablets (containing 10 μg of drug for amphotericin B, tioconazole, miconazole, and isoconazole; 15 μg for ketoconazole and fluconazole; and 50 μg for nystatin). Results, which are representative of two independent experiments, correspond to the diameter of growth inhibition zones. M, presence of resistant mutants within the inhibition zone. TR, total resistance.

  • TABLE 3.

    MICs of polyenes and azoles for C. glabrata parent isolates and their derived petite mutantsa

    Antifungal agentMIC (μgml)
    Isolate 90.1085Isolate 94.5579
    ParentResistant mutantETB-induced mutantParentResistant mutantETB-induced mutant
    Amphotericin B0.0320.012*0.016*0.0470.012*0.023*
    Tioconazole<0.125<0.125†<0.125†<0.1250.125†<0.125†
    Miconazole<0.12522<0.12521
    Econazole<0.12511<0.12520.5
    Ketoconazole0.38680.1251612
    Fluconazole32>256>25664>256>256
    • ↵ a Results are representative of two independent experiments performed in triplicate. Data were obtained by the Etest procedure for amphotericin B, ketoconazole, and fluconazole. For tioconazole, miconazole, and econazole, Etest strips were not available, and for these drugs, sensitivity was tested by a microbroth dilution method. Data were analyzed with the Kruskall-Wallis test. For all values, P < 0.001 except *, P < 0.02, and †, no differences observed.

  • TABLE 4.

    Mutation rates of C. glabrata parent isolates in the presence of azole antifungals

    Azole antifungal (μg/ml)Mutation rate
    Parent isolate 90.1085Parent isolate 94.5579
    Tioconazole (20)NDaND
    Miconazole (1)7 × 10−65 × 10−5
    Econazole (1)1 × 10−52 × 10−4
    Ketoconazole (1)1.4 × 10−42 × 10−3
    Fluconazole (100)4 × 10−41 × 10−3
    • ↵ a ND, azole-resistant petite mutants were not detected (mutation rate, <10−6).

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Mechanisms of Azole Resistance in Petite Mutants of Candida glabrata
Sophie Brun, Thierry Bergès, Pascal Poupard, Carole Vauzelle-Moreau, Gilles Renier, Dominique Chabasse, Jean-Philippe Bouchara
Antimicrobial Agents and Chemotherapy Apr 2004, 48 (5) 1788-1796; DOI: 10.1128/AAC.48.5.1788-1796.2004

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Mechanisms of Azole Resistance in Petite Mutants of Candida glabrata
Sophie Brun, Thierry Bergès, Pascal Poupard, Carole Vauzelle-Moreau, Gilles Renier, Dominique Chabasse, Jean-Philippe Bouchara
Antimicrobial Agents and Chemotherapy Apr 2004, 48 (5) 1788-1796; DOI: 10.1128/AAC.48.5.1788-1796.2004
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KEYWORDS

antifungal agents
azoles
Candida albicans

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