Article Figures & Data
Figures
- 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.
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.
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.
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.
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.
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.
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
- TABLE 1.
Oligonucleotides used for CgERG11 sequencing
Primer Nucleotide sequence (5′-3′) Nucleotide coordinatesa 1 sense CTACAATCGAGTGAGCTTG +17 to +35 1 antisense GTAGAACACAAGTGGTGG +729 to +746 2 sense CCATCACATGGCAATTGC +688 to +705 2 antisense GGTCATCTTAGTACCATCC +1445 to +1463 3 sense GGTCGTTGAACTATTGGAG +584 to +602 3 antisense GGACCCAAGTAGACAGTC +863 to +880 4 sense CGTGAGAAGAACGATATCC +1380 to +1398 4 antisense CACCTTCAGTTGGGTAAC +2047 to +2064 5 sense CGCTTACTGTCAATTGGG +1991 to +2008 5 antisense GTCATATGCTTGCACTGC +2397 to +2414 - TABLE 2.
Susceptibility to polyenes and azoles of C. glabrata parent isolates and their derived petite mutantsa
Antifungal agent Diam (mm) of growth inhibition zone Isolate 90.1085 Isolate 94.5579 Parent Resistant mutant ETB-induced mutant Parent Resistant mutant ETB-induced mutant Amphotericin B 28 40 40 26 30 30 Nystatin 28 40 40 26 35 32 Tioconazole 40 32 32 40 40 33 Miconazole 24 (M) TR 11 25 (M) 17 15 Isoconazole 24 (M) TR TR 24 (M) 11 11 Ketoconazole 28 (M) 11 TR 30 (M) 12 12 Fluconazole 26 (M) TR TR 28 (M) TR TR ↵ 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 agent MIC (μgml) Isolate 90.1085 Isolate 94.5579 Parent Resistant mutant ETB-induced mutant Parent Resistant mutant ETB-induced mutant Amphotericin B 0.032 0.012* 0.016* 0.047 0.012* 0.023* Tioconazole <0.125 <0.125† <0.125† <0.125 0.125† <0.125† Miconazole <0.125 2 2 <0.125 2 1 Econazole <0.125 1 1 <0.125 2 0.5 Ketoconazole 0.38 6 8 0.125 16 12 Fluconazole 32 >256 >256 64 >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 90.1085 Parent isolate 94.5579 Tioconazole (20) NDa ND Miconazole (1) 7 × 10−6 5 × 10−5 Econazole (1) 1 × 10−5 2 × 10−4 Ketoconazole (1) 1.4 × 10−4 2 × 10−3 Fluconazole (100) 4 × 10−4 1 × 10−3 ↵ a ND, azole-resistant petite mutants were not detected (mutation rate, <10−6).
