Journal of Antimicrobial Chemotherapy (1999) 44, 109-112
© 1999 The British Society for Antimicrobial Chemotherapy
Brief report |
Effects of voriconazole on Candida glabrata in vitro
Center for Medical Mycology, University Hospitals of Cleveland and Case Western Reserve University, 11 100 Euclid Avenue, Cleveland, OH 44106-5028, USA
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Abstract |
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The effects of voriconazole on the growth, morphology and lipids of Candida glabratawere studied. MIC data showed that voriconazole was up to 32- to 64-fold more active than fluconazole in its ability to inhibit various C. glabrata strains. Voriconazole inhibited the growth of C. glabrata in a dose-dependent fashion. Electron microscope examination showed that voriconazole treatment affected the external and internal morphology of C. glabrata. Treatment of C. glabrata with voriconazole inhibited ergosterol synthesis and led to accumulation of methylated sterols. In contrast, no significant difference in phospholipid composition was observed between treated and untreated cells.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Candida glabrata is increasingly recognized as an important nosocomial pathogen. This fungus is clinically important not only because of its increasing frequency but also because it is associated with high complication and mortality rates.1 These observations reinforce the need for new antifungal agents effective against C. glabrata.
Voriconazole (UK 109,496) is a new triazole (mol. wt 349.3) with potent broad-spectrum activity against fungi.2,3 The present investigation compares the effects of voriconazole and fluconazole on the growth, morphology, and sterols and phospholipids of C. glabrata.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Antifungal agents
Voriconazole and fluconazole powders were obtained from Pfizer Central Research (Sandwich, UK). Fluconazole was dissolved in distilled water and voriconazole in dimethyl sulphoxide to make stock solutions of 1 g/L.
Determination of minimum inhibitory concentration (MIC80 and MIC100)
The MIC80s for seven C. glabrataclinical isolates obtained from non-neutropenic patients were determined using the National Committee for Clinical Laboratory Standards (NCCLS) reference method, M-27A.4 The MIC 80 was defined as the lowest drug concentration necessary to inhibit 80% of growth compared with the control, while MIC100 (equivalent to MFC) was the concentration in which no visible growth was observed following plating on Sabouraud dextrose agar.
Effect of voriconazole and fluconazole on C. glabrata growth kinetics
C. glabrata (106 cells/mL) from an overnight culture were used to inoculate 50 mL of yeast nitrogen base medium with amino acids supplemented with 0.5% glucose (YNB) containing different concentrations of antifungals (0.5–8.0 x MIC 80). The organisms were incubated at 37°C with shaking. At various time intervals, aliquots were taken and optical densities were measured at 600 nm.2
Lipid and sterol extraction and analysis
Lipids and sterols from yeast cells grown in the presence and absence of antifungals (4 x MIC80) were extracted using previously described methods.5,6 Sterols were analysed by gas liquid chromatography,5 and phospholipids were resolved by two-dimensional thin layer chromatography.6
Scanning and transmission electron microscopy (SEM, TEM)
To determine the effect of antifungal agents on the morphology of C. glabrata,yeast cells (106/mL) were grown for 24 h at 37°C in the presence or absence of 4 x MIC80 of either fluconazole or voriconazole. Cells were fixed, prepared, and examined using SEM and TEM as described previously.7
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Minimum inhibitory concentrations
Voriconazole was more effective than fluconazole in inhibiting the growth of C. glabrata. Voriconazole MIC80s ranged from 0.0625 to 1 mg/L, and fluconazole MIC 80s ranged from 2 to >64 mg/L. The MIC80s of voriconazole were 32- to 64-fold lower than those of fluconazole. Similarly, lower concentrations of voriconazole were required to cause 100% inhibition of the isolates studied (data not shown).
Effect of voriconazole and fluconazole on the growth of C. glabrata
Voriconazole and fluconazole showed a dose-dependent growth inhibitory effect on C.
glabrata. Lower concentrations of voriconazole and fluconazole (0.5 x MIC
80) had small or no detectable effects on the growth rate of both C. glabratastrains used. However, candidal growth was markedly inhibited by concentrations 
4 x
MIC80 of voriconazole and fluconazole (Figure).
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),
Control; (
), 0.5 x MIC; (
), MIC; (
), 2 x MIC; (
), 4
x MIC;
(•), 6 x MIC; +, 8 x MIC.Effect of voriconazole and fluconazole on the morphology of C. glabrata
SEM analysis showed that, unlike control cells which had normal oval to spherical yeast cells, treatment of C. glabrata with voriconazole (4 x MIC80) affected the external morphology of this yeast: some voriconazole-treated cells exhibited deformation and protrusions on the cell surface (data not shown). Furthermore, TEM analysis revealed that control C. glabrata cells had typical yeast morphology with regular distinct cytoplasmic membranes surrounded by a multilayered cell wall, and intact cytoplasmic content (data not shown). Treatment of C. glabrata with voriconazole affected the outer cell envelope, causing cell-wall thinning and membrane degradation (data not shown). Voriconazole treatment also caused cell shrinkage: an intervening electron-lucent zone appeared between the cell wall and cytoplasm (data not shown).
Fluconazole treatment (4 x MIC80) caused gross ultrastructural alterations. Cells showed irregularities in cell-wall structure and apparent loss of internal cohesion (data not shown). The cytoplasmic contents of fluconazole-treated cells were coagulated, giving rise to electron-dense and electron-thin areas.
Influence of voriconazole on sterol and phospholipids of C. glabrata
The Table summarizes the sterol composition of antifungal treated and untreated C. glabrata strains 2255 and 2256. Ergosterol was the main sterol in untreated control cells. Unlike Candida albicans, where a number of sterol intermediates are present, in this yeast lanosterol was the only sterol intermediate detected (Table). Treatment of C. glabrata with either voriconazole or fluconazole inhibited ergosterol synthesis and led to the accumulation of methylated sterols such as lanosterol and 4,14-dimethyl zymosterol. Accumulation of squalene, a lanosterol precursor, was also observed following treatment with either triazole (Table).
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Lipid analysis of C. glabrata showed that this yeast contained a mixture of phospholipids including phosphatidylcholine (the predominant phospholipid), and phosphatidylinositol, phosphatidylethanolamine and phosphatidylserine in decreasing order of content (data not shown). Treatment of C. glabrata with either azole did not result in a significant difference in the phospholipid composition of the treated and control cells (P > 0.05).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Our results show that voriconazole exhibits potent antifungal activity for several clinical strains of C. glabrata. We demonstrated that growth inhibition by voriconazole is concentration dependent. Voriconazole also showed a significant effect on cell ultrastructure. Our data extend previous findings on voriconazole for C. albicansand Candida krusei. 2,5
Sterol analysis of untreated control C. glabrata isolates showed that, as for C.
albicans and C. krusei, the predominant sterol was ergosterol. However, unlike
these two species which had 24-methylenedihydrolanosterol, lanosterol, obtusifoliol and
calciferol as sterol intermediates, C. glabrata had lanosterol as the only sterol
intermediate. Treatment of C. glabrata with either voriconazole or fluconazole resulted
in the inhibition of ergosterol synthesis and accumulation of methylated sterols. Neither
voriconazole nor fluconazole treatment altered C. glabrata’s phospholipid
pattern. These findings are consistent with the premise that voriconazole inhibits fungal growth
primarily by interfering with the cytochrome P-450 dependent 14
-sterol demethylase,
which is a key enzyme in the biosynthesis of ergosterol.8
Phenotypic changes such as growth inhibition and deleterious morphological alterations observed
following treatment with voriconazole are secondary effects attributed to ergosterol inhibition
and accumulation of methylated sterols.8 In all our studies,
voriconazole was shown to be more active than fluconazole.
In conclusion, our studies demonstrate that voriconazole has a potent inhibitory activity for C. glabrata making it a viable therapeutic option for the treatment of infections caused by this organism.
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Acknowledgments |
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This work was supported by a grant from Pfizer Pharmaceuticals Group, New York, NY, USA.
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Notes |
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* Corresponding author. Tel: +1-216-844-8580; Fax: +1-216-844-1076.
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References |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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1 . Nguyen, M. H., Peacock, J. E., Morris, A. J., Tanner, D. C., Nguyen, M. L., Snydman, D. R. et al. (1996). The changing face of candidemia: emergence of non-Candida albicans species and antifungal resistance. American Journal of Medicine 100, 617 –23.[Web of Science][Medline]
2 . Belanger, P., Nast, C. C., Fratti, R., Sanati, H. & Ghannoum, M. (1997). Voriconazole (UK-109, 496) inhibits the growth and alters the morphology of fluconazole-susceptible and -resistant Candida species. Antimicrobial Agents and Chemotherapy 41, 1840–2.[Abstract]
3 . Hitchcock, C. A., Pye, L. W., Oliver, G. P. & Troke, P. F. (1995). UK-109, 496, a novel, wide-spectrum triazole derivative for the treatment of fungal infections. In Program and Abstracts of the Thirty-Fifth Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, CA, 1995. Abstract F72, p. 125. American Society for Microbiology, Washington, DC.
4 . National Committee for Clinical Laboratory Standards. (1997). Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts: Approved Standard M27-A. NCCLS, Wayne, PA.
5 . Sanati, H., Belanger, P., Fratti, R. & Ghannoum, M. (1997). A new triazole, voriconazole (UK-109, 496), blocks sterol biosynthesis in Candida albicans and Candida krusei. Antimicrobial Agents and Chemotherapy 41, 2492–6.[Abstract]
6 . Koul, A., Chandra, J. C. & Prasad, R. (1995). Status of membrane lipids and amino acid transport in morphological mutants of Candida albicans. Biochemistry and Molecular Biology International 35, 1215–22.[Web of Science][Medline]
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Ghannoum, M. A., Abu Elteen, K., Ellabib, M. &
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8 . Hitchcock, C. A. & Whittle, P. J. (1993). Chemistry and mode of action of fluconazole. In Cutaneous Antifungal Agents: Selected Compounds in Clinical Practice and Development (Ripen, J. W. & Fromtling, R. A., Eds), pp. 183–97. Marcel Dekker, New York, NY.
Received 20 October 1998; returned 26 January 1999; revised 24 February 1999; accepted 24 March 1999
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