This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Arthington-Skaggs, B. A.
Right arrow Articles by Morrison, C. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Arthington-Skaggs, B. A.
Right arrow Articles by Morrison, C. J.

 Previous Article  |  Next Article 

Journal of Clinical Microbiology, October 1999, p. 3332-3337, Vol. 37, No. 10
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Quantitation of Ergosterol Content: Novel Method for Determination of Fluconazole Susceptibility of Candida albicans

Beth A. Arthington-Skaggs, Hoda Jradi, Tejal Desai, and Christine J. Morrison*

Mycotic Diseases Branch, Division of Bacterial and Mycotic Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 30333

Received 11 May 1999/Returned for modification 29 June 1999/Accepted 21 July 1999

MIC end points for the most commonly prescribed azole antifungal drug, fluconazole, can be difficult to determine because its fungistatic nature can lead to excessive "trailing" of growth during susceptibility testing by National Committee for Clinical Laboratory Standards broth macrodilution and microdilution methods. To overcome this ambiguity, and because fluconazole acts by inhibiting ergosterol biosynthesis, we developed a novel method to differentiate fluconazole-susceptible from fluconazole-resistant isolates by quantitating ergosterol production in cells grown in 0, 1, 4, 16, or 64 µg of fluconazole per ml. Ergosterol was isolated from whole yeast cells by saponification, followed by extraction of nonsaponifiable lipids with heptane. Ergosterol was identified by its unique spectrophotometric absorbance profile between 240 and 300 nm. We used this sterol quantitation method (SQM) to test 38 isolates with broth microdilution end points of <= 8 µg/ml (susceptible), 16 to 32 µg/ml (susceptible dose-dependent [SDD]), or >= 64 µg/ml (resistant) and 10 isolates with trailing end points by the broth microdilution method. No significant differences in mean ergosterol content were observed between any of the isolates grown in the absence of fluconazole. However, 18 susceptible isolates showed a mean reduction in ergosterol content of 72% after exposure to 1 µg of fluconazole/ml, an 84% reduction after exposure to 4 µg/ml, and 95 and 100% reductions after exposure to 16 and 64 µg of fluconazole/ml, respectively. Ten SDD isolates showed mean ergosterol reductions of 38, 57, 73, and 99% after exposure to 1, 4, 16, and 64 µg of fluconazole/ml, respectively. In contrast, 10 resistant isolates showed mean reductions in ergosterol content of only 25, 38, 53, and 84% after exposure to the same concentrations of fluconazole. The MIC of fluconazole, by using the SQM, was defined as the lowest concentration of the drug which resulted in 80% or greater inhibition of overall mean ergosterol biosynthesis compared to that in the drug-free control. Of 38 isolates which gave clear end points by the broth microdilution method, the SQM MIC was within 2 dilutions of the broth microdilution MIC for 33 (87%). The SQM also discriminated between resistant and highly resistant isolates and was particularly useful for discerning the fluconazole susceptibilities of 10 additional isolates which gave equivocal end points by the broth microdilution method due to trailing growth. In contrast to the broth microdilution method, the SQM determined trailing isolates to be susceptible rather than resistant, indicating that the SQM may predict clinical outcome more accurately. The SQM may provide a means to enhance current methods of fluconazole susceptibility testing and may provide a better correlation of in vitro with in vivo results, particularly for isolates with trailing end points.

* Corresponding author. Mailing address: Centers for Disease Control and Prevention, 1600 Clifton Rd., NE Mailstop G-11, Atlanta, GA 30333. Phone: (404) 639-3098. Fax: (404) 639-3546. E-mail: cjm3{at}cdc.gov.

Journal of Clinical Microbiology, October 1999, p. 3332-3337, Vol. 37, No. 10
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


This article has been cited by other articles:

  • De Seta, F., Schmidt, M., Vu, B., Essmann, M., Larsen, B. (2009). Antifungal mechanisms supporting boric acid therapy of Candida vaginitis. J Antimicrob Chemother 63: 325-336 [Abstract] [Full Text]  
  • Hoot, S. J., Oliver, B. G., White, T. C. (2008). Candida albicans UPC2 is transcriptionally induced in response to antifungal drugs and anaerobicity through Upc2p-dependent and -independent mechanisms. Microbiology 154: 2748-2756 [Abstract] [Full Text]  
  • Oliver, B. G., Silver, P. M., White, T. C. (2008). Polyene susceptibility is dependent on nitrogen source in the opportunistic pathogen Candida albicans. J Antimicrob Chemother 61: 1302-1308 [Abstract] [Full Text]  
  • Blum, G., Perkhofer, S., Haas, H., Schrettl, M., Wurzner, R., Dierich, M. P., Lass-Florl, C. (2008). Potential Basis for Amphotericin B Resistance in Aspergillus terreus. Antimicrob. Agents Chemother. 52: 1553-1555 [Abstract] [Full Text]  
  • Oliver, B. G., Silver, P. M., Marie, C., Hoot, S. J., Leyde, S. E., White, T. C. (2008). Tetracycline alters drug susceptibility in Candida albicans and other pathogenic fungi. Microbiology 154: 960-970 [Abstract] [Full Text]  
  • Coen, M., Bodkin, J., Power, D., Bubb, W. A., Himmelreich, U., Kuchel, P. W., Sorrell, T. C. (2006). Antifungal Effects on Metabolite Profiles of Medically Important Yeast Species Measured by Nuclear Magnetic Resonance Spectroscopy. Antimicrob. Agents Chemother. 50: 4018-4026 [Abstract] [Full Text]  
  • Pinto, E., Pina-Vaz, C., Salgueiro, L., Goncalves, M. J., Costa-de-Oliveira, S., Cavaleiro, C., Palmeira, A., Rodrigues, A., Martinez-de-Oliveira, J. (2006). Antifungal activity of the essential oil of Thymus pulegioides on Candida, Aspergillus and dermatophyte species.. J Med Microbiol 55: 1367-1373 [Abstract] [Full Text]  
  • Navarro-Martinez, M. D., Garcia-Canovas, F., Rodriguez-Lopez, J. N. (2006). Tea polyphenol epigallocatechin-3-gallate inhibits ergosterol synthesis by disturbing folic acid metabolism in Candida albicans. J Antimicrob Chemother 57: 1083-1092 [Abstract] [Full Text]  
  • Silver, P. M., Oliver, B. G., White, T. C. (2004). Role of Candida albicans Transcription Factor Upc2p in Drug Resistance and Sterol Metabolism. Eukaryot Cell 3: 1391-1397 [Abstract] [Full Text]  
  • da Silva Ferreira, M. E., Capellaro, J. L., dos Reis Marques, E., Malavazi, I., Perlin, D., Park, S., Anderson, J. B., Colombo, A. L., Arthington-Skaggs, B. A., Goldman, M. H. S., Goldman, G. H. (2004). In Vitro Evolution of Itraconazole Resistance in Aspergillus fumigatus Involves Multiple Mechanisms of Resistance. Antimicrob. Agents Chemother. 48: 4405-4413 [Abstract] [Full Text]  
  • Song, J. L., Harry, J. B., Eastman, R. T., Oliver, B. G., White, T. C. (2004). The Candida albicans Lanosterol 14-{alpha}-Demethylase (ERG11) Gene Promoter Is Maximally Induced after Prolonged Growth with Antifungal Drugs. Antimicrob. Agents Chemother. 48: 1136-1144 [Abstract] [Full Text]  
  • Young, L. Y., Hull, C. M., Heitman, J. (2003). Disruption of Ergosterol Biosynthesis Confers Resistance to Amphotericin B in Candida lusitaniae. Antimicrob. Agents Chemother. 47: 2717-2724 [Abstract] [Full Text]  
  • Pinjon, E., Moran, G. P., Jackson, C. J., Kelly, S. L., Sanglard, D., Coleman, D. C., Sullivan, D. J. (2003). Molecular Mechanisms of Itraconazole Resistance in Candida dubliniensis. Antimicrob. Agents Chemother. 47: 2424-2437 [Abstract] [Full Text]  
  • Mukhopadhyay, K., Kohli, A., Prasad, R. (2002). Drug Susceptibilities of Yeast Cells Are Affected by Membrane Lipid Composition. Antimicrob. Agents Chemother. 46: 3695-3705 [Abstract] [Full Text]  
  • Arthington-Skaggs, B. A., Lee-Yang, W., Ciblak, M. A., Frade, J. P., Brandt, M. E., Hajjeh, R. A., Harrison, L. H., Sofair, A. N., Warnock, a. D. W. (2002). Comparison of Visual and Spectrophotometric Methods of Broth Microdilution MIC End Point Determination and Evaluation of a Sterol Quantitation Method for In Vitro Susceptibility Testing of Fluconazole and Itraconazole against Trailing and Nontrailing Candida Isolates. Antimicrob. Agents Chemother. 46: 2477-2481 [Abstract] [Full Text]  
  • Kohli, A., Smriti, N., Mukhopadhyay, K., Rattan, A., Prasad, R. (2002). In Vitro Low-Level Resistance to Azoles in Candida albicans Is Associated with Changes in Membrane Lipid Fluidity and Asymmetry. Antimicrob. Agents Chemother. 46: 1046-1052 [Abstract] [Full Text]  
  • Rex, J. H., Pfaller, M. A., Walsh, T. J., Chaturvedi, V., Espinel-Ingroff, A., Ghannoum, M. A., Gosey, L. L., Odds, F. C., Rinaldi, M. G., Sheehan, D. J., Warnock, D. W. (2001). Antifungal Susceptibility Testing: Practical Aspects and Current Challenges. Clin. Microbiol. Rev. 14: 643-658 [Abstract] [Full Text]  
  • Arthington-Skaggs, B. A., Warnock, D. W., Morrison, C. J. (2000). Quantitation of Candida albicans Ergosterol Content Improves the Correlation between In Vitro Antifungal Susceptibility Test Results and In Vivo Outcome after Fluconazole Treatment in a Murine Model of Invasive Candidiasis. Antimicrob. Agents Chemother. 44: 2081-2085 [Abstract] [Full Text]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»