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

Abstract

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.

Atorvastatin-induced cell toxicity in yeast is linked to disruption of protein isoprenylation

“…Ergosterol assays were carried out on 40 mL of yeast cultures grown in SC medium following treatment with atorvastatin and supplements. Ergosterol was extracted according to the method of Arthington-Skaggs et al (1999), involving digestion of the cell pellet in an alcoholic potassium hydroxide solution, followed by sterol extraction with n-heptane. The heptane layer was collected, diluted (up to fivefold) in 100% ethanol and scanned spectrophotometrically between 240 and 300 nm (Arthington-Skaggs et al, 1999) using a Shimadzu UV-1601 spectrophotometer.…”

Simvastatin reduces ergosterol levels, inhibits growth and causes loss of mtDNA inCandida glabrata

“…Ergosterol levels were assayed using a modification of the sterol quantitation methods of Arthington-Skaggs et al (1999) and Breivik & Owades (1957). Yeast strains were grown to stationary phase in 50 mL of minimal medium in the presence or absence of simvastatin.…”

The Thm1 Zn(II)2Cys6 transcription factor contributes to heat, membrane integrity and virulence in the insect pathogenic fungus Beauveria bassiana

“…Ergosterol content was measured as described (Arthington-Skaggs et al, 1999;Yarden et al, 2014). Briefly, 1 ml of conidial suspensions (10 7 conidia/ml) were inoculated in 100 ml of SDB and incubated at room temperature for 3 days with aeration.…”