Analysis of fluconazole effect on Candida albicans viability during extended incubations

Fluconazole is an azole agent with primarily fungistatic activity in standard in vitro susceptibility tests. However, recent work has demonstrated that this drug can reduce Candida albicans viability during prolonged incubations under non-growing conditions. The present study was undertaken to examine more closely some of the parameters of this killing activity. Fungicidal effects of 1.0 microg ml-1 of fluconazole were found during 7-14-day exposures in each of two media that prevented proliferation, distilled water and metal-depleted RPMI 1640 tissue-culture medium. Fluconazole appeared to be stable after being incubated at 37 degreesC for either 7 or 14 days. Strains of C. albicans resistant to fluconazole in standard short-term growth-inhibition assays were also found to be resistant to fluconazole's effect on viability in prolonged culture, suggesting similar mechanisms of action for these effects. C. albicans yeast cells pre-incubated for 7 days in distilled water were not more sensitive to the drug in short-term susceptibility assays. Although all proliferation of the organisms in distilled water cultures appeared to cease after 3 days, fluconazole added at 7 days still reduced C. albicans viability. Therefore, the drug appeared to kill the non-proliferating organisms directly rather than preventing growth and thereby the emergence of younger organisms that would live longer. Transmission electron microscopy demonstrated damage to the cell wall-cell membrane complex and interior contents of yeast cells incubated in distilled water alone; fluconazole appeared to increase the percentages of cells so affected. In summary, extended-incubation susceptibility tests demonstrated that fluconazole has direct fungicidal activity of non-proliferating C. albicans yeast cells. These results may be relevant to the manner in which this drug promotes clearance of chronic fungal infections.