Multiple molecular mechanisms contribute to a stepwise development of fluconazole resistance in clinical Candida albicans strains

From each of two AIDS patients with oropharyngeal candidiasis, five Candida albicans isolates from recurrent episodes of infection which became gradually resistant against fluconazole during antimycotic treatment were analyzed for molecular changes responsible for drug resistance. In both patients, a single C. albicans strain was responsible for the recurrent infections, but the CARE-2 fingerprint pattern of the isolates exhibited minor genetic alterations, indicating that microevolution of the strains took place during fluconazole therapy. In the isolates from patient 1, enhanced mRNA levels of the MDR1 gene, encoding a multiple drug resistance protein from the superfamily of major facilitators, and constitutive high expression of the ERG11 gene, coding for the drug target enzyme sterol 14alpha-demethylase, correlated with a stepwise development of fluconazole resistance. The resistant strains exhibited reduced accumulation of fluconazole and, for the last in the series, a slight increase in drug needed to inhibit sterol 14alpha-demethylation in vitro. In the isolates from patient 2, increased MDR1 mRNA levels and the change from heterozygosity to homozygosity for a mutant form of the ERG11 gene correlated with continuously decreased drug susceptibility. In this series, reduced drug accumulation and increased resistance in the target enzyme activity, sterol 14alpha-demethylase, were observed. These results demonstrate that different molecular mechanisms contribute to a gradual development of fluconazole resistance in C. albicans.