Changes in susceptibility to posaconazole in clinical isolates of Candida albicans

Abstract

Objectives: To characterize the molecular mechanisms responsible for reduced susceptibility to azoles in Candida albicans clinical isolates. Materials and methods: Seven sequential C. albicans isolates were cultured from an AIDS patient treated with posaconazole for refractory oropharyngeal candidiasis. Expression levels of the CDR1, CDR2 and MDR1 genes, encoding efflux pumps previously implicated in azole resistance, and ERG11, encoding the azole target site, were monitored using northern blot and real-time PCR. The ERG11 genes from all seven isolates were sequenced. Results: The seven closely related isolates exhibited significant decreases in susceptibility to fluconazole (MIC ≥ 32 mg/L) and voriconazole (MIC ≥ 2 mg/L) and progressive decreases in susceptibility to both posaconazole (isolates 1–4 MIC 0.25 mg/L, isolates 5–7 MIC 2 mg/L) and itraconazole (isolates 1–4 MIC 1 mg/L, isolates 5–7 MIC > 8 mg/L). None of the isolates exhibited any significant changes in the expression levels of ERG11 or the efflux pump genes. All seven isolates had multiple mutations in ERG11; isolates one through four each had five missense mutations; four of the resultant amino acid changes were previously associated with azole resistance. The fifth isolate had an additional novel mutation in one copy of ERG11, resulting in a Pro-230 to Leu substitution. This mutation was present in both ERG11 genes in the last two isolates. Select ERG11 genes were expressed in Saccharomyces cerevisiae, the ERG11 allele with all six mutations conferred the highest level of posaconazole resistance. Conclusions: Multiple mutations in ERG11 are required to confer decreased susceptibility to posaconazole.