Reduced susceptibility in laboratory-selected mutants of Aspergillus fumigatus to itraconazole due to decreased intracellular accumulation of the antifungal agent

To study the mechanism of resistance of Aspergillus fumigatus to itraconazo le, spontaneous mutants with reduced susceptibility were selected by spread ing 2 x 10(8) conidia from a clinical isolate (W73355) susceptible to micon azole (MIC 2 mg/l), itraconazole (MIC 0.25 mg/l) and amphotericin B (MIC 0. 5 mg/l) on 40 peptone yeast extract glucose agar plates containing miconazo le (32 mg/l). The 19 colonies that grew (frequency 0.95 x 10(-7)) in the pr esence of miconazole were screened by broth macrodilution technique for the ir susceptibility to itraconazole. A total of two isolates (frequency 1 x 1 0(-8)) MCZ14 and MCZ15 had MICs of 16 and 8 mg/l, respectively, for itracon azole. Both MCZ14 and MCZ15 showed concomitant increases in MICs for ketoco nazole and miconazole, but not for amphotericin B. Growth inhibition studie s as well as kill curve experiments revealed that MCZ14 and MCZ15 were less susceptible to itraconazole compared to the parental strain. The intracell ular accumulation of itraconazole in A. fumigatus was time and concentratio n dependent. Maximum accumulation was obtained within 30 min at 5 mu M itra conazole. In MCZ14 and MCZ15 intracellular accumulation of [H-3]itraconazol e was reduced by approximately 80 and 60%, respectively, compared to the su sceptible parent. The respiratory inhibitor carbonyl cyanide m-chlorophenyl hydrazone at 200 mu M reduced the intracellular accumulation of itraconazo le by approximately 36.2% (P less than or equal to 0.05) in the parent and in the mutant strains. These results suggest that (i) the reduced accumulat ion of itraconazole in MCZ14 and MCZ15 is due to diminished permeability of the drug and perhaps not due to efflux, (ii) the uptake of itraconazole in A. fumigatus may be an energy dependent process, and (iii) decreased accum ulation of itraconazole is at least in part responsible for the reduced sus ceptibility of the mutant isolates to itraconazole. (C) 1999 Elsevier Scien ce B.V. and International Society of Chemotherapy. All rights reserved.