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

Elias K. Manavathu, Jose Antonio Vazquez, Pranatharthi H. Chandrasekar

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Abstract

To study the mechanism of resistance of Aspergillus fumigatus to itraconazole, spontaneous mutants with reduced susceptibility were selected by spreading 2x108 conidia from a clinical isolate (W73355) susceptible to miconazole (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 miconazole (32 mg/l). The 19 colonies that grew (frequency 0.95x10-7) in the presence of miconazole were screened by broth macrodilution technique for their susceptibility to itraconazole. A total of two isolates (frequency 1x10-8) MCZ14 and MCZ15 had MICs of 16 and 8 mg/l, respectively, for itraconazole. Both MCZ14 and MCZ15 showed concomitant increases in MICs for ketoconazole and miconazole, but not for amphotericin B. Growth inhibition studies as well as kill curve experiments revealed that MCZ14 and MCZ15 were less susceptible to itraconazole compared to the parental strain. The intracellular accumulation of itraconazole in A. fumigatus was time and concentration dependent. Maximum accumulation was obtained within 30 min at 5 μM itraconazole. In MCZ14 and MCZ15 intracellular accumulation of [3H]itraconazole was reduced by approximately 80 and 60%, respectively, compared to the susceptible parent. The respiratory inhibitor carbonyl cyanide m-chlorophenyl hydrazone at 200 μM reduced the intracellular accumulation of itraconazole by approximately 36.2% (P≤0.05) in the parent and in the mutant strains. These results suggest that (i) the reduced accumulation 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 accumulation of itraconazole is at least in part responsible for the reduced susceptibility of the mutant isolates to itraconazole. Copyright (C) 1999 Elsevier Science B.V.

Original languageEnglish (US)
Pages (from-to)213-219
Number of pages7
JournalInternational Journal of Antimicrobial Agents
Volume12
Issue number3
DOIs
StatePublished - Aug 1 1999

Fingerprint

Aspergillus fumigatus
Itraconazole
Antifungal Agents
Miconazole
Amphotericin B
Carbonyl Cyanide m-Chlorophenyl Hydrazone
Peptones
Ketoconazole
Fungal Spores
Agar
Permeability

Keywords

  • Antifungal agents
  • Aspergillus fumigatus
  • Drug resistance
  • Itraconazole
  • Resistant mutants

ASJC Scopus subject areas

  • Applied Microbiology and Biotechnology
  • Microbiology
  • Parasitology
  • Virology
  • Immunology and Allergy
  • Infectious Diseases

Cite this

@article{8f7a2506851d452e91793d3112113dab,
title = "Reduced susceptibility in laboratory-selected mutants of Aspergillus fumigatus to itraconazole due to decreased intracellular accumulation of the antifungal agent",
abstract = "To study the mechanism of resistance of Aspergillus fumigatus to itraconazole, spontaneous mutants with reduced susceptibility were selected by spreading 2x108 conidia from a clinical isolate (W73355) susceptible to miconazole (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 miconazole (32 mg/l). The 19 colonies that grew (frequency 0.95x10-7) in the presence of miconazole were screened by broth macrodilution technique for their susceptibility to itraconazole. A total of two isolates (frequency 1x10-8) MCZ14 and MCZ15 had MICs of 16 and 8 mg/l, respectively, for itraconazole. Both MCZ14 and MCZ15 showed concomitant increases in MICs for ketoconazole and miconazole, but not for amphotericin B. Growth inhibition studies as well as kill curve experiments revealed that MCZ14 and MCZ15 were less susceptible to itraconazole compared to the parental strain. The intracellular accumulation of itraconazole in A. fumigatus was time and concentration dependent. Maximum accumulation was obtained within 30 min at 5 μM itraconazole. In MCZ14 and MCZ15 intracellular accumulation of [3H]itraconazole was reduced by approximately 80 and 60{\%}, respectively, compared to the susceptible parent. The respiratory inhibitor carbonyl cyanide m-chlorophenyl hydrazone at 200 μM reduced the intracellular accumulation of itraconazole by approximately 36.2{\%} (P≤0.05) in the parent and in the mutant strains. These results suggest that (i) the reduced accumulation 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 accumulation of itraconazole is at least in part responsible for the reduced susceptibility of the mutant isolates to itraconazole. Copyright (C) 1999 Elsevier Science B.V.",
keywords = "Antifungal agents, Aspergillus fumigatus, Drug resistance, Itraconazole, Resistant mutants",
author = "Manavathu, {Elias K.} and Vazquez, {Jose Antonio} and Chandrasekar, {Pranatharthi H.}",
year = "1999",
month = "8",
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doi = "10.1016/S0924-8579(98)00102-2",
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TY - JOUR

T1 - Reduced susceptibility in laboratory-selected mutants of Aspergillus fumigatus to itraconazole due to decreased intracellular accumulation of the antifungal agent

AU - Manavathu, Elias K.

AU - Vazquez, Jose Antonio

AU - Chandrasekar, Pranatharthi H.

PY - 1999/8/1

Y1 - 1999/8/1

N2 - To study the mechanism of resistance of Aspergillus fumigatus to itraconazole, spontaneous mutants with reduced susceptibility were selected by spreading 2x108 conidia from a clinical isolate (W73355) susceptible to miconazole (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 miconazole (32 mg/l). The 19 colonies that grew (frequency 0.95x10-7) in the presence of miconazole were screened by broth macrodilution technique for their susceptibility to itraconazole. A total of two isolates (frequency 1x10-8) MCZ14 and MCZ15 had MICs of 16 and 8 mg/l, respectively, for itraconazole. Both MCZ14 and MCZ15 showed concomitant increases in MICs for ketoconazole and miconazole, but not for amphotericin B. Growth inhibition studies as well as kill curve experiments revealed that MCZ14 and MCZ15 were less susceptible to itraconazole compared to the parental strain. The intracellular accumulation of itraconazole in A. fumigatus was time and concentration dependent. Maximum accumulation was obtained within 30 min at 5 μM itraconazole. In MCZ14 and MCZ15 intracellular accumulation of [3H]itraconazole was reduced by approximately 80 and 60%, respectively, compared to the susceptible parent. The respiratory inhibitor carbonyl cyanide m-chlorophenyl hydrazone at 200 μM reduced the intracellular accumulation of itraconazole by approximately 36.2% (P≤0.05) in the parent and in the mutant strains. These results suggest that (i) the reduced accumulation 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 accumulation of itraconazole is at least in part responsible for the reduced susceptibility of the mutant isolates to itraconazole. Copyright (C) 1999 Elsevier Science B.V.

AB - To study the mechanism of resistance of Aspergillus fumigatus to itraconazole, spontaneous mutants with reduced susceptibility were selected by spreading 2x108 conidia from a clinical isolate (W73355) susceptible to miconazole (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 miconazole (32 mg/l). The 19 colonies that grew (frequency 0.95x10-7) in the presence of miconazole were screened by broth macrodilution technique for their susceptibility to itraconazole. A total of two isolates (frequency 1x10-8) MCZ14 and MCZ15 had MICs of 16 and 8 mg/l, respectively, for itraconazole. Both MCZ14 and MCZ15 showed concomitant increases in MICs for ketoconazole and miconazole, but not for amphotericin B. Growth inhibition studies as well as kill curve experiments revealed that MCZ14 and MCZ15 were less susceptible to itraconazole compared to the parental strain. The intracellular accumulation of itraconazole in A. fumigatus was time and concentration dependent. Maximum accumulation was obtained within 30 min at 5 μM itraconazole. In MCZ14 and MCZ15 intracellular accumulation of [3H]itraconazole was reduced by approximately 80 and 60%, respectively, compared to the susceptible parent. The respiratory inhibitor carbonyl cyanide m-chlorophenyl hydrazone at 200 μM reduced the intracellular accumulation of itraconazole by approximately 36.2% (P≤0.05) in the parent and in the mutant strains. These results suggest that (i) the reduced accumulation 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 accumulation of itraconazole is at least in part responsible for the reduced susceptibility of the mutant isolates to itraconazole. Copyright (C) 1999 Elsevier Science B.V.

KW - Antifungal agents

KW - Aspergillus fumigatus

KW - Drug resistance

KW - Itraconazole

KW - Resistant mutants

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U2 - 10.1016/S0924-8579(98)00102-2

DO - 10.1016/S0924-8579(98)00102-2

M3 - Article

VL - 12

SP - 213

EP - 219

JO - International Journal of Antimicrobial Agents

JF - International Journal of Antimicrobial Agents

SN - 0924-8579

IS - 3

ER -