A novel mechanism of fluconazole resistance associated with fluconazole sequestration in Candida albicans isolates from a myelofibrosis patient.
A series of 10 strains of Candida albicans, from TIMM 3309 to TIMM 3318, were repeatedly isolated in one myelofibrosis-complicated patient with recurrent candidemia. The latter five isolates, from TIMM 3314 to TIMM 3318, became suddenly resistant to fluconazole during the 10 to 16 weeks after antimycotic therapy. We investigated the resistant mechanism of fluconazole using one susceptible isolate and two of the five resistant isolates in the series. The ergosterol synthesis by cell-free extracts from the two resistant isolates was less susceptible to fluconazole partly as a result of a decreased affinity of cytochrome P-450. Unexpectedly, these two resistant isolates showed higher levels of an intracellular accumulation of [H]fluconazole than the susceptible isolate and the control strain of C. albicans ATCC 10231. In the resistant isolate, TIMM 3318, most intracellular incorporated fluconazole was distributed in the 12,000 X g pellet (P-120) fraction by centrifugation unlike the two susceptible strains. An observation of the ultrastructure of TIMM 3318 showed the most notable alteration to be the characteristic appearance of numerous vesicular vacuoles (diameter, 150 to 400 nm); these vacuoles were not observed, however, in either of the susceptible strains. A direct observation of the subcellular fraction prepared from TIMM 3318 by the electron microscopy negative-staining method suggests that most of the vesicular vacuoles were recovered in the P-120 fraction. These results suggest that fluconazole sequestration caused by vesicular vacuoles of the resistant isolate might act as a novel mechanism of fluconazole resistance besides the decreased affinity of cytochrome P-450.
Maebashi
Kazunori
K
Teikyo University Institute of Medical Mycology, Hachioji, Tokyo, Japan. kazunori_maebashi@meiji.co.jp
Kudoh
Michinari
M
Nishiyama
Yayoi
Y
Makimura
Koichi
K
Uchida
Katsuhisa
K
Mori
Takeshi
T
Yamaguchi
Hideyo
H
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics / Q000378:metabolism; D000935:Antifungal Agents / Q000378:metabolism / Q000494:pharmacology*; D002176:Candida albicans / Q000378:metabolism* / Q000648:ultrastructure; D002177:Candidiasis / Q000150:complications / Q000188:drug therapy / Q000382:microbiology*; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D025141:Drug Resistance, Fungal / Q000502:physiology; D004875:Ergosterol / Q000096:biosynthesis; D015725:Fluconazole / Q000378:metabolism / Q000494:pharmacology*; D005656:Fungal Proteins; D006801:Humans; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism; D008854:Microscopy, Electron; D010088:Oxidoreductases / Q000378:metabolism; D055728:Primary Myelofibrosis / Q000150:complications / Q000382:microbiology*; D012331:RNA, Fungal / Q000737:chemistry / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013104:Spheroplasts / Q000378:metabolism*; D058886:Sterol 14-Demethylase
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000935:Antifungal Agents; C094693:CDR1 protein, Candida albicans; C548418:CYP51A1 protein, human; D005656:Fungal Proteins; D026901:Membrane Transport Proteins; D012331:RNA, Fungal; D015725:Fluconazole; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D058886:Sterol 14-Demethylase; D004875:Ergosterol