Rapid, transient fluconazole resistance in Candida albicans is associated with increased mRNA levels of CDR

Abstract

Fluconazole-resistant Candida albicans, a cause of recurrent oropharyngeal candidiasis in patients with human immunodeficiency virus infection, has recently emerged as a cause of candidiasis in patients receiving cancer chemotherapy and marrow transplantation (MT). In this study, we performed detailed molecular analyses of a series of C. albicans isolates from an MT patient who developed disseminated candidiasis caused by an azole-resistant strain 2 weeks after initiation of fluconazole prophylaxis (K. A. Marr, T. C. White, J. A. H. vanBurik, and R. A. Bowden, Clin. Infect. Dis. 25:908-910, 1997). DNA sequence analysis of the gene (ERG11) for the azole target enzyme, lanosterol demethylase, revealed no difference between sensitive and resistant isolates. A sterol biosynthesis assay revealed no difference in sterol intermediates between the sensitive and resistant isolates. Northern blotting, performed to quantify mRNA levels of genes encoding enzymes in the ergosterol biosynthesis pathway (ERG7, ERG9, and ERG11) and genes encoding efflux pumps (MDR1, ABC1, YCF, and CDR), revealed that azole resistance in this series is associated with increased mRNA levels for members of the ATP binding cassette (ABC) transporter superfamily, CDR genes. Serial growth of resistant isolates in azole-free media resulted in an increased susceptibility to azole drugs and corresponding decreased mRNA levels for the CDR genes. These results suggest that C. albicans can become transiently resistant to azole drugs rapidly after exposure to fluconazole, in association with increased expression of ABC transporter efflux pumps.

FIG. 1

Susceptibilities of isolates and exposure to fluconazole. MICs of fluconazole (○), ketoconazole (□), and itraconazole (◊) for each isolate are shown on the left y axis, and the total cumulative doses of fluconazole administered to the patient (▴) are shown on the right y axis. Isolates 1 through 9 were obtained on days −9, −2, 6, 15, 17, 18, 22, 23, and 28 relative to transplantation (day 0), respectively. Fluconazole was administered from days −7 to 7 and from days 12 to 21, and AmB was administered from days 7 to 10 and from days 20 to 25.

FIG. 2

Results of sterol intermediate assay. Relative intensities of intermediates, measured as described in the text, for susceptible isolate 1 (open symbols) and for resistant isolate 5 (solid symbols) are shown. Cell extracts were incubated in increasing concentrations of fluconazole (x axis). Relative intensities of the intermediates lanosterol (○) squalene (□), 2,3-oxidosqualene (◊), and ergosterol (▵) were measured by phosphorimaging.

FIG. 3

Northern analysis of mRNA of efflux pump genes. Signal intensities of mRNA levels of CDR (■), ABC1 (○), and MDR1 (▿) were quantified by phosphorimaging relative to actin probe and normalized to the intensity of the first isolate (as described in the text).

FIG. 4

Stability of fluconazole resistance after growth in drug-free media. (A) Fluconazole MICs for isolates 2 (■), 5 (○), and 8 (▵), serially transferred in drug-free media, are shown. Each transfer represents approximately 12 generations of growth. MICs were determined by E test and confirmed by the NCCLS method (see text). (B) Susceptibilities of transferred isolates to other azoles. MICs for the initial fluconazole-resistant (isolates 5 and 8) and -susceptible (isolates 5T and 8T) isolates are shown. 5T, 5T33; 8T, 8T12. Fluconazole (□), ketoconazole (▵), and itraconazole (○) MICs were determined by E test, and additional MICs of fluconazole (◊) and AmB (■) were determined by macrodilution methods.

FIG. 5

Analysis of revertant isolates. Resistant isolates 5 and 8 are compared with the sensitive isolates obtained after serial transfer (isolates 5T and 8T). (A) RFLP analysis of genomic DNA from isolates, after digestion with EcoRI, and hybridization with the Ca3 probe. 5T, 5T33; 8T, 8T12. (B) Intensities of mRNA signals from Northern blots, probed with a CDR probe (upper panel). The blot was stripped and rehybridized with an actin probe (lower panel). (C) Levels of CDR mRNA were quantified by phosphorimaging of the Northern blot signals, calculated relative to actin controls, and normalized to the level for the resistant isolate for each sample.