Experimental induction of fluconazole resistance in Candida tropicalis ATCC 750

Abstract

Candida tropicalis is less commonly isolated from clinical specimens than Candida albicans. Unlike C. albicans, which can be occasionally found as a commensal, C. tropicalis is almost always associated with the development of fungal infections. In addition, C. tropicalis has been reported to be resistant to fluconazole (FLC). To analyze the development of FLC resistance in C. tropicalis, an FLC-susceptible strain (ATCC 750) (MIC = 1.0 microg/ml) was cultured in liquid medium containing increasing FLC concentrations from 8.0 to 128 microg/ml. The strain developed variable degrees of FLC resistance which paralleled the concentrations of FLC used in the medium. The highest MICs of FLC were 16, 256, and 512 microg/ml for strains grown in medium with 8.0, 32, and 128 microg of FLC per ml, respectively. Development of resistance was rapid and could be observed already after a single subculture in azole-containing medium. The resistant strains were cross-resistant to itraconazole (MIC > 1.0 microg/ml) and terbinafine (MIC > 512 microg/ml) but not to amphotericin B. Isolates grown in FLC at concentrations of 8.0 and 32 microg/ml reverted to low MICs (1.0 microg/ml) after 12 and 11 passages in FLC-free medium, respectively. The MIC for one isolate grown in FLC (128 microg/ml) (128 R) reverted to 16 microg/ml but remained stable over 60 passages in FLC-free medium. Azole-resistant isolates revealed upregulation of two different multidrug efflux transporter genes: the major facilitators gene MDR1 and the ATP-binding cassette transporter CDR1. The development of FLC resistance in vitro correlated well with the results obtained in an experimental model of disseminated candidiasis. While FLC given at 10 mg/kg of body weight/day was effective in reducing the fungal burden of mice infected with the parent strain, the same dosing regimen was ineffective in mice infected with strain 128 R. Finally, the acquisition of in vitro FLC resistance in strain 128 R was related to a loss of virulence. The results of our study elucidate important characteristics and potential mechanisms of FLC resistance in C. tropicalis.

FIG. 1

Variations of FLC, ITC, TRB, and AMB MICs for isolates grown in medium containing 8.0 (A), 32 (B), and 128 (C) μg of FLC per ml. Each datum point represents one passage.

FIG. 2

Variations of FLC and ITC MICs for isolates previously grown in medium containing 8.0 (A), 32 (B), and 128 (C) μg of FLC per ml. Each datum point represents one passage.

FIG. 3

Variation of CtMDR1 and CDR1 expression in FLC-susceptible and -resistant phenotypes of C. tropicalis ATCC 750. Lanes: ATCC 750, parent strain; 8-14-IND, isolate cultured in FLC at 8.0 μg/ml (14th passage); 8-13-REV, isolate previously cultured in FLC at 8.0 μg/ml and then passaged in FLC-free medium (13th passage); 32-30-REV, isolate previously cultured in FLC at 32 μg/ml and then passaged in FLC-free medium (30th passage); 128-13-IND, isolate cultured in FLC at 128 μg/ml (13th passage); 128-21-REV, isolate previously cultured in FLC at 128 μg/ml and then passaged in FLC-free medium (21st passage); 128-M1, 128-M2, and 128-M3, isolates cultured in FLC at 128 μg/ml and recovered from the kidneys of three mice 30 days postinfection.

FIG. 4

Virulence of FLC-susceptible (S [parent strain]; FLC MIC, 1.0 μg/ml) and FLC-resistant (R; organism cultured in FLC at 128 μg/ml; FLC MIC, 256 μg/ml) phenotypes of C. tropicalis ATCC 750 in an immunocompetent mouse model. Mice were inoculated with 10⁷ CFU via the lateral tail vein. (A) Survival curves. There were 15 mice per group (B) CFU of strain ATCC 750-infected kidneys recovered from animals sacrificed on days 5 and 30 postinfection. There were four to seven mice per time interval or group. Error bars show standard deviations from the mean.