Heteroresistance of Cryptococcus gattii to fluconazole

Abstract

We analyzed 71 clinical and environmental Cryptococcus gattii strains that had been isolated before or after the advent of azole antifungals to determine their level of heteroresistance to fluconazole (LHF). All strains of C. gattii manifested heteroresistance, with LHFs that ranged between 4 microg/ml and 32 microg/ml. A considerably higher proportion of the C. gattii strains (86%) than Cryptococcus neoformans strains (46%) exhibited LHFs that were > or =16 microg/ml. No significant correlation was observed between the molecular type or serotypes of strains and their respective LHF. The strains which expressed a higher LHF were also more resistant to xenobiotics than the strains with a low LHF, and the level of resistance to xenobiotics was significantly higher than that reported for C. neoformans. The heteroresistant subpopulation, whose level of drug resistance had been raised in a stepwise manner to 64 microg/ml, reverted to the original LHF upon daily transfers in drug-free medium. Importantly, the strains with high LHFs were significantly more virulent than those with low LHFs. Since all the clinical isolates that had not been exposed to azole drugs as well as the environmental strains manifested heteroresistance to fluconazole, heteroresistance of C. gattii to azoles is an intrinsic mechanism as in C. neoformans and is associated with the strain's virulence.

FIG. 1.

Determination of fluconazole resistance by Etest analysis. Three representative cultures, exhibiting resistance to 8, 16, and 32 μg/ml fluconazole, are shown.

FIG. 2.

Determination of LHF by spot test analysis. Spot test analysis representing growth of C. gattii strains on YPD agar medium alone (A) and YPD agar medium supplemented with 8 μg/ml (B) or 16 μg/ml (C) fluconazole. Lanes 1 to 4 represent 10-fold serial dilutions of culture. This example shows the spot test of a strain determined to have the LHF of 16 μg/ml FLC.

FIG. 3.

(A) Examples of PCR fingerprint patterns obtained by using the M13 primer for clinical and environmental strains of C. gattii that represent the four different molecular types. Lane 1, size marker; lanes 2 to 5, reference strains representing the four molecular types VGI to VGIV. (B) Examples of URA5 gene restriction fragment length polymorphism profiles from clinical and environmental isolates of C. gattii representing the four different molecular types. Lanes are as in Fig. 1A.

FIG. 4.

Loss of the acquired tolerance to fluconazole by heteroresistant subpopulations of C. gattii strains upon daily transfer of the cultures grown at 30°C in YPD medium without fluconazole.

FIG. 5.

Spot test analysis to determine resistance of the C. gattii strains to different xenobiotics: gliotoxin (Glio., 3.2 μg/ml), rhizoxin (Rhizo., 0.5 μg/ml), and trichostatin A (Trich. A, 64 μg/ml). The strains selected represent the four groups of strains that exhibited heteroresistance at 4, 8, 16, and 32 μg/ml fluconazole (boldface in Table 2).

FIG. 6.

Virulence in BALB/c mice following intranasal inoculation of 5 × 10⁷ cells per mouse with the selected C. gattii strains from different geographic regions that exhibited different levels of heteroresistance to fluconazole.