Heteroresistance to fluconazole and voriconazole in Cryptococcus neoformans

Cryptococcus neoformans isolates that exhibited unusual patterns of resista nce to fluconazole and voriconazole were isolated from seven isolates from two different geographical regions: one isolate from an Israeli non-AIDS pa tient and six serial isolates fi om an Italian AIDS patient who had suffere d six recurrent episodes of cryptococcal meningitis. Each isolate produced cultures with heterogeneous compositions in which most of the cells were su sceptible, but cells highly resistant to fluconazole (MICs, greater than or equal to 64 mu g/ml) were recovered at a variable frequency (7 x 10(-3) to 4.6 x 10(-2)), Evidence showed that this type of resistance is innate and is unrelated to drug exposure since the Israeli patient had never been trea ted with azoles or any other antimycotic agents. Analysis of clonal subpopu lations of these two strains showed that they exhibited heterogeneous patte rns of resistance. The number of subpopulations which grew on fluconazole o r voriconazole agar declined progressively with increasing azole concentrat ion without a sharp cutoff point, For the Italian serial isolates, the numb er of clonal populations resistant to fluconazole (64 mu g/ml) and voricona zole (1 mu g/ml) increased steadily, yielding the highest number for the is olate from the last episode. Attempts to purify a sensitive subpopulation f ailed, but clones highly resistant to fluconazole (100 mu g/ml) and moderat ely resistant to voriconazole (1 mu g/ml) always produced a homogeneous pop ulation of resistant cells. Upon maintenance on drug-free medium, however, the majority of the homogeneously resistant cells of these subclones lost t heir resistance and returned to the stable initial heteroresistant phenotyp e. The pattern of heteroresistance was not affected by the pH or osmolarity of the medium but was influenced by temperature. The resistance appeared t o be suppressed at 35 degrees C and was completely abolished at 40 degrees C. Although heterogeneity in azole resistance among subpopulations of singl e isolates has been reported for Candida species, the transient changes in expression of resistance under different growth conditions reported here ha ve not been observed in fungal pathogens.