Characterization of Heteroresistance to Fluconazole among Clinical Isolates of Cryptococcus neoformans

Abstract

Strains of Cryptococcus neoformans expressing heteroresistance to fluconazole have been described previously. The present study was conducted to investigate the prevalence of heteroresistance among clinical isolates of C. neoformans and to characterize the heteroresistant phenotypes. A total of 107 clinical isolates of C. neoformans for which the MICs of fluconazole ranged from 0.25 to 32 g/ml were selected. The isolates were chosen to represent a broad geographic distribution. Of the 107 C. neoformans isolates tested, 4 grew on medium containing fluconazole at concentrations that were four to eight times higher than the MICs for each strain. A fifth isolate, for which the fluconazole MIC was 32 g/ml, grew on agar with 64 g of fluconazole per ml. These five isolates (4.7% of the total number) were confirmed to exhibit heteroresistant compositions by population analysis. The degree and frequency of resistance varied among the isolates. Stepwise selection by exposure to fluconazole resulted in subclones of all five strains for which the fluconazole MIC was >64 g/ml. Subclones of three strains demonstrated a homogenous population of resistant cells on medium containing 64 g of fluconazole/ml. The resistance was sensitive to incubation temperature, that is, heteroresistance was demonstrable only at 30°C by agar-based tests, and was reversible through serial transfers on fluconazole-free medium over a period of 8 days. These results suggest that the fluconazole-heteroresistant phenotype of C. neoformans exists in a significant proportion of clinical isolates and that fluconazole resistance can be developed by selection from heteroresistant clones and induction by exposure to fluconazole.

Stress, Drugs, and Evolution: the Role of Cellular Signaling in Fungal Drug Resistance

“…Strikingly, the resistance is affected by temperature, as it is suppressed at 35°C and abolished at 40°C. A subsequent study examining 107 clinical isolates found fluconazole heteroresistance in 5 isolates and confirmed both that fluconazole resistance could be developed by selection of heteroresistant clones in the presence of fluconazole and that heteroresistance was suppressed at increased temperatures (236). Notably, separate from the phenomenon of heteroresistance, the patterns of mutation to fluconazole resistance in C. neoformans are complex.…”

Pathophysiology of Escherichia coli ventilator-associated pneumonia: implication of highly virulent extraintestinal pathogenic strains

“…This finding may explain clinical failure of antimicrobial treatment when the more susceptible strain has been picked for the antibiogram. We recently described the same phenomenon in other E. coli extraintestinal infections [43], and it has been reported for other microorganisms [44][45][46][47]. We, therefore, believe that several colonies of a single respiratory sample should be tested for antimicrobial susceptibility in case of GNB VAP.…”

Regulatory Circuitry Governing Fungal Development, Drug Resistance, and Disease

“…The resistant subpopulations can adapt to increasing concentrations of the azoles in a stepwise manner; however, the original susceptibility is restored after passage in the absence of an antifungal. This mechanism of resistance has been observed among clinical isolates and is unrelated to prior drug exposure and unaffected by pH or osmolarity (387,639). Notably, resistance is affected by temperature, as heteroresistance phenotypes can be suppressed at 35°C and are abolished at 40°C (387,639).…”

“…This mechanism of resistance has been observed among clinical isolates and is unrelated to prior drug exposure and unaffected by pH or osmolarity (387,639). Notably, resistance is affected by temperature, as heteroresistance phenotypes can be suppressed at 35°C and are abolished at 40°C (387,639). Most recently, a study analyzing 130 C. neoformans strains isolated from clinical and environmental sources before 1979, prior to azole use, and 16 fluconazole-resistant strains isolated from AIDS patients undergoing fluconazole therapy from 1990 to 2000 found that all the strains manifested heteroresistance, confirming that this phenomenon is universal and unrelated to prior drug exposure (537).…”