Resumo

C. gattii strains have high potential to develop in vitro resistance to fluconazole (FLC), which could explain the constant therapeutic failures and relapses in patients affected by Cryptococcosis under FLC long-term therapy. Heteroresistance is the phenomenon described as the emergence of a minor subpopulation of resistant cells within a single colony of the susceptible strain that can tolerate FLC concentrations higher than the strain’s MIC levels. We aimed in this study investigate the level of FLC-heteroresistance exhibited by 18 C. gattii VGII clinical strains (1 per patient) from the culture collection (1994-2004) of Institute Adolfo Lutz (SP, Br). Firstly, the MIC of FLC was determined by Etest^® method for each strain, and then its level of heteroresistance to FLC (LHF) was assessed. Cell suspensions of all strains were inoculated onto YPD plates containing various concentrations of FLC (4 to 128 mg/L). The lowest FLC concentration at which minor resistant subpopulations emerged was identified as each isolate’s LHF. To obtain the highly resistant subclones, the heteroresistant colonies were isolated and inoculated on YPD agar containing stepwise (twofold) increases in the concentrations of FLC (up to 256 mg/L). The culture plates of each passage were incubated at 30°C for 72 to 96h. Finally, we have analyzed if the transferring of the highly resistant subclones on drug-free media caused reversion to the original level of heteroresistance. The MICs for these 18 strains ranged between 1 to 24 mg/L. Similarly to the unique published study we have demonstrated that all C. gattii tested strains manifested FLC-heteroresistance with LHFs that ranged between 32mg/L and 128 mg/L. The highly resistant subclones have been raised in a stepwise manner to 256 mg/L. All strains reverted to the original LHF upon daily transfers in drug-free medium. In conclusion we observed that in C. gattii the level of FLC-heteroresistance was high (LHF ≥32 mg/L) and varies between strains. Moreover, all 18 tested strains (100%) showed highly resistant subclones (growth at 256 mg/L). We confirmed it is a reversible adaptive response to the presence of the drug. The study on heteroresistance may reveal a novel adaptive mechanism for survival under the azole stress and can offer helpful insights into the management of long-term fluconazole therapy cases. Master Fellowship from Fundação de Amparo à Pesquisa do Estado de São Paulo-FAPESP