Investigation of fluconazole heteroresistance in clinical and environmental isolates of Cryptococcus neoformans complex and Cryptococcus gattii complex in the state of Amazonas, Brazil

Abstract

Heteroresistance, defined as the occurrence of apparently homogeneous subpopulations of microbial cells showing different levels of antimicrobial susceptibility is a problem that has been associated with therapeutical failure in cryptococcosis. The purpose of the study was an investigation on the level of heteroresistance to fluconazole (LHF) as observed in clinical and environmental C. neoformans/C. gattii complex species isolates from Amazonas State (AM), Brazil. A total of 45 isolates and 9 type strains were analyzed. The assessments comprised testing for minimal inhibitory concentrations (MICs), for LHFs, for the strains' capacity of adaptation to high fluconazole (FLC) concentrations above the LHF, and for the stability of the heteroresistance phenomenon. The mean MICs for clinical isolates of C. gattii (6.4 µg/ml) were higher than those observed for environmental C. gattii strains (1.7 µg/ml) and clinical (3.7 µg/ml) as well as environmental (1.5 µg/ml) C. neoformans isolates. The phenomenon of heteroresistance to FLC was recorded for all isolates. On average, the LHF (8-256 µg/ml) of the isolates was 16 times higher than the FLC MICs (0.5-16 µg/ml) and a proportion of 85% isolates showed LHFs ≥ 16 µg/ml, 40% even ≥ 32 µg/ml. According to the adaptation assay, a considerable number of isolates (58%) showed the capacity of adaptation to MICs even higher than the initially recorded LHF. After the adaptation experiment, the adaptative-LHF values (8-512 µg/ml) were about 60 times higher than the original MIC values. After nine subsequent passages in drug-free broth, the isolates had their adaptative-LHF reduced. However, the LHF did not revert to the initially measured level. Our findings challenge the clinical interpretation of the antifungal MIC testing and motivate future studies correlating the levels of heteroresistance and parameters like LHF and adaptative-LHF with cryptococcosis-associated morbidity and mortality.

Lay summary: Cryptococcosis affects many people and is caused by fungi of the Cryptococcus neoformans/Cryptococcus gattii complexes. These agents appear to become more resistant to antifungals when exposed to increasing concentrations of antifungals due to a phenomenon called heteroresistance.

Keywords: Cryptococcus gattii complex; Cryptococcus neoformans complex; MIC; cryptococcosis; heteroresistance; susceptibility.