Heteroresistance to Itraconazole Alters the Morphology and Increases the Virulence of Cryptococcus gattii

Abstract

cCryptococcus gattii is the main etiological agent of cryptococcosis in immunocompetent individuals. The triazole drug itraconazole is one of the antifungals used to treat patients with cryptococcosis. Heteroresistance is an adaptive mechanism to counteract the stress of increasing drug concentrations, and it can enhance the ability of a microorganism to survive under antifungal pressure. In this study, we evaluated the ability of 11 C. gattii strains to develop itraconazole heteroresistance. Heteroresistant clones were analyzed for drug susceptibility, alterations in cell diameter, capsule properties, and virulence in a murine model. Heteroresistance to itraconazole was intrinsic in all of the strains analyzed, reduced both the capsule size and the cell diameter, induced molecular heterogeneity at the chromosomal level, changed the negatively charged cells, reduced ergosterol content, and improved the antioxidant system. A positive correlation between surface/volume ratio of original cells and the level of heteroresistance to itraconazole (LHI) was observed in addition to a negative correlation between capsule size of heteroresistant clones and LHI. Moreover, heteroresistance to itraconazole increased the engulfment of C. gattii by macrophages and augmented fungal proliferation inside these cells, which probably accounted for the reduced survival of the mice infected with the heteroresistant clones and the higher fungal burden in lungs and brain. Our results indicate that heteroresistance to itraconazole is intrinsic and increases the virulence of C. gattii. This phenomenon may represent an additional mechanism that contributes to relapses of cryptococcosis in patients during itraconazole therapy.

Heteroresistance and fungi

“…Another study addressed the fact that stress caused by itraconazole alters the virulence in C. gattii . We observed a positive correlation between surface/volume ration (S/V ratio) of original cells and the level of heteroresistance to itraconazole, where high S/V ratios occur in small cells.…”

“…The resistant colonies grown on medium with the same concentration of drug generate a homogeneous population of resistant cells. Upon stepwise exposure to increasing concentrations of drug, subpopulations are observed to adapt to higher concentrations of the drug . Some authors compared the two methods .…”

“…They are considered a heterogeneous resistant population if upon maintenance on drug‐free medium the clones lost their resistance and returned to the stable initial susceptible phenotype. It is important to observe that the size of inoculum and temperature can alter the pattern of heteroresistance, but the same have not been seen for pH and osmolarity …”

“…For fungi, some studies show that heteroresistance to azoles seems to be intrinsic and others indicate that it is acquired. Several studies focused on Cryptococcus heteroresistance showed that all strains tested in repeated exposure to fluconazole or itraconazole led to a subset of resistant subpopulations . However, different data were found for Candida glabrata, Aspergillus fumigatus and even for Cryptococcus neoformans (Table ).…”

Effects of Alterations in Staphylococcus aureus Cell Membrane and Cell Wall in Antimicrobial Resistance

“…These resistant subpopulations are able to adapt to increasing drug concentrations in a stepwise manner [12]. This phenomenon has been described in a wide range of bacteria and fungi, but atention has been directed toward S. aureus [13].…”