Hypervirulence and cross-resistance to a clinical antifungal are induced by an environmental fungicide in Cryptococcus gattii.

The increasing human population requires ongoing efforts in food production. This is frequently associated with an increased use of agrochemicals, leading to environmental contamination and altering microbial communities, including human fungal pathogens that reside in the environment. Cryptococcus gattii is an environmental yeast and is one of the etiological agents of cryptococcosis. Benomyl (BEN) is a broad-spectrum fungicide used on several crops. To study the effects of agrochemicals on fungal pathogens, we first evaluated the susceptibility of C. gattii to BEN and the interactions with clinical antifungals. Antagonistic interaction between BEN and fluconazole was seen and was strain- and concentration-dependent. We then induced BEN-resistance by culturing strains in increasing drug concentrations. One strain demonstrated to be more resistant and showed increased multidrug efflux pump gene (MDR1) expression and increased rhodamine 6G efflux, leading to cross-resistance between BEN and fluconazole. Morphologically, BEN-adapted cells had a reduced polysaccharide capsule; an increased surface/volume ratio; increased growth rate in vitro and inside macrophages and also higher ability in crossing an in vitro model of blood-brain-barrier. BEN-adapted strain demonstrated to be hypervirulent in mice, leading to severe symptoms of cryptococcosis, early mortality and higher fungal burden in the organs, particularly the brain. The parental strain was avirulent in murine model. In vivo cross-resistance between BEN and fluconazole was observed, with mice infected with the adapted strain unable to present any improvement in survival and behavior when treated with this antifungal. Furthermore, BEN-adapted cells cultured in drug-free media maintained the hypervirulent and cross-resistant phenotype, suggesting a persistent effect of BEN on C. gattii. In conclusion, exposure to BEN induces cross-resistance with fluconazole and increases the virulence of C. gattii. Altogether, our results indicate that agrochemicals may lead to unintended consequences on non-target species and this could result in severe healthy problems worldwide.

Effect of non-antifungal agrochemicals on the pathogenic fungus Cryptococcus gattii.

The chemical control of pests and weeds is employed to improve crop production and the quality of agricultural products. The intensive use of pesticides, however, may cause environmental contamination, thus altering microbial communities. Cryptococcus gattii is an environmental yeast and the causative agent of cryptococcosis in both humans and animals. Up to this day, the effects of agrochemicals on human pathogens living in nature are still widely unknown. In this work, we analyzed the susceptibility of C. gattii to nonfungicide agrochemicals (herbicides and insecticides). Microdilution and drug-combination susceptibility tests were performed for the herbicides flumioxazin (FLX), glyphosate (GLY), isoxaflutole (ISO), pendimethalin (PEND), and also for the insecticide fipronil (FIP). Moreover, these compounds were combined with the clinical antifungals amphotericin B and fluconazole. The MIC values found for the agrochemicals were the following: < 16 µg/ml, for flumioxazin; 128 to 256 µg/ml, for FIP, ISO, and PEND; and >256 µg/ml, for GLY. Synergistic and antagonistic interactions, depending on the strain and concentration tested, were also observed. All strains had undergone adaptation to increasing levels of agrochemicals, in order to select the less susceptible subpopulations. During this process, one C. gattii strain (196 L/03) tolerated high concentrations (50 to 900 µg/ml) of all pesticides assessed. Subsequently, the strain adapted to flumioxazin, isoxaflutole and pendimethalin showed a reduction in the susceptibility to agrochemicals and clinical antifungals, suggesting the occurrence of cross-resistance. Our data point to the risk of exposing C. gattii to agrochemicals existing in the environment, once it might impact the susceptibility of clinical antifungals.

From the environment to the host: How non-azole agrochemical exposure affects the antifungal susceptibility and virulence of Cryptococcus gattii.

Agrochemicals such as the non-azoles, used to improve crop productivity, poses severe undesirable effects on the environment and human health. In addition, they induce cross-resistance (CR) with clinical drugs in pathogenic fungi. However, till date emphasis has been given to the role of azoles on the induction of CR. Herein, we analyzed the effect of a non-azole agrochemical, pyraclostrobin (PCT), on the antifungal susceptibility and virulence of the human and animal pathogens Cryptococcus gattii and C. neoformans. We determined the minimum inhibitory concentration (MIC) of fluconazole (FLC), itraconazole, ravuconazole, amphotericin B, and PCT on colonies: (i) that were not exposed to PCT (non-adapted-NA-cultures), (ii) were exposed at the maximum concentration of PCT (adapted-A-cultures) and (iii) the adapted colonies after cultivation 10 times in PCT-free media (10 passages-10p-cultures). Our results showed that exposure to PCT induced both temporary and permanent CR to clinical azoles in a temperature-dependent manner. With the objective to understand the mechanism of induction of CR through non-azoles, the transcriptomes of NA and 10p cells from C. gattii R265 were analyzed. The transcriptomic analysis showed that expression of the efflux-pump genes (AFR1 and MDR1) and PCT target was higher in resistant 10p cells than that in NA. Moreover, the virulence of 10p cells was reduced as compared to NA cells in mice, as observed by the differential gene expression analysis of genes related to ion-metabolism. Additionally, we observed that FLC could not increase the survival rate of mice infected with 10p cells, confirming the occurrence of permanent CR in vivo. The findings of the present study demonstrate that the non-azole agrochemical PCT can induce permanent CR to clinical antifungals through increased expression of efflux pump genes in resistant cells and that such phenomenon also manifests in vivo.

Environmental Triazole Induces Cross-Resistance to Clinical Drugs and Affects Morphophysiology and Virulence of Cryptococcus gattii and C. neoformans.

Cryptococcus gattii and Cryptococcus neoformans are environmental fungi that cause cryptococcosis, which is usually treated with amphotericin B and fluconazole. However, therapeutic failure is increasing because of the emergence of resistant strains. Because these species are constantly isolated from vegetal materials and the usage of agrochemicals is growing, we postulate that pesticides could be responsible for the altered susceptibility of these fungi to clinical drugs. Therefore, we evaluated the influence of the pesticide tebuconazole on the susceptibility to clinical drugs, morphophysiology, and virulence of C. gattii and C. neoformans strains. The results showed that tebuconazole exposure caused in vitro cross-resistance (CR) between the agrochemical and clinical azoles (fluconazole, itraconazole, and ravuconazole) but not with amphotericin B. In some strains, CR was observed even after the exposure ceased. Further, tebuconazole exposure changed the morphology, including formation of pseudohyphae in C. neoformans H99, and the surface charge of the cells. Although the virulence of both species previously exposed to tebuconazole was decreased in mice, the tebuconazole-exposed colonies recovered from the lungs were more resistant to azole drugs than the nonexposed cells. This in vivo CR was confirmed when fluconazole was not able to reduce the fungal burden in the lungs of mice. The tolerance to azoles could be due to increased expression of the ERG11 gene in both species and of efflux pump genes (AFR1 and MDR1) in C. neoformans Our study data support the idea that agrochemical usage can significantly affect human pathogens present in the environment by affecting their resistance to clinical drugs.

Atorvastatin as a promising anticryptococcal agent.

Cryptococcosis caused by Cryptococcus gattii leads to pneumonia and meningoencephalitis, and has a high mortality rate worldwide due to the inadequacy of available therapy and increasing drug resistance. There is a need to develop effective treatments, and drug repositioning is an interesting alternative to achieve new strategies to treat cryptococcosis. Atorvastatin (ATO), a statin currently used to treat hypercholesterolaemia, was tested in this study as an adjuvant to control infections caused by C. gattii. Several aspects of the effect of ATO on the host and the yeast were evaluated, with particular focus on the association of ATO with fluconazole (FLC), which (i) reduced ergosterol content in the cell membrane and altered properties of the polysaccharide capsule of C. gattii; (ii) increased the production of reactive oxygen species by macrophages; and (iii) reduced yeast phagocytosis and the intracellular proliferation rate. In an animal model, infected mice treated with ATO + FLC showed increased survival, improved clinical condition, and reduced fungal burden in the lungs and brain. This study is the first to perform in vivo tests with ATO + FLC for the treatment of cryptococcosis. The results suggest that ATO may be an important adjuvant for the treatment of cryptococcosis.

A subdose of fluconazole alters the virulence of Cryptococcus gattii during murine cryptococcosis and modulates type I interferon expression.

Cryptococcosis is an invasive infection caused by yeast-like fungus of the genera Cryptococcus spp. The antifungal therapy for this disease provides some toxicity and the incidence of infections caused by resistant strains increased. Thus, we aimed to assess the consequences of fluconazole subdoses during the treatment of cryptococcosis in the murine inflammatory response and in the virulence factors of Cryptococcus gattii. Mice infected with Cryptococcus gattii were treated with subdoses of fluconazole. We determined the behavior of mice and type 1 interferon expression during the treatment; we also studied the virulence factors and susceptibility to fluconazole for the colonies recovered from the animals. A subdose of fluconazole prolonged the survival of mice, but the morbidity of cryptococcosis was higher in treated animals. These data were linked to the increase in: (i) fluconazole minimum inhibitory concentration, (ii) capsule size and (iii) melanization of C. gattii, which probably led to the increased expression of type I interferons in the brains of mice but not in the lungs. In conclusion, a subdose of fluconazole altered fungal virulence factors and susceptibility to this azole, leading to an altered inflammatory host response and increased morbidity.

Photodynamic inhibition of Trichophyton rubrum: in vitro activity and the role of oxidative and nitrosative bursts in fungal death.

OBJECTIVES: Antimicrobial photodynamic inhibition (aPI) is based on the use of a light source and a photosensitizer to kill pathogens. Little is known about aPI of dermatophytic fungi and its mechanism of action. We aimed to evaluate aPI of Trichophyton rubrum. METHODS: We performed tests using toluidine blue (TBO) as a photosensitizer and a 630 nm light-emitting diode (LED) as a source of light to target 12 T. rubrum isolates. Susceptibility testing with cyclopiroxolamine, time-kill curves and quantification of reactive oxygen species (ROS), peroxynitrite (ONOO·) and nitric oxide (NO·) were performed. RESULTS: The optimal conditions for in vitro aPI were 10 mg/L for TBO and 48 J/cm(2) for LED; these conditions were fungicidal or inhibited >98% of fungal growth depending on the strain tested. LED or TBO treatment alone did not inhibit growth. The MICs of cyclopiroxolamine were 2.0 mg/L for 90% of the strains. Analysis of time-kill curves revealed that pathogen death occurred 24 h post-treatment. Quantification of ROS, ONOO· and NO· revealed improvement after aPI. CONCLUSIONS: Photodynamic inhibition was more efficient in promoting cell death than the antifungal cyclopiroxolamine against T. rubrum. ROS, ONOO· and NO· were important in the fungicidal activity of aPI. A suggested mechanism for this activity is that TBO is excited by LED light (630 nm), reacts with biomolecules and increases the availability of transition electrons and substrates for nitric oxide synthase, thereby increasing the oxidative and nitrosative bursts in the fungal cell.