Easy storage strategies for Sporothrix spp. strains.

The present study evaluated the maintenance of Sporothrix spp. (6 Sporothrix brasiliensis; 6 S. schenckii; 5 S. mexicana, and 3 S. globosa) in saline at 4°C, and in 10% glycerol plus either 10% lactose or 10% sucrose, at -20°C and -80°C. Viability was assessed after 3, 6, and 9 months of storage, through the recovery of strains on potato dextrose agar and analysis of macro- and micromorphological features. Conidium quantification was performed before and after storage, at 3, 6 and 9 months. 100% viability was observed, regardless of storage conditions or time period. Storage at 4°C and at -20°C did not alter the number of conidia, but lower conidium counts were observed at -80°C. This study shows that the combination of glycerol with lactose or sucrose is effective to maintain Sporothrix spp. at freezing temperatures.

Histoplasma capsulatum in planktonic and biofilm forms: in vitro susceptibility to amphotericin B, itraconazole and farnesol.

It is believed that most microbial infections are caused by pathogens organized in biofilms. Recently, it was shown that the dimorphic fungus Histoplasma capsulatum, estimated to be the most common cause of fungal respiratory diseases, is also able to form biofilm. Although the antifungal therapy commonly used is effective, refractory cases and recurrences have been reported. In the search for new compounds with antimicrobial activity, the sesquiterpene farnesol has gained prominence for its antifungal action. This study aimed to evaluate the in vitro susceptibility of H. capsulatum var. capsulatum to the antifungal agents itraconazole and amphotericin B, and farnesol alone and combined, as well as to determine the in vitro antifungal activity of these compounds against biofilms of this pathogen. The results show that farnesol has antifungal activity against H. capsulatum in the yeast and filamentous phases, with MIC values ranging from 0.0078 to 0.00312 µM. A synergistic effect (fractional inhibitory concentration index ≤0.5) between itraconazole and farnesol was found against 100 and 83.3 % of the isolates in yeast and mycelial forms, respectively, while synergism between amphotericin B and farnesol was only observed against 37.5 and 44.4 % of the isolates in yeast and filamentous forms, respectively. Afterwards, the antifungal drugs, itraconazole and amphotericin B, and farnesol alone, and the combination of itraconazole and farnesol, were tested against mature biofilms of H. capsulatum, through XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide) metabolic assay, and the itraconazole and amphotericin B showed lower antibiofilm activity when compared to farnesol alone and farnesol combined with itraconazole. In conclusion, farnesol showed promising results as an antifungal agent against H. capsulatum and also showed adjuvant action, especially when combined with itraconazole, increasing the fungal susceptibility to this drug.

In vitro inhibitory activity of terpenic derivatives against clinical and environmental strains of the Sporothrix schenkii complex.

Sporotrichosis is a subacute or chronic subcutaneous infection, caused by the fungus Sporothrix schenkii complex, occurring in human and animal tissues. Potassium iodide and itraconazole have been used as effective therapy for first-choice treatment, while amphotericin B may be indicated for disseminated infection. However, the adverse effects of potassium iodide and amphotericin B or the long duration of therapy with itraconazole often weigh against their use, leading to the search for alternatives for the treatment of severe infections. Terpinen-4-ol and farnesol are components of essential oils present in many plant species and have been described to have antifungal activity against microorganisms. In this study, 40 strains of Sporothrix spp. were tested for the susceptibility to terpinen-4-ol and farnesol. Changes in cytoplasmic membrane permeability were also investigated. Terpenes inhibited all Sporothrix strains with MIC values ranging from 87.9 to 1,429.8 µg/ml for terpinen-4-ol and from 0.003 to 0.222 µg/ml for farnesol. The MFC values ranged from 177.8 to 5,722.6 µg/ml and from 0.027 to 0.88 µg/ml, respectively, for terpinen-4-ol and farnesol. Farnesol was the most active compound for the Sporothrix strains. Significant loss of 260 and 280 nm-absorbing material did not occur after treatment with concentrations equivalent to the MIC and sub-MIC of the tested terpenes, when compared to corresponding untreated samples. The failure of terpenes to lyse Sporothrix cells suggests that their primary mechanism of action is not by causing irreversible cell membrane damage. Thus, new studies are needed to better understand the mechanisms involved in the antifungal activity.

In vitro inhibitory effect of miltefosine against strains of Histoplasma capsulatum var. capsulatum and Sporothrix spp.

Miltefosine (MIL), originally developed for use in cancer chemotherapy, has been shown to have important antifungal activity against several pathogenic fungi. Our aim in this study was to determine the in vitro activity of MIL against the dimorphic fungi Histoplasma capsulatum and Sporothrix spp. This was done using the broth microdilution method. MIL had an in vitro inhibitory effect against all strains of H. capsulatum var. capsulatum and Sporothrix spp. analyzed. The minimal inhibitory concentrations (MIC) varied from 0.25 µg/ml to 2 µg/ml for H. capsulatum var. capsulatum in the filamentous phase and from 0.125 µg/ml to 1 µg/ml in the yeast phase. The MIC interval for Sporothrix spp. in the filamentous phase was 0.25-2 µg/ml. The minimal fungicidal concentrations (MFCs) were ≤4 µg/ml for isolates of both analyzed species. This study demonstrates that MIL has an antifungal effect in vitro against two potentially pathogenic fungi and that more studies should be performed in order to evaluate its applicability in vivo.