Ultraviolet light C prototype device against Sporothrix brasiliensis as a potential physical method for surface disinfection.

Sporothrix brasiliensis is recognized as an emergent fungal pathogen and the high amount of fungal propagules in the lesions of infected cats allows the contamination of surfaces by direct contact. Given that the environment can play a role in the transmission of this fungus, effective methods to eliminate this pathogen from contaminated surfaces are necessary. Physical methods, such as ultraviolet light C (UVC), are broad used for surfaces disinfection, however, non-data about its activity against S. brasiliensis is reported. Therefore, we aimed to evaluate an easy handled prototype of a UVC device, in the inhibition of S. brasiliensis. Three doses and times of exposure of irradiance were tested: 3.5 mJ/cm2 (1 s), 5.25 mJ/cm2 (1.5 s) and 329 mJ/cm2 (94 s) against a standardized inoculum of yeast and mold phase of S. brasiliensis. A decrease in CFU was shown in all doses of irradiance in both phases of S. brasiliensis, the average reduction ranged from 78 to 100% among doses, being a complete fungicidal activity achieved against the yeast phase after the 94 s exposure (329 mJ/cm2). Our data shows that UVC is a potential physical method for disinfection of surfaces contaminated with S. brasiliensis, and the prototype device developed provides an easy handling, and quickly results.

In vitro photodynamic inactivation of Sporothrix schenckii complex species.

Photodynamic therapy has been applied successfully against cutaneous and subcutaneous mycoses. We applied methylene blue as a photosensitizing agent and light emitting diode (InGaAlP) against Sporothrix schenckii complex species in an in vitro assay. The viability of the conidia was determined by counting colony-forming units. Methylene blue in conjunction with laser irradiation was able to inhibit the growth of all tested samples. The in vitro inhibition of Sporothrix spp. isolates by laser light deserves in vivo experimental and clinical studies since it may be a promising treatment for cutaneous and subcutaneous sporotrichosis.

Biosynthesis and functions of a melanoid pigment produced by species of the sporothrix complex in the presence of L-tyrosine.

Sporothrix schenckii is the etiological agent of sporotrichosis, the main subcutaneous mycosis in Latin America. Melanin is an important virulence factor of S. schenckii, which produces dihydroxynaphthalene melanin (DHN-melanin) in conidia and yeast cells. Additionally, l-dihydroxyphenylalanine (l-DOPA) can be used to enhance melanin production on these structures as well as on hyphae. Some fungi are able to synthesize another type of melanoid pigment, called pyomelanin, as a result of tyrosine catabolism. Since there is no information about tyrosine catabolism in Sporothrix spp., we cultured 73 strains, including representatives of newly described Sporothrix species of medical interest, such as S. brasiliensis, S. schenckii, and S. globosa, in minimal medium with tyrosine. All strains but one were able to produce a melanoid pigment with a negative charge in this culture medium after 9 days of incubation. An S. schenckii DHN-melanin mutant strain also produced pigment in the presence of tyrosine. Further analysis showed that pigment production occurs in both the filamentous and yeast phases, and pigment accumulates in supernatants during stationary-phase growth. Notably, sulcotrione inhibits pigment production. Melanin ghosts of wild-type and DHN mutant strains obtained when the fungus was cultured with tyrosine were similar to melanin ghosts yielded in the absence of the precursor, indicating that this melanin does not polymerize on the fungal cell wall. However, pyomelanin-producing fungal cells were more resistant to nitrogen-derived oxidants and to UV light. In conclusion, at least three species of the Sporothrix complex are able to produce pyomelanin in the presence of tyrosine, and this pigment might be involved in virulence.

Gamma radiation effects on Sporothrix schenckii yeast cells.

Sporotrichosis is a subcutaneous mycosis caused by Sporothrix schenckii. Zoonotic transmission to man can occur after scratches or bites of animals, mainly cats. In this study, the gamma radiation effects on yeast of S. schenckii were analyzed with a view of developing a radioattenuated vaccine for veterinary use. The cultures were irradiated at doses ranging from 1.0 to 9.0 kGy. The reproductive capacity was measured by the ability of cells to form colonies. No colonies could be recovered above 8.0 kGy, using inocula up to 10(7) cells. Nevertheless, yeast cells irradiated with 7.0 kGy already were unable to produce infection in immunosuppressed mice. Evaluation by the FungaLight™ Kit (Invitrogen) indicated that yeast cells remained viable up to 9.0 kGy. At 7.0 kGy, protein synthesis, estimated by the incorporation of [L-(35)S] methionine, continues at levels slightly lower than the controls, but a significant decrease was observed at 9.0 kGy. The DNA of 7.0 kGy irradiated cells, analyzed by electrophoresis in agarose gel, was degraded. Cytoplasmic vacuolation was the main change verified in these cells by transmission electron microscopy. The dose of 7.0 kGy was considered satisfactory for yeast attenuation since irradiated cells were unable to produce infection but retained viability, metabolic activity, and morphology.

Biosynthesis and functions of melanin in Sporothrix schenckii.

Sporothrix schenckii is a human pathogen that causes sporotrichosis, an important cutaneous mycosis with a worldwide distribution. It produces dark-brown conidia, which infect the host. We found that S. schenckii synthesizes melanin via the 1,8-dihydroxynaphthalene pentaketide pathway. Melanin biosynthesis in the wild type was inhibited by tricyclazole, and colonies of the fungus were reddish brown instead of black on tricyclazole-amended medium. Two melanin-deficient mutant strains were analyzed in this study: an albino that produced normal-appearing melanin on scytalone-amended medium and a reddish brown mutant that accumulated and extruded melanin metabolites into its medium. Scytalone and flaviolin obtained from cultures of the reddish brown mutant were identified by thin-layer chromatography, high-performance liquid chromatography, and UV spectra. Transmission electron microscopy showed an electron-dense granular material believed to be melanin in wild-type conidial cell walls, and this was absent in conidial walls of the albino mutant unless the albino was grown on a scytalone-amended medium. Melanized cells of wild-type S. schenckii and the albino grown on scytalone-amended medium were less susceptible to killing by chemically generated oxygen- and nitrogen-derived radicals and by UV light than were conidia of the mutant strains. Melanized conidia of the wild type and the scytalone-treated albino were also more resistant to phagocytosis and killing by human monocytes and murine macrophages than were unmelanized conidia of the two mutants. These results demonstrate that melanin protects S. schenckii against certain oxidative antimicrobial compounds and against attack by macrophages.