Comparison of the antifungal activity of the pyrimidine analogs flucytosine and carmofur against human-pathogenic dematiaceous fungi.

Chromoblastomycosis (CBM) and pheohyphomycosis (PHM) are the most common implantation mycoses caused by dematiaceous fungi. In the past, flucytosine (5-FC) has been used to treat CBM, but development of resistance is common. Carmofur belongs to the same class as 5-FC and has in vitro inhibitory activity against the main agents of CBM and PHM. The aim of this study was to compare the action of these two pyrimidine analog drugs against CBM and PHM agents. The minimum inhibitory concentration (MIC) and the selectivity index based on cytotoxicity tests of these two drugs against some agents of these mycoses were determined, with carmofur presenting a higher selectivity index than 5-FC. Carmofur demonstrated here synergistic interactions with itraconazole and amphotericin B against Exophiala heteromorpha, Fonsecaea pedrosoi, Fonsecaea monophora, and Fonsecaea nubica strains. Additionally, carmofur plus itraconazole demonstrated here synergism against a Phialophora verrucosa strain. To evaluate the development of carmofur resistance, passages in culture medium containing subinhibitory concentrations of this pyrimidine analog were carried out, followed by in vitro susceptibility tests. Exophiala dermatitidis quickly developed resistance, whereas F. pedrosoi took seven passages in carmofur-supplemented medium to develop resistance. Moreover, resistance was permanent in E. dermatitidis but transient in F. pedrosoi. Hence, carmofur has exhibited certain advantages, albeit accompanied by limitations such as the development of resistance, which was expected as with 5-FC. This underscores its therapeutic potential in combination with other drugs, emphasizing the need for a meticulous evaluation of its application in the fight against dematiaceous fungi.

Metabolic and phenotypic plasticity may contribute for the higher virulence of Trichosporon asahii over other Trichosporonaceae members.

BACKGROUND: The Trichosporonaceae family comprises a large number of basidiomycetes widely distributed in nature. Some of its members, especially Trichosporon asahii, have the ability to cause human infections. This ability is related to a series of virulence factors, which include lytic enzymes production, biofilm formation, resistance to oxidising agents, melanin and glucuronoxylomannan in the cell wall, metabolic plasticity and phenotypic switching. The last two are poorly addressed within human pathogenic Trichosporonaceae. OBJECTIVE: These factors were herein studied to contribute with the knowledge of these emerging pathogens and to uncover mechanisms that would explain the higher frequency of T. asahii in human infections. METHODS: We included 79 clinical isolates phenotypically identified as Trichosporon spp. and performed their molecular identification. Lactate and N-acetyl glucosamine were the carbon sources of metabolic plasticity studies. Morphologically altered colonies after subcultures and incubation at 37°C indicated phenotypic switching. RESULTS AND CONCLUSION: The predominant species was T. asahii (n = 65), followed by Trichosporon inkin (n = 4), Apiotrichum montevideense (n = 3), Trichosporon japonicum (n = 2), Trichosporon faecale (n = 2), Cutaneotrichosporon debeurmannianum (n = 1), Trichosporon ovoides (n = 1) and Cutaneotrichosporon arboriforme (n = 1). T. asahii isolates had statistically higher growth on lactate and N-acetylglucosamine and on glucose during the first 72 h of culture. T. asahii, T. inkin and T. japonicum isolates were able to perform phenotypic switching. These results expand the virulence knowledge of Trichosporonaceae members and point for a role for metabolic plasticity and phenotypic switching on the trichosporonosis pathogenesis.

New possibilities for chromoblastomycosis and phaeohyphomycosis treatment: identification of two compounds from the MMV Pathogen Box® that present synergism with itraconazole.

BACKGROUND: Black fungi of the Herpotrichiellaceae family are agents of chromoblastomycosis and phaeohyphomycosis. There are few therapeutic options for these infections and it is common to associate antifungal drugs in their treatment. OBJECTIVES: To investigate the Medicines for Malaria Venture (MMV) Pathogen Box® for possible compounds presenting synergism with antifungal drugs used to treat black fungal infections. METHODS: An initial screening of the Pathogen Box® compounds was performed in combination with itraconazole or terbinafine at sub-inhibitory concentrations against Fonsecaea pedrosoi. Hits were further tested against eight Herpotrichiellaceae using the checkerboard method. FINDINGS: No synergism was observed with terbinafine. MMV687273 (SQ109) and MMV688415 showed synergism with itraconazole against F. pedrosoi. Synergism of these compounds was confirmed with some black fungi by the checkerboard method. SQ109 and itraconazole presented synergism for Exophiala dermatitidis, F. pedrosoi, F. monophora and F. nubica, with fungicidal activity for F. pedrosoi and F. monophora. MMV688415 presented synergism with itraconazole only for F. pedrosoi, with fungicidal activity. The synergic compounds had high selectivity index values when combined with itraconazole. MAIN CONCLUSIONS: These compounds in combination, particularly SQ109, are promising candidates to treat Fonsecaea spp. and E. dermatitidis infections, which account for most cases of chromoblastomycosis and phaeohyphomycosis.

A case-series of bloodstream infections caused by the Meyerozyma guilliermondii species complex at a reference center of oncology in Brazil.

Bloodstream infections (BSI) caused by Candida species are the fourth cause of healthcare associated infections worldwide. Non-albicans Candida species emerged in the last decades as agents of serious diseases. In this study, clinical and microbiological aspects of six patients with BSI due to the Meyerozyma (Candida) guilliermondii species complex from an oncology reference center in Brazil, were evaluated. To describe demographic and clinical characteristics, medical records of the patients were reviewed. Molecular identification of the isolates was performed by ITS1-5.8S-ITS2 region sequencing. Antifungal susceptibility was evaluated by the EUCAST method and the minimal inhibitory concentrations (MIC) assessed according to the epidemiological cutoff values. Virulence associated phenotypes of the isolates were also studied. Ten isolates from the six patients were evaluated. Five of them were identified as Meyerozyma guilliermondii and the others as Meyerozyma caribbica. One patient was infected with two M. caribbica isolates with different genetic backgrounds. High MICs were observed for fluconazole and echinocandins. Non-wild type isolates to voriconazole appeared in one patient previously treated with this azole. Additionally, two patients survived, despite infected with non-wild type strains for fluconazole and treated with this drug. All isolates produced hemolysin, which was not associated with a poor prognosis, and none produced phospholipases. Aspartic proteases, phytase, and esterase were detected in a few isolates. This study shows the reduced antifungal susceptibility and a variable production of virulence-related enzymes by Meyerozyma spp. In addition, it highlights the poor prognosis of neutropenic patients with BSI caused by this emerging species complex. LAY ABSTRACT: Our manuscript describes demographic, clinical and microbiological characteristics of patients with bloodstream infection by the Meyerozyma guilliermondii species complex at a reference center in oncology in Brazil.

Production of Secreted Carbohydrates that Present Immunologic Similarities with the Cryptococcus Glucuronoxylomannan by Members of the Trichosporonaceae Family: A Comparative Study Among Species of Clinical Interest.

Glucuronoxylomannan (GXM) participates in several immunoregulatory mechanisms, which makes it an important Cryptococcus virulence factor that is essential for the disease. Trichosporon asahii and Trichosporon mucoides share with Cryptococcus species the ability to produce GXM. To check whether other opportunistic species in the Trichosporonaceae family produce GXM-like polysaccharides, extracts from 28 strains were produced from solid cultures and their carbohydrate content evaluated by the sulfuric acid / phenol method. Moreover, extracts were assessed for cryptococcal GXM cross-reactivity through latex agglutination and lateral flow assay methods. Cryptococcus neoformans and Saccharomyces cerevisiae were used as positive and negative controls, respectively. In addition to T. asahii, the species Trichosporon inkin, Apiotrichum montevideense, Trichosporon japonicum, Trichosporon faecale, Trichosporon ovoides, Cutaneotrichosporon debeurmannianum, and Cutaneotrichosporon arboriformis are also producers of a polysaccharide immunologically similar to the GXM produced by human pathogenic Cryptococcus species. The carbohydrate concentration of the extracts presented a positive correlation with the GXM contents determined by titration of both methodologies. These results add several species to the list of fungal pathogens that produce glycans of the GXM type and bring information about the origin of potential false-positive results on immunological tests for diagnosis of cryptococcosis based on GXM detection.

Comparative Analysis of Putative Virulence-Associated Factors of Microsporum canis Isolates from Human and Animal Patients.

Microsporum canis is a zoophilic dermatophyte and the most common fungus isolated from dogs and cats worldwide. To invade skin, this pathogen uses different enzymes, which may be associated with virulence, that contribute to the fungal pathogenicity. The aim of this study is to compare the expression of enzymes that may be associated with virulence, and thermotolerance of M. canis strains isolated from dogs, cats, and humans. The in vitro expression of the enzymes keratinase, catalase, urease, hemolysin, and aspartic protease was evaluated in 52 M. canis strains recently isolated from 14 human patients, 12 dogs, 15 symptomatic, and 11 asymptomatic cats. In addition, thermotolerance was assessed by comparative analysis of fungal growth at 25 °C and 35 °C. Keratinase activity was low in 34 and moderate in 18 strains. Aspartic-protease activity was low in 7, moderate in 33, and high in 12 strains. Hemolysin activity was low in 44 and moderate in 8 strains. All strains were classified as low producers of catalase. All but three strains produced urease in vitro, with a broad range of activity. The strains presented in vitro growth at the two studied temperatures were classified as presenting low (36.5%), medium (44.3%), or high (19.2%) thermotolerance. There was no statistically significant difference in the new putative virulence-associated factors studied among the different hosts, which suggests that they may have a similar role on human, cat, and dog infection. Also, no difference was observed between strains isolated from symptomatic and asymptomatic cats. This suggests that these factors have a limited impact on the fate of feline dermatophytosis caused by M. canis.

Evaluation of melanin production by Sporothrix luriei.

There is a paucity of studies on the cell biology of Sporothrix luriei, the less common of the pathogenic Sporothrix species worldwide. The production of DHN-melanin, eumelanin, and pyomelanin were evaluated on the mycelial and yeast forms of the S. luriei ATCC 18616 strain. The mycelial form of this species produced only pyomelanin, which protected the fungus against environmental stressors such as ultraviolet light, heat, and cold. The yeast form was unable to produce any of the tested melanin types. The lack of melanin in the parasitic form of S. luriei may be an explanation for its low frequency in human infections.

Relationship between the Antifungal Susceptibility Profile and the Production of Virulence-Related Hydrolytic Enzymes in Brazilian Clinical Strains of Candida glabrata.

Candida glabrata is a facultative intracellular opportunistic fungal pathogen in human infections. Several virulence-associated attributes are involved in its pathogenesis, host-pathogen interactions, modulation of host immune defenses, and regulation of antifungal drug resistance. This study evaluated the in vitro antifungal susceptibility profile to five antifungal agents, the production of seven hydrolytic enzymes related to virulence, and the relationship between these phenotypes in 91 clinical strains of C. glabrata. All C. glabrata strains were susceptible to flucytosine. However, some of these strains showed resistance to amphotericin B (9.9%), fluconazole (15.4%), itraconazole (5.5%), or micafungin (15.4%). Overall, C. glabrata strains were good producers of catalase, aspartic protease, esterase, phytase, and hemolysin. However, caseinase and phospholipase in vitro activities were not detected. Statistically significant correlations were identified between micafungin minimum inhibitory concentration (MIC) and esterase production, between fluconazole and micafungin MIC and hemolytic activity, and between amphotericin B MIC and phytase production. These results contribute to clarify some of the C. glabrata mechanisms of pathogenicity. Moreover, the association between some virulence attributes and the regulation of antifungal resistance encourage the development of new therapeutic strategies involving virulence mechanisms as potential targets for effective antifungal drug development for the treatment of C. glabrata infections.

Comparison of four molecular approaches to identify Candida parapsilosis complex species.

Since the description of Candida orthopsilosis and C. metapsilosis in 2005, several methods have been proposed to identify and differentiate these species from C. parapsilosis sensu stricto. Species-specific uniplex polymerase chain reaction (PCR) was performed and compared with sequencing of the D1/D2 region of the LSU 28S rDNA gene, microsatellite typing of C. parapsilosis sensu stricto, and PCR-restriction fragment length polymorphism patterns in the ITS1-5.8S-ITS2 region of the rDNA gene. There was agreement between results of testing of 98 clinical isolates with the four PCR-based methods, with 59 isolates identified as C. parapsilosis sensu stricto, 37 as C. orthopsilosis, and two as C. metapsilosis.

Minimal inhibitory concentration distributions and epidemiological cutoff values of five antifungal agents against Sporothrix brasiliensis.

BACKGROUND: Sporothrix brasiliensis is the most virulent sporotrichosis agent. This species usually responds to antifungal drugs, but therapeutic failure can occur in some patients. Antifungal susceptibility tests have been performed on this species, but no clinical breakpoints (CBPs) are available. In this situation, minimal inhibitory concentration (MIC) distributions and epidemiological cutoff values (ECVs) support the detection of identification of resistant strains. OBJECTIVES: To study the MIC distributions of five antifungal drugs against S. brasiliensis and to propose tentative ECVs. METHODS: MICs of amphotericin B (AMB), itraconazole (ITR), ketoconazole (KET), posaconazole (POS), and terbinafine (TRB) against 335 S. brasiliensis strains were determined by the Clinical and Laboratory Standards Institute broth microdilution method. FINDINGS: The proposed ECV, in µg/mL, for AMB, ITR, KET, POS, and TRB were 4.0, 2.0, 1.0, 2.0, and 0.25, respectively. Percentages of wild-type strains in our population for the above antifungal drugs were 98.48, 95.22, 95.33, 100, and 97.67%, respectively. MAIN CONCLUSIONS: These ECVs will be useful to detect strains with resistance, to define CBPs, and to elaborate specific therapeutic guidelines for S. brasiliensis. Rational use of antifungals is strongly recommended to avoid the emergence of resistant strains and ensure the therapeutic effectiveness of sporotrichosis.

Tinea Capitis by Microsporum audouinii: Case Reports and Review of Published Global Literature 2000-2016.

Tinea capitis caused by Microsporum audouinii is reported herein from two Brazilian schoolchildren, which are brothers. Arthroconidia were evidenced on direct examination of scalp hair, and a fungus of the genus Microsporum was isolated from cultures of each patient. The isolated fungi were classified as M. audouinii by visualization of species-specific structures, including: pectinate hyphae, chlamydospores, and fusiform macroconidia, sterile growth with characteristic brown pigment in rice grains, and through DNA sequencing of the internal transcriber spacer region. Patients were refractory to ketoconazole, but the two cases had a satisfactory response to oral terbinafine. All M. audouinii infections described in this century were reviewed, and to our knowledge, this is the first literature description of this species from South America. Misidentification of M. audouinii with Microsporum canis can occur in this area, leading to erroneous data about the occurrence of this species.

Hepatic Disease with Portal Hypertension and Acute Juvenile Paracoccidioidomycosis: A Report of Two Cases and Literature Review.

Paracoccidioidomycosis (PCM) is a neglected systemic mycosis endemic to Latin America caused by dimorphic fungi of the genus Paracoccidioides. The acute juvenile PCM is a severe type of presentation that usually affects young vulnerable patients and rarely progresses to portal hypertension. Here, two cases of liver disease and portal hypertension as complications of acute juvenile PCM are reported. Diagnosis of PCM was performed by isolation of the fungus and molecular identification of the strains provided through partial sequencing of two protein encoding genes, arf and gp43. Genotypic analysis revealed that Paracoccidioides brasiliensis S1 was the phylogenic species involved in both cases. Patients presented a good clinical response to amphotericin B and sulfamethoxazole-trimethoprim. These results highlight the importance of the interdisciplinary approach in patients with severe forms of PCM to avoid and treat complications, and the necessity of further investigations focusing on host-pathogen interaction in order to explain the broad clinical spectrum in PCM as well as the severity and poor outcome in some clinical cases.

The identification and differentiation of the Candida parapsilosis complex species by polymerase chain reaction-restriction fragment length polymorphism of the internal transcribed spacer region of the rDNA.

Currently, it is accepted that there are three species that were formerly grouped under Candida parapsilosis: C. para- psilosis sensu stricto, Candida orthopsilosis, and Candida metapsilosis. In fact, the antifungal susceptibility profiles and distinct virulence attributes demonstrate the differences in these nosocomial pathogens. An accurate, fast, and economical identification of fungal species has been the main goal in mycology. In the present study, we searched sequences that were available in the GenBank database in order to identify the complete sequence for the internal transcribed spacer (ITS)1-5.8S-ITS2 region, which is comprised of the forward and reverse primers ITS1 and ITS4. Subsequently, an in silico polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was performed to differentiate the C. parapsilosis complex species. Ninety-eight clinical isolates from patients with fungaemia were submitted for analysis, where 59 isolates were identified as C. parapsilosis sensu stricto, 37 were identified as C. orthopsilosis, and two were identified as C. metapsilosis. PCR-RFLP quickly and accurately identified C. parapsilosis complex species, making this method an alternative and routine identification system for use in clinical mycology laboratories.

First description of Candida nivariensis in Brazil: antifungal susceptibility profile and potential virulence attributes.

This study evaluated the antifungal susceptibility profile and the production of potential virulence attributes in a clinical strain of Candida nivariensis for the first time in Brazil, as identified by sequencing the internal transcribed spacer (ITS)1-5.8S-ITS2 region and D1/D2 domains of the 28S of the rDNA. For comparative purposes, tests were also performed with reference strains. All strains presented low planktonic minimal inhibitory concentrations (PMICs) to amphotericin B (AMB), caspofungin (CAS), and voriconazole. However, our strain showed elevated planktonic MICs to posaconazole (POS) and itraconazole, in addition to fluconazole resistance. Adherence to inert surfaces was conducted onto glass and polystyrene. The biofilm formation and antifungal susceptibility on biofilm-growing cells were evaluated by crystal violet staining and a XTT reduction assay. All fungal strains were able to bind both tested surfaces and form biofilm, with a binding preference to polystyrene (p < 0.001). AMB promoted significant reductions (≈50%) in biofilm production by our C. nivariensis strain using both methodologies. This reduction was also observed for CAS and POS, but only in the XTT assay. All strains were excellent protease producers and moderate phytase producers, but lipases were not detected. This study reinforces the pathogenic potential of C. nivariensis and its possible resistance profile to the azolic drugs generally used for candidiasis management.

L-Dihydroxyphenylalanine induces melanin production by members of the genus Trichosporon.

Melanization of members of the genus Trichosporon is poorly described. In this study, six strains, including two clinical isolates, from four different species (Trichosporon asahii, T. asteroides, T. inkin, and T. mucoides) were grown in culture media with or without L-dihydroxyphenylalanine (L-DOPA). Each strain produced a brownish pigment compatible with melanin when cultured in presence of L-DOPA, suggesting that these species are able to produce eumelanin. L-tyrosine was not able to elicit any type of pigment production on cultures. As eumelanin is produced by several fungi during parasitism, this pigment may contribute to Trichosporon virulence.

In vitro activity of the anthelmintic drug niclosamide against Sporothrix spp. strains with distinct genetic and antifungal susceptibility backgrounds.

The drugs available to treat sporotrichosis, an important yet neglected fungal infection, are limited. Some Sporothrix spp. strains present reduced susceptibility to these antifungals. Furthermore, some patients may not be indicated to use these drugs, while others may not respond to the therapy. The anthelmintic drug niclosamide is fungicidal against the Sporothrix brasiliensis type strain. This study aimed to evaluate whether niclosamide also has antifungal activity against Sporothrix globosa, Sporothrix schenckii and other S. brasiliensis strains with distinct genotypes and antifungal susceptibility status. Minimal inhibitory and fungicidal concentrations (MIC and MFC, respectively) were determined using the microdilution method according to the CLSI protocol. The checkerboard method was employed to evaluate niclosamide synergism with drugs used in sporotrichosis treatment. Metabolic activity of the strains under niclosamide treatment was evaluated using the resazurin dye. Niclosamide was active against all S. brasiliensis strains (n = 17), but it was ineffective (MIC > 20 µM) for some strains (n = 4) of other pathogenic Sporothrix species. Niclosamide MIC values for Sporothrix spp. were similar for mycelial and yeast-like forms of the strains (P = 0.6604). Niclosamide was fungicidal (MFC/MIC ratio ≤ 2) for most strains studied (89%). Niclosamide activity against S. brasiliensis is independent of the fungal genotype or non-wild-type phenotypes for amphotericin B, itraconazole, or terbinafine. These antifungal drugs presented indifferent interactions with niclosamide. Niclosamide has demonstrated potential for repurposing as a treatment for sporotrichosis, particularly in S. brasiliensis cases, instigating in vivo studies to validate the in vitro findings.

Non-Zoonotic Transmission of Sporotrichosis: A Translational Study of Forty-Three Cases in a Zoonotic Hyperendemic Area.

Over the past two decades, zoonotic sporotrichosis transmitted by naturally infected cats has become hyperendemic in Rio de Janeiro, Brazil. Sporothrix brasiliensis is the main agent involved. However, there are other forms of transmission of sporotrichosis. The aim of this study was to evaluate and associate the epidemiological, clinical and therapeutic data and the susceptibility of Sporothrix spp. to antifungal drugs in 43 non-zoonotic sporotrichosis cases. Forty-three clinical strains of Sporothrix were identified by partial sequencing of the calmodulin gene. An antifungal susceptibility test of amphotericin B, terbinafine, itraconazole, posaconazole and isavuconazole was performed according to the broth microdilution method. Most patients were male (55.8%). Regarding the source of infection, 21 patients (48.8%) reported trauma involving plants and/or contact with soil. Sporothrix brasiliensis was the predominant species (n = 39), followed by S. globosa (n = 3) and S. schenckii (n = 1). Sporothrix brasiliensis was associated with all the sources of infection, reinforcing previous data showing the presence of this species in environmental sources, as well as with all the clinical forms, including severe cases. One clinical strain of Sporothrix brasiliensis was classified as a non-wild-type strain for amphotericin B and another for itraconazole. S. schenckii was classified as non-WT for all the antifungals tested. In this context, it is important to emphasize that non-zoonotic sporotrichosis still occurs in the state of Rio de Janeiro, with S. brasiliensis as the main etiological agent, primarily associated with infections acquired after traumatic inoculation with plants and/or soil contact, followed by S. globosa and S. schenckii. In addition, non-WT strains were found, indicating the need to monitor the antifungal susceptibility profile of these species. It is crucial to investigate other natural sources of S. brasiliensis to better understand this fungal pathogen and its environment and host cycle.

Cyclosporine Affects the Main Virulence Factors of Cryptococcus neoformans In Vitro.

This study aimed to investigate the effects of cyclosporine on the morphology, cell wall structure, and secretion characteristics of Cryptococcus neoformans. The minimum inhibitory concentration (MIC) of cyclosporine was found to be 2 µM (2.4 µg/mL) for the H99 strain. Yeast cells treated with cyclosporine at half the MIC showed altered morphology, including irregular shapes and elongated projections, without an effect on cell metabolism. Cyclosporine treatment resulted in an 18-fold increase in chitin and an 8-fold increase in lipid bodies, demonstrating changes in the fungal cell wall structure. Cyclosporine also reduced cell body and polysaccharide capsule diameters, with a significant reduction in urease secretion in C. neoformans cultures. Additionally, the study showed that cyclosporine increased the viscosity of secreted polysaccharides and reduced the electronegativity and conductance of cells. The findings suggest that cyclosporine has significant effects on C. neoformans morphology, cell wall structure, and secretion, which could have implications for the development of new antifungal agents.

Repurposing Benzimidazoles against Causative Agents of Chromoblastomycosis: Albendazole Has Superior In Vitro Activity Than Mebendazole and Thiabendazole.

Chromoblastomycosis (CBM) is a neglected human implantation mycosis caused by several dematiaceous fungal species. Currently available therapy is usually associated with physical methods, especially surgery, and with high refractoriness. Therefore, drug discovery for CBM is essential. Drug repositioning is a strategy used to facilitate the discovery of new treatments for several diseases. The aim of this study was to discover substances with antifungal activity against CBM agents from a collection of drugs previously approved for use in human diseases. A screening was performed with the NIH Clinical Collection against Fonsecaea pedrosoi. Ten substances, with clinical applicability in CBM, inhibited fungal growth by at least 60%. The minimum inhibitory concentration (MIC) of these substances was determined against other CBM agents, and the benzimidazoles albendazole, mebendazole and thiabendazole presented the lowest MIC values. The selectivity index, based on MIC and cytotoxicity of these substances, revealed albendazole to be more selective. To investigate a possible synergism of this benzimidazole with itraconazole and terbinafine, the chequerboard method was used. All interactions were classified as indifferent. Our current results suggest that benzimidazoles have repositioning potential against CBM agents. Albendazole seems to be the most promising, since it presented the highest selectivity against all dematiaceous fungi tested.

Comparative Biophysical and Ultrastructural Analysis of Melanins Produced by Clinical Strains of Different Species From the Trichosporonaceae Family.

Melanin is one of the most studied virulence factors in pathogenic fungi. This pigment protects them from a series of both environmental and host stressors. Among basidiomycetes, Cryptococcus neoformans and Trichosporon asahii are known to produce melanin in the presence of phenolic precursors. Other species from the Trichosporonaceae family also produce this pigment, but the extent to this production among the clinically relevant species is unknown. For this reason, the aim of this study was to verify the production of melanin by different Trichosporonaceae species of clinical interest and to compare their pigments with the ones from C. neoformans and T. asahii, which are more prevalent in human infections. Melanin was produced in a minimal medium supplemented with 1 mM L-dihydroxyphenylalanine (L-DOPA). Pigment was evaluated using scanning electron microscopy, Zeta potential measurements, and energy-dispersive X-ray spectroscopy. It was found that, besides C. neoformans and T. asahii, Trichosporon japonicum, Apiotrichum montevideense, Trichosporon inkin, Trichosporon faecale, Cutaneotrichosporon debeurmannianum, and Cutaneotrichosporon arboriformis also produce melanin-like particles in the presence of L-DOPA. Melanin particles have negative charge and are smaller than original cells. Variations in color, fluorescence, and chemical composition was noticed between the studied strains. All melanins presented carbon, oxygen, sodium, and potassium in their composition. Melanins from the most pathogenic species also presented iron, zinc, and copper, which are important during parasitism. Biophysical properties of these melanins can confer to the Trichosporonaceae adaptive advantages to both parasitic and environmental conditions of fungal growth.