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The limited therapeutic options for fungal infections and the increased incidence of fungal strains resistant to antifungal drugs, especially Candida spp., require the development of new antifungal drugs and strategies. Histone deacetylase inhibitors (HDACi), like vorinostat, have been studied in cancer treatment and have antifungal effects, acting alone or synergistically with classical antifungals. Here we investigated the antifungal activity of two novel sustainable HDACi (LDT compounds) based on vorinostat structure. Molecular docking simulation studies reveal that LDT compounds can bind to Class-I HDACs of Candida albicans, C. tropicalis, and Cryptococcus neoformans, which showed similar binding mode to vorinostat. LDT compounds showed moderate activity when tested alone against fungi but act synergistically with antifungal azoles against Candida spp. They reduced biofilm formation by more than 50% in C. albicans (4 µg/mL), with the main action in fungal filamentation. Cytotoxicity of the LDT compounds against RAW264.7 cells was evaluated and LDT536 demonstrated cytotoxicity only at the concentration of 200 µmol/L, while LDT537 showed IC50 values of 29.12 µmol/L. Our data indicated that these sustainable and inexpensive HDACi have potential antifungal and antibiofilm activities, with better results than vorinostat, although further studies are necessary to better understand the mechanism against fungal cells., (© The Author(s) 2023. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

Cryptococcus neoformans/Cryptococcus gattii are fungal pathogens that affect the central nervous system, mainly in immunocompromised individuals. Due to the limited pharmacological arsenal available for the treatment of cryptococcosis associated with cases of antifungal resistance of Cryptococcus spp. reported in some studies, the search for new compounds with antifungal potential becomes relevant. Thus, the objective of this study was to evaluate the inhibitory effect of phenothiazines (promethazine and chlorpromazine) on C. neoformans/C. gattii planktonic cells and biofilms. In vitro planktonic susceptibility testing was performed using the broth microdilution assay. The effect of phenothiazines was evaluated against biofilm formation and mature Cryptococcus biofilms. Biofilm morphology and ultrastructure were also evaluated by scanning electron microscopy. Promethazine and chlorpromazine showed antifungal activity against planktonic cells, with minimum inhibitory concentrations of 8-32 μg/ml and 4-16 μg/ml, respectively. As for biofilm formation, phenothiazines reduced biomass by 60% and metabolic activity by 90% at 64 μg/ml; while in mature biofilms, reductions of 85% and 90% in biomass and metabolic activity, respectively, were observed at 1024 μg/ml. Promethazine and chlorpromazine were also able to disrupt and fragment biofilms. In conclusion, promethazine and chlorpromazine have antifungal activity against planktonic cells and biofilms of Cryptococcus spp. These data show the potential of promethazine and chlorpromazine as antibiofilm drugs., (© The Author(s) 2020. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

The present study evaluated the antifungal activity of the chelators deferiprone (DFP) and ethylenediaminetetraacetic acid (EDTA) and their effect on biofilm formation of the S. schenckii complex. Eighteen strains of Sporothrix spp. (seven S. brasiliensis, three S. globosa, three S. mexicana and five Sporothrix schenckii sensu stricto) were used. Minimum inhibitory concentration (MIC) values for EDTA and DFP against filamentous forms of Sporothrix spp. ranged from 32 to 128 μg/ml. For antifungal drugs, MIC values ranged from 0.25 to 4 μg/ml for amphotericin B, from 0.25 to 4 μg/ml for itraconazole, and from 0.03 to 0.25 μg/ml for terbinafine. The chelators caused inhibition of Sporothrix spp. in yeast form at concentrations ranging from 16 to 64 μg/ml (for EDTA) and 8 to 32 μg/ml (for DFP). For antifungal drugs, MIC values observed against the yeast varied from 0.03 to 0.5 μg/ml for AMB, 0.03 to 1 μg/ml for ITC, and 0.03 to 0.13 μg/ml for TRB. Both DFP and EDTA presented synergistic interaction with antifungals against Sporothrix spp. in both filamentous and yeast form. Biofilms formed in the presence of the chelators (512 μg/ml) showed a reduction of 47% in biomass and 45% in metabolic activity. Our data reveal that DFP and EDTA reduced the growth of planktonic cells of Sporothrix spp., had synergistic interaction with antifungal drugs against this pathogen, and reduced biofilm formation of Sporothrix spp., Lay Summary: Our data reveal that iron chelators deferiprone and ethylenediaminetetraacetic acid reduced the growth of planktonic cells of Sporothrix spp. as well as had synergistic interaction with antifungal drugs against this pathogen and reduced biofilm formation of Sporothrix spp., (© The Author(s) 2020. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

This study aimed to evaluate the yeast biofilm growth kinetics and ultrastructure of Sporothrix schenckii complex and assess their mature biofilm susceptibility in filamentous and yeast forms to potassium iodide (KI) and miltefosine (MIL). Yeast biofilms were evaluated by crystal violet staining, XTT reduction assay and microscopic techniques. Susceptibility of planktonic and sessile cells was analyzed by broth microdilution. S. schenckii complex in yeast form produced biofilms, with an optimum maturation at 96 h, showing multilayered blastoconidia embedded in extracellular matrix. KI and MIL minimum inhibitory concentration (MIC) ranges against planktonic cells were 62,500-250,000 μg/ml and 0.125-4 μg/ml, respectively. KI and MIL reduced biofilm metabolic activity by 75.4% and 67.7% for filamentous form and 55.1% and 51.6% for yeast form, respectively. This study demonstrated that S. schenckii complex forms biofilms in vitro, and potassium iodide and miltefosine inhibit Sporothrix spp. biofilms in both filamentous and yeast forms., (© The Author(s) 2018. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

The aim of this study was to evaluate the effect of promethazine on the antifungal minimum inhibitory concentrations against planktonic cells and mature biofilms of Candida tropicalis, as well as investigate its potential mechanisms of cell damage against this yeast species. Three C. tropicalis isolates (two azole-resistant and one azole-susceptible) were evaluated for their planktonic and biofilm susceptibility to promethazine alone and in combination with itraconazole, fluconazole, voriconazole, amphotericin B, and caspofungin. The antifungal activity of promethazine against C. tropicalis was investigated by performing time-kill curve assays and assessing rhodamine 6G efflux, cell size/granularity, membrane integrity, and mitochondrial transmembrane potential, through flow cytometry. Promethazine showed antifungal activity against planktonic cells and biofilms at concentrations of 64 and 128 μg/ml, respectively. The addition of two subinhibitory concentrations of promethazine reduced the antifungal MICs for all tested azole drugs against planktonic growth, reversing the resistance phenotype to all azoles. Promethazine decreased the efflux of rhodamine 6G in an azole-resistant strain. Moreover, promethazine decreased cell size/granularity and caused membrane damage, and mitochondrial membrane depolarization. In conclusion, promethazine presented synergy with azole antifungals against resistant C. tropicalis and exhibited in vitro cytotoxicity against C. tropicalis, altering cell size/granularity, membrane integrity, and mitochondrial function, demonstrating potential mechanisms of cell damage against this yeast species.

Sporotrichosis, caused by species of Sporothrix schenckii complex, is the most prevalent subcutaneous mycosis in many areas of Latin America. The aim of this study was to evaluate the ability of Sporothrix spp. to form biofilms in vitro and to characterize the growth kinetics, morphology, and antifungal susceptibility of biofilms against classical antifungals. We investigated the ability of strains to produce biofilms in vitro and determined the effects of exposure to amphotericin B, itraconazole, caspofungin, ketoconazole, voriconazole, and fluconazole at minimum inhibitory concentration (MIC) against planktonic form and at 10× MIC and 50× MIC on the biomass and metabolic activity of these biofilms. Biofilm structure was analyzed by optical microscopy using Congo-red staining, confocal and scanning electron microscopy. Strains were classified for biofilm-forming ability, through the analysis of absorbance of crystal violet retained by biomass of mature biofilms. We found that all S. brasiliensis (n = 10), S. schenckii sensu stricto (n = 2), S. globosa (n = 2), and S. mexicana (n = 4) strains were strong biofilm-producers. The analyzed biofilms had dense network of hyphae and conidia immersed in extracellular matrix, with presence of water channels. Antifungal drugs at the three tested concentrations showed different effects on biomass and metabolic activity of biofilms. However, the best inhibitory response was observed with 50× MIC of amphotericin B and caspofungin, which reduced these parameters. Furthermore, high drug concentrations, especially amphotericin B and caspofungin, showed antifungal activity against these biofilms, probably because they damaged the architecture and extracellular matrix, allowing diffusion of the drugs.

This study aimed to identify yeasts from the gastrointestinal tract of scarlet ibises (Eudocimus ruber) and from plant material collected from the environment where they live. Then, the isolates phenotypically identified as Candida famata were submitted to molecular identification of their closely related species and evaluated for their antifungal susceptibility and possible resistance mechanisms to antifungal drugs. Cloacal swabs from 20 scarlet ibises kept in captivity at Mangal das Garças Park (Brazil), pooled stool samples (n = 20) and samples of trunks and hollow of trees (n = 20) obtained from their enclosures were collected. The samples were seeded on Sabouraud agar supplemented with chloramphenicol. The 48 recovered isolates were phenotypically identified as 15 Candida famata, 13 Candida catenulata, 2 Candida intermedia, 1 Candida lusitaniae, 2 Candida guilliermondii, 1 Candida kefyr, 1 Candida amapae, 1 Candida krusei, 8 Trichosporon spp., and 4 Rhodotorula spp. The C. famata isolates were further identified as 3 C. famata, 8 Debaryomyces nepalensis, and 4 C. palmioleophila. All C. famata and C. palmioleophila were susceptible to caspofungin and itraconazole, while one D. nepalensis was resistant to fluconazole and voriconazole. This same isolate and another D. nepalensis had lower amphotericin B susceptibility. The azole resistant strain had an increased efflux of rhodamine 6G and an alteration in the membrane sterol content, demonstrating multifactorial resistance mechanism. Finally, this research shows that scarlet ibises and their environment harbor C. famata and closely related species, including antifungal resistant isolates, emphasizing the need of monitoring the antifungal susceptibility of these yeast species., (© The Author 2017. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

This was a cross-sectional study to investigate the antifungal susceptibility and production of virulence factors in strains of Candida isolated from the outlet and the lumen of the nasolacrimal duct of horses in the state of Ceará, Brazil. The samples were obtained from 103 horses. Sterile cotton swabs were used to collect the material from the outlet of the nasolacrimal duct and urethral probes, for the instillation of 2 ml of saline solution, were used to collect samples from the lumen of the nasolacrimal duct. A total of 77 Candida isolates were obtained, with C. famata, C. tropicalis, C. guilliermondii, and C. parapsilosis sensu lato as the most prevalent species. One isolate (C. glabrata) was resistant to caspofungin. One isolate was resistant only to fluconazole (C. parapsilosis sensu lato), 11 were resistant only to itraconazole (7 C. tropicalis, 2 C. guilliermondii, 1 C. famata, 1 C. parapsilosis sensu lato), while eight C. tropicalis showed resistance to both azoles. Overall, 28 isolates produced phospholipases and 12 produced proteases. These results highlight the importance of investigating the antifungal susceptibility and virulence trends of Candida spp. from the microbiota of the nasolacrimal duct of horses., (© The Author 2015. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Candida tropicalis has been associated with invasive candidiasis, being the first or second most common non-Candida albicans Candida species isolated in humans with candidemia and candiduria, as well as being frequently isolated from healthy animals. This study aimed to characterize C. tropicalis isolates (n = 64) obtained from several animal species regarding antifungal susceptibility and production of virulence factors. The isolates were obtained from the microbiota of healthy animals (goats, n = 25; sheep, n = 6; psittacines, n = 14; rheas, n = 6; horses, n = 2; sirenians, n = 5; shrimp, n = 1), as well as from aquatic mammals found dead in the environment (cetaceans, n = 5). The isolates were subjected to in vitro susceptibility testing by broth microdilution according to the CLSI M27-A3 protocol against amphotericin B, caspofungin, itraconazole, and fluconazole. We also evaluated the virulence attributes, such as proteases and phospholipases, as well as biofilm formation. Resistance to itraconazole (n = 29) and fluconazole (n = 30) was detected among isolates from every source; resistance to both azoles was detected in 24 isolates, but none of them were resistant to amphotericin B and caspofungin. Protease production was detected in the majority of the isolates (n = 59), but phospholipase was produced by only a few of them (n = 6). The isolates showed different patterns in biofilm production, being considered strong producers (n = 41), moderate producers (n = 11), weak producers (n = 9) or non-producers (n = 3). In summary, C. tropicalis isolated from animals showed high rate of resistance to azoles, expressed virulence factors and therefore may represent a potential threat to human and animal health., (© The Author 2014. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)