Influence of Zinc on Histoplasma capsulatum Planktonic and Biofilm Cells.

Histoplasma capsulatum causes a fungal respiratory disease. Some studies suggest that the fungus requires zinc to consolidate the infection. This study aimed to investigate the influence of zinc and the metal chelator TPEN on the growth of Histoplasma in planktonic and biofilm forms. The results showed that zinc increased the metabolic activity, cell density, and cell viability of planktonic growth. Similarly, there was an increase in biofilm metabolic activity but no increase in biomass or extracellular matrix production. N'-N,N,N,N-tetrakis-2-pyridylmethylethane-1,2 diamine (TPEN) dramatically reduced the same parameters in the planktonic form and resulted in a decrease in metabolic activity, biomass, and extracellular matrix production for the biofilm form. Therefore, the unprecedented observations in this study highlight the importance of zinc ions for the growth, development, and proliferation of H. capsulatum cells and provide new insights into the role of metal ions for biofilm formation in the dimorphic fungus Histoplasma, which could be a potential therapeutic strategy.

Alternative Non-Mammalian Animal and Cellular Methods for the Study of Host-Fungal Interactions.

In the study of fungal pathogenesis, alternative methods have gained prominence due to recent global legislation restricting the use of mammalian animals in research. The principle of the 3 Rs (replacement, reduction, and refinement) is integrated into regulations and guidelines governing animal experimentation in nearly all countries. This principle advocates substituting vertebrate animals with other invertebrate organisms, embryos, microorganisms, or cell cultures. This review addresses host-fungus interactions by employing three-dimensional (3D) cultures, which offer more faithful replication of the in vivo environment, and by utilizing alternative animal models to replace traditional mammals. Among these alternative models, species like Caenorhabditis elegans and Danio rerio share approximately 75% of their genes with humans. Furthermore, models such as Galleria mellonella and Tenebrio molitor demonstrate similarities in their innate immune systems as well as anatomical and physiological barriers, resembling those found in mammalian organisms.

Evaluation of the Anti-Histoplasma capsulatum Activity of Indole and Nitrofuran Derivatives and Their Pharmacological Safety in Three-Dimensional Cell Cultures.

Histoplasma capsulatum is a fungus that causes histoplasmosis. The increased evolution of microbial resistance and the adverse effects of current antifungals help new drugs to emerge. In this work, fifty-four nitrofurans and indoles were tested against the H. capsulatum EH-315 strain. Compounds with a minimum inhibitory concentration (MIC90) equal to or lower than 7.81 µg/mL were selected to evaluate their MIC90 on ATCC G217-B strain and their minimum fungicide concentration (MFC) on both strains. The quantification of membrane ergosterol, cell wall integrity, the production of reactive oxygen species, and the induction of death by necrosis-apoptosis was performed to investigate the mechanism of action of compounds 7, 11, and 32. These compounds could reduce the extracted sterol and induce necrotic cell death, similarly to itraconazole. Moreover, 7 and 11 damaged the cell wall, causing flaws in the contour (11), or changing the size and shape of the fungal cell wall (7). Furthermore, 7 and 32 induced reactive oxygen species (ROS) formation higher than 11 and control. Finally, the cytotoxicity was measured in two models of cell culture, i.e., monolayers (cells are flat) and a three-dimensional (3D) model, where they present a spheroidal conformation. Cytotoxicity assays in the 3D model showed a lower toxicity in the compounds than those performed on cell monolayers. Overall, these results suggest that derivatives of nitrofurans and indoles are promising compounds for the treatment of histoplasmosis.

Synthesis and Evaluation of the Antifungal and Toxicological Activity of Nitrofuran Derivatives.

Fungal diseases affect more than 1 billion people worldwide. The constant global changes, the advent of new pandemics, and chronic diseases favor the diffusion of fungal pathogens such as Candida, Cryptococcus, Aspergillus, Trichophyton, Histoplasma capsulatum, and Paracoccidioides brasiliensis. In this work, a series of nitrofuran derivatives were synthesized and tested against different fungal species; most of them showed inhibitory activity, fungicide, and fungistatic profile. The minimal inhibitory concentration (MIC90) values for the most potent compounds range from 0.48 µg/mL against H. capsulatum (compound 11) and P. brasiliensis (compounds 3 and 9) to 0.98 µg/mL against Trichophyton rubrum and T. mentagrophytes (compounds 8, 9, 12, 13 and 8, 12, 13, respectively), and 3.9 µg/mL against Candida and Cryptococcus neoformans strains (compounds 1 and 5, respectively). In addition, all compounds showed low toxicity when tested in vitro on lung cell lines (A549 and MRC-5) and in vivo in Caenorhabditis elegans larvae. Many of them showed high selectivity index values. Thus, these studied nitrofuran derivatives proved to be potent against different fungal species, characterized by low toxicity and high selectivity; for these reasons, they may become promising compounds for the treatment of mycoses.

2-Hydroxychalcone as a Potent Compound and Photosensitizer Against Dermatophyte Biofilms.

Dermatophytes, fungi that cause dermatophytosis, can invade keratinized tissues in humans and animals. The biofilm-forming ability of these fungi was described recently, and it may be correlated with the long treatment period and common recurrences of this mycosis. In this study, we evaluated the anti-dermatophytic and anti-biofilm activity of 2-hydroxychalcone (2-chalcone) in the dark and photodynamic therapy (PDT)-mediated and to determine its mechanism of action. Trichophyton rubrum and Trichophyton mentagrophytes strains were used in the study. The antifungal susceptibility test of planktonic cells, early-stage biofilms, and mature biofilms were performed using colorimetric methods. Topographies were visualized by scanning electron microscopy (SEM). Human skin keratinocyte (HaCat) monolayers were also used in the cytotoxicity assays. The mechanisms of action of 2-chalcone in the dark and under photoexcitation were investigated using confocal microscopy and the quantification of ergosterol, reactive oxygen species (ROS), and death induction by apoptosis/necrosis. All strains, in the planktonic form, were inhibited after treatment with 2-chalcone (minimum inhibitory concentration (MIC) = 7.8-15.6 mg/L), terbinafine (TRB) (MIC = 0.008-0.03 mg/L), and fluconazole (FLZ) (1-512 mg/L). Early-stage biofilm and mature biofilms were inhibited by 2-chalcone at concentrations of 15.6 mg/L and 31.2 mg/L in all tested strains. However, mature biofilms were resistant to all the antifungal drugs tested. When planktonic cells and biofilms (early-stage and mature) were treated with 2-chalcone-mediated PDT, the inhibitory concentrations were reduced by four times (2-7.8 mg/L). SEM images of biofilms treated with 2-chalcone showed cell wall collapse, resulting from a probable extravasation of cytoplasmic content. The toxicity of 2-chalcone in HaCat cells showed higher IC50 values in the dark than under photoexcitation. Further, 2-chalcone targets ergosterol in the cell and promotes the generation of ROS, resulting in cell death by apoptosis and necrosis. Overall, 2-chalcone-mediated PDT is a promising and safe drug candidate against dermatophytes, particularly in anti-biofilm treatment.

A Two-Way Road: Antagonistic Interaction Between Dual-Species Biofilms Formed by Candida albicans/Candida parapsilosis and Trichophyton rubrum.

Dermatomycoses include superficial fungal infections of the skin and its appendages. Trichophyton rubrum, Candida albicans, and Candida parapsilosis are some of the most prevalent species that cause dermatomycoses. Several studies show a variable predominance of Candida spp. in relation to dermatophytes, especially in onychomycosis and the possibility of isolating both from the same site. The ability of dermatophytes to form biofilms recently been explored and there is currently no evidence on the involvement of these filamentous fungi in multi-species biofilms. Thus, this study aims to investigate the probable dual-species interaction between T. rubrum and C. albicans and T. rubrum and C. parapsilosis biofilms, considering variable formation conditions, as well as the susceptibility of these dual-species biofilms against terbinafine and efinaconazole. Three conditions of formation of dual-species biofilms were tested: (a) the suspensions of T. rubrum and Candida albicans or C. parapsilosis placed together; (b) suspensions of C. albicans and C. parapsilosis added the pre-adhesion of T. rubrum biofilms; (c) after the maturation of T. rubrum sessile cells. In the first and second conditions, the quantification of metabolic activities, biomass, and polysaccharide materials of mixed biofilms tended to resemble Candida monospecies biofilms. In the third condition, the profiles were modified after the addition of Candida, suggesting that T. rubrum biofilms served as substrate for the development of Candida biofilms. Scanning electron microscopy showed Candida predominance, however, numerous blastoconidia were noted, most evident in the conditions under which Candida was added after the pre-adhesion and maturation of T. rubrum biofilms. Despite the predominance of Candida, the presence of T. rubrum appears to inhibit C. albicans filamentation and C. parapsilosis development, confirming an antagonistic interaction. Fungal burden assays performed when the biofilms were formed together confirmed Candida predominance, as well as susceptibility to antifungals. Further studies will be needed to identify the components of the Candida and T. rubrum biofilm supernatants responsible for inhibiting dermatophyte growth and C. albicans filamentation.

Biofilm Formation by Histoplasma capsulatum in Different Culture Media and Oxygen Atmospheres.

Histoplasma capsulatum is a dimorphic fungus that causes an important systemic mycosis called histoplasmosis. It is an infectious disease with high prevalence and morbidity that affects the general population. Recently, the ability of these fungi to form biofilms, a phenotype that can induce resistance and enhance virulence, has been described. Despite some efforts, data regarding the impact of nutrients and culture media that affect the H. capsulatum biofilm development in vitro are not yet available. This work aimed to study H. capsulatum biofilms, by checking the influence of different culture media and oxygen atmospheres in the development of these communities. The biofilm formation by two strains (EH-315 and G186A) was characterized under different culture media: [Brain and Heart Infusion (BHI), Roswell Park Memorial Institute (RPMI) with 2% glucose, Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum and nutrient medium HAM-F12 (HAM-F12) supplemented with glucose (18.2 g/L), glutamic acid (1 g/L), HEPES (6 g/L) and L-cysteine (8.4 mg/L)] and oxygen atmospheres (aerobiosis and microaerophilia), using the XTT reduction assay to quantify metabolic activities, crystal violet staining for biomass, safranin staining for the quantification of polysaccharide material and scanning electron microscopy (SEM) for the observation of topographies. Results indicated that although all culture mediums have stimulated the maturation of the communities, HAM-F12 provided the best development of biomass and polysaccharide material when compared to others. Regarding the oxygen atmospheres, both stimulated an excellent development of the communities, however in low oxygen conditions an exuberant amount of extracellular matrix was observed when compared to biofilms formed in aerobiosis, mainly in the HAM-F12 media. SEM images showed yeasts embedded by an extracellular matrix in several points, corroborating the colorimetric assays. However, biofilms formed in BHI, RPMI, and DMEM significantly induced yeast to hyphae reversal, requiring further investigation. The results obtained so far contribute to in vitro study of biofilms formed by these fungi and show that nutrition promoted by different media modifies the development of these communities. These data represent advances in the field of biofilms and contribute to future studies that can prove the role of these communities in the fungi-host interaction.

Influence of lactation stages and rain periods on subclinical mastitis in meat producing ewes / Influência dos estágios de lactação e ocorrência de chuvas sobre a mastite subclínica em ovelhas com aptidão para produção de carne

ABSTRACT: Mastitis negatively influences the survival and weight gain of ovines for meat production. The purpose of this study was to investigate, in sheep for meat production, the occurrence of subclinical mastitis in ewes at the end of lactation and beginning of the consecutive lactation and to assess the composition and cellular characteristics of milk as a function of different rainfall indices. Mammary halves (821) of Santa Ines (479) and Morada Nova (342) ewes were examined. Milk samples were collected in two different moments of lactation: at weaning and postpartum of the consecutive lactation. Sample collection periods were called "dry" or "rainy" according to the rainfall index in the month immediately before the month of collection. The occurrence of subclinical mastitis at weaning in the Santa Ines and Morada Nova ewes were 16.4 and 12.6% in the dry period, and 17.7 and 23.5% in the rainy period, respectively. In the consecutive lactation period, the occurrences were 26.7 and 27.7% in the dry period and 41.8 and 39.1% in the rainy period, for the Santa Ines and Morada Nova ewes, respectively. Postpartum stage was critical for the occurrence of subclinical mastitis, as compared to that at the end of the previous lactation. Occurrence of the disease negatively influenced the SCC in the milk at the beginning of lactation and changed its composition, mainly in the rainiest periods, probably due to a difficulty in maintaining hygiene in the environment where the animals remained.

RESUMO: A mastite influencia negativamente na sobrevivência e no ganho de peso em ovinos com aptidão para produção de carne. O objetivo do presente estudo foi investigar, em ovelhas para produção de carne, a ocorrência de mastite ovina subclínica, no final de lactação e no início da lactação consecutiva, bem como avaliar as características composicionais e celulares do leite em função de diferentes índices pluviométricos. Metades mamárias (821) das raças 'Santa Inês' (479) e 'Morada Nova' (342) foram analisadas. As amostras de leite foram colhidas em dois estágios diferentes de lactação: no desmame e após o parto da lactação consecutiva. Os períodos de colheita de amostras foram denominados "seco" ou "chuvoso", conforme o índice de chuva no mês imediatamente antes ao mês da colheita. A ocorrência de mastite subclínica na desmama nas raças 'Santa Inês' e 'Morada Nova' foi de 16,4 e 12,6% no período seco, e 17,7 e 23,5% no período chuvoso, respectivamente. Na lactação consecutiva, as ocorrências foram 26,7 e 27,7% no período seco e 41,8 e 39,1% no período chuvoso, nas ovelhas 'Santa Inês' e 'Morada Nova', respectivamente. O período pós-parto foi crítico para a ocorrência de mastite subclínica, comparado àquele no final da lactação anterior. A ocorrência da doença teve uma influência negativa na CCS do leite no início da lactação e alterou sua composição, principalmente nos períodos mais chuvosos, provavelmente por dificuldade em manter a higiene no ambiente onde os animais permaneceram.