Antifungal Activity of Decyl Gallate against Several Species of Pathogenic Fungi

This work aims to demonstrate that the gallic acid structure modification to the decyl gallate (G14) compound contributed to increase the antifungal activity against several species of pathogenic fungi, mainly, Candida spp., Cryptococcus spp., Paracoccidioides spp., and Histoplasma capsulatum, according to standardized microdilution method described by Clinical Laboratory Standard Institute (CLSI) documents. Moreover this compound has a particularly good selectivity index value, which makes it an excellent candidate for broad-spectrum antifungal prototype and encourages the continuation of subsequent studies for the discovery of its mechanism of action

Caracterização dos biofilmes de dermatófitos e desenvolvimento de protótipos anti-dermatófitos nas formas planctônica e biofilme

Dermatófitos são fungos que apresentam a capacidade de invadir os tecidos queratinizados do homem e dos animais, produzindo a dermatofitose. Biofilmes são comunidades estruturadas de microrganismos que se agrupam de maneira organizada a uma superfície ou que se aderem uns aos outros, dentro de uma matriz extracelular que eles mesmos produzem. Pela primeira vez, neste trabalho, foi descrita a formação de biofilmes por isolados clínicos e cepas ATCC de dermatófitos. Também se avaliou a sensibilidade das cepas frente aos antifúngicos convencionais e derivados sintéticos do ácido protocatecuico, determinando sua toxicidade e eficiência, tanto nas formas planctônicas como de biofilme. A identificação molecular dos isolados clínicos mostrou concordância com a identificação convencional. Todas as cepas e isolados foram capazes de formar biofilmes nos quatro meios testados. Trichophyton. rubrum ATCC 28189, ATCC MYA-4438, T. rubrum 143 e Trichophyton. mentagrophytes 66 produziram mais biomassa e matriz extracelular que T. mentagrophytes ATCC 11481 (p<0.05). RPMI 1640, BHI e DMEM estimularam maior produção de biomassa e matriz extracelular que o meio de queratinócitos. Todas as espécies foram capazes de formar biofilmes maduros em 72 h e uma rede de hifas circundadas em vários pontos por uma matriz extracelular foi mostrada por microscopia eletrônica de varredura (MEV). Na microscopia confocal, aparentemente os meios RPMI e DMEM produziram biofilmes mais espessos e o meio BHI, biofilmes mais densos e compactos. As melhores atividades anti-dermatófitos foram expressas pelos protocatecuatos de butila, pentila, hexila, nonila e decila, que exibiram baixa toxicidade para as células HaCat, NHOK e HepG2 nas regiões da concentração inibitória mínima (CIM). Nonila foi o composto com melhor índice de seletividade para as células HaCat e HepG2; para as células NHOK os compostos mais seletivos foram hexila e nonila. Fluconazol, griseofulvina, terbinafina e nonila inibiram o crescimento das células planctônicas para todas as cepas. No entanto, os biofilmes pré-formados foram resistentes a todos os fármacos, com exceção do composto nonila. As células em formato biofilme foram mais resistentes aos antifúngicos que as células planctônicas. A maioria dos biofilmes tratados teve redução da espessura quando comparada ao não tratado. Nenhum biofilme tratado apresentou 100% de morte celular. Na sequência deste estudo, foi verificado o potencial antifúngico in vitro de nanopartículas de óxido nítrico (NO-np) e de efinaconazol a 10% (Jublia®) contra as formas planctônicas e biofilmes de T. rubrum (cepa MYA-4438 ATCC e um isolado clínico). As NO-np foram capazes de inibir in vitro, o crescimento de T. rubrum. O efinaconazol foi o fármaco que melhor inibiu o crescimento de T. rubrum, seguido pela terbinafina e o fluconazol. As combinações, entre as NO-np, o efinaconazol e a terbinafina produziram interações sinérgicas. A combinação entre as NO-np e o efinaconazol teve um comportamento fungistático na cepa ATCC de T. rubrum. As NO-np não apresentaram toxicidade para as células HaCat. Todos os fármacos testados foram capazes de inibir o crescimento das células planctônicas de T. rubrum; já as NOnp não foram capazes de inibir o crescimento e reduzir a atividade metabólica. Os biofilmes pré-formados foram resistentes a todos os fármacos testados, porém, o biofilme formado pelo isolado clínico de T. rubrum foram sensíveis às NO-np na concentração de 40 mg/mL. Já os biofilmes da cepa ATCC foram resistentes à mesma concentração de nanopartículas. A grande maioria dos resultados da MEV dos biofilmes tratados foi concordante aos resultados mostrados pelo ensaio de redução do XTT. Os tratamentos com o efinaconazol e as NO-np causaram diminuição das espessuras dos biofilmes formados pelo isolado clínico e pela cepa ATCC. Esses resultados representam um avanço para a pesquisa no campo dos dermatófitos e de biofilmes e podem contribuir de maneira significativa para a pesquisa de novos fármacos para o tratamento dessas micoses.Dermatophytes are fungi that have the ability to invade keratinized tissues of human and animals, producing a condition called dermatophytosis. Biofilms are structured microbial communities that are organized by adhering to a surface, as well as to each other via an extracellular polymeric matrix. For the first time, in this work, the biofilm formation by ATCC strains and clinical isolates of dermatophytes was described. Also, the susceptibilities of the strains to conventional antifungal drugs and to the synthetic derivatives of protocatechuic acid were evaluated, determining its toxicity and efficacy. The molecular identification corroborated with the conventional one. All strains were able to form biofilms in the four media tested. Trichophyton. rubrum ATCC 28189, Trichophyton. mentagrophytes 66, and T. rubrum 143 produced more biomass, polysaccharide structures and extracellular matrix than T. mentagrophytes ATCC 11481 (p <0.05). RPMI 1640, BHI and DMEM stimulated increased production of biomass and extracellular matrix when compared to the medium for keratinocytes. All species were able to form mature biofilms in 72 hours. The SEM results showed a coordinated network of hyphae in all directions, surrounded by an extracellular matrix. In confocal microscopy, apparently RPMI and DMEM media produced thicker biofilms, while the BHI produced denser and compact biofilms. The greatest antidermatophytes activities were expressed by butyl, pentyl, hexyl, nonyl and decyl, protocatechuates, which exhibit low toxicity to HaCat, HepG2 and NHOK cells in the regions of minimal inhibitory concentrations (MIC). Nonyl was the compound with improved selectivity index for HaCat and HepG2 cells; for NHOK cells, the most selective compounds were nonyl and hexyl. Fluconazole, griseofulvin, terbinafine and nonyl inhibited the growth of planktonic cells for all strains. However, the pre-formed biofilms were resistant to all antifungal drugs tested except to the nonyl compound. The cells in biofilm form were more resistant to antifungal drugs than planktonic cells. Most of treated biofilms had reduced thickness when compared to the untreated biofilms. In the sequence of this study, the in vitro antifungal activity of nitric oxide nanoparticles (NO-np) and efinaconazole 10% (Jublia®) was checked against planktonic and biofilm forms of T. rubrum (strain ATCC MYA-4438 and one clinical isolate). The NO-np was able to inhibit the in vitro's growth of T. rubrum. Efinaconazole was the best drug able to inhibit the growth of T. rubrum, followed by terbinafine and fluconazole. The combinations between NO-np, efinaconazole and terbinafine, produced synergistic interactions. The combination between NO-np and efinaconazole was fungistatic against the ATCC strain of T. rubrum. The NO-np showed no toxicity to HaCat cells. Both clinical isolate and the ATCC strain ATCC MYA-4438 were able to form biofilms and 72 hours was the ideal time for biofilm maturation. The biofilms formed by ATCC strain produced more biomass than the clinical isolate. All the antifungal drugs tested were able to inhibit the growth of planktonic cells of T. rubrum; the NO-np concentrations in which the reading on the spectrophotometer was possible, were not able to inhibit growth and to reduce the metabolic activity. The pre-formed biofilms were resistant to all tested drugs; however, the biofilms formed by the clinical isolate were susceptible to NO-np in a concentration of 40 mg/ml. The biofilms formed by the ATCC strain were resistant to the same concentration of nanoparticles. The vast majority of the SEM results of the treated biofilms were consistent with the results shown by the XTT reduction assay. Treatments with efinaconazole and NO-np reduced the thickness of the biofilms formed by the clinical isolate and by ATCC MYA-4438 strain. These results represent an advance for the research in biofilms field and can contribute significantly to the research of new drugs for the treatment of dermatophytosis.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Nitric Oxide-Releasing Nanoparticles Are Similar to Efinaconazole in Their Capacity to Eradicate

Filamentous fungi such as Trichophyton rubrum and T. mentagrophytes, the main causative agents of onychomycosis, have been recognized as biofilm-forming microorganisms. Nitric oxide-releasing nanoparticles (NO-np) are currently in development for the management of superficial and deep bacterial and fungal infections, with documented activity against biofilms. In this context, this work aimed to evaluate, for the first time, the in vitro anti-T. rubrum biofilm potential of NO-np using standard ATCC MYA-4438 and clinical BR1A strains and compare it to commonly used antifungal drugs including fluconazole, terbinafine and efinaconazole. The biofilms formed by the standard strain produced more biomass than those from the clinical strain. NO-np, fluconazole, terbinafine, and efinaconazole inhibited the in vitro growth of planktonic T. rubrum cells. Similarly, NO-np reduced the metabolic activities of clinical strain BR1A preformed biofilms at the highest concentration tested (SMIC(50) = 40 mg/mL). Scanning electron and confocal microscopy revealed that NO-np and efinaconazole severely damaged established biofilms for both strains, resulting in collapse of hyphal cell walls and reduced the density, extracellular matrix and thickness of the biofilms. These findings suggest that biofilms should be considered when developing and testing new drugs for the treatment of dermatophytosis. Development of a biofilm phenotype by these fungi may explain the resistance of dermatophytes to some antifungals and why prolonged treatment is usually required for onychomycosis

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 &micro;g/mL against H. capsulatum (compound 11) and P. brasiliensis (compounds 3 and 9) to 0.98 &micro;g/mL against Trichophyton rubrum and T. mentagrophytes (compounds 8, 9, 12, 13 and 8, 12, 13, respectively), and 3.9 &micro;g/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

Topical nitric oxide releasing nanoparticles are effective in a murine model of dermal Trichophyton rubrum dermatophytosis

Systemic therapies are preferred for treating dermal dermatophytosis due to inadequate penetration of topical agents. However, systemic antifungals are associated with off-target effects and limited tissue penetration, and antimicrobial resistance is a growing concern. To address this, we investigated topical nitric oxide-releasing nanoparticles (NO-np), which have been used against superficial fungal infections and bacterial abscesses. In addition to enhanced penetration and permeation conferred by nanoparticles, nitric oxide, a broad-spectrum multimechanistic antimicrobial agent, offers decreased likelihood of resistance development. In the current study, NO-np inhibited Trichophyton rubrum in vitro, as well as in a murine model of dermal dermatophytosis. In mice, NO-np reduced fungal burden after three days, with complete clearance after seven. Furthermore, NO-np decreased tissue IL-2, 6, 10 and TNF alpha, indicating earlier attenuation of the host inflammatory response and decreased tissue morbidity. Thus, topical NO-np represent an attractive alternative to systemic therapy against dermal T. rubrum infection.Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior 99999.007910/2014-02 Conselho Nacional de Desenvolvimento Cientifico e Tecnologico 150261/2016-

Anti-Trichophyton Activity of Protocatechuates and Their Synergism with Fluconazole

Dermatophytosis and superficial mycosis are a major global public health problem that affects 20-25% of the world's population. The increase in fungal resistance to the commercially available antifungal agents, in conjunction with the limited spectrum of action of such drugs, emphasises the need to develop new antifungal agents. Natural products are attractive prototypes for antifungal agents due to their broad spectrum of biological activities. This study aimed to verify the antifungal activity of protocatechuic acid, 3,4-diacetoxybenzoic, and fourteen alkyl protocatechuates (3,4-dihydroxybenzoates) against Trichophyton rubrum and Trichophyton mentagrophytes and to further assess their activities when combined with fluconazole. Susceptibility and synergism assays were conducted as described in M38-A2 (CLSI), with modifications. Three strains of Trichophyton rubrum and three strains of Trichophyton mentagrophytes were used in this work. The pentyl, hexyl, heptyl, octyl, nonyl, and decyl protocatechuates showed great fungicidal effects, with minimum inhibitory concentration (MIC) values ranging from 0.97 to 7.8 mg/L. Heptyl showed a synergistic activity (FIC index = 0.49), reducing the MIC of fluconazole by fourfold. All substances tested were safe, especially the hexyl, heptyl, octyl, and nonyl compounds, all of which showed a high selectivity index, particularly in combination with fluconazole. These ester associations with fluconazole may represent a promising source of prototypes in the search for anti-Trichophyton therapeutic agents.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Anti-Trichophyton Activity of Protocatechuates and Their Synergism with Fluconazole

Dermatophytosis and superficial mycosis are a major global public health problem that affects 20-25% of the world&apos;s population. The increase in fungal resistance to the commercially available antifungal agents, in conjunction with the limited spectrum of action of such drugs, emphasises the need to develop new antifungal agents. Natural products are attractive prototypes for antifungal agents due to their broad spectrum of biological activities. This study aimed to verify the antifungal activity of protocatechuic acid, 3,4-diacetoxybenzoic, and fourteen alkyl protocatechuates (3,4-dihydroxybenzoates) against Trichophyton rubrum and Trichophyton mentagrophytes and to further assess their activities when combined with fluconazole. Susceptibility and synergism assays were conducted as described in M38-A2 (CLSI), with modifications. Three strains of Trichophyton rubrum and three strains of Trichophyton mentagrophytes were used in this work. The pentyl, hexyl, heptyl, octyl, nonyl, and decyl protocatechuates showed great fungicidal effects, with minimum inhibitory concentration (MIC) values ranging from 0.97 to 7.8 mg/L. Heptyl showed a synergistic activity (FIC index = 0.49), reducing the MIC of fluconazole by fourfold. All substances tested were safe, especially the hexyl, heptyl, octyl, and nonyl compounds, all of which showed a high selectivity index, particularly in combination with fluconazole. These ester associations with fluconazole may represent a promising source of prototypes in the search for antiTrichophyton therapeutic agents

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 &micro;g/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&ndash;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