A new coumarin derivative, 4-acetatecoumarin, with antifungal activity and association study against Aspergillus spp

Abstract Fungal infections have become a concern for health professionals, and the emergence of resistant strains has been reported for all known classes of antifungal drugs. Among the fungi causing disease, we highlight those that belong to the genus Aspergillus. For these reasons, the search for new antifungals is important. This study examines the effects of a coumarin derivative, 4-acetatecoumarin (Cou-UMB16) both alone and together with antifungal drugs, and its mode of action against Aspergillus spp. Cou-UMB16 was tested to evaluate its effects on mycelia growth, and germination of Aspergillus spp. fungal conidia. We investigated its possible action on cell walls, on the cell membrane, and also the capacity of this coumarin derivative to enhance the activity of antifungal drugs. Our results suggest that Cou-UMB16 inhibits Aspergillus spp. virulence factors (mycelia growth and germination of conidia) and affects the structure of the fungal cell wall. When applying Cou-UMB16 in combination with azoles, both synergistic and additive effects were observed. This study concludes that Cou-UMB16 inhibits mycelial growth and spore germination, and that the activity is due to its action on the fungal cell wall, and that Cou-UMB16 could act as an antifungal modifier.

Pythium insidiosum: inhibitory effects of propolis and geopropolis on hyphal growth

Abstract Propolis and geopropolis are resinous products of bees showing antimicrobial effects. There is no data concerning their action against Pythium insidiosum - the causative agent of pythiosis, a pyogranulomatous disease of the subcutaneous tissue that affects mostly horses, dogs and humans. Fragments of 15 isolates of P. insidiodum were incubated with propolis and geopropolis extracts and evaluated for up to seven days to detect the minimal fungicidal concentration (MFC). Propolis inhibited three isolates at 1.0 mg mL-1 after 24 h and all other isolates at 3.4 mg mL-1. Geopropolis led to more variable results, exerting predominantly a fungistatic action than a fungicidal one. Propolis was more efficient than geopropolis in inhibiting P. insidiosum since lower concentrations led to no growth after 24 h. This effect may be due to propolis chemical composition, which has more active compounds than geopropolis. Propolis seemed to be a good candidate for in vivo studies, since treatment with conventional antifungal compounds is difficult in most of the cases, requiring extensive surgical debridement.

Effect of fungicide on Fusarium verticillioides mycelial morphology and fumonisin B1 production

The effect of fludioxonil + metalaxyl-M on the mycelial morphology, sporulation and fumonisin B1 production by Fusarium verticillioides 103 F was evaluated. Scanning electron microscopy analysis showed that the fungicide caused inhibition of hyphal growth and defects on hyphae morphology such as cell wall disruption, withered hyphae, and excessive septation. In addition, extracellular material around the hyphae was rarely observed in the presence of fludioxonil + metalaxyl-M. While promoting the reduction of mycelial growth, the fungicide increased sporulation of F. verticillioides compared to the control, and the highest production occurred on the 14th day in the treatments and on the 10th day in the control cultures. Fumonisin B1 production in the culture media containing the fungicide (treatment) was detected from the 7th day incubation, whereas in cultures without fungicide (control) it was detected on the 10th day. The highest fumonisin B1 production occurred on the 14th day, both for the control and for the treatment. Fludioxonil + metalaxyl - M can interfere in F. verticillioides mycelial morphology and sporulation and increase fumonisin B1 levels. These data indicate the importance of understanding the effects of fungicide to minimize the occurrence of toxigenic fungi and fumonisins.

Treatment with some anti-inflammatory drugs reduces germ tube formation in Candida albicans strains

Candida albicans is an opportunistic dimorphic fungus that inhabits various host mucosal sites. It can cause both superficial and serious systemic disease. Conversion from the yeast to the hyphal form has been associated with increased virulence and mucosal invasiveness. The aim of this study was to investigate the effect of sodium diclofenac and aspirin on germs tube formation of different Candida albicans strains. Prostaglandins may play an important role in fungal colonization. Nonsteroidal anti-inflammatory drugs are inhibitors of the cyclooxygenase isoenzymes. These drugs specifically block the biosynthesis of mammalian prostaglandins by inhibiting one or both of cyclooxygenase isoenzymes. In tests for germ tube formation sodium diclofenac reduced the filamentation to the 12.5%- 5.1%. In the presence of aspirin the filamentation was reduced up to 85-45% depending on the tested strain. Our results suggest that cyclooxygenase-depending synthesis of fungal prostaglandins is important for morphogenesis and fungal virulence. Inhibitors of cyclooxygenase isoensymes (aspirin and diclofenac) are effective in decreasing germ tube formation of Candida albicans.

Anti-fungal potentials of extracellular metabolites of Western Ghats isolated Streptomyces sp. NII 1006 against moulds and yeasts.

Realization of hazardious effects of chemical fungicides has led to an interest in the usage of biocontrol agents. The present study, therefore, evaluates the biocontrol efficacy of Western Ghats (India) soil bacterial isolates. A potential strain NII 1006 was evaluated for its antagonistic property against a diverse range of moulds and yeasts. The strain was characterized morphologically, biochemically and molecularly, which revealed the isolate belonged to Streptomyces genus. Organic solvent extracts of NII 1006 culture filtrates inhibited the growth of the test pathogens indicating that growth suppression was due to extracellular anti-fungal metabolites present in the culture filtrates. The strain produced extracellular chitinase enzyme in addition to some stable partially purified anti-fungal compounds. Morphological changes such as hyphae degradation into debris and abnormal shapes were observed in test fungi and yeast grown on potato dextrose broth that contained the NII 1006 culture filtrate. The cell free supernatant has a tolerance to wide range of pH, temperature and enzymes such as lipase and protease. The biocontrol potential of NII 1006 strain may be correlated significantly with their ability to produce antibiotics as well as extracellular hydrolytic enzymes particularly chitinolytic enzyme.

Cancer drugs inhibit morphogenesis in the human fungal pathogen, Candida albicans

Candida infections are very common in cancer patients and it is a common practice to prescribe antifungal antibiotics along with anticancer drugs. Yeast to hyphal form switching is considered to be important in invasive candidiasis. Targeting morphogenetic switching may be useful against invasive candidiasis. In this study, we report the antimorphogenetic properties of thirty cancer drugs.

Exopolysaccharide matrix of developed candida albicans biofilms after exposure to antifungal agents

This study aimed to evaluate the effects of fluconazole or nystatin exposure on developed Candida albicans biofilms regarding their exopolysaccharide matrix. The minimal inhibitory concentration (MIC) against fluconazole or nystatin was determined for C. albicans reference strain (ATCC 90028). Poly(methlymethacrylate) resin (PMMA) specimens were fabricated according to the manufacturer's instructions and had their surface roughness measured. Biofilms were developed on specimens surfaces for 48 h and after that were exposed during 24 h to fluconazole or nystatin prepared in a medium at MIC, 10 x MIC or 100 x MIC. Metabolic activity was evaluated using an XTT assay. Production of soluble and insoluble exopolysaccharide and intracellular polysaccharides was evaluated by the phenol-sulfuric method. Confocal laser scanning microscope was used to evaluate biofilm architecture and percentage of dead/live cells. Data were analyzed statistically by ANOVA and Tukey's test at 5% significance level. The presence of fluconazole or nystatin at concentrations higher than MIC results in a great reduction of metabolic activity (p<0.001). At MIC or 10 x MIC, fluconazole showed high amounts of intracellular polysaccharides (p<0.05), but did not affect the exopolysaccharide matrix (p>0.05). The exposure to nystatin also did not alter the exopolysaccharide matrix at all the tested concentrations (p>0.05). Biofilm architecture was not affected by either of the antifungal agents (p>0.05). Nystatin promoted higher proportion of dead cells (p<0.05). It may be concluded that fluconazole and nystatin above the MIC concentration reduced the metabolic activity of C. albicans biofilms; however, they were not able to alter the exopolysaccharide matrix and biofilm architecture.

Este estudo avaliou o efeito da exposição de fluconazol ou nistatina a biofilmes de Candida albicans desenvolvidos, considerando a matriz de polissacarídeos extracelulares. Inicialmente uma cepa referência de C. albicans (ATCC 90028) foi submetida ao teste de concentração inibitória mínima (CIM) utilizando-se o fluconazol ou nistatina como agentes antifúngicos. Após, espécimes foram confeccionados em resina acrílica de polimetilmetacrilato (PMMA) de acordo com as recomendações do fabricante e tiveram sua rugosidade de superfície padronizada. Após, biofilmes de C. albicans foram desenvolvidos na superfície dos espécimes durante 48 h. Em seguida, os biofilmes foram expostos a fluconazol ou nistatina nas concentrações de CIM, 10 x CIM ou 100 x CIM, por 24 h. A atividade metabólica dos biofilmes foi avaliada pelo teste de XTT. A produção de polissacarídeos extracelulares solúveis e insolúveis, bem como dos polissacarídeos intracelulares foi avaliada pelo método fenol-sulfúrico. A arquitetura dos biofilmes e proporção de células vivas e mortas foi investigada utilizando-se microscopia confocal a laser. Os resultados foram analisados por ANOVA seguido do teste de Tukey, utilizando-se o nível de significância de 5%. A presença do fluconazol ou nistatina em concentrações maiores que CIM resultaram em uma redução significativa da atividade metabólica (p<0,001). Nas concentrações de CIM e 10 x CIM, biofilmes expostos ao fluconazol apresentaram quantidades significativas de polissacarídeos intracelulares (p<0,05), enquanto não houve alterações na quantidade de polissacarídeos extracelulares (p>0,05). A presença de nistatina também não alterou a matriz de polissacarídeos extracelulares em todas as concentrações investigadas (p>0,05). A arquitetura dos biofilmes não foi afetada por ambos os agentes antifúngicos, em qualquer concentração testada (p>0,05). A nistatina apresentou maior proporção de células mortas (p<0,05). Conclui-se que tanto para o fluconazol quanto para a nistatina, concentrações maiores que CIM reduziram a atividade metabólica dos biofilmes de C. albicans; no entanto, tais concentrações não alteraram a matriz de polissacarídeos extracelulares nem a arquitetura dos biofilmes.

Molecular analysis and dimorphism of azole-susceptible and resistant Candida albicans isolates / Análise molecular e dimorfismo de isolados de Candida albicans suscetíveis e resistentes aos azólicos

INTRODUCTION: Candida albicans is responsible for superficial or systemic infections known as candidiasis, which may be found in infected tissue as unicellular budding yeasts, hyphae, or pseudohyphae. In this study, the effects of both fluconazole and itraconazole antifungal agents on the hyphal formation and genotypic characterization of C. albicans isolates classified as either susceptible or resistant were investigated. METHODS: The hyphal production of five C. albicans isolates under the action of antifungal agents was investigated by culturing yeast on growth medium and on hyphal induction medium. The genotypic characterization was carried out for 13 isolates of C. albicans using the random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) method. RESULTS: The dimorphism analysis showed that the hyphal formation was higher in resistant than in the susceptible isolates to both azoles. The RAPD-PCR method identified the formation of two different groups. In group A, four resistant and two susceptible isolates were clustered, and in group B, one resistant and six susceptible isolates were clustered. CONCLUSIONS: Considering that hyphal formation was higher in resistant isolates in the presence of azole drugs, we confirmed that the hyphal production is closely related to susceptibility to azoles. These drugs may affect the morphogenesis of C. albicans depending on their susceptibility to these drugs. In relation to RAPD-PCR, most resistant isolates classified in group A and susceptible isolates in group B demonstrated that this method presented a similar standard between the two groups, suggesting that by this technique, a strong correlation between genotypes and fluconazole-resistant samples may be found.

INTRODUÇÃO: Candida albicans é responsável por infecções superficiais ou sistêmicas conhecidas como candidíase, encontrada em tecidos infectados na forma de leveduras brotantes unicelulares, hifas ou pseudohifas. Neste estudo, os efeitos de agentes antifúngicos como o fluconazol e o itraconazol sobre a formação de hifas e caracterização genotípica de isolados de C. albicans suscetíveis ou resistentes foram investigados. MÉTODOS: A produção de hifas de cinco isolados de C. albicans, sob a ação de antifúngicos foi investigada pelo cultivo da levedura em meios de crescimento e de indução de hifas. A caracterização genotípica foi realizada para 13 isolados de C. albicans pelo método de RAPD-PCR. RESULTADOS: A análise do dimorfismo mostrou que a formação de hifas foi maior nos isolados resistentes do que nos suscetíveis aos antifúngicos. O método de RAPD-PCR identificou a formação de dois diferentes grupos. No grupo A, foram agrupados quatro isolados resistentes e dois suscetíveis e no grupo B um resistente e seis suscetíveis. CONCLUSÕES: Considerando que a formação hifal foi maior em isolados resistentes na presença de azólicos, concluimos que a produção hifal está muito relacionada a suscetibilidade a estes fámacos. Estes antifúngicos podem alterar a morfologia de C. albicans em dependência da sua suscetibilidade. No método de RAPD-PCR, o encontro da maioria dos isolados resistentes classificados como pertencentes ao grupo A e suscetíveis ao grupo B demonstrou que este método apresentou um padrão semelhante entre os dois grupos, sugerindo que por este método pode ser detectado uma estreita correlação entre genótipos e amostras resistentes ao fluconazol.

Alterations in growth and branching of Neurospora crassa caused by sub-inhibitory concentrations of antifungal agents / Alteraciones de crecimiento y ramificación en Neurospora crassa provocadas por concentraciones subinhibitorias de agentes antimicóticos

Six antifungal agents at subinhibitory concentrations were used for investigating their ability to affect the growth and branching in Neurospora crassa. Among the antifungals herein used, the azole agent ketoconazole at 0.5 μg/ml inhibited radial growth more than fluconazole at 5.0 μg/ml while amphotericin B at 0.05 μg/ml was more effective than nystatin at 0.05 μg/ml. Morphological alterations in hyphae were observed in the presence of griseofulvin, ketoconazole and terbinafine at the established concentrations. The antifungal agents were more effective on vegetative growth than on conidial germination. Terbinafine markedly reduced growth unit length (GU) by 54.89%, and caused mycelia to become hyperbranched. In all cases, there was a high correlation between hyphal length and number of tips (r > 0.9). All our results showed highly significant differences by ANOVA, (p < 0.001, α = 0.05). Considering that the hyphal tip is the main interface between the fungus and its environment /through which enzymes and toxins are secreted and nutrients absorbed, it would not be desirable to obtain a hyperbranched mycelia with inefficient doses of antifungal drugs.

Se investigó el efecto de seis agentes antimicóticos en concentraciones subinhibitorias sobre el crecimiento y la ramificación en Neurospora crassa. El agente azólico ketoconazol a la concentración de 0,5 μg/ml inhibió el crecimiento radial más que el fluconazol a 5,0 μg/ml, y la anfotericina B a 0,05 μg/ ml fue más eficiente que 0,05 μg/ml de nistatina, entre los agentes poliénicos usados. En presencia de griseofulvina, ketoconazol y terbinafina a las concentraciones establecidas se observaron alteraciones morfológicas en las hifas. Los agentes antimicóticos fueron más eficientes sobre el crecimiento vegetativo que sobre la germinación conidial. La terbinafina redujo marcadamente (54,89%) la longitud de la unidad de crecimiento y provocó la hiperramificación del micelio. En todos los casos, existió gran correlación entre la longitud y el número de ápices de las hifas (r > 0,9). Todos los resultados mostraron diferencias altamente significativas de acuerdo con ANOVA (p < 0,001, α = 0,05). Considerando que el ápice de la hifa es la principal interfase entre el hongo y su ambiente, a través de la cual las enzimas y las toxinas son secretadas y los nutrientes son absorbidos, un micelio hiperramificado resultante de dosis ineficientes de agentes antimicóticos sería perjudicial.