Abstract Candida infection is an important cause of morbidity and mortality in immunocompromised patients. The increase in its incidence has been associated with resistance to antimicrobial therapy and biofilm formation. The aim of this study was to evaluate the efficacy of tea tree oil (TTO) and its main component – terpinen-4-ol – against resistant Candida albicans strains (genotypes A and B) identified by molecular typing and against C. albicans ATCC 90028 and SC 5314 reference strains in planktonic and biofilm cultures. The minimum inhibitory concentration, minimum fungicidal concentration, and rate of biofilm development were used to evaluate antifungal activity. Results were obtained from analysis of the biofilm using the cell proliferation assay 2,3-Bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) and confocal laser scanning microscopy (CLSM). Terpinen-4-ol and TTO inhibited C. albicans growth. CLSM confirmed that 17.92 mg/mL of TTO and 8.86 mg/mL of terpinen-4-ol applied for 60 s (rinse simulation) interfered with biofilm formation. Hence, this in vitro study revealed that natural substances such as TTO and terpinen-4-ol present promising results for the treatment of oral candidiasis.

Abstract: We evaluated the antifungal and antibiofilm potential of the hydroalcoholic extract of bark from Anadenanthera colubrina (vell.) Brenan, known as Angico, against Candida spp. Antifungal activity was evaluated using the microdilution technique through the Minimum Inhibitory and Fungicide Concentrations (MIC and MFC). The antibiofilm potential was tested in mature biofilms formed by Candida species and analyzed through the counting of CFU/mL and scanning electron micrograph (SEM). In vivo toxicity and therapeutic action was evaluated in the Galleria mellonella model. The treatment with the extract, in low doses, was able to reduce the growth of planktonic cells of Candida species. MIC values range between 19.5 and 39 µg/mL and MFC values range between 79 and 625 µg/mL. In addition was able to reduce the number of CFU/mL in biofilms and to cause structural alteration and cellular destruction, observed via SEM. A. colubrina showed low toxicity in the in vivo assay, having not affected the viability of the larvae at doses below 100mg/kg and high potential in the treatment of C. albicans infection. Considering its high antifungal potential, its low toxicity and potential to treatment of infections in in vivo model, A. colubrina extract is a strong candidate for development of a new agent for the treatment of oral candidiasis.

Resumo O objetivo desse estudo foi avaliar a atividade antifúngica do Terpinen4-ol associado à nistatina em biofilmes simples e misto, formados por Candida albicans e Candida tropicalis, bem como o efeito do terpinen-4-ol na adesão em células orais e atividade enzimática. As concentrações inibitórias mínimas e as concentrações fungicidas mínimas do terpinen-4-ol e da nistatina em Candida albicans e Candida tropicalis foram determinadas pelo método de microdiluição em caldo, juntamente com a atividade sinérgica (método do tabuleiro de “xadrez”). Biofilmes simples e misto foram preparados usando o modelo de placa de microtitulação estática e quantificados por unidades formadoras de colônias (CFU/mL). O efeito do Terpinen-4-ol na adesão de Candida albicans e Candida tropicalis em co-cultura com queratinócitos orais (NOK Si) foi avaliado, bem como a atividade enzimática, medindo o tamanho da zona de precipitação, após o crescimento em ágar fosfolipase, protease e hemolisina. O terpinen-4-ol (4.53 mg mL-1) e a nistatina (0,008 mg mL-1) conseguiram inibir o crescimento de biofilmes e um efeito antifúngico sinérgico foi demonstrado com a associação de fármaco, reduzindo a concentração inibidora de nistatina até 8 vezes em biofilme simpes de Candida albicans e 2 vezes em biofilme misto. Uma pequena diminuição na adesão de Candida tropicalis em células NOK Si foi mostrada após o tratamento com terpinen-4-ol e a nistatina teve um efeito maior para ambas as espécies. Para a atividade enzimática, as drogas não apresentaram ação. O efeito potencializado pela combinação de terpinen-4-ol e nistatina e a redução de adesão evidenciam seu potencial como agente anti-fúngico.

Abstract The aim of this study was to evaluate the antifungal activity of Terpinen-4-ol associated with nystatin, on single and mixed species biofilms formed by Candida albicans and Candida tropicalis, as well as the effect of terpinen-4-ol on adhesion in oral cells and the enzymatic activity. The minimum inhibitory concentrations and minimum fungicide concentrations of terpinen-4-ol and nystatin on Candida albicans and Candida tropicalis were determined using the microdilution broth method, along with their synergistic activity (“checkerboard” method). Single and mixed species biofilms were prepared using the static microtiter plate model and quantified by colony forming units (CFU/mL). The effect of Terpinen-4-ol in adhesion of Candida albicans and Candida tropicalis in coculture with oral keratinocytes (NOK Si) was evaluated, as well as the enzymatic activity by measuring the size of the precipitation zone, after the growth agar to phospholipase, protease and hemolysin. Terpinen-4-ol (4.53 mg mL-1) and nystatin (0.008 mg mL-1) were able to inhibit biofilms growth, and a synergistic antifungal effect was showed with the drug association, reducing the inhibitory concentration of nystatin up to 8 times in single biofilm of Candida albicans, and 2 times in mixed species biofilm. A small decrease in the adhesion of Candida tropicalis in NOK Si cells was showed after treatment with terpinen-4-ol, and nystatin had a greater effect for both species. For enzymatic activity, the drugs showed no action. The effect potentiated by the combination of terpinen-4-ol and nystatin and the reduction of adhesion provide evidence of its potential as an anti-fungal agent.

Millions of people and animals suffer from superficial infections caused by a group of highly specialized filamentous fungi, the dermatophytes, which only infect keratinized structures. With the appearance of AIDS, the incidence of dermatophytosis has increased. Current drug therapy used for these infections is often toxic, long-term, and expensive and has limited effectiveness; therefore, the discovery of new anti dermatophytic compounds is a necessity. Natural products have been the most productive source for new drug development. This paper provides a brief review of the current literature regarding the presence of dermatophytes in immunocompromised patients, drug resistance to conventional treatments and new anti dermatophytic treatments.