The increasing recognition and importance of fungal infections, the difficulties encountered in their treatment and the increase in resistance to antifungals have stimulated the search for therapeutic alternatives. Essential oils have been used empirically. The essential oils of Thymus (Thymus vulgaris, T. zygis subspecies zygis and T. mastichina subspecies mastichina) have often been used in folk medicine. The aim of the present study was to evaluate objectively the antifungal activity of Thymus oils according to classical bacteriological methodologies - determination of the minimal inhibitory concentration (MIC) and the minimal lethal concentration (MLC) - as well as flow cytometric evaluation. The effect of essential oils upon germ tube formation, an important virulence factor, was also studied. The mechanism of action was studied by flow cytometry, after staining with propidium iodide. The chemical composition of the essential oils was investigated by gas chromatography (GC) and gas chromatography/mass spectroscopy (GC/MS). The antifungal activity of the major components (carvacrol, thymol, p-cymene and 1,8-cineole) and also possible interactions between them were also investigated. The essential oils of T. vulgaris and T. zygis showed similar antifungal activity, which was greater than T. mastichina. MIC and MLC values were similar for all the compounds tested. At MIC values of the essential oils, propidium iodide rapidly penetrated the majority of the yeast cells, indicating that the fungicidal effect resulted primarily from an extensive lesion of the cell membrane. Concentrations below the MIC values significantly inhibited germ tube formation. This study describes the potent antifungal activity of the essential oils of Thymus on Candida spp., warranting future therapeutical trials on mucocutaneous candidosis.

The composition of the essential oil of Thymus pulegioides and its antifungal activity on Candida, Aspergillus and dermatophyte fungal strains were studied. Essential oil from the aerial parts of the plant was obtained by hydrodistillation and analysed by GC and GC-MS. The oil showed high contents of carvacrol and thymol. The MIC and minimal lethal concentration were used to evaluate the antifungal activity against Candida (seven clinical isolates and four ATCC type strains), Aspergillus [five clinical isolates, and two Colecció n Españ ola de Cultivos Tipo (CECT) and two ATCC type strains] and five clinical dermatophyte strains. Antifungal activity was evaluated for the essential oil and for its main components. To clarify its mechanism of action on yeasts and filamentous fungi, flow-cytometric studies of cytoplasmic membrane integrity were performed, and the effect on the amount of ergosterol was investigated. Results showed that T. pulegioides essential oil exhibited a significant activity against clinically relevant fungi, mainly due to lesion formation in the cytoplasmic membrane and a considerable reduction of the ergosterol content. The present study indicates that T. pulegioides essential oil has considerable antifungal activity, deserving further investigation for clinical applications.

Candida albicans represents the most frequent isolated yeast from bloodstream infections. Despite the remarkable progress in diagnostic and therapeutic approaches, these infections continue to be a critical challenge in intensive care units worldwide. The economic cost of bloodstream fungal infections and its associated mortality, especially in debilitated patients, remains unacceptably high. Candida albicans is a highly adaptable microorganism, being able to develop resistance following prolonged exposure to antifungals. Formation of biofilms, which diminish the accessibility of the antifungal, selection of spontaneous mutations that increase expression or decreased susceptibility of the target, altered chromosome abnormalities, overexpression of multidrug efflux pumps and the ability to escape host immune defenses are some of the factors that can contribute to antifungal tolerance and resistance. The knowledge of the antifungal resistance mechanisms can allow the design of alternative therapeutically options in order to modulate or revert the resistance. We have focused this review on the main factors that are involved in antifungal resistance and tolerance in patients with C. albicans bloodstream infections.

Invasive fungal infections, specifically candidemia, constitute major public health problems with high mortality rates. Therefore, in the last few years, the development of novel diagnostic methods has been considered a critical issue. Herein we describe a multiplex PCR strategy allowing the identification of 8 clinically relevant yeasts of the Candida genus, namely C. albicans, C. glabrata, C. parapsilosis, C. tropicalis, C. krusei, C. guilliermondii, C. lusitaniae and C. dubliniensis. This method is based on the amplification of two fragments from the ITS1 and ITS2 regions by the combination of 2 yeast-specific and 8 species-specific primers in a single PCR reaction. Results from the identification of 231 clinical isolates are presented pointing to the high specificity of this procedure. Furthermore, several Candida isolates were identified directly from clinical specimens which also attests to the method's direct laboratory application. The results from the multiplex reactions with other microorganisms that usually co-infect patients also confirmed its high specificity in the identification of Candida species. Moreover, this method is simple and presents a sensitivity of approximately 2 cells per ml within 5 hours. Furthermore, it allows discrimination of individual Candida species within polyfungal samples. This novel method may therefore provide a clinical diagnostic procedure with direct applicability.