BackgroundCryptococcal infections, besides being a problem for immunocompromised patients, are occasionally being a problem for immunocompetent patients. In addition, the lower susceptibility of this yeast to azoles is a growing problem in health care. To date, there are very few molecules with any activity towards Cryptococcus neoformans, leading to heightened interest in finding new alternatives or adjuvants to conventional drugs for the treatment of mycosis caused by this yeast. Since the essential oils (EOs) are considered as a potential rich source of bioactive antimicrobial compounds, we evaluated the antifungal activity of Origanum vulgare (oregano), Pinus sylvestris (pine), and Thymus vulgaris (thyme red) EOs, and their components (α-pinene, carvacrol, thymol) compared with fluconazole, itraconazole, and voriconazole, against C.neoformans clinical strains. Then, we investigated the effect of EOs and components in combination with itraconazole.MethodsEO composition was analysed by Gas chromatography-mass spectrometry (GC-MS). A broth microdilution method was used to evaluate the susceptibility of C.neoformans to azoles, EOs and components. Checkerboard tests, isobolograms and time-kill assays were carried out for combination studies.ResultsSix C.neoformans isolates were susceptible to azoles, while one C.neoformans exhibited a reduced susceptibility to all tested azole drugs. All EOs exerted a good inhibitory activity against all C.neoformans strains. Pine EO was the most effective. Among components, thymol exerted the most remarkable activity. By checkerboard testing and isobolographic analysis, combinations of itraconazole with oregano, pine, or thyme EOs, and carvacrol were found to be synergistic (FICI≤0.5) against azole susceptible C.neoformans. Regarding the azole not susceptible C.neoformans strain, the synergistic effect with itraconazole was observed with thyme EO (chemotype: thymol 26.52%; carvacrol 7.85%), and carvacrol. Time-kill assays confirmed the synergistic effects of itraconazole and oregano or thyme EO against azole susceptible C.neoformans. Binary mixtures of itraconazole/thyme EO or carvacrol yielded additive effects on the azole not susceptible C.neoformans.ConclusionsOur findings highlight the potential effectiveness of thyme, oregano EOs, and carvacrol as natural and cost-effective adjuvants when used in combination with itraconazole. Identification of EOs exerting these effects could be one of the feasible ways to overcome drug resistance, reducing drug concentration and side effects.

BackgroundThe management of Candida infections faces many problems, such as a limited number of antifungal drugs, toxicity, resistance of Candida to commonly antifungal drugs, relapse of Candida infections, and the high cost of antifungal drugs. Though azole antifungal agents and derivatives continue to dominate as drugs of choice against Candida infections, there are many available data referring to the anticandidal activity of essential oils. Since we have previous observed a good antimicrobial activity of some essential oils against filamentous fungi, the aim of this study was to extend the research to evaluate the activity of the same oils on Candida albicans, C.glabrata and C.tropicalis clinical strains, as well as the effects of related components. Essential oils selection was based both on ethnomedicinal use and on proved antibacterial and/or antifungal activity of some of these oils. Fluconazole and voriconazole were used as reference drugs.MethodsThe minimum inhibitory concentration (MIC) and the minimal fungicidal concentration (MFC) of essential oils (thyme red, fennel, clove, pine, sage, lemon balm, and lavender) and their major components were investigated by the broth microdilution method (BM) and the vapour contact assay (VC).ResultsUsing BM, pine oil showed the best activity against all strains tested, though C.albicans was more susceptible than C.glabrata and C.tropicalis (MIC50-MIC90 = 0.06 %, v/v). On the contrary, sage oil displayed a weak activity (MIC50-MIC90 = 1 %, v/v). Thyme red oil (MIC50-MIC90 ≤ 0.0038 %, v/v for C.albicans and C.tropicalis, and 0.0078- < 0.015 %, v/v for C.glabrata), followed by lemon balm, lavender and sage were the most effective by VC. Carvacrol and thymol showed the highest activity, whereas linalyl acetate showed the lowest activity both by two methods. α-pinene displayed a better activity by BM than VC.ConclusionResults show a good activity of essential oils, mainly thymus red and pine oils, and their components carvacrol, thymol and α-pinene against Candida spp., including fluconazole/voriconazole resistant strains. These data encourage adequately controlled and randomized clinical investigations. The use in vapour phase could have additional advantages without requiring direct contact, resulting in easy of environmental application such as in hospital, and/or in school.

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The promising antimicrobial activity of essential oils (EOs) has led researchers to use them in combination with antimicrobial drugs in order to reduce drug toxicity, side effects, and resistance to single agents. Mentha x piperita, known worldwide as “Mentha of Pancalieri”, is produced locally at Pancalieri (Turin, Italy). The EO from this Mentha species is considered as one of the best mint EOs in the world. In our research, we assessed the antifungal activity of “Mentha of Pancalieri” EO, either alone or in combination with azole drugs (fluconazole, itraconazole, ketoconazole) against a wide panel of yeast and dermatophyte clinical isolates. The EO was analyzed by GC-MS, and its antifungal properties were evaluated by minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) parameters, in accordance with the CLSI guidelines, with some modifications. The interaction of EO with azoles was evaluated through the chequerboard and isobologram methods. The results suggest that this EO exerts a fungicidal activity against yeasts and a fungistatic activity against dermatophytes. Interaction studies with azoles indicated mainly synergistic profiles between itraconazole and EO vs. Candida spp., Cryptococcus neoformans, and Trichophyton mentagrophytes. Thus, the “Mentha of Pancalieri” EO may act as a potential antifungal agent and could serve as a natural adjuvant for fungal infection treatment.

The influence of caspofungin on polymorphonuclear leukocyte (PMN) phagocytosis and intracellular killing of Candida albicans was investigated. Caspofungin, at all of the concentrations tested (2, 3.2, and 8 g/ml), significantly increased intracellular killing by PMNs through its direct action on both yeast cells and PMNs, indicating the potential ability of caspofungin to synergize with phagocytes for candidal killing. Caspofungin may therefore constitute an effective therapeutic option for the treatment of invasive fungal infections, including those refractory to conventional treatment with azole agents.Echinocandins, such as caspofungin, are new drugs that broaden the available therapeutic arsenal for invasive fungal infection (IFI) treatment (6,7,11). Caspofungin displays favorable pharmacodynamic and pharmacokinetic characteristics and has an excellent toxicological profile and antifungal activity against Candida spp., Aspergillus spp., Histoplasma spp., Blastomyces spp., and Coccidioides spp. (3,7,11,12,15). As the current trend in therapy requires drugs with high in vitro activity associated with the capacity to potentiate host defense mechanisms, especially in immunocompromised hosts (2, 19), the interaction of caspofungin with human polymorphonuclear leukocytes (PMNs) was evaluated, focusing on both the phagocytosis and intracellular killing of Candida albicans.(This study was presented in part at the Congress of the Italian Society of Pharmaceutical Microbiology, Turin, Italy, 20 to 22 June 2008.)A clinical C. albicans strain isolated from blood and identified by biochemical methods was subcultured on Sabouraud dextrose agar (Oxoid S.p.A., Milan, Italy) to ensure viability and purity. Yeast cultures consisted entirely of blastoconidia and had a slight tendency to differentiate into pseudohyphae during the course of the experiments.Caspofungin acetate (Merck Sharp & Dohme Ltd., Hoddesdon, United Kingdom) was dissolved in pyrogen-free water and stored at Ϫ20°C. Antifungal susceptibility testing was performed with an inoculum of 10 3 CFU/ml, in accordance with CLSI M27-A3 (4), and an inoculum of 10 6 CFU/ml was used to perform tests with phagocytes.PMNs were separated from lithium heparinized venous blood using Ficoll-Paque (Pharmacia S.p.A., Milan, Italy) and adjusted to 10 6 cells/ml in RPMI 1640 medium (Gibco Laboratories, Grand Island, NY) (1, 5). Viability, determined by trypan blue exclusion, was greater than 95%.The effect of caspofungin on the phagocytosis of radiolabeled C. albicans ([ 3 H]uracil [specific activity, 1,270 GBq/ mmol; NEN Life Science Products, Milan, Italy]) by PMNs was investigated by incubating the yeast cells (10 6 invasive fungal cells/ml) and PMNs (10 6 cells/ml) at 37°C in a shaking water bath in the presence of 2 g/ml (MIC), 3.2 g/ml, or 8 g/ml caspofungin; the last two concentrations were within the range achieved clinically (8, 9). Caspofungin-free controls were included. After 30, 60, or 90 min, phagocytosis was assessed (18,19). PMNs were centrifuged twice at 200 ϫ g fo...