Mandras et al. BMC Complementary and Alternative Medicine (2016) 16:330 DOI 10.1186/s12906-016-1316-5 RESEARCH ARTICLE Open Access Liquid and vapour-phase antifungal activities of essential oils against Candida albicans and non-albicans Candida Narcisa Mandras1†, Antonia Nostro2†, Janira Roana1, Daniela Scalas1, Giuliana Banche1, Valeria Ghisetti3, Simonetta Del Re3, Giacomo Fucale4, Anna Maria Cuffini1 and Vivian Tullio1* Abstract Background: The 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. Methods: The 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). Results: Using 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. Conclusion: Results 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. Keywords: Antifungal activity, Yeasts, Essential oils, Broth microdilution method, Vapour contact assay Abbreviations: BM, Broth microdilution method; EOs, Essential oils; MFC, Minimal fungicidal concentration; MIC, Minimum inhibitory concentration; VC, Vapour contact assay; WT, Wild type * Correspondence: [email protected] †Equal contributors 1Department of Public Health and Paediatrics, Microbiology Division, University of Turin, via Santena 9, 10126 Turin, Italy Full list of author information is available at the end of the article © 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Mandras et al. BMC Complementary and Alternative Medicine (2016) 16:330 Page 2 of 7 Background without any further purification. EOs and related com- Candidiasis is the most common opportunistic yeast in- ponents were storage at 4 °C until use. fection and encompasses infections that range from superficial mucosal infections, such as oral thrush and Essential oils and their components stock solutions vaginitis, to systemic and potentially life-threatening dis- Stock solutions of each EO and its components were eases, such as disseminated candidiasis. In the last two prepared in ethanol (1:25) and diluted (1:20) to obtain a decades, it has been observed a considerable increase in final concentration of 2 % (v/v) in RPMI-1640 without the incidence of deep fungal infections, not only in im- sodium bicarbonate and with L-glutamine (Invitrogen, munocompromised patients related to nosocomial infec- San Giuliano Milanese, Milano, Italy), buffered to pH 7.0 tions, but also in healthy subjects. Moreover, the with 0.165 M morpholinepropanesulfonic acid (MOPS) − incidence of C. albicans, the leading pathogenic Candida (Sigma) at a concentration of 0.165 mol 1 1 and supple- species so far, has declined while that of non-albicans mented with glucose 18 g/L. To enhance EOs solubility, Candida is increased [1]. Tween-80 (Sigma-Aldrich) was included at a final con- The most commonly used classes of antifungal agents centration of 0.001 % (v/v). to treat Candida infections are the azoles, polyenes, and echinocandins; however, the management of Candida Antifungal drugs infections faces many problems, such as toxicity, resist- Fluconazole and voriconazole powders (≥98 % purity by ance of Candida to commonly used antifungal drugs, HPLC) were purchased from Sigma-Aldrich (n° F8929 relapse of Candida infections, and the high cost of anti- and PZ0005, respectively). Fluconazole stock solutions fungal drugs [2, 3]. To elude these problems, investiga- were prepared in sterile distilled water, while voricona- tors are exploiting alternative therapeutic strategies, zole in 100 % dimethyl sulfoxide (Sigma-Aldrich), and such as the use of natural products, especially essential stored at −20 °C until use. oils (EOs) [4–7]. EOs have long been used in ethnomedicine as effective Yeasts and safe antifungal agents; however, good scientific and Forty-six yeasts, including C. albicans (n = 26), C. glab- clinical data that either supports or contravenes the ef- rata (n = 10) and C. tropicalis (n = 10) were collected fectiveness of these alternative therapies are still needed from various specimens (blood, normally sterile body before consumers can be sure they will enjoy any bene- fluids, deep tissue, genital tract, gastrointestinal tract, re- fits. Previously we have studied the antimicrobial activity spiratory tract) from hospitalized patients in Turin (Italy). of thyme red, clove, pine, sage, lemon balm, fennel, lav- The strains were identified by standard methods (corn- ender EOs against filamentous fungi [8]. Hence, the ob- meal for blastoconidia, germ-tube formation, pseudohy- jective of this study was to extend the research to phae and true hyphae, and growth on CHROMagar™ evaluate the activity of the same EOs on Candida albi- Candida (BD, Milan, Italy) and with commercially avail- cans and non-albicans Candida strains, as well as the able yeast identification system (API ID32C panels, bio- effects of related EO components, by using two investi- Mérieux, Rome, Italy) [10]. gative tools, such as the broth microdilution method (BM) and the vapour contact assay (VC). EOs selection Inoculum preparation was based both on ethnomedicinal use and on proved Yeasts inocula were prepared by picking two to three antibacterial and/or antifungal activity of some of these colonies of >1 mm diameter from an overnight culture oils [8, 9]. Fluconazole and voriconazole were used as of Candida spp. on Sabouraud dextrose (SAB) agar at reference drugs to compare EOs activity. 35 °C, and suspending them in 2 mL of 0.85 % normal saline, to yield a yeast stock suspension of ≈ 5×106 Methods cells/mL by 0.5 McFarland standard. A working suspen- Essential oils and their components sion was made by a 1:100 dilution followed by a 1:20 di- The EOs have directly been purchased from Azienda lution of the stock suspension with RPMI 1640 broth Agricola Aboca (Sansepolcro, Arezzo) as steam distilled medium (0.2 % glucose) (Sigma, Milan, Italy), which re- samples obtained from Thymus vulgaris L. (thyme red), sulted in ≈ 2.5 × 103 cells/mL, confirmed by colony counts Foeniculum vulgare Mill. var. dulce DC (fennel), Eugenia in triplicate. The cell density was adjusted to 2.0 × 103 caryophyllata Thumb. (clove), Pinus sylvestris L. (pine), cells/mL, confirmed by colony counts in triplicate. Salvia officinalis L. (sage), Melissa officinalis L. (lemon balm) and Lavandula vera DC (lavender). The compo- In vitro antimicrobial assays nents carvacrol, eugenol, linalool, linalyl acetate, thymol Broth microdilution method and α-pinene (≥98 % purity) were supplied by Sigma The antimicrobial activity of EOs and their components Aldrich (Steinheim, Germany) and used as received was determined using a BM susceptibility assay, Mandras et al. BMC Complementary and Alternative Medicine (2016) 16:330 Page 3 of 7 according to CLSI document M27-A3 for yeasts with the cover of each Petri dish, so that it did not directly some modifications [11]. Minimum inhibitory concen- touch the surface of the agar medium, and various tration (MIC) determination was performed by serial di- amounts of pure EOs or their components were added to lution using 96-well microtitre plates (Sarstedt, Milan, obtain final concentration ranging from 1 to 0.0019 %, v/v Italy). Doubling dilutions of the EOs ranging from 2 to air space. The space inside of the sealed Petri dish was cal- 0.0038 % (v/v) were prepared in 96-well microtitre trays culated to be 70 cm3 air. The plates were sealed with vinyl in RPMI-1640 with MOPS buffered to pH 7. Further- tape immediately after inoculation, and incubated at 37 °C more, each yeast strain included in the study was tested for 48 h. The control, consisting of RPMI medium with for its sensitivity to fluconazole and voriconazole follow- MOPS, was included.