Effects of Rosmarinus offi cinalis essential oil on germ tube formation by Candida albicans isolated from denture wearers

Introduction: The aim of this study was to investigate the effects of Rosmarinus offi cinalis essential oil on germ tube formation by Candida albicans isolated from denture wearers. Methods: Ten C. albicans isolates recovered from denture wearers were tested using 10% fetal bovine serum with or without 4% R. offi cinalis essential oil. Results: The essential oil from R. offi cinalis completely inhibited germ tube formation in the investigated C. albicans isolates. Conclusions: The results demonstrate that the essential oil of R. offi cinalis modulates C. albicans pathogenicity through its primary virulence factor (i.e., germ tube formation was suppressed).

Candida albicans is the primary fungal species associated with oral candidiasis, can adhere to and colonize denture resins, and contributes to denture-related stomatitis, during which the yeast may form germ tubes and hyphae 1 .Candida albicans virulence depends on its ability to transform between the yeast and fi lamentous (pseudohyphae or true hyphae) forms 2 ; the latter is induced in human infections 3 .Recent studies have aimed at developing novel therapies to control denture surface colonization by Candida species 4 .Additionally, investigating the antifungal activities 5,6 of medicinal plants might facilitate the identifi cation of compounds that could potentially be developed into novel antifungal agents.Lima et al. 7 found that C. albicans (ATCC-76615) was sensitive to 8% Rosmarinus offi cinalis essential oil, resulting in a 10-mm inhibition zone.Based on such considerations, the aim of this study was to investigate the effects of R. offi cinalis essential oil on germ tube formation by C. albicans isolated from denture wearers.
Ten C. albicans isolates were recovered from the oral cavities of 10 denture wearers (Table 1).The yeast was isolated from the denture base or the palatal mucosa using sterile swabs.The samples were collected from March to October 2012 at the School of Clinical Dentistry in the Federal University of Pará (Brazil).The study was approved by the Research Ethics Committee of the Evandro Chagas Institute (Comitê de Ética em Pesquisa do Instituto Evandro Chagas -CEP/IEC 032/10).The volunteers were informed of the study aims and procedures and signed an informed consent form.The isolates were identifi ed based on their carbohydrate assimilation profi les using the Vitek 2 system (bioMérieux, Marcy l'Etoile, France).Next, the isolates were grown on Sabouraud dextrose agar (Difco, Laboratories, Detroit MI) under aerobic conditions at 37ºC for over 24h.A suspension containing 10 6 cells/ml in sterile PBS (phosphate-buffered saline) (pH 7.2) was generated and used for the germ tube inhibition assay.Rosmarinus offi cinalis was grown in the Jacques Huber botanical garden, which is located at the Coordination of Botany on the research campus of the Goeldi Museum, under 50% shade in black polyethylene pots, with dimensions of 20 × 25cm, fi lled with black soil substrate; the plants were irrigated as needed to maintain the humidity of the substrate.The specimens were cultured during the rainy season and did not receive any chemical treatment, such as pesticides or fertilizers.The collections of botanic material (by Gauch, L.M.R 01) were held at the growing site, the phenophase of fl ower buds adults.R. offi cinalis was identifi ed by Ely Simone Cajueiro Gurgel (Emilio Goeldi Museum, Pará, Brazil) using MG 204.248.The essential oil was extracted from fresh leaves (350g) through steam distillation for over 240min using the Clevenger system.The process yielded 2mL of essential oil, which was stored in refrigerated and dark conditions.
The effects of the R. offi cinalis essential oil on germ tube formation were assessed as described by Bernardes et al. 8 with Gauch LMR et al -R.offi cinalis inhibits germ tube by C. albicans some modifi cations.Briefl y, germ tube formation was rapidly induced in Sabouraud dextrose broth using 10% fetal bovine serum and either 4% R. offi cinalis essential oil and 0.02% Tween 80 (assay tube) or nothing (control tube), with 10ml as a fi nal volume.The yeast suspension (100µL) was inoculated, and the test was conducted at 37ºC for over 3h.The number of cells was determined using a Neubauer chamber, and germ tube formation was expressed as the percentage of germ tube-forming cells relative to the total cell number.All isolates were tested in duplicate, and the C. albicans INCQS 49175 strain was used as a positive control.The effect of R. officinalis essential oil on germ tube formation was assessed in 10 C. albicans isolates.In the samples that were treated with 10% fetal bovine serum and no essential oil, the germ tube-forming cells exhibited a range of reactions, including fi ve isolates (5/10) that exhibited 100% germ tube formation; however, the percentage varied from 63% to 94% in the remaining fi ve isolates (5/10).The type of stomatitis exhibited by the denture wearers was not related to the number of germ tube-forming cells.When 4% R. offi cinalis essential oil was added to the incubation, germ tube formation was entirely inhibited in the investigated isolates.All of these results are shown in Table 1.
The literature includes many studies assessing the susceptibility of yeasts of the genus Candida to medicinal plant derivatives 6 ; however, few studies have investigated the plant derivatives' effects on C. albicans morphology and germ tube formation.Herein, the complete inhibition of germ tube formation was observed in the investigated isolates when 4% R. offi cinalis essential oil was added.Several studies investigated the antifungal activity of various R. offi cinalis derivatives against C. albicans 5,7 ; however, its effect on the primary fungal virulence factor (i.e., the germ tube) has been neglected.According to Silva et al. 9 , a compound's ability to inhibit germ tube formation could be a means to assess its antifungal activity.In this context, the results of this study clearly demonstrate the antifungal activity of R. offi cinalis essential oil and its capacity to modulate this fungal species' pathogenicity.
Consistent with our results, Bernardes et al. 8 observed a 95% reduction in germ cell formation when C. albicans cells (ATCC 10-231) were treated with raw glycolic 10% (v/v) extract from fresh Aloe vera leaves.Interestingly, Pinto et al. 10 demonstrated an effect of the Ferulago capillaris essential oil on cells during inhibition of germ tube formation in C. albicans, which demonstrated that oxidative stress affects the enzymatic activity as well as cell mitochondrial membrane potential and results in growth inhibition and death.Given the high cost of the required methods, it was impossible to analyze the composition of the essential oil investigated in this study, particularly for the two primary active components, α-pinene and 1,8-cineole 11 .Knowledge of the essential oil composition is necessary to identify the mechanism underlying the inhibition of germ tube formation in C. albicans.Previously, Pozzatti et al. 12 demonstrated that R. officinalis extract was able in to inhibit germ tube formation in both C. albicans and C. dubliniensis.Thus, our results were in agreement with this information and illustrated that this essential oil also exhibits antifungal properties.
Because oral mucosa and denture resin colonization by C. albicans is a predisposing factor for oral mucosa infections, such as denture-related stomatitis, the ability to reduce yeast viability is a desired quality in candidate products and/or compounds that could be developed into novel therapeutic agents.A study conducted by Ellepola et al. 13

TABLE 1 -Data for the denture wearers who provided the Candida albicans isolates.
a stomatitis type according to Newton's classifi cation.b percentage of germ tubes formed.M: male; F: female.