Background
Candidiasis is the most common opportunistic yeast infection and encompasses infections that range from superficial mucosal infections, such as oral thrush and vaginitis, to systemic and potentially life-threatening diseases, such as disseminated candidiasis. In the last two decades, it has been observed a considerable increase in the incidence of deep fungal infections, not only in immunocompromised patients related to nosocomial infections, but also in healthy subjects. Moreover, the incidence of
C. albicans, the leading pathogenic Candida species so far, has declined while that of non-
albicans Candida is increased [
1].
The most commonly used classes of antifungal agents to treat Candida infections are the azoles, polyenes, and echinocandins; however, the management of Candida infections faces many problems, such as toxicity, resistance of Candida to commonly used antifungal drugs, relapse of Candida infections, and the high cost of antifungal drugs [
2,
3]. To elude these problems, investigators are exploiting alternative therapeutic strategies, such as the use of natural products, especially essential oils (EOs) [
4‐
7].
EOs have long been used in ethnomedicine as effective and safe antifungal agents; however, good scientific and clinical data that either supports or contravenes the effectiveness of these alternative therapies are still needed before consumers can be sure they will enjoy any benefits. Previously we have studied the antimicrobial activity of thyme red, clove, pine, sage, lemon balm, fennel, lavender EOs against filamentous fungi [
8]. Hence, the objective of this study was to extend the research to evaluate the activity of the same EOs on
Candida albicans and non-
albicans Candida strains, as well as the effects of related EO components, by using two investigative tools, such as the broth microdilution method (BM) and the vapour contact assay (VC). EOs selection was based both on ethnomedicinal use and on proved antibacterial and/or antifungal activity of some of these oils [
8,
9]. Fluconazole and voriconazole were used as reference drugs to compare EOs activity.
Results and discussion
The research on EOs and closely related components has been recently intensified, due to their biological, antioxidant and antimicrobial properties [
5,
15‐
18]. Moreover, there is a growing evidence that EOs in vapour phase are effective antimicrobial systems and that they could have advantages over the use of EOs in liquid phase, especially in a hospital environment. In fact, our previously studies demonstrated a better activity in vapour phase of some oils (thyme red, fennel, clove, pine, sage, lemon balm and lavender) against human and plant pathogen filamentous fungi [
8].
In this study, we evaluated by two methods (BM and VC) the antifungal activity of seven EOs including thyme red, fennel, clove, pine, sage, lemon balm and lavender (Table
1) and some their components (Table
2), towards 46 clinical isolates of
C. albicans,
C. glabrata and
C. tropicalis. In literature, there are some available data referring to the anticandidal activity of EOs such as thyme, clove, pine, fennel, sage, and lemon balm [
5‐
7,
19,
20].
Our results, reported as concentrations of EO, major components, fluconazole and voriconazole, are showed as MIC
50 (Minimum Inhibitory Concentration required to inhibit the growth of 50 % of yeasts) and MIC
90 (Minimum Inhibitory Concentration which inhibits the growth of 90 % of yeasts), respectively (Tables
1,
2).
Table 1
Minimum inhibitory concentration (MIC) of EOs (%, v/v) and drugs (μg/mL) against Candida spp. evaluated by the broth microdilution (BM) and vapour contact (VC) methods
Thyme red | BM | 0.03–0.25 | 0.03 | 0.125 | 0.06–0.25 | 0.06 | 0.125 | 0.06–0.25 | 0.06 | 0.25 |
VC | <0.0038 | <0.0038 | <0.0038 | <0.0019–0.03 | 0.0078 | 0.015 | 0.0075–0.015 | 0.0038 | 0.0038 |
Fennel | BM | 0.25–1 | 0.25 | 1 | 0.25–1 | 0.25 | 1 | 0.5–1 | 1 | 1 |
VC | 0.25–1 | 0.25 | 0.5 | 0.25–1 | 0.25 | 0.25 | 0.25–1 | 0.25 | 0.5 |
Clove | BM | 0.25–1 | 0.25 | 1 | 0.25–1 | 0.25 | 1 | 0.125–1 | 0.25 | 1 |
VC | 0.25–1 | 0.5 | 0.5 | 0.25–0.5 | 0.5 | 0.5 | 0.06–1 | 0.06 | 0.25 |
Pine | BM | 0.03–0.06 | 0.06 | 0.06 | 0.0075–0.5 | 0.015 | 0.25 | 0.015–0.5 | 0.03 | 0.5 |
VC | 0.5–1 | 1 | 1 | 0.5–1 | 1 | 1 | 0.5–1 | 1 | 1 |
Sage | BM | 0.5–1 | 1 | 1 | 0.5–1 | 1 | 1 | 0.5–1 | 1 | 1 |
VC | 0.06 | 0.06 | 0.06 | 0.125–0.25 | 0.125 | 0.25 | 0.06 | 0.06 | 0.06 |
Lemon balm | BM | 0.5–1 | 1 | 1 | 0.5–1 | 0.125 | 1 | 0.06–0.5 | 0.25 | 0.25 |
VC | 0.015–0.03 | 0.015 | 0.015 | 0.015–0.06 | 0.03 | 0.06 | 0.0038–0.015 | 0.015 | 0.015 |
Lavender | BM | 0.5–1 | 1 | 1 | 0.5–1 | 0.25 | 1 | 0.125–1 | 0.25 | 1 |
VC | 0.0019–0.06 | 0.06 | 0.06 | 0.0075–0.06 | 0.03 | 0.06 | 0.015–0.06 | 0.03 | 0.06 |
Fluconazole | BM | 0.5- > 64 | 0.50 | 4.00 | 0.125- > 64 | 4.00 | 64.00 | 0.25- > 64 | 2.00 | 16 |
Voriconazole | BM | 0.008–8 | 0.06 | 0.12 | 0.008–8 | 0.25 | 4.00 | 0.032–8 | 0.12 | 2.00 |
Table 2
Minimum inhibitory concentration (MICs) of EOs components (%, v/v) by the broth microdilution (BM) and vapour contact (VC) methods
Carvacrol | BM | 0.06–0.5 | 0.125 | 0.125 | 0.06–0.25 | 0.125 | 0.25 | 0.25–0.5 | 0.25 | 0.25 |
VC | 0.0038 | 0.0038 | 0.0038 | <0.0019 | <0.0019 | <0.0019 | <0.0019–0.0038 | <0.0019 | <0.0019 |
Eugenol | BM | 0.25 | 0.25 | 0.25 | 0.125–0.25 | 0.125 | 0.25 | 0.25–0.5 | 0.25 | 0.25 |
VC | 0.125–0.5 | 0.125 | 0.125 | 0.06–0.25 | 0.125 | 0.125 | 0.125–0.25 | 0.125 | 0.125 |
Linalool | BM | 0.125–1 | 0.25 | 0.25 | 0.125–1 | 0.5 | 0.25 | 0.25–0.5 | 0.25 | 0.25 |
VC | 0.0075 | 0.0075 | 0.0075 | 0.0075–0.03 | 0.015 | 0.03 | 0.0015–0.03 | 0.03 | 0.03 |
Linalyl acetate | BM | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 |
VC | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 |
Thymol | BM | 0.06–0.125 | 0.06 | 0.06 | 0.06–0.25 | 0.125 | 0.125 | 0.125–0.5 | 0.125 | 0.125 |
VC | 0.0038 | 0.0038 | 0.0038 | 0.0019–0.0038 | 0.0019 | 0.0019 | <0.0019 | <0.0019 | <0.0019 |
α-Pinene | BM | 0.06–0.125 | 0.06 | 0.06 | 0.06–0.125 | 0.06 | 0.125 | 0.125–0.5 | 0.125 | 0.125 |
VC | 0.25–0.5 | 0.25 | 0.5 | 0.25–0.5 | 0.25 | 0.5 | 0.25–1 | 0.5 | 0.5 |
As regard BM, thyme red and pine EOs showed the best activity against all strains tested, though
C.albicans proved to be more susceptible than
C.glabrata and
C.tropicalis to pine oil (MIC
50 - MIC
90 = 0.06 %, v/v). On the contrary, fennel, clove, sage and lavender EOs showed the highest MICs (Table
1), whereas lemon balm oil displayed a weak activity against both
C.tropicalis (MIC
50 - MIC
90 = 0.25 %, v/v) and
C. glabrata (MIC
50 - MIC
90 = 0.125–1 %, v/v, respectively).
Interestingly, the MIC values obtained with VC were lower than those in liquid medium for all the EOs tested, except for pine. Specifically, thyme red was the oil with the highest activity (MIC
90 < 0.0038 %, v/v) against
C.albicans, followed by lemon balm, lavender, and sage (Table
1).
The different antifungal activity in liquid and vapour phase could be due to the characteristics of EOs such as high hydrophobicity and volatility. In fact, when added to a medium, the EO distributes more or less into the aqueous phase depending on its relative hydrophobicity. In the liquid phase, the activity depends upon the diffusibility and solubility of the EOs in the medium while in the vapour assay it depends upon the volatility [
2].
MIC values showed that EOs activity is higher than that obtained with the conventional antifungal drugs tested against
C. glabrata e
C. tropicalis, both resistant to fluconazole and voriconazole (fluconazole MIC90 = 64 and 16 μg/mL, respectively and voriconazole MIC90 = 4 and 2 μg/mL, respectively) (Table
1).
The data of the present study can be explained because of chemical composition of EOs, we had already reported in a previous study [
8]. These EOs contained the following major components determined by gas chromatography–mass spectrometry: thymol (26.5 %, v/v), ρ-cymene (16.2 %, v/v), limonene (13.2 %, v/v), α-pinene (13.2 %, v/v), carvacrol (7.8 %, v/v) in thyme red; anethole (72.1 %, v/v), fenchone (14.2 %, v/v) in fennel; eugenol (77.5 %, v/v), eugenyl acetate (7.6 %, v/v) in clove; α-pinene (55.7 %, v/v), β-pinene, (10 %, v/v), limonene (9.7 %, v/v) in pine; cis-thujone (29.4 %, v/v), camphor (22.6 %, v/v), 1,8 cineole (7.7 %, v/v) in sage; citronellal (29.4 %, v/v), limonene (22.6 %, v/v), geranial (8.8 %, v/v) in lemon balm; linalool (41.9 %, v/v), linalyl acetate (32.7 %, v/v) in lavender. The EOs tested in this study showed the presence of significant amounts of monoterpenes, mainly represented by thymol, α-pinene, and linalool compounds, in accordance with previous published data even if with different percentile [
21] (Table
2). Carvacrol and thymol exerted an interesting anti-Candida in vitro activity both by BM and VC, similar to previous findings [
22‐
24], even against fluconazole-resistant
C. glabrata and
C. tropicalis strains. α-pinene showed a better activity by BM than VC; conversely, linalyl acetate showed the lowest activity against all strains tested both by two methods. Generally, MFCs were one or more concentrations higher than MICs (Table
3), suggesting a fungicidal activity of the EOs at low concentrations against yeasts cells, probably due to their related components, such as terpenoids and phenolics known for their broad-spectrum antimicrobial activity [
25,
26]. However, the mechanisms behind the antifungal activity of EOs are not fully understood.
Table 3
Minimum fungicidal concentration (MFC) of EOs (%, v/v) and their components (%, v/v) in comparison with drugs (μg/mL)
Thyme red | 0.06-0.25 | 0.06-0.25 | 0.06-0.5 |
Fennel | 0.5- > 1 | 0.25- > 1 | 1- > 1 |
Clove | 0.25- > 1 | 0.25- > 1 | 0.5- > 1 |
Pine | 0.03-0.125 | 0.03-0.5 | 0.06-1 |
Sage | >1 | >1 | >1 |
Lemon balm | >1 | >1 | >1 |
Lavender | >1 | >1 | >1 |
Carvacrol | 0.125-1 | 0.25-1 | 0.25-1 |
Eugenol | 0.5 | 0.25 | 0.5 |
Linalool | 0.5 | 0.5 | 1 |
Linalyl acetate | 1- > 1 | 1- > 1 | 1- > 1 |
Thymol | 0.125-0.25 | 0.25 | 0.25-0.5 |
α-Pinene | 0.5-1 | 0.5-1 | 1 |
Fluconazole | 2 - >64 | 0.5 - >64 | 0.5- > 64 |
Voriconazole | 0.06-8 | 0.12-8 | 0.12-8 |
High antifungal activity of examined EOs, also against antibiotic-resistant isolates is according to recent evidence of some authors [
20,
27,
28]. However, it is difficult to compare the data with the literature because the antimicrobial activity of EOs and their components are influenced by the several factors including chemical compositions and experimental conditions.
The composition of EOs varies significantly because of plant different species and chemotypes, geographical origin, season and extraction procedure [
29]. In this context, Tampieri et al. [
30] studied a
T. vulgaris EO based on carvacrol (41.33 %), p-cymene (17.53 %) and thymol (5.34 %) and observed a fungistatic activity of the EO tested [
30]. Conversely, we studied a
T. vulgaris EO with different composition; in fact, thymol was 26.5 %, p-cymene 16.2 % and carvacrol 7.8 % and we observed a fungicide activity of the EO tested.
Regarding experimental conditions, it is important to underline that EOs antimicrobial activity data depend on the methodology used, the variability of which includes factors such as inoculum size, medium used, and use of sealants, surfactants and solvents such as Tween, dimethylsulphoxide and ethanol. In part, these may explain the differences in results obtained by different research groups [
31].
Results obtained in this study highlight the activity of the main compounds, thymol in thyme red oil (26.5 %) and α-pinene in pine oil (55 %). However, some data suggest that components presented in small amounts in EOs, such as carvacrol, also could play an important role in antimicrobial activity due to the possible synergistic action with other components [
24,
25,
32,
33].
Thymol, known to be lipophilic, together with carvacrol can enter between the fatty acyl chains making up membrane lipid bilayers, thus altering the fluidity and permeability of cell membranes [
34].
Some authors indicate that this action on fungi, particularly on
C. albicans, affects the regulation and function of important membrane-bound enzymes that catalyze the synthesis of a number of major cell wall polysaccharide components, such as β-glucans, chitin and mannan, thus disturbing cell growth and envelope morphogenesis [
26].
Our data on thymol are in agreement with those of Fontenelle et al. [
7] who demonstrated its potent antimicrobial activity against
C. albicans with MIC = 39 μg/mL. On the contrary, our data do not support those of Zore et al. [
35], who reported the considerable activity of linalyl acetate against 39/48 yeast isolates with MIC = 0.064 % (v/v), but they are in agreement with D’Auria et al. against
C. albicans (Table
2) [
7,
35,
36].
In accordance with our previous findings against clinical filamentous fungi and since active compounds of EOs are highly volatile, EOs possess high antimicrobial activity in vapour phase [
8].
Phenols, such as thymol and carvacrol, are among the most active natural antioxidants and antimicrobials found in EOs [
2,
7]. However, due to their poor water solubility and the requirement for high concentrations to reach a therapeutic effect, the efficiency of these compounds in treatment is limited. It is important to emphasize that thymol is a smaller and more volatile molecule than the ether-containing eugenol from clove. According to Suhr et al. [
29], thyme was also generally more effective than clove in the volatile experiment (Table
2) [
29].