Antifungal activity of essential oils against filamentous fungi determined by broth microdilution and vapour contact methods
Vivian Tullio, Department of Public Health and Microbiology, University of Turin, Via Santena 9, 10126 Turin, Italy.
Aims: The in vitro activity of some essential oils (EO) (thyme red, fennel, clove, pine, sage, lemon balm and lavender) against clinical and environmental fungal strains was determined.
Methods and Results: The minimal inhibitory concentrations were determined by a microdilution method in RPMI 1640 and by a vapour contact assay. The composition of oils was analysed by gas chromatography (GC) and GC/mass spectrometry. The results indicated that the oils antifungal activity depended on the experimental assay used. The inhibiting effects of EO in vapour phase were generally higher than those in liquid state. According to both methods thyme red and clove were found to be the oils with the widest spectrum of activity against all fungi tested.
Conclusions: Despite the differences between the two methods, our results demonstrate that some EO are very active on dermatophytes and dematiaceous fungi. However, more data will be necessary to confirm this good in vitro efficacy.
Significance and Impact of the Study: This study could identify candidates of EO for developing alternative methods to control environmental and clinically undesirable filamentous fungi.
The increasing recognition and importance of fungal infections, the difficulties encountered in their treatment and the increase in resistance to antifungal agents have stimulated the search for therapeutic alternatives (Pina-Vaz et al. 2004). The essential oils (EO) and products of plant secondary metabolism had a wide application in folk medicine, fragrance industries, food flavouring and preservation but only in recent years they have started to be recognized for their potential antimicrobial role (Kalemba and Kunicka 2003). Although numerous studies have documented the EO antibacterial and anticandidal effect (Inouye et al. 2001a; Oliva et al. 2003; Burt 2004; Chami et al. 2004; Friedman et al. 2004; Hammer et al. 2004), there have been few comprehensive in vitro studies of the effects exerted by EO on filamentous fungi, probably due to the difficulties encountered in standardized susceptibility methods for these mycetes (Hammer et al. 2002, 2003; Koc et al. 2005; Silva et al. 2005). Recently, National Committee for Clinical Laboratory Standards (CLSI) (2002) has approved a method for broth dilution antifungal susceptibility testing of filamentous fungi, including aspergilli, Fusarium spp., Rhizopus arrhizus, Pseudallescheria boydii and Sporothrix schenkii, but not penicilli, dermatophytes and dematiaceous fungi. EO antimicrobial activity in vitro could also be tested using a vapour contact (VC) method; recently, some studies have reported the higher potency of EO in vapour state than that in solution contact (Inouye et al. 2000, 2001b).
The aim of this study was to determine the in vitro activities of seven EO against some environmental and clinically important fungi, i.e. dermatophytes, Zygomycetes, penicilli, aspergilli and dematiaceous moulds, by using two different investigative tools, such as the modified (CLSI) NCCLS method and the VC assay.
Materials and methods
Essential oils and gas chromatography-flame ionization detector and gas chromatography-mass spectrometry analysis
The EO used, purchased from Azienda Agricola Aboca (Sansepolcro, Arezzo), were obtained by steam distillation from Thymus vulgaris L. (thyme red), Foeniculum vulgare Mill. var. dulce DC (fennel), Eugenia caryophyllata Thumb. (clove), Pinus sylvestris L. (pine), Salvia officinalis L. (sage), Melissa officinalis L. (lemon balm) and Lavandula vera DC (lavender). All gas chromatography-flame ionization detector (GC-FID) analyses were carried out with either a Shimadzu GC-2010 gas chromatograph with a split/splitless injector or a Shimadzu autosampler AOC-20is (Shimadzu, Milan, Italy). In details: column MDN-5S, 30 m × 0·25 mm ID × 0·25 mm film thickness (5% diphenyl + 95% polydimethylsiloxane) (Supelco, Milan, Italy); temperature program, 50°C (2 min) to 250°C (10 min) at 3°C min−1; injection temperature, 250°C; injection volume, 1 ml; inlet pressure, 100 kPa; carrier gas, H2; linear velocity (), 30 cm s−1; split ratio, 100 : 1; detector, flame ionization detector at 275°C; H2 flow, 50 ml min−1; air flow, 400 ml min−1; make up (N2/air), 50 ml min−1; sampling rate, 200 ms. All GC-mass spectrometry (MS) analyses were carried out with a Shimadzu GC-MS QP-2010 model GC-MS equipped with an AOC-20is series autosampler, under the same conditions used for GC-FID analyses. In detail: inlet pressure, 37·1 kPa; carrier gas, H2; linear velocity (), 32·4 cm s−1; split ratio, 50 : 1; detector, MS (250°C); interface T, 250°C; MS mode, EI; detector voltage, 0·9 kV; mass range, 40–400 u; scan speed, 769 u s−1; interval, 0·5 s (2 Hz). All data were acquired by a GC/MS solution software (Shimadzu). The components were identified by comparison of their mass spectra with commercial mass spectral libraries.
A total of 44 strains were used. These included: dermatophytes (Microsporum canis, Microsporum gypseum, Trichophyton mentagrophytes, Epidermophyton floccosum); clinical isolates from onychomycosis (Scopulariopsis brevicaulis, Fusarium oxysporum) and from bronchial alveolar lavage (Aspergillus niger, Aspergillus flavus, Asp. flavus var. columnaris, Aspergillus fumigatus); environmental strains: Zygomycetes (Mucor spp. and Rhizopus spp.), penicilli (Penicillium lanosum and Penicillium frequentans) and dematiaceous fungi (Alternaria alternata and Cladosporium cladosporioides). Trichophyton mentagrophytes strains were isolated from both human and animal clinical specimens. Human dermatophytes, Scop. brevicaulis and Fus. oxysporum were provided by Clinica Dermatologica II – Discipline Medico-Chirurgiche Department, University of Turin, San Lazzaro Hospital (Turin, Italy), whereas aspergilli by A.S.L. San Giovanni Battista-Molinette (Turin); animal strains were provided by Giancarlo Baralis veterinary (Turin). Fungi were identified according to macroscopic and microscopic morphological procedures and maintained on potato dextrose agar (PDA) slopes stored at room temperature. Before the experiments the fungi were transferred to fresh media and incubated for 7 days, in twice.
Preparation of non-germinated conidial suspensions
Inocula were prepared by growing aspergilli, penicilli and Fus. oxysporum isolates on PDA (Merck, Darmstadt, Germany) slopes as described by the reference method M38-A recommended by the National Committee for Clinical Laboratory Standards (CLSI) (2002). Conversely, dermatophytes, dematiaceous fungi, Scop. brevicaulis and Zygomycetes were incubated on Sabouraud dextrose (SAB; Merck) agar at room temperature for 7 days, except for Zygomycetes (3 days). Slopes were flooded with 0·85% saline and conidia gently probed; the resulting suspensions were removed and vortexed thoroughly. After the settling of the larger particles, suspensions were adjusted by nephelometry and diluted in saline to obtain inocula of 2 × 104 CFU ml−1, as confirmed by colony counts in triplicate on SAB agar.
Broth microdilution method
Broth microdilution (BM) testing was based on National Committee for Clinical Laboratory Standards (CLSI) (2002) method, with some modifications. A volume of 40% (v/v) stock solution of each EO in ethanol (1 : 2·5) was diluted 1 : 20 in RPMI-1640 (with l-glutamine and phenol red, without bicarbonate) (Invitrogen, San Giuliano Milanese, Milan, Italy), containing 3-(N-morpholino) propane sulfonic acid (MOPS) (Sigma-Aldrich, Steinheim, Germany) at a concentration of 0·165 mol l−1 for pH 7·0, to obtain 2% v/v solution.
Doubling dilutions of the EO ranging from 2% to 0·0038% (v/v) were prepared in 96-well microtitre trays in RPMI-1640 with MOPS. After the addition of 0·1 ml of inoculum, the trays were incubated at 30°C for 7 days. The final EO concentrations ranging from 1% to 0·0019% (v/v) and ethanol maximum concentration was 1·5% (v/v). Growth controls consisting of RPMI-1640 medium and RPMI-1640 with 1·5% (v/v) ethanol were included for each isolate tested. The minimal inhibitory concentration (MIC) of EO was defined as the lowest concentration of compound that completely inhibited visible growth after 3 and 7 days of incubation. The minimal fungicidal concentration (MFC) of EO was determined by spot inoculating 10 μl from wells not visibly turbid onto SAB agar plates that were incubated for 3 days at 30°C (Hammer et al. 2002). MFC was defined as the lowest concentration resulting in no growth on subculture.
Vapour contact assay
The effect of volatile oil fraction was studied with an invert Petri dishes method. Double-strength concentrated RPMI 1640 broth supplemented with l-glutamine, phenol red and MOPS, adjusted to pH 7·0 was mixed with molten 3·0% (w/v) agar in an equal ratio immediately before the assay. The RPMI agar was poured into a 90 mm Petri dish and spot inoculated with 1 μl of standardized suspension of each fungal strain tested (final concentration of 104 conidia per ml). A glass slide (1-cm size) was placed in the cover of each Petri dish, so that it did not directly touch the surface of the agar medium, and various amounts of pure EO were added to obtain final concentration ranging from 1% to 0·0019% v/v air space. The space inside of the sealed Petri dish was calculated to be 70 cm3 air. The plates were sealed with vinyl tape immediately after inoculation, and incubated at 30°C for 7 days. The control, consisting of RPMI medium, was included. The MIC (%, v/v air space) was determined by comparison with the control and was defined as the lowest concentration of EO inhibiting the visible growth.
The major components of the EO are reported in Table 1.
Table 1. Essential oils used and their major constituents
|Major constituents (content)|
|p-Cymene||16·2||Fenchone||14·2||Eugenyl acetate||7·6||β-Pinene||10||Camphor||22·6||Limonene||12·4||Linalyl acetate||32·7|
|α-Pinene||11·5||Methyl chavicol||3·7|| || ||δ-3-Carene||6·9||α-Humulene||5·8||α-Terpineol||6·3||β-Caryophyllene||2·9|
|Carvacrol||7·8||Limonene|| 3|| || ||Myrcene||3·4||Camphene||5·4||Geraniol||6·3||Terpinen-4-ol|| 2|
|γ-Terpinene|| 4|| || || || || || ||trans-Thujone||4·2||Neral||5·4|| || |
| || || || || || || || ||β-Caryophyllene||3·5||α-Terpinenyl acetate||4·5|| || |
| || || || || || || || ||α-Pinene||2·7||Isopulegol||4·3|| || |
| || || || || || || || ||Borneol||2·2||Citronellol||3·5|| || |
| || || || || || || || ||Limonene||2·2||Linalool||2·3|| || |
Tables 2 and 3 compare the MICs of the seven EO tested by the two methods used. Using BM method, the MICs values ranged from 0·0078% to 0·5% after 3 days, and from 0·25% to >1% after 7 days for dermatophytes and Scop. brevicaulis; MICs for Fus. oxysporum ranged from 0·0156% to 0·125% after 3 and 7 days (Table 2). MICs for aspergilli and penicilli ranged from 0·125% to 1% after 3 and 7 days, whereas for dematiaceous they ranged from 0·0312% to 0·5% after 3 days, and from 0·125% to 1% after 7 days (Table 3). Zygomycetes showed the lowest susceptibility (MIC ≥ 1%) and were not inhibited by any oil. Thyme red was found to be the oil with the better spectrum of activity against all fungi tested, followed by clove and fennel. Among the 44 fungi tested, the most susceptible were dermatophytes, Fus. oxysporum and Pen. frequentans (Table 2), followed by dematiaceous fungi (Table 3). Almost all EO exhibited good antifungal activity, which generally decreased in the following order: thyme, clove, fennel and pine. The pine EO displayed a good activity against dermatophytes and onychomycosis fungi (Table 2). Sage, lemon balm and lavender EO were the weakest in activity against aspergilli (Table 3).
Table 2. Essential oils – minimal inhibitory concentration (MIC) values for dermatophytes, Scopulariopsis brevicaulis and Fusarium oxysporum by the broth microdilution (BM) and vapour contact (VC) methods
|Microsporum canis (4)||BM (3)||0·06–0·125||0·06–0·125||0·06–0·125||0·125||0·0312–0·125||0·0625–0·125||0·0625–0·25|
|Microsporum gypseum (6)||BM (3)||0·0078–0·125||0·015–0·25||0·0625–0·25||0·125–0·25||0·125–0·5||0·125–0·5||0·125–0·5|
|Trichophyton mentagrophytes (animal) (2)||BM (3)||0·25||0·25||0·25||0·25||0·5||0·25||0·0625|
|Trichophyton mentagrophytes (human) (3)||BM (3)||0·0625–0·25||0·0625–0·5||0·0625–0·25||0·125–0·25||0·125–0·25||0·25–0·5||0·0625–0·5|
|Epidermophyton floccosum (1)||BM (3)||0·0312||0·125||0·125||0·125||0·25||0·25||0·5|
|Scopulariopsis brevicaulis (3)||BM (3)||0·25||0·25||0·25||0·25||0·25||0·25||0·25–0·5|
|Fusarium oxysporum (2)||BM (3)||0·125||0·0625||0·0312||0·0312||0·0156||0·0312||0·0156|
Table 3. Essential oils – minimal inhibitory concentration (MIC) values for filamentous fungi by the broth microdilution (BM) and vapour contact (VC) methods
|Mucor spp. (2)||BM (3)||>1||>1||>1||>1||>1||>1||>1|
|Rhizopus spp. (2)||BM (3)||>1||>1||>1||>1||>1||>1||>1|
|Aspergillus niger (3)||BM (3)||0·125–0·25||0·25||0·25||0·25–0·5||1||1||1|
|Aspergillus flavus (3)||BM (3)||0·25||0·25–0·5||0·25||0·5||1||0·5||0·5–1|
|Aspergillus flavus var. columnaris (1)||BM (3)||0·125||0·125||0·125||0·125||0·125||0·125||0·125|
|Aspergillus fumigatus (4)||BM (3)||0·25||0·25||0·25||0·25–1||0·5–1||0·5–1||0·5–1|
|Penicillium lanosum (2)||BM (3)||0·25||0·25||0·25||0·5||1||0·25||0·5|
|Penicillium frequentans (2)||BM (3)||0·0625||0·125||0·0625||0·125||0·25||0·125||0·125|
|Alternaria alternata (3)||BM (3)||0·125–0·5||0·25–0·5||0·125–0·5||0·125–0·25||0·125–0·25||0·25–1||0·0625–0·5|
|Cladosporium cladosporioides (3)||BM (3)||0·0625||0·0625–0·125||0·125–0·25||0·03–0·25||0·0625–0·25||0·03–0·25||0·015–0·25|
A comparison of values obtained at 3 and 7 days showed that EO MICs did not change for Fus. oxysporum, whereas those for aspergilli, penicilli, dermatophytes and Scop. brevicaulis were one or more concentrations higher at 7 days compared with those observed at 3 days. Similarly, MICs obtained with dematiaceous fungi were one or more concentrations higher at 7 days, with the exception of clove and pine EO.
By the VC method the MICs were lower than those in liquid medium for five of the seven EO tested. Among these, thyme red was found to be the oil with the highest activity, followed by lemon balm, clove, lavender and sage. The activity of fennel and pine oils was weaker. Interestingly, Mucor spp. and Rhizopus spp. exhibited susceptibility to all EO vapours whereas they were not susceptible in liquid state (Tables 2 and 3).
Table 4 depicts MFC values obtained from BM wells method after 7 days. In general, MFCs were one or more concentrations higher than MICs at 7 days, with some exceptions for dematiaceous fungi and Fus. oxysporum.
Table 4. Essential oils – minimal fungicidal concentration (MFC) values for dermatophytes and filamentous fungi
|Microsporum canis (4)||0·25–0·5||0·25–0·5||0·25–1||0·25–1||0·5 to >1||0·25 to >1||0·5 to >1|
|Microsporum gypseum (6)||0·5–1||0·5–1||0·25–1||0·5–1||>1||1 to >1||1 to >1|
|Trichophyton mentagrophytes (animal) (2)||0·5||1||0·5||1||>1||>1||>1|
|Trichophyton mentagrophytes (human) (3)||0·25–0·5||0·25–1||0·5||0·25–1||0·5–1||1||1|
|Epidermophyton floccosum (1)||0·0625||0·25||0·25||0·25||0·25||0·25||0·5|
|Scopulariopsis brevicaulis (3)||>1||>1||>1||1||>1||>1||>1|
|Fusarium oxysporum (2)||0·125||0·125||0·0312||0·0625||0·0625||0·0312||0·125|
|Aspergillus niger (3)||0·5||0·5||0·5–1||1||>1||>1||>1|
|Aspergillus flavus (3)||1||>1||0·5||1||1||1||1|
|Aspergillus flavus var. columnaris (1)||0·5||1||0·5||0·5||ND||ND||ND|
|Aspergillus fumigatus (4)||1||>1||1||>1||>1||>1||>1|
|Penicillium lanosum (2)||1||>1||1||>1||>1||>1||>1|
|Penicillium frequentans (2)||1||>1||0·5||0·5||1||0·5||>1|
|Alternaria alternata (3)||>1||>1||0·5||>1||0·5||1 to >1||1|
|Cladosporium cladosporioides (3)||0·5–1||0·25–0·5||0·125–0·5||0·25–0·5||1||0·25–1||0·25 to >1|
Recently, the scientific interest into biological properties of EO has been increasing. In particular, a large number of papers have been published on the EO antimicrobial activity, but many of them focused on the activity of EO in solution against bacteria and yeasts while little investigation on the effect of EO in vapour state has been performed (Inouye et al. 2000, 2001a, 2001b). In the present study, the antifungal activity of seven EO were compared by microdilution and VC methods against dermatophytes and other moulds. Quantification of the EO antimicrobial activity appears to be influenced by the test method used, as evidenced by the differences between the MICs obtained by BM and VC methods. The results revealed that five of the seven EO tested were stronger inhibitory in vapour state than in liquid phase. The efficacy of EO in vapour state was probably attributable to the direct deposition of EO on lipophilic fungal mycelia together with an indirect effect via adsorption through the agar medium (Inouye et al. 2000). The high antifungal activity of vapours evidenced in this study was in accordance with previous findings (Suhr and Nielsen 2003). Although a comparison of studies with different methodologies is difficult, our data correlate with those of Suhr and Nielsen (2003), who showed that thyme and clove oils were more effective in vapour state against Asp. flavus.
The EO are complex mixtures of many different aromatic components with various degrees of lipophilicity and relative hydrophilicity given by the presence of constituents with polar functional groups (Griffin et al. 1999). When added to a medium, the EO distributes more or less into the aqueous phase depending on its relative hydrophilicity. Hence, an EO with constituents with low water solubility should dissolve little in aqueous medium, and consequently should show a weak activity. This is the case of lemon balm and lavender oils whose constituents (linalool, linalyl-acetate and citronellal, limonene respectively) have been reported to possess low water solubility (Griffin et al. 1999). These EO, however, showed very good activity when assayed by VC and this might related to their high volatility (Inouye et al. 2000). These observations suggest that the physical and chemical properties (solubility and volatility) can have considerable effect on the in vitro antimicrobial activity, but it must be pointed out that the intrinsic activity of a compound is of paramount importance for the evaluation of its efficacy. In this context, the EO-containing aldehyde or phenol was reported to exhibit a high inhibitory effect, followed by those containing alcohol, ketone and ether (Dorman and Deans 2000; Inouye et al. 2001a). Hence it is not surprising that thyme and clove oil proved to be the overall best inhibitors because of phenolic thymol, carvacrol and eugenol as the major contributors to their bioactivity. Although thymol and carvacrol possess moderate solubility and low volatility, their vapours have been reported to accumulate in great amounts into agar layers (Inouye et al. 2001a). Chami et al. (2004) reported the high antifungal activity of carvacrol and eugenol on experimental oral candidiasis in immunosuppressed rats and interestingly demonstrated that the fungi disappeared completely from the tongue mucosa in all treated animals whereas many hyphae were found when nystatin was used as control. The EO clinical application is often discouraged because of their potential harsh or caustic effects when used at high concentrations due to their lipophilic character (Inouye et al. 2000). Indeed, it is true that these problems could be partly solved making use of their specific volatility at room temperature. Overall, our results show the higher potency of EO in vapour state than that in solution contact, suggesting that they could have distinct advantages including the reduction of effective dose and potential chemical irritation.
Despite the differences between the two methods, our results demonstrate that some EO are very active on dermatophytes and dematiaceous fungi. Similarly, the higher sensibility of dermatophytes was observed by other Authors with other EO (Salgueiro et al. 2004; Cavaleiro et al. 2006). However, more data will be necessary either to confirm this good in vitro efficacy or to explain the EO mechanisms of action. Further studies are required to study the effect and toxicity of these compounds in experimental animals (in vivo) and to establish if they could be safely used as antifungal agents against these fungi. Our findings showing that some EO are effective against Scop. brevicaulis and Fus. oxysporum which are often resistant to available antifungal agents, open important perspectives in alternative antifungal therapies.
Moreover, this study could identify candidate EO for developing alternative methods to control environmental undesirable filamentous fungi.
The authors are grateful to Professor O. Cervetti and Dr M. Panzone (Clinica Dermatologica II – Department of Discipline Medico-Chirurgiche, University of Turin, Italy), Dr R. Serra (A.S.L. San Giovanni Battista-Molinette, Turin) and Dr Giampiero Baralis for providing fungal strains.