Antifungal activity of thyme (Thymus vulgaris L.) essential oil and thymol against moulds from damp dwellings

Abstract

Aims:  To characterize antifungal activities of essential oil of thyme (Thymus vulgaris L.) and pure thymol, as comparative substance, on different mould species isolated from damp dwellings.

Methods and Results:  Fifty samples of wall scrapes were collected from damp dwellings in Zagreb, the capital of Croatia. The members of the following mould genera were recovered from the samples: Aspergillus (44%), Penicillium (18%) Alternaria, Ulocladium, Absidia and Mucor (8%) Cladosporium, Trichoderma and Rhizopus (6%), and Chaetomium (2%). Two strains of Stachybotrys chartarum were isolated from damp dwellings in Slovakia. Antifungal activities of the thyme essential oil, which contains p-cymene (36.5%), thymol (33.0%) and 1,8-cineole (11.3%) as main components, and pure thymol were determined by the dilution method and exposure to vaporous phase of the oil. Minimum inhibitory concentrations (MIC) of both thymol and essential oil were bellow 20 μg ml⁻¹, except for Mucor spp. (50.20 μg ml⁻¹). Thymol exhibited approximately three-times stronger inhibition than essential oil of thyme. The vaporous phase of the thyme essential oil (82 μg l⁻¹) in glass chambers strongly suppressed the sporulation of moulds during 60 days of exposure.

Conclusion:  The thyme essential oil possesses a wide range spectrum of fungicidal activity. The vaporous phase of the oil exhibited long-lasting suppressive activity on moulds from damp dwellings.

Significance and Impact of the Study:  Essential oil of thyme and thymol could be used for disinfection of mouldy walls in the dwellings in low concentration.

INTRODUCTION

Microscopic filamentous fungi (moulds) are ubiquitous micro-organisms with a great capacity to colonize many kinds of substrates and to develop under extreme environmental conditions. Moulds and their airborne particles in the indoor and outdoor environment have been recognized as possible causative agents of various diseases in humans including the airway infections, irritation of respiratory mucous membranes, acute and chronic damage of respiratory organs and mycotoxicoses (Nielsen 2003). One of the mould sources in the indoor environment is damp building material of the dwellings. Studies in the USA and European countries (the UK, the Netherlands, Finland and Denmark) showed that the proportion of dwellings with mould growth varies between 20% and 40% (Nielsen 2003). Mould growth on damp-building materials mostly depend on water activity value (a_w). Materials with an a_w < 0·8 are usually colonized by Penicillium chrysogenum and Aspergillus versicolor followed by A. fumigatus, A. niger, Eurotium spp., Wallemia sebi and Paecilomyces variotii, materials with an a_w between 0·8 and 0·9 by species of Alternaria, Cladosporium, Phoma and Ulocladium, and materials with an a_w > 0·9 by Chaetomium globosum, Stachybotrys chartarum and Trichoderma spp. (Grant et al. 1989; Nielsen 2003). As various agents from moulds including proteins causing allergy (Gravesen et al. 1999), β-1,3-glucans (Rylander 1997), microbial volatile compounds (Pasanen et al. 1998), mycotoxins and other secondary metabolites (Piecková and Jesenská 1999) can be implicated in diverse health effects in humans, it is of great importance to develop the applicable method for suppression of their growth in the dwellings.

Essential oils as well as compounds derived from them possess wide range of activities with the antimicrobial as the most studied (Hammer et al. 2003; Pepeljnjak et al. 2003). One of the potent antimicrobial compounds isolated from essential oils are lipophilic monoterpens such as thymol, carvacrol, linalool, citral, geraniol and 1,8-cineole. Their applications as preservatives in food or antiseptics and disinfectants are widely studied (Pauli 2001; Burt 2004). The main objective of this work was to characterize the in vitro antifungal activities of essential oil of thyme (which is reach in thymol) and pure thymol, as comparative substance, on different mould species isolated from damp dwellings.

MATERIALS AND METHODS

Oil analysis

The essential oil of thyme was obtained from local essential oils store. Gas chromatography (GC) and GC–mass spectrometry (MS) analysis of essential oil of thyme were performed.

Gas chromatography (GC–FID)

Gas chromatography analysis was performed on a Hewlett-Packard Model 5890 Series II gas chromatograph equipped with flame ionization detector and capillary column HP-101 (Methyl silicone fluid), 25 m × 0·2 mm i.d., coating thickness 0·2 μm. Chromatographic conditions were as follows: helium as carrier gas at 1·0 ml min⁻¹; injector and detector temperatures, 250°C and 300°C. Oven temperature was isothermal at 70°C for 2 min, then increased to 200°C, at a rate of 3°C min⁻¹ and held isothermal for 15 min. Volume injected 1 μl. Split ratio 1 : 50.

Gas chromatography–mass spectrometry

Essential oil was also analysed by Hewlett Packard GC–MS (model 5890 series II) with mass selective detector (model 5971A). Two columns of different polarity were used: an HP-101 column (Methyl silicone fluid, Hewlett Packard; 25 m × 0·2 mm i.d., film thickness 0·2 μm) and an HP-20M column (Carbowax 20M, Hewlett Packard; 50 m × 0·2 mm i.d., film thickness 0·2 μm). Oven temperature was programmed as follows: isothermal at 70°C for 4 min, then increased to 180°C, at a rate of 4°C min⁻¹ and subsequently held isothermal for 15 min (for HP-20M column); isothermal at 70°C for 2 min, then increased to 200°C, at a rate of 3°C min⁻¹ and held isothermal for 15 min (for HP-101 column). Carrier gas was helium, flow rate: 1 ml min⁻¹; injector temperature: 250°C; volume injected: 1 μl; split ratio: 1 : 50. MS conditions: ionization voltage: 70 eV; ion source temperature: 280°C; mass range: 30–300 mass units.

Qualitative and quantitative determination

The individual peaks were identified by comparison of their retention indices to those of authentic samples, as well as by comparing their mass spectra with the Wiley 6·0 library (Wiley, New York, NY, USA) and NIST98 (National Institute of Standards and Technology, Gaithersburg, MD, USA) mass spectral database and literature (Adams 1995).

The percentage composition of the samples was computed from the GC peak areas by using the normalization method (without correction factors). Quantitative results are mean of data derived from duplicate GC-FID analyses.

Isolation and detection of moulds

From February to March (2005), fifty samples of wall scrapes were collected from damp dwellings in Zagreb, the capital of Croatia. Samples were plated on Sabouraud 2% (w/v) glucose agar (SGA), supplemented with 50 mg l⁻¹ of streptomycin and 20·000 IU of penicillin, and incubated at 25 ± 2°C for the 5–7 days. Moulds were identified on the basis of their macro- and microscopic characteristics after subculturing on Czapek and Malt agar (Raper and Thom 1949; Samson et al. 1981; de Hoog et al. 2000). Two strains of S. chartarum were isolated in Slovakia by inoculation of 1-ml sample of wall scrapes diluted in saline into a Petri dish with sterile paper (Pieckováet al. 2004). Before analysis, mould species were cultured on SGA slants until sporulation at 25 ± 2°C.

For the antifungal activity testing, thymol and thyme essential oil were dissolved in 96% (v/v) ethanol and then diluted with 30% (v/v) ethanol in distilled water with 0·1% (w/v) Tween 80. Final concentrations of thymol and thyme essential oil were 2% (w/v) and 2% (w/v), respectively.

Antifungal activity testing by dilution method

Minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) were determined by using the serial broth dilution method described by Pepeljnjak et al. (2003). MIC was determinated by the broth twofold macro-dilution method in Sabouraud 2% (w/v) glucose broth starting with 2% (w/v) of the thyme essential oil or thymol. MIC is defined as the lowest concentration of extract or essential oil that allows no more than 20% growth of the fungus, visualized as a reduced number of colonies after removing the loop with approx. 10 μl of each dilution in Sabouraud 2% (w/v) glucose broth, and then inoculated on SGA and incubated at 25 ± 2°C for 7 days. MFC is defined as the lowest concentration of thymol or essential oil of thyme that completely inhibited the growth of fungi. Each experiment was performed in duplicate.

Antifungal activity testing by vaporous contact in glass chamber

The activity of the vapour phase of essential oil of thyme was tested by a modified micro-atmosphere method (Inouye et al. 1998). Isolated mould strains were separated in two experimental groups.

First group: in a 40-mm Petri plate, 100 μl of spore suspension (10⁶ spores ml⁻¹ of each strain) was spread on the surface of SGA. One millitre of essential oil of thyme (1% w/v) was added with a micro-syringe on the filter-paper in a 40-mm Petri plate. Open seeded Petri plates and plate with oil were placed on the bottom of 12-l glass chambers, which were hermetically sealed and incubated at 25°C for 60 days at room temperature.

Second group: each mould strain was seeded on the SGA plates, as it was previously described and incubated at 25°C until sporulation. Open plates with sporulating mould cultures and plate with essential oil were placed in glass chambers and incubated as it was previously described. After 60 days of exposure to the vapour of essential oil, mould samples were cultured on fresh SGA 7 days at 25 ± 2°C. Each experiment in the test chamber was performed in duplicate.

Statistics

The data obtained as MIC and MFC of thymol and thyme essential oil, expressed in μg ml⁻¹, were statistically analysed by using the Wilcoxon matched pairs test. The level of P < 0·05 was considered statistically significant.

RESULTS

Composition of thyme essential oil

The content and chemical composition of the essential oil is given in Table 1. Seventeen compounds were identified representing 98·3% of the total oil. The main components were p-cymene (36·5%), thymol (33·0%) and 1,8-cineole (11·3%). The essential oil also contained smaller quantities of α-terpineol (4·8%), carvacrol (3·9%), borneol (2·1%), linalool (2·0%) and other compounds.

Table 1.   Identified constituents and percentage composition of thyme (Thymus vulgaris L.) essential oil

Compound	RI*	RI**	%
RI*, retention index on HP-101 column; RI**, retention index on HP-20M column; /, not identified on this column.
α-Pinene	945	1038	1·2
Camphene	953	1059	1·7
β-Pinene	957	1092	0·8
p-Cymene	962	1247	36·5
1,8-Cineole	971	1191	11·3
Linalool	1102	1507	2·0
Borneol	1166	1654	2·1
β-Citronellol	1185	1709	0·3
α-Terpineol	1188	1652	4·8
Nerol	1240	1752	0·7
Geraniol	1265	1796	0·5
trans-Anethole	/	1790	0·2
Caryophyllene oxyde	1328	2007	1·4
Thymol	1362	2115	33·0
Carvacrol	1372	2140	3·9
trans-Caryophyllene	1405	1578	1·4
α-Humulene	1437	1638	0·5
Total identified	98·3%

Mould composition of wall scrapes

Mould species isolated from 50 samples of wall scrapes collected from the dwellings are listed in Table 2. Species of Aspergillus (44%) followed by Penicillium spp. (18%), as primary colonizers, dominated in the samples. Among secondary colonizers Alternaria spp. and Ulocladium spp., as well as Absidia spp. and Mucor spp. have been found in 8% of the samples, while Cladosporium spp., Trichoderma spp. and Rhizopus spp. equally contribute the mycoflora composition with 6% of the frequency. Chaetomium globosum, as tertiary colonizer, was detected in the only one sample of wall scrapes. Two strains of S. chartarum were isolated from damp dwellings in Slovakia (Pieckováet al. 2004). This particular species was not detected in the samples from indoor spaces in Zagreb.

Table 2.   Antifungal activities of thyme essential oil (Thymus vulgaris L.) and thymol against moulds recovered from 50 samples of wall scrapes collected in Zagreb

Mould species/% of recovery from 50 samples of wall crapes	Thyme essential oil (μg ml^−1) ( ± SD)		Thymol (μg ml^−1) ( ± SD)
	MIC	MFC	MIC	MFC
± SD, mean ± standard deviation; *P < 0·05; **P < 0·01; †, S. chartarum collected in damp dwellings in Slovakia.
Aspergillus (total)/44	9·85 ± 6·19	22·30 ± 14·70	5·52 ± 2·83**	10·89 ± 5·9**
A. versicolor/18	9·60 ± 9·25	23·60 ± 20·7	4·86 ± 2·13	9·64 ± 4·20
A. niger/12	9·35 ± 3·45	22·50 ± 5·60	6·72 ± 3·10	13·74 ± 6·86
A. sulphureus/8	10·88 ± 3·10	21·88 ± 6·25	6·22 ± 4·37	12·45 ± 8·86
A. flavus/4	9·35 ± 4·40	25·00 ± 0·00	3·20 ± 0·00	6·25 ± 0·00
Aspergillus spp./2	3·20 ± 0·00	6·25 ± 0·00	3·00 ± 0·00	6·25 ± 0·00
Penicillium (total)/18	19·17 ± 9·00	31·76 ± 11·05	5·91 ± 2·89**	11·84 ± 5·82**
P. chrysogenum/2	19·60 ± 0·00	39·00 ± 0·00	3·20 ± 0·00	6·20 ± 0·00
P. brevicompactum/2	19·60 ± 0·00	39·00 ± 0·00	6·20 ± 0·00	12·60 ± 0·00
P. griseofulvum/2	19·60 ± 0·00	39·00 ± 0·00	6·20 ± 0·00	12·60 ± 0·00
Penicillium spp./12	18·95 ± 11·40	28·13 ± 12·18	6·27 ± 3·43*	12·53 ± 6·86*
Alternaria (total)/8	7·05 ± 3·96	14·10 ± 7·86	2·95 ± 0·94	4·75 ± 2·80
A. alternata/4	4·70 ± 2·10	9·40 ± 4·52	2·40 ± 1·13	4·70 ± 2·12
Alternaria spp./4	9·40 ± 4·50	18·80 ± 8·75	3·50 ± 0·42	5·00 ± 4·20
Ulocladium spp./8	5·45 ± 1·50	11·00 ± 3·20	2·40 ± 0·93	4·70 ± 1·80
Absidia spp./8	7·00 ± 4·00	14·00 ± 8·00	5·00 ± 2·40	10·00 ± 0·40
Mucor spp./8	50·20 ± 8·40	116·60 ± 17·20	6·20 ± 0·00**	12·20 ± 4·0**
Cladosporium (total)/6	15·20 ± 8·00	30·2 ± 11·00	4·20 ± 1·80*	5·20 ± 1·60**
C. sphaerospermum/2	19·60 ± 0·00	39·00 ± 0·00	3·20 ± 0·00**	6·20 ± 0·00**
Cladosporium spp./1	12·80 ± 0·00	25·80 ± 0·00	4·60 ± 0·00*	5·00 ± 0·00**
Trichoderma spp./6	16·80 ± 0·00	20·80 ± 0·00	4·20 ± 0·00*	5·00 ± 1·80**
Rhizopus spp./6	12·60 ± 0·00	25·00 ± 0·00	4·80 ± 1·80	9·40 ± 3·60**
Chaetomium globosum/2	1·60 ± 0·00	3·20 ± 0·00	1·60 ± 0·00	3·20 ± 0·00
Stachybotrys chartarum†	6·20 ± 0·00	12·60 ± 0·00	3·20 ± 0·00	6·20 ± 0·00

Screening of thymol and thyme essential oil for antifungal activity by dilution method

According to the results of dilution method (Table 2), both thymol and thyme essential oil showed strong fungicidal activity with the MIC below 20·00 μg ml⁻¹ and MFC below 40·00 μg ml⁻¹ on the most of primary, secondary and tertiary fungal colonizers of damp walls. Thymol exhibited significantly stronger inhibition on the species of Aspergillus, Penicillium, Cladosporium, Trichoderma, Rhizopus and Mucor comparing with essential oil of thyme (P < 0·05, P < 0·01, respectively). The most sensitive on both thymol and thyme essential oil activity was C. globosum for which MIC values of tested substances were equal (1·60 μg ml⁻¹). MIC values of thymol, in the range from 2·40 μg ml⁻¹ to 6·72 μg ml⁻¹, suppressed the growth of other isolated fungi. Comparing with thymol, thyme essential oil was less effective. The growth of Ulocladium spp. S. chartarum, Absidia spp., Alternaria spp. and species of Aspergillus was inhibited by essential oil of thyme in the range of MICs from 5·45 μg ml⁻¹ to 9·85 μg ml⁻¹ while suppression of Cladosporium spp., Trichoderma spp. and Penicillium spp. was observed after treatment with 15·20, 16·80 and 19·17 μg ml⁻¹, respectively. The highest MIC value of essential oil of thyme was recorded for Mucor spp. (50·20 μg ml⁻¹). Comparing the mean of MIC and MFC values of all tested mould strains, thymol exhibited approximately three-times stronger inhibition than essential oil of thyme.

Antifungal activity of essential oil of thyme by vaporous contact in glass chambers

Vaporous phase of the thyme essential oil in concentration of 82 μg l⁻¹ exhibited fungistatic and/or fungicidal activity on primary, secondary and tertiary colonizers during 60 days of exposure in glass chambers. Suppression of development of fructification organs and sporulation was determined for following species from the first experimental group: A. niger, A. flavus, A. vericolor, A. sulphureus, P. chrysogenum, P. brevicompactum, Penicillium spp., Trichoderma spp., Mucor spp., Rhizopus spp. and Absidia spp. (Table 3). Sporocidal effect against fungal cultures in sporulation phase from the second experimental group was observed in all tested mould species.

Table 3.   Fungistatic activity of vaporous phase (82 μg l⁻¹) of the thyme essential oil

Mould species	Mycelial growth	Sporulation
+, observed; −, not observed.
A. niger	+	−
A. versicolor	+	−
A. sulphureus	+	−
A. flavus	+	−
Aspergillus spp.	−	−
P. chrysogenum	+	−
P. brevicompactum	+	−
P. griseofulvum	+	−
Penicillium spp.	+	−
A. alternata	−	−
Alternaria spp.	−	−
Ulocladium spp.	−	−
Absidia spp.	+	−
Mucor spp.	+	−
C. sphaerospermum	−	−
Cladosporium spp.	−	−
Trichoderma spp.	+	−
Rhizopus spp.	+	−
Chaetomium globosum	−	−
Stachybotrys chartarum	−	−

DISCUSSION

Many studies showed that occupants of mouldy dwellings might have physical impairments because of inhalation of mould components (live or dead conidia and mycelial fragments), usually manifested as irritations of respiratory tract, conjunctivitis or systemic manifestations. These symptoms could be attributed to allergic and/or toxic effects of mould cell wall components and especially mycotoxins (Flappan et al. 1999; Schulz et al. 2004). Secondary toxic metabolites (e.g. satratoxins, aflatoxins, ochratoxin A and sterigmatocystin), which are produced by Stachybotrys, Aspergillus and Penicillium species, exerted their various toxic effects in cell cultures as well as in vivo toxicity studies (Piecková and Jesenská 1999; Bünger et al. 2004; Hossain et al. 2004; Pieckováet al. 2004; Schulz et al. 2004). In this study, the most abundant fungi originated from wall scrapes were species of Aspergillus and Penicillium. These species can grow at a_w values below 0·8, and thus were found more frequently on building materials. Secondary colonizers including Alternaria spp. and Ulocladium spp. together with Absidia spp. and Mucor spp. constituted the 8% of recovered mould isolates. Cladosporium spp., Trichoderma spp. and Rhizopus spp. were found in three samples while C. globosum was recovered from the only one sample. Studies in Slovakia also showed that the most frequently isolated species from wall scrapes were Penicillium spp., Aspergillus spp. (A. versicolor, A. flavus), as well as Cladosporium spp. (C. sphaerospermum, C. cladosporioides) (Piecková and Kunová 2002). Many literature reports focused on S. chartarum because of production of highly toxic metabolites, such as satratoxins. However, this species is not frequently isolated from the walls, and it was not found in this study because it usually grows in water-damaged dwellings at high a_w values (Piecková and Jesenská 1999), from which we did not collect our scrapings. Two strains of S. charatrum employed in this study were found in Slovakia among seven isolates recovered from 147 collected samples of wall scrapes, house dust and air samples (Pieckováet al. 2004). All of these findings indicate a certain health hazard during long-lasting exposure to airborne microfungi particles because of toxic effects of mycotoxins and other toxic metabolites, which could be found in them. Recently, there has been considerable interest in essential oils with antifungal activity, which are more acceptable, ecological safe and less hazardous than disinfecting synthetic compounds that are used in air cleaning. Essential oil of thyme (Thymus vulgaris L.), used in this study contains p-cymene (36·5%), thymol (33·0%) and 1,8-cineole (11·3%) as main components. The thyme essential oil exhibited strong antifungal activity against isolated microfungi with MIC values ranged between 1·60 and 50·20 μg ml⁻¹, and MFC values ranged between 1·60 and 116·6 μg ml⁻¹, respectively. Essential oils from other Thymus species, such as Thymus eriocalyx and Thymus × porlock, which contained thymol as major component (63·8, 31·7%), exhibited fungistatic (250 ppm) and fungicidal activity (500 ppm) on aflatoxigenic A. parasiticus strain (Rasooli and Owlia 2005). In addition, essential oil of Thymus spathulifolius, which contained similar amount of thymol (36·5%) as essential oil used in this study, suppressed the growth of Trichophyton spp., Fusarium spp., Penicillium spp., Rhizopus spp., Alternaria spp. and Aspergillus spp., with MICs ranged between 31 and 250 μg ml⁻¹ (Sokeman et al. 2004). The thymol itself exhibited approximately three-times stronger inhibition than essential oil of thyme. MIC values of thymol were between 1·60 and 6·72 μg ml⁻¹, and MFC values were between 3·20 and 13·74 μg ml⁻¹, respectively. According to MIC values of both essential oil of thyme and pure thymol, the most sensitive was C. globosum while the most resistant were species of Mucor. Thymol expressed strong fungicidal activity against opportunistic yeasts Cryptococcus neoformans and Candida spp., as well as against dermatophytes (Viollon and Chaumont 1994; Pauli 2001). Few studies reported on antifungal activity of pure thymol as well as p-cymene and 1,8-cineole. Pure p-cymene and 1,8-cineole were much less effective against Aspergillus spp. and Penicillium spp., (MIC values were ≥4 or 8% v/v), when compared with pure thymol (Hammer et al. 2003). However, thymol and p-cymene exhibited strong antifungal activity against number of Candida spp., with similar MICs ranged 0·08 and 0·32% (v/v), while 1,8-cineole was effective only in higher concentrations (5–10% v/v). In addition, the combination of thymol and p-cymene as well as thymol and 1,8-cineole showed synergistic antifungal effect against Candida spp. (Pina-Vaz et al. 2004). According to these observations, we can speculate that strong antifungal activity of thyme essential oil could be attributed to thymol itself or could be a result of synergism of other main components in the oil. Vaporous phase of essential oil of thyme (82 μg l⁻¹) employed in this study suppressed the development of fructification organs and sporulation and exhibited the sporocidal effect on sporulating cultures after 2 months of exposure of tested moulds. Rakotonirainy and Lavédrine (2005) reported on fungistatic but not fungicidal activity of linalool (295 and 415 ppm) after 21 days of exposure of mouldy books in glass chamber, volume 10·2 l. Comparing with these results, vaporous phase of essential oil of thyme happens to be more effective. Rasooli and Owlia (2005) showed that main target of thyme oils were cell wall and cell membrane of A. parasiticus. The plasma membrane was irregular, dissociated from cell wall, invaginated and associated with the formation of lomasomes, which were found in fungi treated with imidazole components. At low concentrations, phenolic lipophilic compounds such as thymol altered the microbial cell permeability permitting the loss of macromolecules. Exact cause–effect relation for the mode of action of phenols has not been determined, but they may inactivate essential enzymes, react with cell membrane proteins or disturb genetic material functionality (López-Malo et al. 2005). From this point of view, essential oil of thyme, which is rich in thymol and other antifungal components, could be used for disinfection of mouldy walls in the dwellings in low concentration.

ACKNOWLEDGEMENTS

We wish to thank Ms. Štefica Babić for technical assistance and Boris Filipaj, BSc (Bioaromatica Ltd, Zagreb) for donation of essential oil. This work was supported by a grant from the Ministry of Science, Education and Sports of the Republic of Croatia (No. 0006641).