Antimicrobial potential of the Ethiopian Thymus schimperi essential oil in comparison with others against certain fungal and bacterial species

Background To evaluate the in vitro activities of Ethiopian Thymus schimperi with other three hydro distilled essential oils against Dermatophytes (Tricophyton spp. and Microsporum spp.) and other pathogenic micro organisms. Methods The studies were carried out using Agar disk diffusion method for screening the most effective essential oils and Agar dilution to determine Minimum Inhibitory Concentration (MIC) of the essential oils. Results Essential oils of T. schimperi and Cinnamomum zeylanicum were highly active against tested organisms. The MIC were in the range of 0.08 μl/ml to 0.31 μl/ml for T. schimperi, 0.31 μl/ml to 0.16 μl/ml for C. zeylanicum, 2.5 μl/ml to1.25 μl/ml for Citrus limon and 5 μl/ml to 2.5 μl/ml for Eucalyptus camaldulensis against Tricophyton spp. and Microsporum spp. T. schimperi and C. zeylanicum oils also showed antimicrobial effect against Candida albicans, Aspegilus niger, Rhodotorula rubra, Escherichia coli, Shigella spp., Bacillus spp. and Streptococci. Conclusions The Ethiopian T. schimperi oil had pronounced antifungal and antibacterial activities against all the tested microbes. Therefore, it is required further investigation in order to identify the active compounds and their clinical applications for treatment of tested organisms.


Background
Antimicrobial properties have been reported more frequently in a wide range of plant extracts and essential oils and natural products in an attempt to discover new chemical classes of antifungal and antibacterial drugs that could resolve strains expressing resistance to the available antifungal and antibacterial drugs [1,2].
Essential oils are volatile, natural, complex compounds characterized by a strong odor and are formed by aromatic plants as secondary metabolites. They are usually obtained by steam or hydro-distillation [3,4]. At present, approximately 3000 essential oils are known, 300 of which are commercially important especially for the pharmaceutical, agronomic, food, sanitary, cosmetic and perfume industries [5].
T. pulegioides essential oil has potential as a topical antifungal agent against Dermatophytes, Aspergilus, and Candida [6]. Other species of the genus Thymus, such as T. zygis and T. vulgaris, with high amounts of phenols, also show a broad spectrum of activity against a variety of pathogenic yeasts and filamentous fungi, including fungi with decreased susceptibility to fluconazole [6].
The Thymus species in Ethiopia are restricted to afromontane and afro-alpine regions and are represented by the two endemic species, T. schimperi Ronninger and T. serrulatus Hochst [7]. They have a crowded inflorescence with pink corollas and have ovate to elliptic leaves with entire margins [8]. They are extensively used by local people as food preservatives, cure for various ailments and food flavoring and seasonings [8]. However, the antimicrobial activity of T. schimperi essential oil has not been widely investigated. Therefore, in order to fill this gap of information, essential oil of this plant is to be investigated for its potential in comparison with other essential oils in the control of Trichophyton spp., Microsporum spp., C. albicans, A. niger, R. rubra and different bacteria strains (E. coli, Bacillus spp., Shigella spp. and Streptococci) grown in vitro using Agar disk diffusion and Agar dilution method.

Plant material and preparation of essential oils
Four various types of herbs and spice plants were collected around Addis Ababa: T. schimperi (leaf with aerial parts), C. zeylanicum (bark), C. limon (peel), and E. camaldulensis (leaf) and identified by the national herbarium of Ethiopia. The plants were brought to the laboratory and thoroughly washed in running tap water to remove debris and dust particles and then rinsed in distilled water. The collected plant materials were dried at room temperature and ground into a semi powder using a grinder.
Each semi powdered plant materials were hydro distilled by a Clevenger apparatus at 70°C -80°C for 3-5 hrs (approximately four times water (w/v) in volume than the plant material). The extracted fractions of plant parts exhibited two distinct layers an upper oily layer and the lower aqueous layer. Both the layers were separated and oils were collected in clear vials, after removing water traces using anhydrous sodium sulfate, and they were stored at 4°C until needed.

Microorganisms
Tricophyton spp., Microsporum spp. and C. albicans were obtained from Ethiopian Health and Nutrition Institute (EHNRI) while local isolates of A. niger and R. rubra and four bacteria strains (E. coli, Bacillus spp., Shigella spp. and Streptococci) were obtained from Addis Ababa University, Mycology and Bacteriology laboratory. Sabouraud Dextrose Agar was used as growing medium for Tricophyton spp., Microsporum spp. and C. albicans while Potato dextrose agar (PDA; Himedia Ltd., India) for A. niger and R. rubra. The bacteria were grown in nutrient broth and Mueller -Hinton agar. Periodic transfers were done to keep the micro organism viable.

Disk diffusion assay
The antimicrobial activity test of the essential oils were carried out by the agar diffusion method, which is normally used as a preliminary screening of efficient essential oils [9] following the procedure approved by NCCLS (2002) with little modification. The agar plate was prepared for each organism as follows: a suspension of the organism (20 μl and 0.1 ml of inoculums suspension for fungi and bacteria respectively) of the standardized inoculums was mixed with 20 ml of sterile Sabouraud Dextrose ® Agar for Tricophyton spp., Microsporum spp. and C. albicans, PDA for A. niger and R. rubra and Nutrient agar for bacteria then poured into sterilized Petri dishes and set aside. After solidified, sterile filter paper disc (Whatman no.1, 6 mm in diameter) impregnated with 5 μl of each essential oil were placed over the middle of the plates already seeded test organism. Sterile distilled water but not oil was added on the discs to provide negative control and as a positive control Griseofulvin (0.03 mg/ml) and Chloramphenicol (0.01 mg/ml) for fungi and bacteria respectively were provided by Addis Ababa University, Department of Microbiology; Mycology Laboratory. The plates were incubated at 37°C for 10 days for Dermatophytes and plates with Candida, Aspergillus and Rhodotorula for 48 h and plates with bacteria were incubated for 24 h. Each test was done in triplicates. The antimicrobial activity was evaluated by measuring the diameter of the zone of inhibition (including diameter of the disk) [9].

Determination of minimum inhibitory concentration (MIC)
The essential oils that previously showed antimicrobial activity were screened for determination of MIC by Agar dilution method [10]. The agar dilution method followed that approved by the National Committee for clinical laboratory standards NCCLS (2000) with little modification. Two fold serial dilutions in DMSO ranging from 0.019 to 20 μ/ml were tested for each essential oil. The prepared plates were incubated and evaluated for the presence or absence of microbial growth. The MIC was determined as the lowest concentration of oil inhibiting the visible growth of each organism on the plates [10].

Statistical analysis
All the measurements were replicated three times for each assay and the results are presented as mean standard deviation. The statistical analysis was performed by one-way analysis of variance (ANOVA) and P values < 0.05 were considered as significant.

Results
Preliminary screening of the antifungal activity in vitro of the four essential oils was studied against fungal pathogens using the filter paper disc agar diffusion technique as shown in (Table 1). All the oils tested exhibited different degrees of antifungal activity against Tricophyton spp, Microsporum spp. and other tested fungi. The maximum antimycotic activity was shown by T. schimperi followed by C. zeylanicum. T. schimperi has shown the greatest inhibition zone diameter of 88.66 mm against R. rubra and 73.33 mm against Clinical isolates of Ttricophyton spp. and Microsporum spp. The least inhibition zone exhibited by T. schimperi was 35.66 mm against C. albicans. Similarly, C. zeylanicum inhibited with the range of 31.33 mm to 62.66 mm against all the tested fungi and considered as a high result next to T. schimperi. The oils of C. limon (lemon) exhibited moderate activity and the oils of E. camaldulensis showed comparatively low activity against all the tested organisms. Control disks with distilled water showed no inhibition in a preliminary test. The difference between the essential oils was statistically significant as proofed from the results of one-way ANOVA (Table 1).
Bacteria susceptibility to the essential oils also determined by the agar diffusion method and showed that T. schimperi oil possessed potential antibacterial activity against E. coli, Bacillus spp., Shigella spp. and Streptococci. The highest antibacterial activity of T. schimperi oil was 90 mm in E.coli, Shigella spp. and Striptococci and relatively least activity was recorded in Bacillus sp. measured 63 mm ( Table 2). C. zeylanicum exhibited almost the highest activity against all the tested bacteria which measured in the range of 36 mm-43 mm as shown in (Table 2) next to Thymus oil. The oils of C. limon exhibited moderate activity and E. camaldulensis which has less or no effect with similar concentration (5 μl).
As to the standard drugs used in the test, the inhibition zone for Grisofulvin was 36 mm, 34 mm and 32.66 mm for Tricophyton spp. (scalp isolate), Microsporum spp and Tricophyton spp. (nail isolate) respectively. The highest inhibition diameter recorded for Chloramphenicol was 44.33 mm against Shigella spp., and the list was 34 mm against Bacilus spp. (Tables 1 and 2).
Evaluation of MIC showed that the Thymus and Cinnamon oils were active against all the tested isolates. T. schimpri oil showed the highest MIC values (0.08 μl/ml) against Tricophyton spp. (scalp isolate), Microsporum spp. and R. rubra. Cinnamon oil also has similar effect on C. albicans, A. niger and R. rubra (Table 3). The lowest concentration of the lemon essential oil at which the tested organisms (Microsporum spp. and R. rubra) were unable to grow was found to be ≥1.25 μl/ml. As can be noted from the Table 3, Eucalyptus essential oil inhibited growth of the Tricophyton spp. (scalp isolate) and A. niger at 2.5 μl/ml and for the rest of tested fungal strain at 5 μl/ml that were higher concentration when compared to the other oils.
The data presented in Table 4 revealed variability in the inhibitory concentrations (MIC) of each extracts for   (Table 4).

Discussion
In the preliminary screening of the essential oils, T. schimperi at 5 μl concentration caused higher inhibition zone with the range of 35.66 mm to 88.66 mm against tested fungi and 63 mm to 90 mm against bacteria. In similar study antimicrobial activity of seven essential oils from three Thymus species against Salmonella enteritidis, Salmonella typhimurium, Eschericcha coli, Shigella flexneri and Listeria monocytogens [11]. The result showed essential oil from T. hyemalis inhibited with the range of 19.6 mm to 45 mm. The existence of variations in the inhibition zone can be assumed that due to differences in the number of molecules and chemical type of molecules in the plant materials [12,13]. In this study the essential oil extracted from C. zeylanicum bark demonstrated strong antifungal and antibacterial activity next to T. schimperi. The antifungal activity of C. zeylanicum exhibited 61 mm, 62.66 mm, 62 mm, 31.33 mm, 54.66 mm and 54 mm inhibition zone against Tricophyton spp. (scalp isolate), Tricoophyton spp. (nail isolate), Microsporum spp., C.albicans, A. niger and R.rubra respectively. It also showed inhibition zone of 42.66 mm, 43.33 mm, 36.66 mm and 41.33 mm against E. coli, Bacillus spp., Shigella spp. and Streptococci respectively. The antimycotic activity of C. zeylanicum bark is due to presence of cinnamaldehyde [14].
In our work, the C. limon peel oil inhibited the tested fungi and bacteria in the range of 14.66 mm to 42 mm and 20.33 mm to 27 mm respectively and the antimicrobial effect is taken as a moderate activity when compared to Thymus and Cinnamon oils. However, the present study results were better than reported by (Gulay-Kirbasilar et al., [15]). This may be due to variations in climate and soil composition in which the plant growing [12,13].
In this study, E. camaldulensis showed less inhibition zone against almost all the tested organisms. However, the result of E. coli and Streptococci were 16 mm and 14.3 mm respectively and taken as a moderate activity. In contrast to the present study, Babayi, et al., [16]) tested the efficacy of methanol extract of E. camaldulensis against Salmonella typhi, Staphylococcus aureus and Bacillus subtilis and reported a higher inhibition zone in a range of 15 mm-16 mm. In this case, the solvent extract of E. camaldulensis is better than the hydrodistiled essential oil against tested organism. According to Masotti et al., [13]) and Angioni et al., [12]), the extraction product can vary in quality, quantity and in composition according to the type of extraction method.
Some oils appeared more active with respect to Gram reaction, exerting a greater inhibitory activity against Gram positive bacteria [17]. However, the result obtained in the present study showed that the oil of T. schimperi did not show any selectivity towards the tested gram-positive and gram-negative bacteria. The substances extracted from thyme especially the phenolic components thymol and carvacrol showed antibacterial activity against gram-positive and gram-negative bacteria due to their effects on the bacterial membrane [7]. In this case, T. schimperi seems to better than other essential oils.
The MIC values of T. schimperi were with the range of 0.08 μl/ml to 0.031 μl/ml against Tricophyton spp. (scalp and nail isolate); Microsporum spp. and R. rubra. These results are in line with the work of Eugenia et al., [6] who had reported that Thymys pulegioides essential oil showed the lowest MIC value in the range of 0.16 μl/ml to 0.32 μ l/ml for Dermatophyte and 0.32 μl/ml to 0.64 μl/ml for Candida. This indicates that plants rich in a wide variety of secondary metabolites such as tannins, alkaloids, terpenoids which have  been found invitro to have antimicrobial property [18]. C. zeylanicum oil also showed similar result with the same concentration on Microsporum spp., C. albicans, A.niger and R. rubra.
The MIC of C. limon peel oil was 1.25 μl/ml against R. rubra and Microsporum spp. On the other hand, 2.5 μL/ml was recorded for the rest of the tested organisms. Therefore, Rhodotorula and Microsporum were more susceptible to C. limon peel oil than other tested organisms. However, the result obtained in limon oil was not comparable from the result of T. schimperi and C. zeylanicum which showed better antimicrobial activity for all tested organisms. This variation was due to the difference in plant type and part (organ) used in the experiment [12,13]. In the present study, MIC of the essential oil of E. camaldulensis was in the range of 2.5 μl/ml to 5 μl/ml against all the tested organisms. The obtained results were in contrast to Morteza et al., [19]) who had recorded 1.95 μl/ml against Staphylococcus aureus. This variation may due to the difference in the tested organisms and the method used to assess antimicrobial activity (Janssen et al., [20]).
Our study has several limitations. There were no controls for C. albicans, A. niger and R. rubra, because the purpose was not comparison of the standard antimicrobial agent with plant materials. Moreover, all the tested organisms were not standards. However, the organisms were identified by cellular, cultural and biochemical characteristics. Finally, this study also did not include the chemical composition of plant materials due to financial shortage and lack of materials.

Conclusion
The present study confirmed antimicrobial properties of essential oil from Ethiopian T. schimperi and showed significant growth inhibition for all the tested organisms. Therefore, these results will invite further study in the chemical components and the target cellular structure of the organisms.