Broad-Spectrum Antibacterial Properties of Thymus fallax.

Abstract

Following the crude extraction of aerial parts of Thymus fallax. with methanol, the soluble fraction was tested for antibacterial activity using the disk diffusion assay. Four antibiotics, ofloxacin, sulbactam, cafoperazona, and netilmicin, were used as controls. The extract was shown to have pronounced antibacterial effects against 27 of 52 bacteria species. The results showed that the methanol extract of Thymus fallax. had great potential for antibacterial activity, in particular against Arthrobacter atrocyaneus., Bacillus sphaericus., Enterobacter hormaechei., Staphylococcus cohni cohni., Pseudomonas syringae., and Kocuria rosea., and the extract inhibited growth of these bacteria at concentrations of 31.25–500 µg/ml. Broad-spectrum antibacterial activity of the methanol extracts against known etiologic agents of diseases traditionally treated with Thymus fallax. of similar preparations provides scientific justification for the use of the herb in ethnomedical practice in Turkey.

Keywords:

• Antibacterial screening
• crude extract
• Thymus fallax
• Turkey

Introduction

Incidences of foodborne illnesses are still a major problem, even in developed countries. It has been estimated that 6 to 81 million cases of illnesses and up to 9000 deaths annually were attributed to foodborne pathogens in the United States alone (Mead et al., 1999). In fact, food poisoning is still a threat for both consumers and the food industry, despite the use of preserving processes. Meanwhile, consumers are concerned about the safety of foods containing preservatives. Thus, there has been a growing interest in new and effective techniques to reduce cases of foodborne illnesses. Antimicrobial substances from natural sources like plants have been investigated to achieve higher levels of food safety. For centuries, indigenous plants have been used in herbal medicine for curing various diseases (Cowan, 1999). Plants are known to produce certain bioactive molecules that react with other organisms in the environment, inhibiting bacterial or fungal growth (antibiotic activity). Traditionally used medicinal plants produce a variety of compounds of known therapeutic properties (Chopra et al., 1992; Bruneton, 1995). The substances that can inhibit pathogens and have little toxicity to host cells are considered candidates for developing new antimicrobial drugs. Recently, the acceptance of traditional medicine as an alternative form for health care and the development of microbial resistance to the available antibiotics have led authors to investigate the antimicrobial activity of medicinal plants (Scrinivasan et al., 2001; Kumarasamy et al., 2002).

Turkey is recognized as a one of the main diversity centers of Thymus.. Thymus. (Labiatae) is a polymorphic genus with 60 taxa belonging to 39 species in Turkey. The ratio of endemism is 45%. The studies have indicated that 49% of all Thymus. taxa are considered as oil-rich (> 1%). The survey has also revealed that 24 Thymus. taxa contain thymol and 11 taxa contained carvacrol as main constituents (Baser, 2002). Most of Thymus. species are traditionally used by Turkish people as folk remedies as tonic, carminative, digestive, antispasmodic, anti-inflammatory, antitussive, or expectorant, and for the treatment of colds (Baytop, 1984). Recent studies have showed that Thymus. species have strong antibacterial, antifungal, antiviral, antiparasitic, and antioxidant activities (Cosentino et al., 1999; Karaman et al., 2001; Rasooli & Mirmostafa, 2002; Stahl-Biskup & Saez, 2002; Vardar-Unlu et al., 2003). In particular, in the eastern part of Turkey, it is added in a special cheese, namely “herby cheese.” To make herby cheese, sheep milk is first filtered immediately after milking and then coagulated with calf rennet at the milking temperature. After cutting the coagulum, whey is removed, and previously prepared herbs are added into the curd. About 25 kinds of herbs can be used to make herby cheese. (e.g.., Falcaria vulgaris., Allium. spp., Thymus. spp., Ferula. spp., Anthriscus nemorosa., etc.). From these herbs, single herbs or mixtures of some herbs can be added. The rate of the addition of herbs changes between 0.5 and 2 kg per curd obtained from 100 l of milk (Tarakci et al., 2004).

Available literature indicates that no previous studies have been carried out on the antibacterial properties of Thymus fallax.. The purpose of the current study was to investigate the antibacterial activity of the aerial parts of Thymus fallax. that is naturally growing in Turkey. It is expected that screening of plant extracts against a wide variety of test microorganisms will be helpful in obtaining broad-spectrum herbal formulation as well as new antimicrobial substances.

Materials and Methods

Plant material

The plant (aerial parts) used for the current study was collected locally in the Erzurum region of Turkey. Plant materials were further identified by senior taxonomist, Avni Ozturk, in the Department of Botany, Yuzuncu Yil University (Van, Turkey), and a voucher specimen was deposited in the herbarium of the Horticulture Department of Agricultural Faculty of Ataturk University (Erzurum, Turkey). The dried and powdered plant materials (400 g) were extracted successively in a Soxhlet with methanol (MeOH) for 72 h at a temperature not exceeding the boiling point of the solvent (Lin et al., 1999). The extracts were filtered using Whatman filter paper (no. 1) and then concentrated in vacuo. at 40°C using a rotary evaporator. The residues obtained were stored in a freezer at − 80°C until further tests.

Biological materials

A total of 100 bacterial strains belonging to 52 bacteria species (listed in Table 1) were used in this study. The bacteria, from herby cheese collected from retail markets in Van and maintained on nutrient agar (Merck, Darmstadt, Germany), were supplied by Microbiology Laboratory of Agricultural Faculty of Ataturk University (Erzurum, Turkey). The food-associated bacteria were selected because they are frequently reported in foods. Identity of the bacteria used in this study was confirmed by the Microbial Identification System in the Biotechnology Application and Research Center at Ataturk University.

Table 1.. Antibacterial activity of Thymus fallax. extracts against the bacteria

		Inhibition zone in diameter (mm/sensitive strains)^a.
Bacterial species	Number of strains/origins	Thymus fallax. extracts (300 µg/disk)	Negative control MeOH	Positive controls (mm)^b. standard antibiotic disk
Acidovorax facilis.	1/food	11 mm	—	28 (OFX)
Arthrobacter agilis.	2/food	—	—	31 (SCF)
Arthrobacter atrocyaneus.	1/food	11 mm	—	15 (OFX)
Arthrobacter ilicis.	1/food	7 mm	—	20 (OFX)
Arthrobacter protophormiae.	2/food	—	—	21 (NET)
Bacillus cereus.	3/food	—	—	21 (OFX)
Bacillus dipsauri.	4/food	—	—	26 (OFX)
Bacillus flexus.	3/food	—	—	27 (SCF)
Bacillus lentimorbus.	1/food	—	—	30 (OFX)
Bacillus lichemiformis.	4/food	—	—	29 (OFX)
Bacillus marinus.	4/food	—	—	14 (OFX)
Bacillus megaterium.	2/food	—	—	26 (OFX)
Bacillus psychrosaccharolyticus.	6/food	—	—	15 (OFX)
Bacillus pumilus.	7/food	—	—	24 (SCF)
Bacillus sphaericus.	1/food	13 mm	—	21 (OFX)
Bacillus. spp.	1/food	8 mm	—	20 (SCF)
Bacillus subtilis.	1/food	8 mm	—	29 (OFX)
Brevibacillus agri.	5/food	—	—	27 (OFX)
Brevibacillus brevis.	1/food	9 mm	—	32 (NET)
Brevibacterium linen.	2/food	—	—	22 (SCF)
Chryseomonas luteola.	2/food	—	—	30 (OFX)
Citrobacter amalonaticus.	3/food	—	—	23 (NET)
Corynebacterium ammoniagenes.	1/food	12 mm	—	20 (OFX)
Corynebacterium cystitidis.	2/food	—	—	18 (OFX)
Corynebacterium flavescens.	1/food	10 mm	—	24 (OFX)
Enterococcus faecalis.	3/food	—	—	10 (SCF)
Enterobacter hormaechei.	1/food	13 mm	—	22 (OFX)
Enterobacter intermedius.	2/food	7–8 mm	—	16 (SCF)
Enterobacter sakazakii.	1/food	9 mm	—	21 (NET)
Erwinia carotovora.	4/food	—	—	20 (NET)
Erwinia chrysanthemi.	2/food	—	—	17 (SCF)
Exiguobacterium acetylicum.	1/food	10 mm	—	20 (OFX)
Flavimonas oryzihabitans.	2/food	—	—	30 (OFX)
Kocuria kristinae.	2/food	—	—	24 (NET)
Kocuria rosea.	1/food	8 mm	—	15 (OFX)
Micrococcus luteus.	5/food	—	—	28 (OFX)
Micrococcus lylae.	3/food	—	—	30 (OFX)
Moraxella catarrhali.	1/food	10 mm	—	18 (OFX)
Neisseria subflava.	1/food	11 mm	—	24 (OFX)
Paenibacillus apiarius.	1/food	—	—	30 (OFX)
Paenibacillus macerans.	1/food	13 mm	—	30 (OFX)
Paenibacillus polymyxa.	1/food	—	—	30 (OFX)
Pantoea agglomerans.	1/food	7 mm	—	30 (OFX)
Proteus vulgaris.	1/food	11 mm	—	20 (OFX)
Pseudomonas putida.	1/food	9 mm	—	17 (OFX)
Pseudomonas syringae syringae.	2/food	8–13 mm	—	15 (OFX)
Psychrobacter immobilis.	1/food	12 mm	—	20 (OFX)
Salmonella typhimurium.	1/food	9 mm	—	28 (OFX)
Serratia liquefaciens.	3/food	—	—	30 (OFX)
Shigella dysenteriae.	1/food	10 mm	—	21 (NET)
Staphylococcus cohnii cohnii.	1/food	8 mm	—	12 (OFX)
Xanthomonas arb. corylina.	1/food	9 mm	—	22 (OFX)
Total 52 bacterial species	100

^a.MeOH, methanol extract.

^b.OFX, ofloxacin (5 µg/disk); SCF, sulbactam (30 µg) + cefoperazona (75 µg) (105 µg/disk); NET, netilmicin (30 µg/disk) were used as positive reference standards antibiotic disks (oxoid).

Table 2.. The MIC values of Thymus fallax. extracts against microorganisms tested in the microdilution assay (MIC in µg/ml)

Bacterial species	Number of strains/origins	Thymus fallax. extracts	Standard drug (maxipime)
Acidovorax facilis.	1	> 500 µg/ml	250 µg/ml
Arthrobacter atrocyaneus.	1	250 µg/ml	62.50 µg/ml
Arthrobacter ilicis.	1	62.50 µg/ml	7.81 µg/ml
Bacillus. spp.	1	31.25 µg/ml	7.81 µg/ml
Bacillus sphaericus.	1	> 500 µg/ml	31.25 µg/ml
Bacillus subtilis.	1	125 µg/ml	7.81 µg/ml
Brevibacillus brevis.	1	250 µg/ml	7.81 µg/ml
Corynebacterium ammoniagenes.	1	250 µg/ml	7.81 µg/ml
Corynebacterium flavescens.	1	250 µg/ml	7.81 µg/ml
Enterobacter hormaechei.	1	250 µg/ml	7.81 µg/ml
Enterobacter intermedius.	2	250 µg/ml	31.25 µg/ml
Enterobacter sakazakii.	1	> 500 µg/ml	7.81 µg/ml
Exiguobacterium acetylicum.	1	250 µg/ml	7.81 µg/ml
Kocuria rosea.	1	125 µg/ml	31.25 µg/ml
Moraxella catarrhalis.	1	250 µg/ml	7.81 µg/ml
Neisseria subflava.	1	> 500 µg/ml	7.81 µg/ml
Paenibacillus macerans.	1	> 500 µg/ml	7.81 µg/ml
Pantoea agglomerans.	1	> 500 µg/ml	7.81 µg/ml
Proteus vulgaris.	1	250 µg/ml	62.50 µg/ml
Psychrobacter immobilis.	1	62.50 µg/ml	7.81 µg/ml
Pseudomonas putida.	1	> 500 µg/ml	> 500 µg/ml
Pseudomonas syringae syringae.	2	> 500 µg/ml	250 µg/ml
Salmonella typhimurium.	1	> 500 µg/ml	NT
Shigella dysenteriae.	1	> 500 µg/ml	7.81 µg/ml
Staphylococcus cohni cohni.	1	125 µg/ml	7.81 µg/ml
Xanthomonas arboricola corylina.	1	250 µg/ml	> 500 µg/ml

OFX, ofloxacin (5 µg/disk); SCF, sulbactam (30 µg) + cefoperazona (75 µg) (105 µg/disk); NET, netilmicin (30 µg/disk) were used as positive reference standards antibiotic disks (oxoid). NT, not tested.

Antibacterial activity

The antibacterial activity of the extracts was carried out by disk diffusion test (Kim et al., 1995) using 100 µl of suspension containing 10⁸ CFU/ml of bacteria spread on nutrient agar (NA) medium. Sterile 6-mm-diameter filter paper disks were impregnated with 300 µg of sterile test material and placed onto nutrient agar. Negative controls were prepared using the same solvents employed to dissolve the plant extracts. Ofloxacin (5 µg/disk), sulbactam (30 µg) + cefoperazona (75 µg) (105 µg/disk), and/or netilmicin (30 µg/disk) were used as positive reference standards to determine the sensitivity of one strain in each bacterial species tested. The inoculated plates with food-associated bacteria were incubated at 27°C for 24 h. The antibacterial activity was measured as the diameter (mm) of clear zone of growth inhibition. Five disks per plate and three plates were used, and each test was run in triplicate (Djipa et al., 2000).

Microdilution assays

The minimal inhibition concentration (MIC) values were also studied for the bacteria, which were determined as sensitive to the extracts in disk diffusion assay. The inocula of bacteria were prepared from 12 h broth cultures, and suspensions were adjusted to 0.5 McFarland standard turbidity. Thymus fallax. extracts dissolved in 0.5% dimethylsulfoxide (DMSO) were first diluted to the highest concentration (500 µg/ml) to be tested, and then serial twofold dilutions were made in a concentration range from 7.80 to 500 µg/ml in 10-ml sterile test tubes containing nutrient broth. MIC values of radish extracts against bacterial strains were determined based on a micro-well dilution method (Swanson et al., 1992). The 96-well plates were prepared by dispensing into each well 95 µl of nutrient broth and 5 µl of the inoculum. A 100-µl sample from Thymus fallax. extracts initially prepared at the concentration of 500 µg/ml was added into the first wells. Then, 100 µl from their serial dilutions was transferred into six consecutive wells. The last well, containing 195 µl of nutrient broth without compound and 5 µl of the inoculum on each strip, was used as negative control. The final volume in each well was 200 µl. Maxipime (Bristol-Myers Squibb) at the concentration range of 500–7.8 µg/ml was prepared in nutrient broth and used as standard drug for positive control. Contents of each well were mixed on a plate shaker at 300 rpm for 20 s and then incubated at appropriate temperatures for 24 h. Microbial growth was determined by absorbance at 600 nm using the ELx 800 universal microplate reader (Biotek Instrument Inc., Highland Park, VT, USA) and confirmed by plating 5 µl samples from clear wells on nutrient agar medium. The extract tested in this study was screened two-times against each organism. The MIC, value of each extract was taken as the lowest concentration that showed no growth.

Results and Discussion

The aromatic and medicinal properties of the genus Thymus. have made it one of the most popular plants throughout the world. The current study was conducted to study the in vitro. antibacterial activity of aerial parts of Thymus fallax. used by Turkish people for medicinal purposes to determine if the therapeutic properties of this species used in traditional medicine coincide with laboratory findings. The highest antibacterial activity of this plant was determined against Arthrobacter atrocyaneus., Bacillus sphaericus., Enterobacter hormaechei., Staphylococcus cohni cohni., Pseudomonas syringae., and Kocuria rosea., respectively (Table 1). Based on MIC values, MICs of the tested extract show bacteriostatic activity between 31.25 and 500 µg/ml against 27 bacteria species. Although the MICs obtained with the methanol extracts are high compared with those of maxipime, in general between 7.81 and 62.50 µg/ml, these results are of interest because they have been obtained with methanol extracts and are not a pure product, and it could be considered to have a good potency level. With phytochemical studies, Thymus. was found to be rich in monoterpene, phenols, especially thymol, carvacrol, c-terpinene, and p.-cymene (Baser, 2002).

The obtained results might be considered sufficient for further studies to isolate and identify active principles, and to evaluate possible synergism among extract components for their antibacterial activity. Investigations are in progress to determine the degree of toxicity of these extracts.

Based on these results, it is possible to conclude that aerial parts of Thymus fallax. have a stronger and broader spectrum of antibacterial activity against many foodborne bacteria. This is the first study to provide data that the extracts of Thymus fallax. evaluated against a wide range of bacteria possess potential antibacterial activities.