Antibacterial Activity of Sideritis curvidens. and Sideritis lanata. from Turkey

Abstract

In this study, the antibacterial activity of Sideritis curvidens. Stapf. and Sideritis lanata. L. were investigated. The antibacterial effects of the essential oils, which were obtained from voucher specimens of the plants by the hydrodistilled method, were tested by a disk diffusion method using Streptococcus mutans. CNCTC 8/77, Staphylococcus aureus. ATCC 6538 P, Staphylococcus aureus. MU 38, Staphylococcus epidermidis. MU 30, Micrococcus luteus. NRRL B-4375, Bacillus subtilis. ATCC 6633, Bacillus cereus. RSKK 863, Escherichia coli. ATCC 11230, Escherichia coli. ATCC 35218, Pseudomonas aeruginosa. ATCC 27853, Shigella sonnei. RSKK 878, Enterobacter aerogenes. RSKK 720, and Salmonella typhimurium. CCM 5445. Both essential oils showed similar potencies against all tested microorganisms. The results showed that the essential oils of Sideritis curvidens. Stapf. and Sideritis lanata. L. had a strong antimicrobial effect against Gram-positive bacteria, especially methicillin-resistant Staphylococcus aureus. (MRSA) and oxacillin-resistant coagulase negative Staphylococcus epidermidis.. In additional, B. cereus., B. subtilis., and M. luteus. were found as the most sensitive bacteria to essential oils of Sideritis curvidens. Stapf. and Sideritis lanata. L., and both essential oils were more effective against these bacteria than the reference antibiotics used in this study.

Keywords:

• Antibacterial activity
• coagulase-negative staphylococci
• methicillin-resistant Staphylococcus aureus
• Sideritis curvidens
• Sideritis lanata

Introduction

An increase in bacterial resistance to antimicrobial agents has been seen worldwide during the past decades (Livermore, 1995; Jones et al., 1997; Kolar et al., 2001). As with antibiotics and other chemotherapeutic drugs, acquired resistance to antiseptics and disinfectants can arise by either mutation or the acquisition of genetic material in the form of plasmids or transposons (McDonnell & Russell, 1999). Antimicrobial resistance rates among key Gram-positive pathogens continue to grow at an alarming rate in distinct geographic regions worldwide. It is evident that new drug-resistant strains have emerged and proliferated in some areas of the world (Pfaller et al., 1998; Sujata & Ballow, 2000). For example, the occurrence of methicillin-resistant Staphylococcus aureus. (MRSA) in hospitals has risen from less than 3% in the early 1980s to as much as 40% now. It has been reported that coagulase-negative Staphylococcus. spp. (CoNS) are becoming increasingly important in nosocomial infections and that they may cause serious infections (Kloos & Bannermann, 1994). Therapeutic options for CoNS infections caused by methicillin-oxacillin-resistant strains are limited to vancomycin-based regimens. However, vancomycin therapy of staphylococcal infections has been associated with a slow and inadequate response in many instances (Levine et al., 1991). For this reason, there is a continuous need for alternative inhibitors. New chemical entities with such activities may be identified through a variety of approaches, one of them being screening of natural products (Bedoya et al., 2001). Natural products can be selected for biological screening based on ethnomedical use of plants, because many infectious diseases are known to have been treated with herbal remedies throughout the history of mankind. Even today, plant materials continue to play a major role in primary health care as therapeutic remedies in many developing countries (Zakaria, 1991; Sökmen et al., 1999).

The genus Sideritis. (Lamiaceae) comprises about 140 species distributed in several countries of the Mediterranean region (Tomas-Barberan et al., 1988). Certain biological activities, such as anti-inflammatory (Villar et al., 1984; Yeşilada & Ezer, 1989; De Las Heras et al., 1990; Manez et al., 1990; Godoy et al., 2000; Villena et al., 2000), antiulcer (Villar et al., 1984), antioxidant (Rios et al., 1992), anticataract (Tomas-Barberan et al., 1986), antimicrobial (Diaz et al., 1988; Gergis et al., 1990), immunomodulating (Navarro et al., 2000), as well as inhibition of NOS-2 expression in macrophages (De Las Heras et al., 2000) were also reported in the several species of Sideritis. genus.

The genus Sideritis. is widely used in folk medicine for anti-inflammatory, antirheumatic, digestive, and antimicrobial activities (Ezer et al., 1995). Sideritis. species are a group of plants known as “mountain tea” in Anatolia. Local names are dağ çayι. or yayla çayι. in Turkish (Kιrιmer et al., 1991). Some species are used as tea, flavoring agents, and for medicinal purposes in several regions. Infusion of aerial parts of a number of Sideritis. species are used as tonics, carminatives, antispasmodics, diuretics, digestives, and in the treatment of colds (Ezer et al., 1991; Yeşilada & Ezer, 1989; Koedam, 1986; Villar et al., 1984). Previous results on the pharmacological activities of Turkish Sideritis. revealed their diuretic, anti-inflammatory, antispasmodic, and antibacterial activities (Perez de Paz & Hernandez Padron, 1999; Ezer et al., 1991; Darias et al., 1990; Yeşilada & Ezer, 1989; Koedam, 1986; Villar et al., 1984). However, as far as can be ascertained by a survey of the literature, the in vitro. antibacterial activity of S. curvidens. Stapf. and S. lanata. L. has not been reported before. Therefore, this may be considered as the first report on the antibacterial activity of the two plant species mentioned above. We hope that our results will provide a starting point for discovering new compounds with better activity than agents currently available.

Materials and Methods

Plant materials

Sideritis curvidens. Stapf. and Sideritis lanata. L. were collected at the flowering stage in March–June 2002 from the Muğla region of Turkey. Voucher specimens of the plants were collected and taxonomically identified by Dr. Ömer Varol at the Faculty of Science, University of Muğla. These voucher specimens have been deposited at the Herbarium of the Department of Biology, University of Muğla, Turkey.

Isolation of the essential oil

The volatile oil of air-dried aerial parts of S. curvidens. and S. lanata. were obtained by hydrodistillation by using a Clevenger-type apparatus.

Antimicrobial activity

Microorganisms and condition for cultivation

Staphylococcus aureus. MU 38 and Staphylococcus epidermidis. MU 30 (obtained from the culture collection of the microbiology Department in Mugla University) were identified by API Staph (Biomerieux). Staphylococcus aureus. MU 38 is resistant to penicillin, amikacin, clindamycin, gentamicin, methicillin, teicoplanin, tetracycline, and oxacillin antibiotics and contains one plasmid with molecular size of 15.3 kb. Coagulase-negative Staphylococcus. (CoNS) Staphylococcus epidermidis. MU 30 is resistant to penicillin, amikacin, clindamycin, gentamicin, teicoplanin, tetracycline, and oxacillin and contains five plasmids with molecular sizes of 2.2, 3.6, 7.7, 12.1, and 13.5 kb, respectively. Staphylococcus aureus. ATCC 6538 P, Bacillus subtilis. ATCC 6633, Escherichia coli. ATCC 11230, E. coli. ATCC 35218, and Pseudomonas aeruginosa. ATCC 27853 were obtained from the American Type Culture Collection. Shigella sonnei. RSKK 878, Bacillus cereus. RSKK 863, and Enterobacter aerogenes. RSKK 720 were obtained from the Culture Collection of Refik Saydam Central Hygiene Institute. Micrococcus luteus. NRRL B-4375 was obtained from the Agricultural Research Service Culture Collection (USA). Streptococcus mutans. CNCTC 8/77 was obtained from the Czechoslovak Collection of Type Cultures Institute of Hygiene and Epidemiology, and Salmonella typhimurium. CCM 5445 was obtained from the Czechoslovak Collection of Microorganisms.

The above-mentioned bacteria (except S. mutans.) were cultured in nutrient broth (NB) (Difco, Detroit, MI, USA) at 37 ± 0.1°C; S. mutans. were cultured in blood agar (Difco) at 28 ± 0.1°C.

Inocula prepared by adjusting the turbidity of the medium to match the 0.5 McFarland standard dilutions of this suspension in 0.1% peptone (w/v) solution in sterile water were inoculated on NB to check the viability of the preparation. The cultures of bacteria were maintained in nutrient agar (NA) (Difco) slants, in the dark at 4°C during the study.

Antimicrobial assays

The antimicrobial activities of the essential oils of S. lanata. and S. curvidens. were assayed by the disk diffusion method (Collins et al., 1995; Murray et al., 1995). The inoculum size of each group of bacteria was prepared by using a no. 0.5 McFarland tube to give a concentration of 1 × 10⁸ organisms per ml. From the bacterial culture (except S. mutans.), 1 ml was aseptically inoculated into 15 ml of Mueller Hinton agar (MHA) (Difco) tubes that had been warmed to 48–50°C. S. mutans. was inoculated into 15 ml of MHA with 5% blood above. These mixtures were transferred in sterile Petri dishes and medium was distributed in Petri dishes homogeneously. The plates were held for 15–20 min at room temperature. Each essential oil (5, 10, and 25 µl) was applied under suction to the sterile 6 mm disks (Schleicher & Schuell) using the multipoint inoculator. Prepared disks were added aseptically onto the agar with cooled and flamed pliers. The plates containing bacteria were incubated at 37°C for 24 h. At the end of the incubation periods, diameters of no-growth zones around the disks were measured to the nearest 0.1 mm using Vernier calipers. Disks of cefadroxil (30 µg, Oxoid, Basingstoke, Hampshire, UK), chloramphenicol (30 µg, Oxoid), tetracycline (30 µg, Oxoid), streptomycin (10 µg, Oxoid), sulbactam + ampicillin (10 µg/10 µg, Oxoid) and vancomycin (30 µg, Oxoid) were used as positive controls. Studies were performed in triplicate, and the developing inhibition zones were compared with those of reference disks.

Results

Antimicrobial activity of different quantities of essential oils of Sideritis curvidens. Stapf. and Sideritis lanata. L. has been evaluated in vitro. against six Gram-negative bacteria (Table 1) and eight Gram-positive bacteria (Table 2) that are known to cause infections in humans. As summarized in these tables, the effect of the essential oils of S. curvidens. and S. lanata. were increased by the amount of essential oils. Essential oil of S. curvidens. and S. lanata. (5 µl) had no effect on any Gram-negative bacteria. Although 10 µl of S. curvidens. was effective on Gram-negative bacteria except E. coli., the same amount of essential oil of S. lanata. did not show any effect on E. coli. strains and E. aerogenes.. Twenty-five micro liters of essential oil had an inhibition effect on all Gram-negative organisms. Both oils (10 and 25 µl) had an important effect on all Gram-positive bacteria used in the study. A 25-µl amount of essential oils was greatly effective for M. luteus, B. subtilis., and B. cereus.. The strains S. aureus. MU 38 and CoNS S. epidermidis. MU 30, which are resistant to various antibiotics and carry plasmid, were highly effected by different amounts of both essential oils, especially by 25 µl. In Table 3, the sensitivities of studied organisms to the various antibiotics are given.

Table 1.. Antimicrobial activity of essential oils of Sideritis curvidens. and Sideritis lanata. against Gram-negative bacteria.

	Inhibitory zones (mm)
	Sideritis curvidens.			Sideritis lanata.
Microorganisms	5 µl/disk	10 µl/disk	25 µl/disk	5 µl/disk	10 µl/disk	25 µl/disk
E. coli. ATCC 11230	–	–	12	–	–	12
E. coli. ATCC 35218	–	–	11	–	–	12
E. aerogenes. RSKK 720	–	10	15	–	–	16
S. sonnei. RSKK 878	–	11	14	–	12	14
S. typhimurium. CCM 5445	–	11	13	–	10	14
P. aeruginosa. ATCC 27853	–	8	12	–	10	13

–:No inhibition.

Table 2.. Antimicrobial activity of essential oils of Sideritis curvidens. and Sideritis lanata. against Gram-positive bacteria.

	Inhibitory zones (mm)
	Sideritis curvidens.			Sideritis lanata.
Microorganisms	5 µl/disk	10 µl/disk	25 µl/disk	5 µl/disk	10 µl/disk	25 µl/disk
S. mutans. CNCTC 8/77	8	17	22	9	18	23
S. aureus. (MU 38)	8	14	20	9	16	20
S. epidermidis. (MU 30)	–	16	23	10	16	22
S. aureus. ATCC 6538 P	–	15	21	9	14	21
M. luteus. NRRL B-4375	12	16	38	11	18	38
B. subtilis. ATCC 6633	8	14	30	–	15	35
B. cereus. RSKK 863	9	12	27	10	12	31

–:No inhibition.

Table 3.. Sensitivity of microorganisms to control antibiotics (inhibitory zone diameter, mm).

	Antibiotics
Microorganisms	CFR	VA	SAM	TE	C	S
E. coli. ATCC 11230	12	NT	10	32	8	11
E. coli. ATCC 35218	15	NT	–	27	–	–
E. aerogenes. RSKK 720	15	17	16	28	20	21
S. sonnei. RSKK 878	20	11	17	34	18	16
S. typhimurium. CCM 5445	19	10	15	20	21	19
P. aeruginosa. ATCC 27853	15	NT	16	26	19	14
S. mutans. CNCTC 8/77	14	11	17	14	19	NT
S. aureus. (MU 38)	21	11	12	8	21	17
S. epidermidis. (MU 30)	15	12	14	12	13	17
S. aureus. ATCC 6538 P	16	16	13	17	18	19
M. luteus. NRRL B-4375	22	25	17	28	23	24
B. subtilis. ATCC 6633	25	22	11	27	20	19
B. cereus. RSKK 863	27	16	10	30	25	16

CFR, cefadroxil (30 µ g, Oxoid); C, chloramphenicol (30 µg, Oxoid); TE, tetracycline (30 µ g, Oxoid); S, streptomycin (10 µ g, Oxoid); SAM, sulbactam + ampicillin (10 µ g/10 µ g, Oxoid); VA, vancomycin (30 µ g, Oxoid); NT, not tested; –, no inhibition.

Discussion

No studies on antimicrobial activities of essential oils of these two Sideritis. species have been found in the literature, but the antibacterial activity of essential oils belonging to other species of the Sideritis. genus has been reported in many studies (Gergis et al., 19901991; Rodriguez-Linde et al., 1994; Ezer & Abbasoğlu, 1996; Sökmen et al., 1999; Aboutabl et al., 2002; Hernandez-Perez & Rabanal, 2002). Although there are some studies related to the chemical compositions of essential oils of many Sideritis. spp., it was found that these two species were not studied (Ezer et al., 1995; Kirimer et al., 1999).

In the E. coli. strains where the lowest effect was observed in Gram-negative bacteria, it was found that 25 µl of the essential oils of S. curvidens. and S. lanata. had a greater inhibitory effect than sulbactam + ampicillin, chloramphenicol, and streptomycin antibiotics. The other Gram-negative bacteria were affected by 10 and 25 µl of essential oils, except E. aerogenes., with a 10 µl essential oil of S. lanata.. But these effects were not significant.

When the results were taken into general consideration, it was seen that the antimicrobial effect was lower in Gram-negative bacteria (Tables 1 and 2). In another study on 22 endemic Sideritis. spp. collected in Spain, Rodriguez et al. (1994) reported that no higher antimicrobial activity was observed against Gram-negative bacteria. Gergis et al. (1990) in Greece reported that essential oils extracted from S. sipylea., S. euboea., S. clandestina. subsp. cyllenea., and S. clandestina. subsp. clandestina. were less effective for Gram-negative bacteria than the gram-positive ones. Hernandez-Perez and Rabanal (2002) suggested that the pure ethanol and chloroform extracts of Sideritis canariensis. var. pannosa. had no significant antimicrobial activity against Gram-negative bacteria.

It was determined in this study that essential oils showed higher antimicrobial activity against Gram-positive bacteria. This effect was found to be striking especially on M. luteus, B. cereus., and B. subtilis.. The inhibition effects of control antibiotics in the study were found lower than essential oils except for their tetracycline effect against B. cereus.. In previous antimicrobial activity studies with the pure ethanol and chloroform extracts of Sideritis canariensis. var. pannosa., Hernandez-Perez and Rabanal (2002) reported that they found antimicrobial activity in S. aureus, B. cereus., and B. subtilis.. Gergis et al. (1991) indicated that the essential oils of Sideritis. spp. showed high antimicrobial activity against S. aureus, B. cereus, B. subtilis, M. luteus., and S. mutans. was influenced by essential oils of S. curvidens. and S. lanata. at the same level. These oils (25 µl) were found more effective than antibiotics for S. mutans..

MRSA is an emerging cause of failure of antimicrobial treatments. In the hospital environment, MRSA resistant to a variety of classes of agents other than β-lactams are responsible for many life-threatening infections (Cormican & Jones, 1996). Alternative phytotherapeutic compounds against MRSA might become available as a result of research on new targets for antimicrobials for which no inhibitors are currently in use, preventing cross-resistance to already existing compound classes. In our study, It was also found that 25 µl amounts of both essential oils were more effective against S. aureus. MU 38 than vancomycin, sulbactam + ampicillin, tetracycline, and streptomycin antibiotics. So, this result was found to be very significant to evaluate the in vitro. activity of two essential oils and their bactericidal effect over time for MRSA isolates. In addition, this effect was observed higher than that of all test antibiotics used for reference S. aureus. ATCC 6538 P strain. In our experiments, 25 µl amounts of essential oils were found more effective than the antibiotics used against CoNS S. epidermidis. MU 30, which is clinically important in this study. CoNS have become increasingly recognized as important agents of nosocomial infection (Pfaller & Herwaldt, 1988; Archer, 1990). One of the characteristics of CoNS is their resistance to multiple antimicrobial agents, including methicillin and other drugs commonly used for the treatment of staphylococcal infections (Archer & Climo, 1994; Woodford et al., 1995). For this reason, these results were found relevant for treatment of nosocomial infections.

In conclusion, essential oils of S. curvidens. Stapf. and S. lanata. L., selected based on relevant ethnomedical use, has shown strong activity against bacteria, especially Gram-positive, including those resistant to various antibiotics. The results of this study support the traditional use of these plants. The clinical strains in the species of Staphylococcus. were found to be sensitive to S. curvidens. and S. lanata. essential oils. Therefore, these results may suggest that essential oils of S. curvidens. and S. lanata. possess compounds with antimicrobial properties that can be used as antimicrobial agents in new drugs for therapy of infectious diseases in human beings.