Antimicrobial Effect of Zataria Essential Oil on the Skin Bacteria in Wistar Rats

Objective Zataria is one of the native plants of Iran which is widely used for the treatment of diseases among Iranians. In this study, we investigated the antimicrobial effects of Zataria essential oil on the skin bacteria in rats. Methods Bacterial strains were isolated from the skin of 6 wistar rats and the antimicrobial effects of Zataria essential oil were evaluated by disk diffusion and microbroth dilution methods. In-vivo tests were performed to evaluate the antimicrobial effect of the essential oil by microbial culture as well as allergy tests on the skin of experimental rats compared to controls. Results Three bacterial strains were isolated from the skin of rats identified as Staphylococcus aureus, Corynebacterium and Staphylococcus epidermidis. Minimum Growth Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) for the two strains of Staphylococcus aureus and Corynebacterium were obtained 0.39 and 0.78 mg/ml, while for Staphylococcus epidermidis, they were 0.195 and 0.39 mg/ml, respectively. In-vivo test results showed the antibacterial effect of the essential oil on the skin bacteria and no inflammatory effects were observed under the allergy test. Conclusion Zataria essential oil has antimicrobial effects on the skin infections in lower concentrations. The use of this essential oil as an antiseptic and preservative in cosmetics is recommended instead of chemical preservatives that generally have skin side effects.


Introduction
n cosmetics, preservatives such as parabens, benzyl alcohol, salicylic acid, alcohol sterols and formaldehyde are used to inhibit the growth of bacteria and fungi. Microbial contamination may be present in non-standard cosmetics from the beginning and may be transmitted to the consumer during production and pack-I aging or during consumption and improper storage. The percentage of preservatives in the final product is usually between 0.01% and 5%. These chemicals can cause glandular and hormonal disorders. With the continuation of consumption, the possibility of their carcinogenicity also increases. Recent studies have emphasized the low efficacy of these substances. In this regard, the investigation for herbal ingredients as a preservative in cosmetics has recently become a priority. Zataria is one of the native plants of Iran and its consumption for treatment of diseases is common among Iranians. In this study, the effect of Zataria essential oil on skin bacteria was evaluated to examine its applicability to cosmetics.

Materials and Methods
In this study, 6 adult Wistar rats were used. The rats were first temporarily anesthetized by chloroform and then were given an intraperitoneal injection of Ketamine/Xylazine (2:1 dose ratio) to increase the anesthesia time proportional to their weight. The hair on the back of the rats was shaved and disinfected after anesthesia. This place was contaminated with three isolated bacteria including corynebacterium, staphylococcus epidermidis and staphylococcus aureus using a sterile swab with 0.5 McFarland standard concentration. In the three tested rats, 120 minutes after the skin was impregnated with the three bacteria, the essential oil with Minimum Bactericidal Concentration (MBC) was sprayed to the microbial site. After 60 minutes, the spraying was repeated. At the end, 60 minutes after the second time of spraying, the skin of experimental rats impregnated with the microbe and treated with the essential oil, was sampled and cultured on the nutrient agar medium. Then, the colonies that had grown in this medium were laminated and compared with the lams of the previous stage. Moreover, after 30, 60 and 90 minutes of spraying the essential oil on the skin of rats in the experimental group, their skin reactions were examined.
Bacterial strains were isolated from the skin and the antimicrobial effects of Zataria essential oil were evaluated by disc diffusion and microbroth dilution methods. For isolation and identification of skin bacteria, the back of the rat's hand was washed with soap and dried. A sterile swab moistened with distilled water was applied to the back of the hand, and grass cultivation was performed on the Mueller-Hinton Agar. By conventional microbiological methods including cultivation in different mediums and colonial and staining studies, isolated bacteria were identified. For disk diffusion method, blank discs were placed at appropriate distances on the cultured medium and 20 μl of diluted Zataria essential oil was poured on each disc.
After 24 hours of incubation, the diameters of the zone of inhibitions were measured. Minimum Inhibitory Concentration (MIC) was determined in sterile 96-well microplates. First, 100 μl of culture media containing bacteria were poured into the all wells. Then, 100 μl of antimicrobial solution was added to the first well. From the second to the third well until the tenth well, 100 μl of solution was transferred each time. Next, 100 μl of pure microbial suspension was added to the 11th well and 100 μl of the antimicrobial solution to the 12th well. The turbidity which indicates the growth or non-growth of the bacterium, was measured by a microplate (Synergy HTX, BioTek Instruments, China) at a wavelength of 545 nm for all three bacteria. The concentration of the last non-growth well was recorded as MIC. The well concentrations were arranged in descending order, from 50 to 0.097 μl/ml. In-Vivo experiments were performed to evaluate the antimicrobial properties of microbial culture and its allergenicity on the skin of 6 rats compared with control mice.

Results
Three bacteria isolated from the skin were identified as staphylococcus aureus, corynebacterium, and staphylococcus epidermidis. The MIC and MBC for the two strains of Staphylococcus aureus and Corynebacterium were obtained 0.39 and 0.78 mg/ml, respectively and for Staphylococcus epidermidis, they were 0.195 and 0.39 mg /ml. The results of the MicroBroth dilution showed that Zataria essential oil in very low concentrations was able to inhibit the growth of all three bacteria. It should be noted that Staphylococcus aureus and Corynebacterium had higher MIC than Staphylococcus epidermidis. This means that the essential oil has inhibited the growth of Staphylococcus epidermidis with greater strength and speed. In-vivo test results showed the antibacterial effect of Zataria essential oil on the skin of rats, and no allergic effects were observed in the allergy assessment. In cultures of samples taken from the skin of control rats, all three bacteria grew during culture, but in the cultures of samples taken from the skin of experimental rats, none of them grew, which indicates that they were killed by Zataria essential oil. After 30, 60 and 90 minutes of treatment with Zataria essential oil, no allergic reaction caused by sensitivity to this essential oil was observed in the skin of experimental rats.

Conclusion
The Zataria essential oil in low concentrations has longterm antimicrobial effects on the infectious bacteria.

Compliance with ethical guidelines
This study was conducted with the tracking code of: 2545562 and registered in the national committee of ethics in medical researchs: (Code: IR.PNU.REC.1396,6).

Funding
The present paper was extracted from the master thesis of the first author Department of Biology, Payame-Noor University.