SCREENING AND EVALUATION OF ANTIMICROBIAL ACTIVITY OF THYMUS VULGARIS USING IN VIVO AND IN VITRO METHODS

1. Department of Microbiology, Faculty of Medicine, Rabigh, King Abdulaziz University, Saudi Arabia. 2. Department of Animal Health, Infectious Diseases Unit, Desert Research Center, Cairo, Egypt. 3. Department of Medicinal & Aromatic Plants, Desert Research Center, Cairo, Egypt. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History

In vivo assessment of antibacterial activity of thyme oil:-Forty-eight Swedish mice (Musmusculus) were divided into six groups of eight mice each as the following: Control (injected with 100 µl saline), S. aureus infected (injected with 100 µl of 1 × 10 8 colony-forming units (CFU) of S. aureus suspension), S. aureus infected plus antibiotic (Gentamycin, Sigma, USA) in dose 40 mg/kg/day, S. aureus infected plus 1 x MIC, S. aureus infected plus 2 x MIC and S. aureus infected plus 4 x MIC. To facilitate the infection, all treated groups were injected I/P with cyclophosphamide (Sigma, USA) (30mg/kg body weight) at 72 hours prior to infection. All groups were infected via subcutaneous injection. Five days later, the animals were sacrificed and spleen from individual animals was weighted and homogenized in phosphate-buffered saline (PBS), serially diluted 10-fold and streaked on Baird Parker plates (Oxoid, Basingstoke, Hampshire, UK), and incubated at 37°C for 24 hours to measure the bacterial load. The results were expressed as log (CFU/gm) of the spleen (Barquero-Calvoet al., 2013). Blood was collected and sera were prepared for estimation of hematological parameters and organ function tests, respectively.

Haematological and Biochemical assays:-
The collected blood was subjected to analysis by using automated blood analyzer (Abbott Labs Cell-Dyn 1800, USA) to determine the white blood cells count (WBCs), red blood corpuscles (RBCs), and haemoglobin (Hb) concentration. The above-prepared sera were subjected to estimate the following parameters using commercially available kits: alanine aminotransferase (ALT), total protein (TP), Albumin (Alb) (TECO DIAGNOSTICS, ANAHEIM, CA), urea and serum creatinine (BIOASSAY SYSTEM, HAYWARD, CA).
Histopathological assessment:-Liver and kidneys morphology were characterized by light microscopy using routine hematoxylin and eosin (H&E) staining. After sacrificing the mice, small pieces of liver and kidney were fixed in 10% formol-saline solution. The fixed tissues were embedded in paraffin wax, sectioned, and stained.

Statistical analysis:-
The obtained data were expressed as mean ± SEM. The means of different groups were compared by using one-way analysis of variance (ANOVA) with Turkey's post-hoc test. The statistical analysis was performed by using SPSS version 21.0 (Chicago, IL). A p-value of <0.05 was considered statistically significant.

Ethics:-
The work was done with the respect recommended by WHO*for the animal welfare.

Result:-
The chromatographic analysis of the thyme essential oil revealed that Thymol (45.15%) and ρ-cymene (25.31%) are the major constituents as illustrated in Table (1).  Table 2. The activity of the thyme oil as antimicrobial agent against S. aureus strain was determined by using disc diffusion and MIC techniques. It is obvious that the oil had a strong antibacterial activity particularly at the concentration 100% whereas the inhibition zone was 28 mm in diameter. As the MIC was calculated as the lowest concentration causing inhibition of the visible growth of tested organism, the MICin our study was 0.025%, as shown in Table 3.  ( 1 ), thyme essential oil confirmed to have in vivo antibacterial effect on tested S. aureus strain. Whereas there is no significant difference (P>0.05) between the result obtained from 2MIC and 4MIC treated groups with those of antibiotic treated group while 1MIC treated group showed lesser activity.

Effect of Thyme essential oil on hematologicalparameters:-
As it is shown in fig ( 2   Effect of thyme extract on liver and kidney function tests:-As illustrated in fig 3, concerning ALT, there was no significant difference (P > 0.05) between treated groups except in case of 4MIC group whereas it recorded the highest level. It was found that the infected group (INF) recorded the highest activity of the enzyme when compared with the other groups and followed by 4MIC group. Regarding serum creatinine, there was significant difference (P < 0.05) between the infected (INF) and the all treated groups. However, 2MIC group recorded the nearest enzyme activity when compared with the antibiotic treated (positive control) and normal (neutral) groups and followed by 4MIC group.   Almost the same trend of hepatic sections was shown in renal sections ( fig 5) where all treated groups resemble to large extent the control group especially 4MIC group while 1MIC and 2MIC groups showed moderate signs of inflammation in the form of congestion of the interstitial blood vessels and degeneration of some convoluted tubules whereas infected group displayed the highest vacuolar degeneration of renal tubules.

Discussion:-
Essential oils have many applications in veterinary medicine such as insecticidal, anti-parasitic and treatment of infectious diseases in animals. Studies deal with antimicrobial properties of herbal plants essential oils against microorganisms with veterinary importance in vitro and in vivo are as yet restricted. Essential oils were applied on a large scale, in spite of their mechanism of action was not well clear. Because of their antimicrobial activity, they were mainly used practically to treat infections (Tiwariet al., 2009; Bakkaliet al., 2008 and Burt 2004). From our foregoing findings on antimicrobial sensitivity testing of thyme essential oil extract against S. aureus, it was detected that the thyme essential oil has bactericidal activity with MIC 0.025% but their effectiveness varied according to the concentration (Table 3) In the present work, the antimicrobial activity of thyme essential oil may be attributed mainly to the high content of thymol (45.15%) and p-cymene (25.31%). Thymol had been established as bactericidal (Sikkemaet al., 1995) by a variety of mechanisms of action result in structural and functions modifications in the cytoplasmic membrane by affecting its permeability causing the release of K+ ions and ATP. This in turn leads to cytoplasm coagulation, the denaturation of many enzymes involved in various carbon and nitrogen sources catabolism as well as the loss of ions and metabolites and then cellular death (Nazzaroet al., 2013; Burt and Reinders., 2003;Xuet al., 2008;Walsh et al., 2003). Regarding p-cymene, it is a monoterpene, some previous work revealed that it has antimicrobial activity when it is used alone (Bagamboulaet al., 2004;Aligianniset al., 2001 andMann et al., 2000) and others had a different opinion when they described its antimicrobial effect as cofactor by increasing the antibacterial activity of other compounds such as, its derivative carvacrol (Rattanachaikunsoponet al., 2010 andUlteeet al., 2002). Pcymene does not affect the membrane permeability but accepts its antimicrobial activity as it has a high affinity for bacterial membranes causing them to expand and decreasing their melting temperature (Cristaniet al., 2007), and affecting the membrane potential of intact bacteria (Ulteeet al., 2002).
According to other studies (Sivropoulouet al., 1997; Carson andRiley., 1995 andSur et al., 1991) camphor, terpinen and 1, 8-cineole was principally responsible for the antibacterial activities of the plant essential oils. It is worth mentioning that the antimicrobial activities of essential oils are difficult to correlate to a specific compound due to their complexity and variability where their constituents concentrations may be affected by several factors (Nevaset al.,2004;Cakmakcıet al., 2009 andOsman et al., 2009) such as water stress, the seasonal condition, chemical polymorphism, the origin, the stage of the collected plant material, and the method of extraction where there is no an international standardized technique for the evaluation of essential oils allows the use of different protocols, hampering the comparison of the results (Smith-Palmer et al., 1998 and Viuda-Martoset al., 2008). So, we used the M31-A3 protocol (CLSI; 2008) in our work as it is the most recommended among the technique found in the literatures. What is more, the activity of the essential oil was superior to the compound alone (Santurioet al., 2014). Such finding is explained by the fact that the high antimicrobial activity showed by essential oils results from the synergism of the major components (Höferlet al., 2009)  It is obvious from the result of disk diffusion test that the used strain of S. aureus considered a multi-drug resistant bacterium. This resistant may be attributed to many factors; one of those is possessing penicillinase enzyme (Kumar et al., 2011) which renders the bacteria resistant to β-lactam antibiotics (MRSA). Other factors of antibiotic resistance in the bacteria may be back to the genetically presence of antibiotic resistance genes on its chromosomes or plasmids, or getting them via a variety of genetic means as transformation, conjugation, and transduction or selecting the bacteria containing resistance genes by unwise use of penicillin or other antibiotics. All these processes promote and enhance the presence of antibiotic resistance and pathogenic S. aureus strains (Kumar et al., 2011).
Depending on the established data provided by in vitro antibacterial test results, in vivo study was conducted with a view to confirming the therapeutic effectiveness of the extract. The present study results revealed that the administration of thyme essential oil in different doses inhibited the growth, and reduced the viable S. aureus numbers. This ability of inhibition may be attributed to the existence of phytochemical groups as aforementioned in Table 1. This result recommended that the antibacterial activity against S. aureus obtained in vitro was continued in vivo.
To check whether there were any statistically significance differences in the spleen bacterial load achieved by different treatments against S. aureus, two-way analysis of variance followed by Turkey`s test was applied. This analysis discovered significance variations (5% level) among the different treatments where the effect of this essential oil against S. aureus gave a significant (P<0.05) result as an antibacterial agent when comparing infected control groups with all treatments where 4MIC group was the best group in inhibiting the bacteria followed by 2MIC and 1MIC. This indicates that the thyme essential oil has inhibitory effect on S. aureus and the bacterial inhibition increased directly with the thyme essential oil concentration, as shown in Fig. 1 On the other hand, it must be taken into account that the highest thyme oil concentration (4MIC) had no bad effect on the hemoglobin concentration and RBCs count (Fig. 2) as well as the vital organs as liver and kidneys as indicated by the normal ALT and creatinine levels which commonly used as markers of liver and kidney degeneration (Olaleyeet al., 2014; Suganthiet al., 2013 and Bidieet al., 2010), respectively (Fig. 3). High level of this enzyme in the infected untreated group was backed to the infection. The hepatic damage was further supported by histopathological findings in liver and kidney sections ( Fig. 4 and 5). The hepatic sections of infected untreated groups revealed signs of inflammation in the form of dilatation and congestion of hepatoportal blood vessels with ballooning degeneration of hepatocytes. At doses of 1MIC, 2MIC and 4MIC liver and kidney sections revealed a significant difference (P<0.05) from the infected untreated group and showed the normal appearance similar to large extent the control group particularly in the case of 4MIC group. Therefore, the thyme essential oil at these doses not only overcome the infection but might correct the harmful effect of infection on hepatic and renal cells. Nonetheless, the major signs of hepatic and renal damage as necrosis and apoptosis (Eroschenko, 2000) were not detected in our study, the same findings were observed in another previous study (Talaet al., 2015).

Conclusion:-
The inclusive results of our study provide baseline data for the possible use of essential oils in the remedy of Staphylococcus aureus infection and can be considered the best alternative to the antimicrobial drugs in facing the drug resistance development. Beside the antimicrobial activity, the information recorded from using doubled doses showed that the thyme essential oil considered safe in the treatment of bacterial infection. This effect was dose dependent, therefore, there is need to conduct experiments on large scale and on large animals, both in vitro and in vivo, to establish the maximum safe level of this EO against bacteria and also, it is necessary to further investigate the interaction between the pathogenic bacteria and thyme essential oil and its impact on the development of resistance.