EVALUATION OF THE ANTIBACTERIAL ACTIVITY OF ESSENTIAL OILS AGAINST E. COLI ISOLATED FROM RABBITS

The antibacterial activity of essential oils extracted from Origanum compactum, Thymus capitatus, Foeniculum vulgare, and Rosmarinus officinalis was assessed with the well diffusion method and a microbroth dilution assay against E. coli isolated from the carcasses of rabbits. The chemical composition of these essential oils was also determined by gas chromatography coupled with mass spectrometry (GC/MS). The results of this study indicate that essential oils with high phenol content exert a strong antibacterial activity against E. coli. Essential oils of Origanum compactum and Thymus capitatus containing high amounts of the monoterpenoid phenols thymol and carvacrol (68.99% and 95.25% carvacrol composition, respectively) were particularly effective against E. coli with low values of MIC = 0.3125% v/v and MBC = 0.625% v/v to report. The essential oil of Foeniculum vulgare also possessed moderate antibacterial activity (MIC = 50 % v/v) with a non-bactericidal effect, while the essential oil of Rosmarinus officinalis was ineffective at the concentrations tested.


INTRODUCTION
Rabbit colibacillosis is an infectious disease of rabbit caused by an infection of pathogenic Escherichia coli. This disease affects animals that have just been weaned resulting in severe diarrhea after infection including some cases that exhibit phases of mucoid diarrhea (24). Death can sometimes precede these symptoms. Antibiotics are often employed by rabbit breeders to treat and prevent the disease, however this approach can be problematic for human health. While antibiotics are capable of eliminating susceptible bacteria, this can have the unintended effect of promoting the proliferation of resistant strains in bacteria and lead to the greater occurrence of infections that are impossible to treat with conventional antibiotics (36). Each year, antibiotics-resistant strains of bacteria cause the death of 25,000 people in Europe and 19,000 in the United States (7). The essential oils of plants have been traditionally used for medicinal purposes and have proven their efficacy in several areas, which suggests one promising alternative to the routine use of antibiotics in animal agriculture. In order to better understand the ability of plants to control infections in rabbit breeding, the essential oils of four medicinal and aromatic plants recognized for their digestive and antispasmodic properties (2 , 9 , `12 , 23) were tested for their bacteriostatic and bactericidal qualities against E. coli isolated from the carcasses of dead rabbits. Plants that were featured in this study include: Foeniculum Vugaris, Origanum compactum, Rosmarinus officinalis, and Thymus capitatus. Essential oils were extracted from the seeds and leaves of Foeniculum Vugaris independently.

MATERIALS AND METHODS Plant and essential oil
Large quantity of seeds from the plant Foeniculum vulgare were purchased while leaves of the plant were collected, cleaned and then dried without light in at room temperature.
Specimens of Origanum compactum, Rosmarinus officinalis, and Thymus capitatus were collected in northern Morocco during the month of June. Species identification was done by Professor Bakkali, a specialist in botany, in the laboratory of ERGB. The essential oils (EO) of each plant were extracted via steam distillation for 3 hours using a Clevenger-type apparatus. Essential oil yield was determined as a percentage of the weight of the dry plant matter. Chemical analysis of essential oils was done by gas chromatography coupled with mass spectrometry (GC/MS). Isolation of E. coli bacteria Specimens were collected from the carcasses of rabbits that showed previous signs of diarrhea and bloating. Specimens from the intestinal mucosa and cecum along with their liquid contents were put into a ringer solution. Culture and isolation of E. coli was done on MacConkey agar. Petri dishes containing the culture medium were inoculated in depth and incubated at 37 °c for 24 hours. Miniature biochemical tests can conveniently and simultaneously performed on a colony for E. coli identification using an API 20 e gallery. Antibacterial activity A preliminary assay was performed with the agar diffusion method to compare the antibacterial effects of the essential oils against the performance of the antibiotic Oxytetracycline. The diameters of the resulting inhibition zones were measured in centimeters, including the diameter of the well. The results are expressed as an average by three determinations (+/-) standard deviation. The minimum inhibitory concentration (MIC) is defined as the smallest essential oil concentration capable of producing a total inhibition of growth after an incubation period of 24 to 48 hours (30). The minimum bactericidal concentration (MBC) is defined as the minimum bactericidal concentration of the oil capable of killing the inoculum. The MIC 1 and MBC 2 values were determined by microbroth dilution assay using resazurin as an indicator (21) of bacterial growth.

RESULTS AND DISCUSSION
The yield of each essential oil extracted are summarized in Table 1. The species of plant in order of percentage yield are: Origanum Compactum, seeds of Foeniculum vulgare, Thymus capitatus, leaves of Foeniculum vulgare, and Rosmarinus officinalis. The chemical composition of essential oils is very complex and diversified. An understanding of their constituents is very important in evaluating their properties and predicting their potential toxicity. The chemical composition of the essential oils of Foeniculum vulgare, Origanum compactum, Thymus capitatus and Rosmarinus officinalis are shown in Table 2. consists mainly of 1-8 cineol (51,62%), αpinene (18,94%), and α-Campholène aldehyde (10,65%). The results of the antibacterial activity of essential oils against E. coli are shows in Table 3. Table 3

. Diameter of inhibition zones of essential oil against E.coli (cm)
E. coli showed resistance to oxytetracycline with an inhibition diameter of 0,6 cm, while the essential oil of the Thymus capitatus showed high antibacterial activity (5,1cm), followed by the essential oil of Origanum compatum (4,2 cm). Rosmarinus officinalis essential oil appears to be ineffective towards this strain (0,6 cm). Low activity was observed with the essential oils of the leaves and seeds of the Foeniculum vulgare (1,1 and 1,4 cm successively). The combination of the two essential oils Thymus capitatus and Foeniculum vulgare seed was found to be more effective against E. coli (2,1 cm) than the antibiotic oxytetracycline (0,6 cm).
The minimum inhibitory concentrations (MIC) and bactericidal (MBC) of essential oils against E. coli are grouped in table 4.  (13) found a low percentage in carvacrol (13,4%) and high in P-Cymene (18,9%) for the essential oil of Thymus capitatus (harvested in February), the harvest period does not only influence the yield but also the Composition. The essential oil of the Algerian Thymus capitatus consists of carvacrol (55%) and γterpinene (11%) ( 34) and that of Tunisia is also composed by carvacrol (70%), accompanied by other constituents βcaryophyllene (8.5%), γ-terpinen (4.3%) and P-cymen (3.8%) (26). Whereas Turkish Capitatus Coridothymus is characterized by an average content of carvacrol (35,6%) and Pcymene (21%) and a moderate proportion in thymol (18.6%) and γ-terpinene (12.3%) (15 (26) in this essential oil is 1-8 cineol (44,2%), camphor (12%) and α-pinene (11,6%). These results are similar to our results. The antibacterial activity of essential oils has been demonstrated by several studies (17 , 28), including that of Origanum compatum and Thymus capitatus (5 , 8), Foeniculum vulgare, and Rosmarinus officinalis (18,19,25). However, in the literature few studies address the effect of essential oils on pathogenic bacteria isolated from farmed animals. The low antibacterial power of the essential oil of Rosmarinus officinalis against E. coli 011 compared with that of Origanum Compatum has already been reported by Mathlouthi et al.(22). The presence of Monoterpene phenols in the two essential oils of Thymus capitatus and Origanum Compactum seems to be responsible for the important antibacterial activity demonstrated in our work by these two oils. Carvacrol and thymol have proven antibacterial power against a wide range of bacteria (20, 36).The essential oil of Rosmarinus officinalis was ineffective against our isolated bacterium despite its capacity in oxygenated monoterpenes (51%). Indeed, Ait-Ouazzou et al., (1) studied the antimicrobial effect of 11 major constituents of essential oils and found that the 1.8 cineole had moderate activity compared with other oxygenated monoterpèniques compounds such as carvacrol, thymol, and linalol. The low antibacterial activity recorded by essential oils of seeds and leaves of Foeniculum vulgare has already been demonstrated by the study of Grigore et al., (16). According to this study, the essential oil of Foeniculum vulgare (80% d'anéthol and 13% de limonène) has a low antibacterial activity compared to that of Thymus vulgare. The De et al. Team (9) found that the anethole isolated from anise is responsible for the antibacterial power of this plant. Phenylpropanoids have a lower antibacterial coefficient than terpene phenols. The low presence of oxygenated monoterpenes such as fenchone and limonene, respectively, in the essential oil of seeds and leaves, may also be responsible for their low antibacterial activity (35). The complex composition of essential oils with all its majority and minority products offers this antibacterial power. Some studies have shown that the mixture of the majority constituents of the essential oil has a low antibacterial activity compared to the whole essential oil (27) which shows that the effect of the compounds quantitatively minority is sometimes not negligible and this supports the presence of additive or synergistic effect of all the compounds of the essential oil. Mathlouthi et al., (22) showed that the bacterial activity of the essential oil of Rosmarinus officinalis against E. coli 011 is moderate (MIC = 4, 4mg/ml) compared with that of Origanum compatum (MIC = 0.9 mg/ml). According to Sienkiewicz et al., 2013, the essential oil of Rosmarinus officinalis has antibacterial activity against clinical strains of E. coli isolated from the human abdominal cavity (MIC = 18 Μ L/ml). In the present study, the potency of the antibacterial action of essential oils varies according to the chemical profile of their majority constituents. The modes of action of essential oils and their main constituents described so far, all seem to affect the wall or cytoplasmic membrane. Indeed, the attack of the bacterial wall by the essential oil and the damage of the plasma membrane causes an increase in permeability, a loss of the cellular constituents and a coagulation of the cytoplasmic content (4). The inhibition of the resultant proton motor force and the alteration of the membrane proteins block the production of the cell energy resulting in the death of the bacterium .In fact, the chemical variability of essential oils suggests the existence of molecules that can act by new cellular mechanisms.

Conclusion
In this work, a relationship between the biochemical families of the active constituents of essential oils and their antibacterial powers has been revealed. Indeed, the essential oils of Origanum compactum and Thymus capitatus rich in terpene phenols (thymol, carvacrol) demonstrated great antibacterial power, followed by Foeniculum vulgare oil rich in phenols phenylpropanoidic (anethole) which revealed Moderate antibacterial activity. The lowest activity is recorded by Rosmarinus officinalis oil rich in terpene oxides (1.8 cineole). The essential oil of oregano, thyme, and fennel can be suggested as phytobiotics to prevent, treat colibacillosis and reduce the mortality of rabbit.