Evaluation of the antibacterial activity of three essential oils extracted from plants used in traditional medicine in Algeria (Salvia officinalis L, Melissa officinalis L and Origanum vulgare L)

This study consists in highlighting the antibacterial activity of three essential oils extracted from aromatic plants used in traditional medicine in Algeria which are: Salvia officinalis L, Melissa officinalis L and Origanum vulgare L, in order to demonstrate the therapeutic value, we evaluated their antibacterial activity using the antibiogram method. We previously extracted the essential oil from the flowering stems and dried leaves. This is done using a standard device. To mount the antibiogram, we used pathogenic germs resistant to many antibiotics: Enterococcus faecalis ATCC 29212, Staphylococcus aureus ATCC 43300, Escherichia coli ATCC 25922, Klebsiella pneumoniae carbapénemase positif (KPC+), Klebsiella pneumoniae carbapénemase négatif (KPC-), Enterobacter cloacea, Serratia marcescens, Acinétobacter baumanii, Pseudomonas aeruginosa ATCC 27853, Citrobacter freundi. The results that we have obtained indicate that the three raw and diluted essential oils exhibit significant antibacterial activity on pathogenic strains which are all very sensitive to raw and diluted essential oils. The three essential oils studied are precious natural bioactive substances that can be used to fight against infectious diseases.


Introduction
Phytotherapy continue to be a popular healthcare choice with the general public not only for health maintenance and wellbeing, minor ailments (e.g. coughs and colds), chronic conditions (e.g. back pain) and serious chronic diseases (e.g. asthma, cancer, depression, diabetes) [1]. Salvia officinalis is used for its tonic, choleretic and antiperspirant properties. Melissa officinalis is used as stomachics and antispasmodics. Origanum vulgare is used as a stomachic and antispasmodic [2]. For our study we have selected these spontaneous species used in traditional medicine in Algeria. These three species have essential oils rich in citral (Melissa officinalis), carvacrol (Salvia officinalis) and thyone (Origanum vulgare) [3]. The extraction of essential oils is carried out according to a standardized process, by the hydrodistillation method [4]. The purpose of our study is to assess the antimicrobial activity of the three essential oils against multidrug-resistant pathogens. The pathogenic strains we have chosen come from the microbiology laboratory at the University Hospital Center (C.H.U) in Annaba (a city in eastern Algeria). These strains are made up of: Enterococcus faecalis ATCC 29212, Staphylococcus aureus ATCC 43300, Escherichia coli ATCC 25922, Klebsiella pneumoniae carbapénemase positif (KPC+), Klebsiella pneumoniae carbapénemase négatif (KPC −), Enterobacter cloacea, Serratia marcescens, Acinétobacter baumanii, Pseudomonas aeruginosa ATCC 27853 and Citrobacter freundi. The method of studying the antibacterial activity that we have recommended is that of the antibiogram. This method makes it possible to evaluate the sensitivity of a bacterium with regard to antibiotics [5]. The results of the aromatogram show us that these three raw and diluted essential oils exhibit significant antibacterial activity on the pathogenic strains studied.

Vegetal material and extraction of essential oil
The flowering stems of Salvia officinalis, Melissa officinalis and Origanum vulgare were harvested in the Annaba region (northeastern Algeria). The extraction of the essential oils is carried out by the method of steam distillation, which uses a Likens Nickerson type extraction apparatus, from the selected flowering stems and dried. 100 grams of dried flower stems are introduced into a balloon distilled water impregnated until quarters.

Reactivation of bacterial strains
The bacterial strains are reactivated by subculture from the preservation medium on a solid and nonselective culture medium (nutrient agar) previously melted, poured into petri dishes 4 cm thick (these cans are cooled and dried before to be sown) for 24 hours of incubation in the oven. The petri dishes are then removed and the raw and young cultures previously prepared will be used for the preparation of bacterial suspensions.

Preparation of bacterial suspensions
The method of preparation of the inoculum is that recommended by the SFM (French Society of Microbiology) which consists of preparing, from an 18 to 24 hour culture of the bacterium studied on the agar medium. A suspension in saline solution (0.9% NaCl) equivalent to the standard Mac Farland 0.5 (~106 CFU / ml can be obtained by measuring the optical density (OD) ranging from 0.08 to 0.1 read at 625 nm.

Environment of culture
Mueller-Hinton agar prepared as follows: We introduced 35 g of agarose powder into one liter of water which was heated until completely dissolved. Then we sterilized the solution obtained by autoclaving at 121 ° C. for 15 minutes. We then poured the solution into Petri dishes 4 cm thick and cooled to 45˚C.

Seeding proper
The petri dishes are seeded from the bacterial suspensions using the sterile swabs. The swab is impregnated with the bacterial suspension, wrung out against the inner wall of the test tube. Seeding is by tight streaks, from top to bottom. The operation is repeated two to three times, turning the box 60˚ each time. The swab should be passed on the periphery of the agar plate.

Choice of solvent for diluting essential oils
The aprotic organic solvent (DMSO) is chosen for its innocuity against bacteria (lacking antibacterial activity). In the presence of the drug, it does not give interference (since the desired activity is that of the drug).

Preparation of the dilutions of the essential oils
A range of five dilutions has been prepared and the raw essential oil.
Going from ½, ¼, ⅛, ¹/16 from the raw essential oil by adding 0.5 ml of DMSO in the five tubes, 1ml of pure oil in one tube then take 0.5 ml of the tube of the Raw oil and pour it into the second tube and each time we take 0.5 ml of the previous tube and add to the next tube to the last tube where we throw the last 0, 5 ml so each tube contains 0.5 ml.

Disk distribution
Sterile discs of blotting paper 6 millimeters in diameter are grasped with sterile forceps and impregnated with a small amount of our essential oil previously prepared at different concentrations. The discs are applied to previously seeded culture media. A maximum of 6 disks is arranged per box of 8.5 centimeters in diameter, with a gap of 24 millimeters between the disks.

Reading
After 24 hours of incubation, the dishes are removed and the results are read by measuring the diameter, in millimeters, of the possible inhibition area around the discs.

Results and discussion
By comparing the results of the antibiogram with the critical values, we were able to classify the bacteria in the following categories: sensitive, intermediate and resistant. We measured in millimeters the diameter of the inhibition halo or clear area surrounding the discs. This clear area represents the destruction of pathogenic germs on which we have deposited our essential oils. This allows us to assess the degree of antibacterial activity of the three essential oils (Table 1). The antibiogram obtained from the essential oil of Salvia officinalis shows us that the majority of the germs tested are sensitive to the essential oil but their sensitivity differs from one germ to another but other germs are resistant. We give the case of Staphylococcus aureus ATCC 43300 and Escherichia coli ATCC 25922 which are very sensitive to raw oil and oil diluted to ½. We also give the case of Enterobacter cloacea which is moderately sensitive to raw oil; but it is resistant to diluted oil. Enterococcus faecalis ATCC 29212, Klebsiella pneumoniae, (KPC+) and Klebsiella pneumoniae (KPC −) ( Table 2).  (Table 3).
The majority of the strains tested are very sensitive to raw essential oil from Origanum vulgare and moderately sensitive to diluted oil. However, strains like Serratia marcescens, Acinétobacter baumanii and Pseudomonas aeruginosa ATCC 27853 are resistant to raw and diluted oil (Table 4). The areas of inhibition observed around these germs are large with diameters ranging from 35 mm to 44.8 mm. But the raw essential oils of Salvia officinalis and Melissa officinalis are weakly active against these germs since the areas of inhibition observed around these strains are insignificant with diameters ranging from 8.5 mm to 10.5 mm (Figure 1).

Figure 1
Antibacterial activity of raw three essential oil The essential oil diluted to ½ of Origanum vulgare is very active against most of the germs tested. The area of inhibition has a diameter which arrives at 38 mm. It is weakly active against Serratia marcescens Acinétobacter baumanii and Citrobacter freundii. The essential oils of Salvia officinalis and Melissa officinalis diluted to ½, lose their activity on most strains. But remain weakly active towards Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 43300 ( Figure 2).

Figure 2
Antibacterial activity of the three essential oils after dilution with ½ The essential oil of Origanum vulgare diluted to ¼ is very active against most of the strains tested. The inhibition area arrives at a diameter of 30 mm. On the other hand, it is inactive against Serratia marcescens, Acinétobacter baumanii and Pseudomonas aeruginosa ATCC 27853. The essential oils of Salvia officinalis and Melissa officinalis diluted to ¼ are inactive against all the strains tested ( Figure 3).

Figure 3
Antibacterial activity of the three essential oils after dilution with ¼ The essential oil of Origanum vulgare diluted in ⅛ is very active against Escherichia coli ATCC 25922 with a area of inhibition of 29 mm. However, it is not very active to inactive against other germs. The essential oils of Salvia officinalis and Melissa officinalis diluted in ⅛ are inactive against all the strains tested ( Figure 4).

Figure 4
Antibacterial activity of the three essential oils after dilution with 1/8 The essential oil of Origanum vulgare diluted to 1 /16 is weakly active against Escherichia coli ATCC 25922 and Enterobacter cloacea with a maximum inhibition area of 14 mm diameter. On the other hand, it is inactive against other germs, as is the case for the essential oils of Salvia officinalis and Melissa officinalis ( Figure 5).

Figure 5
Antibacterial activity of the three essential oils after dilution with 1/16 Marino and Bersani [6] obtained a similar result for the essential oil of Salvia officinalis which showed inhibition against the bacteria tested at low concentrations, the most sensitive was Escherichia coli and that Salvia officinalis has limited activity against these germs ». « Mimica-Dukic and Bozin [7] obtained a different result for the essential oil of Melissa officinalis which has an important activity against Pseudomonas aeruginosa and Escherichia coli which is particularly sensitive to this essential oil.

Conclusion
We have demonstrated that three essential oils obtained from aromatic plants have significant antibacterial activity on multi-resistant pathogenic germs. These tested germs are very sensitive to these three raw essential oils but their sensitivity is variable when these oils are diluted.