Antibacterial power of Olea Europaea extracts from different Moroccan regions

. The discovery of antibiotics has always been a medical advance that has improved the prognosis of infections. However, resistance to these products has evolved into a major health problem. Some diseases are even resistant to all antibiotics currently available on the market. A reduction in the use of antibiotics must be based on other approaches, namely the use of alternative treatments to antibiotics, such as phytotherapy. In the same objective our study focused on the research of the antibacterial power of Olea Europaea known for its various medicinal properties. 31 bacterial strains were tested for their susceptibility using the agar diffusion method, and we determined their MIC by the micro titration technique on microplates. Five bacteria belonged to the American collection (ATCC) and 26 strains isolated from nosocomial infections between the period of 2011 and 2015. We noticed that our aqueous, methanolic, ethanolic and ethyl acetate extracts are active against all tested bacteria. On the other hand, we observed that the hexane and dichloromethane extracts showed no inhibition effect on all the bacteria tested. The methanolic extract showed a higher MIC against ESBL enterobacteria (E. coli, E. cloacae, P. mirabilis) and imipenem-resistant A. baumanii. The lowest MIC was 1.56 ug / ml.


Introduction
Hospital infections are a major public health problem worldwide, including Morocco [1]. The use of drugs is extremely important for the treatment of these infections, but it is also subject to an important selection pressure that affects many bacteria and the development of resistance to the most prescribed antibiotics. The use of new and more durable antibiotics is the most effective solution to this problem [2]. Plant extracts and essential oils have been a natural source of antimicrobial mixtures or pure compounds for centuries [3]. As for Olea europaea (Olive), it is a most important fruit tree. It is native to the Mediterranean region such as Palestine, Syria, Spain, Italy, Greece, France, Turkey, Algeria and Morocco [4, 5]. In addition, the leaf is a main site of plant metabolism best known at the level of primary and secondary plant products and can be considered as a potential source of bioactive compounds [7]. Antimicrobial activity was evaluated by measuring the zone of inhibition against the tested microorganism.

Minimum inhibitory concentration (MIC)
The determination of the MIC of plant extracts against microbial strains was performed according to the microtitration technique described by Eloff [8].

Minimum bactericidal concentration (MBC)
To assess the MBC, 100 μL of each sample, in which no microbial growth was observed, was spread in Muller-Hinton agar (MH). The plates were incubated at the appropriate temperature for 24 h. BMC was defined as the lowest concentration at which bacterial growth was completely inhibited [9-10].

Diameter of inhibition
During the screening of the antibacterial activity of the different extracts of olea europeae, the inhibition diameters obtained are mentioned in the tables (2, and 3).

Minimum inhibitory concentration of extracts
The minimum inhibitory concentration of our extracts was performed in microplates, the results obtained are shown in tables (3 and 4) for ethanolic, dichloromethane, hexanolic, and ethyl acetate extracts.

The minimum bactericidal concentration of the extracts
When the test was applied to determine the minimum bactericidal concentration and to number the surviving bacteria, it was suggested that for all the

Discussion
In this study, we worked on the different ATCC strains, among which we find Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853; they served as test microorganisms (quality control) [11].
The results of antibacterial activity of aqueous and methanolic extracts of Olea Europaea leaves from different regions are presented in Table 3.

It was noticed that both extracts had an
inhibitory effect on all the tested organisms: but Gram-positive bacteria are more sensitive to the extracts than Gram-negative bacteria.

The aqueous extract of leaves from Meknes is the most active; it has a strong activity against all tested bacteria, with a maximum inhibitory zone for Methicillin-resistant S. aureus (MRSA) (20 mm) and a minimum inhibitory zone for E. Coli (8) with a diameter of (11 mm).
It is also active against ESBL bacteria: E. coli (1 and 3) and P. aeruginosa (1) with inhibition zones (16 mm and 14 mm respectively). It is effective against imipenem-resistant A. baumannii (18 mm) which are pathogenic germs of nosocomial origin.
The methanolic extract also showed activity against the different microorganisms tested, it has the highest inhibitory potential on imipenem-resistant A. baumanii (4) and multiresistant S.aureus (2) with inhibitory zones (16 mm and 15 mm respectively). It is effective against P. mirabilis (2) ESBL and P. aeruginosa (1) ESBL by 12.25 mm and 11.5 mm respectively. Table 5. The observed MIC from the aqueous and methanolic leaf extract is 12.5-50 μg / ml and 1.56-12.5 μg / ml respectively, thus showing that the plant extracts are effective on the tested bacteria. Table 4 and 5, we reported that the dichloromethane and hexane extracts of olive leaves studied in this work shows no inhibitory effect on all tested bacteria.

From the analysis of the results recorded in
The ethanolic extract is active against all tested bacteria by a maximum inhibition zone for staphylococcus aureus ATCC (17 mm) which was shown by Meknes extract, while the minimum inhibition zone is reported for klebsiella pneumoniae (1) (9 mm). It is also active against ESBL bacteria: Proteus Mirabilis (2) and Pseudomonas aeruginosa (1) with inhibition zones (15 mm and 13 mm respectively) presented by the extract of fquih ben salah.
The MIC observed from the ethanolic extracts of the leaves is between 0.78 and 12.5 μg / ml, showing that our olive leaf extracts are effective on the bacteria tested with a small dose.

On the other hand, ethyl acetate extract is active against all the bacteria tested by a maximum inhibition zone observed against Citrobacter Freundii ATCC 8090 (30 mm), this zone is presented by the extract of Meknes, while the minimum inhibition zone is against Escherichia coli (5) (10 mm) presented by the extract of Ouezzane
Ethyl acetate extract is also active against ESBL bacteria: klebsiella pneumoniae (2) and Proteus Mirabilis (2) by one of inhibition (20 mm) for both bacteria, and which is presented by Meknes extract. In addition, it has a high activity against Acinetobacter Baumannii (1) and (4) resistant to Imipenem with a zone of inhibition for both bacteria (25 mm and 22 mm respectively).
The MIC observed from ethyl acetate leaf extracts is 0.78 to 12.5 μg / ml. For all the strains tested, bactericidal activity was not observed for any extract used. Finally, we can say that all our extracts are bacteriostatic.
The results obtained in this study indicated the antibacterial efficacy of aqueous and methanolic extracts of O. Europaea leaves on tested isolates. A major challenge of using water for extraction is that non-polar bioactive compounds cannot be extracted [12]. Morteza Azizollahi reported that the aqueous extract of olive leaves showed good antibacterial capabilities and maximum inhibition of 11.5 mm against Salmonella typhimurium PTCC 1639 [13].

Peter Masoko and David M. Makgapeetja
showed that out of nine solvents used, methanol was the excellent extractant, as it carried out a larger amount of plant material than the other solvents used [11].
In addition, the minimum inhibitory concentrations observed from the methanolic extract of the leaves are between 1.56 and 12.5 and between 12.5 and 50 mg / ml for the aqueous extract. The results of our study have a similarity between several other studies [15], [16].
The bactericidal effect (minimum bactericidal concentration) of aqueous and methanolic extracts were not detected in our work; this result is similar to a result presented in the study conducted by Daoud Ziad et al [14].
In our study, Staphylococcus aureus was found to be the most sensitive microorganism, presenting a maximum zone of inhibition (20 mm).
The successful inhibition of this bacterium and its contemporary etiology of gastroenteritis is a good development, especially when considering the appearance of its resistance to various conventional antibiotics [17], [18] [19]. This is consistent with previous studies reporting that the spectrum of antibacterial activity varied depending on the type of extract and the Gram of the bacteria. Nevertheless, Gram-positive bacteria are globally the most sensitive to the effects of these polyphenolic extracts. This is in agreement with a previous study indicating that the highest antibacterial activity against ESBL-producing bacteria was mainly manifested by Olea europaea [14].

This generally higher resistance in Gram
The MIC observed from ethanolic extracts is 0.78 to 12.5 μg / ml, which shows that these extracts have a very remarkable effectiveness on all these bacteria. Daoud Ziad postulated that the results showed significant inhibition with MICs ranging from 3.125 to 6.250 mg / ml [14].
It could be said that the nature and composition of the solvent, the ratio between the volume and mass of the solvent, the pressure and temperature of extraction, the number of extraction cycles and the duration of each cycle are factors influencing the efficiency of the extraction. The majority of pressurized liquid extraction applications reported in the literature use organic solvents, specifically ethanol and water [24]. Some researchers explained that oleuropein, which is included in these products, has many pharmacological properties, including antioxidant, antimicrobial, antiviral activities... They also mentioned that the zones of inhibition that have a diameter of less than 12 mm were considered to have low antibacterial activity. The diameters included between 12 and 16 mm were considered moderately active and those above 16 mm were reported as highly active [15].

In relation to this situation, our ethyl acetate extract from Meknes is very active towards the majority of the tested bacteria
This result correlates with the study conducted by Daoud Ziad et al, they showed that ethyl acetate extracts of all the selected plants, showed a very potent antibacterial activity against Escherichia coli producing extended spectrum betalactamase and Klebsiella pneumoniae [14]. Furthermore, Altaf Hussain et al confirmed that the maximum activities were found for methanolic extracts in both plants against both types of bacterial strains. This could be due to the good extraction efficiency of methanol compared to other solvents, as it allows the extraction of whole phenolic compounds. The second and third effective solvents were ethanol and water, respectively.
Other extracts of (ethyl acetate, n-hexane, chloroform, and diethyl ether) had no significant difference regarding activities against the selected bacterial strains [25] In the two previous studies, these results can be explained by the fact that, like many natural products, the variation in effectiveness due to differences such as geographical location, plant nutrition. It can be suggested that the collection site affects the antimicrobial activity in relation to the soil composition.
The results of our study, have a similarity between other studies regarding the antibacterial activity of olive leaf extracts [26], [27], [28].

Conclusion
The data obtained in this study reveal that the use of olive leaves can reduce the risk of bacterial infections, especially in the intestinal and respiratory tracts. The observed antibacterial effects of these medicinal plants on the microorganisms used, although in-vitro, seem interesting and promising and may be effective as a potential source of new antibacterial drugs. Further research is needed to obtain information regarding the practical effectiveness of Olea europaea L. extracts in inhibiting the growth of a broad spectrum of bacteria under specific application conditions. applications.