Antimicrobial activity of essential oils extracted from leaves of native Moroccan plants against clinical bacterial isolates

Introduction: Medicinal plants possess plentiful of bioactive molecules effective against multi-resistance bacteria. The aim of this study is to assess the in vitro antimicrobial activities of essential oils extracted from three Moroccan aromatic plants. Method: Analysis of essential oils of Origanum compactum, Rosmarinus officinalis and Pelargonium asperum, collected from different localities in Morocco was performed using a GC-MS spectrophotometry. Antibacterial activity by disc diffusion assay was evaluated in vitro against five clinical multi-resistant isolates. Results: Origanum showed strong antibacterial activity against tested strains except Pseudomonas aeruginosa while Rosmarinum showed a bactericidal effect against Acinetobacter baumanii, Escherichia coli and Staphylococcus aureus. Pelargonium presented only slight growth inhibition of Staphylococcus aureus on solid medium. Interestingly, fractions F7 and F8 of Pelargonium which represented only 0.3% and 0.1% respectively of the total mass were found to be bactericidal against Klebsiella pneumoniae and Pseudomonas aeruginosa. Conclusions: Ours results showed that the antimicrobial activities were variables depending on the chemical composition of essential oils, the fraction used and the microorganism tested. Essential oils fractionation allows detection of bioactive substances, especially those owning antimicrobial activity and present in small quantities. Antimicrobial activity of essential oils extracted from leaves of native Moroccan plants against clinical bacterial isolates Fatima El Malki1,4, Kamal Eddaraji2, Rajae Alloudane2, Hassane Greche3, Haiat Essalmani2, Saïd Barrijal2 1 Institut Pasteur du Maroc, Laboratory of Medical Microbiology, Tangier 90000, Morocco. 2 Department of Biology, Faculty of Science and Techniques of Tangier, University Abdelmalek Essaadi, Morocco. 3 Université Sidi Mohamed Ben Abdellah, Fès, Agence National des Plantes Médicinales et Aromatiques, Taounate, BP8875, 30100 Al Atlas-Fès, Maroc. 4 Institut Supérieur des Professions Infirmiers et Techniques de Santé (ISPITS). Tétouan; Annexe Tanger, Maroc. Contact information: Prof. Said Barrijal. Address: BP 416, FST Tangier, Morocco. Tel: (00212) 539393954/55.


Introduction
The overuse of antibiotics to treat infectious diseases resulted in the emergence of multidrug resistance (MDR) bacteria.Actually, this increased phenomenon is currently considered as a major threat to global public health.This has prompted the World Health Organization (WHO) to direct research into new alternatives and especially to plants that have always constituted a source of ongoing bioactive molecules and current inspiration for new medicines.Delacroix in 1881 showed for the first time the essential oils (Eos) action against bacteria [1].Since then, numerous studies have been reported the in vitro antimicrobial activities of certain chemical compounds of various plant extracts [2].The capacity of certain essential oils to neutralize germs is now irrefutable [3].
Origanum compactum, Rosmarinus officinalis and Pelargonium asperum are wildly used plants over the world and particularly in Mediterranean countries.They are known for their aromatic, gastronomic and phytotherapeutic features.Hence, EO extracted from these plants is actively investigated for antimicrobial effect [4], as they could substitute antibiotics in the treatment of infectious diseases caused by multi-resistant bacteria (MRB).
The mode of action of EOs on bacteria is not clearly elucidated, but for the variety of molecules in these oils, it seems that their effect results from a combination of several modes of action that involve various cell targets [5].
This study was carried out aiming to perform qualitative and quantitative analyses and to assess the in vitro antimicrobial activities of EOs extracted from O. compactum, R. officinalis and P. asperum in order to contribute to the development of the Moroccan native flora by identifying new potentially interesting therapeutic molecules.

Plant material and essential oil extraction
Plant specimens used in this study were harvested in March 2013 from different Moroccan localities.O. compactum collected from Taounate, R. officinalis from Debdou and P. asperum from Rabat.Authentication and extraction were made by the National Institute of Plant Medicinal and Aromatic (NIPMA) in Taounate (Morocco).Selected fresh leaves were oven dried at 40°C for 15 h, ground into fine powder, then passed through an 80 mesh sieve with 0.177 mm of diameter and stored at 20°C in sealed plastic bags for future use.
Large-scale extraction was performed by vapo hydro-distillation in the Technological Hall of NI-PMA.Small scale extraction was done in the laboratory of chemistry at the NIPMA-Taounate by hydro-distillation (Clevenger).100 g of plant material were extracted with 500 ml distilled water for 3 h.Obtained EOs were stored at 4°C and protected from light.The yield of EOs was calculated using the following formula: R= (Px/Py) x 100 [6] R: Oil yield (%); Px: Oil weight (g); Py: Plant oil weight (g).

Identification of essential oils compounds by GC-MS spectrophotometry
Qualitative and quantitative analysis of EOs chemical composition was conducted at the Plant Biotechnology Laboratory applied to aromatic and medicinal plants (LBVpam) in Faculty of Sciences and Techniques, Saint Etienne, France.Analytical study was carried out using Gas Chromatography Mass Spectrometer (GC-MS Agilent 6850 GC 5973) equipped with a capillary column DB-5 (30 m x 0.25 mm id.x 0.25 µm film thickness) by injecting 2 µl of extract using hexane as solvent.Carrier gas was helium at 1 ml/min.Oven temperature programmed from 60°C to 245°C at 3°C/min.The system is connected to a MSDCHEM program and a Wiley data bank to identify compounds.

Pre-fractionation of the EOs
Each EO was fractionated away from light and air stream at room temperature into 10 fractions by chromatography on silica Type 60 column as previously described [7].Purification and concentration of each fraction eluted in 350 ml of the mobile phase was carried out by vacuum distillation using a rotary evaporator BUCHI type Rotavapor R -205.The house-bath temperature was set at 33°C.

Microorganisms
EO antibacterial activity was performed on five clinical isolates resistant to at least five antibiotics including methicillin resistant Staphylococcus aureus PN15 (MRSA) and Escherichia coli extended spectrum beta-lactamase producing TF2 (ESBL).Resistance profiles were previously determined following Clinical and Laboratory Standards Institute (CLSI) and are shown in Table 1.

Determination of antibacterial activity by disc diffusion assay
Bacteria was cultured on Plate Count Agar (PCA, Biokar) and incubated overnight at 37°C to obtain individual colonies.Colonies were suspended in distilled water to achieve turbidity equal to 0.5 McFarland standard (10 6 -10 8 CFU/ml).Petri dish containing 20 ml of Mueller Hinton Agar (MHA, Biokar) was flooded with the bacterial suspension and excess inoculum was removed.10 µl of each EO were applied to 6 mm diameter sterile paper Wattman discs and were then carefully placed onto the surface of Petri dish containing MHA medium.An empty disc soaked with distilled water was used as negative control and a 30 µg amikacin disc as positive control (Sigma-Aldrich).The dishes were maintained at 4°C for 15 to 20 min to allow diffusion of EOs and then, incubated at 37°C for 18 to 22 h under normal atmosphere [8].Antimicrobial activities were expressed as inhibition diameter zones in millimeters (mm) and interpreted [9].Experiments were carried out in triplicate and the average zone of inhibition was calculated.

Determination of Minimal Inhibitory Concentration (MIC) by broth microdilution assay [10]
The MIC was determined by broth micro-dilution assay [10] using resazurin (Sigma-Aldrich) as a survival indicator.Resazurin was prepared by dissolving 1 mg per 1 ml of sterile distilled water and then filtered through a micro-filter (0.2 µm, Millipore).
A twofold serial dilution (ranging from 16 -0.05µl/ml) for each EO was performed in Mueller Hinton Broth (MHB, Biokar) containing 3% of ethanol and 50 µl of each dilution were distributed in a 96-well plate.Fresh bacterial colonies were selected and suspended in Mueller Hinton broth at turbidity equal to 0.5 McFarland and mixed with an equal volume of the previous serial dilution of each EO.After an overnight incubation at 37°C, 5 µl of resazurin (1 mg/ml) were added and further incubated for 2 h at 37°C to visualize bacterial growth.The MIC value corresponds to the lowest concentration of EO which produces no resazurin discoloration.

Determination of the Minimal Bactericidal Concentration (MBC) [10]
On PCA plates, 5 µl of bacterial culture picked up from wells were no resazurin discoloration observed was streaked.Plates were incubated for 18 to 20 h at 37°C and MBC corresponds to the lowest EO concentration which gives no bacterial growth.

Determination of the inhibitory fraction by broth dilution assay
Test was performed using 0.5 ml Eppendorf tube with a final volume of 200 µl MHB mixture containing 3% of ethanol; 20 µl bacterial suspension (10 6 to 10 8 CFU/ml) and 1.2 µl of fraction extract (6 µl/ml).After incubation at 37°C for 18 to 20 h, bacterial growth was visualized by adding 5 µl of resazurin to each tube and incubated for 2 h at 37°C.Fractions inhibiting bacterial growth showed no resazurin discoloration.To assess the effect of this inhibition, 5 µl picked from no resazurin discoloration mixture was streaked on PCA plates and incubated for 18 to 20 h at 37°C.A fraction has a bactericidal activity when no bacterial growth on Petri dishes was observed.Fractions exhibiting bacterial growth were considered bacteriostatic.

Pre-fractionation by liquid chromatography
The fractional separation of essential oil extracts obtained from O. compactum, R. officinalis and P. asperum was performed by vacuum distillation column.EO was separated into 10 fractions (F1, F2… F10) containing different chemical families (Hydrocarbons, Esters, Ethers, Aldehydes, Ketones, Alcohols and Acid).Each fraction exhibits smell and color except fractions F1 and F2 of each EO.Fractions F3 and F4 represented together about 50% of total yield of each essential oil.Yields and densities of crude extracts and their fractions are given in Table 5.It should be noted that the total mass of eluted fractions was slightly superior to the initial mass.In addition, density of some fractions was higher than the corresponding total oil.

Antibacterial
Antibacterial activity was evaluated against five multi-drug resistant clinical isolates including Gram negative (E. coli, K. pneumoniae, A. baumanii and P. aeruginosa) and Gram positive (S.aureus) bacteria.The total results obtained using disc diffusion method as well as MIC and MBC are given in Table 6.
O. compactum oil showed a very significant antibacterial activity against the strains tested.It is strongly inhibitory a determined zone inhibition diameter ranging from 29.33 ± 1.15 to 48.5 ± 0.7 mm and bactericidal effect in liquid medium at concentrations less or equal to 0.5 µl/ml.The action of this oil is slightly inhibitory for P. aeruginosa in a solid medium with a diameter of inhibition equal to 14.61 ± 1.99 mm and bacteriostatic at a concentration of 1 µl/ml in liquid medium.
On solid medium, EO of R. officinalis had no effect on P. aeruginosa (7.67 ± 1.15 mm) and slightly inhibit the other species with inhibition diameters varying between 10.02 ± 0.1 and 14, 75 ± 0.7 mm.Dilution method showed bactericidal effect, especially against A. baumanii, S. aureus and E. coli, at concentra-  tions less or equal to 8 µl/ml, but it is bacteriostatic against P. aeruginosa and K. pneumoniae.P. asperum EO, are considered not inhibitory on solid medium with diameters not exceeding 13.67 ± 1,52mm.Whereas, in liquid medium this oil had a bactericidal effect against S. aureus and A. baumanii at concentrations less than 4 µl/ml and a bacteriostatic effect even at a concentration of 16 µl/ ml for other bacteria.
As shown in Table 6, dilution method in liquid medium revealed that activities of certain oils on tested bacteria were bactericidal with MBC equal to MIC and the ratio MBC/MIC = 1, while for others, the MBC value capable of killing the inoculums was twice the MIC value.
The 10 fractions of each EO were assessed for antibacterial activity using MH broth medium.Inhibiting fractions were identified using 6 µl/ml and their antimicrobial effects were interpreted as shown in Table 7.
For O. compactum only F3, F4 and F5, that represents 67.4% of the total weight of this oil, showed bacterial growth inhibition and with only a bacteriostatic effect.
Fractions F4, F5 and F7 together represent 9.5% of the total mass of R. officinalis oil (Table 5).Bacterial growth inhibition was detected with F1, F4, F5 and F7 with a bacteriostatic effect.P. asperum oil had various fractions exhibiting inhibitory effect against tested bacteria (F2 to F9) and which represent 86.2% of the total composition (Table 5).All these fractions showed a bacteriostatic effect except fractions F7 and F8 which were respectively bactericidal against K. pneumoniae, and P. aeruginosa (Table 7).The mass fraction of F7 and F8 were respectively 0.3 % and 0.1% of total mass.

Discussion
Three Moroccan native plants (O.compactum, R. officinalis and P. asperum), were subjected to phytochemical and antimicrobial activity studies.Comparing our results with others cited in literature, differences were found and these probably attributed to factors related to genetic, environment and techniques used.
The obtained yields values were more or less comparable to other published studies.Indeed, the same yield (2.1 ± 07) has been reported for O. compactum [11], while for R. officinalis obtained yield (1.2 ± 10) was lower than various reported values ranging from 1.74 ± 0.38% to 2.58 ± 0.75% [12].Important yield was obtained for P. asperum (0.8 ± 10) compared to previous studies that consisted of approximately 0.15 to 0.40% [13].These differences in performance can be attributed to several intrinsic and extrinsic factors including genotype and environment [7].
Chemical compositions were slightly different from those described in the literature with a predominance of Carvacrol as major component of the O. compactum essential oil [11,14] , while for R. officinalis our results were different from those described in the literature with the regular presence of the same major components [7,15].Regarding P. asperum, our values were slightly different from those described in the literature with Citronellol and Geraniol being the main elements [13].
The exact composition of oils is not always specified for plant sections used and generally, EOs chemical compositions are depending on their derivation [16].This variability may affect the physicochemical properties and bioactivity of these essential oils [13].
From the results shown above (Table 5), there is a performance difference between the fractions obtained for the same EO, which may be proportional to a difference in polarity between molecules Furthermore, we can underline that 30% of the fractions obtained are denser than O. compactum crude extract and that fraction F9 had the same density than water.All of R. officinalis fractions showed lower density than of the crude extract.For P. asperum 70% of fractions were denser than total extract while fractions F1 and F10 showed higher density than water.
These differences in densities are related to the chemical composition that characterizes each fraction.Density provides information on the quality of the chemical composition, since a density less than 0.9 g/cm 3 indicates the presence in this oil of terpene and aliphatic compounds at high rates, whereas a density greater than 1 indicates a highly varied composition polycyclic terpene compounds [17].The obtained inhibition diameters, CIM and CBM varied depending on the oil used and the bacterial strain tested.Thus, essential oil is considered active if it produces superior inhibition diameters or equal to 15 mm [18].The comparison of the numerical values of our results with other publications, often remain qualitative, since the authors come to express the results in case of MIC and MBC with various units, either µl/ml, in % (v / v) (Bourkhis et al. 2007) or µg/ml (Oussou et al. 2008).This study demonstrated that O. compactum have significantly high antimicrobial activity against tested strains except P. aeruginosa.This was expressed by large zone inhibition diameters ranging from 29.33 mm to 48.50 mm as well as low as by low MIC values of 0.025% (v/v) and 0.05% (v/v) and MBC/MIC ratios of 1 and 2. On the other hand, only slight inhibition was observed for P. aeruginosa with a MIC of 0.1% (v/v) and a MBC superior to 4. Hence, our results correlated with those reported in the literature [7].Therefore, MIC and MBC values ranging from 0.0625% to 0.125% and from 0.0625 to 0.25% have been reported for E. coli and from 0.0078 to 0.031% and from 0.0078 to 0.125% for S. aureus [14].
The high activity shown by the O. compactum EO is probably associated with its high content of Carvacrol (48%), γ-Terpinen (17%) and Thymol (16%) representing the major constituents of this oil.These compounds are phenols and considered to have a high antibacterial action [5,21].In addition, oxygenated terpenes and especially terpene alcohols are highly active antimicrobial agents [22].Moreover, several studies have shown that the total antimicrobial activity of EOs is not always attributed to the major compounds, but may be due to synergism [23] or antagonism [24] interactions with others minor compounds.
Although R. officinalis exhibited only slight inhibitions of bacterial growth on solid medium, MIC values were 0.2% (v/v) and 0.4% (v/v) with bactericidal effect against A. baumanii, E. coli and S. aureus.Previous study reported bactericidal effect of R. officinalis against E. coli and S. aureus [12] which is in agreement with our result.For K. pneu-moniae, a bacteriostatic effect was observed while no antimicrobial activity was detected against P. aeruginosa.
In the case of P. asperum, no inhibition growth could be obtained in solid medium for examined strains except a slight inhibition observed for S. aureus.Whereas a MIC value of 0.2% (v/v) was obtained for A. baumanii and S. aureus with bactericidal effect.Previous study reported a weak effect of P. asperum oil from Comoros islands on Escherichia coli and Staphylococcus aureus.This effect can be attributed to terpene alcohols, mainly Citronellol (25.07%) and Geraniol (10.46%) that is considered to own antibacterial activity [13].
P. aeruginosa has an outer membrane composed of lipopolysaccharides which form an impermeable barrier to hydrophobic compounds which may explain its resistance to EOs tested [3,26].
The discrepancy between the methods used in microbiology to study the activity of EOs is relatively linked to the degree of solubility and volatility of each EO [27,28].
Antimicrobial activities of fractionated EOs were performed using directly liquid medium (Table 7).Numerous fractions inhibited bacterial growth but showed often bacteriostatic effect except fractions F7 and F8 of P. asperum which were respectively bactericidal against K. pneumoniae, and P. aeruginosa.The mass fraction for F7 and F8 was respectively 0.3 % and 0.1% of the total mass.This suggests the presence of a certain antagonism between compounds avoiding the expression of any bactericidal effect in mixed oil.
Generally, the difference in the action between these EOs is likely due to the difference in chemical composition, nature and cell wall composition of microorganisms tested and also the influence of the reaction medium which requires studying each compound alone and in combination to avoid false results [14,29].

Conclusion
The present study provides additional data of the chemical composition and antimicrobial activities of the essential oils extracted from O. compactum, R. officinalis and P. asperum obtained from Moroccan aromatic plants.
Among the fractions obtained, some of them provided interesting antibacterial activities, especially against P. aeruginosa which exhibited high resistance to assayed EOs.
This allows deducing that certain molecules present in small quantities, even in trace in the essential oil can be more active if they are concentrated and purified.Important features illustrated in this work consider these three plants, and generally all medicinal plants, as a heritage to be preserved and developed as source of discovering more effective bioactive molecules.Further studies should be carried out to overcome the limitations of employing essential oils as alternative way for health care.

Table 3 .
Chemical composition of Rosmarinus officinalis essential oil.

Table 4 .
Chemical composition of Pelargonium asperum essential oil.

Table 5 .
Yield and density of the essential oil fractions.
a : diameter of zone inhibition in millimeters including the paper disc; b : disc of Amikacin antibiotic; *: Mutai et al. (2009).

Table 7 .
Antibacterial activities of the fractionated EOs on different clinical bacterial strains.