Assessment of the antibacterial activity of four essential oils and the

The purpose of this study was to assess citratus, Eucalyptus citriodora, Lippia multiflora, Melaleuca quinquenervia biobactericide The aromatogram and antibiogram were assessed by the agar well diffusion method and the Muller Hinton disk concentration and the minim microdilution method in liquid medium. The aromatogram showed that the biobactericide


Introduction
In recent years, the emergence and spread of antibacterial resistance mechanisms, the growing importance of care infections, combined with the virtual absence of new antibiotics, have generated a real health problem beyond individual health 2]. We also witness the unprecedented emergence of the multi resistance of Gram-positive bacteria (Gram+) in both hospital and community environments [2][3][4][5], placing them in the forefront of bacteria resistant to antibacterials [3,5]. Indeed, phenotypic and genotypic isolation and characterization of methicillin Staphylococcus aureus (MRSA) and other antibiotics have reported in the literature [6][7][8]. Similarly, the European Organization for Research and Treatment of Cancer Trials has also highlighted changes within isolated Gram positive microorganisms. Indeed, the percentage of isolated streptococci increased, in particular In recent years, the emergence and spread of antibacterial resistance mechanisms, the growing importance of care-associated infections, combined with the virtual absence of new antibiotics, have generated a real health problem beyond individual health [1, We also witness the unprecedented emergence of the multipositive bacteria (Gram+) in both hospital and placing them in the forefront of Indeed, phenotypic and genotypic isolation and characterization of methicillin-resistant (MRSA) and other antibiotics have been Similarly, the European Organization tment of Cancer Trials has also highlighted changes within isolated Gram positive microorganisms. Indeed, the percentage of isolated streptococci increased, in particular β-haemolytic streptococci and Streptococcus bacteria are responsible for numerous nosocomial and/or community infections. Faced with this worrying situation, it seems urgent to ward off and propose new therapeutic approaches in order to efficient with the various bacterial ailments. So far, several approaches have been proposed, but the one concerning the antimicrobial properties of essential oils from aromatic and medicinal plants seems to be the most envisaged and of major interest. Indeed, numerous works have demonstrated antimicrobial and antifungal activity of multiple species Moreover, these essential oils, concentrated odorous substances, have found applications in the field of food pharmaceutical and cosmetic industries Recent studies, the antibacterial activity of Cymbopgon citrarus, Lippia multiflora species, and the biobactericide Neco® has been demonstrated on This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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Assessment of the antibacterial activity of four essential oils and the , Alain Souza 1 , Alexis Nicaise the activity of four essential oils (Cymbopogon citratus, Eucalyptus citriodora, Lippia multiflora, Melaleuca quinquenervia) and the The aromatogram and antibiogram were assessed by the agar well diffusion method and the agar diffusion method, respectively. Also, the minimum inhibitory um bactericidal concentration were determined by the Neco® induced the largest inhibition 5.38 mm) of all strains combined, followed by the essential 1.77 mm) and Lippia multiflora (20. Streptococcus viridans [9,10]. These bacteria are responsible for numerous nosocomial and/or Faced with this worrying situation, it seems urgent to ward off and propose new therapeutic approaches in order to efficiently cope So far, several approaches have been proposed, but the one concerning the antimicrobial properties of essential oils from aromatic and medicinal plants seems to be the most envisaged and of major interest. Indeed, numerous works have demonstrated bial and antifungal activity of multiple species [11][12][13][14][15]. Moreover, these essential oils, concentrated odorous substances, have found applications in the field of food-processing, chemical, pharmaceutical and cosmetic industries [16,17]. Gram negative (Gram-) bacteria producing or not extendedspectrum β-lactamases [18]. However, it seemed important in this study to assess the antibacterial activity of these plant species on Gram + bacteria. Thus, this study proposes to assess the antibacterial activity of four essential oils (Cymbopogon citratus, Eucalyptus citriodora, Lippia multiflora, Melaleuca quinquenervia) and the biobactericide Neco® on Enterococcus faecalis, Staphylococcus aureus1 and 2, Micrococcus spp. This work is in line with the prospect of valorization of aromatic molecules in the medical field.

Material and methods
Essential oils (EH) Essential oils of leaf samples from four plant species (Eucalyptus citriodora, Cymbopgon citrarus, Lippia multiflora and Melaleuca quinquinervia L.) were obtained by distillation. These extractions were carried out in the Laboratory of Plant Physiology, UFR (Faculty of) Biosciences, University Félix HOUPHOUËT-BOIGNY, using steam distillation as previously described by numerous authors. The biobactericide Neco®, is a product used as a trademark and sold by the University Félix HOUPHOUËT-BOIGNY [14,19,20].

Bacterial strains
The microbiological tests were performed in the Laboratory of Molecular and Cellular Biology (LABMC) of the Biology department of the University of Sciences and techniques of MASUKU (Franceville, Gabon). The four (4) bacterial strains teste table-1 were cryopreserved and selected according to several criteria as described by Fontanay, et al., [1]. The latter are not only frequently isolated in hospital environment, but also, responsible for various pathologies. Assessment of the antibacterial activities of essential oils by the diffusion method.
In order to determine the activity of these essential oils, over-night cultures were prepared from 50 μl of the cryopreserved stocks and transferred into tubes containing Brain Heart Infusion (BHI) Broth (Biomérieux, France). The different tubes were incubated at 37°C for 18-24 hours. From these preparations, an aromatogram was produced according to the same principle as that of the agar medium diffusion method of a classical antibiogram. The different essential oils were tested without any prior dilution.

Assessment of antibiotic sensitivity: comparative test
The sensitivity of the bacterial strains was assessed by the Mueller Hinton (MH) agar medium diffusion method. For this purpose, an antibiogram was made with discs of five different families of antibiotics (ATB) table-2.  [22]. Moreover, for any value of α greater than 16, the activity is said to be tolerant.

Statistical analysis
Single-factor analysis of variance was used to compare the inhibitory activity of essential oils. The Duncan test helped achieve the pair wise comparison of the different essential oils. All this, was made using the software XLSTAT 2014 and Excel 2013.

Assessment of EH activities
The inhibitory effects of the four (4) essential oils (EHs) and of the biobactericide on the growth of four bacterial strains are shown in

Assessment of the sensitivity of the bacterial germs tested
The phenotypic profile of the different bacterial germs studied against antibiotics (ATB) is recorded in table-4.

Discussion
The antimicrobial properties of the biobactericide Neco® and the essential oils of Eucalyptus citriodora (E. citriodora) andLippia multiflora (L. multiflora) Melaleuca quinquinervia (M. quinquinervia) and Cympopogon citratus (C. citratus), although already known [19,[23][24][25][26], have been further corroborated by this study. Indeed, these authors through their works have demonstrated mainly an antifungal activity. In contrast, this study focused on the antibacterial particularities of these species.

Demonstration of the activity and justification thereof
To this end, all the strains tested showed growth inhibition in the presence of the essential oil from the selected plantspecies, which could be explained by their intrinsic chemical compositions. By analogy with other studies, these oils, like other essential oils, are composed of molecules belonging to the groups of terpenes and oxygenated molecules (aldehydes, phenols, alcohols, ketones) [27][28][29]. The latter might provide their antibacterial power [30,25]. The antibacterial activity of the oils might also be correlated, on the one hand, by the proportions and the chemical structure of the aforementioned components. Among them are the so-called majority components and other minority ones [31].Indeed, the works of Zayyad et al., [32], on the EH of thymus showed that the combined action of majority components such as thymol and terpinen and minority ones (carvacrol and borneol), had inhibited the growth of Gram positive and negative bacteria (Streptococcus pneumoniae, Staphylococcus aureus, Escherichia coli, Bacillus subtilis, and Erwinia chrysanthemi). And on the other hand, this activity might also be due to the different chemotypes present in this study. To this end, the scientific literature reports several examples [33][34][35]. These authors demonstrated in their various works, a causal link between the spectrum of activity and the abundance of specific chemical elements of the species.Also, Djenaneet al., in 2011 asserted that EHs containing large proportions of 1.8-cineole are more excellent antibacterial agents than those which do not. In analyzing these results, the strong antibacterial activities of the EHs obtained in this work could be attributed to their chemotypes.

Aromatogram and antimicrobial comparison
In view of the results obtained, the bacterial strains studied appear to be resistant to most of the antibiotics (ATB) tested, with resistance percentage of 58.33%. By comparing the activity spectra of the antibiogram and the aromatogram, the best inhibition diameters are obtained with the essential oils of the selected plant species. This could be explained by the multiple mechanisms and sites of action that interact [36]. They are related to the number of molecules present and the chemical complexity of the volatile extracts.In contrast, antibiotics have a well-defined spectrum of action in the bacterial cell. The latter act either by inhibition of protein synthesis, bacterial wall, nucleic acids or by inhibition of metabolism [37]. On the other hand, although not explicit enough, essential oils have a very wide spectrum of action. Indeed, by their chemical variability and the synergistic effects of the components, several cellular actions are set up within the bacterium [17,31].

MIC, MBC, ratio
The high activity obtained in this study was confirmed by the microdilution method. The MIC values for the biobactericide Neco® and the essential oils generally range between 3 and 96 μl.ml -1 .
For the essential oil of Cymbopogon citratus, with the smallest MIC (3±1.41 μl.ml -1 ) obtained for S. aureus 1; in this study, is contrary  [19,39]. In contrast, this study confirmed the antibacterial activity of Neco®. Although having a very excellent inhibitory activity, this Neco species is bacteriostatic against the bacterial strains tested and can be adapted in case of bacterial infection.
Similarly, for the oil stemming from Melaleuca quinquinervia L., the works of Camara et al., [14] and Doumbouya et al., [19]  By comparing these two studies, the action of these species on Gram (-) appears to be lower than the one on Gram (+). This finding corroborates the arguments of many previous studies [20,29,40].
The activity of EHs Neco and E. citriodora appears to be bacteriostatic on cocci, while L. multiflora and C. citratus seem to be both bactericidal and bacteriostatic. And this depends on the strain present. Furthermore, the EH M. quinquinervia L. appears to be bactericidal on the latter. Also, this bactericidal activity is more important on the strain Staphylococcus aureus, tested whose MBCs are equal to MICs.

Conclusion
The analysis of the antimicrobial activity of the essential oils of Cymbopogon citratus, Eucalyptus citriodora, Lippia multiflora and Melaleuca quiquinervia L. and the biobactericide Neco® revealed a proven effect on Gram positive cocci showing resistance to Some antibiotics. This activity is generally bacteriostatic in all species, although bactericidal activity has been recorded on the strains of Enterococcus faecalis, Micrococcus spp. and Staphylococcus aureus 2 for C. citratus, L. multiflora and M. quinquinervia L.
The relatively average MICs show a certain therapeutic interest in spite of the fact that the cytotoxic studies have not been carried out.
Also, based on these results in vivo studies are being conducted to explore the full pharmacological potential of these essential oils.