In vitro antibacterial activity of Cymbopogon citratus , Eucalyptus citriodora , Lippia multiflora , Melaleuca quinquenervia essential oils and Neco ® on extended-spectrum ß-lactamases producing or non-producing bacterial strains

1 Laboratory of Molecular and Cellular Biology, Microbiology-Immunology team; Agrobiology Research Unit, University of Sciences and Techniques of Masuku (USTM), BP 067 Franceville, Gabon. 2 Laboratory of Plant Physiology, Faculty of Biosciences, University Félix HOUPHOUET-BOIGNY Cocody, 22 BP 582 Abidjan 22, Côte d’Ivoire. 3 Laboratory of Plant Physiology, Plant Pathology and Plant Breeding, Agrobiology Research Unit, University of Sciences and Techniques of Masuku (USTM), BP 067 Franceville, Gabon.


INTRODUCTION
The third millennium is marked by the emergence of cases of bacterial resistance to antibiotics.Indeed, many studies have showed the presence of resistant bacteria (RB), highly resistant bacteria (HRB) and multi-bacteria (RB), highly resistant bacteria (HRB) and multiresistant bacteria (MRB) (Fournier et al., 2012;Jing-yi Zhao et al., 2014;Li et al., 2015) to antibiotics, in hospitals as well as in communities.These resistances have a direct impact on the effectiveness of the antibiotherapy by making it difficult.Thus, β-lactamase producing bacteria, by their multi-resistance to antibiotics pose a serious public health problem.
Building on this alarming fact, it seems wise to focus research on the discovery of new molecules.These should be different from conventional antibiotics and have different mechanisms of action (Falconner et al., 2009).Clearly, in recent years, natural resources and more particularly aromatic plants have been the subject of renewed interest in the search for antimicrobial molecules.The choice was directed to essential oils, natural complex mixtures of volatile secondary metabolites, extracted from aromatic plants (Kalemba and Kunicka, 2003) which present numerous bioactive properties.Indeed, essential oils of Chrysocoma ciliata L. (Afolayan and Ashafa, 2009), Cymbopogon proximus and Ocimum canum (Bassolet et al., 2001), of Prangos asperula Boissier (Hilan et al., 2009) Inula viscosa, Salvia officinalis and Laurus nobilis (Kheyar et al., 2014) and Lavandula officinalis showed antimicrobial activities.Some showed antifungal activity (Kalemba and Kunicka, 2003;Koné et al., 2010;Doumbouya et al., 2012;Kassi et al., 2014;Kouamé et al., 2015).Thus, they are used in food preservation (Mahanta et al., 2007;Kouamé et al., 2015).
In this study, we propose to assess in vitro the antibacterial activity of C. citratus, E. citriodora, Lippia multiflora, Melaleuca quinquenervia essential oils and the biobactericide Neco® on beta-lactamase producing or non-producing Gram-bacteria.This work falls within the scope of promotion of aromatic molecules for the purpose of using them in medicine.

Essential oils (EH)
In this work, we were provided with five (5) essential oils (EH) by the Laboratory of Plant Physiology, Faculty of Biosciences, University Félix HOUPHOUET-BOIGNY of Cocody-Abidjan (Côte d'Ivoire).The extraction of these species was performed according Yala et al. 797 to the method described by several authors in previous studies (Camara et al., 2010;Koné et al., 2010;Doumbouya et al., 2012).Their different characteristics are noted in Table 1.Among these oils, the biobactericide Neco®, is a product used as a trademark and sold by the University Félix HOUPHOUËT-BOIGNY (Kassi et al., 2014).

Bacterial strains
For this study, eight bacterial strains cryopreserved in the laboratory of Molecular and Cellular Biology (LABMC) were tested (Table 2).
Their selection criteria were based on the fact that these bacteria are frequently isolated in hospital environment, and are responsible for various pathologies.Thus, these strains are carrier or noncarrier of antibiotic resistance genes (ATB).

Bacterial cultures or overnight cultures
From cryopreserved stocks, 50 µl were taken and transferred into tubes containing brain-heart infusion broth (BHI) (Bioméreux, France).Then, the tubes were incubated at 37°C for 18 to 24 h.

Assessment of the antibacterial activity of essential oils by the diffusion method
The aromatogram method was used to determine the inhibitory activity of EH.From overnight cultures, successive decimal dilutions were performed at (10 -1 to 10 -3 ), in order to obtain standardized inocula (approximately 10 5 to 10 6 cells/ml).These were seeded by flooding on Mueller-Hinton agar (MH).Then the plates were dried in the laminar flow hood.Afterwards, wells were made in the agar using Pasteur pipettes and 50 µl of the different pure extracts were deposited.Finally, the dishes were incubated at 37°C for 18 to 24 h.After incubation, the inhibition diameters were measured.

Assessment of antibiotic sensitivity: Comparative test
The determination of resistance phenotypes based on the sensitive, intermediate and resistant trilogy of bacterial strains was made by the diffusion method on MH agar medium.For this purpose, an antibiogram was conducted with antibiotic discs (ATB) belonging to five different families (Table 3).

Determination of the minimum inhibitory concentration (MIC)
The microdilution method in liquid medium in 96-well microplates was used to assess bacteria growth inhibition parameters by essential oils (EH).Indeed, two-fold geometric dilutions were performed (from 1/2 to 1/256).In order to obtain a homogeneous solution, the EHs were diluted in a Tween 20 solution added with the culture medium.The microplate was covered with parafilm and incubated at 37°C for 18 to 24 h.The lowest concentration of essential oils inhibiting any growth visible to the naked eye after the incubation period was the MIC.Thus, three trials were conducted for each of the strains and the MIC value was the average of both tests.

Determination of minimum bactericidal concentration (MBC)
The minimum bactericidal concentration or MBC was determined by seeding on Lauria-Bertani (LB) medium by spreading 100 µl samples of wells not showing growth in the microplate.The lowest essential oil concentration having decimated 99.99% of the starting population, after 24 h of incubation at 37°C corresponded to the MBC.The intrinsic activity of the different EHs was determined based on the ratio MBC/MIC named α (alpha).In fact, if 1 ≤ α ≤ 2, the effect is bactericidal and if 4 ≤ α ≤ 16, the effect is bacteriostatic (Kpodekon et al., 2013;El amri et al., 2014).Moreover, for any value of α superior to 16, the activity is said to be tolerant.

Statistical analysis
The single-factor analysis of variance was used for comparing the inhibitory capacity of essential oils.Duncan's multiple range test was used to compare in pairs the different essential oils.All these were done with the XLSTAT 2014 software and Excel 2013.

Assessment of essential oil (EH) activities
In this study, the antimicrobial activity was assessed by observing the inhibiting capacity of the five (5) essential oils at the same concentration on the bacteria.The results obtained are shown in Table 4.The values recorded are the averages of the three tests.Indeed, all the essential oils showed a significant inhibitory effect against the studied microorganisms.However, a variation in efficiency in terms of inhibition zones depending on the type of germ and oil concerned was observed.

Activities of oils on E. coli strains
The results obtained in this study showed that the 5 tested oils had an inhibitory activity on the ESBL producing or non-producing E. coli strains.Indeed, inhibition diameters ranging from 6.96 ± 1.67 to 41.42 ± 8.12 mm and 6.82 ± 1.42 to 28.42 ± 7.08 mm respectively were observed for E. coli ESBL+ and E. coli BLSE-.Also, the single-factor analysis of variance showed that there was a significant difference (p <0.0001 and p = 0.0002) between the different oils used against E. coli ESBL+ and BLSE-bacteria.As for Duncan's test, it showed that there was no significant difference between the biobactericide Neco® and E. citriodora and L. multiflora oils.However, a difference was found between C. citrates and M. quinquenervia essential oils and the other abovementioned oils.Similarly, the parity check showed no significant difference between the biobactericide Neco® and E. citriodora oil, between L. multiflora and M. quinquenervia oils and between those of M. quinquenervia and C. citrates for E. coli BLSE-.Overall, no significant difference (p = 0.33> 0.05) existed between the behavior of ESBL-producing and non-producing strains.

Activities of oils on Klebsiella
Considering the results, an inhibitory effect was recorded for all oils regarding the two tested Klebsiella.Obviously, inhibition zones ranging between 7.54 ± 1.87 and 29.34 ± 5.22 mm were observed for K. pneumoniae carrying resistance genes with diameters ranging from 8.96 ± 2.69 to 31.40 ± 5.61 mm for the other Klebsiella.Moreover, for the 5 oils, significant differences (p≥0,002) were obtained for both strains.Thus, Duncan's test showed a significant difference against K. pneumonia ESBL+ respectively between the biobactericide Neco® and E. citriodora oil, C. citratus, M. quinquenervia and L. multiflora oils.Moreover, the same result was obtained between species L. multiflora and C. citratus.Moreover, for K. pneumoniae BLSE-, the paired comparison between oils, showed a significant difference between the biobactericide Neco® and C. citratus, M. quinquenervia and L. multiflora oil.Also, the same observation was made between E. citriodora, L. multiflora and M. quinquenervia species.No significant differences (p> 0.05) existed between the behavior of ESBL+ strains and the other BLSE-.

Activity of oils on bacteria of the genus Enterobacter
The average inhibition diameters of species obtained regarding bacteria of the genus Enterobacter ranged from 7.86 ± 1.86 to 43.27 ± 9.32 mm.
Concerning the species Ent.cloacae BLSE-, the value of diameters ranged between 7.86 ± 1.86 -31.00 ± 12.94 mm.From a statistical point of view, there was a significant difference (p = 0.011 <0.05) between the 5 species against this bacterium.The Duncan's test conducted showed that there was a significant difference for Ent.cloacae between Neco® volatile and E. citriodora, and C. citratus and M. quiquenervia oils respectively on the one hand.On the other hand, between L. multiflora and C. citratus.
For the species Ent.aerogenes, which is ESBL+, diameters in the range of 8.12 ± 2.56 to 43.27 ± 9.32 mm were observed.Also, the statistical analysis revealed a significant difference (p <0.0001).Duncan's multiple range test performed, showed no significant difference for species L. multiflora, M. quinquenervia and C. citratus.
And also, for M. quinquenervia and C. citrates oils against Ent.aerogenes.In general, no significant difference (p = 0.33) existed between the behavior of ESBL-producing bacteria and the other bacteria.

Activity of oils on P. aeruginosa BLSE-
The results revealed that all the species tested showed an activity against P. aeruginosa (Table 4).Thus, the diameters observed ranged between 14.97 ±13.29 and 36.12 ± 10.82 mm.Furthermore, the statistical analysis showed that there was no significant difference (p = 0.109) between these oils for this strain.

Activity of oils on A. baumanii ESBL+
The five species also showed an effective activity on A. baumanii.Diameters varying between 11.50 ± 0.57 and 42.62 ± 11.89 mm were recorded.The best diameter was obtained with the biobactericide Neco® followed by the E. citriodora, M. quinquenervia, L. multiflora and C. citrates species which showed a significant difference (p = 0.005).The parity test showed that there was no significant difference between the biobactericide and E. citriodora, M. quinquenervia and L. multiflora oils.The same result was also observed between M. quinquenervia and C. citratus.

Comparison between aromatogram and antibiogram
The sensitivity of bacteria against six (6) antibiotics (ATB) was assessed by the standard disc diffusion method.The measures of inhibition halos obtained are summarized in Table 5.
Considering the results, it appears that both E. coli strains showed a resistance profile to the different antibiotics tested, except for gentamicin for E. coli BLSE-.The Klebsiella strains, ESBL+ strains as for it, were resistant to chloramphenicol and cefepime while for BLSE-it was resistant to doxycycline only.In contrast, Ent.aerogenes was resistant to cephalosporins, to doxycycline and ofloxacin.Similarly, Ent.cloacae was resistant to chloramphenicol only.Concerning the species A. baumanii, resistance to two ATB (cefepime and chloramphenicol) was recorded.Furthermore, a resistance phenotype was observed for P. aeruginosa against ATBs of the cephalosporin family, doxycycline, chloramphenicol and ofloxacin.
By comparing the activity of essential oils and that of ATBs, the oils showed better inhibition diameters.Indeed, the results recorded revealed that the diameters of antibiogram ranged from 0.00 ± 0.00 and 31.68 ± 0.21 mm (Table 5), while those of aromatogram ranged from 6.82 ± 1.42 to 43.27 ± 9.32 mm for all strains put together (Table 4).

Determination of MICs
The different minimum inhibitory concentrations (MICs) obtained for each of the oils tested are shown in Table 6.Each of these oils showed different activities depending on their nature, but also on the bacterial strains tested.Thus, the biobactericide Neco® and the natural species E. citriodora and L. mutiflora showed strong inhibitory capacities except for K. pneumoniae and P. aeruginosa respectively for Neco® and L. mutiflora.Furthermore, these three (3) oils, gave interesting MICs ranging between 3 ± 1.41 and 96 ± 45.25 μl.ml -1 . In contrast, C. citrates and M. quinquenervia species showed very high MICs or even an absence for some strains (Table 6).And therefore, they had low and very low inhibitory capacities (Table 6).

Determination of MCB and assessment of the MBC/MIC
In general, the biobactericide Neco® and the E. citriodora species had a bacteriostatic activity against the strains tested (Table 8).The values of minimum bactericidal concentrations (MBCs) were included in a range of 16 ± 0.00 to 96 ± 45.25 µl/ml for the first oil.Similarly, the MBC values were in the range 12 ± 5.66 to 192 ± 90.51 µl/ml, respectively (Table 7).
Considering the results, the M. quinquenervia species therefore had a bactericidal intrinsic activity overall.However, the values of its concentrations were very high and were between 64 ± 0.00 and ≥ 256 ± 0.00 µl/ml.However, the EH of L. multiflora also had a bactericidal activity against most strains studied.With the difference that the assessment of MBCs showed values ranging from 32 ± 0.00 to 192 ± 90.51 μl/ml, which are close to the MIC values obtained for this essential oil (Table 6).Furthermore, the only oil of which no effect was observed during this study was C. citratus.Thus, no MBC was recorded for all tested bacteria.

DISCUSSION
Through this study, the antibacterial activities of five (5) essential oils (HE) were assessed.In the light of the

*
All the values shown are the averages of 3 trials (n=3) avec ± standard deviation.⃰ On the same line the values having the same letter are not significantly different from a<b<c.

Table 1 .
Features of the essential oils studied.

Table 3 .
Names of the different antibiotic discs.

Table 4 .
Antibacterial activity of different essential oil against bacterial strains.

Table 8 .
Nature of the intrinsic activity of the essential oils studied.
results, it appears that the inhibitory effect of these species, against the studied bacteria, is heterogeneous.Thus, the biobactericide Neco® and the Eucalyptus citriodora (E.citriodora) and Lippia multiflora (L.multiflora) oils present the highest efficiencies while Melaleuca quinquinervia (M.quinquinervia) and Cympopogon citratus (C.citratus) oils have lower efficiencies as compared to the first three ones.