Available online on 15.06.2023 at http://jddtonline.info

Journal of Drug Delivery and Therapeutics

Open Access to Pharmaceutical and Medical Research

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Open Access  Full Text Article                                                                                                                              Research Article 

Synergistic Effects of Essential Oils and Antibiotics Against Some Bacterial Strains

Feudjieu Egoume Gaizirene*^1,2,3, Kemegne Gislaine Aurelie^1,2, Tchinda Fossi Cedric^1,2,3, Tchamgoue Deutou Armelle¹, Moni Ndedi Esther Del Florence ^2,3, Matchuenkam Gayap Sonia¹, Agbor Gabriel ¹

¹ Pharmacology and Drugs Discovery Laboratory, Center For Research on Medicinal Plants and Traditional Medicine, Institute of Medical Research and Medicinal Plants Studies, P.O. Box 13033, Yaounde, Cameroon;

² Department of Microbiology, Faculty of Sciences, University of Yaounde I, P.O. Box 812, Yaounde, Cameroon; 

³ Laboratory for Tuberculosis Research, The Biotechnology Center, University of Yaounde I, P.O. Box 812, Yaounde, Cameroon

Since 1980s, the number of antibiotics resistant strains has increased exponentially, making difficult it in the elimination of pathogens¹. One alternative explored by the scientific community to overcome multi-drug resistant is antimicrobials combination². This combination approach based on the concept that the more targets a drug have on a microorganism the more difficult it will be for the cell to develop resistance strategies^3,4,5,6. For this reason, research of medicinal and aromatic plants containing active compounds is increasing^7,8. 

Since antiquity, medicinal plants have been used in phytotherapy against several diseases, given their richness in hundreds components with therapeutic virtues⁹, but  their use was based on traditional practices and applications without scientific bases¹⁰. Aromatic plants have antimicrobials properties known and used for a long time^11,12, they, are able to synthetize aromatics compounds called essential oils (EOs)¹³. These EOs have a very broad inhibition spectrum including Gram-positive and Gram-negative bacteria, fungi, as well as viruses¹. Currently, they are used in the fields of food industry,  cosmetology and hygiene products sector¹⁵. In  pharmaceutical industry, bacteriostatic, bactericidal, vermicidal, fungicidal, antiseptic, insecticidal, antiviral, anti-inflammatory, antioxidant and many more EOs properties are used^15,16. antimicrobial power of EOs are due to the diversity of active compounds with antimicrobial properties makes resistances to these EOs difficult or impossible by micoorganisms, because they cannot undergo resistances mutations to all active molecules simultaneously present in EOs¹⁷. Based on this observation, the combinations of several EOs with each other or with antibiotics would therefore be an effective way to reduce the resistance risk and also increase antimicrobial activity effectiveness ¹⁸.  

Drypetes Gosswelleri, Echinops giganteus and Melaleuca leucadendron are Cameroonian’s pharmacopoeia plants. In Cameroon and Congo the powdered stem bark of D. gosswelleri is eaten to treat sexual weakness and venereal diseases¹⁹. The roots of E. giganteus are widely used in the traditional medicine of Cameroon and Nigeria for the treatment of various diseases such as heart disease, gastric disorders, stomach pain, and asthma attacks²⁰. M. leucadendron is used for antiseptic, antipasmodics, antineuralgics and antirheumatics properties²¹. It would be interesting to assess if these three EOs can potentiate the activity of some antibiotics in the combination approach. Thus, The designed study aimed to evaluate the combined effects of essentials oils from Drypetes Gosswelleri, Echinops giganteus, Melaleuca leucadendron and antibiotics against strains implicated in infectious deseases.

1.  MATERIALS AND METHODS
   1.  Preparation of essential oils

The plants were collected in Cameroon. The bark of Drypetes gosswelleri (D. gosswelleri) harvested in Littoral Region, especially in Gwei near Edea. The roots of  Echinops giganteus (E. giganteus) were bought at Mokolo market in the Center Region (Yaoundé-Cameroon). The leaves of Melaleuca leucadendron (M. leucadendron) were harvested in Yaoundé. EOs were obtained by hydrodistyllation. The plant part used was crushed and immersed in water, then subject to hydrodistillation using Clevenger-type apparatus during 6 to 8 hours. EO was collected, and dried with anhydrous sodium sulfate¹⁸. The solutions were prepared at 6000 µg/mL by dilution of 12  µL EOs with a mixture of tween (11%), DMSO (5%) and sterile distilled water for a total volume of 2 mL.

For this study, two antimicrobials were used : injectable Ciproflaxacin (CIP) N°RA/DRUGS/RAJ/1594 (BONCIPRO^(R)) and injectable Ceftriaxone (CEF) N°GUJ/DRUGS/G/198 (XONE). Antibiotics tested solutions were prepared at 100 µg/mL.

Five bacterials strains have been used in this study. Two Gram positive: Bacillus cereus ATCC 11966 and Staphylococcus aureus ATCC 25923 and three Gram negative: Salmonella enteritidis 155A, Pseudomonas aeruginosa ATCC 27853 and isolate of Klebsiella pneumoneae spp,. The strains were obtained from the Pharmacology and Drugs Discovery Laboratory (at the Institute of Medical Research and Medicinal Plants Studies), except isolate of Klebsiella pneumoneae spp obtained from the University Teaching Hospital of Yaounde Cameroon. All was stored at -80°C. Before starting, the strains were sub-cultured on solid medium Mueller Hinton Agar (MHA) followed by Mueller Hinton Broth (MHB). 

Microdilution method was carried out according to the Microplate Alamar Blue Assay as described previously by Collins and Franzblau and modified by Jimenez-Arellanes et al.^22,23. A stock of EOs solution initialy prepared at 6000 μg/mL was added in the wells containing culture medium MHB to reach final samples concentrations ranging from 3000 μg/mL to 2.92 μg/mL for EOs, from 50 μg/mL to 0.048 μg/mL for CIP and CEF. The wells were inoculated with bacteria inocula (6log UFC/mL prepared from the MHB medium) to obtain concentration ranging from 1500 μg/mL to 1.46 μg/mL for EOs, from 25 μg/mL to 0.024 μg/mL for CIP and CEF. Each 96 wells microtiter plates were mixed and incubated at 37 °C for 24 hours. After incubation periods, 20μL of 0.02 % resazurin salt solution were added to individual wells and the plates were reincubated for 20 minutes and color changing was checked. The resazurin changing colour from blue to pink indicated reduction of the indicator and thus bacterial growth. The Minimal Inhibitory Concentration (MIC) was defined as the lowest concentration of samples at which the microorganism did not demonstrate growth²⁴. The Minimal Bactericidal Concentration (MBC) of promising EOs was assessed by sub-culturing MIC test microtiter plates on MHB medium. The MBC was considered as lowest concentration at which no growth occurred in the medium. The value of the ratio MBC/MIC was used to classify antimicrobial activity : MBC/MIC ≤ 4 for a bactericidal effect, 4<MBC/MIC≤16 for a bacteriostatic effect  and MIC/MBC>16 for tolerance effect²⁵.

2.5.1 Determination of the minimal inhibitory concentration of combinations

The MICs of combination was determined using isobologram method^18,26,27. This method is base on the inhibitory concentration determination of two antimicrobials combination on a 96-wells microplate followed by the isobologram construction. The tests was done against the two bacterials strains showing the best sensitivity to EOs one Gram positive and one Gram negative (Staphylococcus aureus and Salmonella enteritidis). The combinations was realized between D. gossweilleri  and E. giganteus EOs, which showed the best activities against bacteria and with two antibiotics (Ciprofloxacin and Ceftriaxone). For the combined solutions, one antibacterial was at a variable concentrations (MIC/16, MIC/8, MIC/4, MIC/2 and MIC) and another at a fixed concentration (MIC).

Thirty solutions was obtained and tested, of which five solutions of each concentration of EOs and antibiotics combinations below: Dg*Eg (variable concentration of D. gossweilleri  and a fixed concentration of E. giganteus), Eg*Dg (variable concentration E. giganteus and a fixed concentration for D. gossweilleri), Dg*CIP (variable concentration of D. gossweilleri and a fixed concentration of CIP), Eg*CIP (variable concentration of E. giganteus and a fixed concentration of CIP), Dg*CEF (variable concentration of D. gossweilleri and a fixed concentration of CEF) and finally Eg*CEF (variable concentration of E. giganteus and fixe concentration of CEF).

In the width direction of microplate, from wells 1 to 10, after adding 100 µl of culture medium MHB, the solutions initially prepared at different concentrations of each combination were added in duplicate in the following order : MIC/16 *MIC, MIC/8*MIC , MIC/4* MIC, MIC/2* MIC, and MIC*MIC. Successive dilutions following a geometric progression of 1/2 ratio were carried out from wells 1 to 10 to the well G. 100 μL of bacterial inoculum at 6 Log CFU/mL were added. The wells of columns 11 and 12 were used as a negative control for combined solutions which contained MHB culture medium and solutions at decreasing concentrations. The wells of row H were used as a positive control for bacterial growth and contained MHB medium and inoculum (Figure 1). The microplate was closed, covered with film paper and incubated at 37°C for 24 hours. The results were read by turbidity observation of wells: turbidity indicating bacterial growth. 

Figure 1: protocol of antimicrobials combination on 96 wells microplate.

The wells of line A are those in which differents solutions of combination (A, B, C, D and E) are deposited, the series of dilutions are carried out in  direction A to G (these wells contain bacterial strains, MHB medium and antimicrobial solutions). wells H1 to H8 are used as a growth control (without antimicrobials, just MHB medium and bacterial innoculum), the negative controls of solutions (just antimicrobial and MHB medium) are represented in wells A11 to C11, A12 to C12, D11 to G11, D12 to G12 and H9 to H12 respectively for solutions A,B,C,D and E.

2.5.2 Isobolograms construction

Isobologram is a two-dimensional graph with, on the axis, the concentrations of each compound used in the association are represented^18,28,29. This method aims to define on a geometric graph the type of interaction between substances. According to this method, the points connecting to form diagonal represent the MIC values of antimicrobials tested individually, one on x-axis and the other on y-axis. This diagonal is the isobole of additivity. The junction points position of  MICs of two antimicrobials in combination (at different concentration : five points) allow us to conclude on interaction type. Thus, a point located at the top of the right side of the isobole indicates an antagonism. Those below indicates a synergy and the point near or on isobole indicates additivity^18,26,30.

1. RESULTS
   1.  Antimicrobial activity

Antibacterial parameters (MIC and MBC) of essential oils (EOs) against bacterials cells are reported in Table 1. EOs from D. gosswelleri showed the strongest activity against all the bacterials strains with MICs of 1.46 µg/mL against S. aureus, S. enteritidis and B. Cereus, 5.88 µg/mL against K. pneumoneae and 11.71 µg/mL against P. aeruginosa. Followed by E. giganteus EO with MICs ranging from 2.91 µg/mL to 23.43 µg/mL. M. leucadendron had the lowest activity with MICs ranging from 5.88 µg/mL to 750µg/mL.

CEF did not showed any activity against P. aeruginosa at the maximal concentrations tested against others strains (1500 µg/mL), against the others strains, the MICs of CEF equal to 0.19 µg/mL. The MICs of CIP ranging from 0.048 µg/mL to 0.19 µg/mL. It should also be notice that P. aeruginosa is a strains against which EOs had a less strong activity. 

For all antibacterial tested the bactericidal effect was obtained against P. aeruginosa , except for CEF. D. Gosswelleri EO that had a bactericidal effect against most strains except against B. Cereus.

The more sensitive strains at EOs was S. aureus strain among the Gram positive bacteria and  S. enteritidis among the Gram negative bacteria.  against these strains, D. gosswelleri had MIC equal to 1.46 µg/mL and 2.91 µg/mL and E. giganteus EOs, MIC equal to 1.46 µg/mL and 5.88 µg/mL for.

Table 1: inhibition parameters against bacterial strains in μg/mL.

Legend : MIC : minimal inhibitory concentration, MBC : minimal bactericidal concentration, >1500 : no activity at consentration tested, ND : no determined.

The MIC combinations results were reported in Tables 2. In the combinationsm the MICs of antibacterial combined was reduces compared to MICs obtained when they were tested individually. The best reduction of MICs was obtained by the combination using D. gosswelleri EO against the two strains. For the combination of D. gosswelleri EO and CEF, the MIC obtained against the strains equal to 0.04 µg/mL for each antibacterial initialy equal to  1.46 µg/mL  and 0.09 µg/mL respectively. The combination of D. gosswelleri EO with CIP showed MIC equal to  0.19 µg/mL for D. gosswelleri EO and 0.02 µg/mL for CIP against the strains, initialy equal to 1.46 µg/mL et  0.01 µg/mL respectively.

Table 2: Minimal inhibitory concentration of the Combination on bacterials strains in μg/mL.

Legend : Dg: D. gosswelleri., Eg: E. giganteus and Ml: M. leucadendron, CIP: Ciprofloxacin, CEF: Ceftriaxone, Dg*Eg: combination of D. gosswelleri EO at varying concentrations and E. giganteus EO at a fixed concentration equal to the MIC. Eg*Dg: combination of E. giganteus EO at varying concentrations and D. gosswelleri EO at a fixed concentration equal to the MIC. Dg*CIP: combination of D. gosswelleri Eo at varying concentrations and Ciprofloxacin at a fixed concentration equal to the MIC, Dg*CEF: combination of Eo from D. gosswelleri at varying concentrations and Ceftriaxone at a fixed concentration equal to the MIC, Eg*CIP: combination of E. giganteus EO at varying concentrations and ciprofloxacin at a fixed concentration equal to the MIC, Eg*CEF: combination of E. giganteus EO at varying concentrations and Ceftriaxone at a fixed concentration equal to the MIC.

Using these MICs, the type of interaction of different combined concentration was determined using the isobologram construction (figure 2, 3 and 4). The results obtained showed the synergistic action of all contrations of the combinations tested against S. aureus and S. enteritidi strains. 

Figure 2 represent the isobolograms for the combination between the EOs. Synergistic action was obtained for the combined concentrations against S. aureus and S. enteritidis. For the combinaison Eg*Dg, the MICs obtained with D. gosswelleri EO was the same against S. enteritidi  (0,09 µg/mL) and against S. aureus (0.18 µg/mL) for all the solutions. While, the MICs values of E. giganteus EO ranging from 0.09 µg/mL at 0.73 µg/mL S. aureus and S. enteritidis for all the solutions, the interaction type was the same (synergistic) in any case. It for this reason, all point appear in the same posistion on the  isobolograms of this combination (figure 2 : C and D).

Figure 2 : synergytic effect of combination between D. gosswelleri EO and E. giganteus EO.

A : Dg*Eg: combination of D. gosswelleri EO at varying concentrations with E. giganteus EO at a fixe concentration equal to the MIC against S. aureus. B: Dg*Eg: combination of D. gosswelleri EO at varying concentrations with E. giganteus EO at a fixe concentration equal to the MIC against S. enteritidis.C: Eg*Dg: combination of E. giganteus EO at varying concentrations with D. gosswelleri EO at a fixe concentration equal to the MIC against S. aureus. D: Eg*Dg: combination of E. giganteus EO at varying concentrations with D. gosswelleri EO at a fixe concentration equal to the MIC against S. Enteritidi.

Figure 3 represent the isobolograms for the combination of EOs with CIP. Synergistic action was obtained for the combined concentrations against S. aureus and S. enteritidi strains. Against  S. aureus strain, the range of the MICs values of CIP for the combinaison of Eg*CIP (0.006 µg/mL to 0.01 µg/mL) was lower than MICs obtained by combination of Dg*CIP (0.003 µg/mL to 0.02 µg/mL). While against S. enteritidi strains, the range of the MICs values of CIP for the combinaison of Dg*CIP (0.003 µg/mL to 0.023 µg/mL) was lower than MICs obtained by combination of Eg*CIP (0.003 µg/mL to 0.09 µg/mL).

Figure 3 : synergytic effect of the combination between Eos and CIP.

 E: Dg*CIP: combination of D. gosswelleri EO at varying concentrations with Ciprofloxacin at a fixe concentration equal to the MIC against S. aureus, F: Dg*CIP: combination of D. gosswelleri EO at varying concentrations with Ciprofloxacin at a fixe concentration equal to the MIC against S. enteritidis, G : Eg*CIP: combination of E. giganteus EO at varying concentrations with Ciprofloxacin at a fixe concentration equal to the MIC against S. aureus, H: Eg*CIP: combination of E. giganteus EO at varying concentrations with Ciprofloxacin at a fixe concentration equal to the MIC against S. enteritidis.

Figure 4 : synergytic effect of combination between EOs and CEF.

I: Dg*CEF: combination of D. gosswelleri EO at varying concentrations with Ceftriaxone at a fixe concentration equal to the MIC against S. aureus, J: Dg*CEF: combination of D. gosswelleri EO at varying concentrations with Ceftriaxone at a fixe concentration equal to the MIC against S. enteritidis, K Eg*CEF: combination of E. giganteus EO at varying concentrations with Ceftriaxone at a fixe concentration equal to MIC against S. aureus, L Eg*CEF: combination of of E. giganteus EO at varying concentrations with Ceftriaxone at a fixe concentration equal to the MIC against S. enteritidis.

Figure 4 represent the isobolograms for the combination of EOs with CEF. Synergistic action was obtained for the combined concentrations against S. aureus and  S. enteritidis. For the combination Eg*CEF, the range of the MICs obtained with E. giganteus EO against S. enteritidi and S. aureus strains (ranging from 0,001 µg/mL to 0,01 µg/mL) are lower than the MICs obtained with the combination Dg*CEF (raging from 0.005 µg/mL to 0.04 µg/mL against S. aureus  and from 0.02 µg/mL to 0.045 µg/mL against S. enteritidis) for the solutions tested.While, the MICs values of E. giganteus EO ranging from 0.09 µg/mL at 0.73 µg/mL S. aureus and S. enteritidis for all the solutions, the interaction type was the same (synergistic) in any case. It for this reason, all point appear in the same position on the  isobolograms of this combination (figure 2 : C and D).

In this study, the antimicrobial activity of three EOs was determined by microdillution method against five strains (B. cereus, S. aureus, S. enteritidis, P. aeruginosa and K. pneumoneae). The results showed anti-bacterial effects of D. gossweileri, E. giganteus and M. leucadendron EOs.

The ability of the plants to inhibit microbial growth and the difference in activity that exists between EO of the same plant and plants of the same family or different families would be influenced by chemical composition of each EO also influenced by several harvest factors^31,32. Synergy action between these different majority and minority compound would therefore be responsible for this microbial inhibition³³. 

D. gosswelleri EO showed MICs ranging from 1.46 µg/mL to 11.71 µg/Ml. Moni et al^34,35 studied the activity of D. gosswelleri EO against Mycobacterium tuberculosis strain and isolates. They had obtained MICs ranging from 4.88 µg/mL to 9.86 µg/mL, showing the strong activity of this EO. Similarly, in another study, the activity of this EO against yeasts showed MICs equal to 62.5µg/mL³⁶.  D. gosswelleri EO is mainly composed of benzyl isothiocyanate noted in previous work of our research team^34,35  at 91.28% and, also by the others studies (86.7%³⁷ and 63.19%³⁸) in their studies. Isothiocyanates (ITCs) are compounds with the great antimicrobials prospects. Particular ITCs, have long been known to have biologicals activities including various pharmaceutical benefits to human health (anticarcinogenic, antimicrobial and antioxidant properties) and plants defense (against insects, fungi and microbial infections)^39,40,41. Kaiser et al.⁴², studied the activity against P. aeruginosa strain of benzyl isothiocyanate present in D. gosswelleri and obtained the MIC equal to 2.14 µg/mL⁴², similar to MICs obtained in this study against S. aureus, S enteritidis and B. cereus strains (1.46 µg/mL), this represents the best inhibition among all isothiocyanates inhibitions tested in their study.

Regarding the E. giganteus EO, the MICs value obtained ranging from 2.91 µg/mL to 23.43 µg/mL. Tekwu et al⁴³studied the extracts activites of E. giganteus plant against Mycobacterium tuberculosis and obtained MICs between 16µg/mL to 32µg/mL, inhibition by E. giganteus extract was the best observed among the plants extracts tested. Sesquiterpenes are the compounds mainly present in E. giganteus. The presence of these compounds at 94.3% was revealed by Menut et al.²⁰ and at 93% by Pavela et al.,⁴⁴. 

The activity obtaines with M. leucadendron EO ranging from 5.88µg/mL to 750µg/mL respectively against S. enteritidis and P. aeruginosa. The antibacterial and anti-inflammatory properties of M. leucadendron have been demonstrated against various pathogens with MICs between 4µg/mL and 8µg/mL by Monzote et al.⁴⁵ (2020). This EO are mainly composed to Sesquiterpenes like E. giganteus EO. Rajendra et al was revealed the presence of thesecoumpounds in this EO at percentage between 81.23% and 93.50% from different locations and harvest seasons⁴⁶. These compounds could be of great pharmacological utility²⁰. Their antimicrobials activities have been the subject of the preview studies, while, one showed the antimicrobial activity of sesquiterpenes on fungi with MICs between 128µg/mL and 256µg/mL⁴⁷.

The MICs obtained with ciprofloxacin (CIP) against the five strains in this study (0,05 µg/mL to 0.19 µg/mL) allow us to conclude on the sensitivity of these strains to this antibiotic. One strain is declared sensible at CIP when the MIC of this is <1mg/l⁴⁸.

Ceftriaxone (CEF) have an activity against all strains except P. aeruginosa. The sensitivity of strains to CEF are declare when MIC ranging from 0.125 µg/mL to 8 µg/mL⁴⁹ this is the case in this study with MICs equal to 0.10 µg/mL. Against P. aeruginosa, CEF was not active, that explains the lack of inhibition obtained in this study. This resistance seems to be due to the natural presence of cephalosporinase, efflux mechanisms and the impermeabily developed which protects these bacteria against action of the antibiotics belonging to the family of cephalosporine like CEF^50,51. This mecanism made also the action of the EOs difficult. P. aeruginosa are generally ranked among the least sensitive bacteria to EOs compared to other Gram-negative bacteria^51,52 which explain the results of this study with EOs from D. Gosswelleri, E. Giganteus, and M. leucadendron (MIC equal to 5.88 µg/mL, 23.43 µg/mL and 750µg/mL respectively). CIP are the fluoroquinolone the most active against P. aeruginosa ⁵³, this is justifies by MIC obtained (0.048 µg/mL) in this study with CIP against this strains, represente the best inhibition of this antibiotic. 

The activities of EOs against Gram-negative bacteria (P. aeruginosa, S. enteritidis and K. pneumoneae) and against Gram-positive bacteria (B. cereus and S. aureus) are not difference in this study. Preview study showed that, the antibacterial activity of EOs does not depend on the Gram type bacteria⁵⁴.

In this study, Synergistic effect of D. gosswelleri, E. giganteus and antibiotics are study using isobologram method. The combinations realized between the two EOs and the EOs with antibiotics showed a synergistic action for different concentration combined. In their comparative study of methods for evaluating the interaction type of antimicrobial combinations, Kemegne et al concluded on a best reliability of isobologram method which was used in this study¹⁸. The reduction of MICs of antibacterials combinations compared when they are tested alone was obtained with synergistic interaction. 

Indeed, several research have been the subject of the combinations between EOs and with antibiotics against several pathogens with the aim to potentiate their respective actions^55,56,57. Malik et al, studied the synergistic action between P. graveolens EO and CIP against bacteria responsible for urinary tract infections: Klebsiella pneumoniae, Proteus mirabilis and S. aureus all initially resistant to CIP, they obtained synergy between two antimicrobials⁵³. another study evaluated the antibacterial activity of 11 EOs against S. aureus, E. coli, K. pneumoniae and P. aeruginosa as well as 50 clinical strains isolated from different infections, alone and in combination with standard antibiotics. The results found were significant because most EO/antibiotic combinations had a synergistic effect⁵⁸. The development of the resistances against CIP increase over time, this is due to its frequently use for her strong antimicribial power with  broad spectrum. To potentilised the action of this antibiotic, the combinations between plants products are much studied in various works and a synergistic activity is frequently obtained.^53.

The mechanisms by which EOs inhibit bacterial growth involve several modes of action. Their hydrophobic nature and their richness in several phenolic compounds allows them to divide the lipid bilayer of bacterial wall making it more permeable. This permeability facilitate antibiotics penetration and improves their action, hence the synergy obtained⁵⁹. The combinations between EOs and with the antibiotics allows to obtain the set of components, affect multiple biochemical processes in the bacteria, producing a plethora of interactive antibacterial effects^9,18. Moreover, somes EOs act by inhibiting the efflux pumps bacterial, which cause the bacterial resistances to antibiotics if they are used alone. This inhibition increases antibiotic action on bacteria with a reduced effective dose.

Based on the results obtained in vitro, the antibacterial activity of D. gosswelleri, E. giganteus and M. leucadendron EOs could serve as a source of new antibacterials agents. The reduction of effective dose of antibiotics obtained in this sudy, allows us to reduce undesirable effects attributed to it, this also makes it possible to overcome bacterial multi-resistance problem. 

The results of this study indicated that, aromatherapy as an interesting alternative to antibiotics. That is due to the antimicrobial activities demonstrated against five microbial strains responsible for common infections. The synergistic action of EOs between them and  with antibiotics makes more effective for reisistance treatment. The importance of this synergistic action between essential oils and with antibiotics is contributing to the broadening of microbiocidal spectrum and bactericidal activities against several microbial strains and simultaneously, increasing the effectiveness of antimicrobial activity. Therefore, it would be an effective means of reducing the risk of resistance emergence.

Competing interests

The authors declare that there is no conflict of interest regarding the publication of this paper.

Acknowledgments 

We are profoundly grateful to the laboratory of pharmacology and Drugs Discovery of Institute of Medical Research and Medicinal Plants Studies (IMPM), Cameroon for technical support.

Fundings

The authors declare that they have received no funding for the research reported.

Ethic approval and consent to participate

Not applicable in this section.

Availability of data and materials

The datasets supportings the conclusions of this article are presented in this main paper. Plant material used in this study have been identifieds at Cameroon National Herbarium where voucher specimen are deposited.

Author’s contributions

FEG carried out the research work, prepared the first draft of the manuscript and participated in revision and formating of the final version. KGA designed and supervised laboratory works and revised the manuscript. TFC participated in the behavioud experiment. MNE guided to choices the plants and design study. TDA guided to laboratory work and revised manuscript. MGS participated at laboratory works. GAA conceived the study and revised the final form manuscript. All authors have read, revised and approved the final version of the manuscript.

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