In vitro Antibacterial Effects of Salvia sclarea, Eucalyptus Globulus and Eugenia Caryophyllata Essential oils Against Multidrug Resistant Corynebacterium spp Clinical Isolates

Objectives: Multidrug resistant Corynebacterium species are increasingly reported as the ethiological agent of various clinical infections. Thus, the purpose of this research was to evaluate the in vitro antimicrobial activity of three essential oils Salvia sclarea, Eucalyptus globulus and Eugenia caryophyllata against Corynebacterium species. Methods: Twenty-four multidrug resistant strains including C. striatum, C. amycolatum, C. urealyticum, C. aurimucosum, C. imitans, and C. jeikeium were used in the study. Inhibition diameter zone, minimum inhibitory concentration and minimum bactericide concentration of these oils were determined using agar disc diffusion method and microdilution method. Tigecycline was used as positive control. Results: Our study showed that Eugenia caryophyllata had the best activity. Eucalyptus globulus extract exhibited a moderate activity and Salvia sclarea was inactive against all the species tested. We found that C. amycolatum was more resistant to the essential oils than other species. On the other hand, tigecycline was effective on the majority of the strains (37.5%), but his action was lower than Eugenia caryophyllata oil. Conclusion: These results support the use of clove oil as a natural alternative to treat infections caused by multidrug resistant corynebacteria.


Introduction
Essential oils from aromatic and medicinal plants have been known since antiquity to possess biological activity, notably antibacterial properties [1,2]. Nowadays, the use natural, effective and nontoxic antimicrobial agents have been developed interestingly in particular with the emergence of many drug resistances among a wild range of bacteria. In fact, the prolonged exposure to antibiotics in clinic significantly increases the manifestation of opportunistic pathogens notably Corynebacterium spp. The spread of antimicrobial resistance Corynebacterium species is a known problem worldwide [3][4][5]. Thus, there is growing interest in using natural antimicrobial compounds to treat patients suffering from infections due to multidrug resistance Corynebacterium species. Previous studies reported the antimicrobial activity of essential oils from clove (Eugenia caryophyllata), eucalyptus (Eucalyptus globulus), and sage (Salvia sclarea) against bacteria [6][7][8][9]. Thereby, in this study we evaluated the antimicrobial potency of Eos extracted from these plants against MDR Corynebacterium strains responsible for clinical infections and we compared their activity to tigecycline.

Essential oils obtention
Three medicinal plants selected for this study were Salvia sclarea, Eucalyptus globulus and Eugenia caryophyllata. Brut EOs were purchased from Gandiva Cosmética Vegetal (Spain) and stored at room temperature until tested.

Determination of diameter of inhibition zone
The susceptibility of MDR pathogens to the 3 essential oils was tested by disc diffusion method Muller Hinton Agar. Sterile blank discs of 6 mm diameter (Biomérieux, Marcy-l'Etoile, France) loaded with 20μl of each oil were placed on solidified medium, previously inoculated on the surface agar with 200 μL of 10 6 cfu/mL suspension. The plates were incubated at 37 ºC for 24 hours. Diameter of zone of inhibition of growth was measured.

Determination of the MIC and MBC
The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) for The essential oils, were determined using broth dilution method. Serial dilutions (10 dilutions) of essential oils (50µl/ml) were prepared with BHI broth medium. One colony of each bacterial strain was sampled with a loop, then inoculated in 25 mL BHI broth and incubated for 18-24 h at 37°C. The suspension was diluted to obtain 10 6 cfu/mL and 50µl of the solution were placed in each well. Final solutions were incubated at 37°C overnight. The MIC was considered as the lowest concentration that prevented the visible growth. The MBC was determined by subculturing 100 μL from each test well ≥ MICs onto plate count agar plates. MBC was defined as the lowest concentration resulting in a negative subculture or giving presence of only one colony after incubation. Bacteriostatic activity has been defined as a ratio of MBC to MIC >4 [11].

Results and Discussion
The antimicrobial activities of S. sclarea, E. globulus and E. caryophyllata against 24 MDR clinically relevant Corynebacterium spp strains was evaluated. Bacterial susceptibility to the essential oils, as determined by the agar diffusion method, showed that the selected essential oils have variable antibacterial activity. E. caryophylatta EO showed remarkable activity against the all the tested strains (Table I). Our study was in concordance with other findings reporting the potent antibacterial effect of this EO [12]. It was active against food-borne bacteria [13,14] and H. pylori [15,16]. This activity is due to the eugenol, the major compount of E. caryophylatta EO [17] . Our results revealed that E. globulus EO had a moderate activity against the strains tested. The inhibition diameters zones were lower than those obtained with the clove oil, ranging from 7 mm to 20 mm (Table II). Tigecyclin   1  30  18  15  20  2  25  10  9  20  3  25  16  10  20  4  23  20  10  20  5  25  12  10  30  6  20  15  15  22  7  22  15  10  22  8  25  18  10  24  9  20  15  8  22  10  20  7  9  20  11  20  18  10  24  12  18  13  10  18  13  22  8  8  20  14  22  10  10  20  15  20  13  8  18  16  8  8  8  24  17  28  17  15  26 Auctores Publishing -Volume 2(3)-020 www.auctoresonline.org ISSN: 2690-1919  Page 3 of 5   18  28  18  15  30  19  22  10  8  26  20  24  7  10  28  21  20  15  10  24  22  22  10  10  20  23  10  12  10  18  24  10  13  10  20  Table II. Inhibition diameter zone (mm) of different plant essential oil on Corynebacterium spp strains For the majority of the strains (52%), the diameters were comprised between 10 and 18 mm. The lowest values were exhibited by C. amycolatum strains. The MICs values ranges proportionally to the inhibition diameter. They ranged from 0.05µl to 50µl. MBCs results showed either bacteriostatic or bactericide effect of this EO (Table III) Similarly, eucalyptus oil was used in various studies where its activity was found either moderate [8,18,19] or low [20]. Singh et al [8] found that Eucalyptus oil inhibited growth in S. indicum and E. coli but the area of inhibition zone was much less in comparison to clove oil, while Staphylococcus and Bacillus strains were found completely insensitive to eucalyptus oil. However, other studies reported that the extract of E. globulus potently inhibited the growth of E. coli, S. aureus [21] and fish pathogenic bacteria [22]. The antibacterial activity of Eucalyptus extracts has been due to his principal constituents oxygenated monoterpenes and terpinen-4-ol and other components such as α-pinene, β-pinene, α-phellandrene, 1,8cineole, limonene, terpine-4-ol, aromadendrene, epiglobulol, piperitone and globulol [23]. It seemed that the presence of 1,8-cineole and α-phellandrene coupled with low antioxidant activity and high cytotoxic effect makes eucalyptus oil less effective against the control of bacterial growth. Thus, eucalyptus oil is not much suited for medicinal purposes, but can be used as repellant or anti-feedant in insecticidal formulation [8]. In this study, sage oil has shown the lowest effect against our strains. All the isolates tested exhibited diameter ≤15mm. Only undiluted oil was active against the strains. The MICs value obtained were high for almost all the strains. Sepahvand et al [24] has demonstrated that S. sclarea essential oil did have different anti-bacterial effects among the microorganisms tested. In accordance to our study, it has a low effect on P. aeruginosa. Unlikely, Cui et al [9] proved that this oil was highly lethal to E. coli, S. aureus, B. pumilus, B. subtilis, K. pneumoniae, S. typhimurium and P. aeruginosa. S. sclarea oil was an effective bacterial inhibitor and bactericide with a broad antibacterial spectrum. It damaged the cell membrane and changed its permeability, leading to the release of some cytoplasm such as macromolecular substances, ATP and DNA. The antimicrobial action of S. sclarea essential oil is not only attributable to a unique pathway, but also involves a series of events both on the cell surface and within the cytoplasm. Two diterpenoids, salvipisone and aethiopinone contained in Salvia sclarea are known for their high antibacterial activity [24]. In our study, we noted that C. amycolatum was the most insensitive specie among the strains tested. This may be explained by the particular structure of his cell wall which lacked mycolic acid but contains a cation-selective cell wall channel that may be responsible for the limited permeability of the cell wall to EO and notably to different antibiotics. The structure of the cellular walls justifies also the differences observed between Grampositive and Gram-negative bacteria [9]. We observed that the susceptibility an EO varied among different species included in the study and among strains belonging to the same species. These discrepancies may be explained by the same mechanisms of antibiotic resistance including adaptation of a strain to ecological environment, selection pression and cross breeding [25]. Among the 24 strains tested, 9 (37.5%) strains showed resistance to tigecycline. Salas et al. [26] and Fernandez-Roblas et al. [27] reported good activity of this antibiotic against coryneform bacteria. Tigecyclin Auctores Publishing -Volume 2(3)-020 www.auctoresonline.org ISSN: 2690-1919 Page 4 of 5 was more potent than E. globulus and S. sclarea. However, the diameters of inhibition zone of E. caryophyllata were higher than the tigecycline for the majority of the strains. This fact proved the potential activity of clove oil against Corynebacterium spp.

Conclusion
In this study, results obtained showed that clove EO had the highest activity against MDR Corynebacterium species. His activity was slightly higher than tigecycline. However, the sage oil was inactive on all the species tested. E. caryophyllata EO seems to be an important alternative treatment in nosocomial Corynebacterium infections ranging from cutaneous infections to urinary tract infections.

Conflict of interest:
None.