Synergistic effects of Ruta montana ( Clus . ) L . essential oil and antibiotics against some pathogenic bacteria

Antibiotic resistance has been called one of the world’s most pressing public health threats. The combination of essential oils with conventional antibiotics is one of the emerging approaches that could help prevent this problem. In light of this, the present study aimed to investigate the impact of the combination of Ruta montana essential oil with conventional antibiotics on some pathogenic bacteria. The essential oil isolated by hydrodistillation was first analyzed using GC-MS then tested alone and in combination with five recommended antibiotics against three bacterial strains by the agar disc diffusion and broth micro-dilution methods. Out of forty-nine peaks, thirty-eight components were identified representing 98.17% of the total oil composition. The major components were 2-Undecanone (63.39%), 2-Nonanone (5.65%), 2-Acetoxytetradecane (4.94%), 2-Decanone (4.47%) and 2-Dodecanone (3.35%). While R. montana essential oil showed only weak antibacterial activity compared to the antibiotics tested alone, unexpectedly, the combination of RM essential oil with antibiotics remarkably increased the antibacterial activity of the antibiotics through synergistic effects in up to 70% of cases. These results suggest that combining antibiotics with essential oils, even those with low antibacterial activity, may be effective in overcoming problems caused by increasing bacterial resistance.


INTRODUCTION
The increased prevalence of bacterial resistance is one of the major problems of global health today ( Levy & Marshall, 2004). Faced with such a situation, the high antimicrobial potential of essential oils (EOs), their ability to improve the effectiveness of antibiotics (ABs) and the lack of any apparent emergence of bacterial resistance to them or their components make them valuable subjects of research (Bakkali et al., 2008;Sienkiewicz et al., 2017). In fact, essential oils are made up of many molecules, which make bacteria vulnerable to antibiotics (Sienkiewicz et al., 2017). The occurrence of synergism is thought to be the key to the bioactivity of EOs (Boonyanugomol et al., 2017;Chouhan et al., 2017). Therefore, the association between EOs and ABs has emerged as a novel approach in controlling multidrug-resistant (MDR) strains and modulating the activity of ABs (Boonyanugomol et al. 2017;Chouhan et al., 2017;Boudjedjou et al., 2018Boudjedjou et al., , 2019. Ruta montana is one of the four species of the genus Ruta, from Rutaceae family; grow wild in Algeria (Quézel & Santa, 1963). All are perennial herbaceous plants with yellow flowers, characterized by a strong, foul-smelling, nauseating odor, due to an essential oil contained in enormous secretory pockets (Hammiche et al., 2013). In folk medicine, R. montana is used for the treatment of persistent cough by fumigation. The infusion or decoction of the aerial parts in milk is used for all problems related to the female genital system such as painful periods and after childbirth. Their decoction in olive oil is used for rheumatism and body aches. Their infusions are used as eye drops for corneal ulcers, as ear drops for otitis and tinnitus, as nasal drops to treat atrophic rhinitis, for fever and vomiting in infants and children (Hammiche et al., 2013).
The chemical composition of Ruta montana essential oils (RMEOs) from various localities and harvested at different seasons have been reported in several papers. The results showed that RMEOs are characterized by the predominance of 2-ketones, such as 2-undecanone and 2-decanone, whereas terpene components were present in lower amounts, with the exception of caryophyllene oxide which occurs as a major component of RMEOs (Bennaoum et al., 2017). The variation in the content of terpene components can be ascribed to many factors, such as the harvesting period (Bennaoum et al., 2017), plant organ (Khadhri et al., 2014), and geographical origin (Mohammedi et al., 2020).
The antibacterial activity of RMEOs seems a bit controversial. Yet, several studies have reported a moderate to the strong antibacterial activity of RMEOs (Belkessame et al., 2011;Zellagui et al., 2012;Hazzit et al., 2015;Daoudi et al., 2016;Fekhar et al., 2017;Benali et al., 2020;Drioiche et al., 2020;Mohammedi et al., 2020). However, to our knowledge, there are no reports on the combination of RMEOs with antibiotics. Hence, this study aimed to investigate the chemical composition and to compare the antibacterial activity of R. montana essential oil alone and in combination with some recommended antibiotics against three pathogenic bacteria.

Plant Material
Arial parts of Ruta montana (Clus.) L. were collected in May 2015, during the period of full flowering, from T'kout, department of Batna in Algeria, at 35.0547 (latitude in decimal degrees) and 6.2235 (longitude in decimal degrees). The identification of plant material has been demonstrated according to the flora of Quezel & Santa (1963). A voucher specimen has been deposited in the Herbarium of our laboratory under the code RUT-001-1-2015. The plant material was cleaned of impurities, dried at room temperature in the dark for two weeks, then cut into small pieces not exceeding 1 cm and kept in paper bags to be used for the extraction of essential oils.

Extraction and Analysis of the Essential Oil
The air-dried plant material (150 g) was subjected to steam hydrodistillation for 3 h using a Clevenger-type apparatus (Clevenger, 1928). The oil obtained was stored in a sealed vial in the dark at 4 °C until analysis. Quantitative and qualitative analyses of the collected oil were determined by gas chromatography coupled with mass spectrometry (GC/MS). Mass spectra of EO were obtained using a GC focus (Thermo) with BR5-MS column (5% phenyl methyl siloxane), 30 m long and 0.32 mm i.d., with 0.25 µm film thickness (Bruker). Coupled to a mass spectrometer (MS) type DSQII (Thermo) with a detector impact of electrons, 70 eV. The carrier gas is helium at a rate of 1.2 mL min -1 ; the injection volume was 0.1 µL; injector split mode 1:100. The initial temperature of the column was kept at 70 ºC for 1 min and programmed to 300 ºC at a rate of 10 ºC.min -1 and kept constant at 300 ºC for 5 min. The mass spectrum of each compound was recorded between 40 and 500 Da (m/z equivalent unit). Identification of compounds was achieved by comparison of their recorded mass spectra with those of a computer library (NIST2008 v2.0/ Xcalibur data system) provided by the instrument software, and of their retention indices with literature data (Adams, 2001). Retention indices (RI) were calculated by the retention times of a series of n-alkanes.

Antibacterial Screening
The antibacterial activity of R. montana EO was tested against three bacterial strains (Staphylococcus aureus ATCC25923, Pseudomonas aeruginosa ATCC27853, and Escherichia coli ATCC25922) using two different methods: the agar disc diffusion and the Broth micro-dilution methods respectively.

Agar disc diffusion assay
The agar disc diffusion assay was performed as recommended by the Food and Drug Administration (FDA) and the Clinical and Laboratory Standards Institute (CLSI) (de Sousa Eduardo et al., 2018). Sterile filter discs (6 mm in diameter) impregnated with 10 µL/disk of the RMEO were placed onto the Petri dishes containing 20 mL of Mueller Hinton Agar (MHA) and inoculated with the tested bacteria (10 8 CFU mL -1 ). The plates were then incubated at 37 °C for 24 h. The antibacterial activity was evaluated by measuring the diameter of the inhibition zone around the discs. Standard discs of five conventional antibiotics: gentamycin 10 µg disk -1 , amoxicillin 25 µg disk -1 , cefazolin 30 µg disk -1 , tetracycline 30 µg disk -1 , and Amoxicillin/Clavulanic Acid (claventin) 20/10 µg disk -1 , were used as positive control, whilst discs soaked with 10 µLdimethyl sulfoxide (DMSO) were used as the negative control (no zone inhibition was observed).

Determination of the MIC and MBC
The in vitro minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of RMEO were determined using a microdilution assay as described by de Sousa Eduardo et al. (2018) with modification. The serial two-fold dilutions of the tested EO were prepared in standard sterile 96-well flat bottom microplates and the layout was designed so that each row covered the final dilution of 500 to 3.9 µL mL -1 . One hundred microliters of MH broth media and 10 µL (10 8 CFU mL -1 ) of the bacterial culture were added to each well containing 100 µL of the serially diluted test Zeraib et al. EO, giving a final concentration of the bacteria in the well of approximately 10 5 CFU mL −1 . After inoculation overnight at 37 °C, the optical density (OD) was measured. The minimal concentration that has OD less than the OD of the control was defined as the MIC. The MBC was determined as the lowest concentration at which 99.9% of the bacterial population was killed.

Synergistic Interaction
The combinatory effect of RMEOwith conventional ABs was evaluated using an agar-disc diffusion method as previously described (Moussaoui and Alaoui, 2015). The standard discs of antibiotics were impregnated with 10 µL/disc of the RMEO and were put onto the surface of the inoculated MHA. The plates were incubated for 24h at 37°C after which the inhibition zones were measured. If the value of the inhibition zone of the EO/ AB combination is significantly higher (P <0.05) than the sum of the individual values, this is considered to be a synergistic effect, but if they are equal (P ≥0.05), then this is considered an additive effect. The antagonistic effect occurs when the values of the inhibition zones of both treatments separately EO/AB are significantly greater than the value of their combination (Boudjedjou et al., 2018(Boudjedjou et al., , 2019.

Statistical Analysis
All experiments were carried out in triplicate and the data were reported as the mean ± stander deviation of three samples. Statistical analysis was performed using Statistica 8.0 software, StatSoft Inc., USA (Hill and Lewicki, 2007). Differences were tested for significance by using the one-way ANOVA test (P<0.05).
The obtained EO was chemically characterized using GC-MS (Table 1, Figure 1). Table 1 depicts the component's identification and their percentages, as well as the RT and RI values, which are listed in order of their elution from the BR5-MS capillary column. Thirty-eight components were identified, representing 98.17% of the total essential oil components. In addition, eleven unidentified compounds were present in the sample, representing 1.7% (from 0.09 to 0.43%) of the total oil. As expected, the aliphatic ketone 2-Undecanone was found to be the major component of RMEO (63.39%), followed by 2-Nonanone (5.65%), 2-Acetoxytetradecane (4.94%), 2-Decanone (4.47%), and 2-Dodecanone (3.35%). Among the main compounds identified, there is also the sesquiterpene caryophyllene oxide (2.34 %). This is in agreement with most of the previous literature on the chemical composition of RMEOs confirming the predominance of 2-Undecanone (Kambouche et al., 2008;Boutoumi et al., 2009;Belkessam et al., 2011;Zellagui et al., 2012;Ferhat et al., 2014;Khadhri et al., 2014;Hazzit et al., 2015;Bennaoum et al., 2017;Benali et al., 2020;Drioiche et al., 2020;Mohammedi et al., 2020). Nevertheless, the difference in the percentage of some minor and major compounds could be attributed to the status of the plant material (dry or fresh), period of harvesting, geographic origin, and the kind of plant material (Kambouche et al., 2008;Boutoumi et al., 2009;Zellagui et al., 2012;Ferhat et al., 2014, Khadhri et al., 2014Hazzit et al., 2015;Bennaoum et al., 2017;Negri et al., 2020;Mohammedi et al., 2020). Bennaoum et al. (2017), from a study conducted on EOs extracted from 11 samples belonging to three species of the genus Ruta, it has been suggested that the main factors able to influence the chemical composition of RMEO was the harvested period and the geographical origin (Bennaoum et al., 2017). The EOs extracted from plants harvested in spring and winter, as in our case, were characterized by the predominance of ketones, whereas those harvested on summer and autumn seasons were characterized by the predominance of sesquiterpenes and monoterpenes (Bennaoum et al., 2017).

Antibacterial Activity
The results of the antibacterial activity of RMEO and five standard antibiotics against the three selected pathogenic bacteria are compiled in Table 2. Compared to antibiotics for which all the tested bacteria showed more or less significant sensitivity with the exception of claventin against P. aeruginosa, RMEO was found to be ineffective against all the tested strains.
In line with this, the evaluation of MIC and MBC of RMEO (Table 3) showed no or very slight antibacterial activity against E. coli and S. aureus, with MICs of 125µL/mL and 250 µL/mL for S. aureus and E. coli respectively. These results are consistent with some previous studies, which reported that essential oils of Ruta genus displayed no or less antibacterial activity (Merghache et al., 2008;Bnina et al., 2010;Haddouchi et al., 2013). This weak antibacterial activity could be attributed to the high percentage of ketones in the oils (Gibka et al., 2009;Haddouchi et al., 2013). Indeed, the antimicrobial activity of 2-undecanone, the most abundant ketone in this oil, is known to be weak against the bacterial strains (Gibka et al., 2009).
On the other hand, Zellagui et al. (2012) and Bouzidi et al. (2012) reported that the essential oil of R. montana has a strong antibacterial activity against all tested bacterial strains (E. coli, S. aureus, P. aeruginosa, Klebsiella pneumoniae, Mycobacterium kansasii, and Mycobacterium vaccae), with an inhibition diameter that increases as the concentration of the oil extract increases. This activity appears to be related to the relative amount of hydrocarbon and alcohol terpenes (Haddouchi et al., 2013). It is widely accepted that essential oils rich in aldehydes or phenols have the highest antibacterial activity, followed by those containing alcoholic terpenes. Essential oils containing high levels of ketones or esters have much weaker activity, while those containing terpene hydrocarbons are often inactive (Bassolé & Juliani, 2012).

Synergistic Interaction
In order to assess whether or not the addition of essential oils improves the effectiveness of antibiotics, the combined effect of RMEO and five antibiotics recommended against three pathogenic strains were investigated. The results indicated a pronounced antibacterial activity of almost all   ABs/RMEO combinations against the three tested bacterial strains, even against P. aeruginosa (gram-negative) the most resistant strain among the tested ones (Table 4). RMEO showed significant synergistic effects, making bacterial strains more sensitive when combined with gentamycin, amoxicillin, cefazolin, and claventin, as evidenced by the significant increase in the inhibition zone diameters (Figure 2). It should, however, be noted that the GEN/RMEO and TET/RMEO combinations induced antagonistic effects against P. aeruginosa.
It is widely accepted that synergy can occur if the components of a mixture affect different targets (Lewis % Ausubel, 2006;Wagner and Ulrich Merzenich, 2009). Considering the weak RMEO's antibacterial activity, the synergism observed between RMEO and the different ABs is likely due to the fact that EOs facilitates the penetration of ABs into the bacterial cells, since the lipophilic nature of oils can causes expansion of the membrane, increased membrane fluidity and permeability (Sienkiewicz et al., 2017). On the other hand, given the great synergistic effect induced by the interaction between RMEO and the β-lactam antibiotics (amoxicillin, cefazolin, and claventin), which are known to act by inhibiting the synthesis of the peptidoglycan layer of bacterial cell walls (Elander, 2003), it can be also assumed that these ABs facilitates the diffusion of essential oils into the cell.

CONCLUSION
In the present study, the chemical composition and antimicrobial activity of R. montana essential oil alone and in combination with some antibiotics were investigated. The results showed that the combination of R. montana EO with conventional antibiotics, particularly with amoxicillin and cefazolin induced significant synergistic effects against all pathogenic strains tested. Therefore, the combination of antibiotics and essential oils have the potential to be used as an alternative therapeutic treatment, not only to reduce possible adverse effects and the cost of antibiotic-based treatments but also to prevent the development of bacterial resistance. Values were expressed as mean ± standard deviation. Ns, not significant; *, significant (P˂0.05); **, highly significant (P˂0.01); ***, very highly significant (P˂0.001); RMEO: R. montana essential oil; GEN: gentamycin; AMX: amoxicillin; CEF: cefazolin; TET: tetracycline; CLA: claventin; ATCC: American type culture collection.