Introduction

Bacterial resistance to conventional antimicrobial agents (AMR) has been described as one of the world’s major healthcare concerns, as it is associated with increased morbidity, mortality, and economic loss [1]. AMR is estimated to be result in around 33,000 deaths in the EU and 700,000 deaths globally per year, with annual economic costs of 1.5 billion Euros in the EU [2]. It has been estimated that globally AMR might cause more deaths than cancer by 2050 with a projected cost to be around 100 trillion dollars [1]. This healthcare problem requires the development of novel effective antimicrobial materials tools to limit the usage of current antibiotics, and to relieve the burden of infection [3]. With no new drugs in the pipeline, essential oils (EOs) have emerged as alternative antimicrobial products due to their strong and wide-spectrum activity against microorganisms, in addition to their ecofriendly and human safety status [4, 5]. Many medicinal plants with their secondary metabolites (including EOs) form the basis for commercial pharmaceutical drugs and herbal remedies. However, the doses of these EOs required to achieve an effective antimicrobial activity may exceed consumer’s preference [4]. To counteract this problem in the context of AMR, combination of EOs with traditional antibiotics to enhance their effectiveness and consequently to decrease their efficacious doses has been investigated [6,7,8]. Many recent studies report that the synergistic combination of EOs and conventional antimicrobials has proved to be an effective solution and a practical alternative to develop drugs with increased efficacy and low toxicity [5, 7, 9]. Their inherent ability to suppress development of resistance has been attributed to some plant EOs being able to sensitize bacteria to the action of antibiotics [7]. In addition, EOs with their complex mixtures containing different compounds with variable chemical structures, can act through different mechanisms and against target sites compared to antibiotics which commonly target relatively few sites [7]. Different mechanisms of synergy exhibited by EO-antibiotic combinations have been suggested, including the inhibition of efflux pumps implicated in exporting antimicrobials outside the bacterial cells, the inhibition of the production or activity of enzymes (e.g., beta lactamases, lipases, coagulases, and/or amino acid decarboxylases), the permeabilization and disruption of bacterial membranes by inhibiting ATPase activity, the alteration of the cell wall composition, the inhibition of membrane-located metabolic events leading to inhibition of respiration, leakage of cell ions, and reduction of proton motive force, among others [5, 10,11,12,13].

Thymus species are of special interest with respect to the investigation of their natural antimicrobial agents and the possible synergistic effect of their plant volatile extracts with antibiotics, mainly due to their richness in EOs, and their strong antimicrobial activity [14, 15]. Morocco is rich in Thymus species with 22 species, of which 15 are endemic [16]. Among the endemic species, T. broussonnetii Boiss., known locally as “Zaater Essouiri,” is a spontaneous, perennial aromatic shrub extensively used in Moroccan folk medicine, and as an excellent food preservative [17]. Previous investigations have shown interesting antimicrobial activity and a high level of synergism by T. broussonnetii EO with some antibiotics [13, 18]. To our knowledge, no information is available on the antibacterial synergistic potential of T. broussonnetii EO with some other medicinal plant EOs, or on the combinational effect of EO mixtures based on this valuable Thymus species with conventional antimicrobials. As far as we know, this is the first study to improve the antibiotic efficacy by some effective EO mixtures based on T. broussonnetii EO. Thus, the present work aimed to investigate the combined antimicrobial potential of T. broussonnetii EO with some selected medicinal plant EOs, namely Myrtus communis, Artemisia herba alba, Thymus pallidus, Thymus satureioides, Teucrium polium, and Rosmarinus officinalis. We also investigated the antibacterial effect of the most effective EO mixtures based on T. broussonnetii EO in combination with two commonly used antibiotics (streptomycin and ciprofloxacin).

Materials and Methods

Plant material and EO Extraction

Aerial parts of M. communis, A. herba alba, T. pallidus, T. satureioides, T. polium, R. officinalis, and T. broussonnetii were harvested from different rural locations in Morocco (Table 1). The medicinal plants used in the present work were selected based on their therapeutic usages and antimicrobial properties. In fact, Thymus species, in particular, T. broussonnetii, T. pallidus, and T. satureioides aerial parts are well known in Moroccan folk medicine for their high antimicrobial and antiseptic properties and for the treatment of many infections such as gastrointestinal, respiratory, genital, and skin diseases. Because of their antiseptic activities, the aerial part of R. officinalis, M. communis, and A. herba alba are also widely used as herbal remedies for the treatment of chronic wounds, eye, and gastrointestinal infections, respectively [17]. The identification of plant species was undertaken by one of the authors (A. Abbad), and voucher specimens have been deposited at the Laboratory of Microbial Biotechnologies, Agrosciences and Environment of the Faculty of Science Semlalia, University Cadi Ayyad, Marrakech. The plant samples were dried at ambient temperature (≈25 °C) for EO isolation. Each dried plant material was submitted to three successive steam distillations (3 × 200 g) for about 3 h using a Clevenger-type apparatus, and the resulting oils were dried with anhydrous sodium sulfate (Na2SO4), weighed, and kept at 4 °C for further analysis. The EO yields were expressed in % (v/w) of the dry material using the following equation: % Yield of the EO = (Volume of the extracted EO (mL) ∕ Weight of the dried plant material (g)) × 100.

Table 1 Collection site and geographical coordinates of the studied Moroccan aromatic plants and their EO yields
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GC/FID and GC/MS Analysis

The qualitative and quantitative identifications of the EO constituents were conducted using gas chromatography with Flame Ionization Detection (GC/FID) and gas chromatography coupled to mass spectrometry (GC/MS) analysis. The GC/FID was performed using a Shimadzu GC-2010 instruments equipped with an SLB-5MS Supelco (Milan, Italy), 30 m × 0.25 mm i.d. × 0.25 μm d, coated with 5% diphenyl-95% polydimethylsiloxane. Helium (flow rate 1 mL/mn) was used as a carrier gas. The initial oven temperature was 50 °C, the final temperature was 270 °C, and the temperature gradient was 4 °C/min. The injector and detector temperature were 280 °C and the injected volume of EO was 2.0 μL using the split mode. The GC/MS analysis was carried out using a gas chromatograph equipped with a TG-5MS column (length: 30 m; internal diameter: 0.25 mm, thickness film: 0.25 µm), and coupled to a mass selective detector ISQ (Single Quadrupole Mass spectrometer). The oven temperature was programmed to rise from 60 to 246 °C at 3 °C/min. Transfer, source, and quadrupole temperatures were 280 °C, 230 °C, and 150 °C, respectively, operating at 70 eV ionization energy and scanning the m/z range 41–450. EOs samples (20 μL) were diluted with hexane (2mL). 1 μL was injected at 260 °C in split mode (1/50). Identification of the individual components was based on the comparison of their mass spectra with NIST MS Search database and Adams terpene library, and comparison of their retention indices (RI) on a TG-5MS. The RI was calculated relative to the retention times of a series of n-alkanes (C5-C30), using the Van den Dool and Kratz equation [19], with linear interpolation with those of authentic compounds or literature data. For semi-quantification purposes, the normalized peak area of each compound was used without any correction factors to establish abundances. RI and peak area percentages were calculated as mean values of three injections.

Determination of Antibacterial Activity

Chemicals and Culture Media

Antibiotics selected for this study (streptomycin and ciprofloxacin) and chemicals (DMSO) were purchased from Sigma-Aldrich, while culture media, MHA, and MHB were obtained from Biokar.

Antibacterial Screening

The individual EOs and their binary combinations were first screened for their antibacterial activity against six bacterial pathogenic strains provided by the American type culture collection through the QualiUp Groupe (Agadir, Morocco), known for their low susceptibility to most conventional antimicrobials [13]: Staphylococcus aureus (ATCC 6538), Bacillus subtilis (ATCC 6633), Escherichia coli (ATCC 25922), Enterococcus faecalis (ATCC19433), Listeria monocytogenes (ATCC13932), and Salmonella enterica (ATCC 14028).

The screening of antibacterial activity of each EO was evaluated using the agar disk diffusion method following the CLSI instructions with some modifications [20]. For this, sterile 6 mm diameter disks impregnated with 10 µL of each EO were applied to the surface of MHA plates previously seeded with 0.1 mL of bacterial suspensions at 108 CFU/mL, equivalent to 0.5 McFarland. All plates were kept at 4°C for 2 h in order to allow EOs diffusion, and then incubated at 37 °C for 18–24 h. After incubation, IZ were measured in mm (including disk diameter). Streptomycin (10 μg/disk) and ciprofloxacin (5 μg/disk) were used as positive controls. All assays were carried out in triplicate.

Determination of MIC and MBC

Quantitative analyses of EO antibacterial activity were evaluated using the broth microdilution method according to the CLSI guidelines M07-A10 [21], with slight modifications. Briefly, 100 µL of twofold serial dilutions of each EO (previously prepared in MHB using 1% DMSO) were added to microwells containing 100 µL of bacterial suspensions at 108 CFU/mL. DMSO was used as negative control. The microplates were incubated for 24 h at 37 °C, and MICs (mg/mL) were determined as the lowest concentration of the oils contributing to inhibition of macroscopic bacterial growth. To determine the MBC (mg/mL), 0.1 mL of all clear wells which did not show any visible growth during MIC assays were spread on MHA plates, and incubated at 37 °C for 24 h. The MBC was defined as the lowest concentration of the EO required to kill 99.99% of the incubated bacteria [20]. All assays were conducted in triplicate. Streptomycin and ciprofloxacin were used as standard antibacterial drugs. A negative control was prepared using a concentration of 1% DMSO.

Checkerboard Method

The Checkerboard method is an in vitro technique used to determine the activity of combination of antimicrobial substances. In this study, the binary antibacterial interactions between T. broussonnetii EO at MIC/4 as previously determined [14], and other selected medicinal plant EOs against some pathogenic bacteria were investigated as described by Kwiatkowski et al. [22]. 50 µL of T. broussonnetii EO at MIC/4 was added to each well, containing 50 µL of eight serial twofold dilutions (from MIC to MIC/128) of each other plant EO, in such a way that each well contained a unique combination of the two different EOs. Then, 100 µL of bacterial suspensions at 108 CFU/mL, equivalent to 0.5 McFarland, were added to each well and incubated at 37 °C for 24h. The FICI used for the determination of the nature of the interaction between two EOs was calculated following the formula described by Skroza et al. [23]. The interactions between the most effective EO binary combinations based on T. broussonnetii EO, and the antibiotics were carried out using the same methodology. Briefly, 50 µL of each effective EO combination at MIC/4 was added to each well, containing 50 µL of eight serial twofold dilutions (from MIC to MIC/128) of antibiotics in such a way that each well contained a unique combination of a binary EO mixture (50 µL) and an antibiotic (50 µL). Then, 100 µL of bacterial suspensions at 108 CFU/mL, equivalent to 0.5 McFarland, was added to each well, and incubated at 37 °C for 24h.

FICI was calculated as follows:

$${\text{FICI}} = {\text{ FIC }}\left( {{\text{EO}}_{{1}} } \right) \, + {\text{ FIC }}\left( {{\text{EO}}_{{2}} } \right)$$

With FIC (EO1) = MIC of EO1 in combination with EO2/MIC of EO1 alone, and FIC (EO2) = MIC of EO2 in combination with EO1/MIC of EO2 alone.

The results were interpreted as following: synergism (FICI ≤ 0.5), addition (0.5 < FICI ≤ 1.0), neutral (1 < FICI ≤ 4.0), or antagonism (FICI > 4.0).

The MIC gain of the antibiotics was calculated as the antibiotic MIC alone divided by the MIC of antibiotic combined with EO.

The FICI values of the effective EO combinations and antibiotics against the tested bacterial strains were determined according to the same formula.

Statistical Analysis

Data were expressed as the mean ± Standard deviation, group means were compared by one-way ANOVA and Tukey test to identify significance (p < 0.05) among groups using SPSS 21.0 statistical software.

Results

Yield and Chemical Composition of EOs

The yield of EOs isolated by steam distillation from aerial parts of the studied plants varied from 0.2% ± 0.21 to 2.80% ± 0.45 (Table 1). The EO chemical compositions analyzed by GC-SM led to the identification of 56 compounds accounting for between 86.69 and 99.16% of the total oils. Monoterpene hydrocarbons constituted the principal sub-class in T. satureioides EO, while oxygenated monoterpenes were dominant in M. communis, A. herba alba, T. pallidus, R. officinalis, and T. broussonnetii EOs. In T. polium EO, the dominant chemical family was sesquiterpene hydrocarbons.

With respect to particular chemical constituents, M. communis and R. officinalis were dominated by 1,8-cineole at 42.00% and 45.79%, respectively. In addition to this oxygenated monoterpene, M. communis EO was characterized by the presence of α-pinene (19.73%) and myrtenyl acetate (23.63%) as other major constituents, while camphor (21.86%) was the other main component of R. officinalis EO (Table 2). EOs obtained from T. pallidus, T. satureioides, and T. broussonnetii were dominated, respectively, by thymol (25.12%)/p-cymene (18.74%)/γ-terpinene (15.99%), borneol (25.10%)/camphene (23.20%)/p-cymene (13.70%)/α-pinene (11.50%), and carvacrol (44.39%)/thymol (21.47%)/γ-terpinene (9.09%). The most abundant compounds in A. herba alba were β-thujone (37.83%), α-thujone (23.70%), and camphor (12.70%), while the main constituents of T. polium EO were germacrene (29.4%), α-pinene (18.05%), and bicyclogermacrene (14.5%).

Table 2 Chemical composition of the EOs isolated from the aerial parts of studied Moroccan aromatic plants
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Antibacterial Activity of Individual EOs

The in vitro antibacterial activity of the tested EOs against S. aureus, B. subtilis, E. coli, E. faecalis, L. monocytogenes, and S. enterica was determined using disk diffusion and broth microdilution assays. All tested EOs exhibited varying levels of activity against these bacterial species. In general, the EOs obtained from Thymus species showed excellent antibacterial activity, with IZ and MIC values ranging from 13.99 ± 0.15 to 40.09 ± 0.13 mm and 0.140 to 1.125 mg/mL, respectively (Tables 3 and 4). T. broussonnetii EO displayed the highest antibacterial efficacy (IZ = 21.61 ± 0.03–40.09 ± 0.02 mm and MIC = 0.140–0.280 mg/mL), which was surprisingly, more active than the tested antibiotics against some bacteria strains (Tables 3 and 4). M. communis and R. officinalis EOs exhibited moderate inhibitory effects (IZ = 7.77 ± 0.19–14.38 ± 0.13 mm and MIC = 4.5–144 mg/mL for M. communis, and IZ = 6.47 ± 0.21–14.00 ± 0.06 and MIC = 1.125 mg/mL–> 144 mg/mL for R. officinalis). The weakest antibacterial activity against the tested bacteria was observed with A. herba alba and T. polium EOs (Tables 3 & 4). The antibacterial results also showed that the all EOs were more effective against the Gram-positive bacteria than the Gram-negative ones. Among the Gram-positive bacteria screened, S. aureus was the most sensitive to all tested EOs.

Table 3 Inhibition zone of tested EOs and selected antibiotics against six bacterial strains
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Table 4 Minimal inhibition concentration (MIC) and minimal bactericidal concentration (MBC) of tested EOs and antibiotics against six bacterial strains
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Antibacterial Activities of Different EO Combinations

Synergistic interactions between T. broussonnetii at MIC/4 and selected plant EOs were determined using the checkerboard method. The binary combinations of these EOs were tested against the six bacterial species assayed for sensitivity test to EOs (viz. S. aureus, B. subtilis, E. coli, E. faecalis, L. monocytogenes, and S. enterica). The results showed that from the 34 tested combinations, 9 (26.47%) displayed synergism and 15 (44,12%) had neutral interactions (Table 5). The greatest synergistic effect was observed with the EO combination of T. broussonnetii and T. pallidus against S. aureus, E. coli, and S. enterica, with FICI = 0.258 (Table 5). The synergistic effect of T. broussonnetii in combination with the other plant EOs varied in relation to the bacterial species tested. Against S. aureus, except for A. herba alba EO, all other EOs showed synergism in combination with T. broussonnetii EO. In addition, the combination of T. broussonnetii and R. officinalis or M. communis EOs showed synergism against E. faecalis and L. monocytogenes, respectively (Table 5).

Table 5 Fractional inhibitory concentration index (FICI) of most effective binary combinations of T. broussonnetii EO (at MIC/4) with other plant EOs
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Synergistic Interaction Between Effective EOs Combinations and Antibiotics

The synergistic antibacterial properties of effective EO mixtures based on T. broussonnetii EO (at MIC/4) with two conventional antibiotics (streptomycin and ciprofloxacin) against S. aureus, E. coli, E. faecalis, L. monocytogenes, and S. enterica were investigated using the checkerboard method. The results showed varying degrees of synergistic interaction between the antibiotics and the EO mixtures depending on bacterial strains and combination type (Tables 6 & 7). The greatest synergistic antibacterial effects were obtained with the combination of T. broussonnetii and T. pallidus EOs with the two tested antibiotics streptomycin and ciprofloxacin. The association of this EO mixture with streptomycin achieved gains of 8-, 64-, and 128-fold for S. aureus, S. enterica, and E. coli, respectively, while with ciprofloxacin, the gains reached 64-and 128-fold for S. enterica and E. coli, respectively (Tables 6 and 7). Interestingly, the T. broussonnetii and M. communis EO mixture showed high synergy with the two antibiotics with a gain of 128-fold against L. monocytogenes (Tables 6 and 7). Other synergisms between the mixtures of T. broussonnetii with R. officinalis, T. polium, or T. satureioides Eos, and the antibiotics were also observed, with a gain of fourfold against S. aureus.

Table 6 Fractional inhibitory concentration index (FICI) of streptomycin and most effective EO combinations of T. broussonnetii with some selected medicinal plants
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Table 7 Fractional inhibitory concentration index (FICI) of ciprofloxacin and most effective EO associations of T. broussonnetii with some selected medicinal plants
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Discussion

The emergence of antibiotic-resistant bacterial pathogens has rendered most available antibiotics ineffective [24]. Alternative strategies are, therefore, needed to counteract drug-resistant bacterial infections. Combination therapies between EOs and conventional drugs to enhance their efficacy appear to be the most effective solution. In this study, the antibacterial effect of Moroccan endemic Thymus broussonnetii EO alone, and in combination with EOs obtained from some other medicinal plants namely, Myrtus communis., Artemisia herba alba., Thymus pallidus., Thymus satureioides., Teucrium polium., and Rosmarinus officinalis were evaluated for the first time against some well-known multidrug-resistant bacteria, namely S. aureus, B. subtilis, E. coli, E. faecalis, L. monocytogenes, and S. enterica. The synergistic interactions between the most effective EO combinations (based on T. broussonnetii EO) with two conventional antibiotics (streptomycin and ciprofloxacin) were also investigated.

The EOs extracted by steam distillation from aerial parts of the investigated plants yielded variable values, which are consistent with those reported in several previous studies [25,26,27,28,29,30]. The chemical analyses showed different qualitative and quantitative EO profiles. 1,8-cineole was found to be the major constituent characterizing M. communis and R. officinalis EOs, which is in agreement with results previously reported by Farah et al. [28] and Pesavento et al. [31]. Thymol was the major compound in the T. pallidus EO, while borneol and carvacrol were the most abundant constituents in T. satureioides and T. broussonnetii EOs, respectively, which are in accordance with findings of Mohamed et al. [30], El Asbahani et al. [32], and Tagnaout et al. [33]. The most abundant compound in A. herba alba was β-thujone, while the main constituents of T. polium EO were germacrene and α-pinene; these findings are in line with those reported by Barroso et al. [26] and Belmekki et al. [34].

Thymus species EOs exhibited significant antibacterial activity against all tested bacteria. Interestingly, T. broussonnetii EO exhibited the highest antibacterial efficacy, which is mainly attributable to high levels of its bioactive isomeric monoterpenoid constituents, carvacrol and thymol [35]. The antibacterial activity of these phenolic monoterpenes has been attributed to their chemical structure characterized by the presence of the hydroxyl group, and their strong synergistic effect when applied together [36]. It has been well established that these phenolic monoterpenes can exert their antimicrobial effect through several modes of action against different sites at the cellular level including: disruption of bacterial inner membranes making the structures more permeable, inhibition of microbial motility, inhibition of microbial ATPase, and/or inhibition of efflux pump [13, 37, 38]. The presence of other compounds, including p-cymene, borneol, and γ-terpinene may also contribute to the activity of these oils [39]. The strong antibacterial activity observed in Thymus EOs is consistent with reports from previous studies [13, 40, 41]. The EOs of M. communis and R. officinalis were found to be have antimicrobial activity but at levels lower than Thymus spp. EO. This moderate activity mainly due to their richness in some monoterpenes (e.g., 1,8-cineole and α-pinene), well known for their moderate antibacterial potency [42]. The results overall showed that the studied EOs were more effective against Gram-positive bacteria than Gram-negative ones, which is consistent with many previous reports, likely related to their different bacterial cell coating structures [13, 43, 44]. Among the Gram-positive bacteria tested, S. aureus was the most sensitive strain to all EOs.

With regard to synergistic interactions between T. broussonnetii EO and the other plant EOs, the combination of T. broussonnetii and T. pallidus showed the highest synergistic antibacterial effects against S. aureus, E. coli, and S. enterica. This result may be attributable to the positive interaction between their major constituents, in particular the phenolic monoterpenes thymol and carvacrol, and with their precursors γ-terpinene and p-cymene [38, 45]. The synergistic antibacterial action between carvacrol and thymol has been ascribed to their capacity to act at different microbial targets [46]. The positive interaction between carvacrol and/or thymol and the main precursors in their biogenetic pathway, p-cymene, and γ-terpinene has been demonstrated in previous reports [47]. Indeed, it has been reported that the hydrophobic nature of p-cymene and γ-terpinene, leads to a swelling of the cytoplasmic membrane that can permit carvacrol and/or thymol to be more easily transported into the bacterial cells [48]. Synergistic interaction associated the binary EO combinations of T. broussonnetii and M. communis, R. officinalis, T. pallidus, T. satureioides, or T. polium were demonstrated against S. aureus. In addition, the combinations of T. broussonnetii and R. officinalis, and T. broussonnetii and M. communis EOs showed synergism against E. faecalis and L. monocytogenes, respectively. This can be explained by the chemical profiles of these EOs, which are characterized by the abundance of carvacrol and 1,8-cineole. The synergistic interaction between carvacrol and 1,8-cineole has been previously reported detected against several microorganisms [49, 50], and it has been demonstrated that the combination of carvacrol and 1,8-cineole can disrupt cell membrane permeability of bacterial cells, resulting in the outflow of intracellular functional substances, disturbing normal cell functions, and destroying the cell structure, leading to inactivation of the cells [51]. The synergistic effects of combined T. broussonnetii and T. polium EOs against S. aureus may be the result of a possible positive interaction between carvacrol and α-pinene and/or germacrene.

The interaction of EOs with antibiotics is one of the promising ways to overcome the counter resistance in pathogenic bacteria. A strong synergistic effect was demonstrated between T. broussonnetii and T. pallidus EO mixture with the two tested antibiotics. This results is likely to be a result of the previously reported positive interactions between the phenolic monoterpenes carvacrol and thymol with the antibiotics, [52, 53]. There was also high synergistic activity from the combination of T. broussonnetii and M. communis EO mixture with each of the antibiotics.

Conclusions

Our results demonstrate that EO obtained from T. broussonnetii has substantial antibacterial activity, even higher than the two tested antibiotics against some bacterial strains. Combining the EO of this Moroccan endemic Thymus species with some other selected plant EOs showed strong synergistic activity against some multidrug-resistant bacteria, with superior synergism when T. broussonnetii EO was combined with T. pallidus EOs. Furthermore, this effective EO mixture synergistically enhanced the antimicrobial activity of the two antibiotics streptomycin and ciprofloxacin against a range of bacterial strains. This interesting EO mixture is, therefore, suggested as a potential natural adjuvant to potentiate these two conventional antimicrobials, and possibly others. Other effective EO combinations containing T. broussonnetii EO were also recorded. Most of these efficacious EO mixtures enhanced the antimicrobial activity of the two tested antibiotics, but to varying degrees and also differed with different bacterial strains. Overall, these results represent the basis for further in vivo investigations, which could ultimately lead to development of new antimicrobial agents based on T. broussonnetii EO, and therefore, contribute to enhancing the efficacy of antibiotics in controlling multidrug-resistant pathogenic bacteria. However, additional investigations are needed to determine the compounds responsible for the antibacterial activity in complex mixtures and to elucidate the likely modes of action responsible for their synergistic effect when combined with antibiotics.