In-Depth Study of Thymus vulgaris Essential Oil: Towards Understanding the Antibacterial Target Mechanism and Toxicological and Pharmacological Aspects

Questions have been raised apropos the emerging problem of microbial resistance, which may pose a great hazard to the human health. Among biosafe compounds are essential oils which captured consumer draw due to their multifunctional properties compared to chemical medication drugs. Here, we examined the chemical profile and the mechanism(s) of action of the Thymus vulgaris essential oil (TVEO) against a Gram-negative bacterium Salmonella enterica Typhimurium ATTCC 10028 (S. enterica Typhimurium ATTCC 10028) and two Gram-positive bacteria Staphyloccocus aureus ATCC 6538 (S. aureus ATCC 6538) and Listeria monocytogenes ATCC 19117 (L. monocytogenes ATCC 19117). Findings showed that TVEO was principally composed of thymol, o-cymene, and γ-terpinene with 47.44, 16.55, and 7.80%, respectively. Molecular docking simulations stipulated that thymol and β-sesquiphellandrene (a minor compound at 1.37%) could target multiple bacterial pathways including topoisomerase II and DNA and RNA polymerases of the three tested bacteria. This result pointed plausible impairments of the pathogenic bacteria cell replication and transcription processes. Through computational approach, the VEGA quantitative structure–activity relationship (QSAR) model, we revealed that among twenty-six TVEO compounds, sixteen had no toxic effects and could be safe for human consumption as compared to the Food and Drug Administration (FDA) approved drugs (ciprofloxacin and rifamycin SV). Assessed by the SwissADME server, the pharmacokinetic profile of all identified TVEO compounds define their absorption, distribution, metabolism, and excretion (ADME) properties and were assessed. In order to predict their biological activity spectrum based on their chemical structure, all TVEO compounds were subjected to PASS (Prediction of Activity Spectra for Substances) online tool. Results indicated that the tested compounds could have multiple biological activities and various enzymatic targets. Findings of our study support that identified compounds of TVEO can be a safe and effective alternative to synthetic drugs and can easily combats hazardous multidrug-resistant bacteria.


Introduction
In recent years, antimicrobial-resistant (AMR) bacteria have been admitted as a public health risk that could cause an increase in the global burden of infectious disease [1][2][3][4]. For instance, each year, more than 670,000 infections and 700,000 deaths worldwide were provoked by AMR [5,6]. Mutually controlled by host immune condition, microflora organization, and antimicrobial interventions, AMR evolu-tion occurs with the multidrug resistance [7,8]. While AMR could not be pragmatically eradicated, antimicrobials will endure to miss their potency, and, in the close future, more people may die from infections [9][10][11]. Therefore, it is crucial to explore original effective and broad-spectrum antibacterial agents to control bacteria, which can be antibiotic resistant, highly virulent, and high costs for the medication. Among the leading strategies for the exploration and detection of new targets in pathogens are the masterfully using bioinformatics tools. These tools can provide useful information for better comprehension of the interactions between targets and biomolecules and therefore help to anticipate new treatment targets for pathogenic microorganisms [12,13].
Extracted from natural plants, essential oils (EOs) can be exploited as a practical alternative. More specifically, thyme (Thymus vulgaris L.), appertained to the family of Lamiaceae and the genus of Thymus, was consumed for centuries because of its medicinal properties and was generally recognized as safe (GRAS) by the FDA [14][15][16][17]. In addition, essential oil acquired from Thymus vulgaris L. had an extensive range of biological activities [17]. Thyme EO contains high levels of phenolic compounds, like thymol, carvacrol, p-cymene, and γ-terpinene [17,18]. Interestingly, numerous studies reported that thymol, the major antibacterial component occurring in TEO, can destroy the bacterial cell membrane [19,20]. Liu et al. revealed that thymol powerfully inhibit Pseudomans aeruginosa and directly change the cell structure [15]. P. aeruginosa cell membrane integrity is destroyed as evidenced by an increase in permeability of the inner/outer membranes. In other studies, Wang et al. and Lade et al. noted that thymol disrupts the Staphylococcus aureus membrane integrity to achieve the inner structure of the bacterial cell and joints to the minor groove of bacterial DNA, ensuring in a destabilization of the DNA secondary structure [21,22].
In extension of our research to disclose the potential of natural therapeutic agents [13], this current investigation is aimed at elucidating the molecular docking interactions of all components of Thymus EO with bacterial DNA and RNA polymerases, as well as the topoisomerase II of S. aureus, S. enterica Typhimurium, and L. monocytogenes were profoundly reviewed in silico. As a part of our endeavor to increase the potential and exploration of these activities, a pharmacokinetics and computational toxicological studies were well discussed. was helium and has been maintained at 1.2 mL/min flow rate. The oven temperature program was as follows: 1 min at 100°C ramped from 100 to 280°C at 5°C/min and 25 min at 280°C. The chromatograph was equipped with a split/split less injector used in the split less mode. Identification of TVEO components was achieved by matching their mass spectra with Wiley Registry of Mass Spectral Data 7th edition (Agilent Technologies) and National Institute of Standards and Technology 05 MS (NIST) library data.

Materials and Methods
2.2. Antibacterial Activity 2.2.1. Microorganisms and Growth Conditions. In order to evaluate TVEO antibacterial activity, two Gram-positive bacteria: S. aureus ATCC 6538 and L. monocytogenes ATCC 19117, and two Gram-negative bacteria: S. enterica Typhimurium ATCC 14028 and E. coli ATCC 8739, were selected. Bacterial cultures were deposited in 3 mL of Luria-Bertani (LB) agar medium composed of (g/L): peptone, 10; yeast extract, 5; NaCl, 5; and agar, 20 at pH 7.2; then, the bacterial strains were incubated at 37°C according to the method described by [23].

Agar Diffusion
Method. Antimicrobial activity of TVEO was evaluated by agar-well diffusion assay according to a method proposed by Güven et al. [24]. Fifteen milliliters of the molten agar (45°C) were flowed into sterile Petri dishes (Ø 90 mm). Bacterial cell suspensions were prepared, and 100 μL was evenly deposited onto the surface of plates containing LB agar medium. Plates were aseptically dried, and then, 5 mm wells were punched into the agar with a sterile Pasteur pipette. TVEO was dissolved in DMSO, water (1/ 9; v/v) to a final concentration of 1 mg/mL and then filtered through 0.22 μm pore size black polycarbonate filters. 100 μL of this filtered solution was placed into each well, and the plates were incubated at 37°C. Gentamicin (10 μg/wells) was used as a positive control.

Minimal Inhibitory Concentration (MIC)
. MIC is defined as the lowest concentration that could inhibit the visible growth of the tested microorganism. In this context, MIC of TVEO was tested against four pathogenic bacteria which are as follows: two Gram-positive bacteria: S. aureus ATCC 6538 and L. monocytogenes ATCC 19117, and two Gramnegative bacteria: S. enterica Typhimurium ATCC 14028 and E. coli ATCC 8739, using the microdilution method with serial dilution described by Chandrasekaran and Venkatesalu [25]. Then, bacterial suspension was added with a final inoculum concentration of 10 6 CF/mL. The contents of the tubes were mixed by pipetting and were incubated for 24 h at 37°C. For the antibacterial activity determination (inhibition zones and CMIs), each experiment was carried out simultaneously in triplicate under same conditions. The obtained diameters of inhibition zones were measured in mm and the MIC values were reported in mg/mL.  2.7. Statistical Analysis. All tests were assayed in triplicate and expressed as the mean ± standard deviation of the measurements. The statistical program SPSS version 21.00 for Windows (SPSS Inc., Chicago, IL, USA) was used to analyze the data. Variance was analyzed by one-way ANOVA and Student's t-test was applied to compare each parameter at p < 0:05.  Table 1). The main components were thymol, the most abundant compound (47.44%), followed by o-cymene (16.55%), γ-terpinene (7.80%), and linalool (4.41%). Moreover, it detected the presence of 4 compounds which their percentages (EO %) were more than 2%. These later are the α-zingibirene (3.40%), β-myrcene (2.36%), caryophyllene (2.09%), and borneol (2.02%).
On the other hand, linalool, known as 3,7-dimethyl-1,6octadien-3-ol [77], is a naturally occurring acyclic monoterpenoid and tertiary alcohol which is commonly found as a major active component in the essential oil of several aromatic plant species [78] principally in Lamiaceae and Table 2: Antibacterial activity of TVEO and the control gentamicin. Zones of growth inhibition was expressed in mm, and minimum inhibitory concentrations (MICs) were expressed in mg/mL.
3.2. Antibacterial Activity. As represented in Table 2, TVEO displayed an interesting antibacterial activity against the four tested bacterial strains with inhibition zones ranging from of 21 to 23 mm against Gram-negative and Gram-positive bacteria, respectively. In addition, it is important to note that monoterpenes exhibit a broad-spectrum antibacterial activity against pathogenic bacteria [92,93]. This fact could be explained by the presence of a lipophilic character which provides to monoterpenes the ability to adhere to bacterial cell membrane lipids and to deploy their antibacterial action [94]. In our study, the obtained MIC values mentioned in Table 2, indicated that TVEO was more potent (p < 0:05) against Gram-positive bacteria (MICs = 0:097 mg/mL) than Gram-negative bacteria (MICs = 0:195 mg/mL). These findings were in agreement with previous studies which reported that Gram-positive bacteria are susceptible to be more sensitive to plant EOs than Gram-negative bacteria due to the existence of lipopolysaccharides which acts as a hydrophobic barrier [95,96].

Interactions between TVEO Molecules and Bacterial
Topoisomerase II and DNA and RNA Polymerases. Computational modeling is a 3R-based approach and an attractive alternative to experiments in a view to understand bioactive compounds mechanism of action and their antibacterial inhibitory process [97]. Molecular docking was performed to predict different TVEO components that could bind specifically to select bacterial receptors active sites responsible for DNA replication and transcription processes. In this respect, we investigated TVEO antibacterial inhibitory effect on topoisomerase II (DNA gyrase) and DNA and RNA polymerases of three pathogenic bacteria S. aureus ATCC 700699, S. enterica Typhimurium ATCC 700720, and L. monocytogenes ATCC 19115. Molecular homology results indicated that selected templates can be used for molecular modeling (Table 3); their identities (%) are higher than 30% [98], and Ramachandran plot values of favored regions and allowed regions were over 90% [99].
Moreover, it is necessary to remind that DNA and RNA polymerases are crucial enzymes involved in the DNA replication, transcription, and translation as well as nucleic acid formation in bacterial cells [100]. On the other hand, topoisomerase II (DNA gyrase) is also implicated in DNA replication and transcription processes and has an imperative role characterized by its ability to catalyze the unwinding of supercoiled DNA strands [101]. These imperative enzymes constitute attractive and validated targets for antibacterial agents [102]. Molecular docking simulation results are elucidated by (Table 4). These later displayed that TVEO major compound thymol (47.44%) showed a good inhibitory effect on topoisomerase II, RNA polymerase, and DNA polymerase of the selected pathogenic bacteria. A previous study conducted by Liu et al. reported that thymol has a potential antibacterial activity against P. aeruginosa [15]. This monoterpenoid can affect bacterial DNA normal function. It could block gene expression processes including replication, transcription, and expression by intercalation with bacterial DNA leading to bacterial death. The same study indicated that thymol could destroy bacterial membrane integrity by affecting its permeability and it could also hinder biofilm formation. Another research paper displayed that thymol could bind to bacterial DNA, modulate its structure, and prohibit its biological function [103]. Furthermore, dos Santos Barbosa et al. showed that the antibacterial inhibitory activity of Origanum vulgare EO was effective against Salmonella Enteritidis due to the presence of thymol which caused interference in protein regulation as well as DNA synthesis [104]. On the other hand, the minor compound β-sesquiphellandrene (1.34%) showed the lowest free energy of binding (Kcal/mol) and the highest inhibitory potential on topoisomerase II and DNA and RNA polymerases of the selected pathogenic bacteria (Table 4). A previous study reported that β-sesquiphellandrene was responsible for the high antibacterial and antioxidant activities of some pomelo varieties' EOs [105]. Another study indicated that the presence of β-sesquiphellandrene presented in ginger (Zingiber officinale) essential oil could make this later as a potential    BioMed Research International antimicrobial agent by inhibiting mycobacterial acyl carrier protein reductase enzyme and Enoyl acyl carrier protein reductase activities [106].
A recent study showed that Cupressus sempervirens EO had a great inhibitory effect on DNA gyrase and DNA and RNA polymerases of S. aureus and S. enterica owing to the presence of α-pinene, δ-3 carene, and borneol [13]. Moreover, a previous study conducted by [107] evaluated the inhibitory effect of Litsea cubeba EO on topoisomerase and DNA and RNA polymerases of E.coli. Another study revealed that the germacrene B, a minor compound in Siparuna guianensis EO, had an effective inhibitory activity against bacterial DNA and RNA polymerases of multiple pathogenic bacteria including E.coli, P. aeruginosa, S. aureus, and S. pyogenes [108]. Therefore, these findings confirmed that TVEO has a powerful inhibitory effect on pathogenic bacteria based on the inhibition of DNA replication and transcription processes. Consequently, we project in subsequent work to perform further in vitro assays based on the evaluation of protein-molecule binding assays.
Interaction profiles details of thymol and β-sesquiphellandrene with the active sites of the selected targets of S. aureus ATCC 700699, S. enterica Typhimurium ATCC 700720, and L. monocytogenes ATCC 19115 are summarized in Table 5.
Interaction profile results of thymol and β-sesquiphellandrene with topoisomerase II and DNA and RNA polymerases of S. aureus are presented by Figures 1 and 2.

In Silico TVEO Compound
Toxicity Evaluation by VEGA QSAR Model. Toxicity evaluation of different TVEO compounds and the two selected FDA-approved antibiotics, used as controls (rifamycin SV and ciprofloxacin), was performed by the help of QSAR (quantitative structure-activity relationship) approach and using VEGA HUB software. We chose to assess compounds toxicity based on 8 different toxicity measurements. Results are represented by ( Table 6) and revealed that FDA-approved drugs could be toxic in several assays. In this context, rifamycin SV and ciprofloxacin are found to be mutagenic in the mutagenicity test/model (Ames test) and predicted to engender developmental/ reproductive toxicity. These two antibiotics were also predicted to be genotoxic according to the in vitro micronucleus activity model. However, many TVEO compounds such as α-pinene, α-thujene, camphene, β-pinene, 3-carene, D-limonene, γ-terpinene, terpinolene, linalool, borneol, 4-terpineol, α-terpineol, thymol, caryophyllene, α-bisabolene, and β-sesquiphellandrene showed nontoxic effects. Computational toxicity results confirmed that TVEO molecules could be used as a safe antimicrobial agents and economically lowcost alternative as compared to synthetic antibiotics. It is important to mention that research related to toxicological profiles of different essential oil compounds are poorly studied due to experiments complexity, expensive cost, and difficulty to detect toxicity variation because of the chemical function and factor variability [109]. However, previous studies indicated that toxicity is a dose-/concentrationdependent manner and thymol could have a certain limit of toxicity ranging from 36 mg/mL to 49 mg/mL with less risks of accumulation in body tissues and suggested to replace synthetic drugs, which has more side effect [110]. In addition, Schönknecht et al. confirmed the safety and 10 BioMed Research International the effectiveness of the drug containing the extracts of thyme with the addition of thymol (Bronchosol®) in cough treatment instead of using the synthetic drug ambroxol [111]. Concerning β-sesquiphellandrene, it was reported that this sesquiterpenoid could have a great anticancer activity and can be safe to use as compared to synthetic chemotherapeutic agents velcade, thalidomide, and capecitabine [112]. Nevertheless, more toxicological in vitro/in vivo data are needed to validate the safety of these phytochemicals. Thus, computational toxicity assessment could be the best alternative that would give robust data, avoid unnecessary waste of reagents, and minimize cruelty and sacrifices of lab animal testing.
3.5. TVEO Component ADME Analysis. In the present study, SwissADME server was used to determine some pharmacokinetics parameters included in absorption, distribution, metabolism, and excretion (ADME), drug-likeness, and medicinal chemistry characteristics for all TVEO compounds as represented in (Table 7). Results revealed that all TVEO components possess slow passive gastrointestinal absorption (GI) except linalool, camphor, borneol, 4-terpineol, α-terpineol, thymol methyl ether, and thymol which were predicted to have high GI permeability. Additionally, only sesquiterpenes including caryophyllene, α-humulene, α-amorphene, α-curcumene, α-zingibirene, αbisabolene, and β-sesquiphellandrene were found not to be blood-brain barrier (BBB) permanent due to their heavy molecular weight. However, rest of compounds could easily cross the blood-brain barrier for that reason it could be suggested as potent central nervous system antioxidants and effective drug candidates in the treatment of neurodegenerative diseases like Alzheimer's and Parkinson's [113]. On the other part, none of the tested compounds was predicted to be P-gp transporter substrate. Concerning Cytochrome p450 (CYP) isoenzymes which are involved in 50-90% of therapeutic molecules biotransformation processes in a view to reduce metabolites accumulation in blood/tissues and drug-drug interaction risks [114], thymol methyl ether and thymol were predicted to be CYP1A2 inhibitors. Many TVEO molecules were predicted to inhibit CYP2C9 and CYP2D6 inhibitors. However, none of the compounds showed an inhibitory effect towards the CYP3A4. Moreover, skin permeation coefficient (log Kp) indicated that all compounds were impermeable through the skin barrier. Interestingly, TVEO drug-likeness score was acceptable with good bioavailability score (>10%) and the absence of violations related to known rules such as Lipinski's rule of five that predicts drug permeability and absorption based on H-bond

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BioMed Research International donors, H-bond acceptors, molecular weight (MW > 500), and a calculated log p) [115], as well as Veber. Both rules are a mainstay of decision-making in drug design and development and in the present study; both were validated. Furthermore, medicinal chemistry parameters revealed that none of the selected molecules returns any pan-assay interference compounds (PAINS) alert. The synthetic accessibility values of TVEO compounds indicated that these later could be synthesized for pharmaceutical uses.

Prediction of Possible Activity Spectra of TVEO
Components. All TVEO compounds were subjected to PASS (Prediction of Activity Spectra for Substances) online tool intending to predict their biological activity spectrum based on their chemical structure. Results indicated that the tested compounds could have multiple biological activities and various enzymatic targets (Table 8). We have selected the top three activities which showed a Pa ≥ 0:7. Pa and Pi values indicated the probability of the selected molecule to be active/ inactive towards the targeted receptor. Mojumdar et al. reported that the probability of experimental pharmacological action is high when Pa > 0:7; however, the chance of finding the activity experimentally is less when Pa < 0:5 [116]. Interestingly, TVEO major compounds such as thymol which were predicted, in earlier section by molecular docking, to have a potent antibacterial activity on pathogenic bacteria by inhibiting DNA replication and transcription processes, displayed the existence of other biological activities including the ability to inhibit bacterial membrane permeability (Pa = 0:876) and it was anticipated to enhance AP0A1 expression (Pa = 0:830) involved in the cellular synthesis of beneficial HDL [117]. In addition, O-cymene was predicted to be mitochondria ubiquinol-cytochrome-c reductase inhibitor (Pa = 0:924) (antifungal activity), mucomembranous protector (Pa = 0:842), and a fibrinolytic agent (Pa = 0:778) which could stimulate the dissolution of blood clots. Concerning γ-terpinene, it was predicted to treat skin eczema (Pa = 0:854) and phobic disorders (Pa = 0:803) owing to its ability to cross the blood-brain barrier (BBB). Finally, PASS prediction revealed that the minor compound β-sesquiphellandrene has other possible biological activities including antineoplastic effect (Pa = 0:827) and antipsoriatic (Pa = 0:750) and could be also used as immunosuppressant agent during organ transplant (Pa = 0:702) instead of using synthetic compounds such as cyclosporin A which was demonstrated to cause severe cholestatic liver disease [118]. These findings could provide more insights towards further in vivo and in vitro assays to validate the computational predictions.

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Conclusion
The dramatical increase of antibiotic resistance urged scientists to diverge towards the use of aromatic medicinal plants essential oils to tackle the spread of superbugs. In that regard, the present study is aimed at investigating the TVEO chemical composition and antibacterial mechanism of action against S. aureus ATCC 6538, S. enterica Typhimurium ATCC 14028, and L. monocytogenes ATCC 19117. In addition, chemocomputational toxicological profile and pharmacological proprieties were developed. Interestingly, molecular docking simulations revealed that TVEO compounds such as thymol and β-sesquiphellandrene had an effective antibacterial activity against the tested bacteria by inhibiting topoisomerase II and DNA and RNA polymerase functions leading to vigorous impairment of bacterial DNA replication and transcription processes. Additionally, through VEGA QSAR, we demonstrated that TVEO could be a safe resource for potential antibacterial agents. Moreover, ADME analysis showed that both compounds fulfill the Lipinski's rule of five and could be used as potential candidate to overcome antibiotic resistance. Likewise, the in silico PASS prediction studies disclosed the presence of other useful bioactivities and possible enzymatic targets of TVEO which would be applied in the future to reduce the impact of several lethal diseases.

Data Availability
All the relevant data have been provided in the manuscript. The authors will provide additional details if required.