Antibacterial and Synergistic Effects of Shirazi Thyme (Zataria multiflora) Essential Oil Against Methicillin-Resistant Staphylococcus aureus

Antibacterial and Synergistic Effects of Shirazi Thyme (Zataria multiflora) Essential Oil Against Methicillin-Resistant Staphylococcus aureus | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Antibacterial and Synergistic Effects of Shirazi Thyme (Zataria multiflora) Essential Oil Against Methicillin-Resistant Staphylococcus aureus Kimia Kazemi, Mitra Dadgar, Zahra Dargahi, Armin Khaleghjoo, Ehsan Ghasemi, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7411474/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 04 Feb, 2026 Read the published version in Molecular Biology Reports → Version 1 posted 7 You are reading this latest preprint version Abstract Background Methicillin-resistant Staphylococcus aureus (MRSA) is a significant public health concern due to its resistance to multiple antibiotics, primarily mediated by the mecA gene, which encodes penicillin-binding protein 2a (PBP2a), given the urgent need for alternative antimicrobial agents. Materials and Methods A clinical MRSA strain harboring the mecA gene was identified. Antimicrobial susceptibility testing was performed to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of Shirazi thyme ( Zataria multiflora ) essential oil. The strain’s susceptibility to multiple antibiotics was assessed and the synergistic effect of Zataria multiflora essential oil and cefoxitin was also evaluated. Gas chromatography-mass spectrometry (GC-MS) was employed to identify the bioactive compounds in the essential oil. Molecular docking studies were performed to evaluate the competitive binding affinity of those compounds to PBP2x. Results The MRSA strain exhibited resistance to all tested antibiotics except linezolid. The MIC and MBC values for Zataria multiflora essential oil were 3.125 mg.mL − 1 and 6.25 mg.mL − 1 for the reference S. aureus strain (ATCC 25923), and 6.25 mg.mL − 1 for both MIC and MBC for the clinical MRSA strain. The synergy assay demonstrated an enhanced inhibition zone for cefoxitin in combination with Zataria multiflora essential oil, indicating a synergistic interaction. Molecular docking studies revealed strong binding interactions between spathulenol, isospathulenol, and aromadendrene comparable to clinically used β-lactam antibiotics. Conclusion The findings highlight the significant antibacterial activity of Zataria multiflora essential oil against MRSA and its potential to enhance the efficacy of cefoxitin through synergistic interactions as natural inhibitors of PBP2x. Methicillin-resistant Staphylococcus aureus Zataria multiflora mecA gene PBP2X Antibiotic resistance Molecular Docking Figures Figure 1 Figure 2 Figure 3 1. Introduction Antimicrobial resistance is one of the most serious global public health threats, leading to a concerning increase in mortality due to infections resistant to treatment [ 1 ]. Staphylococcus aureus ( S. aureus ) is a Gram-positive pathogen responsible for a wide range of infections and poses a significant public health challenge [ 2 ]. Before the introduction of penicillin in the 1940s, Staphylococcus infections were often untreatable. However, shortly after the widespread use of this drug, penicillin-resistant strains emerged, marking the first documented β-lactam resistance mechanism in S. aureus , which involved β-lactamase production. Methicillin-resistant Staphylococcus aureus (MRSA) is recognized as a major cause of both hospital-acquired and community-associated infections [ 3 ]. According to the Centers for Disease Control and Prevention (CDC), MRSA is classified as a serious threat, accounting for more than 80,461 infections and 11,285 deaths annually in the United States [ 3 ]. Traditionally, β-lactam antibiotics have been used to treat S. aureus infections; however, due to the emergence of resistance mechanisms, their effectiveness against resistant strains has significantly declined [ 4 ]. Penicillin-binding proteins (PBPs) are key enzymes involved in bacterial cell wall synthesis and serve as primary targets for β-lactam antibiotics. The production of penicillin-binding protein 2a (PBP2a) is one of the key mechanisms conferring β-lactam resistance in MRSA [ 5 ]. The mecA gene, which encodes PBP2a, is incorporated into the bacterial genome via the staphylococcal cassette chromosome mec (SCCmec). This gene transfer enables the bacterium to produce an enzyme with transpeptidase activity that has a low affinity for β-lactam antibiotics [ 6 ]. Resistance to β-lactams and carbapenems poses a severe threat to public health. If left unaddressed, it is projected that by 2050, antimicrobial resistance could result in millions of deaths annually [ 7 ]. Given the inefficacy of conventional antibiotics and the rapid spread of resistant bacterial strains, the exploration of natural compounds as alternative antimicrobial agents has gained considerable attention. Plant-derived compounds, particularly essential oils, have been recognized as promising candidates for combating drug-resistant bacteria due to their diverse chemical compositions and antimicrobial properties [ 8 ]. Essential oils are commonly volatile, aromatic mixtures extracted from various plant parts. Due to their accessibility, cost-effectiveness, safety profile, and the low likelihood of microbial resistance, they are considered viable alternatives to synthetic drugs for the treatment of infectious diseases [ 9 ]. However, their antimicrobial mechanisms and chemical compositions are not uniform across all essential oils, these compounds can effectively eliminate bacteria and fungi while inducing minimal adverse effects in consumers [ 10 ]. Zataria multiflora , commonly known as Shirazi thyme, belongs to the Lamiaceae family. Traditionally, this medicinal plant has been used to treat respiratory infections, alleviate bloating, and serve as an antiseptic and anesthetic agent. Previous studies have demonstrated that Z. multiflora essential oil exhibits antimicrobial properties against both Gram-positive and Gram-negative bacteria, largely attributed to its phenolic constituents such as thymol and carvacrol [ 11 ]. With advancements in technology, bioinformatics approaches such as molecular docking have emerged as powerful tools for simulating and analyzing the interactions between proteins and plant-derived compounds. Molecular docking enables the precise evaluation of the inhibitory potential of various compounds against target proteins, facilitating the identification of the most effective inhibitors against drug-resistant proteins [ 12 ]. This study aims to comprehensively assess the antimicrobial activity of Z. multiflora essential oil against S. aureus strains, evaluate its synergistic effects with cefoxitin, and elucidate its mechanism of action through molecular docking analysis. In this study, the bioactive compounds identified in Z. multiflora essential oil are assessed for their interactions with PBP2x using molecular docking techniques (PDB ID: 5OJ0) [ 13 ]. This computational approach allows for the simulation of molecular interactions between PBP2x and essential oil components, leading to the identification of the most promising ligands with the lowest binding free energies. It is hypothesized that some of these compounds can effectively bind to PBP2x, inhibiting its function and thereby preventing bacterial cell wall synthesis. Furthermore, the combination of these natural compounds with antibiotics such as cefoxitin, imipenem, and meropenem may not only reduce treatment costs but also mitigate the side effects of conventional antibiotics, ultimately improving patient outcomes. Finally, this study aims to provide evidence-based scientific insights that can contribute to the development of practical strategies for combating antibiotic resistance. 2. Materials and Methods 2.1. Materials and Bacterial Strains Used One clinical strain of MRSA and the standard strain ATCC 25923 were obtained from the Microbial Bank of the Faculty of Medical Sciences, Jundishapur University of Ahvaz. [ 14 ]. Phenotypic identification was confirmed by Gram staining and standard biochemical tests, including catalase, coagulase, and DNase assays, as well as characteristic growth patterns on blood agar and mannitol salt agar (MSA). The chemicals and reagents were bought from Merck (Darmstadt, Germany) except indicated. 2.2. Bacterial Culture and Viability Confirmation The bacterial strains were cultured on Mueller-Hinton agar using a sterile loop under flame sterilization. Following incubation at 37° C for 24 hours, microbial growth was examined. The presence of convex, cream-colored colonies of S. aureus confirmed bacterial viability. Methicillin resistance was determined using a cefoxitin (30 µg) disk and disk diffusion method according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. Staphylococcus aureus ATCC 33591 was used as the methicillin-resistant (positive) control strain, and ATCC 25923 was used as the methicillin-sensitive (negative) control. 2.3. Bacterial DNA Extraction and Assessment of DNA Quality In the process of DNA extraction, bacterial colonies from a 24-hour culture on Mueller-Hinton agar were suspended in 500 µL of distilled water. The suspension was then incubated at 95°C for 10 minutes using a Thermo block to lyse the cells. Following heat treatment, the microtubes were placed in a − 20°C freezer for 5 minutes to enhance cell lysis. Subsequently, the samples were centrifuged at 14,000 rpm for 10 minutes. The supernatant containing the genomic DNA was carefully collected and used as the DNA template for subsequent analyses [ 15 ]) 2.4. Primer Design, PCR, and Electrophoresis of PCR Products The primer was selected based on previous studies [ 14 ], and its specificity was confirmed using the NCBI Primer-BLAST tool. Following the manufacturer's protocol (SinaClon), sterile distilled water was added to the lyophilized primer vial to achieve a stock concentration of 100 µM. For each experiment, a working solution of 10 µM was freshly prepared by diluting 10 µL of the stock with 90 µL of sterile distilled water in a sterile microtube. After thorough vortexing, the primer solution was utilized for PCR amplification. Details of the designed primers are provided in Table S1 [ 16 ] PCR reactions were prepared in a final volume of 25 µL in sterile microtubes. Each reaction contained 1 µL of purified genomic DNA from the target isolate. DNA amplification was performed in a thermal cycler (Eppendorf, Germany) using the following program: initial denaturation at 95°C for 5 minutes; 35 cycles of denaturation at 95°C for 35 seconds, annealing at 55°C for 35 seconds, and extension at 72°C for 35 seconds; followed by a final extension at 72°C for 5 minutes. PCR products were analyzed by electrophoresis on a 2% agarose gel pre-stained with SafeStain. A 100 bp DNA ladder was used as a molecular size marker. Electrophoresis was performed at 70 V for approximately 30 minutes. After completion, the gel was visualized under UV light at 560 nm using a Gel Documentation System (Vilber, France) to detect DNA bands based on their size (Fig. 1 ). 2.5. Preparation of Plant Samples and Essential Oil Extraction The leaves of Zataria multiflora (Shirazi thyme) were obtained from the Pharmaceutical Research Center of Jundishapur University. The plant identity was confirmed by experts at the university’s herbarium. To prevent enzymatic degradation and chemical alterations, the leaves were dried in the shade. The dried leaves were then ground into a fine powder and stored in sterile, dark laboratory containers at 4° C until use. Essential oils were extracted using the Clevenger apparatus via hydro-distillation. Briefly, 100 g of dried powdered plant material was placed in a glass boiling flask and submerged in distilled water. After 30–45 minutes of heating, the essential oil was collected. The extracted essential oils were stored in sealed, dark containers at 4° C for further analysis. The extracted essential oil was subsequently analyzed using Gas Chromatography-Mass Spectrometry (GC-MS) to identify its chemical constituents. The prepared essential oil ingredients were analyzed as following: 40 mg of essential oil was dissolved in 4 ml of ethyl acetate and 0.2 µl of the produced solution was directly injected to gas chromatography (Agilent, 7890 A GC/5975 MSD Model) using a HP5-MS (30M×250µM×0.25µM) column and analyzed by a mass spectrometer analyzer. The inlet and auxiliary temperatures were set at 280°C and the split ratio was set to 50/1. 2.6. Antibacterial Activity Assessment and Determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) To determine the Minimum Inhibitory Concentration (MIC) of Zataria multiflora , a stock solution was prepared in methanol. The MIC was assessed using the broth microdilution method, following CLSI guidelines. So, Serial two-fold dilutions of the extract were prepared. A bacterial suspension with a turbidity equivalent to 0.5 McFarland standard was prepared and subsequently diluted 1:100 in Tryptic Soy Broth (TSB). Then, 100 µL of this diluted suspension was added to each well, resulting in a final bacterial concentration of 5×10⁵ CFU/mL. Two additional wells served as controls: the positive control contained bacterial suspension and TSB without extract, while the negative control contained extract and TSB without bacteria. The microplates were incubated at 35 ± 2°C for 16–20 hours. Following incubation, the MIC was defined as the lowest extract concentration at which no visible turbidity was observed. To determine the Minimum Bactericidal Concentration (MBC), 10 µL from each clear well (showing no visible growth) was plated onto Mueller-Hinton Agar. After incubation at 35 ± 2°C for 24 hours, colonies were counted. The MBC was defined as the lowest concentration of the extract resulting in at least a 3-log₁₀ reduction in viable bacterial count compared to the initial inoculum (5×10⁵ CFU/mL). 2.7. Evaluation of Synergistic Effects Between Extract and Cefoxitin Against MRSA and MSSA To assess the synergistic effects of plant extract and the antibiotic cefoxitin, both methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-susceptible Staphylococcus aureus (MSSA) strains were tested. A cefoxitin disc with a concentration of 30 µg was used in this experiment. For synergy testing, sub-minimum inhibitory concentration (sub-MIC) levels of the extract were used separately. A volume of 10 µL of the extract at its sub-MIC concentration was individually loaded onto a 30 µg cefoxitin disc. Additionally, a cefoxitin disc without any extract, as well as individual sub-MIC concentrations of the extract, were placed separately on inoculated Mueller-Hinton agar plates containing bacterial cultures. Following incubation at 37° C for 24 hours, the inhibition zones around each disc were measured in millimeters to determine the antimicrobial and potential synergistic effects. 2.8. Retrieval and Preparation of PBP2X Crystal Structure In the initial phase of this study, the crystallographic structure of PBP2X with appropriate resolution in the presence of cefepime will be obtained from the RCSB Protein Data Bank (PDB) [ 13 ] via https://www.rcsb.org (Fig. 2 ). Based on the retrieved crystal structure, the sequence, secondary structures, and interactions of PBP2X with the cefepime ligand will be analyzed in detail. Next, the protein structure of PBP2X will be extracted from the 5oJ0.pdb file. Using AutoDock Tools [ 17 ], all water molecules will be removed, missing polar hydrogens will be added, and the structural grid of PBP2X will be precisely defined. On the other hand, the structure of cefepime will be retrieved in SDF format from Pubchem data base ( https://pubchem.ncbi.nlm.nih.gov/ ) [ 18 ] and converted to PDB format. Using AutoDock Tools, the molecule will be further prepared by adding charged hydrogen atoms and determining its atomic charges through the Compute Gasteiger algorithm. The final ligand structure will be stored in PDBQT format for docking studies. Following these preparations, validation docking will be performed between cefepime and PBP2X. If the docking results yield a suitable ΔG of binding, the generated complexes will be analyzed in three-dimensional format using Chimera [ 19 ] and VMD [ 20 ] and in two-dimensional format using LigPlot [ 21 ]. 2.9. Molecular Docking of Essential Oil Constituents Against PBP2X In the next phase, the identified compounds in Zataria multiflora essential oil will be retrieved in SDF format from PubChem [ 18 ] and converted to PDBQT format for molecular docking studies. The molecular docking will be performed against the three-dimensional structure of PBP2X, targeting the cefepime-binding site in 5oJ0.pdb, using AutoDock Tools 4.2 and AutoDock Vina [ 22 ] (Table S3). The binding affinities (ΔG of binding) will be evaluated, and the compounds will be ranked based on their docking scores for further investigation. After identifying the most promising PBP2X inhibitors, the physicochemical properties, pharmacokinetics, and toxicity of the top three compounds with the highest binding affinities will be predicted using SwissADME [ 23 ] and PASS Online [ 24 ]. 2.10. Statistical Analysis Statistical analyses were performed using student's t-test. A significance level of p < 0.05 was considered statistically significant. Data analysis was conducted using SPSS version 2. 3. Results and Discussion 3.1. Identification and Confirmation of MRSA Clinical Strain The clinical isolates used in this study were identified as MRSA based on phenotypic and genotypic characteristics. Those isolates that induced a color change in MSA to yellow were selected for further testing. Final confirmation was based on positive results of coagulase, catalase, and DNase tests. Additionally, according to the 2023 CLSI guidelines, isolates exhibiting an inhibition zone diameter of ≤ 21 mm around the cefoxitin disk were classified as MRSA. Further validation was achieved by the detection of the mecA gene via PCR (Table S1 and Fig. 1 ), confirming the presence of the methicillin resistance determinant responsible for the synthesis of penicillin-binding protein 2a (PBP2a). Electrophoresis results revealed a distinct 310 bp DNA band corresponding to the expected size of the mecA gene (Fig. 1 ), verifying the identity of the MRSA isolates. This molecular validation was crucial, as mecA is a key genetic marker distinguishing methicillin resistance in S. aureus . Consistent with findings by KB Anand and his colleagues (2009), the presence of mecA correlates strongly with resistance to all β-lactam antibiotics, especially cefoxitin [ 25 ]. This initial characterization laid the foundation for subsequent antimicrobial and synergistic assays. 3.2. Antibacterial Activity of Zataria multiflora Essential Oil The broth microdilution method was used to evaluate the antibacterial activity of Zataria multiflora essential oil against both the standard strain (ATCC 25923) and clinical MRSA strains. Results showed significant inhibitory effects, with the clinical MRSA strain exhibiting slightly lower susceptibility than the standard strain (Table S2 ). The minimum inhibitory concentration and minimum bactericidal concentration were determined via dilution and disk placement assays. For the ATCC 25923 strain, the MIC was 3.125 mg.mL − 1 and MBC was 6.25 mg.mL − 1 . However, for the MRSA isolate, both MIC and MBC were recorded at 6.25 mg.mL − 1 , indicating a higher threshold required for inhibition and killing. These findings may align with the known resistance mechanisms of MRSA, particularly the expression of PBP2a, which diminishes the efficacy of β-lactams and likely contributes to decreased essential oil sensitivity. Notably, these results are consistent with the antimicrobial properties reported for Z. multiflora , a plant rich in bioactive compounds like thymol, carvacrol, and other phenolics. Previous reports by Maryam Motevasel et al. (2013) also document its potent antibacterial activities against Gram-positive pathogens, including S. aureus [ 26 ]. The differential response between MSSA and MRSA suggests that Z. multiflora essential oil may exert effects independent of β-lactam targets, which becomes especially relevant in the context of synergistic assays. 3.3. Synergistic Interaction Between Cefoxitin and Essential Oil One of the study's most promising findings was the enhanced antibacterial effect observed when sub-MIC concentrations of Z. multiflora essential oil were combined with cefoxitin. In disk diffusion assays, cefoxitin alone displayed a limited inhibition zone against MRSA, confirming resistance. However, the co-application of the essential oil significantly increased the diameter of the inhibition zone, indicating a synergistic interaction. Quantitatively, the increase in the inhibition zone was statistically significant (p < 0.05), reflecting enhanced bacterial susceptibility. This synergistic effect suggests that essential oil constituents may facilitate cefoxitin access to its target site, possibly by disrupting the cell membrane or altering the expression/conformation of PBP2a [ 27 ]. Although the precise molecular mechanism remains speculative, this observation echoes findings from Aribisala et al. (2024), who demonstrated similar synergy between phenolic compounds and β-lactam antibiotics [ 28 ]. The clinical implications of such synergy are substantial. The restoration or enhancement of cefoxitin efficacy through natural compounds could reduce reliance on high-dose β-lactam therapy, lower the risk of toxicity, and delay the emergence of further resistance. 3.4. GC-MS Analysis of Zataria multiflora Essential Oil Gas chromatography–mass spectrometry analysis of the essential oil revealed several bioactive constituents (Table S3). Among them, o-Cymene (11.00%), Thymol (37.76%), and Carvacrol (18.59%) were identified as major components with potential antibacterial properties. The molecular diversity of these compounds contributes to the broad-spectrum antibacterial effect of the essential oil [ 29 ]. The identification of these compounds served as the basis for subsequent in silico docking studies. Their molecular weights, hydrogen bond acceptors/donors, and structural compatibility with biological targets suggested possible interactions with bacterial proteins involved in resistance mechanisms, particularly PBP2x. 3.5. Molecular Docking with PBP2x To understand the molecular interactions between essential oil components and bacterial resistance proteins, molecular docking studies were conducted using AutoDock Vina [ 22 ]. The crystal structure of PBP2x (PDB ID: 5OJ0) was used as the target, and the docking site was defined based on the binding pocket of cefepime (Fig. 2 A), a fourth-generation cephalosporin [ 30 ]. Cefepime served as the positive control and exhibited a binding energy of -7.5 kcal.mol − 1 (Fig. 2 B and 2 C). Among the essential oil constituents, spathulenol and isospathulenol both displayed binding energies of -6.7 kcal.mol − 1 , while aromadendrene showed a slightly weaker interaction at -6.6 kcal.mol − 1 (Table S3). The docking results are summarized in Table 1 . Despite having weaker affinities compared to cefepime, the essential oil constituents demonstrated comparable binding energies, suggesting that they may competitively inhibit PBP2x. Notably, spathulenol and isospathulenol in spite of smaller and more simple structure exhibited ligand efficiencies higher than cefepime (0.4188 vs . 0.2344 kcal/mol/non-H atom), indicating a strong binding per atom, a desirable trait in drug development. Visual inspection of the ligand-protein interactions (Figs. 2 and 3 ) confirmed that these molecules occupied similar spatial positions as cefepime within the PBP2x active site (Figure S1 ). This spatial mimicry suggests that the essential oil compounds might interfere with cell wall synthesis, a hallmark mechanism of β-lactam antibiotics. Table 1 Chemical name of the substance, Pubchem code, binding free energy, inhibition constant, ligand binding efficiency and chemical structure of cefepime and compounds present in the essential oil obtained from Shirazi thyme (Zataria multiflora) with the highest binding energy docked against the PBP2x protein obtained from the crystallographic structure 5OJ0.pdb. Name of the ligand Pubchem ID Binding Free Energy (kcal/mol) pKi Ligand Efficiency (kcal/mol/non-H atom) Chemical Structure Cefepime 5479537 -7.5 5.5 0.2344 Spathulenol 13854255 -6.7 4.91 0.4188 Isospathulenol (14038848) -6.7 4.91 0.4188 (+)-Aromadendrene 11095734 -6.6 4.84 0.44 3.6. Structure-Activity Relationship and Drug-Likeness Further evaluation using SwissADME [ 23 ] and PASS online tools [ 24 ] indicated favorable physicochemical and pharmacokinetic profiles for the docked compounds (Table S4). All three compounds adhered to Lipinski’s Rule of Five, indicating drug-likeness. Additionally, predicted properties included good gastrointestinal absorption, absence of P-glycoprotein efflux susceptibility, and significant blood-brain barrier permeability—suggesting these compounds might be suitable for systemic for CNS infection. Toxicity assessments predicted low mutagenic and carcinogenic risks for these molecules, further supporting their potential as antimicrobial adjuvants. The ability of these compounds to act synergistically with existing antibiotics, coupled with favorable pharmacokinetics, reinforces their potential role in future anti-MRSA therapeutic formulations. 4. Conclusion This study comprehensively demonstrated the significant antibacterial and synergistic properties of Zataria multiflora (Shirazi thyme) essential oil against methicillin-resistant Staphylococcus aureus . In current study we concluded, clinical validation of MRSA resistance, effective antibacterial activity against both MRSA and the standard S. aureus strain, synergistic enhancement of cefoxitin efficacy which could enhance clinical efficacy while potentially reducing cefoxitin dosage, minimizing adverse effects and delaying resistance, spathulenol, isospathulenol, and aromadendrene were identified as major active constituents, these terpenoids are known for their antimicrobial properties and were selected for molecular docking, the essential oil compounds showed strong binding affinities to PBP2x, comparable to cefepime, their physicochemical profiles combined with predicted low toxicity encourage further in vivo and clinical evaluation, and the integration of essential oils with standard antibiotics represents a promising strategy to revitalize old antibiotics and combat multidrug-resistant organisms. In summary, the use of Z. multiflora essential oil—rich in spathulenol, isospathulenol, and aromadendrene—offers a promising adjunctive treatment approach for MRSA infections. By combining natural compounds with conventional antibiotics, we can not only restore lost efficacy but also reduce treatment costs and slow the global rise of antibiotic resistance. Future work should explore the in vivo efficacy, formulation stability, and clinical pharmacokinetics of these combinations to facilitate their translation into medical practice. Abbreviations Centers for Disease Control and Prevention CDC Methicillin-resistant Staphylococcus aureus MRSA Methicillin-susceptible Staphylococcus aureus MSSA Minimum Inhibitory Concentration MIC Penicillin-binding proteins PBPs Penicillin-binding protein 2a PBP2a Staphylococcus aureus S. aureus Staphylococcal cassette chromosome mec SCCmec. Declarations Ethics Statement: Ethical approval for this study was obtained from Abadan University of Medical Sciences, Abadan, Iran (ID: IR.ABADANUMS.REC.1402.055). Consent to Participate: This study did not involve human participants. Consent to participate was therefore not applicable. Consent to Publish declarations: All authors have reviewed and approved the final version of this manuscript and consent to its publication. This study did not involve human participants or personal data requiring individual consent for publication. Funding: The authors did not receive support from any organization for the submitted work. No funding was received to assist with the preparation of this manuscript. No funding was received for conducting this study. No funds, grants, or other support was received. Conflicts of interest/Competing interests: All authors certify that they have no affiliations with or involvement in any organization or entity with any financial or non-financial interest in the subject matter or materials discussed in this manuscript. Availability of data and material: The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request. Author contributions: In this research, all authors have been in charge of research, data collection and analysis, and article writing. References Turner NA et al (2019) Methicillin-resistant Staphylococcus aureus: an overview of basic and clinical research. Nat Rev Microbiol 17(4):203–218 Jensen SO, Lyon BR (2009) Genetics of antimicrobial resistance in Staphylococcus aureus. Future Microbiol 4(5):565–582 Watkins RR, Bonomo RA (2020) Overview: the ongoing threat of antimicrobial resistance. 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Supplementary Files SupplementaryFigure.docx SupplementareyTable.docx Cite Share Download PDF Status: Published Journal Publication published 04 Feb, 2026 Read the published version in Molecular Biology Reports → Version 1 posted Editorial decision: Revision requested 02 Oct, 2025 Reviews received at journal 14 Sep, 2025 Reviewers agreed at journal 01 Sep, 2025 Reviewers invited by journal 27 Aug, 2025 Editor assigned by journal 27 Aug, 2025 Submission checks completed at journal 26 Aug, 2025 First submitted to journal 19 Aug, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7411474","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":508800491,"identity":"de15dfa6-dd10-4df6-b7b9-1cad11a06482","order_by":0,"name":"Kimia Kazemi","email":"","orcid":"","institution":"Ahvaz Jundishapur University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Kimia","middleName":"","lastName":"Kazemi","suffix":""},{"id":508800492,"identity":"2844f1bf-5db4-4b6e-91c7-c8722e7fb72e","order_by":1,"name":"Mitra Dadgar","email":"","orcid":"","institution":"Ahvaz Jundishapur University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Mitra","middleName":"","lastName":"Dadgar","suffix":""},{"id":508800493,"identity":"12a56108-98ce-4cd9-bdc6-6728fa0344fc","order_by":2,"name":"Zahra Dargahi","email":"","orcid":"","institution":"Ahvaz Jundishapur University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Zahra","middleName":"","lastName":"Dargahi","suffix":""},{"id":508800494,"identity":"b2ab2b05-cd43-4747-b65e-f57fe789cfb5","order_by":3,"name":"Armin Khaleghjoo","email":"","orcid":"","institution":"Abadan University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Armin","middleName":"","lastName":"Khaleghjoo","suffix":""},{"id":508800495,"identity":"82d9588a-b8ff-4e17-adbd-60a666091ab6","order_by":4,"name":"Ehsan Ghasemi","email":"","orcid":"","institution":"Abadan University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Ehsan","middleName":"","lastName":"Ghasemi","suffix":""},{"id":508800496,"identity":"d1a80fd7-4701-426d-989e-9c5b20ea1fe3","order_by":5,"name":"Forouzan Absalan","email":"","orcid":"","institution":"Abadan University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Forouzan","middleName":"","lastName":"Absalan","suffix":""},{"id":508800497,"identity":"d2551ca9-819b-413c-9a9e-740cfb138309","order_by":6,"name":"Ebrahim Barzegari","email":"","orcid":"","institution":"Kermanshah University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Ebrahim","middleName":"","lastName":"Barzegari","suffix":""},{"id":508800498,"identity":"b2b4ef0b-1f8b-4de5-b374-4390a714e197","order_by":7,"name":"Mostafa Jamalan","email":"data:image/png;base64,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","orcid":"","institution":"Abadan University of Medical Sciences","correspondingAuthor":true,"prefix":"","firstName":"Mostafa","middleName":"","lastName":"Jamalan","suffix":""}],"badges":[],"createdAt":"2025-08-19 19:53:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7411474/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7411474/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11033-025-11391-5","type":"published","date":"2026-02-04T15:59:52+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":90695326,"identity":"d1dd13bb-8065-464b-9949-bb0c5818e8cc","added_by":"auto","created_at":"2025-09-05 19:56:51","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":117638,"visible":true,"origin":"","legend":"\u003cp\u003eDetection of the mecA gene \u003cem\u003evia\u003c/em\u003e PCR. Electrophoresis results revealed a distinct 310 bp DNA band corresponding to the expected size of the mecA gene.\u003c/p\u003e","description":"","filename":"Picture0.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7411474/v1/395d527402c412d2af4c81bc.jpg"},{"id":90695327,"identity":"1f6944c0-48dc-4b03-9c87-771550bf66a0","added_by":"auto","created_at":"2025-09-05 19:56:51","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":232268,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eChemical structure of the antibiotic cefepime, \u003cstrong\u003e(B) \u003c/strong\u003eTwo-dimensional interaction of the antibiotic cefepime with the PBP2x protein derived from the crystallographic structure 5OJ0.pdb, \u003cstrong\u003e(C) \u003c/strong\u003eThree-dimensional interaction of the antibiotic cefepime (red) with the PBP2x protein obtained from the crystallographic structure 5OJ0.pdb and three-dimensional interaction of the antibiotic cefepime (blue) docked against the PBP2x protein from Staphylococcus aureus obtained from the crystallographic structure 5OJ0.pdb (ΔG\u003csub\u003ebinding\u003c/sub\u003e=-8.7 kcal/mol)\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7411474/v1/2b667309d0e4560ee79b3bde.jpg"},{"id":90695450,"identity":"b286c2ed-1af0-4afd-87bd-9b6d204a9058","added_by":"auto","created_at":"2025-09-05 20:04:51","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":226068,"visible":true,"origin":"","legend":"\u003cp\u003eThe 3D position of the antibiotic cefepime (blue) with the PBP2x protein obtained from the crystallographic structure 5OJ0.pdb compared to the 3D position of Spathulenol (13854255) (ΔG\u003csub\u003ebinding\u003c/sub\u003e=-6.7 kcal/mol), Isospathulenol (14038848) (ΔG\u003csub\u003ebinding\u003c/sub\u003e=-6.7 kcal/mol), Aromadendrene (11095734) (ΔG\u003csub\u003ebinding\u003c/sub\u003e=-6.6 kcal/mol) docked against the PBP2x protein in the presence of the PBP2x protein \u003cstrong\u003e(A)\u003c/strong\u003e and without the presence of PBP2x protein \u003cstrong\u003e(B) \u003c/strong\u003efor more clarify presentation.\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7411474/v1/9c48fa70a513a09e492c0b75.jpg"},{"id":102234308,"identity":"03fa2fd1-7c32-4bf0-b065-866def7131e5","added_by":"auto","created_at":"2026-02-09 16:09:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1680246,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7411474/v1/8ad597fb-337c-4526-9985-581f51170bd5.pdf"},{"id":90695454,"identity":"a9582906-d148-4591-93d2-f98892e0f418","added_by":"auto","created_at":"2025-09-05 20:04:51","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":254582,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure.docx","url":"https://assets-eu.researchsquare.com/files/rs-7411474/v1/6bc8a4d8a251570927fd51cd.docx"},{"id":90695452,"identity":"97d4c786-d443-46a3-9f95-a7f7eaa4189f","added_by":"auto","created_at":"2025-09-05 20:04:51","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":32022,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementareyTable.docx","url":"https://assets-eu.researchsquare.com/files/rs-7411474/v1/305bf8f3036e9f5cba3a4c52.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Antibacterial and Synergistic Effects of Shirazi Thyme (Zataria multiflora) Essential Oil Against Methicillin-Resistant Staphylococcus aureus","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAntimicrobial resistance is one of the most serious global public health threats, leading to a concerning increase in mortality due to infections resistant to treatment [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (\u003cem\u003eS. aureus\u003c/em\u003e) is a Gram-positive pathogen responsible for a wide range of infections and poses a significant public health challenge [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Before the introduction of penicillin in the 1940s, Staphylococcus infections were often untreatable. However, shortly after the widespread use of this drug, penicillin-resistant strains emerged, marking the first documented β-lactam resistance mechanism in \u003cem\u003eS. aureus\u003c/em\u003e, which involved β-lactamase production. Methicillin-resistant \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (MRSA) is recognized as a major cause of both hospital-acquired and community-associated infections [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. According to the Centers for Disease Control and Prevention (CDC), MRSA is classified as a serious threat, accounting for more than 80,461 infections and 11,285 deaths annually in the United States [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Traditionally, β-lactam antibiotics have been used to treat \u003cem\u003eS. aureus\u003c/em\u003e infections; however, due to the emergence of resistance mechanisms, their effectiveness against resistant strains has significantly declined [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Penicillin-binding proteins (PBPs) are key enzymes involved in bacterial cell wall synthesis and serve as primary targets for β-lactam antibiotics. The production of penicillin-binding protein 2a (PBP2a) is one of the key mechanisms conferring β-lactam resistance in MRSA [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The mecA gene, which encodes PBP2a, is incorporated into the bacterial genome \u003cem\u003evia\u003c/em\u003e the staphylococcal cassette chromosome mec (SCCmec). This gene transfer enables the bacterium to produce an enzyme with transpeptidase activity that has a low affinity for β-lactam antibiotics [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eResistance to β-lactams and carbapenems poses a severe threat to public health. If left unaddressed, it is projected that by 2050, antimicrobial resistance could result in millions of deaths annually [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Given the inefficacy of conventional antibiotics and the rapid spread of resistant bacterial strains, the exploration of natural compounds as alternative antimicrobial agents has gained considerable attention. Plant-derived compounds, particularly essential oils, have been recognized as promising candidates for combating drug-resistant bacteria due to their diverse chemical compositions and antimicrobial properties [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Essential oils are commonly volatile, aromatic mixtures extracted from various plant parts. Due to their accessibility, cost-effectiveness, safety profile, and the low likelihood of microbial resistance, they are considered viable alternatives to synthetic drugs for the treatment of infectious diseases [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. However, their antimicrobial mechanisms and chemical compositions are not uniform across all essential oils, these compounds can effectively eliminate bacteria and fungi while inducing minimal adverse effects in consumers [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. \u003cem\u003eZataria multiflora\u003c/em\u003e, commonly known as Shirazi thyme, belongs to the Lamiaceae family. Traditionally, this medicinal plant has been used to treat respiratory infections, alleviate bloating, and serve as an antiseptic and anesthetic agent. Previous studies have demonstrated that \u003cem\u003eZ. multiflora\u003c/em\u003e essential oil exhibits antimicrobial properties against both Gram-positive and Gram-negative bacteria, largely attributed to its phenolic constituents such as thymol and carvacrol [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eWith advancements in technology, bioinformatics approaches such as molecular docking have emerged as powerful tools for simulating and analyzing the interactions between proteins and plant-derived compounds. Molecular docking enables the precise evaluation of the inhibitory potential of various compounds against target proteins, facilitating the identification of the most effective inhibitors against drug-resistant proteins [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThis study aims to comprehensively assess the antimicrobial activity of \u003cem\u003eZ. multiflora\u003c/em\u003e essential oil against \u003cem\u003eS. aureus\u003c/em\u003e strains, evaluate its synergistic effects with cefoxitin, and elucidate its mechanism of action through molecular docking analysis. In this study, the bioactive compounds identified in \u003cem\u003eZ. multiflora\u003c/em\u003e essential oil are assessed for their interactions with PBP2x using molecular docking techniques (PDB ID: 5OJ0) [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. This computational approach allows for the simulation of molecular interactions between PBP2x and essential oil components, leading to the identification of the most promising ligands with the lowest binding free energies. It is hypothesized that some of these compounds can effectively bind to PBP2x, inhibiting its function and thereby preventing bacterial cell wall synthesis. Furthermore, the combination of these natural compounds with antibiotics such as cefoxitin, imipenem, and meropenem may not only reduce treatment costs but also mitigate the side effects of conventional antibiotics, ultimately improving patient outcomes. Finally, this study aims to provide evidence-based scientific insights that can contribute to the development of practical strategies for combating antibiotic resistance.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Materials and Bacterial Strains Used\u003c/h2\u003e\u003cp\u003eOne clinical strain of MRSA and the standard strain ATCC 25923 were obtained from the Microbial Bank of the Faculty of Medical Sciences, Jundishapur University of Ahvaz. [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Phenotypic identification was confirmed by Gram staining and standard biochemical tests, including catalase, coagulase, and DNase assays, as well as characteristic growth patterns on blood agar and mannitol salt agar (MSA). The chemicals and reagents were bought from Merck (Darmstadt, Germany) except indicated.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Bacterial Culture and Viability Confirmation\u003c/h2\u003e\u003cp\u003eThe bacterial strains were cultured on Mueller-Hinton agar using a sterile loop under flame sterilization. Following incubation at 37\u0026deg; C for 24 hours, microbial growth was examined. The presence of convex, cream-colored colonies of \u003cem\u003eS. aureus\u003c/em\u003e confirmed bacterial viability. Methicillin resistance was determined using a cefoxitin (30 \u0026micro;g) disk and disk diffusion method according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. \u003cem\u003eStaphylococcus aureus\u003c/em\u003e ATCC 33591 was used as the methicillin-resistant (positive) control strain, and ATCC 25923 was used as the methicillin-sensitive (negative) control.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Bacterial DNA Extraction and Assessment of DNA Quality\u003c/h2\u003e\u003cp\u003eIn the process of DNA extraction, bacterial colonies from a 24-hour culture on Mueller-Hinton agar were suspended in 500 \u0026micro;L of distilled water. The suspension was then incubated at 95\u0026deg;C for 10 minutes using a Thermo block to lyse the cells. Following heat treatment, the microtubes were placed in a \u0026minus;\u0026thinsp;20\u0026deg;C freezer for 5 minutes to enhance cell lysis. Subsequently, the samples were centrifuged at 14,000 rpm for 10 minutes. The supernatant containing the genomic DNA was carefully collected and used as the DNA template for subsequent analyses [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e])\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Primer Design, PCR, and Electrophoresis of PCR Products\u003c/h2\u003e\u003cp\u003eThe primer was selected based on previous studies [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], and its specificity was confirmed using the NCBI Primer-BLAST tool. Following the manufacturer's protocol (SinaClon), sterile distilled water was added to the lyophilized primer vial to achieve a stock concentration of 100 \u0026micro;M. For each experiment, a working solution of 10 \u0026micro;M was freshly prepared by diluting 10 \u0026micro;L of the stock with 90 \u0026micro;L of sterile distilled water in a sterile microtube. After thorough vortexing, the primer solution was utilized for PCR amplification. Details of the designed primers are provided in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/p\u003e\u003cp\u003ePCR reactions were prepared in a final volume of 25 \u0026micro;L in sterile microtubes. Each reaction contained 1 \u0026micro;L of purified genomic DNA from the target isolate. DNA amplification was performed in a thermal cycler (Eppendorf, Germany) using the following program: initial denaturation at 95\u0026deg;C for 5 minutes; 35 cycles of denaturation at 95\u0026deg;C for 35 seconds, annealing at 55\u0026deg;C for 35 seconds, and extension at 72\u0026deg;C for 35 seconds; followed by a final extension at 72\u0026deg;C for 5 minutes. PCR products were analyzed by electrophoresis on a 2% agarose gel pre-stained with SafeStain. A 100 bp DNA ladder was used as a molecular size marker. Electrophoresis was performed at 70 V for approximately 30 minutes. After completion, the gel was visualized under UV light at 560 nm using a Gel Documentation System (Vilber, France) to detect DNA bands based on their size (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5. Preparation of Plant Samples and Essential Oil Extraction\u003c/h2\u003e\u003cp\u003eThe leaves of \u003cem\u003eZataria multiflora\u003c/em\u003e (Shirazi thyme) were obtained from the Pharmaceutical Research Center of Jundishapur University. The plant identity was confirmed by experts at the university\u0026rsquo;s herbarium. To prevent enzymatic degradation and chemical alterations, the leaves were dried in the shade. The dried leaves were then ground into a fine powder and stored in sterile, dark laboratory containers at 4\u0026deg; C until use. Essential oils were extracted using the Clevenger apparatus \u003cem\u003evia\u003c/em\u003e hydro-distillation. Briefly, 100 g of dried powdered plant material was placed in a glass boiling flask and submerged in distilled water. After 30\u0026ndash;45 minutes of heating, the essential oil was collected. The extracted essential oils were stored in sealed, dark containers at 4\u0026deg; C for further analysis. The extracted essential oil was subsequently analyzed using Gas Chromatography-Mass Spectrometry (GC-MS) to identify its chemical constituents. The prepared essential oil ingredients were analyzed as following: 40 mg of essential oil was dissolved in 4 ml of ethyl acetate and 0.2 \u0026micro;l of the produced solution was directly injected to gas chromatography (Agilent, 7890 A GC/5975 MSD Model) using a HP5-MS (30M\u0026times;250\u0026micro;M\u0026times;0.25\u0026micro;M) column and analyzed by a mass spectrometer analyzer. The inlet and auxiliary temperatures were set at 280\u0026deg;C and the split ratio was set to 50/1.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6. Antibacterial Activity Assessment and Determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC)\u003c/h2\u003e\u003cp\u003eTo determine the Minimum Inhibitory Concentration (MIC) of \u003cem\u003eZataria multiflora\u003c/em\u003e, a stock solution was prepared in methanol. The MIC was assessed using the broth microdilution method, following CLSI guidelines. So, Serial two-fold dilutions of the extract were prepared. A bacterial suspension with a turbidity equivalent to 0.5 McFarland standard was prepared and subsequently diluted 1:100 in Tryptic Soy Broth (TSB). Then, 100 \u0026micro;L of this diluted suspension was added to each well, resulting in a final bacterial concentration of 5\u0026times;10⁵ CFU/mL. Two additional wells served as controls: the positive control contained bacterial suspension and TSB without extract, while the negative control contained extract and TSB without bacteria. The microplates were incubated at 35\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C for 16\u0026ndash;20 hours. Following incubation, the MIC was defined as the lowest extract concentration at which no visible turbidity was observed. To determine the Minimum Bactericidal Concentration (MBC), 10 \u0026micro;L from each clear well (showing no visible growth) was plated onto Mueller-Hinton Agar. After incubation at 35\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C for 24 hours, colonies were counted. The MBC was defined as the lowest concentration of the extract resulting in at least a 3-log₁₀ reduction in viable bacterial count compared to the initial inoculum (5\u0026times;10⁵ CFU/mL).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7. Evaluation of Synergistic Effects Between Extract and Cefoxitin Against MRSA and MSSA\u003c/h2\u003e\u003cp\u003eTo assess the synergistic effects of plant extract and the antibiotic cefoxitin, both methicillin-resistant \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (MRSA) and methicillin-susceptible \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (MSSA) strains were tested. A cefoxitin disc with a concentration of 30 \u0026micro;g was used in this experiment. For synergy testing, sub-minimum inhibitory concentration (sub-MIC) levels of the extract were used separately. A volume of 10 \u0026micro;L of the extract at its sub-MIC concentration was individually loaded onto a 30 \u0026micro;g cefoxitin disc. Additionally, a cefoxitin disc without any extract, as well as individual sub-MIC concentrations of the extract, were placed separately on inoculated Mueller-Hinton agar plates containing bacterial cultures. Following incubation at 37\u0026deg; C for 24 hours, the inhibition zones around each disc were measured in millimeters to determine the antimicrobial and potential synergistic effects.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8. Retrieval and Preparation of PBP2X Crystal Structure\u003c/h2\u003e\u003cp\u003eIn the initial phase of this study, the crystallographic structure of PBP2X with appropriate resolution in the presence of cefepime will be obtained from the RCSB Protein Data Bank (PDB) [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] \u003cem\u003evia\u003c/em\u003e \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.rcsb.org\u003c/span\u003e\u003cspan address=\"https://www.rcsb.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Based on the retrieved crystal structure, the sequence, secondary structures, and interactions of PBP2X with the cefepime ligand will be analyzed in detail. Next, the protein structure of PBP2X will be extracted from the 5oJ0.pdb file. Using AutoDock Tools [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], all water molecules will be removed, missing polar hydrogens will be added, and the structural grid of PBP2X will be precisely defined. On the other hand, the structure of cefepime will be retrieved in SDF format from Pubchem data base (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pubchem.ncbi.nlm.nih.gov/\u003c/span\u003e\u003cspan address=\"https://pubchem.ncbi.nlm.nih.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] and converted to PDB format. Using AutoDock Tools, the molecule will be further prepared by adding charged hydrogen atoms and determining its atomic charges through the Compute Gasteiger algorithm. The final ligand structure will be stored in PDBQT format for docking studies. Following these preparations, validation docking will be performed between cefepime and PBP2X. If the docking results yield a suitable ΔG of binding, the generated complexes will be analyzed in three-dimensional format using Chimera [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] and VMD [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] and in two-dimensional format using LigPlot [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.9. Molecular Docking of Essential Oil Constituents Against PBP2X\u003c/h2\u003e\u003cp\u003eIn the next phase, the identified compounds in \u003cem\u003eZataria multiflora\u003c/em\u003e essential oil will be retrieved in SDF format from PubChem [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] and converted to PDBQT format for molecular docking studies. The molecular docking will be performed against the three-dimensional structure of PBP2X, targeting the cefepime-binding site in 5oJ0.pdb, using AutoDock Tools 4.2 and AutoDock Vina [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] (Table S3). The binding affinities (ΔG of binding) will be evaluated, and the compounds will be ranked based on their docking scores for further investigation. After identifying the most promising PBP2X inhibitors, the physicochemical properties, pharmacokinetics, and toxicity of the top three compounds with the highest binding affinities will be predicted using SwissADME [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] and PASS Online [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.10. Statistical Analysis\u003c/h2\u003e\u003cp\u003eStatistical analyses were performed using student's t-test. A significance level of p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant. Data analysis was conducted using SPSS version 2.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results and Discussion","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e3.1. Identification and Confirmation of MRSA Clinical Strain\u003c/h2\u003e\u003cp\u003eThe clinical isolates used in this study were identified as MRSA based on phenotypic and genotypic characteristics. Those isolates that induced a color change in MSA to yellow were selected for further testing. Final confirmation was based on positive results of coagulase, catalase, and DNase tests. Additionally, according to the 2023 CLSI guidelines, isolates exhibiting an inhibition zone diameter of \u0026le;\u0026thinsp;21 mm around the cefoxitin disk were classified as MRSA.\u003c/p\u003e\u003cp\u003eFurther validation was achieved by the detection of the mecA gene \u003cem\u003evia\u003c/em\u003e PCR (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e), confirming the presence of the methicillin resistance determinant responsible for the synthesis of penicillin-binding protein 2a (PBP2a). Electrophoresis results revealed a distinct 310 bp DNA band corresponding to the expected size of the mecA gene (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e), verifying the identity of the MRSA isolates. This molecular validation was crucial, as mecA is a key genetic marker distinguishing methicillin resistance in \u003cem\u003eS. aureus\u003c/em\u003e. Consistent with findings by KB Anand and his colleagues (2009), the presence of mecA correlates strongly with resistance to all β-lactam antibiotics, especially cefoxitin [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. This initial characterization laid the foundation for subsequent antimicrobial and synergistic assays.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.2. Antibacterial Activity of \u003cem\u003eZataria multiflora\u003c/em\u003e Essential Oil\u003c/h2\u003e\u003cp\u003eThe broth microdilution method was used to evaluate the antibacterial activity of \u003cem\u003eZataria multiflora\u003c/em\u003e essential oil against both the standard strain (ATCC 25923) and clinical MRSA strains. Results showed significant inhibitory effects, with the clinical MRSA strain exhibiting slightly lower susceptibility than the standard strain (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). The minimum inhibitory concentration and minimum bactericidal concentration were determined \u003cem\u003evia\u003c/em\u003e dilution and disk placement assays. For the ATCC 25923 strain, the MIC was 3.125 mg.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and MBC was 6.25 mg.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. However, for the MRSA isolate, both MIC and MBC were recorded at 6.25 mg.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, indicating a higher threshold required for inhibition and killing. These findings may align with the known resistance mechanisms of MRSA, particularly the expression of PBP2a, which diminishes the efficacy of β-lactams and likely contributes to decreased essential oil sensitivity. Notably, these results are consistent with the antimicrobial properties reported for \u003cem\u003eZ. multiflora\u003c/em\u003e, a plant rich in bioactive compounds like thymol, carvacrol, and other phenolics. Previous reports by Maryam Motevasel et al. (2013) also document its potent antibacterial activities against Gram-positive pathogens, including \u003cem\u003eS. aureus\u003c/em\u003e [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The differential response between MSSA and MRSA suggests that \u003cem\u003eZ. multiflora\u003c/em\u003e essential oil may exert effects independent of β-lactam targets, which becomes especially relevant in the context of synergistic assays.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.3. Synergistic Interaction Between Cefoxitin and Essential Oil\u003c/h2\u003e\u003cp\u003eOne of the study's most promising findings was the enhanced antibacterial effect observed when sub-MIC concentrations of \u003cem\u003eZ. multiflora\u003c/em\u003e essential oil were combined with cefoxitin. In disk diffusion assays, cefoxitin alone displayed a limited inhibition zone against MRSA, confirming resistance. However, the co-application of the essential oil significantly increased the diameter of the inhibition zone, indicating a synergistic interaction. Quantitatively, the increase in the inhibition zone was statistically significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), reflecting enhanced bacterial susceptibility. This synergistic effect suggests that essential oil constituents may facilitate cefoxitin access to its target site, possibly by disrupting the cell membrane or altering the expression/conformation of PBP2a [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Although the precise molecular mechanism remains speculative, this observation echoes findings from Aribisala et al. (2024), who demonstrated similar synergy between phenolic compounds and β-lactam antibiotics [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The clinical implications of such synergy are substantial. The restoration or enhancement of cefoxitin efficacy through natural compounds could reduce reliance on high-dose β-lactam therapy, lower the risk of toxicity, and delay the emergence of further resistance.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e3.4. GC-MS Analysis of \u003cem\u003eZataria multiflora\u003c/em\u003e Essential Oil\u003c/h2\u003e\u003cp\u003eGas chromatography\u0026ndash;mass spectrometry analysis of the essential oil revealed several bioactive constituents (Table S3). Among them, o-Cymene (11.00%), Thymol (37.76%), and Carvacrol (18.59%) were identified as major components with potential antibacterial properties. The molecular diversity of these compounds contributes to the broad-spectrum antibacterial effect of the essential oil [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The identification of these compounds served as the basis for subsequent \u003cem\u003ein silico\u003c/em\u003e docking studies. Their molecular weights, hydrogen bond acceptors/donors, and structural compatibility with biological targets suggested possible interactions with bacterial proteins involved in resistance mechanisms, particularly PBP2x.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e3.5. Molecular Docking with PBP2x\u003c/h2\u003e\u003cp\u003eTo understand the molecular interactions between essential oil components and bacterial resistance proteins, molecular docking studies were conducted using AutoDock Vina [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The crystal structure of PBP2x (PDB ID: 5OJ0) was used as the target, and the docking site was defined based on the binding pocket of cefepime (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), a fourth-generation cephalosporin [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Cefepime served as the positive control and exhibited a binding energy of -7.5 kcal.mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eB and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Among the essential oil constituents, spathulenol and isospathulenol both displayed binding energies of -6.7 kcal.mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, while aromadendrene showed a slightly weaker interaction at -6.6 kcal.mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Table S3). The docking results are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Despite having weaker affinities compared to cefepime, the essential oil constituents demonstrated comparable binding energies, suggesting that they may competitively inhibit PBP2x. Notably, spathulenol and isospathulenol in spite of smaller and more simple structure exhibited ligand efficiencies higher than cefepime (0.4188 \u003cem\u003evs\u003c/em\u003e. 0.2344 kcal/mol/non-H atom), indicating a strong binding per atom, a desirable trait in drug development. Visual inspection of the ligand-protein interactions (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e) confirmed that these molecules occupied similar spatial positions as cefepime within the PBP2x active site (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). This spatial mimicry suggests that the essential oil compounds might interfere with cell wall synthesis, a hallmark mechanism of β-lactam antibiotics.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eChemical name of the substance, Pubchem code, binding free energy, inhibition constant, ligand binding efficiency and chemical structure of cefepime and compounds present in the essential oil obtained from Shirazi thyme (Zataria multiflora) with the highest binding energy docked against the PBP2x protein obtained from the crystallographic structure 5OJ0.pdb.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eName of the ligand\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePubchem ID\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eBinding Free Energy (kcal/mol)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003epKi\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eLigand Efficiency (kcal/mol/non-H atom)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eChemical Structure\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCefepime\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e5479537\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e-7.5\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e5.5\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e0.2344\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSpathulenol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e13854255\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e-6.7\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e4.91\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e0.4188\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIsospathulenol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e(14038848)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e-6.7\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e4.91\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e0.4188\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e(+)-Aromadendrene\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e11095734\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e-6.6\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e4.84\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e0.44\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e3.6. Structure-Activity Relationship and Drug-Likeness\u003c/h2\u003e\u003cp\u003eFurther evaluation using SwissADME [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] and PASS online tools [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] indicated favorable physicochemical and pharmacokinetic profiles for the docked compounds (Table S4). All three compounds adhered to Lipinski\u0026rsquo;s Rule of Five, indicating drug-likeness. Additionally, predicted properties included good gastrointestinal absorption, absence of P-glycoprotein efflux susceptibility, and significant blood-brain barrier permeability\u0026mdash;suggesting these compounds might be suitable for systemic for CNS infection. Toxicity assessments predicted low mutagenic and carcinogenic risks for these molecules, further supporting their potential as antimicrobial adjuvants. The ability of these compounds to act synergistically with existing antibiotics, coupled with favorable pharmacokinetics, reinforces their potential role in future anti-MRSA therapeutic formulations.\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eThis study comprehensively demonstrated the significant antibacterial and synergistic properties of \u003cem\u003eZataria multiflora\u003c/em\u003e (Shirazi thyme) essential oil against methicillin-resistant \u003cem\u003eStaphylococcus aureus\u003c/em\u003e. In current study we concluded, clinical validation of MRSA resistance, effective antibacterial activity against both MRSA and the standard \u003cem\u003eS. aureus\u003c/em\u003e strain, synergistic enhancement of cefoxitin efficacy which could enhance clinical efficacy while potentially reducing cefoxitin dosage, minimizing adverse effects and delaying resistance, spathulenol, isospathulenol, and aromadendrene were identified as major active constituents, these terpenoids are known for their antimicrobial properties and were selected for molecular docking, the essential oil compounds showed strong binding affinities to PBP2x, comparable to cefepime, their physicochemical profiles combined with predicted low toxicity encourage further \u003cem\u003ein vivo\u003c/em\u003e and clinical evaluation, and the integration of essential oils with standard antibiotics represents a promising strategy to revitalize old antibiotics and combat multidrug-resistant organisms. In summary, the use of \u003cem\u003eZ. multiflora\u003c/em\u003e essential oil\u0026mdash;rich in spathulenol, isospathulenol, and aromadendrene\u0026mdash;offers a promising adjunctive treatment approach for MRSA infections. By combining natural compounds with conventional antibiotics, we can not only restore lost efficacy but also reduce treatment costs and slow the global rise of antibiotic resistance. Future work should explore the \u003cem\u003ein vivo\u003c/em\u003e efficacy, formulation stability, and clinical pharmacokinetics of these combinations to facilitate their translation into medical practice.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCenters for Disease Control and Prevention\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCDC\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eMethicillin-resistant \u003cem\u003eStaphylococcus aureus\u003c/em\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eMRSA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eMethicillin-susceptible \u003cem\u003eStaphylococcus aureus\u003c/em\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eMSSA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eMinimum Inhibitory Concentration\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eMIC\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ePenicillin-binding proteins\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePBPs\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ePenicillin-binding protein 2a\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePBP2a\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003e\u003cem\u003eStaphylococcus aureus\u003c/em\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cem\u003eS. aureus\u003c/em\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eStaphylococcal cassette chromosome mec\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eSCCmec.\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics Statement:\u003c/strong\u003e Ethical approval for this study was obtained from Abadan University of Medical Sciences, Abadan, Iran (ID: IR.ABADANUMS.REC.1402.055).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Participate:\u0026nbsp;\u003c/strong\u003eThis study did not involve human participants. Consent to participate was therefore not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Publish declarations:\u0026nbsp;\u003c/strong\u003eAll authors have reviewed and approved the final version of this manuscript and consent to its publication. This study did not involve human participants or personal data requiring individual consent for publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e The authors did not receive support from any organization for the submitted work. No funding was received to assist with the preparation of this manuscript. No funding was received for conducting this study. No funds, grants, or other support was received.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest/Competing interests:\u003c/strong\u003e All authors certify that they have no affiliations with or involvement in any organization or entity with any financial or non-financial interest in the subject matter or materials discussed in this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material:\u003c/strong\u003e The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions:\u003c/strong\u003e In this research, all authors have been in charge of research, data collection and analysis, and article writing.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eTurner NA et al (2019) Methicillin-resistant Staphylococcus aureus: an overview of basic and clinical research. Nat Rev Microbiol 17(4):203\u0026ndash;218\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJensen SO, Lyon BR (2009) Genetics of antimicrobial resistance in Staphylococcus aureus. Future Microbiol 4(5):565\u0026ndash;582\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWatkins RR, Bonomo RA (2020) Overview: the ongoing threat of antimicrobial resistance. Infect Disease Clin 34(4):649\u0026ndash;658\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFoster TJ (2019) Can β-lactam antibiotics be resurrected to combat MRSA? Trends Microbiol 27(1):26\u0026ndash;38\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePeacock SJ, Paterson GK (2015) Mechanisms of methicillin resistance in Staphylococcus aureus. Annu Rev Biochem 84(1):577\u0026ndash;601\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFuda C et al (2004) The basis for resistance to β-lactam antibiotics by penicillin-binding protein 2a of methicillin-resistant Staphylococcus aureus. J Biol Chem 279(39):40802\u0026ndash;40806\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDing D et al (2023) The spread of antibiotic resistance to humans and potential protection strategies. Ecotoxicol Environ Saf 254:114734\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAribisala JO, S\u0026rsquo;thebe NW, Sabiu S (2024) silico exploration of phenolics as modulators of penicillin binding protein (PBP) 2\u0026times; of Streptococcus pneumoniae. Sci Rep 14(1):8788\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVahabi S, Najafi E, Alizadeh S (2011) In vitro antimicrobial effects of some herbal essences against oral pathogens. J Med Plant Res 5(19):4870\u0026ndash;4878\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNgamsurach P, Praipipat P (2022) Antibacterial activities against Staphylococcus aureus and Escherichia coli of extracted Piper betle leaf materials by disc diffusion assay and batch experiments. RSC Adv 12(40):26435\u0026ndash;26454\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAli MS et al (2000) Chemistry of zataria multiflora (lamiaceae). Phytochemistry 55(8):933\u0026ndash;936\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eStanzione F, Giangreco I, Cole JC (2021) Use of molecular docking computational tools in drug discovery. Prog Med Chem 60:273\u0026ndash;343\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBernardo-Garc\u0026iacute;a N et al (2018) Allostery, recognition of nascent peptidoglycan, and cross-linking of the cell wall by the essential penicillin-binding protein 2x of Streptococcus pneumoniae. ACS Chem Biol 13(3):694\u0026ndash;702\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHasanian S et al (2020) The Comparison of cefoxitin disk disffusion method with other phenotypic and molecular methods in identification of methicillin-resistant Staphylococcus aureus in clinical specimens of patients admitted to Ahvaz educational hospitals. Jundishapur Sci Med J 19(3):253\u0026ndash;266\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eQueipo-Ortu\u0026ntilde;o MI et al (2008) Preparation of bacterial DNA template by boiling and effect of immunoglobulin G as an inhibitor in real-time PCR for serum samples from patients with brucellosis. Clin Vaccine Immunol 15(2):293\u0026ndash;296\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePimenta LKL et al (2023) Staphylococcus spp. causatives of infections and carrier of blaZ, femA, and mecA genes associated with resistance. Antibiotics 12(4):671\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMorris GM et al (2009) AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility. J Comput Chem 30(16):2785\u0026ndash;2791\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKim S et al (2023) PubChem 2023 update. Nucleic Acids Res 51(D1):D1373\u0026ndash;D1380\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePettersen EF et al (2004) UCSF Chimera\u0026ndash;a visualization system for exploratory research and analysis. J Comput Chem 25(13):1605\u0026ndash;1612\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHumphrey W, Dalke A, Schulten K (1996) VMD: visual molecular dynamics. J Mol Graph 14(1):33\u0026ndash;38\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWallace AC, Laskowski RA, Thornton JM (1995) LIGPLOT: a program to generate schematic diagrams of protein-ligand interactions. Protein Eng Des selection 8(2):127\u0026ndash;134\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEberhardt J et al (2021) AutoDock Vina 1.2. 0: New docking methods, expanded force field, and python bindings. J Chem Inf Model 61(8):3891\u0026ndash;3898\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDaina A, Michielin O, Zoete V (2017) SwissADME: a free web tool to evaluate pharmacokinetics, drug-likeness and medicinal chemistry friendliness of small molecules. Sci Rep 7:42717\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFilimonov D et al (2014) Prediction of the biological activity spectra of organic compounds using the PASS online web resource. Chem Heterocycl Compd 50:444\u0026ndash;457\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAnand K et al (2009) Comparison of cefoxitin disc diffusion test, oxacillin screen agar, and PCR for mecA gene for detection of MRSA. Ind J Med Microbiol 27(1):27\u0026ndash;29\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMotevasel M et al (2013) A study of the effect of Zataria multiflora extract on methicillin resistant staphylococcus aureus. Jundishapur J Microbiol 6(5):1A\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePoole K (2004) Resistance to β-lactam antibiotics. Cell Mol Life Sci CMLS 61:2200\u0026ndash;2223\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAribisala JO, Aruwa CE, Sabiu S (2024) Progressive approach of phenolic acids toward the advancement of antimicrobial drugs. Advancement of Phenolic Acids in Drug Discovery. Elsevier, pp 177\u0026ndash;210\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRadulovic N et al (2013) Antimicrobial plant metabolites: structural diversity and mechanism of action. Curr Med Chem 20(7):932\u0026ndash;952\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOkamoto MP et al (1994) Cefepime: a new fourth-generation cephalosporin. Am J Health-System Pharm 51(4):463\u0026ndash;477\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"molecular-biology-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mole","sideBox":"Learn more about [Molecular Biology Reports](https://www.springer.com/journal/11033)","snPcode":"11033","submissionUrl":"https://submission.nature.com/new-submission/11033/3","title":"Molecular Biology Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Methicillin-resistant Staphylococcus aureus, Zataria multiflora, mecA gene, PBP2X, Antibiotic resistance, Molecular Docking","lastPublishedDoi":"10.21203/rs.3.rs-7411474/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7411474/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eMethicillin-resistant \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (MRSA) is a significant public health concern due to its resistance to multiple antibiotics, primarily mediated by the \u003cem\u003emecA\u003c/em\u003e gene, which encodes penicillin-binding protein 2a (PBP2a), given the urgent need for alternative antimicrobial agents.\u003c/p\u003e\u003ch2\u003eMaterials and Methods\u003c/h2\u003e\u003cp\u003eA clinical MRSA strain harboring the \u003cem\u003emecA\u003c/em\u003e gene was identified. Antimicrobial susceptibility testing was performed to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of Shirazi thyme (\u003cem\u003eZataria multiflora\u003c/em\u003e) essential oil. The strain\u0026rsquo;s susceptibility to multiple antibiotics was assessed and the synergistic effect of \u003cem\u003eZataria multiflora\u003c/em\u003e essential oil and cefoxitin was also evaluated. Gas chromatography-mass spectrometry (GC-MS) was employed to identify the bioactive compounds in the essential oil. Molecular docking studies were performed to evaluate the competitive binding affinity of those compounds to PBP2x.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eThe MRSA strain exhibited resistance to all tested antibiotics except linezolid. The MIC and MBC values for \u003cem\u003eZataria multiflora\u003c/em\u003e essential oil were 3.125 mg.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 6.25 mg.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for the reference \u003cem\u003eS. aureus\u003c/em\u003e strain (ATCC 25923), and 6.25 mg.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for both MIC and MBC for the clinical MRSA strain. The synergy assay demonstrated an enhanced inhibition zone for cefoxitin in combination with \u003cem\u003eZataria multiflora\u003c/em\u003e essential oil, indicating a synergistic interaction. Molecular docking studies revealed strong binding interactions between spathulenol, isospathulenol, and aromadendrene comparable to clinically used β-lactam antibiotics.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eThe findings highlight the significant antibacterial activity of \u003cem\u003eZataria multiflora\u003c/em\u003e essential oil against MRSA and its potential to enhance the efficacy of cefoxitin through synergistic interactions as natural inhibitors of PBP2x.\u003c/p\u003e","manuscriptTitle":"Antibacterial and Synergistic Effects of Shirazi Thyme (Zataria multiflora) Essential Oil Against Methicillin-Resistant Staphylococcus aureus","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-05 19:56:46","doi":"10.21203/rs.3.rs-7411474/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-02T08:53:28+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-14T16:54:35+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"54997811009593338389635965544275568563","date":"2025-09-02T02:49:32+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-27T19:50:34+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-27T17:28:59+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-27T03:58:31+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular Biology Reports","date":"2025-08-19T19:38:39+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-biology-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mole","sideBox":"Learn more about [Molecular Biology Reports](https://www.springer.com/journal/11033)","snPcode":"11033","submissionUrl":"https://submission.nature.com/new-submission/11033/3","title":"Molecular Biology Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"6b39c02b-050f-4a38-90c4-45779d221980","owner":[],"postedDate":"September 5th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-02-09T16:05:11+00:00","versionOfRecord":{"articleIdentity":"rs-7411474","link":"https://doi.org/10.1007/s11033-025-11391-5","journal":{"identity":"molecular-biology-reports","isVorOnly":false,"title":"Molecular Biology Reports"},"publishedOn":"2026-02-04 15:59:52","publishedOnDateReadable":"February 4th, 2026"},"versionCreatedAt":"2025-09-05 19:56:46","video":"","vorDoi":"10.1007/s11033-025-11391-5","vorDoiUrl":"https://doi.org/10.1007/s11033-025-11391-5","workflowStages":[]},"version":"v1","identity":"rs-7411474","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7411474","identity":"rs-7411474","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}