A Study of the Synergistic Effects of Essential Oils from Origanum compactum and Origanum elongatum with Commercial Antibiotics against Highly Prioritized Multidrug-Resistant Bacteria for the World Health Organization

The irrational use of antibiotics has favored the emergence of resistant bacteria, posing a serious threat to global health. To counteract antibiotic resistance, this research seeks to identify novel antimicrobials derived from essential oils that operate through several mechanisms. It aims to evaluate the quality and composition of essential oils from Origanum compactum and Origanum elongatum; test their antimicrobial activity against various strains; explore their synergies with commercial antibiotics; predict the efficacy, toxicity, and stability of compounds; and understand their molecular interactions through docking and dynamic simulations. The essential oils were extracted via hydrodistillation from the flowering tops of oregano in the Middle Atlas Mountains in Morocco. Gas chromatography combined with mass spectrometry (GC-MS) was used to examine their composition. Nine common antibiotics were chosen and tested alone or in combination with essential oils to discover synergistic effects against clinically important and resistant bacterial strains. A comprehensive in silico study was conducted, involving molecular docking and molecular dynamics simulations (MD). O. elongatum oil includes borneol (8.58%), p-cymene (42.56%), thymol (28.43%), and carvacrol (30.89%), whereas O. compactum oil is mostly composed of γ-terpinene (22.89%), p-cymene (15.84%), thymol (10.21%), and (E)-caryophyllene (3.63%). With O. compactum proving to be the most potent, these essential oils showed antibacterial action against both Gram-positive and Gram-negative bacteria. Certain antibiotics, including ciprofloxacin, ceftriaxone, amoxicillin, and ampicillin, have been shown to elicit synergistic effects. To fight resistant bacteria, the essential oils of O. compactum and O. elongatum, particularly those high in thymol and (E)-caryophyllene, seem promising when combined with antibiotics. These synergistic effects could result from their ability to target the same bacterial proteins or facilitate access to target sites, as suggested by molecular docking simulations. Molecular dynamics simulations validated the stability of the examined protein–ligand complexes, emphasizing the propensity of substances like thymol and (E)-caryophyllene for particular target proteins, opening the door to potentially effective new therapeutic approaches against pathogens resistant to multiple drugs.


Introduction
The development of antibiotics has revolutionized public health and prevented millions of deaths.However, the excessive and irrational use of these drugs has led to their dispersion in the environment, promoting the emergence of antibiotic-resistant bacteria [1].These multidrug-resistant bacteria are one of the top threats to global food security, development, and health, according to a report by the WHO [2].
The use of antibiotics has been on the rise in recent decades.Global antibiotic consumption increased by 46% between 2000 and 2010 [3].This large quantity of antibiotics ends up in wastewater, and even if it reaches treatment plants, they fail to eliminate these residues, releasing them into water bodies [4].Antibiotic residues in high amounts have been found in effluents; some of these residues are applied as fertilizers to the soil.
This widespread dissemination of antibiotic residues in the environment promotes selective pressure and the spread of resistance genes.Antibiotic resistance can result from mutations that modify the molecular targets of bacterial antibiotics [5].Multidrug-resistant pathogen infections are becoming more difficult to treat, which highlights the need for novel antimicrobial compounds with distinct mechanisms of action that can reduce resistance and, if possible, have fewer negative effects on the health of humans, animals, and the environment following the "One Health" philosophy [6].
To battle strains that are resistant to several drugs, the WHO has created an action plan that centers on the creation of novel antimicrobial products.The search for natural compounds that may be effective as novel antibacterial agents has therefore been quite active.
Numerous natural plant-based medications, such as essential oils (EOs), have been investigated for the treatment and prevention of multidrug-resistant micro-organisms [7][8][9].Unfortunately, natural products generally have weaker antibiotic activity than common antibiotics; therefore, it is challenging for them to effectively replace current antibiotics in clinical practice.Nonetheless, it has been shown that a small number of plant-derived antimicrobial compounds may work in concert to boost antibiotic action [10,11].By lowering the minimum inhibitory concentration (MIC) of both the antibiotic and the natural product, the synergistic interaction of natural compounds with commercial antibiotics can make the combination as effective as the antibiotic alone while preserving the use of commercial antibiotics (ATBs) [12,13].
Since combinations with synergistic effects can lower the likelihood of bacterial resistance emergence while maintaining effective pharmacological outcomes, using lower concentrations of both agents presents significant opportunities for exploring alternative solutions for the treatment of infectious diseases [14].Furthermore, as opposed to the side effects from large doses of synthetic medications, this may result in less toxicity from antibiotics [15].
Polyphenols, alkaloids, carotenoids, terpenes, terpenoids, and sulfur compounds are among the phytochemical substances showing promising antimicrobial activity in vitro [16].Global demands for antibiotics are increasing, but antibiotic research programs are significantly inadequate; for example, no new class of Gram-negative antibiotics has been introduced in over 50 years [17].Therefore, medicinal plants can represent a rapid and safe source of innovation for new antimicrobial agents.
The Lamiaceae family of plants is one of the most significant for yielding essential oils as it has antibacterial and antioxidant characteristics.The majority of aromatic plants that are high in essential oils are found in the Mediterranean region, where the extraction of oil is a profitable industry that promotes both ecological and economic growth.Species from the genus Origanum are among these aromatic and therapeutic plants that are often used as spices due to their abundance of essential oils.Additionally, they display a range of biological activities, the potential of which has been demonstrated by numerous scientific investigations [18][19][20].The Lamiaceae family includes the genus Origanum, which is known as "Oregano" in English.In total, 38 species, including 6 subspecies and 17 hybrids, are found in the Mediterranean, Irano-Turanian, and Euro-Siberian regions.Moreover, it is divided into 10 parts [20].Its medicinal use goes back thousands of years because of its many culinary and therapeutic uses.The leaves were used as an antiseptic and for healing skin blemishes.The ancient Greek and Roman empires also employed them to treat various ailments, like indigestion, diarrhea, and asthma [21].A common treatment for colds and stomachaches in Greece is still an oregano infusion [22].Locally known as "Zaatar" or "Zwi" in Berber, oregano species are highly prized in Morocco.While the Zwi aqueous infusion is used to treat dysentery, colitis, bronchopulmonary diseases, gastric acidity, and gastrointestinal ailments, this plant is traditionally used to cure liver disorders in particular places, such as the Middle Atlas (Jbel-Bouiblane) [23].Additionally, several ethnic groups have been using Origanum species in traditional medicine since antiquity to cure and relieve a wide range of ailments.In addition to treating other illnesses, they have antibacterial, anti-inflammatory, antioxidant, anticancer, antifungal, antiviral, and antileishmanial properties [24][25][26].
Finding combinations of these EOs with various classes of commercial antibiotics to find those that have synergistic effects and may allow the use of lower antibiotic doses is the aim of this study.Origanum compactum and Origanum elongatum EOs were harvested in different regions of Morocco.The chemical composition of the EOs was identified.We investigated the synergistic combinations by comparing the minimum inhibitory concentrations of the EOs under investigation with nine commonly used antibiotics, both in isolation and in combination, against microbial strains that cause a variety of human diseases.Following the WHO's list of priority pathogens, the types of bacteria were selected based on their potential severity and capacity for resistance development, as well as their clinical significance as they cause some of the most prevalent diseases in existence today [27].Lastly, to gain a deeper comprehension of the fundamental mechanisms behind our experimental endeavors, we will employ drug similarity prediction, molecular dynamics, pharmacokinetics (ADME-Tox), and molecular docking simulations.

Plant Material
In July 2022, the flowering tops of Origanum compactum and Origanum elongatum were collected in full bloom in the Moroccan Middle Atlas highlands of Bouyablane and Khenifra.At the Scientific Institute of Rabat's botany lab, species identification was performed.The plants were subsequently identified and verified at the Scientific Institute of Rabat's Department of Botany after being dried in the shade for around ten days.Tables 1 and 2, together with Figure 1, offer comprehensive details on each species.

Extraction of Essential Oils
Clevenger-type apparatus equipment was used to hydrodistillate the essential oils for three hours to extract them.For every 100 g of dry matter, the yield of essential oil was calculated in milliliters.The extracted essential oil was kept out of direct sunlight at 4 °C.

Gas Chromatography Coupled with Mass Spectrometry Analysis of Essential Oils
A Thermo Electron mass spectrometer (Thermo Electron: Trace GC Ultra; Polaris Q MS) and gas chromatograph (Trace GC Ultra, Milan, Italy) were used for the chromatographic examination of the examined essential oil sample.By employing an electron collision with an energy level of 70 eV, fragmentation was accomplished.A flame ionization detector (FID) and a DB-5-type column were fitted to the chromatograph.The temperature of the column was set to rise for five minutes, from 50 to 200 °C at a pace of 4 °C per minute.The carrier gas was nitrogen, which was injected in split mode at a rate of 1 mL/min (leak ratio: 1/70).The Kovats indices (KI) of compounds were calculated and compared to those of standard products listed in the Kovats [28], Adams [29], and Hübschmann [30], databases to determine the chemical composition of the essential oil.Each molecule was identified by comparing its retention time to genuine standards that were known to be kept in the laboratory of the authors.Their stated KI and MS data were also verified against the published literature and standards included in the WILEY and NIST 14 standard mass spectrum databases.This was carried out using the Kovats index.To compare the retention periods of any products to the retention times of linear alkanes with an equivalent carbon content, Kovats indices were employed.By simultaneously infusing a traditional C7-C40 alkane combination under the same operating circumstances, they were established.

Extraction of Essential Oils
Clevenger-type apparatus equipment was used to hydrodistillate the essential oils for three hours to extract them.For every 100 g of dry matter, the yield of essential oil was calculated in milliliters.The extracted essential oil was kept out of direct sunlight at 4 • C.

Gas Chromatography Coupled with Mass Spectrometry Analysis of Essential Oils
A Thermo Electron mass spectrometer (Thermo Electron: Trace GC Ultra; Polaris Q MS) and gas chromatograph (Trace GC Ultra, Milan, Italy) were used for the chromatographic examination of the examined essential oil sample.By employing an electron collision with an energy level of 70 eV, fragmentation was accomplished.A flame ionization detector (FID) and a DB-5-type column were fitted to the chromatograph.The temperature of the column was set to rise for five minutes, from 50 to 200 • C at a pace of 4 • C per minute.The carrier gas was nitrogen, which was injected in split mode at a rate of 1 mL/min (leak ratio: 1/70).The Kovats indices (KI) of compounds were calculated and compared to those of standard products listed in the Kovats [28], Adams [29], and Hübschmann [30], databases to determine the chemical composition of the essential oil.Each molecule was identified by comparing its retention time to genuine standards that were known to be kept in the laboratory of the authors.Their stated KI and MS data were also verified against the published literature and standards included in the WILEY and NIST 14 standard mass spectrum databases.This was carried out using the Kovats index.To compare the retention periods of any products to the retention times of linear alkanes with an equivalent carbon content, Kovats indices were employed.By simultaneously infusing a traditional C 7 -C 40 alkane combination under the same operating circumstances, they were established.

Antimicrobial Compounds
The nine antibiotics that were examined were chosen because they represent various modes of action and are among the most widely used antibiotics on the market today.The suppliers of all antibiotics were Sigma Aldrich in Darmstadt, Germany, and Acofarma in Barcelona, Spain.Table 3 presents a comprehensive overview of every chemical.

Micro-Organisms
Twenty-nine bacterial strains (Gram-positive cocci and Gram-negative bacilli) and eight fungal strains (yeasts and molds) were subjected to the antibacterial activity (Table 4).These particular microbes are highly pathogenic and are well known for their invasiveness, potency, and toxicity to humans.They are often seen in Morocco in a variety of infections that present both clinical and treatment challenges.These strains were identified at the Mohamed V Provincial Hospital in Meknès.Furthermore, isolates from Ibn Sina Hospital's parasitology laboratory mycology collection at CHIS Rabat were subjected to antifungal activity.Each strain was reconstituted on Mueller Hinton and Sabouraud broths using a 20% glycerol stock that was kept at −80 • C. Before usage, each strain was subcultured.The minimum inhibitory concentration (MIC) was found in 96-well microplates using the reference microdilution technique [31].Based on the amount of growth visible to the unaided eye during incubation, the minimum inhibitory concentration (MIC) of an EO is the lowest value needed to stop the tested micro-organism's development.A stock solution of each EO produced in 10% DMSO was used for a series of dilutions to achieve concentrations of 5 to 0.93 × 10 −2 mg/mL.These dilutions were made in Sabouraud broth for fungus and Mueller-Hinton medium for bacteria, with a final volume of 100 µL for each concentration.After this, 100 µL of microbial inoculum was added to each of the several dilution steps, with the ultimate concentrations of bacteria and fungus being 10 6 or 10 4 CFU/mL, respectively.Ten microliters of resazurin was applied to each well to measure the growth of bacteria after a 24 h incubation at 37 • C. The color shifted from purple to pink after a second incubation at 37 • C for two hours, indicating microbial growth.The MIC value was discovered to be the lowest concentration that stops resazurin from changing color.The growth and sterility controls were located in wells 11 and 12, respectively.This oil was used for two runs of the test.Terbinafine 250 mg, the typical antifungal employed in this study, was ground into a powder and mixed with 2 milliliters of 10% DMSO.To calculate the minimum bactericidal concentration (MBC) and minimum fungicidal concentration (MFC), 10 µL was extracted from every well that did not exhibit any growth.The samples were then plated on Mueller-Hinton (MH) agar for bacteria or in Sabouraud broth for fungi for 24 h at 37 • C. The sample concentrations that resulted in a 99.99% decrease in CFU/mL relative to the control are MBC and MFC.The MFC/MIC or MBC/MIC ratio may be used to calculate the antimicrobial efficacy of each extract.Consequently, the EO exhibits bactericidal/fungicidal activity if the ratio is less than 4 and bacteriostatic/fungistatic impact if the ratio is more than 4 [32].

Determination of the Product Combination Behavior: Checkerboard Assays and Fractional Inhibitory Concentration Index
Using the checkerboard test, the bactericidal activities of EOs and antibiotic combinations were investigated.Using this procedure, all potential combinations between the concentration ranges of 1.2, 0.6, 0.3, 0.15, and 0.075 mg/mL were studied.The microdilution method on a microplate is another way to find the MIC of a combination (EOs/ATBs).It is associated with the lowest mixture concentrations that prevent microbial development.The FIC was computed to ascertain the impact of a combination.It serves as a gauge for a mixture's (EOs/ATBs) effectiveness against various microbial strains [33].For the combination of EOs (A) and ATBs (B), the calculation was carried out according to Equation (1): where The principal EO constituents (borneol, carvacrol, (E)-caryophyllene, γ-terpinene, linalool, p-cymene, and thymol) derived from the examined oreganos were chosen for ADMET (absorption, distribution, metabolism, excretion, and toxicity) and PASS prediction tests.To choose these chemicals' SMILES format, ChemBioDraw (PerkinElmer Informatics, Waltham, MA, USA, v13.0) [35] was utilized.Then, simulations were run utilizing the PASS-Way2Drug web prediction tool [36], in addition to the SwissADME and pkCSM web tools for ADMET prediction [37].The term "drug-like" substances' potential activity (Pa) and probable inactivity (Pi) are referred to as PASS [38].
A useful tool created especially for this purpose, ProTox II was utilized to examine toxicity levels and gather pertinent data on several toxicological parameters including LD50 and toxicity class [39].With the ADMET program (SwissADME, pkCSM, and ProTox II), which was used to assess the chosen substances (ligands), the physical-chemical properties, lipophilicity, water solubility, pharmacokinetics, drug similarity, medicinal chemistry, and toxicological properties of the selected chemicals (ligands) were predicted.Credible findings on possible therapeutic uses and possible adverse effects connected to the primary chemical components found in the essential oils of the examined oreganos were achieved by employing these approaches and analytical instruments.

Molecular Docking
The protein targets mentioned in Table 5 have their three-dimensional structures sourced from the RCSB protein database (accessible at https://www.rcsb.org/,accessed on 13 March 2024).UCSF Chimera was utilized for the viewing of protein structure.Using Chimera software (Chimera-1.17)and Autodock Tools (version 1.5.6,The Scripps Research Institute, La Jolla, CA, USA), the protein structures were ready to be used as suitable docking targets.Protein structures were preprocessed, meaning that water molecules, heteroatoms (hetatoms), undesired protein chains, and co-crystallized ligands were removed before analysis.After adding polar hydrogen atoms and Gesteiger charges, the structures were transformed into pdbqt format so that more analyses could be performed.
After obtaining their structures from PubChem, the seven compounds' three-dimensional structures were represented, and structural energy reduction was used.The ligands were then processed using the OpenBabel program to convert their SDF files to pdbqt formats, using the Python Prescription Virtual Screening program (AutoDock Vina).
The AutoDock Vina scoring function was used during the compound docking procedure.A grid box was placed over the protein structure once the ligand and protein molecules were chosen for docking in the Vina control.It is possible to modify the grid size under the selected active site residues before initiating the AutoDock Vina software 1.2.0 for the docking procedure.The grid box, whose dimensions and location were established using coordinates to exactly coincide with the active binding site, surrounded the search region (Table 5).PyMOL was used to analyze the properties of ligand-protein binding after the docked molecules' resultant data, represented as free binding energy values, were collected.Removing the crystallized ligand from the protein and performing a fresh docking study in the same area where the ligand was previously present is a dependable way to confirm molecular docking.With software like PyMOL version 2.5.5, we may use this method to determine if the docked ligand overlaps with the crystalline ligand by looking at the RMSD parameter value.2.9.Molecular Dynamics Simulation GROMACS 2019.3 software was utilized to conduct molecular dynamics simulations to assess the stability of the protein-ligand combination [40].The protein was subjected to the all-atom CHARMM36 force field, and ligand topologies were produced via the CGenFF service.The systems were neutralized by adding ions to balance the net charges after all complexes were submerged in a rectangular box filled with TIP3P water molecules.A force threshold (Fmax) of 1000 kJ/mol/nm was selected to obtain both minimal energy and maximum force using the steepest descent approach.Ensembles were kept at constant NVT and NPT (number of atoms, volume, pressure, and temperature) for molecular dynamics simulation studies.Each molecule was then subjected to 100 ns molecular dynamics simulations.In-depth knowledge of protein behavior was obtained by analyzing the files that were produced after output trajectories were formed

Quality Control of the Investigated Oreganos' EO
The dried plant material taken from the flowering tops of the species under study was used to compute the average essential oil output.In comparison to O. elongatum, O. compactum yielded the best EOs (Table 6).A fragrant scent and a yellowish tint define this oil.

GC-MS Analysis of the EOs from the Studied Oregano Species
Chromatograms related to the analysis of the essential oils from the flowering tops of Origanum compactum and Origanum elongatum collected from Bouyablane and El Hamam municipalities are presented in Figure 2.These analyses allowed the deduction that both essential oils are composed of several chemical compounds in different proportions.

Quality Control of the Investigated Oreganos' EO
The dried plant material taken from the flowering tops of the species under study was used to compute the average essential oil output.In comparison to O. elongatum, O. compactum yielded the best EOs (Table 6).A fragrant scent and a yellowish tint define this oil.

GC-MS Analysis of the EOs from the Studied Oregano Species
Chromatograms related to the analysis of the essential oils from the flowering tops of Origanum compactum and Origanum elongatum collected from Bouyablane and El Hamam municipalities are presented in Figure 2.These analyses allowed the deduction that both essential oils are composed of several chemical compounds in different proportions.

O. compactum
Metabolites 2024, 14, 210 10 of 35 The chemical compositions of the EOs is shown in Table 7, from which it was determined that O. compactum and O. elongatum contain, respectively, 28 and 32 compounds, which account for an astounding 99.82% and 99.57% of their total content.The majority of the composition of the EOs in these two Origanum species is made up of oxygenated monoterpenes (46.74% and 43.71%) and monoterpene hydrocarbons (47.05% and 51.02%), respectively.
Furthermore, according to the results presented in Figure 3, we observe that the EOs of O. compactum and O. elongatum are rich in hydrocarbons (51.84% and 53.45%), phenols (41.10% and 28.71%), and non-aromatic alcohols (4.68% and 13.95%), and we also note the presence, in a low proportion, of epoxides (1.60% and 3.05%) and others (0.15% and 0.10%), respectively.In addition, low quantities of esters (0.14%) are recorded for O. elongatum only.

Study of the Sensitivity of Microbial Strains to Antibiotics and Antifungals
The antibiogram and antifungigram aim to identify and predict the sensitivity of micro-organism to one or more antibiotics or antifungals.This sensitivity is determine with BD Phoenix and VITX 2 (Systems 9.02) for bacteria, and on the microplate for fun The MIC (µg/mL) measurements are presented in Tables 8 and 9.The antibiogram and antifungigram aim to identify and predict the sensitivity of a micro-organism to one or more antibiotics or antifungals.This sensitivity is determined with BD Phoenix and VITX 2 (Systems 9.02) for bacteria, and on the microplate for fungi.The MIC (µg/mL) measurements are presented in Tables 8 and 9.   10 and 11.  10 summarizes the values of the minimum inhibitory and bactericidal concentrations of EOs from both oregano species determined against the tested bacterial strains.
Overall, both oregano EOs have shown their effectiveness against all bacteria, except for the multidrug-resistant strain of S. aureus STAIML/MRS/mecA/HLMUP/BLACT (S. aureus 2220), which showed some resistance to the evaluated EO concentrations.Grampositive cocci showed some resistance to the evaluated EOs, except for Streptococcus acidominimus and Streptococcus group D, which are very sensitive.
Oregano essential oils exposed Gram-negative bacteria to sensitivity.All bacteria except Enterococcus faecium require a concentration of EOs up to 5000 µg/mL to achieve 100% inhibition, with the majority of strains being inhibited by concentrations of less than 2500 µg/mL.Additionally, it looks like the Origanum compactum EO is more effective than the Origanum elongatum EO in inhibiting the growth of Gram-positive cocci and Gram-negative bacilli.
The comparison of the MBC/MIC ratio showed that EOs exert a bactericidal action against all bacterial strains.

Antifungal Activity of EOs from Oregano Alone
Table 11 summarizes the values of the minimum inhibitory and fungicidal concentration of EOs from the two oregano species determined against the evaluated fungal strains.
Based on the results obtained from the antifungal activity of EOs from the two oregano species, we observe that the Origanum compactum EO collected from Bouyablane is more potent compared to the Origanum elongatum EO collected from the commune of Al Hamam (Khenifra region).Aspergillus niger is the most sensitive strain to oregano EOs, followed by Candida dubliniensis and other candidoses.However, Saccharomyces cerevisiae, Candida kyfer, and Candida krusei showed some resistance to the studied EO concentrations.Despite this resistance, oregano EOs can be classified as having bactericidal and fungicidal effects.

Antimicrobial Activity (FICI) of Oregano EOs in Combination with Antibiotics
Table 12 presents the FICI of combinations of EOs from O. compactum and O. elongatum with antibiotics (ATBs) from the checkerboard test.Among the 108 combinations of EOs studied with ATBs, 37 showed synergy (FICI < 0.5), 25 showed additivity (0.5 ≤ FICI < 1), and 44 showed no interaction (1 ≤ FICI < 2).Two combinations showed antagonistic effects (FICI > 4).The strain that stands out the most is Staphylococcus aureus (S. aureus 2220), for which we found 14 additive and synergistic combinations.The combination of O. elongatum with amoxicillin (FICI = 0.375) and erythromycin (FICI = 0.281) demonstrated a synergistic impact against A. baumannii, which is interesting to note.A single synergistic combination of O. compactum and ceftriaxone (FICI = 0.375) against E. coli ESBL was another interesting finding.Five synergistic combinations were also observed to be effective against E. cloacae.Of these, O. compactum and tetracycline (FICI = 0.258) and O. elongatum and gentamicin (FICI = 0.375) were found to be particularly effective.Other combinations included imipenem (FICI = 0.281), ampicillin (FICI = 0.5), and ciprofloxacin (FICI = 0.375).These results show that these combinations may be useful in the fight against antibiotics.
Moreover, among the most significant synergistic combinations between the essential oils of the studied oregano species with antibiotics against the K. pneumoniae strain, four showed synergies.The findings demonstrated a strong synergy (FICI of 0.281) between O. compactum and amoxicillin.Further research revealed three further synergies between O. elongatum and the antibiotics: ciprofloxacin (FICI = 0.129), erythromycin (FICI = 0.156), and gentamicin (FICI = 0.156).The results of this study demonstrate the possibility that O. elongatum may increase the antibacterial activity of these drugs.

PASS, ADME-Tox, and the Estimation of the Effectiveness of Possibly Active Components Extracted from the Examined Oregano EO
The websites pkCSM and SwissADME aid in understanding the pharmacokinetic properties and drug-likeness of certain compounds.All of the chosen compounds' lipophilicity ratings indicated that they were quite soluble in water (Table 13).The chosen substances have good skin permeability values (log Kp) and strong Caco-2 permeability values.Hence, the majority of drugs have excellent intestine absorption (HIA > 30%).P-glycoprotein, or P-gp, is necessary for the distribution and absorption of drugs.None of the investigated chemicals operate as P-gp I and P-gp II inhibitors, nor are the main constituents of the two essential oils under investigation P-gp substrates.All of the isolated compounds from the essential oils under study had a CNS score > −3.0 and a log BB > 0.3, suggesting their ease of passage over the blood-brain barrier (BBB) and weak penetration of the central nervous system (CNS).Within tissues, their distribution volumes (logVDss) vary from 0.152 L/kg to 0.697 L/kg.The primary constituents of the examined essential oils are unlikely to have negative effects when taken orally due to drug interactions; nonetheless, cytochrome P450 (CYP) enzymes and molecular interactions are crucial for medication clearance.Retinal organic cation transporters 2 (OCT2) and hepatic and renal substrates' total clearance (CLTOT) were represented as log mL/min/kg anticipated to forecast the excretion pathway.All of the phytochemical elements under study had a good overall clearance value and were excretable, according to the data.
To evaluate the possible toxicity of O. compactum and O. elongatum essential oils in terms of AMES, hepatotoxicity, hERG potassium channel inhibition, skin sensitization, and cytotoxicity, the phytochemical components of these oils were investigated (Table 14).With the possible exception of carvacrol and thymol compounds, which may have very little hepatic effects, the data demonstrate the lack of cytotoxicity and liver toxicity effects.

Molecular Docking
For molecular docking investigations, chemicals discovered via CG/MS were used to identify the essential oils of O. compactum and O. elongatum, which have demonstrated antibacterial activity and strong synergy with antibiotics in vitro.Using molecular docking studies, the antibacterial properties of the compounds were inferred together with their probable mechanism of action on the target proteins 1JZQ, 2VEG, 2ZDQ, 3RAE, 3SRW, 3UDI, 1KZN, 5J8G, 5OE3, and 4URN.To assess the impacts of different compounds isolated from O. compactum and O. elongatum on their particular activities, molecular docking simulations were run on a variety of systems.The docking results for the top-ranked compounds were prioritized based on their binding score, revealing remarkable interaction patterns with the selected protein targets (Table 15).Certain chemicals including carvacrol, thymol, p-cymene, γ-terpinene, and (E)-caryophyllene form substantial interactions with different protein targets, according to the examination of the docking data.This study specifically emphasized the exceptional affinity of p-cymene and carvacrol with important proteins like D-alanine ligase.The compounds exhibited the capacity to establish stable and resilient complexes with D-alanine ligase, indicating favorable possibilities for their possible engagement in modifying these particular systems.These substances may be good candidates for the creation of antibacterial medications that target D-alanine ligase, as this enzyme is essential to the growth and maintenance of bacterial cell walls.Moreover, it is significant that D-alanine ligase has a strong binding affinity for all investigated antibiotics, particularly ampicillin and amoxicillin (Figure 4).
Thymol also showed a strong affinity for the N-terminal domain structures of PqsA in complexes with anthraniloyl-AMP and 6-fluoroanthraniloyl-AMP and the Penicillinbinding protein 1a (PBP1a) protein.These findings emphasize thymol's capacity to form significant interactions with these molecular targets and show its potential for inhibition within the framework of these particular systems.These findings raise additional questions about thymol's possible application in the development of tailored treatments by indicating that it may have a significant role as an inhibitory agent in the context of processes connected to these particular molecular targets.The protein targets isoleucyl-tRNA synthetase, dihydropteroate synthase, D-alanine ligase, dihydrofolate reductase, E. coli crystalline structure, Staph crystalline structure, E. cloacae nitroreductase structure, and Penicillin-binding protein 1a (PBP1a) protein have all been found to have a strong binding affinity with (E)-caryophyllene.These findings demonstrate caryophyllene's capacity to assemble strong, stable complexes with a variety of proteins, highlighting its potential for modifying these particular systems.Furthermore, the residue interactions between the target proteins and the ligands (antibiotics and chemicals from essential oils) under study are shown in Figure 5 below.Thymol also showed a strong affinity for the N-terminal domain structures of PqsA in complexes with anthraniloyl-AMP and 6-fluoroanthraniloyl-AMP and the Penicillinbinding protein 1a (PBP1a) protein.These findings emphasize thymol's capacity to form significant interactions with these molecular targets and show its potential for inhibition within the framework of these particular systems.These findings raise additional questions about thymol's possible application in the development of tailored treatments by indicating that it may have a significant role as an inhibitory agent in the context of processes connected to these particular molecular targets.The protein targets isoleucyl-tRNA synthetase, dihydropteroate synthase, D-alanine ligase, dihydrofolate reductase, E. coli crystalline structure, Staph crystalline structure, E. cloacae nitroreductase structure, and Penicillin-binding protein 1a (PBP1a) protein have all been found to have a strong binding affinity with (E)-caryophyllene.These findings demonstrate caryophyllene's capacity to assemble strong, stable complexes with a variety of proteins, highlighting its potential for modifying these particular systems.Furthermore, the residue interactions between the target proteins and the ligands (antibiotics and chemicals from essential oils) under study are shown in Figure 5 above.Thymol also showed a strong affinity for the N-terminal domain structures of PqsA in complexes with anthraniloyl-AMP and 6-fluoroanthraniloyl-AMP and the Penicillinbinding protein 1a (PBP1a) protein.These findings emphasize thymol's capacity to form significant interactions with these molecular targets and show its potential for inhibition within the framework of these particular systems.These findings raise additional questions about thymol's possible application in the development of tailored treatments by indicating that it may have a significant role as an inhibitory agent in the context of processes connected to these particular molecular targets.The protein targets isoleucyl-tRNA synthetase, dihydropteroate synthase, D-alanine ligase, dihydrofolate reductase, E. coli crystalline structure, Staph crystalline structure, E. cloacae nitroreductase structure, and Penicillin-binding protein 1a (PBP1a) protein have all been found to have a strong binding affinity with (E)-caryophyllene.These findings demonstrate caryophyllene's capacity to assemble strong, stable complexes with a variety of proteins, highlighting its potential for modifying these particular systems.Furthermore, the residue interactions between the target proteins and the ligands (antibiotics and chemicals from essential oils) under study are shown in Figure 5 above.The main substances analyzed form connections that support the stabilization of the ligand-protein complex, improving binding affinity.Stronger and more specific binding between the ligand and the protein is promoted by specific interactions like well-positioned hydrogen bonds, suitable electrostatic interactions, and compatible hydrophobic regions.This could have a positive effect on the compound's efficacy in modulating the biological systems under study.

Molecular Dynamics
For MD simulation, the optimal protein-ligand complexes were chosen based on the outcomes of molecular docking.The molecular docking study and the PASS biological activity prediction accord well.We checked the simulation trajectory data for hydrogen bonding, radius of gyration (Rg), root mean square fluctuation (RMSF), and root mean square deviation (RMSD).In the examined essential oils, thymol, and (E)-caryophyllene were shown to have the best binding energies with the target proteins.Thus, during 100 ns MD simulations, the complexes 5J8G + caryophyllene, 3UDI + thymol, 1JZQ + caryophyllene, 1KZN + caryophyllene, and 1KZN + thymol were chosen.The main substances analyzed form connections that support the stabilization of the ligand-protein complex, improving binding affinity.Stronger and more specific binding between the ligand and the protein is promoted by specific interactions like well-positioned hydrogen bonds, suitable electrostatic interactions, and compatible hydrophobic regions.This could have a positive effect on the compound's efficacy in modulating the biological systems under study.

Molecular Dynamics
For MD simulation, the optimal protein-ligand complexes were chosen based on the outcomes of molecular docking.The molecular docking study and the PASS biological activity prediction accord well.We checked the simulation trajectory data for hydrogen bonding, radius of gyration (Rg), root mean square fluctuation (RMSF), and root mean square deviation (RMSD).In the examined essential oils, thymol, and (E)-caryophyllene were shown to have the best binding energies with the target proteins.Thus, during 100 ns MD simulations, the complexes 5J8G + caryophyllene, 3UDI + thymol, 1JZQ + caryophyllene, 1KZN + caryophyllene, and 1KZN + thymol were chosen.

Structural Dynamics of 1KZN
The complexes of the DNA gyrase protein (1KZN) with thymol and caryophyllene compounds were investigated using molecular dynamics simulations (Figure 6).We computed the root mean square deviation (RMSD) to evaluate these complexes' stability.The RMSD of the protein fluctuated within an average range of 0.17 nm.More stable protein structures are correlated with lower RMSD values.We looked at RMSF diagrams to find ligand-bound states.The average RMSF of protein 1KZN was 0.08 nm.Nonetheless, thymol's and caryophyllene's average RMSD values decreased, suggesting that the complexes remained stable throughout the experiment.An indication of the protein's structural compactness can be found in the radius of gyration (Rg).In the case of the complexes, the average Rg values remained practically unchanged or slightly decreased, indicating that the ligands did not significantly alter the structural compactness of protein 1KZN (Figure 6).
puted the root mean square deviation (RMSD) to evaluate these complexes' stability.The RMSD of the protein fluctuated within an average range of 0.17 nm.More stable protein structures are correlated with lower RMSD values.We looked at RMSF diagrams to find ligand-bound states.The average RMSF of protein 1KZN was 0.08 nm.Nonetheless, thymol's and caryophyllene's average RMSD values decreased, suggesting that the complexes remained stable throughout the experiment.An indication of the protein's structural compactness can be found in the radius of gyration (Rg).In the case of the complexes, the average Rg values remained practically unchanged or slightly decreased, indicating that the ligands did not significantly alter the structural compactness of protein 1KZN (Figure 6).

Structural Dynamics of 5J8G
We studied protein 5J8G in detail, which is a representation of the structure of E. cloacae nitroreductase attached to para-nitrobenzoic acid.A balanced simulation was demonstrated by the dynamic variations in the structure of the 5J8G + caryophyllene complex that were observed (Figure 7).Throughout the 100 ns simulation, protein 5J8G's RMSD fluctuations stayed quite near to the thermal average.Furthermore, caryophyllene's heavy atoms' RMSD values showed how stable it was in comparison to the protein; they were significantly lower than the protein's, indicating that caryophyllene stayed close to its original binding location.Less than 3 Å was found to be the average RMSF fluctuation during a 100 ns period, suggesting a robust interaction between caryophyllene and protein 5J8G without a discernible change in the protein structure caused by caryophyllene.Moreover, caryophyllene attached to the 5J8G active site with an average of 0.14 hydrogen bonds and 0.13 pairs across a 0.35 nm radius, according to hydrogen bond analysis.

Structural Dynamics of 5J8G
We studied protein 5J8G in detail, which is a representation of the structure of E. cloacae nitroreductase attached to para-nitrobenzoic acid.A balanced simulation was demonstrated by the dynamic variations in the structure of the 5J8G + caryophyllene complex that were observed (Figure 7).Throughout the 100 ns simulation, protein 5J8G's RMSD fluctuations stayed quite near to the thermal average.Furthermore, caryophyllene's heavy atoms' RMSD values showed how stable it was in comparison to the protein; they were significantly lower than the protein's, indicating that caryophyllene stayed close to its original binding location.Less than 3 Å was found to be the average RMSF fluctuation during a 100 ns period, suggesting a robust interaction between caryophyllene and protein 5J8G without a discernible change in the protein structure caused by caryophyllene.Moreover, caryophyllene attached to the 5J8G active site with an average of 0.14 hydrogen bonds and 0.13 pairs across a 0.35 nm radius, according to hydrogen bond analysis.

Structural Dynamics of 3UDI
Our study's findings demonstrate a balanced simulation of the 3UDI + thymol complex, with variations in the 3UDI protein's root mean square deviation staying quite near to the thermal average throughout the simulation's 100 ns (Figure 8).Additionally, the RMSD values of thymol's heavy atoms show how stable it is in comparison to the protein; they are noticeably lower than the protein's, suggesting that thymol stayed securely fixed to its original binding site.Without appreciably changing the structure of the protein, the root mean square fluctuations throughout a 100 ns time stay below 3 Å, demonstrating a tight connection between thymol and the 3UDI protein.Thymol binds to the active pocket of 3UDI firmly, creating an average of 0.11 hydrogen bonds and 0.10 pairs within a radius of 0.35 nm, according to hydrogen bond studies.Following the interaction of the 3UDI protein with thymol, the radius of gyration values varied within a tight range of 2.75 to 3.2 Å until the simulation's conclusion, indicating the protein's high flexibility.These results demonstrate a persistent contact that thymol maintains with the binding pockets of 3UDI, while also highlighting a strong relationship between thymol and the crystalline structure of Acinetobacter baumannii PBP1a in association with penicillin G, without appreciably altering the protein's shape.

Structural Dynamics of 3UDI
Our study's findings demonstrate a balanced simulation of the 3UDI + thymol complex, with variations in the 3UDI protein's root mean square deviation staying quite near to the thermal average throughout the simulation's 100 ns (Figure 8).Additionally, the RMSD values of thymol's heavy atoms show how stable it is in comparison to the protein; they are noticeably lower than the protein's, suggesting that thymol stayed securely fixed to its original binding site.Without appreciably changing the structure of the protein, the root mean square fluctuations throughout a 100 ns time stay below 3 Å, demonstrating a tight connection between thymol and the 3UDI protein.Thymol binds to the active pocket of 3UDI firmly, creating an average of 0.11 hydrogen bonds and 0.10 pairs within a radius of 0.35 nm, according to hydrogen bond studies.Following the interaction of the 3UDI protein with thymol, the radius of gyration values varied within a tight range of 2.75 to 3.2 Å until the simulation's conclusion, indicating the protein's high flexibility.These results demonstrate a persistent contact that thymol maintains with the binding pockets of 3UDI, while also highlighting a strong relationship between thymol and the crystalline structure of Acinetobacter baumannii PBP1a in association with penicillin G, without appreciably altering the protein's shape.

Structural Dynamics of 1JZQ
The study we conducted on the protein 1JZQ complexed with caryophyllene demonstrates that the simulation was balanced and that, across the 100 ns of simulation, fluctuations in the root mean square deviation were pretty near to the thermal average (Figure 9).This is acceptable for small globular proteins.Caryophyllene's stability in the protein is further demonstrated by the RMSD values of its heavy atoms, which suggest that the compound stayed near its original binding site.Protein 1JZQ and caryophyllene exhibit a strong binding without creating major changes in the protein's structure, as indicated by the RMSF values, which show that all fluctuations were below 3 Å.Caryophyllene attaches to the active site of 1JZQ with an average of 1.98 hydrogen bonds and 0.14 pairs across a radius of 0.35 nm, according to the hydrogen bonding study as well.After interacting with caryophyllene, protein 1JZQ exhibited good flexibility, as seen by the radius of gyration values fluctuating within a narrow range of 1.85 to 2.2 Å until the simulation's conclusion.These findings imply that protein 1JZQ and caryophyllene have a strong interaction that does not significantly alter the protein's structure.

Structural Dynamics of 1JZQ
The study we conducted on the protein 1JZQ complexed with caryophyllene demonstrates that the simulation was balanced and that, across the 100 ns of simulation, fluctuations in the root mean square deviation were pretty near to the thermal average (Figure 9).This is acceptable for small globular proteins.Caryophyllene's stability in the protein is further demonstrated by the RMSD values of its heavy atoms, which suggest that the compound stayed near its original binding site.Protein 1JZQ and caryophyllene exhibit a strong binding without creating major changes in the protein's structure, as indicated by the RMSF values, which show that all fluctuations were below 3 Å.Caryophyllene attaches to the active site of 1JZQ with an average of 1.98 hydrogen bonds and 0.14 pairs across a radius of 0.35 nm, according to the hydrogen bonding study as well.After interacting with caryophyllene, protein 1JZQ exhibited good flexibility, as seen by the radius of gyration values fluctuating within a narrow range of 1.85 to 2.2 Å until the simulation's conclusion.These findings imply that protein 1JZQ and caryophyllene have a strong interaction that does not significantly alter the protein's structure.The molecular dynamics simulation results corroborate findings from molecular docking and earlier in vitro studies.The ligands under examination exhibit the highest level of inhibitory activity and form stable connections with certain amino acids present in the target proteins.Remarkably, these interactions exhibit little variations in their parameters throughout the 100 ns simulation time.

Discussion
As one of the top three health issues identified by the World Health Organization, antibiotic resistance has become a major factor in worldwide mortality in the twenty-first century [41].It happens when bacteria find methods to resist the effects of antibiotics; this is a problem that is made worse by the overuse and poor application of antibiotics in agricultural and medical settings [42,43].Since the 1940s, when modern antibiotics became widely available, the problem has become worse, and almost all bacterial types now exhibit some degree of resistance.Therefore, it is imperative to quickly ascertain alternative materials capable of efficiently suppressing bacteria that have developed resistance to carbapenems.These include methicillin-resistant Staphylococcus aureus (MRSA), Acinetobacter baumannii, Enterobacteriaceae (especially Klebsiella pneumoniae, Escherichia coli, and Enterobacter spp.), and Pseudomonas aeruginosa.
The quest for efficacious antimicrobial therapies has conventionally centered on using the therapeutic characteristics of naturally occurring compounds derived from a wide The molecular dynamics simulation results corroborate findings from molecular docking and earlier in vitro studies.The ligands under examination exhibit the highest level of inhibitory activity and form stable connections with certain amino acids present in the target proteins.Remarkably, these interactions exhibit little variations in their parameters throughout the 100 ns simulation time.

Discussion
As one of the top three health issues identified by the World Health Organization, antibiotic resistance has become a major factor in worldwide mortality in the twenty-first century [41].It happens when bacteria find methods to resist the effects of antibiotics; this is a problem that is made worse by the overuse and poor application of antibiotics in agricultural and medical settings [42,43].Since the 1940s, when modern antibiotics became widely available, the problem has become worse, and almost all bacterial types now exhibit some degree of resistance.Therefore, it is imperative to quickly ascertain alternative materials capable of efficiently suppressing bacteria that have developed resistance to carbapenems.These include methicillin-resistant Staphylococcus aureus (MRSA), Acinetobacter baumannii, Enterobacteriaceae (especially Klebsiella pneumoniae, Escherichia coli, and Enterobacter spp.), and Pseudomonas aeruginosa.
The quest for efficacious antimicrobial therapies has conventionally centered on using the therapeutic characteristics of naturally occurring compounds derived from a wide array of organisms, including bacteria, fungi, plants, algae, and mammals.Exploring plant-based bioactive molecules as possible alternatives to conventional antibiotics has gained traction in recent times.The antibacterial qualities of essential oils, for example, have been the focus of much research due to their ability to combat common human diseases [44,45].Their antibacterial capabilities can be strengthened by mixing them with other substances, such as essential oils and medicines [46].To determine the effectiveness of O. compactum and O. elongatum essential oils in preventing infection, we developed cultures of bacteria that were resistant to several drugs.We then exposed these strains to the essential oils alone and in combination with antibiotics.
The essential oils under examination underwent a thorough investigation using GC-MS, which revealed their unique chemical signatures.Notably, O. compactum essential oil showed trace components, including €-caryophyllene and linalool together, with a substantial presence of beneficial compounds like carvacrol, γ-terpinene, p-cymene, and thymol.On the other hand, the main constituents of O. elongatum essential oil were p-cymene, thymol, and borneol.These results are consistent with earlier studies on the topic.
Numerous research studies examining the characteristics of O. compactum's flowering tops have focused on Morocco.Two studies, in particular, have drawn notice due to their findings.In 2018, Laghmouchi and colleagues conducted an analysis of fourteen essential oils collected from six distinct zones in northern Morocco, spanning diverse geographic areas [26].The findings showed considerable quantities of p-cymene (6.69 to 42.64%), thymol (0.16 to 34.29%), and γ-terpinene (2.95 to 22.97%), followed by a high concentration of carvacrol (range from 2.18 to 63.65%).Building on this understanding, a follow-up research study was carried out in 2021 by Ez-zriouli and associates [47].Their findings demonstrated the prevalence of carvacrol (72.97%) in O. compactum essential oil together with significant concentrations of p-cymene (14.5%) and γ-terpinene (6.01%).However, in other countries, several studies have shown that O. compactum essential oil is characterized by a predominance of carvacrol [48][49][50][51][52].
A recent Moroccan research study discovered that O. elongatum essential oil exists in four chemical forms: carvacrol, carvacrol/thymol, carvacrol/p-cymene, and thymol [49,53,54].We detected the p-cymene/thymol/borneol form in our investigation.Given that p-cymene is a precursor in the biosynthesis pathways of both carvacrol and thymol, it is plausible that this variance results from harvesting at a different time.
The emergence of antibiotic-resistant bacterial pathogens has rendered most available antibiotics ineffective [55].Alternative strategies are therefore necessary to combat drugresistant bacterial infections.Combined therapies between EOs and conventional antibiotics to enhance their effectiveness appear to be the most effective solution.Indeed, oregano species' essential oils have shown significant antibacterial activity against all tested bacteria.It is noteworthy that O. compactum essential oil exhibited the highest antibacterial efficacy, primarily attributable to the high levels of its bioactive constituents, including Carvacrol, γ-Terpinene, p-Cymene, Thymol, and (E)-Caryophyllene.These substances have been praised for their antibacterial properties because of their distinct chemical composition and strong synergy when combined [56][57][58].
Rosato, Scandorieiro, Oumam, and colleagues concluded that oregano essential oils act against bacteria (Gram-positive and Gram-negative) and fungi (yeasts and molds), including multidrug-resistant strains [59][60][61].In our study, Gram-negative bacteria showed greater sensitivity to both evaluated oregano species than Gram-positive bacteria and fungi, corroborating the results of Lambert's and Amakran's studies [62,63].The different sensitivity profiles of the tested strains could be justified by the structure of the cell wall.
Essential oils and antibiotics work synergistically, which is one of the newest tactics to tackle bacterial resistance [10].When two substances work better together than they do apart, this is known as a synergistic effect.It also occurs when the observed inhibition of a combination is greater than the expected inhibitions of the individual compounds.The presence of multiple active antibacterial constituents and interactions between different components of both oils are responsible for this synergy phenomenon.These interactions can disrupt the internal membranes of bacteria, increasing the permeability of structures, inhibiting microbial motility, inhibiting microbial ATPase, and/or inhibiting efflux pumps.They can also increase the solubility and/or availability of one or more oil constituents or act on different targets, resulting in enhanced antibacterial effects [64].
This study has revealed synergistic interactions between oregano essential oils and selected antibiotics.The works of Wendy et al., 2012 [14,[65][66][67], on the use of Origanum essential oils in combination with antibiotics have shown significant synergistic interactions.Our results support these studies by showing that Origanum essential oils have a synergistic effect on antibiotic-resistant bacteria, such as Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Acinetobacter baumannii, which are frequently responsible for infections linked to healthcare settings.Moreover, the correlation between the composition profile and antibacterial activity can elucidate the synergistic effect.This was especially noticeable when O. compactum and O. elongatum essential oils were combined with antibiotics, suggesting that the concentration of monoterpenes in the studied essential oils may be related to their potency.
The FICI values obtained in our study on the 108 interactions examined in vitro, as well as the strong binding affinities to the protein targets studied in silico, reveal promising synergies in the use of oregano essential oils in combination with different antibiotics.When combined with ampicillin against P. aeruginosa, amoxicillin against strains of S. aureus, A. baumannii, and K. pneumoniae, and ciprofloxacin against P. aeruginosa, E. cloacae, and K. pneumoniae, it has been observed that the essential oils of O. elongatum and O. compactum are highly effective.Furthermore, when coupled with the essential oils, ceftriaxone has demonstrated encouraging synergistic benefits against P. aeruginosa and E. coli ESBL.Additionally, when combined with tetracycline, the essential oils elicit synergistic activity against strains of E. cloacae, P. aeruginosa, and S. aureus.Furthermore, the results of molecular docking demonstrate a noteworthy affinity of these optimal combinations, particularly with EOs high in (E)-caryophyllene and thymol.Our investigation demonstrated a considerable increase in antibacterial activity when ampicillin was combined with the essential oils of O. compactum and O. elongatum, with FICI values of 0.188 and 0.066, respectively.Recent research has demonstrated that a potent combination of ampicillin and oregano essential oil is very successful in treating lung infections brought on by bacteria that are resistant to drugs [68,69].The mechanism of action of this potent medication is to directly target and inhibit the transpeptidase enzyme, which is in charge of binding the essential peptidoglycan molecules that makeup bacteria's cell walls.Ampicillin interferes with this process, making the cell wall brittle and weak, which eventually leads to its rupture.Studies have shown that some essential oils work well when paired with amoxicillin, in addition to displaying encouraging fractional inhibitory concentration index values.O. compactum and O. elongatum had FICI values of 0.125 and 0.123, respectively, against S. aureus.On the other hand, O. elongatum showed a FICI of 0.375 against A. baumannii, and O. compactum demonstrated a FICI of 0.281 against K. pneumoniae.Amoxicillin works by binding to the transpeptidase enzyme, which breaks the bonds between peptidoglycan molecule-essential components of the bacterial cell wall.Its stability in the acidic environment of the stomach further increases its effectiveness when taken orally.Because of its decreased vulnerability to degradation by beta-lactamase enzymes, it is a more effective treatment for bacteria resistant to antibiotics [70].These findings demonstrate how effective essential oils may be used in tandem with medicines to treat bacterial illnesses.Regarding the FICI against P. aeruginosa (FICI = 0.250), E. cloacae (FICI = 0.375), and K. pneumoniae (FICI = 0.129), the combination of O. elongatum essential oil and ciprofloxacin has shown promising results.The drug ciprofloxacin works by blocking DNA gyrase and topoisomerase IV.It is used for some bacterial infections, including respiratory, urinary, gastrointestinal, and skin infections.Ciprofloxacin inhibits these enzymes' ability to operate through their interactions, which causes DNA supercoils to accumulate.Compounds present in O. elongatum essential oils and the antibiotic ciprofloxacin work together to impair DNA repair, transcription, and replication forks, making it impossible for the bacterium to proliferate and survive.While the specific reaction of bacterial cells to fluoroquinolone exposure is still not fully understood, a DNA-damage-induced SOS response is thought to be important [71,72].When combined with ceftriaxone, oregano EO has demonstrated encouraging FICI values against P. aeruginosa (FICI = 0.188 and 0.125 for O. elongatum and O. compactum, respectively) and E. coli ESBL (FICI = 0.375 for O. compactum).This partnership suggests that treating bacterial illnesses may be more successful.Ceftriaxone is a more potent alternative against bacterial strains resistant to antibiotics due to its higher stability when compared to firstand second-generation cephalosporins [73].It can also be used to treat infections of the central nervous system due to its capacity to cross the blood-brain barrier.Furthermore, it differs from other cephalosporins that are usually only administered intravenously in that they can be administered intramuscularly or intravenously [74].The effectiveness of the combination of oregano essential oils (EOs) with tetracycline was studied against different multidrug-resistant bacterial strains.The results obtained revealed promising values of the FICI, highlighting significant synergies between oregano EOs and tetracycline.The found FICI value for P. aeruginosa (O.elongatum) is 0.094, 0.258 for E. cloacae (O.compactum), and 0.313 and 0.490 for S. aureus (O.compactum and O. elongatum), respectively.To stop messenger RNA from being converted into essential proteins, tetracycline binds to the ribosomal RNA of the bacterium.This inhibition gives tetracycline a bacteriostatic property, preventing bacterial growth without directly killing them, due to its ability to penetrate cell membranes because of its lipophilicity.Furthermore, tetracycline demonstrates particular efficacy against bacteria-producing beta-lactamase enzymes capable of breaking down penicillin antibiotics [75].
Studies utilizing molecular docking are widely utilized to ascertain the possible interaction between a protein and a ligand.These investigations seem to be beneficial in revealing significant information on the antibacterial qualities of natural sources.They also provide important insights into the complex interactions and possible mechanisms of action at the binding sites of various bacterial proteins [76].We carried out docking analyses on the main constituents of the essential oils of the two oregano species under investigation to better comprehend these mechanisms.The receptors that we selected for our study were 1JZQ, 2VEG, 2ZDQ, 3RAE, 3SRW, 3UDI, 1KZN, 5J8G, 5OE3, and 4URN.These essential chemicals are vital in stabilizing the ligand-protein complex and eventually raising binding affinity through the establishment of connections.The compound's ability to regulate biological systems is ultimately improved by the specific arrangement of hydrogen bonds and corresponding hydrophobic areas, which promotes a deeper and more focused link between the ligand and protein.Furthermore, there may be increased potential for permeability [77], absorption, and bioavailability in substances with smaller molecular weights, less lipophilicity, and decreased hydrogen bonding capacity [78,79].
The results of molecular docking investigations and in vitro trials are further validated by the results of molecular dynamics simulations conducted on complex models, such as 5J8G + caryophyllene, 3UDI + thymol, 1JZQ + caryophyllene, 1KZN + caryophyllene, and 1KZN + thymol.This demonstrates the significance of these investigations.They validate that caryophyllene and thymol interact steadily with target proteins, bolstering the theory that these substances are the most potent inhibitors.In light of the possible creation of novel therapeutic antimicrobial medicines, our data provide credence to the theory that these chemicals form dynamically stable complexes throughout 100 ns simulations.
The main constituents of an essential oil as well as the mixture of all of its constituents determine how resistant bacteria are to antimicrobial agents.An essential oil's ability to combat germs is directly related to its chemical makeup, the main compounds' functional groups, and the way these components work together.This synergy can arise from several processes, including the inhibition of metabolic pathways or the agents involved, as well as the disruption of the bacterial cell membrane, which makes it easier for additional antibacterial agents to work against the bacterium [80,81].
Based on the findings of molecular docking studies and molecular dynamics (MD) simulations, it is possible to explain the potential mechanism of action of compounds from Origanum compactum and Origanum elongatum essential oils (EOs), such as thymol, (E)-caryophyllene, carvacrol, p-cymene, borneol, and linalool, with antibiotics against the multidrug-resistant bacterial strains (K.pneumoniae, E. coli, E. cloacae, P. aeruginosa, S. aureus, and A. baumannii) that are studied.
According to molecular docking studies, thymol, carvacrol, (E)-caryophyllene, and other EO components have substantial interaction with a variety of bacterial protein targets.For instance, D-alanine ligase, an enzyme required for the construction of bacterial cell walls, has demonstrated a significant affinity for carvacrol and p-cymene.The (E)-caryophyllene has demonstrated great affinity with many protein targets, including isoleucyl-tRNA synthetase and dihydropteroate synthase, whereas thymol has demonstrated considerable affinity with protein PBP1a.These interactions imply that these substances may block these proteins, interfering with processes that are necessary for the survival of bacteria.
Molecular dynamics simulations have confirmed the stability of the complexes formed between essential oil compounds and target proteins.For example, in the case of the 1KZN + caryophyllene complex involving the DNA gyrase protein, the root means square deviation (RMSD) fluctuations of the protein remained stable throughout the simulation, indicating a robust interaction between caryophyllene and the protein.The root means square fluctuations and radius of gyration values also remained within acceptable ranges, confirming the stability of the protein-ligand complex.This work demonstrates the important impact that essential oils have on bacterial cells because of their hydrophobic characteristics, which increase the permeability of the cells and cause cytoplasmic components to seep out.Remarkably, some other pathways have also been discovered, such as cell wall breakdown, ATP generation interference, pH level disruption, suppression of protein synthesis, and even DNA damage [82,83].These results point to a potential relationship between the antibiotics under investigation and oregano essential oils, either by focusing on a common mechanism or by making the target location easier to reach.

Conclusions
This study investigated the potential use of essential oils from the flowering tops of O. compactum and O. elongatum in treating diseases brought on by drug-resistant bacteria.There were significant variances in the wide variety of chemical structures exhibited by both essential oils.O. elongatum essential oil stood out for having a lot of p-cymene and thymol, whereas O. compactum essential oil had large quantities of carvacrol.The results demonstrated the strong antibacterial qualities of these essential oils against resistant bacteria, such as Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus.Their high content of potent substances, including carvacrol, thymol, and (E)-caryophyllene, is mostly to blame for this.These essential oils show remarkable synergistic benefits when used with conventional antibiotics, especially against Gram-negative bacteria.There was significant synergy between the antibiotics ampicillin, amoxicillin, ciprofloxacin, ceftriaxone, and tetracycline.The isolated molecules from essential oils also showed encouraging pharmacokinetic qualities, indicating that they may be used in medicine.These substances astonishingly demonstrated the capacity to cross the blood-brain barrier, in addition to their low toxicity.Intriguingly, studies conducted in silico have shown that the substances included in essential oils establish robust connections with bacterial proteins that are important for vital functions, including DNA replication, cell wall building, and antibiotic resistance.These interactions could explain the synergistic improvements in antibiotic efficacy that were seen.An approach that is very encouraging in the fight against drug-resistant bacteria is the use of essential oils of Origanum compactum and Origanum elongatum rather than conventional antibiotics.These oils' cooperative activity suggests great potential for novel therapeutic opportunities.This is explained by their distinct chemical makeup and capacity to interact with certain bacterial proteins.To confirm the safety and therapeutic efficacy of these synergistic effects, more research is required.Furthermore, maintaining essential oils' ability to effectively treat antibiotic resistance over time depends on their careful and controlled use.

Figure 2 .
Figure 2. GC/MS chromatographic profiles of the EOs of the oreganos studied.

Figure 2 .
Figure 2. GC/MS chromatographic profiles of the EOs of the oreganos studied.

Figure 5 .
Figure 5. Interaction of target proteins with ligands having the highest binding affinities.

Figure 5 .
Figure 5. Interaction of target proteins with ligands having the highest binding affinities.

Table 1 .
Distribution of individuals in the populations of the oregano species studied and their harvest sites by region.

Table 2 .
The taxonomic classification of the genus Origanum.

Table 2 .
The taxonomic classification of the genus Origanum.

Table 3 .
List of antibiotics studied.

Table 4 .
List of tested bacterial and fungal strains, together with references.

Table 5 .
Protein targets and molecular docking parameters.

Table 7 .
The chemical compositions of the EOs of the oreganos studied.

Table 8 .
MIC (µg/mL) of antibiotics evaluated by BD Phoenix and VITEX 2 for selected species.

Table 9 .
MIC (µg/mL) of Terbinafine.Study of the Sensitivity of Microbial Strains to the Studied EO The values of MIC, MBC, and MFC of the EOs extracted from the flowering tops of Origanum elongatum and Origanum compactum species are presented in Tables

Table 11 .
MIC and MFC (µg/mL) of EOs of oregano studied.

Table 12 .
FICI values of EO combinations studied with antibiotics.

Table 13 .
In silico PASS and ADME analysis of oregano EOs studied.

Table 14 .
In silico analysis of the predictive toxicity (ProTox II) of the oregano EOs studied.

Table 15 .
Details of binding affinities of antimicrobial target proteins to selected ligands.