GC/MS Profiling, Antibacterial, Anti-Quorum Sensing, and Antibiofilm Properties of Anethum graveolens L. Essential Oil: Molecular Docking Study and In-Silico ADME Profiling

Anethum graveolens L. has been known as an aromatic, medicinal, and culinary herb since ancient times. The main purpose of this study was to determine the chemical composition, antibacterial, antibiofilm, and anti-quorum sensing activities of the essential oil (EO) obtained by hydro-distillation of the aerial parts. Twelve components were identified, representing 92.55% of the analyzed essential oil. Limonene (48.05%), carvone (37.94%), cis-dihydrocarvone (3.5%), and trans-carvone (1.07%) were the main identified constituents. Results showed that the obtained EO was effective against eight bacterial strains at different degrees. Concerning the antibiofilm activity, limonene was more effective against biofilm formation than the essential oil when tested using sub-inhibitory concentrations. The results of anti-swarming activity tested against P. aeruginosa PAO1 revealed that A. graveolens induced more potent inhibitory effects in the swarming behavior of the PAO1 strain when compared to limonene, with a percentage reaching 33.33% at a concentration of 100 µg/mL. The ADME profiling of the identified phytocompounds confirms their important pharmacokinetic and drug-like properties. The in-silico study using molecular docking approaches reveals a high binding score between the identified compounds and known target enzymes involved in antibacterial and anti-quorum sensing (QS) activities. Overall, the obtained results highlight the possible use of A. graveolens EO to prevent food contamination with foodborne pathogenic bacteria.


Introduction
The use of plants in alternative medicine has increased during the last 25 years [1]. Medicinal and aromatic plants (MAPs) are a rich reservoir of bioactive molecules, able to

Chemical Composition of A. graveolens EO
The chemical composition of A. graveolens EO identified by using the GC/MS technique is listed in Table 1. Twelve components were identified, representing 92.55% of the analyzed essential oil. In fact, the results obtained highlighted that A. graveolens EO was rich in limonene (48.05%), carvone (37.94%), dihydro carvone cis (3.5%), and trans-carvone (1.07%). a Kovats retention index determined relative to the tR of a series of n-alkanes (C10-C35) on a HP-5 MS column; b Kovats retention index determined relative to the tR of a series of n-alkanes (C10-C35) on HP Innowax.
The total number of identified compounds in the EO of A. graveolens is divided into several chemical groups that are essentially cyclic monoterpenes, such as limonene, pinene, and cymene. The terpenoids group is represented by carvone. The chemical structures of all identified compounds are represented in Figure 1 below.  a Kovats retention index determined relative to the tR of a series of n-alkanes (C10-C35) on a HP-5 MS column; b Kovats retention index determined relative to the tR of a series of n-alkanes (C10-C35) on HP Innowax.
The total number of identified compounds in the EO of A. graveolens is divided into several chemical groups that are essentially cyclic monoterpenes, such as limonene, pinene, and cymene. The terpenoids group is represented by carvone. The chemical structures of all identified compounds are represented in Figure 1 below.  Table 1.

Antibacterial Potential of A. graveolens EO and Limonene
The antibacterial effect of A. graveolens EO and its main compound, limonene, was first assessed by the determination of the growth inhibition zone (GIZ) on Mueller Hinton (MH) agar plates. The obtained results are summarized in Table 2. In fact, A. graveolens EO showed a potent antibacterial effect against all tested microorganisms (except for P.  Table 1.

Antibacterial Potential of A. graveolens EO and Limonene
The antibacterial effect of A. graveolens EO and its main compound, limonene, was first assessed by the determination of the growth inhibition zone (GIZ) on Mueller Hinton (MH) agar plates. The obtained results are summarized in Table 2. In fact, A. graveolens EO showed a potent antibacterial effect against all tested microorganisms (except for P. aeruginosa PAO1) as compared to limonene, with GIZ ranging from 10 ± 0.01 to 28.5 ± 0.71 mm. The main Plants 2023, 12, 1997 4 of 21 compound was found to be active only against three pathogenic strains (L. monocytogenes, S. aureus, and S. enterica) with ZI > 10 mm. The results of minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs) showed that both tested agents exerted a bacteriostatic effect (MBC/MIC > 4) against all tested strains. Using the microdilution technique (Table 2), MIC values of A. graveolens EO ranged from 0.048 to 0.097 mg/mL for bacterial strains, and MBC values ranged from 12.5 to >50 mg/mL. For limonene, the main component identified in A. graveolens EO, all MIC values were about 0.048 mg/mL, and the MBCs were about 50 mg/mL. Interestingly, a weak concentration of A. graveolens EO and limonene (0.048 mg/mL) exhibited an inhibitory effect against Gram-positive and Gram-negative pathogenic bacteria used in this study. Moreover, the tested essential oil showed bacteriostatic activity against almost all tested foodborne pathogenic bacteria, as the calculated values of MBC/MIC ratios were higher than 4 [30,31].

Adhesive Properties of Bacterial Strains
The results of slime production on Congo red agar (CRA) plates revealed that five (62.5%) strains were able to produce exopolysaccharides, displaying black or red colonies with black center colonies ( Figure 2, Table 3).  Using the microdilution technique (Table 2), MIC values of A. graveolens EO ranged from 0.048 to 0.097 mg/mL for bacterial strains, and MBC values ranged from 12.5 to >50 mg/mL. For limonene, the main component identified in A. graveolens EO, all MIC values were about 0.048 mg/mL, and the MBCs were about 50 mg/mL. Interestingly, a weak concentration of A. graveolens EO and limonene (0.048 mg/mL) exhibited an inhibitory effect against Gram-positive and Gram-negative pathogenic bacteria used in this study. Moreover, the tested essential oil showed bacteriostatic activity against almost all tested foodborne pathogenic bacteria, as the calculated values of MBC/MIC ratios were higher than 4 [30,31].

Adhesive Properties of Bacterial Strains
The results of slime production on Congo red agar (CRA) plates revealed that five (62.5%) strains were able to produce exopolysaccharides, displaying black or red colonies with black center colonies ( Figure 2, Table 3).    Additionally, among tested strains, four out of eight bacteria were moderately adherent to glass tubes (noted 2+), and two strains were highly adherent (noted 3+; Table 3). Regarding the bacterial adhesiveness on polystyrene surfaces, evaluated with the CV staining assay, our results showed that all strains are moderate biofilm producers (0.1 ≤ OD 570 < 1), except for S. aureus ATCC 6538, which was found to be a highly biofilm producer (OD 570 ≥ 1) ( Table 3).
The biofilm formation on various abiotic surfaces revealed that most tested bacteria are able to produce biofilm structures on glass and polyvinyl chloride (PVC) materials. However, they showed low-grade biofilm formation on the stain-steel surface (OD 570 < 1). In addition, the tested Gram-positive bacteria showed more potent biofilm formation abilities as compared to the Gram-negative ones ( Figure 3). Moderate producer CRA: Congo red agar; S+: slime-positive: S-: slime-negative; OD: optical density; SD: standard deviation; Weak slime production (+), moderate slime production (++), or strong slime production (+++).
Additionally, among tested strains, four out of eight bacteria were moderately adherent to glass tubes (noted 2+), and two strains were highly adherent (noted 3+; Table 3). Regarding the bacterial adhesiveness on polystyrene surfaces, evaluated with the CV staining assay, our results showed that all strains are moderate biofilm producers (0.1 ≤ OD570 < 1), except for S. aureus ATCC 6538, which was found to be a highly biofilm producer (OD570 ≥ 1) ( Table 3).
The biofilm formation on various abiotic surfaces revealed that most tested bacteria are able to produce biofilm structures on glass and polyvinyl chloride (PVC) materials. However, they showed low-grade biofilm formation on the stain-steel surface (OD570 < 1). In addition, the tested Gram-positive bacteria showed more potent biofilm formation abilities as compared to the Gram-negative ones ( Figure 3).

Anti-Adhesion and Anti-Biofilm Formation Activities
The anti-adhesion effect of A. graveolens EO and its main compound (limonene) was also tested. After treatment with different sub-inhibitory concentrations (1/16 × MIC to 1 × MIC) bacterial adhesion was more affected by EO than limonene, since at a concentration of MIC/16 (0.003 mg/mL), A. graveolens EO exhibited an anti-attachment effect in comparison with the untreated bacteria (control). While MIC/2 of limonene has anti-adhesion activity against the tested strains ( Figure 4).

Anti-Adhesion and Anti-Biofilm Formation Activities
The anti-adhesion effect of A. graveolens EO and its main compound (limonene) was also tested. After treatment with different sub-inhibitory concentrations (1/16 × MIC to 1 × MIC) bacterial adhesion was more affected by EO than limonene, since at a concentration of MIC/16 (0.003 mg/mL), A. graveolens EO exhibited an anti-attachment effect in comparison The anti-biofilm effect of A. graveolens EO and limonene on mature biofilm (48 h) subjected to different concentrations (ranging from MIC to 4 × MIC) revealed that both tested agents showed high biofilm eradication on polystyrene and glass surfaces, with percentage reduction exceeding 50% at low concentrations (1 × MIC) ( Figure 5). Additionally, limonene was more effective against biofilm formation than the essential oil, showing the highest percentage of biofilm eradication.

Anti-Swarming Activity
The results of anti-swarming activity tested against P. aeruginosa PAO1 revealed that A. graveolens induced a more potent inhibitory effect in the swarming behavior of PAO1 strains when compared to its mean compound limonene, with a percentage reaching 33.33% at a concentration of 100 µg/mL (Table 4). The anti-biofilm effect of A. graveolens EO and limonene on mature biofilm (48 h) subjected to different concentrations (ranging from MIC to 4 × MIC) revealed that both tested agents showed high biofilm eradication on polystyrene and glass surfaces, with percentage reduction exceeding 50% at low concentrations (1 × MIC) ( Figure 5). Additionally, limonene was more effective against biofilm formation than the essential oil, showing the highest percentage of biofilm eradication. The anti-biofilm effect of A. graveolens EO and limonene on mature biofilm (48 h) subjected to different concentrations (ranging from MIC to 4 × MIC) revealed that both tested agents showed high biofilm eradication on polystyrene and glass surfaces, with percentage reduction exceeding 50% at low concentrations (1 × MIC) ( Figure 5). Additionally, limonene was more effective against biofilm formation than the essential oil, showing the highest percentage of biofilm eradication.

Anti-Swarming Activity
The results of anti-swarming activity tested against P. aeruginosa PAO1 revealed that A. graveolens induced a more potent inhibitory effect in the swarming behavior of PAO1 strains when compared to its mean compound limonene, with a percentage reaching 33.33% at a concentration of 100 µg/mL (Table 4). The results of anti-swarming activity tested against P. aeruginosa PAO1 revealed that A. graveolens induced a more potent inhibitory effect in the swarming behavior of PAO1 strains when compared to its mean compound limonene, with a percentage reaching 33.33% at a concentration of 100 µg/mL (Table 4).  Limonene was able to inhibit violacein production by 6.33% until the concentration of 0.156 mg/mL (MIC/8) (Table 5, Figure 6).   Limonene was able to inhibit violacein production by 6.33 % until the concentration of 0.156 mg/mL (MIC/8) (Table 5, Figure 6).

Draggability and Pharmacokinetic Properties of A. graveolens Main Compounds
The ADME properties of the twelve identified compounds were studied (Table 6). Interestingly, the studied compounds displayed a nil alert. Using the results from the boiled egg, β-pinene and δ-2-carene were out of the obtained model ( Figure 7). Moreover, all compounds had acceptable consensus Log Po/w ranging from 1.56 to 3.5. In addition, Lipinski's rule was confirmed, and good gastro-intestinal absorption, lipophilicity, and bioavailability scores (0.55-0.58) were reported. In addition, all selected compounds exhibited good topological polar surface area values (TPSAs) lower than 125 Å 2 , suggesting that they are expected to be orally absorbed. The drug-like behavior of all identified compounds represented by the bioavailability radar showed that they fit within the pink area of the polygon (Figure 8).  Table 1.
The drug-like behavior of all identified compounds represented by the bioavailability radar showed that they fit within the pink area of the polygon (Figure 8).   Table 1.

Molecular Docking Study
The study of molecular docking delivers an understanding of molecular binding affinities and the binding approach of the phyto-constituents within the targeted protein.
The molecular docking study was carried out against five different receptors, namely the human peroxiredoxin 5 receptor (PDB: 1HD2), TyrRS from S. aureus (PDB: 1JIJ), type IIA topoisomerase from S. aureus (PDB: 2XCT), and the LasR protein receptor of P. aeruginosa (PDB ID: 2UV0 and 3IX3), to determine the binding affinities. The outcomes of the docking studies are mentioned concisely in Table 7. As shown in Table 7, all compounds had negative binding energies (ranging from −3.52 to −7.07 kcal/mol) with the different target receptors, with dihydrocarvone cis showing the most promising docking score of all the  Table 1.

Molecular Docking Study
The study of molecular docking delivers an understanding of molecular binding affinities and the binding approach of the phyto-constituents within the targeted protein. The molecular docking study was carried out against five different receptors, namely the human peroxiredoxin 5 receptor (PDB: 1HD2), TyrRS from S. aureus (PDB: 1JIJ), type IIA topoisomerase from S. aureus (PDB: 2XCT), and the LasR protein receptor of P. aeruginosa (PDB ID: 2UV0 and 3IX3), to determine the binding affinities. The outcomes of the docking studies are mentioned concisely in Table 7. As shown in Table 7, all compounds had negative binding energies (ranging from −3.52 to −7.07 kcal/mol) with the different target receptors, with dihydrocarvone cis showing the most promising docking score of all the receptors investigated. On the other hand, caranone trans (−5.622 kcal/mol) and meta-cymene (−6.875 kcal/mol) have promising docking scores on the 1JIJ and 2UV0 receptors, respectively. The binding affinity of 1HD2 protein, which ranges from −3.03 to −5.105 kcal/mol, is less than that of the target co-crystallized ligand, benzoic acid, which has the highest binding affinity (−7.245 kcal/mol). In the 3IX3 target, α-pinene, β-pinene, δ-2-carene, ρ-cymene, meta-cymene, dill ether, trans-caranone, and dihydro carveol (neoiso) have the highest docking score, which is better than the co-crystalized inhibitor (−6.05 kcal/mol).
Dill was used in food and drugs. Several studies focused on the biological activities of compounds present in EO or/and extracts. It has been demonstrated that A. graveolens has many medicinal uses: antibacterial, antispasmodic, antitumor, digestive, carminative, and could be used as a cardioprotective agent [37]. The most important activity studied was the effect on pathogenic microorganisms due to their resistance to commercialized antibiotics [38]. Essential oils and plant extracts can be used as a solution for the treatment of many infectious diseases [39]. Dill is a rich plant in chemical constituents with several biological effects, especially against multidrug-resistant microorganisms [37]. A relationship between the chemical composition of dill EO and its antimicrobial properties has been proven, and it can be used in the pharmaceutical and food industries as a natural additive [40]. Our results showed that A. graveolens EO showed an important antibacterial effect against foodborne pathogenic bacteria compared to limonene using both solid and liquid methods. The MIC values of A. graveolens EO ranged from 0.048 to 0.097 mg/mL for bacterial strains, and the MBCs ranged from 12.5 to >50 mg/mL. The results of MICs and MBCs showed that both tested agents (EO and limonene) exerted a bacteriostatic effect (MBC/MIC > 4) against all tested strains [30,31].
In our study, commercial dill seed essential oil, rich in limonene (48.05%) and carvone (37.94%), showed antimicrobial activity against almost all tested Gram positive and Gramnegative bacteria. The essential oil of A. graveolens seeds exerted antimicrobial activity against Staphylococcus aureus, Bacillus cereus, Enterococcus faecalis, Listeria monocytogenes, Escherichia coli, Yersinia enterocolitica, Salmonella typhimurium, Shigella flexneri, Pseudomonas aeruginosa, and Mycobacterium [37,39]. It was demonstrated that D-limonene and D-carvone possess strong antifungal activity against several fungi, such as Aspergillus niger, Saccharomyces cerevisiae, and Candida albicans [41,42]. In some studies, it has been shown that S. aureus is implicated in alimentary toxic infections [43]. In addition, dill EO can be used as a natural antimicrobial agent in milk products to kill S. aureus and its enterotoxins [44].
Salmonella is Gram-negative bacteria that cause gastroenteritis. The most common sources of food infections are milk, eggs, meat, and meat products [45]. The dill seeds were studied for their antimicrobial activity against Salmonella typhimurium [46]. L. monocytogenes causes listeriosis after food consumption. Listeriosis can contribute to death in 30% of cases [45]. Many studies focused on the effect of essential oils on L. monocytogenes [46]. de Carvalho et al. [47] exhibited the antimicrobial effect of carvone, a major component of dill oil, against L. monocytogenes. The high amount of carvone, a compound known for its numerous biological properties, indicates the possible application of dill seed essential oil for medical purposes outside the food industry as a bioactive product of natural origin [48]. There is also data on limonene's antimicrobial activity [49].
Gene expression is regulated by the mechanisms of bacterial cell communication. It has been demonstrated that the signal molecules of bacteria and the suppression of QS can be affected by some plant compounds [50]. The interaction between plants and bacteria has been reported for many years [50]. During our study, we investigated in vitro the anti-QS potential of A. graveolens EO and limonene. This plant is known for its culinary uses as a spice and flavor, as well as its medicinal uses. The results showed that A. graveolens EO and limonene inhibited violacein production in C. violaceum ATCC 12472, highlighting their ability to inhibit bacterial cell-to-cell communication and consequently interfere with the QS system regulating the production of virulence traits responsible for bacterial disease [51]. Computational studies are commonly used to correlate the in vitro activities of natural compounds with key target proteins involved in human disorders. In fact, in silico docking studies can provide useful insights into the molecular basis of the biological activity of natural products and the possible mechanisms of action and binding modes of active compounds. Therefore, all compounds from the GC-MS analysis of compounds from the tested essential oil were docked with specific target proteins involved in antibacterial and antioxidant activities [52,53].
In this target, the co-crystallized ligand benzoic acid has the highest docking score (−7.245 kcal/mol), while docking values ranging from −5.105 to −4.332 kcal/mol were found to have significant binding affinities with dihydrocarvone cis, trans-caranone, and dihydrocarvone (neoiso). The human peroxiredoxin family has one cysteine residue, Cys47, that is shared by all peroxiredoxins and has been attributed to peroxide catalysis. Cys47, a conserved cysteine residue, is located at the N-terminus of the kinked helix 2. This active pocket comprises conserved amino acid residues such as Thr44, Gly46, Cys47, and Arg127 that assist in docked chemical identification through hydrogen bonding and hydrophobic interactions. Furthermore, Cys47, Thr44, Gly46, Thr147, Pro40, Pro45, Phe120, Arg127, and Leu149 are all involved in the complex benzoic acid-1 HD2 stabilization [53].
A closer look at the docking poses of the screened phyto-compounds revealed the presence of a hydrogen bond with Cys47 in dihydrocarvone cis, caranone trans, and dihydrocarveol (neoiso), as well as benzoic acid ( Figure 9A), indicating that these three compounds have antioxidant potential.
Antimicrobial medicines often impede cell wall biosynthesis, protein synthesis, nucleic acid synthesis, and anti-metabolism. Antibiotics, in general, disrupt these pathways by interfering with specific cell proteins that perform specific activities [54]. Tyrosyl-tRNA synthetase (TyrRS), a member of the aminoacyl-tRNA synthetase family, can interpret information such as concurrent tRNA molecules and amino acid structures, which are essential for translating coded information into protein structures in nucleic acids. Since this enzyme is highly conserved across prokaryotes, inhibiting it is a promising target for the development of broad-spectrum antibiotics. To better understand the binding interactions, phyto-constituents from dill essential oil compounds were docked in the active site of the crystal structure of S. aureus TyrRS (PDB ID: 1JIJ) [55], which was cocrystallized with the monocyclic SB-239629. In S. aureus tyrosyl-tRNA synthetase (TyrRS) (1JIJ), the docking scores of phyto-constituents ranged from −3.521 to −5.62 kcal/mol, while the co-crystallized ligand had −7.973 kcal/mol. Molecular docking of caranone trans, dihydro carvone cis, and dihydro carveol (neoiso), the top three compounds that have the best binding affinity, was performed to identify their binding sites on the structures of TyrRS S. aureus. In this target, caranone trans has the best docking score, which is −5.622 kcal/mol. The carbonyl group of caranone forms a single hydrogen bond with the amino acid Gly193. The compound trans-caranone interacted with Ser194, Gly192, Val191, Gln190, Asp195, Leu46, His47, and Asp80 at the binding site via van der Waals bonding ( Figure 9B).
DNA gyrase is a bacterial topoisomerase enzyme that controls the structure of DNA during transcription, replication, and recombination by generating transitory breaks in both DNA strands [53]. As a result, this enzyme is critical for bacterial survival and may primarily be used as an antibacterial targeted therapy. The highest scoring ligands in a docking study on topoisomerase II DNA gyrase (PDB ID, 2XCT) protein were ρ-cymene (−6.204 kcal/mol), meta-cymene (−6.238 kcal/mol), dill ether (−6.282 kcal/mol), and trans-caranone (−6.326 kcal/mol). The binding interaction of the most active ligand, transcaranone, shows that there is no direct hydrogen bonding between the ligand atom and protein residues. In fact, trans-caranone is situated on the two DNA helices and makes van der Waals interactions with nucleotide bases such as DT E8, DT E9, DC H14, DA H13, DC H12, and DA H11. Trans-caranone binding contact was unable to exhibit interaction with the Mn +2 ion through a salt bridge, which dramatically increased rates of enzyme-mediated DNA breaking reported in previous research ( Figure 9C).
In order to control the actions of their populations, many Gram-negative bacteria communicate through chemicals known as autoinducers. Quorum sensing is a kind of communication that may control the production of virulence factors, biofilm formation, and drug susceptibility [56,57]. The quorum sensing system in the human opportunistic bacterium P. aeruginosa is now the most extensively studied one. P. aeruginosa pathogenicity may be reduced by using quorum sensing inhibitors. For the quorum sensing inhibition activity, docking studies on the LasR protein receptor of P. aeruginosa (PDB ID: 2UV0 and 3IX3) were performed. Dill ether was chosen as the best natural ligand against 2UV0 and 3IX3 thanks to their good binding affinity within the active site domain, which is −6.976 kcal/mol and −7.077 kcal/mol, respectively, which is comparable to a co-crystallized ligand. Binding interaction shows that dill ether forms van der Waals interactions with Ser40, Gln45, Asp46, Tyr47, Glu48, Asn49, Ala50, Phe51, Ile52, Val76, Ser77, Cys79, and Thr80 in the P. aeruginosa 2UV0 protein target, which is a comparable binding site for co-crystallized ligand. Antimicrobial medicines often impede cell wall biosynthesis, protein synthesis, nucleic acid synthesis, and anti-metabolism. Antibiotics, in general, disrupt these pathways by interfering with specific cell proteins that perform specific activities [54]. Tyrosyl-tRNA synthetase (TyrRS), a member of the aminoacyl-tRNA synthetase family, can interpret information such as concurrent tRNA molecules and amino acid structures, which are essential for translating coded information into protein structures in nucleic acids. Since this enzyme is highly conserved across prokaryotes, inhibiting it is a promising target for the development of broad-spectrum antibiotics. To better understand the binding interactions, phyto-constituents from dill essential oil compounds were docked in the active site of the crystal structure of S. aureus TyrRS (PDB ID: 1JIJ) [55], which was co-crystallized with the monocyclic SB-239629. In S. aureus tyrosyl-tRNA synthetase (TyrRS) (1JIJ), the docking scores of phyto-constituents ranged from −3.521 to −5.62 kcal/mol, while the cocrystallized ligand had −7.973 kcal/mol. Molecular docking of caranone trans, dihydro carvone cis, and dihydro carveol (neoiso), the top three compounds that have the best binding affinity, was performed to identify their binding sites on the structures of TyrRS S. aureus. In this target, caranone trans has the best docking score, which is −5.622 kcal/mol. The carbonyl group of caranone forms a single hydrogen bond with the amino acid Gly193. The compound trans-caranone interacted with Ser194, Gly192, Val191, Gln190, Asp195, Leu46, His47, and Asp80 at the binding site via van der Waals bonding ( Figure 9B).
The presence of dill ether in the LasR receptor's hydrophobic pocket indicated the existence of hydrophobic interactions created by the benzofuran scaffold and hydrophobic amino acids. Thus, molecular docking studies have shown the ability of the dill ether structural structure to inhibit the quorum sensing mechanism.

Chemical Composition Analyses
Anethum graveolens essential oil and its major compound limonene were purchased from Huile & Sens (Crestet, France) and Sigma (Sigma-Aldrich S.r.l., Milan, Italy), respectively. The chemical composition of the essential oil was analyzed by gas chromatographyflame ionization detector (GC-FID) and gas chromatography-mass spectrometry (GC-MS) [58].

Disk Diffusion Test
The antagonistic effects of A. graveolens EO and limonene were evaluated against eight pathogenic bacterial strains: Listeria monocytogenes CECT 933; Vibrio vulnificus CECT 529; Salmonella enterica CECT 443; Shigella flexeneri CECT 4804; Staphylococcus aureus ATCC 6538; Bacillus subtilis CIP 5265; Escherichia coli ATCC 35218; and Pseudomonas aeruginosa PAO1. The disk diffusion method was performed according to Pérez et al. [59]. The results were determined as the diameters of inhibition zones (mm) around discs impregnated with EO. Gentamicin discs were taken as positive controls.

Microdilution Assay
The minimal inhibition concentration (MIC) and the minimal bactericidal concentration (MBC) of A. graveolens EO and its major compound were determined for all bacterial strains as previously described [60]. Serial dilution of the tested agents was performed at concentrations ranging from 50 to 0.048 mg/mL.

Adhesive Potentiality
The ability of the tested microorganisms to secrete exopolysaccharides on Congo red agar plates was determined, and morphotypes obtained were defined on the basis of their color (slime production) using the protocol described by Touati et al. [61]. Qualitative adhesion on glass tubes was carried out following the same protocol described by Davenport et al. [62]. All experiments were done in triplicate.
Quantitative biofilm formation on a polystyrene 96-well plate was determined using the crystal violet technique [63].

Biofilm Formation Capacity on Abiotic Surfaces
Polyvinyl chloride (PVC), stainsteel (SS), and glass (G) strips (1.5 cm 2 ) were disinfected before being used for the biofilm assay. A volume of 100 µL of bacterial suspensions was added to each strip placed into a 12-well tissue culture plate. After incubation (24 h at 37 • C), non-adherent cells were removed from each well by washing with PBS solution. Biofilm quantification was made with crystal violet (1%) staining and then dissolved into acetic acid (33%). The OD at 570 nm was recorded [64].

Biofilm Inhibition
The biofilm inhibition effects of A. graveolens EO and limonene were evaluated according to the protocol of Saising et al. [65]. Each bacterial strain grown in BHI (with 2% glucose) was treated with different subinhibitory concentrations (1/16 to 1 × MIC) of the tested agents. After incubation for 24 h at 37 • C, non-adherent cells were removed, and CV (1%) stained biofilm cells were determined at 570 nm.

Biofilm Eradication
The biofilm eradication properties of A. graveolens EO and limonene were tested as described previously [66]. Pre-established biofilms (48 h) were treated with various concentrations ranging from MIC to 4 × MIC of the selected agents and further incubated for 24 h. Treated biofilm biomass was stained with CV (1%) and measured by the absorbance of CV at 570 nm. The percentage of biofilm eradication was estimated using the following equation (Equation (1)):

Anti-Quorum Sensing Activity
The ability of A. graveolens EO and limonene to inhibit the production of the watersoluble pigment (violacein) in the Chromobacterium violaceum ATCC 12472 starter strain was evaluated. An overnight culture of C. violaceum (OD 600 = 0.4) was added to sterile microtiter plates containing 200 µL of LB broth and incubated at 30 • C supplemented with different concentrations (MIC/32 to MIC) of dill EO and limonene. LB broth containing C. violaceum was used as a positive control [67]. The percentage of violacein reduction was calculated by the following equation (Equation (2)): Violacein inhibition (%) = (OD 585 Control − OD 585 Sample)/OD 585 Control (2)

Anti-Swarming Activity
Anti-swarming activity of A. graveolens EO and limonene was assessed against Pseudomonas aeruginosa PAO1. An overnight culture of PAO1 (OD600 = 0.4) was inoculated on the swarming agar medium at various concentrations of test agents (50,75, and 100 µg/mL). Plates without EOs were considered controls. After incubation for 18 h at 30 • C [68].

Molecular Docking Study
In order to highlight the possibility of binding interactions between phytocompounds identified in A. graveolens essential oil and antimicrobial, antioxidant, and quorum sensing receptors, a docking approach was performed. For antimicrobial activity, S. aureus tyrosyl-tRNA synthetase (PDB ID, 1JIJ) and topoisomerase II DNA gyrase (PDB ID, 2XCT) proteins are promising drug candidates, leading to high selectivity and a broad spectrum of antibacterial agents [72,73]. The human peroxiredoxin 5 (PRDX5) receptor (PDB ID, 1HD2) is a potential target for the evaluation of the antioxidant activity of selected bioactive compounds that permit the reduction of hydrogen peroxide and alkyl peroxide with the help of thiol-containing donor molecules [1,2]. A molecular docking study was also performed against the QS signal receptors LasR (PDB ID: 2UV0) and (PDB ID: 3IX3), from P. aeruginosa as key regulators of P. aeruginosa pathogenesis.
The phyto-constituents 2D structures were obtained using PubChem chemical information resources. The LigPrep module was used to refine the structure obtained. The OPLS3e force field was applied for ligand preparation. Tautomer creation, ionization state at pH 7.0 ± 1.0 utilizing Epik, charged group neutralization, and optimization of the hydrogen bond and ligand 3D geometry are all included in the ligand preparation process [74][75][76]. Protein PDB IDs 1HD2, 1JIJ, 2XCT, 2UV0, and 3IX3 are downloaded by the protein data bank and processed for the modeling study. The protein was imported into the Schrodinger Maestro GUI and refined, optimized, and minimized after undesired water molecules and problem warnings were removed using the protein preparation wizard module [77,78]. The Prime tool was applied to complete the missing side chains and residues. The OPLS3e force field was utilized to construct low-energy state proteins with a default root-mean-square deviation (RMSD) of 0.30 Å, which were then employed for molecular docking.
The grid is constructed from minimized proteins, and the grid box was constructed based on the active site of the protein where the co-crystallized ligand is bound [79,80]. Glide was used to undertake molecular docking simulations using the standard precision (SP) approach, which also produced favorable ligand poses for further evaluating active sites for ligand binding. The docking results included the best positions as well as the dock score.

Statistical Analysis
All experiments were performed in triplicate, and average values were calculated using the SPSS 25.0 statistical package for Windows. Differences in means were calculated using Duncan's multiple range tests for means with a 95% confidence interval (p ≤ 0.05). For anticancer activities, a significance test was carried out among the treatments by a two-way ANOVA followed by a Bonferroni post hoc test at p < 0.001.

Conclusions
Anethum graveolens is a very rich plant in phytochemicals with a pharmacological interest. Docking studies on the identified phyto-compounds in bacteria were studied to reinforce the in vitro results. The results obtained confirm the alternative use of this plant to treat human diseases because of its effectiveness and safety. A. graveolens EO is a considerable natural antibacterial agent and might be used as a natural preservative in the food industry.