Chemical Composition, Antibacterial and Anti-Quorum Sensing Activities of Pimenta dioica L. Essential Oil and Its Major Compound (Eugenol) against Foodborne Pathogenic Bacteria

The Pimenta dioica essential oil and its main compound (eugenol) were tested for their antibacterial potency against eight Gram-negative and Gram-positive bacteria implicated in food intoxication. This essential oil and its main component were evaluated for their ability in inhibiting Quorum sensing (QS)-dependent mechanisms such as motility in Pseudomonas aeruginosa PAO1, production of violacein by Chromobacterium violaceum and biofilm formation on stainless steel and glass surfaces. Our results demonstrated that P. dioica essential oil and eugenol were active against all tested strains with a maximum of inhibition against Listeria monocytogenes CECT 933 (26.66 ± 0.57 mm). The minimal inhibitory concentration (MIC) value of the tested essential oil and eugenol was about 0.048 mg/mL for all strains. The obtained results demonstrated that 4CMI eugenol inhibited foodborne strains biofilm formation on the glass strips by 73.79% and by 75.90% on polystyrene. Moreover, 0.048 mg/mL (MIC) of P. dioica essential oil inhibited the violacein production by 69.30%. At 100 µg/mL, P. dioica oil and eugenol affected the motility of PAO1 by 42.00% and 29.17%, respectively. Low concentrations of P. dioica essential oil are active against the quorum sensing phenomena and biofilm potency. Thus, this essential oil could be further investigated for new molecules useful for the treatment of toxi-alimentary infections.


Introduction
Foodborne pathogenic bacteria are responsible of many human alimentary intoxications. The obvious examples of pathogenic bacteria are Salmonella enterica, Listeria monocytogenes, Vibrio vulnificus, Shigella flexneri, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus. These bacteria are recognized for their high ability to adhere to surfaces and epithelial cells. Food preparations are based on the use of species responsible for their aromatic properties and antimicrobial potency.
The diet based on the consumption of contaminated food with bacteria causes a big problem to public health. Several bacteria accounted for many cases of death [1]. The use of plants (spices) and natural products (herbs), which can be added during the food conception may reduce the risk of contamination with these pathogens by inhibiting their activities and food damage [2][3][4].

Chemical Composition of P. dioica Essential Oil
The chemical composition of P. dioica essential oil is summarized in Table 1. Thirty components with different percentage were identified using HP5 capillary column according to their elution time. P. dioica essential oil was rich in eugenol (48.67%), β-pinene (18.52%) and 2-Propenylphenol (7.61%). Other relevant components were linalool (3.68%) and limonene (3.55%). The structures of the major compounds are represented in Figure 1.

Antibacterial Activity of P. dioica Essential Oil and Eugenol
The results demonstrated that P. dioica essential oil and eugenol were active against all tested strains with a maximum inhibition against L. monocytogenes CECT 933 (21.66 mm with P. dioica and 21.33 mm for eugenol). Eugenol was more active against S. flexeneri CECT 4804. P. aeruginosa PAO1 showed a resistance to P. dioica and eugenol with inhibition zone diameters of 8.00 mm and 7.66 mm, respectively (Table 2).
Low concentrations of P. dioica essential oil and eugenol inhibited the growth of all foodborne pathogenic tested strains. In fact, the MIC value of the tested essential oil was 0.048 mg/mL for all strains. The same concentration (0.048 mg/mL) for the eugenol was able to reduce the growth of all bacterial strains. The MBC values of the tested essential oil were about 1.562 to 12.5 mg/mL and 3.125-12.5 mg/mL (eugenol) are needed to completely inhibit the growth of the Gram-positive and Gram-negative strains tested (Table 2).

Adhesive Properties and Biofilm Formation on Abiotic Materials
Among the isolated strains, five out of eight (62.50%) were slime producing characterized by the black colonies and red with black center, and the remaining strains (P. aeruginosa, B. subtilis and S. enterica) were non-slime producing characterized by red bordeaux colonies ( Figure 2, Table 3).  The study of biofilm formation on glass tubes showed that S. flexeneri and L. monocytogenes were strongly adherent (a, noted +++), and 50% of the tested strains were moderately adherent (b, noted ++) to this material ( Figure 3). The most used materials in culinary preparations (polystyrene, glass, stainless steel and polyvinylchloride) were chosen during this work. The staining assay with 1% crystal violet (CV) showed that all foodborne pathogenic strains form a biofilm (0.1 < OD < 1 or OD 570 > 1) on the selected materials with different degrees depending on the strain and the surface ( Figure 4). All Gram-positive and Gram-negative strains were high biofilm producers on glass with a maximum of adhesion for B. subtilis (OD = 2.65). S. aureus ATCC 6538 was the highest biofilm producer on polystyrene (OD > 1). S. flexneri CECT 4804 showed the lowest values of OD on the four tested materials (Table 3).

Anti-Biofilm Activity of P. dioica and Eugenol on Polystyrene and Glass Surfaces
The study of the anti-biofilm properties of P. dioica and eugenol was carried out on the S. aureus ATCC 6538 strain according to its high potency of biofilm formation.
P. dioica showed an anti-biofilm ability of the S. aureus strain on glass and polystyrene about 55% and 58%, respectively, at the lowest tested concentration (MIC = 0.048 mg/mL) ( Table 4). This effect was stronger when we tested the anti-biofilm effect of eugenol against the same strain showing an inhibition about 73.00% on polystyrene. In fact, low concentrations (0.048, 0.096 and 0.192 mg/mL) of this compound demonstrated an important reduction of the biofilm formation on both tested surfaces. The main compound, eugenol, was more active on sessile S. aureus ATCC 6538 isolate adherent into polystyrene and glass than the essential oil. The effect of P. dioica essential oil and eugenol on biofilm formed on polystyrene and glass was not variable depending on the concentration and tested material. A 4 × MIC concentration of P. dioica EO inhibited the S. aureus biofilm formed on glass and polystyrene with percentages of (64.41 ± 1.4%) and (70.25 ± 1.19%), respectively. All these results are summarized in Table 4.

Violacein Inhibition Assay in C. violaceum
In qualitative analysis, P. dioica essential oil inhibited the production of violacein in C. violaceum ATCC 12472 with a percentage of inhibition more than 50% (71.30 ± 1.5%) at MIC value even at a low concentration (MIC/4) ( Table 5). However, violacein production was inhibited only to an extent of 48.29 ± 0.9% when we tested the eugenol (Table 5, Figure 5).

Anti-Swarming Assay
During this essay, we examined the anti-QS potential of P. dioica essential oil and eugenol on swarming motility in PAO1 strain. The results indicated that P. dioica essential oil and its main compound inhibited the swarming of PAO1 to different extents and at the selected doses (50, 75 and 100 µg/mL). Moreover, 100 µg/mL were able to inhibit swarming about 42.00% and 29.00% for P. dioica oil and eugenol, respectively (Table 6).

Discussion
P. dioica has been used as an important spice for its culinary and medicinal uses [18]. This plant was used to reduce muscle pain, help digestion and stomach gases [19,20]. In Cuba, this species can be cooked or ingested to treat stomach pain and colds [21]. The leaves of Pimenta are used to reduce arthritis, fever and stress and in India [22].
Many compounds have been isolated from this plant such as tannins, glycosides, phenylpropanoids and essential oils [23].
Many constituents found in P. dioica berries and leaves such as galloylglucosides phenylpropanoids [24], tannins and flavonoids [25] showed several properties (antibacterial, analgesic hypotensive and anti-neuralgic). Considering the phytochemical composition and low cost of Pimenta berries, this spice may be used in the food preparation [26] (Table 7).
P. dioica has been described for several years for its biological uses. Our results demonstrated that its essential oil and eugenol were active against all tested foodborne pathogenic strains. The essential oil of P. dioica berries inhibited L. monocytogenes, Salmonella typhimurium, Pseudomonas putida, E. coli and S. aureus [36]. The essential oil of the same plant extracted from its leaves demonstrated a strong antibacterial activity against Pseudomonas and Staphylococcus species [13].
The antimicrobial activity of P. dioica fruits and leaves extracts and essential oil has been proved. P. dioica leaf extracts presented significant antimicrobial properties against many genera of bacteria and fungi such as Escherichia coli, Streptococcus mutans, Staphylococcus aureus, Bacillus cereus, Pseudomonas fluorescens, Salmonella typhimurium, Candida albicans, Aspergillus niger and Penicillium sp. [37][38][39].
Mérida-Reyes et al. [27] tested the antibacterial activity of the essential oil of leaves of P. dioica against B. subtilis, S. aureus, S. enterica and E. coli. Their results showed that this oil is very active against B. subtlis and E. coli was the more resistant strain. The activity of the oil against bacteria depends on the synthesis of the cell wall interfering in the formation of the peptidoglycan molecule [40]. Some authors related to this antibacterial activity to the presence of eugenol and (E)-caryophyllene since these two compounds were currently found in P. dioica oil [40,41]. In addition, it has been demonstrated that eugenol is active on the cytoplasmic membrane [30].
It has been demonstrated that eugenol can reduce the production of pyocyanin and biofilm formation in E. coli and S. aureus [42].
Using the CRA test, 62.50% were slime-producing, characterized by a black colonies and red with black centers. Pigmented colonies were considered as slime-producing strains, whereas unpigmented colonies were classified as non-slime-producing strains [43].
Many works have proved the anti-biofilm potency of monoterpenoids on Gramnegative and Gram-positive bacteria during the biofilm development [44].
Eugenol is largely used as a flavoring agent in the food industry due to its biological properties such as anti-inflammatory, anti-microbial and antioxidant. This compound is used against Gram-positive and Gram-negative bacteria. It is demonstrated that eugenol presents strong inhibition against several anaerobic bacteria (Streptococcus mutans and Prevotella intermedia), Listeria monocytogenes and Candida albicans. Several essential oils are active against bacterial biofilm development such as clove and pimento berry oil [45]. This compound inhibits QS of P. aeruginosa [46]. Some studies reported that thymol and carvacrol were responsible for the anti-QS activity [47]. Burt (2004) [48] demonstrated that essential oils containing phenolic compounds such as eugenol thymol or carvacrol have the strongest antimicrobial activity. Gramnegative bacteria are known to be more resistant to volatile oils than the Gram-positive bacteria [48]. The same scientists proved that monoterpenes (limonene and α-pinene) inhibited biofilm formation more than terpene alcohols such as linalool and terpinene-4-ol. The most foodborne pathogenic bacteria such as the Pseudomonas produce biofilms [49]. The family Lamiaceae is considered as important aromatic plants with constituents having anti-QS properties to combat different food pathogenic microorganisms [50]. Other studies demonstrated that single constituents of essential oils such as eugenol, linalool, γ-terpinene and limonene exhibited anti-QS effects [51].

Bacterial Strains
The antibacterial effect of the volatile oil of P. dioica and its main component the eugenol was tested against eight food-borne pathogenic bacteria including four Grampositive (Staphylococcus aureus ATCC 6538, Bacillus subtilis CIP 5265, Vibrio vulnificus CECT 529, Listeria monocytogenes CECT 933) and four Gram-negative bacterial strains (Pseudomonas aeruginosa PAO1, Escherichia coli ATCC 35218, Salmonella enterica CECT 443, Shigella flexeneri CECT 4804) were procured from American Type Culture Collection (ATCC) USA and Spanish Type Culture Collection (CECT).

Chemical Characterization of the Essential Oil
P. dioica essential oil was purchased from Huile & Sens (Crestet, France) on 27 November 2014 (Product number B750N06). This oil was extracted from the dried unripe fruits by hydrodistillation technique. The main compound (eugenol) was purchased from Sigma (Sigma-Aldrich S.r.l. Milan, Italy). The essential oil was analyzed by gas chromatographyflame ionization detector (GC-FID) and gas chromatography-mass spectrometry (GC-MS) [52][53][54][55] and mass spectra on both columns with those of authentic compounds available in our laboratories by means NIST 02 and Wiley 275 libraries [56].

Disk-Diffusion Assay
Antimicrobial activity testing was performed according to the protocol described by Noumi et al. [57]. Bacterial strains were enriched on a tube containing 9 mL of Mueller-Hinton (MH) broth then incubated at 37 • C for 24 h. The inoculums were streaked onto Mueller-Hinton agar plates using a sterile swab. Tetracycline was used in this study as positive control. The antibiotic susceptibility was determined by using the Kirby-Bauer method and Mueller-Hinton agar plates.

Microdilution Method for the Determination of the MIC and MBC
The MIC and the MBC values were determined for all bacteria as described by Hajlaoui et al. [58]. The inoculums of the bacterial strains were prepared from an overnight broth cultures (37 • C) and suspensions and were adjusted to OD 600 (10 7 CFU/mL). The essential oil dissolved in 10% dimethylsulfoxide (DMSO) with a high concentration about 50 mg/mL. Serial two-fold dilutions of the stock solution of the essential oil were prepared in 96-wells plate containing 95 µL of Mueller-Hinton broth for bacteria. In fact, 100 µL aliquot from the stock solution (50 mg/mL) was added to the first well containing 95 µL of the correspondent broth. Then, a serial two-fold dilutions was prepared by transferring 100 µL from the first well into the 10 consecutive wells. The last well containing 195 µL of Mueller-Hinton broth without essential oil and 5 µL of the inoculum on each strain was used as the negative control. Finally, 5µL of the inoculum of each microorganism was added to the wells with a final volume about 200 µL in each well. We have used the scheme proposed for essential oils by Aligiannis et al. [52]: strong activity (0.05 < MIC < 0.5 mg/mL), moderate activity (0.6 < MIC < 1.5 mg/mL) and weak activity (MIC > 1.5 mg/mL).

Phenotypic Characterization of Bacteria-Producing Slime
Detection of slime producing strains was carried out by culturing the isolates on Congo Red Agar (CRA) plates as previously described by Touati et al. [53]. The plates were prepared by mixing 36 g of saccharose (Sigma Chemical Company, St. Louis, MO, USA) with 0.8 g of Congo red in 1 L of Brain Heart Infusion (BHI) agar (Biorad, Hercules, CA, USA). After incubation for 24 h at 37 • C, black colonies and colonies red with a black center were considered as positive slime producers [53].

Test Tube Method
Slime production on glass tubes was determined using the Safranin staining as described for coagulase negative staphylococci by incubating bacterial culture into a glace test tube containing 10 mL of LB broth supplemented with 8% of glucose [54]. Slime production was interpreted as negative, weak (1+), moderate (2+) or strong (3+).
For biofilm formation on glass, the strips (1.5 cm 2 ) were disinfected by dipping in 70% alcohol for 30 min and was held with sterile distilled water. Biofilm quantification was made with crystal violet 1% staining. Moreover, 125 µL of each well were transferred on 96-well microtiter plate and the OD at 570 nm was measured [16].

Determination of Anti-Biofilm Activity on Polystyrene and Glass
MIC, 2 × MIC and 4 × MIC of P. dioica essential oil and eugenol were tested for their anti-Staphylococcus biofilm formation. Only S. aureus ATCC 6538 strain was selected for this test. The crystal violet staining was employed to test the effects on biofilm formation. One hundred µL of fresh bacterial suspension was added to each well. Growth control, media control and blank control were included. The biofilm formation was evaluated using the crystal violet staining method as described previously [16,57].

Violacein Inhibition Assay
Various concentrations of P. dioica essential oil and eugenol (MIC = 10 mg/mL until MIC/32 = 0.3125 mg/mL) were added to 10 µL of C. violaceum ATCC 12472 and incubated at 30 • C for 18 h for the qualitative screening of violacein inhibition [57,59].

Statistical Analysis
All the experiments were conducted in triplicate and average values were calculated using the SPSS 16.0 statistics package for Windows. The differences in mean were calculated using the Duncan's multiple-range tests for means with 95% confidence limit (p ≤ 0.05). Values were expressed as means ± standard deviations.

Conclusions
In this work, we reported the isolation of eugenol (48.76%) and β-pinene (18.52%) as the main phytocompounds in P. dioica essential oil. Shigella, Vibrio, Listeria, Bacillus, Salmonella, Escherichia, Pseudomonas and Staphylococcus foodborne pathogenic bacteria were highly sensitive to the tested oil with mean diameter of growth inhibition zone ranging from (8.00 ± 0.01) mm to (26.66 ± 0.57) mm. Low concentrations of P. dioica essential oil and eugenol were necessary to inhibit the growth of all tested microorganisms. While concentrations as low as 12.5 mg/mL for Pimenta essential oil and 3.125 mg/mL for eugenol are needed to kill the tested strains. Additionally, P. dioica essential oil and eugenol were able to inhibit the biofilm formation on abiotic surfaces (Polystyrene and glass) by almost all tested foodborne strains. Moreover, the tested essential oil and eugenol were able to regulate the production of some virulence related properties controlled by the quorum sensing mechanism in C. violaceum and P. aeruginosa PAO1 starter strains. Hence, these findings highlighted the potential use of this essential oil as a potential candidate for food preservation, biofilm prevention and bacterial cell to cell communication inhibitor. Data Availability Statement: The data generated and analyzed during this study are included in this article.