Comparative Study of Antibacterial, Antibiofilm, Antiswarming and Antiquorum Sensing Activities of Origanum vulgare Essential Oil and Terpinene-4-ol against Pathogenic Bacteria

Essential oils from aromatic and medicinal plants have many bioactive compounds known for their important biological activities mainly their antibacterial effects. Here we evaluated qualitatively and quantitatively the biofilm formation capability of pathogenic bacterial strains (n = 8). Then, we investigated the antibacterial, antibiofilm, antiquorum-sensing, and antiswarming efficacy of Origanum vulgare essential oil (EO) and terpinene-4-ol. Our results revealed that EO exhibited a more potent inhibitory effect against the tested strains. While the terpinene-4-ol was found to be more effective against developed Staphylococcus aureus biofilm. Regarding the anti quorum-sensing activity, we noticed that O. vulgare displayed better inhibition percentages in violacein production even at a low concentration (MIC/4). Additionally, this EO showed better inhibition of Pseudomonas aeruginosa PAO1 migration in comparison with the terpinene-4-ol. Our findings revealed that using pure O. vulgare EO demonstrated better competitive effects against pathogenic bacteria with a different mode of action when compared to the terpinene-4-ol. Hence, exploration and development of efficient anti-infection agents from natural resources such as full EOs represent promising tools in anti-infective therapy.


Introduction
Pathogenic bacteria are characterized by their ability to produce several virulence factors. One example is, biofilm formation, which provides them protection against the host immune system defense and allows for the acquisition of significant resistance to various antimicrobials [1]. This form of subsistence, well spread in natural ecosystems as well as in medical and industrial area, afford a solid inter-bacterial contact [2]. Numerous pathogenic bacteria ensure the control and monitoring of biofilm formation under a bacterial communication system known as quorum sensing (QS) [3]. In fact, numerous pathogenic bacteria have the ability to control the expression of their virulence factors through the QS system [4], along with the production of secondary metabolites and stress adaptation mechanisms [5].
One of the recent approaches in antibiotic therapy and biofilm dispersal is to target the bacterial QS system [6]. Therefore, developing innovative therapeutic measures based on novel antibiofilm agents with anti-QS properties are needed. Essential oils (EOs), well

Disk Diffusion Assay
The activity of O. vulgare EO and Terpinene-4-ol was evaluated against four Gram negative (P. aeruginosa PAO1, E. coli ATCC 35218, S. enterica CECT 443, S. flexeneri CECT 4804) and four Gram-positive (S. aureus ATCC 6538, B. subtilis CIP 5265, V. vulnificus CECT 529, and L. monocytogenes CECT 933) bacteria, following a standard agar disk diffusion assay [15]. An inoculum of 0.5 McFarland was prepared for each strain and then swabbed onto the surface of Muller Hinton (MH, Bio-rad, Marnes-la-Coquette, France) agar. Whatman discs (Bio-rad, Marnes-la-Coquette, France) were impregnated with 10 µL of the EO and the compound, and then placed onto the surface of inoculated plates (Bio-rad, Marnes-la-Coquette, France). Gentamicin discs (Bio-rad, Marnes-la-Coquette, France) were used as a positive control. After 24 h of incubation at 37 • C, the diameters of inhibition zones (ZOI, in mm) were measured. All experiments were repeated three times.

Minimum Inhibitory and Minimum Bactericidal Concentrations
The minimum inhibitory concentration (MIC) values for O. vulgare EO and terpinene-4-ol against each strain were determined by the broth dilution method [16]. All pathogens were cultured for 24 h and their optical density was adjusted to 0.5 McFarland standards. The tested agents were transferred to sterile 96-well microtiter plates. The inoculum (10 µL) of each strain was added to each well. MIC was defined as the concentration that completely inhibited visible cell growth during a 24 h incubation period at 37 • C. To determine the minimum bactericidal concentration (MBC) values, 10µL of each well medium with no visible growth was removed and pour plated with MH agar. After 24h of incubation at 37 • C, the number of surviving organisms was determined as CFU/mL [17].

Biofilm Formation Ability
The ability to produce slime was carried out by culturing each bacterium on Congo Red Agar (CRA) as previously described [18]. After aerobic incubation at 37 • C for 24 h, Life 2022, 12, 1616 3 of 11 strains with black colonies were identified as slime positive, while red colonies bacteria were classified as slime-negative strains [18].
Qualitative biofilm formation on glass surfaces was determined according to the protocol previously described by Davenport et al. (1986) [19]. Each strain was tested at least three times and biofilm production by each isolate was interpreted as negative, weak (1+), moderate (2+), or strong (3+).

Antiadhesion Effect
The antiadhesion properties of O. vulgare EO and terpinene-4-ol were tested according to the protocol of Saising et al. [21]. A 100 µL aliquot of the bacterial growth in BHI supplemented with 2% glucose was transferred to a microtiter plate (Bio-rad, Marnesla-Coquette, France) and added with 100 µL of different inhibitory concentration (1/16 to 1 × MIC) of the tested agents. After incubation for 24h at 37 • C, the supernatant was discarded and crystal violet (CV) stained biofilm cells were determined at 570nm using a microplate reader (Bio-rad, Marnes-la-Coquette, France).

Reduction of Biofilms Growth and Development
The reduction in biofilms developed for 48h at 37 • C by O. vulgare EO and terpinene-4-ol was evaluated as described previously [22]. A range of concentrations (1 × MIC, 2 × MIC, and 4 × MIC) of the selected agents were added to each well of the microplate per well and then incubated for 24 h. After treatment and staining (CV) assay, the biofilm biomass was measured by the absorbance of CV at 570 nm. The percentage of biofilm eradication was determined as: [(OD growth control-OD sample)/OD growth control] × 100.

Antiquorum Sensing Activity
The qualitative analysis studied the reduction in violacein pigment. An overnight culture (10 µL) of C. violaceum (adjusted to 0.4 OD at 600 nm) was added into sterile microtiter plates containing 200 µL of LB broth and incubated at 30 • C in the presence and absence of various concentrations of tested agents (MIC = 10 mg/mL until MIC/32 = 0.3125 mg/mL). LB broth containing C. violaceum ATCC 12472 was used as a positive control [23]. The percentage of violacein reduction was calculated by following formula: Violacein inhibition (%) = (OD 585 nm Control-OD 585 nm Sample)/OD 585 nm Control.

Essential oil Composition
The chemical composition of O. vulgare EO is summarized in Table 1. Ki: Kovats retention index determined relative to the tR of a series of n-alkanes (C10-C35) on an HP-5 MS column; Kr: Kovats retention index determined relative to the tR of a series of n-alkanes (C10-C35) on HP Innowax.

Antibacterial Activity
Antibacterial effects are reported as inhibition zones, and in vitro activity as MIC and MBC. The obtained results using the disc diffusion method recorded in MH agar are sum-  Table 1.

Antibacterial Activity
Antibacterial effects are reported as inhibition zones, and in vitro activity as MIC and MBC. The obtained results using the disc diffusion method recorded in MH agar are summarized in Table 2. The EO of O. vulgare was active against all the tested strains with an inhibition zone ranging from 10.33 ± 0.57 mm to 41.66 ± 0.57 mm. Its zone of inhibition is larger to the size of the positive control antibiotic (Gentamicin) zone (Exception for P. aeruginosa and S. aureus). Terpinene-4-ol was found to be active again six pathogenic strains, with weak inhibition effect against P. aeruginosa PAO1 (less than 8 mm). However, no activity of this compound was observed against B. subtilis CIP 5265.
Considering the MIC and MBC values, both tested substances present a bacteriostatic effect against all tested pathogens at a concentration of 0.048 mg/mL. The MBC values of O. vulgare EO were found to be 1.562 mg/mL for all strains excepting P. aeruginosa PAO1 (50 mg/mL). The terpinene-4-ol exhibited MBC values weaker than the EO, ranging between 3.125 and 12 mg/mL ( Table 2).

Biofilm Formation Activity
Amongst tested strains, five out of eight bacterial pathogens (62.5%) displayed positive (black colony) and variable phenotype (black center) over CRA plates (Figure 2), indicating slime production (Table 3).
Life 2022, 12, x FOR PEER REVIEW 6 of 12

Biofilm Formation Activity
Amongst tested strains, five out of eight bacterial pathogens (62.5%) displayed positive (black colony) and variable phenotype (black center) over CRA plates (Figure 2), indicating slime production (Table 3).
. Qualitative evaluation of biofilm formation potential on glass tube showed that only S. aureus were highly adherent (noted +++), five strains were moderately adherent (noted ++), and only two strains (B. subtilis and S. enterica) were weakly adherent (noted +, Table  3). The results of quantitative biofilm formation ability evaluated with CV staining assay  Qualitative evaluation of biofilm formation potential on glass tube showed that only S. aureus were highly adherent (noted +++), five strains were moderately adherent (noted ++), and only two strains (B. subtilis and S. enterica) were weakly adherent (noted +, Table 3). The results of quantitative biofilm formation ability evaluated with CV staining assay revealed that among the tested bacteria, only S. aureus ATCC 6538 strain was highly biofilm positive (OD570 ≥ 1) over polystyrene surfaces (Table 3). Other strains showed low-grade biofilm formation (0.1 ≤ OD570 < 1).

Antibiofilm Activity
The highly biofilm forming strain S. aureus ATCC 6538 was selected for the antibiofilm test. For antiadhesion assay, this bacterium was cultured in microtiter plates for 24 h in the presence of sub-inhibitory concentrations of the tested agents (1/16× to 1 × MIC) and the developed biofilm was stained with crystal violet. At a concentration of 1/8 × MIC, corresponding to 0.006 mg/mL against S. aureus ATCC 6538, the EO exerted an antiattachment effect (OD570 < 1), when compared to the control (untreated cells). However, the antiadhesion effect of the main compound was observed from at a concentration of 1/4 × MIC (Figure 3). Regarding the antibiofilm effect of O. vulgare EO and the terpinene-4-ol, a mature biofilm (48h) of S. aureus was subjected to various concentrations (MIC, 2 × MIC, 4 × MIC) of the tested agents. O. vulgare EO and terpinene-4-ol were effective against the development of biofilms with percentage reduction values ranging from 10.36% ± 1.95 to 54.05% ± 1.48 and 62.28% ± 1.42 to 70.97% ± 9.65, respectively (Figure 4). We clearly noted that the terpinene-4-ol was more effective against S. aureus biofilm than the essential oil. ment of biofilms with percentage reduction values ranging from 10.36% ± 1.95 to 54.05% ± 1.48 and 62.28% ± 1.42 to 70.97% ± 9.65, respectively (Figure 4). We clearly noted that the terpinene-4-ol was more effective against S. aureus biofilm than the essential oil. Regarding the antibiofilm effect of O. vulgare EO and the terpinene-4-ol, a mature biofilm (48h) of S. aureus was subjected to various concentrations (MIC, 2 × MIC, 4 × MIC) of the tested agents. O. vulgare EO and terpinene-4-ol were effective against the development of biofilms with percentage reduction values ranging from 10.36% ± 1.95 to 54.05% ± 1.48 and 62.28% ± 1.42 to 70.97% ± 9.65, respectively (Figure 4). We clearly noted that the terpinene-4-ol was more effective against S. aureus biofilm than the essential oil.

Antiquorum Sensing Activity
In order to evaluate the extent of QSI, the violacein pigment production in C. violaceum ATCC 12472, in the absence or presence of O. vulgare EO and terpinene-4-ol at different concentrations was evaluated. Our results revealed that the EO inhibited the violacein production more efficiently, with observed percent inhibition of more than 50%, even

Antiquorum Sensing Activity
In order to evaluate the extent of QSI, the violacein pigment production in C. violaceum ATCC 12472, in the absence or presence of O. vulgare EO and terpinene-4-ol at different concentrations was evaluated. Our results revealed that the EO inhibited the violacein production more efficiently, with observed percent inhibition of more than 50%, even at a low concentration (MIC/4). In contrast, terpinene-4-ol could inhibit violacein production only at high concentration (MIC) to an extent of 42.29±0.9 % (Table 4).

Antiswarming Assay
As swarming migrations play an important role in QS-mediated biofilm formation in uro-pathogens, such as P. aeruginosa PAO1, we examined the anti-QS potential of O. vulgare EO and terpinene-4-ol against QS dependent swarming motility in this strain. The results obtained indicated that the tested agents inhibited the swarming behavior of the test PAO1 pathogen to different extents. In fact, the percentage of antiswarming activity of the main compound reached 25% independently of the concentration. Unlike the O. vulgare EO, which showed more inhibition level in the migration of PAO1 recording 29.17 ± 4.17% (Table 5).

Discussion
The QS is an efficient bacterial cell-cell communication process that controls numerous mechanisms, particularly in pathogenic bacteria, including antibiotic production, biofilm formation, and virulence factor secretion [4]. Recently, there has been a rise in attempts to search for and identify novel antimicrobials of natural origins to control the emergence of dreaded pathogens [25]. In the present study, we characterized the biofilm formation ability of eight pathogenic bacteria. Then, we investigated the antibacterial, antibiofilm, antiquorum-sensing, and antiswarming efficacy of O. vulgare essential oil and its main compound Terpinene-4-ol.
The results of the first part of our study conducted on biofilm characterization, determined by different phenotypic assays, revealed that S. aureus strain exhibited the highest biofilm production capability when compared to the rest of the pathogenic bacteria. As largely documented in the literature, this bacterium is well known for its efficacy to colonize various biotic and abiotic surfaces [1,26]. Secretion of extracellular polymeric substance matrix by S. aureus leads to the establishment of a solid structure called microbial biofilm [27]. Such polymers surrounding the bacterial micro-colonies allow them protective effects against various external agents [28]. In fact, the resistance to antibiotics, disinfectants, and host defense systems increases when the bacteria are implicated into the biofilms [29]. Due to the continuous increase in bacterial infection rates related to microbial biofilm, the necessity to search and develop new active substances with low toxicity remain of interest.
In the second part of our investigation, we tested the antibacterial effects of O. vulgare EO and terpinene-4-ol. Observing the results of the agar disc diffusion method, it is possible to conclude that pure EO exhibited the highest inhibition zones against most of the tested strains when compared to the tested compound and the reference antibiotic (Gentamicin). Interestingly, the EO showed 6 times greater inhibitory effect with ZOI ≥ 28 mm [30] which is statistically significant (p < 0.05) when compared to the terpinene-4-ol (4 times stronger inhibitory effect). Additionally, O. vulgare EO showed more efficient bacteriostatic impact against almost all tested strains with an MBC value of 1.562 mg/mL. The differences in the effects of natural substances can be due to the synergism between the minor and major molecules composing the O. vulgare EO, unlike the unique major compound [31,32]. Moreover, these differences might be attributed to the differences in the susceptibility of tested species, since the outer membrane of Gram-negative bacteria are characterized by the presence of lipopolysaccharide molecules, which provide a hydrophilic surface [33]. Accordingly, Gram-positive strains are relatively more sensitive to hydrophobic compounds such as EOs [34].
Since bacterial cell attachment represents a critical essential step in biofilm formation, we tested the effect of sub-inhibitory concentrations of the O. vulgare EO and Terpinene-4-ol against the adhesion of the selected strains. Our result demonstrated that at a concentration of 0.006 to 0.018 mg/mL, corresponding to 1/8 × MIC and 1/4 × MIC, respectively, the tested agents exerted an antiattachment effect against the highly biofilm forming strain (S. aureus 6538). The effects of EOs and their compounds against the adhesiveness of S. aureus have been frequently reported [22,35]. Interestingly, the use of EOs as natural antibacterial agents, for inhibition of cell attachment of pathogenic bacteria represents a strategic way to prevent the establishment and the development of mature resistant biofilm [36]. The result of biofilm eradication showed that terpinene-4-ol was more effective against the development of preformed S. aureus biofilm (p < 0.05), with a percentage of reduction Life 2022, 12, 1616 9 of 11 values exceeding 60% even at a concentration of 1 × MIC. Our results are in agreement with previous findings recording the effectiveness of various main compounds from EOs such as 1,8 cineol, thymol, and carvacrol in the eradication of staphylococcal biofilms [35,37,38]. Additionally, the efficacy of EOs, as antibiofilm agents from natural origins, was previously documented against several Gram-positive and Gram-negative bacteria [39].
Biologically active substances that interact with bacterial QS systems attenuating their pathogenicity are known as anti-QS compounds. In this part of our study, we evaluated the anti-QS activity O. vulgare EO and terpinene-4-ol, using C. violaceum ATCC12472 as a biomonitor strain. From this test, it was observed that the EO presented better anti-QS activity (p < 0.05) than the compound in a concentration-dependent manner. The inhibition effect of QS by O. vulgare EO from different origins was reported in previous studies [8,10]. The synergistic effects between all the compounds present in this EO leads to the inhibition of bacterial intercellular communication systems and subsequently affects the ability to form biofilms and produce virulence factors in pathogenic bacteria [40,41].
Bacterial swarming motility is one important virulence factor mediated and regulated by the QS system [42]. Our results revealed that a reduction in swarming motility of PAO1 by tested agents was observed in a different manner. In fact, the EO inhibits the migration of PAO1 at higher concentrations (p < 0.05), whereas the effect of terpinene-4-ol is the same regardless of the concentration. It was reported that sub-MICs of various EOs such as tea tree, eucalyptus, and clove revealed a decrease in swarming motility in P. aeruginosa PAO1 in a concentration-dependent manner [35,38]. Interestingly, the inhibition of swarming migration is a promising strategy to fight against pathogenic bacteria since it represents one of the dreaded virulence factors involved in biofilm formation [24].

Conclusions
In summary, O. vulgare EO and terpinene-4-ol exhibit various inhibitory effects against pathogenic bacteria. Considering that O. vulgare EO has the highest antibacterial, antiquorum sensing, and antiswarming potential, it is therefore recommended to be valorized as an efficient antimicrobial agent for the treatment of bacterial infections. More investigations are also necessary to elucidate the biological activities of the main compounds identified in O. vulgare essential oil and their possible use in the industrial formulation of essential oil products.