Chemical Composition, Antibacterial and Antibiofilm Actions of Oregano (Origanum vulgare subsp. hirtum) Essential Oil against Salmonella Typhimurium and Listeria monocytogenes

Essential oils (EOs) are plant mixtures that are known to present strong bioactivities, including a wide antimicrobial action. Biofilms are microbial sessile structures that represent the default mode of growth of microorganisms in most environments. This study focused on the antimicrobial action of the EO extracted from one of the most representative oregano species, that is, Origanum vulgare (subsp. hirtum), against two important foodborne pathogens, Salmonella enterica (serovar Typhimurium) and Listeria monocytogenes. For this, the minimum inhibitory concentrations of the EO against the planktonic and biofilm growth of each bacterium were determined (MICs, MBICs), together with the minimum bactericidal and biofilm eradication concentrations (MBCs, MBECs). The EO was also analyzed for its chemical composition by gas chromatography—mass spectrometry analysis (GC-MS). The influence of EO exposure on the expression of some important virulence genes (hly, inlA, inlB and prfA) was also studied in L. monocytogenes. Results revealed a strong antibacterial and antibiofilm action with MICs and MBICs ranging from 0.03% to 0.06% (v/v) and from 0.06% to 0.13% (v/v), respectively. The application of the EO at 6.25% (v/v) for 15 min resulted in the total eradication of the biofilm cells of both pathogens. The EO was mainly composed of thymol, p-cymene, γ-terpinene and carvacrol. The 3 h exposure of L. monocytogenes planktonic cells to the EO at its MBIC (0.06% v/v) resulted in the significant downregulation of all the studied genes (p < 0.05). To sum, the results obtained advocate for the further exploitation of the antimicrobial action of oregano EO in food and health applications.


Introduction
The genus Origanum L. comprises several species which differ in morphology and chemical characteristics, with most of them being originated in the Mediterranean region [1]. One of the most notable species is Origanum vulgare subsp. hirtum, which is a widely used aromatic plant in foods, feeds, cosmetics, and daily care products, especially due to its content in essential oil (EO), mainly rich in carvacrol and thymol [2]. An increasing number of studies have examined various bioactivities of O. vulgare EO, including strong antimicrobial action against various bacteria and fungi [3]. Such bioactivities may, however, differ between the EO of the same species due to differences in chemical composition which in turn are due to parameters such as the soil and climate conditions of plant growth, cultivation method, harvesting period, anatomical part of the plant used for extraction and method used to extract the oil [4]. at −80 • C in BHI broth (Lab M, Heywood, Lancashire, UK) containing 15% v/v glycerol. When needed, each strain was streaked on Tryptone Soya Agar (TSA; Oxoid, Thermo Fisher Specialty Diagnostics Ltd., Hampshire, UK) and incubated at 37 • C for 24 h (preculture). Working cultures were prepared by inoculating a single colony from each preculture into 10 mL of fresh TSB (Condalab, Torrejón de Ardoz, Madrid, Spain) and then incubating at 37 • C for 18 h. The final concentration of each working culture was ca. 10 9 CFU/mL and its purity was always confirmed by streaking on TSA and incubating it at 37 • C for 24 h.

O. vulgare EO
The EO that was used in this work was extracted from dried plant material by hydrodistillation procedure as previously described [27] and was kindly donated by Dr. Nikolaos Paterakis, owner of the essential oil production and trading company Aegean Organics (Agios Dimitrios, Lemnos, Greece). The year of harvest was 2022. Besides oregano, this company produces EOs and decoctions of various other aromatic plants that are all grown under a certified organic culture. Upon receipt, the EO was kept in a dark glass vial in the refrigerator (4 • C).

Determination of Minimum Inhibitory and Bactericidal Concentrations of EO against Planktonic Bacteria (MICs, MBCs)
The MIC of the EO against the planktonic growth of each bacterial strain was determined following the broth microdilution method as previously described [27]. In brief, ten different concentrations were tested for the EO which were prepared by successive two-fold dilutions in TSB and ranged from 0.5 to 0.001% v/v (a stock solution of the EO at 1% v/v was initially prepared in TSB also containing 9% v/v ethanol). Then, 360 µL of each of those dilutions were placed in duplicate in the wells of a sterile 100-well Bioscreen honeycomb plate (Oy Growth Curves Ab Ltd., Turku, Finland), and inoculated with the tested bacteria so as to have an initial concentration of ca. 10 4−5 CFU/mL. The plate was incubated at 37 • C for 24 h in the Bioscreen Co Pro instrument, which was adjusted to record the absorbance of the contents of each well every 30 min at 600 nm (A 600nm ). Pure sterile growth medium (TSB) was used as a negative control (NC). For each bacterial strain, the MIC of the EO was calculated as its lowest concentration, resulting in no increase in absorbance with respect to the NC. To determine the MBC, 10 µL of broth cultures were aspirated from all the non-growth wells of the MIC assay and spotted (in duplicate) on TSA plates, which were then incubated at 37 • C for 24 h. For each bacterial strain, the MBC of the EO was determined as its lowest concentration that reduced the initial inoculum by more than 99.9% (no appearance of colonies). Each experiment was repeated three times starting from independent bacterial cultures.

Determination of Minimum Biofilm Inhibitory Concentrations (MBICs)
The MBIC of the EO against the biofilm growth of each bacterial strain was determined following the crystal violet (CV) staining method as previously described [27]. In brief, eight different concentrations were tested for the EO which were prepared by successive two-fold dilutions and ranged from 0.25 to 0.002% v/v. These dilutions were done in the respective growth medium for each strain (see below). Each bacterium was left to form biofilm on 96-well polystyrene (PS) microtiter plates (transparent, flat, Cat. No. 30096, SPL Life Sciences, Gyeonggi-do, Korea) for 96 h in the presence of each EO concentration. For that assay, biofilm formation took place under the optimum (biofilm-maximizing) conditions for each strain (with respect to medium, time and incubation temperature), as these had been previously determined [27]. Thus, S. Typhimurium was grown in 1/10 diluted TSB (dTSB) at 20 • C, whereas L. monocytogenes was grown in BHI broth at 37 • C. For both bacteria, the growth medium was renewed at 48 h of incubation. At the end of incubation, the accumulated biomasses were quantified through their staining with CV (0.1% w/v) and subsequently measuring absorbance at 590 nm (A 590nm ). For each bacterial strain, the MBIC of the EO was determined as its lowest concentration that completely inhibited biofilm formation (biomass accumulated did not significantly differ from the negative control). Each experiment was repeated three times starting from independent bacterial cultures.

Determination of Minimum Biofilm Eradication Concentrations (MBECs)
The MBEC of the EO against the already 96 h preformed biofilms of each bacterial strain was determined as previously described [27]. In brief, five different concentrations were tested for the EO which were all prepared in sterile distilled water (dH 2 O) by successive two-fold dilutions and ranged from 6.25 to 0.39% v/v. Each EO disinfection solution was left to act against the biofilm cells for 15 min at room temperature. For each disinfection treatment, the survivors were enumerated by plate counting on TSA following their removal from surfaces through scratching (with a plastic pipette tip). For each bacterial strain, the MBEC of the EO was determined as its lowest concentration leading to at least six log reductions of the biofilm population compared to that of the negative control (dH 2 O also containing 10% v/v ethanol). Each experiment was repeated three times starting from independent bacterial cultures.

Chemical Analysis of EO (GC-MS)
Oregano EO was diluted in pure hexane (1:9) and the mixture was then chemically characterized by GC-MS analysis on a 6890N system (Agilent Technologies, Santa Clara, CA, USA) equipped with an HP-5MS column (30 m, 0.25 mm inner diameter, 0.25 µm film thickness) (Agilent Technologies) and coupled with a 5973Network mass detector (Agilent Technologies), as previously described [27].

Sublethal Exposure of Planktonic L. monocytogenes Cells to EO and RNA Extraction
L. monocytogenes bacteria from the final 18 h working culture were used to inoculate 10 mL of sterile BHI broth at 1:100 dilution (so as to have an initial concentration of ca. 10 7 CFU/mL). The new culture was incubated at 37 • C for 6 h and its cells were then collected by centrifugation (5000× g for 10 min at 4 • C) and the obtained pellet was subsequently suspended in 4 mL of quarter-strength Ringer's solution (Lab M). That freshly saline cellular suspension was aliquoted in two Eppendorf ® tubes (2 mL in each one) and centrifuged (5000× g for 10 min at 4 • C). One of the two bacterial pellets was then suspended in 1 mL of the EO solution (0.063% v/v; equal to the MBIC), while the second pellet was suspended in 1 mL of dH 2 O to be used as the untreated control sample. The dH 2 O of the control sample also contained absolute ethanol at 0.56% v/v. This latter was the concentration that existed in the EO solution (and was used to disolve the oil). Both samples were incubated in a heating dry block for 3 h at 37 • C (sublethal EO exposure) and were then immediately centrifuged (5000× g for 10 min at 4 • C). Supernatants were discarded and each pellet was washed with dH 2 O through an additional centrifugation step (5000× g for 10 min at 4 • C) to remove any EO residues. Washed pellets were then suspended in 1 mL of RNAlater™ Stabilization Solution (AM7021, Invitrogen™, Thermo Fisher Scientific, Carlsbad, CA, USA) and incubated overnight at 4 • C. Next day, RNAlater™ Solution was removed by centrifugation (5000× g for 10 min at 4 • C) and each bacterial pellet was then resuspended in 100 µL of TE buffer (10 mM Tris-HCl, 1 mM EDTA; pH 8) also containing 2.5 mg/mL lysozyme (native, chicken egg white, 4403, Calbiochem ® , Merck KGaA) and incubated at 37 • C for 10 min. Following this enzymatic digest of the bacterial cell walls, the resulting homogenates were directly used for RNA extraction using the NucleoSpin ® RNA Mini Kit (740955, MACHEREY-NAGEL GmbH & Co., KG, Düren, Germany).

cDNA Synthesis and qPCR
The cDNA synthesis was executed starting from 500 ng of each RNA sample (i.e., exposed and not exposed to the EO) using PrimeScript™ RT reagent Kit (RR037A, Takara Bio Inc., Shiga, Japan), according to the manufacturer's instructions. Each cDNA template was then used as substrate in qPCR prepared using the FastGene ® IC Green 2 × qPCR Universal Mix (LS4005, NIPPON Genetics EUROPE GmbH). Four virulence genes of L. monocytogenes were selected (hly, inlA, inlB and prfA) for this analysis, which was done as previously described [28]. Two reference (internal control) genes (tuf, gap) were included in each assay for the normalization of the qPCR data. Each experiment was repeated three times starting from independent bacterial cultures and each reaction was performed in triplicate.

Statistics
Biofilm plate counts (CFU/cm 2 ) were transformed to logarithms before means and standard deviations were computed. The derived data on biofilm biomasses (A 590nm ), planktonic absorbances (A 600nm ), and biofilm populations (log 10 CFU/cm 2 ) were all submitted to analyses of variance (ANOVA), followed by Tukey's multiple range post hoc honestly significant difference (HSD) tests for mean comparison, using the statistical software STATISTICA ® (StatSoft Inc., Tulsa, OK, USA). In ANOVA, the dependent variables were the biofilm biomasses (A 590nm ), planktonic absorbances (A 600nm ) and biofilm populations (log 10 CFU/cm 2 ), while the categorical factor was always the treatment (EO concentration). Regarding gene expression, the data derived from a total of 108 reactions were analyzed. These were the result of six different cDNA samples (i.e., one bacterial strain × two treatments (exposed and not exposed to the EO) × three biological repetitions) × six genes (four targets plus two references)/sample × three technical replicates/gene. For each bacterial strain and target gene, unpaired two-tailed Student's t-tests were applied to check for any significant difference in gene expression (expressed as log 2 (fold difference)) between the two treatments. These tests were performed using the relevant function of Excel ® module of the Microsoft ® Office 365 suite (Redmond, WA, USA). For all statistical analyses, significant differences were reported at a p level of <0.05.

Antimicrobial Actions of O. vulgare EO against Planktonic and Biofilm Cells
The minimum inhibitory and minimum bactericidal concentrations (MICs, MBCs) of O. vulgare EO against the planktonic cells of S. Typhimurium and L. monocytogenes were initially determined. The MICs were found equal to 0.06 and 0.03% v/v against S. Typhimurium and L. monocytogenes, respectively (Table 1; equal to 0.55 and 0.27 mg/mL, respectively). No differences between the MICs and MBCs were observed, indicating the strong bactericidal action of the EO.

Antibacterial Action
Antibiofilm Action Oregano EO, such as many other EOs, may present multiple modes of action against microbial cells. The reader is referred to specialized reviews on the mode of actions of EOs for more info [1,2]. Many other studies have in the past analyzed the antimicrobial action of O. vulgare EOs against planktonic bacteria. For instance, Mazzarrino et al. (2015) determined the MICs of Italian O. vulgare EO against Salmonella spp. and L. monocytogenes and found values ranging from 0.6 to 1.2 µL/mL [29]. Assiri [34]. It is clear that the EO from the Greek organically cultured oregano plants tested in the current study presents strong antimicrobial action that is similar or even superior to that previously described for O. vulgare EOs from some other countries.
Following the calculation of the MICs and MBCs of the O. vulgare EO against the planktonic S. Typhimurium and L. monocytogenes bacteria, we determined its minimum biofilm inhibitory concentrations (MBICs) against the biofilm growth of those bacteria. This is because both pathogens are known to form robust biofilms on various surfaces (both foodcontact and non-food-contact ones) that display increased tolerance to antimicrobial agents and other stresses [35,36]. The protocol we followed to form biofilms had been previously optimized to maximize the sessile growth by each of these two bacterial strains [27]. However, and despite that optimization, the determined MBICs were still quite small and equal to 0.13 and 0.06% v/v against S. Typhimurium and L. monocytogenes, respectively (Table 1). This indicates the ability of the O. vulgare EO to efficiently inhibit biofilm formation by the tested pathogens at very low concentrations and thus its high antibiofilm potential. Figure 1 presents the biofilm biomasses (A 590nm ) accumulated on the PS surface for each one of the eight tested EO concentrations (0.25-0.002% v/v). The densities of the surrounding planktonic suspensions (A 600nm ) found in the same wells at the time of sampling (96 h) are also shown for each treatment (as dotted curved lines). It is worth noting that in the case of L. monocytogenes, the application of the EO at its MBIC (0.06% v/v) resulted in the total inhibition of biofilm formation, whereas the surrounding planktonic cells were still able to multiply, although to a lesser extent compared to the positive control ( Figure 1B). This is quite interesting since it probably indicates that the observed inhibition of biofilm formation should not be a sole result of the reduced growth rate of the bacteria (before and after their attachment to the surfaces), but it could also be associated with the inhibition of some other biofilm-specific mechanisms, such as intercellular interactions and aggregation, motility, production of extracellular polymeric substances (EPS), altered gene expression etc. It is indeed known that various phytochemicals and plant extracts may display antivirulence abilities and anti-quorum sensing behavior upon their application at sub-MICs [37,38]. Such targeted antibiofilm action at sub-MICs is believed to limit the possibilities of the bacteria to develop resistance [39].
Besides the determination of the MBICs, we also determined the Minimum Biofilm Eradication Concentrations (MBECs) of O. vulgare EO against the 96 h preformed biofilms of each target pathogen. Results revealed that the oil needed to be applied at 6.25% v/v to destroy the biofilm communities of both pathogens ( Figure 2). Undoubtedly, this is a quite high concentration that clearly denotes the great recalcitrance of biofilm cells. This is however not surprising since it is well known that the inclusion of bacteria in biofilm structures can greatly increase their tolerance and resistance to biocides, including known antibiotics [40,41]. Thus, in the current experimental setup, O. vulgare EO needed to be applied more than one hundred times more than its MBC to kill S. Typhimurium biofilm cells, while in the case of L. monocytogenes biofilm cells, the required increase in concentration was even more spectacular (more than two hundred times more than its MBC) ( Table 1). Another probably interesting observation is that a dose-response behavior does not seem to exist in the case of the killing of biofilm cells (Figure 2). This is more evident in the case of L. monocytogenes where the application of O. vulgare EO at 0.39% v/v provoked a ca. five-log reduction in the number of biofilm cells, whereas the further ten-fold increase of the EO concentration to 3.13% v/v did not further improve its killing action. The reasons lying behind this last observation are not known but we speculate that these may be somehow related to the presence of EPS and/or the complex biofilm tertiary structure [42,43]. A similar behavior has been previously observed in another similar older study of our group where an increase in the concentrations of sage and spearmint EOs from 5% to 15% v/v did not improve their efficiency against Staphylococcus aureus biofilm bacteria [44]. Besides the determination of the MBICs, we also determined the Minimum Biofilm Eradication Concentrations (MBECs) of O. vulgare EO against the 96 h preformed biofilms of each target pathogen. Results revealed that the oil needed to be applied at 6.25% v/v to destroy the biofilm communities of both pathogens ( Figure 2). Undoubtedly, this is a quite high concentration that clearly denotes the great recalcitrance of biofilm cells. This is however not surprising since it is well known that the inclusion of bacteria in biofilm structures can greatly increase their tolerance and resistance to biocides, including known antibiotics [40,41]. Thus, in the current experimental setup, O. vulgare EO needed to be applied more than one hundred times more than its MBC to kill S. Typhimurium biofilm cells, while in the case of L. monocytogenes biofilm cells, the required increase in  further ten-fold increase of the EO concentration to 3.13% v/v did not further improve its killing action. The reasons lying behind this last observation are not known but we speculate that these may be somehow related to the presence of EPS and/or the complex biofilm tertiary structure [42,43]. A similar behavior has been previously observed in another similar older study of our group where an increase in the concentrations of sage and spearmint EOs from 5% to 15% v/v did not improve their efficiency against Staphylococcus aureus biofilm bacteria [44].  [21]. In that study, O. vulgare EO demonstrated (as expected) a significant antimicrobial action against the planktonic cells (MIC/MBC = 0.16/0.31 µL/mL). Supplementation of EO at concentration 0.25xMIC caused 50% inhibition of biofilm formation for both the tested strains, while its supplementation at 2xMIC resulted in 90% inhibition of their biofilm formation. Contrary to our results, the study on the effectiveness of EO on eradication of preformed 48 h-old biofilms indicated that biofilm reduction occurred in a dose-dependent manner over time (the time of exposure ranged from 15 to 60 min). In another study, Di Vito et al. (2020) compared the in vitro antibiofilm effectiveness of Italian O. vulgare EO against 29 strains of Salmonella spp. isolated from poultry and pig farms belonging to two serotypes (S. Typhimurium and S. Infantis) [45]. O. vulgare EO was active on the disaggregation of mature biofilms of both serotypes, but no inhibiting activity was exerted on biofilm formation. Soni et al. (2013) evaluated EOs of thyme and oregano and their antimicrobial phenolic constituent carvacrol for their ability to inhibit biofilm formation and inactivate preformed Salmonella biofilms [46]. The presence of nonbiocidal concentrations of thyme oil, oregano oil and phenolic carvacrol at 0.006 to 0.012% v/v suppressed Salmonella spp. biofilm formation two-to four-fold, but could not completely eliminate biofilm formation. Treatment of 1-day-old biofilms with thyme oil, oregano oil or carvacrol at 0.05 or 0.1% v/v significantly reduced the amount of preformed biofilms. In a recent study, Vidaković  [48]. For 1-day old biofilms of mixed L. monocytogenes strains produced at 22 • C on PS microtiter plates, only 0.1% v/v of EO was needed to eliminate 7 log CFU per well. On the SS coupons, 0.5% v/v was adequate to completely eliminate 4-day-old biofilms at 7 log CFU per coupon. However, in that older study, the EO was left to act for 24 h against the preformed biofilms, something that probably explains its high efficiency. In our study, we chose a short exposure time (15 min) to try to imitate a short disinfection/biocidal protocol that could be applied in the food industry and/or clinical settings.

Chemical Composition of O. vulgare EO
The GC-MS analysis of the O. vulgare EO revealed the presence of 28 compounds representing 98.1% of the total oil ( Table 2). The EO was mainly composed of the monoterpenes thymol (31.5%), p-cymene (23.7%), γ-terpinene (13.9%), and carvacrol (8%). All these compounds are commonly found as main constituents of O. vulgare EOs and contribute to their bioactivities [2,3,49]. However, in most other studies, the EOs are mainly composed of carvacrol, whereas the EO analyzed in the current study was mainly composed of thymol. This is not strange since some other studies have also described O. vulgare EOs of thymol chemotypes [50][51][52][53][54].

Effect of O. vulgare EO on Virulence Gene Expression of Planktonic L. monocytogenes Cells
The expression of the four target genes (hly, inlA, inlB, and prfA) by the planktonic L. monocytogenes bacteria following their sub-lethal exposure to the O. vulgare EO is shown in Figure 3. All the studied genes were significantly downregulated following the exposure of the cells to the EO at its MBIC (0.06% v/v). This is an intriguing result and seems to agree with and is probably related to the inhibition of biofilm formation that was noticed for that EO concentration ( Figure 1B). Interestingly, in a previous study of our group, all these four genes were found to be significantly overexpressed when the same L. monocytogenes strain (AAL 20107) was left to form biofilm on a PS surface [28]. The hly gene encodes in L. monocytogenes the hemolysin Listeriolysin O (LLO) which is crucial in the manifestation of listeriosis by boosting the cell-to-cell spread and thus successful dissemination of the pathogen during infection [55]. Both inlA and inlB genes encode in L. monocytogenes for important surface proteins that are known to interact with distinct host receptors to cause infection of human cells and crossing of the intestinal, blood-brain or placental barriers [56]. PrfA encodes in L. monocytogenes for the master regulator of virulence that controls the expression of multiple virulence factors that altogether facilitate the transition from extra-to intracellular environments [57]. Rather in agreement with our results, some other previous studies have shown the involvement of all these four genes in attachment, sessile growth, and biofilm formation by L. monocytogenes [58][59][60][61]. In addition, the reduced expression of hly and prfA genes in L. monocytogenes following exposure to some other EOs has been shown in some other previous studies as well [62][63][64]. 06% v/v). Each bar represents the mean ± standard errors (n = 9). The statistically significant differences in genes' expressions between the two treatments (exposed and not exposed to the EO) are indicated with asterisks (*) (p < 0.05).

Conclusions
Origanum vulgare subsp. hirtum EO, extracted from organically cultivated plants on the Greek island of Lemnos was found to present strong antimicrobial and antibiofilm actions against two important foodborne pathogenic bacteria (S. Typhimurium and L. monocytogenes). Its application at 0.13% v/v and 0.06% v/v was sufficient to inhibit biofilm . Relative quantification (log 2 (fold differences)) of the expressions of the four target genes (hly, inlA, inlB, and prfA) by the planktonic L. monocytogenes bacteria following their sub-lethal exposure to the O. vulgare EO at its MBIC (0.06% v/v). Each bar represents the mean ± standard errors (n = 9). The statistically significant differences in genes' expressions between the two treatments (exposed and not exposed to the EO) are indicated with asterisks (*) (p < 0.05).

Conclusions
Origanum vulgare subsp. hirtum EO, extracted from organically cultivated plants on the Greek island of Lemnos was found to present strong antimicrobial and antibiofilm actions against two important foodborne pathogenic bacteria (S. Typhimurium and L. monocytogenes). Its application at 0.13% v/v and 0.06% v/v was sufficient to inhibit biofilm formation by S. Typhimurium and L. monocytogenes, respectively, whereas this needed to be applied at 6.25% v/v (for 15 min) to eradicate the preformed biofilms of the same species. The EO was mainly composed of thymol, p-cymene, γ-terpinene and carvacrol. The sub-lethal exposure of L. monocytogenes planktonic cells to the EO at its MBIC (0.06% v/v) resulted in the significant downregulation of four important virulence genes (hly, inlA, inlB, and prfA). To sum, the results obtained advocate for the further exploitation of the antimicrobial action of oregano EO in food and health applications. Future studies could be conducted on some such applications. In addition, the specific mode of action of this EO against microbial cells could be tested by applying either the crude extract or some of its individual components. Funding: This research received no external funding.

Data Availability Statement:
The data presented in this study are available upon reasonable request form the corresponding author.