Validation of Qualitative Broth Volatilization Checkerboard Method for Testing of Essential Oils: Dual-Column GC–FID/MS Analysis and In Vitro Combinatory Antimicrobial Effect of Origanum vulgare and Thymus vulgaris against Staphylococcus aureus in Liquid and Vapor Phases

Combinatory action of antimicrobial agents such as essential oils (EOs) show to be an effective strategy to overcome the problem with increasing antibiotic resistance of microorganisms, including Staphylococcus aureus. The objective of this study was to evaluate in vitro antimicrobial interactions between Origanum vulgare and Thymus vulgaris EOs against various S. aureus strains in both liquid and vapor phases using the broth volatilization checkerboard method. Fractional inhibitory concentrations (FICs) were determined for both liquid and vapor phases, and the composition of EOs was analyzed by gas chromatography-mass spectrometry using dual-column/dual-detector gas chromatograph. Results of oregano and thyme EOs combination showed additive effects against all S. aureus strains in both phases. In several cases, sums of FICs were lower than 0.6, which can be considered a strong additive interaction. The lowest FICs obtained were 0.53 in the liquid phase and 0.59 in the gaseous phase. Chemical analysis showed that both EOs were composed of many compounds, including carvacrol, thymol, γ-terpinene, and p-cymene. This is the first report on oregano and thyme EOs interactions against S. aureus in the vapor phase. It also confirms the accuracy of the broth volatilization checkerboard method for the evaluation of combinatory antimicrobial effects of EOs in the vapor phase.


Introduction
Staphylococcus aureus is a gram-positive bacterium that has been responsible for a broad spectrum of diseases, ranging from food poisoning and superficial skin and soft tissue infections to life-threatening infections such as bacteremia, endocarditis, osteomyelitis, pneumonia, or toxic shock syndrome [1]. It is notorious for its ability to quickly become resistant to any antibiotic, which makes this bacterium one of the most serious pathogens in humans, and its treatment is often difficult [2]. In humans, S. aureus can occur as both a benign commensal and a harmful pathogen. Besides being a common colonizer of the skin, it also asymptomatically and permanently colonizes the anterior nostrils of up to 30% of the normal human population [3,4], which is widely considered to be a predisposition of invasive infection [5]. Since S. aureus is a microorganism that is associated with a broad spectrum of infections affecting the respiratory tract, taking up antibiotics through inhalation could be one of its possible treatments. Moreover, the combination of two or that the antimicrobial effect of these EOs might be comparable to their main component alone [27]. Due to their antimicrobial properties, EOs (including O. vulgare and T. vulgaris EOs) could be used as alternatives to conventional antimicrobial agents, especially against antibiotic-resistant pathogens [24]. So far, numerous studies regarding the antibacterial activity of O. vulgare and T. vulgaris EOs alone against a wide range of microorganisms, including S. aureus, have been published [28][29][30]. Both EOs have also previously been tested against S. aureus in combination with other EOs [31,32] as well as with classic/conventional antibiotics [33]. Moreover, their synergistic and additive inhibitory activity with each other has previously been reported against S. aureus as well [34,35]. However, although there are numerous articles on the antistaphylococcal activity of O. vulgare and T. vulgaris EOs tested in the broth and agar, substantially fewer articles dealing with their antibacterial effects against S. aureus have been published using the vapor phase [36,37]. Moreover, to the best of our knowledge, the combinatory antistaphylococcal activity of O. vulgare EO and T. vulgaris EO have not previously been studied in the gaseous phase.
Based on the results of our preliminary screenings performed as several combinations of different EOs (Cinnamomum cassia, C. verum, Cymbopogon flexuosus, O. vulgare, Syzygium aromaticum, and T. vulgaris) against S. aureus of the American Type Culture Collection (ATCC) 29213 (the lowest fractional inhibitory concentration (FIC) values in the vapor phase ranged from 0.59 to 1.25), the combination of O. vulgare EO with T. vulgaris EO was selected for more detailed evaluation due to its lowest FIC values that it had produced (unpublished data). Therefore, the aim of the present study was to determine the antibacterial combinatory potential of EOs hydrodistilled from O. vulgare and T. vulgaris against standard strains and clinical isolates of S. aureus in both the vapor and liquid phases. Since the methods currently available for the determination of antimicrobial interactions of EOs in the vapor phase are based on disk diffusion assay, which yields only qualitative information about the antimicrobial agent combination, the accuracy of these techniques is limited because it is difficult to distinguish indifferent from synergistic interaction. For this reason, the validation of the qualitative broth volatilization checkerboard method for testing of combinatory antimicrobial effect of two different EOs was an additional objective of this study.

Antimicrobial Analysis
The detailed results of individual minimum inhibitory concentrations (MICs) of O. vulgare and T. vulgaris EOs against 12 strains of S. aureus including clinical isolates, as well as the MICs of their combinations with corresponding ΣFIC values are summarized in Tables 1 and 2 for the vapor and liquid phases, respectively. Results show that O. vulgare EO exhibited an antistaphylococcal effect with MICs ranging from 427 to 796 µg/mL and from 512 to 1024 µg/mL in agar and broth media, respectively. Similar numbers were observed for T. vulgaris EO with MICs ranging from 427 to 796 µg/mL in the vapor phase and from 512 to 967 µg/mL in the liquid phase.
Considering their combinatory activity, EO of O. vulgare in combination with T. vulgaris EO produced an additive antimicrobial effect against all 12 strains tested. The combination profiles of four S. aureus strains are presented graphically in Figure 1. The isobole curves clearly show the additive effect against S. aureus strains tested, whereas the additive interactions can be read according to the curves indicating the borderline of additivity and synergy. In several cases (i.e., for one combination of these volatile agents in the vapor phase and four combinations in broth), they showed ΣFICs lower than 0.6, which can be considered a strong additive interaction, reaching values close to the synergistic effect. The most effective concentrations inhibiting the growth of S. aureus (SA) were found in the liquid phase against methicillin-resistant clinical isolate SA 2 at 512 µg/mL of O. vulgare EO and 32 µg/mL of T. vulgaris (ΣFIC = 0.53) and in the vapor phase against standard strain SA ATCC 29213 at 242 µg/mL of O. vulgare EO and 128 µg/mL of T. vulgaris EO Plants 2021, 10, 393 4 of 18 (ΣFIC = 0.59). On average, the best FIC values were observed in both the liquid and vapor phases when the concentrations of T. vulgaris EO were 256 and 128 µg/mL. Based on the results, the optimum ratio of T. vulgaris and O. vulgare to achieve bacterial inhibition would be 0.5-2:1 in the vapor phase and 0.4-1.2:1 in the liquid phase.

Gas Chromatography/Mass Spectrometry (GC/MS) Analysis
The yields of O. vulgare and T. vulgaris EOs in the dried weight of plant materials (containing 14.42% and 13.68% of residual moisture) for T. vulgaris were 1.5% and 1.2% (v/w), respectively. The complete chemical compositions of oregano and thyme EOs are provided in Table 3; Table 4, respectively. In EOs isolated from O. vulgare and T. vulgaris, 19 and 28 components have been identified using an HP-5MS column, representing 99.78% and 99.26% of their respective total content. Using DB-HeawyWAX column, 25 and 34 compounds were determined, which constitute 99.90% and 99.53% of the volatile oil, respectively. In total, 26 compounds were identified in the EO of O. vulgare, whereas 37 compounds were found in the EO isolated from T. vulgaris. The analysis showed that the most monoterpene hydrocarbons and oxygenated monoterpenes were the main groups of chemicals in both EOs.

Discussion
In our study, the in vitro growth inhibitory effect of both O. vulgare and T. vulgaris EOs was slightly stronger in the vapor phase than in a liquid medium since the MIC values were for the vast majority of the staphylococcal strains slightly lower on the agar media than in the broth. The only exceptions were standard strain ATCC 33592 and clinical isolate SA 1, where the antimicrobial effect of O. vulgare was stronger in the liquid phase, and staphylococcal strains ATCC 29213, ATCC 43000, SA 1, and SA 6, where the MICs of T. vulgaris EO were the same in both phases. Similar pattern showing that the vapor generated by EOs has a greater antimicrobial effect compared to EOs in liquid form applied by direct contact (in aqueous solutions or on solid agars) can be observed in several previous studies [48,49]. This phenomenon can be explained by the fact that in the aqueous phase, lipophilic molecules associate to form micelles and thus restrain the attachment of EOs to microorganisms, whereas the vapor phase allows for free attachment [50,51].
Values of MICs observed in our study for O. vulgare and T. vulgaris EOs in the liquid phase were similar to numerous previously published data. For example, the investigation carried out by Boskovic et al. [30] [34], where MICs of oregano and thyme EOs against S. aureus were equal to 0.5 µL/mL and 1 µL/mL, respectively.
If mixtures of EOs are used as antimicrobials, they may, according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) [53], show either an antagonistic, additive, indifferent, or synergistic effect, measured by assessment of the FIC values. Several authors have demonstrated additive effects as well as synergistic actions of O. vulgare EO in combination with T. vulgaris EO in the liquid phase. Our results correspond well with Gavaric et al. [35], who reported additive antibacterial action of thyme and oregano against several bacteria, including S. aureus ATCC 25923 (FIC value = 1). Similarly, Gutierrez et al. [54] confirmed the additive effect of these EO combinations against several spoilage organisms, such as Enterobacter cloacae, Pseudomonas fluorescens, and Listeria innocua, using a checkerboard method with FIC values ranging from 0.75 to 1. On the other hand, synergistic activity of oregano and thyme EO combinations have previously been reported as well; for example, in the study of Stojkovic et al. [34], oregano combined with thyme EO produced synergy against S. aureus (FIC value 0.45). However, as the assayed EOs possess similar chemical composition, their combination may exhibit addition rather than a synergistic effect [55].
The disc volatilization method is probably the most frequently used assay for the evaluation of in vitro growth inhibitory effect in the vapor phase. Both EOs have previously been tested individually against S. aureus in the gaseous phase. For example, an investigation carried out by Nedorostova et al. [37] determined antibacterial effects of EOs isolated from O. vulgare and T. vulgaris against S. aureus ATCC 25923 using modified disc volatilization method, and the MIC value of both EOs was 0.017 µg/cm 3 . Similarly, Kloucek et al. [56] used a modified disc volatilization method to assess the antimicrobial activity of various EOs, including those of oregano and thyme. In this study, vapors of O. vulgare EO containing 92% of carvacrol inhibited growth of S. aureus ATCC 25923 with MIC values 62.5 µL/L, whereas three EO samples of T. vulgaris with different chemical composition exhibited antimicrobial activity against the same staphylococcal strain with the MIC ranging from 125 to 250 µL/L. However, a thyme EO where thymol was the predominant compound was not active at all. A study performed by Lopez et al. (2007) [36] determined the growth inhibitory effects of O. vulgare and T. vulgaris vapors against S. aureus ATCC 29213 by a similar method and consequently calculated MIC causing apparent inhibition (17.5 µL/L and 87.3 µL/L, respectively) of the atmosphere above microorganisms. Subsequent research led by Reyes-Jurado et al. [57] assessed the MIC values of T. vulgaris EO vapors against S. aureus and MRSA as >5 µg/mL of air. However, although there has been increasing research interest in the antimicrobial activity of individual EO vapors in recent years, significantly fewer studies have been reported on their combinations. In the case of thyme and oregano EO vapors, to the best of our knowledge, the only study dealing with their combinatory effects in the gaseous phase was published by Cho et al. [58], who reported synergistic activities of gaseous oregano and thyme EOs against Listeria monocytogenes by modified checkerboard assay (FIC = 0.375). Our study is the first report on O. vulgare and T. vulgaris interactions in the vapor phase against S. aureus.
The antimicrobial properties of O. vulgare and T. vulgaris have been attributed to their chemical compositions, which are primarily rich with monoterpene hydrocarbons and oxygenated monoterpenes. The principal terpenes identified in oregano and thyme are usually carvacrol, thymol, γ-terpinene, and p-cymene; while terpinen-4-ol, linalool, β-myrcene, trans-sabinene hydrate, and β-caryophyllene are also present. The proportion of these and other components in oils within the same species defines the chemotype [59]. In our study, the chromatographic profiles of both EOs were analyzed by GC/MS using two detectors and two capillary columns of different polarities to avoid the overlapping of signal peaks observed in the chromatogram and to achieve the best possible resolution of compounds. The internal standard was used for quantitative analysis. Compounds belonging to the classes with monoterpene hydrocarbons and oxygenated monoterpenes were the most numerous identified. Carvacrol was the most abundant compound in oregano EO, followed by p-cymene and γ-terpinene, and the oil is, thus, characterized as a carvacrol chemotype. This finding is in accordance with several previously published studies. For example, Stojkovic et al. [34], Scalas et al. [60], and Stoilova et al. [61] reported carvacrol as the main component of oregano EO (contributing 64.50%, 62.61%, and 66.20% of the EO, respectively), p-cymene as the second the most abundant compound (10.90%; 12.36%, and 9.1%, respectively), and γ-terpinene as third most abundant component (10.80%, 7.60%, and 7.30%, respectively). Thymol, on the other hand, has been in our study detected as the most abundant constituent in thyme EO, also followed by its precursors, p-cymene and γ-terpinene; therefore the present thyme oil belongs to thymol chemotype. This finding is also in accordance with numerous previously published studies [26,29,30,34,62,63], where thymol, p-cymene, and γ-terpinene were reported as the first, second, and third most abundant compounds, respectively. The number of components identified in our study (26 and 37 in total in oregano and thyme EOs, respectively) is within the range of numbers of compounds identified in other reported studies, as eight to 38 compounds have been reported for O. vulgare [30,34,60,61,64], and 16-50 for T. vulgaris [26,29,30,34,60,62,63,65]. Since the used plant material has been obtained from a commercial supplier, the age of the plants as well as their growing conditions, harvest time, transportation, and storage conditions are unknown. Therefore, the chemical composition of EOs analyzed in this study can be influenced by all the above-mentioned factors [66,67]. The qualitative differences (numbers of components) between the two columns are in accordance with previously reported studies on GC/MS analysis of EOs using two columns. For example, Anderson and Parnell [68], who compared cold-pressed orange oil profiles by GC/MS using polar (Zebron ZB-WAX column) and non-polar (Zebron ZB-1ms) GC columns, identified 22 and 29 components on non-polar and polar compounds, respectively. The higher number of volatile components identified on a polar column might have been caused, similarly as in our case, by the better resolution between compounds that were seen to co-elute on the non-polar column. Similarly, quantitative differences between the polar and non-polar columns have previously been reported as well. In our study, the main compound (thymol) in thyme EO showed the highest proportional difference between two columns (more than 6%), which can be, for example, compared to Fan et al. [69], who analyzed the composition of the EO from Dendranthema indicum var. aromaticum and detected α-thujone as the main compound with a difference of 4.88% between columns. Different amounts of the detected compounds are caused by different polarity and material of the used columns. Besides determining of raw percentages of peak areas, the concentration of components in 1 kg of dry plant material was calculated using predicted relative response factors with an objective to increase the reliability and accuracy of the volatile components' quantification [70]. This approach is important in technological processes with various applications in the field of chemical analysis of volatile plant-derived products because it allows the quantification of volatile components by GC/MS with flame-ionization detection when the authentic components are not available, and in addition, it can avoid time-consuming procedures of calibration [71].
Since carvacrol and thymol have been found to be the most abundant compounds in our oregano and thyme EOs, respectively, the additive effects obtained by interactions between our volatile oils might be caused mainly by these two phenolic monoterpenoids. The presumption that the predominant component in both EOs is responsible for the antimicrobial activity of EOs can be supported by our previous research [14], whereas the range of MIC values of carvacrol (370-1593 µg/mL and 484-1024 µg/mL in agar and broth media) and thymol (341-1707 µg/mL and 355-1024 µg/mL in the vapor and liquid phases) were very similar to the MIC values of the O. vulgare (427-796 µg/mL and 512-1024 µg/mL in agar and broth media) and T. vulgaris (427-796 µg/mL in the vapor phase and from 512-967 µg/mL in liquid phase) EOs tested in this study. The occurrence of additive interaction between carvacrol and thymol could be related to the similarity in their molecular structures (they are isomers), suggesting a similar mechanism of action [72]. Both thymol and carvacrol are expected to cause functional and structural damages to the cytoplasmic membranes. The primary mechanism of antibacterial action of thymol is not fully known; however, it is believed to involve outer and inner membrane disruption and interaction with membrane proteins and intracellular targets. Similarly, the primary mechanism of action of carvacrol is its ability to position in the membrane where it increases permeability [73]. In both EOs tested in our study, the principal compounds, carvacrol, and thymol, were followed by their biosynthetic precursors p-cymene and γ-terpinene, which, together with the main compound comprised more than 90% and 77% of the oregano and thyme oils. Their interaction within the tested EOs is presumable and might also contribute to the additive effects. This statement can be supported by Ultee et al. [74], who reported synergistic activity between carvacrol and cymene against Bacillus cereus, or by Delgado et al. [75], who found synergistic effect against the same bacterium when cymene was combined with thymol. The additive antimicrobial effect of carvacrol and thymol has already been previously reported in several studies against different bacteria, including S. aureus in liquid [35,76,77] as well as in the vapor phase [14]. However, further research focused on a better understanding of antimicrobial interactions between major and minor components, which was suggested to play an important role in the synergistic activity of EO gases [78] is warranted.
Although EOs of O. vulgare and T. vulgaris have acquired Generally Recognized as Safe (status from the Flavour and Extract Manufacturers Association and got approved by the US Food and Drug Administration (FDA) for safety food use [79,80]), there is limited published research on the safety of EO vapors per se [51]. As EOs are complex blends of components, individual volatile compounds need to be assessed as potential allergens. Currently, 26 ingredients that may trigger allergic reactions, including, e.g., linalool and limonene, are listed in the seventh amendment of directive 76/768 CEE (directive 2003/15/CE); however, these are all based on skin contact and not inhalation [81,82]. Regarding inhalation toxicity, which is a crucial aspect of inhalation administration, median lethal concentration (LC 50 ) values were determined neither for oregano nor for thyme EOs for the inhalation route. However, the data on their predominant compounds, carvacrol and thymol, might suggest their possible inhalation safety. The European Chemicals Agency reported that the LC 50 of carvacrol in rats was estimated to be greater than 20 mg/L when rats were treated with the given test chemical via inhalation route for 6 h exposure period. Similarly, the reported LC 50 value for thymol was 7.57 mg/L, when mice were exposed to a test chemical via inhalation by vapor for 2 h [83]. Furthermore, neither data from literature nor results from chronic toxicity studies presented in the study by Xie et al. (2019) [84] provide any evidence for chronic toxicity of inhaled thymol. In an acute oral toxicity study, the median lethal dose (LD 50 ) of carvacrol and thymol in rats was found to be 810 and 980 mg/kg of body weight (bw), respectively, and carvacrol-rich EO obtained from the leaves of Origanum spp. showed the oral LD 50 to be 1850 mg/kg bw; therefore, they are all classified as category 4 (H302) according to the Classification, Labelling and Packaging Regulation N • 1272/2008 and the Globally Harmonized System of Classification and Labelling of Chemicals [83] which means that it might be "harmful if swallowed". Moreover, thymol is FDA approved when used as a synthetic flavoring (21 CFR 172.515), a preservative and indirect food additive of adhesives [84], and is a common ingredient in many products such as perfumes, food flavorings, mouthwashes, pharmaceutical preparations, and cosmetics [85]. Similarly, carvacrol is generally considered safe for human consumption. It has been approved by FDA for its use in food and is included by the Council of Europe in the list of chemical flavorings that can be found in several food products, such as alcoholic beverages, baked goods, chewing gums, condiment relish, frozen dairy, gelatine puddings, non-alcoholic beverages, and soft candies [86]. Moreover, EO derived from T. vulgaris has been approved by the Committee on Herbal Medicinal Products of the European Medicines Agency as a traditional herbal medicinal product used for relief of cough associated with cold [87].
The above-mentioned data suggest a low toxicological risk of carvacrol and thymol administration through an inhalation route. Moreover, the rich historical evidence of culinary, medicinal, and pharmaceutical uses of O. vulgare and T. vulgaris could support their use as safe herbal medicinal products. Therefore, due to the considerable antimicrobial activity as well as the presumable safety of O. vulgare and T. vulgaris EOs, it can be assumed that the results of oregano and thyme EO combinations could be potentially applied in the development of various pharmaceutical applications that are based on volatile antimicrobials. These combinations could decrease the minimum effective doses of the agents, thus reducing their possible adverse effects and treatment costs. However, further research to achieve a better understanding of the action mechanisms, further in vivo experiments, and clinical trials on O. vulgare in combination with T. vulgaris are still necessary to determine their pharmacodynamics and pharmacokinetics.

Plant Material and Preparation of Essential Oils
The dried aerial parts of O. vulgare and T. vulgaris were purchased from a commercial supplier (U Salvatora, Prague, Czech Republic). Initially, they were homogenized by a Grindomix apparatus (GM100 Retsch, Haan, Germany). Subsequently, the residual moisture contents of both samples were determined gravimetrically at 130 • C for 1 h by Scaltec SMO 01 analyzer (Scaltec Instruments, Gottingen, Germany) in triplicate, and results were expressed as arithmetic averages according to the Official Methods of Analysis of the Association of Official Agricultural Chemists [88]. Both EOs were obtained by hydrodistillation of dried plant material in 1 L of distilled water using a Clevenger-type apparatus (Merci, Brno, Czech Republic) according to the procedure described in the European Pharmacopeia (2013) [89] and stored in sealed glass vials at 4 • C.

Bacterial Strains and Culture Media
In this study, 12 strains of S. aureus were used, including antibiotic-resistant and sensitive forms. Standard strains of the ATCC 25923, 29213, 33591, 33592, 43300, and BAA 976 were purchased from Oxoid (Basingstoke, UK) on ready-to-use bacteriological Culti-Loops. Clinical isolates (SA 1-6) obtained from Motol University Hospital (Prague, Czech Republic) were selected based on the previous antimicrobial susceptibility testing (data not shown) as representatives of methicillin-sensitive S. aureus (SA 1, SA 5, and SA 6) and methicillin-resistant S. aureus (SA 2, SA 3, and SA 4) strains and were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry as described in Rondevaldova et al. [90].
Mueller-Hinton (MH) broth was used as a cultivation medium, and both MH agar and MH broth purchased from Oxoid (Basingstoke, Hampshire, UK) were used as assay media. The pH of cation-adjusted MH broth was equilibrated to a final value of 7.6 with Trizma base (Sigma-Aldrich, Prague, Czech Republic). Stock cultures of bacterial strains were cultivated in broth medium at 37 • C for 24 h prior to the testing. The bacterial suspension's turbidity used for the inoculation of both plate and lid, was adjusted to 0.5 McFarland standard by Densi-La-Meter II (Lachema, Brno, Czech Republic) to reach the final concentration of 10 7 CFU/mL.

Antimicrobial Assay
The in vitro antibacterial combinatory potential of O. vulgare EO in combination with T. vulgaris EO in the liquid and vapor phase was determined using a broth volatilization checkerboard assay previously developed in our laboratory [17]. The method is based on the combination of classical microdilution checkerboard test and broth microdilution volatilization technique [16], allowing the determination of interactions between EOs and/or plant volatile agents simultaneously in liquid and vapor phase as well as comparison of MIC values and calculation of FICs in both liquid and solid media. Experiments were performed in white, 96-well immunoplates (total well volume = 400 µL) covered by tight-fitting lids with flanges designed to reduce evaporation (SPL Life Sciences, Naechon-Myeon, Korea). In the first part of the procedure, 30 µL of agar was pipetted into every flange on the lid (with the exception of the outermost wells) and inoculated with 5 µL of the bacterial suspension. Subsequently, both O. vulgare and T. vulgaris EOs were dissolved in DMSO and diluted in the broth medium to get the initial concentrations of 2048 µg/mL, with maximum DMSO content of 1%.
The preparation of plate assay and serial dilutions were performed by an automated pipetting platform, Freedom EVO 100, equipped with a four-channel liquid handling arm (Tecan, Mannedorf, Switzerland). In combinations, six two-fold serial dilutions of oregano EO from horizontal rows were subsequently cross-diluted vertically by six two-fold serial dilutions of thyme EO. The final volume in each well was 100 µL, except for the outermost wells, which were left empty to prevent edge leakage effect. The plates were subsequently inoculated by bacterial suspensions using a 96-pin multi-blot replicator (National Institute of Public Health, Prague, Czech Republic). Each plate also contained inoculated and noninoculated broth, which served as growth and sterility controls, respectively. Oxacillin was used as a positive antibiotic control for verification of susceptibility of S. aureus strains in broth medium. The DMSO assayed as the negative control at a concentration of 1% did not inhibit any of S. aureus strains tested either in broth or agar media. After the inoculation, clamps (Lux Tool, Prague, Czech Republic) were used to fasten the plate and lid together, with handmade wooden pads (size 8.5 × 13 × 2 mm) for better fixing, and microtiter plates were incubated for 24 h at 37 • C.
MIC values and combinatory effects in both liquid and the vapor phases (i.e., in plates and on lids) were evaluated by visual assessment of bacterial growth after coloring of metabolically active staphylococcal colonies with 25 µL of MTT dye in a concentration of 600 µg/mL when the interface of color in broth and on agar changed from yellow and purple (relative to that of colors in control wells). The MIC values were defined as the lowest concentration that visually inhibited staphylococcal growth compared to the compoundfree growth control and were expressed in µg/mL. The final MIC values presented in this work are the average of MICs obtained from three independent experiments that were performed in triplicate.
The combinatory effect of EOs was determined based on the value of ΣFIC.

GC/MS Analysis
For determination of the main components of O. vulgare and T. vulgaris EOs, GC/MS analysis was performed using the dual-column/dual-detector gas chromatograph Agilent GC-7890B system equipped with autosampler Agilent 7693, two columns (fused-silica HP-5MS column (30 m × 0.25 mm, film thickness 0.25 µm) and a DB-HeawyWAX column (30 m × 0.25 mm, film thickness 0.25 µm)) and a flame ionization detector (FID) coupled with a single quadrupole mass selective Agilent MSD-5977B detector (Agilent Technologies, Santa Clara, CA, USA). Operational parameters were helium as carrier gas at 1 mL/min, injector temperature 250 • C for both columns. The oven temperature was raised for both columns from 50 • C to 280 • C. Initially, after an isothermic period of 3 min, the heating rate was 3 • C/min until the temperature reached 120 • C. Subsequently, the heating velocity increased to 5 • C/min until it reached 250 • C; and after 5 min of holding time on 250 • C, the heating rate increased to 15 • C/min until it reached 280 • C. Heating was followed by an isothermic period of 20 min. Both EOs were diluted in n-hexane for GC/MS at a concentration of 20 µg/mL, and for quantitative analysis, 1 µL of methyl octanoate was added as an internal standard. One µL of each EO solution was injected in split mode (split ratio 1:50). The mass detector was set to the following conditions: ionization energy 70 eV, ion source temperature 230 • C, scan time 1 s, mass range 40-600 m/z.
Identification of constituents was based on a comparison of their retention indices (RI) and retention times (RT) and spectra with the National Institute of Standards and Technology Library ver. 2.0.f (NIST, USA) [91], as well as with authentic standards and literature [47][48][49][50][51][52][53][54][55][56]. The RI was calculated for compounds separated by both HP5-5MS and DB-HeawyWAX columns using the retention times of n-alkanes series ranging from C 8 to C 40 (Sigma-Aldrich, Prague, Czech Republic). For each analyzed EO, the final number of compounds was calculated as the sum of components simultaneously identified using both columns and the remaining constituents identified by individual columns only. Quantitative data were calculated according to Cachet et al. [70] using the following formula where m i is the mass of the compound i to be quantified, expressed in mg per 1 kg of plant dry weight (DWP); RRF Pred i -predicted relative response factor of compound i (calculated from the molecular formulae of the component using a methyl octanoate as internal standard), m MO -mass of methyl octanoate (internal standard, IS), Ai and A MO are the peak areas of the analyte and the IS, respectively, determined by the FID. Moreover, relative percentage contents of identified components have been determined using the FID data and indicated for both columns.

Conclusions
In summary, the present study reports the results of antistaphylococcal interactions between EOs obtained from O. vulgare and T. vulgaris that were tested by broth volatilization checkerboard assay. This combination of volatile oils exhibited additive effects against all 12 S. aureus strains in both liquid and vapor phases, whereas the best results in the liquid phase were obtained against methicillin-resistant strain (SA 2). To the best of our knowledge, this is the first report on interactions between O. vulgare and T. vulgaris EOs against S. aureus in the gaseous phase. In addition, the results presented in the form of isobologram, a graphical diagram enabling precise and intuitive judgment of the additive effect produced by EOs combination, validates the accuracy of broth volatilization checkerboard method for evaluation of the combinatory antimicrobial effect of EOs in the vapor phase. These results can potentially serve as a base for further research focused on the development of various pharmaceutical applications that are based on volatile microbials. However, since the MICs values obtained in the gaseous phase are only indicative and the real concentrations of evaporated EOs are lower, we believe that our results suggest a potential of thyme and oregano combination for application in the inhalation therapy against respiratory infections caused by S. aureus. However, further research focusing on in vivo evaluation will have to be carried out in order to verify its potential practical use.