2.1 Essential oils and their components

The EO of thyme (batch number H3981/1509), clove (batch number H7352/1602), and peppermint (batch number H7101/1601) were obtained from a Hungarian company (AROMAX Zrt., Budapest, Hungary). The main components of the EOs (thymol, eugenol, and menthol) were bought from Sigma-Aldrich (Budapest, Hungary).

2.2 gas chromatography‒mass spectrometry (GC‒MS) and Gas chromatography‒flame ionization detection (GC‒FID)

The chemical and the percentage compositions of the EO samples were determined with GC‒MS. The parameters were the same as described in our previous paper [8]. An Agilent 6890N/5973N GC-MSD (Santa Clara, CA) system equipped with an Agilent SLB-5MS capillary column (30 m × 250 µm × 0.25 µm) was used. An aliquot of 10 µL EO was diluted in 1 mL ethanol, and from this solution, 1 µL was injected in split mode (the split ratio was 1:50). The injector temperature was 250 °C. The oven temperature was increased at a rate of 8 °C/min from 60 °C (3 min isothermal) to 250 °C, with a final isotherm at 250 °C for 1 min. High purity helium was used as the carrier gas (1.0 mL/min and 37 cm/s, in constant flow mode). The mass selective detector (MSD) was equipped with a quadrupole mass analyzer and was operated in electron ionization mode at 70 eV in full scan mode (41–500 amu at 3.2 scan/s). The data were evaluated using MSD ChemStation D.02.00.275 software (Agilent). The identification of the compounds was carried out by comparing retention times, linear retention indexes, and recorded spectra with the data of authentic standards, and the NIST 2.0 library was also used. Gas chromatography‒flame ionization detection (GC‒FID) was made using a Fisons GC 8000 gas chromatograph (Carlo Erba, Italy). An Rt-β-DEXm (Restek) capillary column, 30 m × 0.25 mm i.d., 0.25 μm film thickness, was used. The carrier gas was nitrogen at 6.8 mL/min flow rate. A 0.2 mL volume of a 0.1% solution of the oil was injected (1 μL EO in 1 mL chloroform). The splitless injection was carried out. The temperatures of the injector and detector were 210 and 240 °C, respectively. The oven temperature was increased at a rate of 8 °C/min from 60 to 230 °C, with a final isotherm at 230 °C for 5 min. Identification of peaks was made by retention data compared with data obtained by GC‒MS and data of standards (Fluka Analytical and Sigma-Aldrich); percentage evaluation was carried out by area normalization. Three parallel measurements were made, and relative standard deviation (RSD) percentages were below 4.5%.

2.3 Microbiological tests2.3.1 Cultivation of test bacteria

In our microbiological experiments, the following strains were used: Salmonella minnesota Re595 was kindly supplied by O. Lüderitz (Freiburg, Germany). The Escherichia coli ReD31m4 strains were kindly supplied by N. Kato, T. Hasegawa (Nagoya University, Japan). Other microorganisms were isolated from our experiments when we studied the cell wall structure of Shigella sonnei strains, for example, S. sonnei phase I (this is a smooth “S” form) and phase II 4303 (this is a rough “R” mutant) [9] and S. sonnei R4341, R4350, and R4351 (these are “R” mutants). Endotoxins were isolated from S. sonnei rough mutants as well. The S. sonnei 4303 strain is a phase II type and is a spontaneous mutant of S. sonnei phase I [9]. After ethyl methanesulfonate treatment of S. sonnei 4303, we could isolate a great number of rough mutants, and we characterized these mutants by core specific phages [10]. The bacteria of these groups were maintained and cultivated in Mueller‒Hinton broth.

2.3.2 Sugar content of core region of “S” form and “R” mutant strains

Phenol‒water method was used for extraction of LPS of S. sonnei phase I (smooth mutant) after Westphal [11]. A phenol‒chloroform‒petroleum ether method was suitable to isolate the endotoxins of rough mutants (R, LPS and LOS, lipooligosaccharide) after Galanos [12] and then was precipitated and freeze-dried. Determination of monosaccharide components in core region of S and R endotoxin was performed with the following method: the LPSs and LOSs were hydrolyzed by sulfuric acid. and the monosaccharides were identified in an alditol acetate form by GC [13]. Table 1 presents these data. R mutant strains were maintained at the Department of Medical Microbiology and Immunology, University of Pécs (Hungary). In the case of the DB assay, bacteria were grown in 100 mL Mueller‒Hinton broth (Sigma Aldrich Ltd., Darmstadt, Germany) at 37 °C in a shaker incubator (at 60 rpm speed for 24 h) [14]. The bacterial suspension was diluted with fresh nutrient broth to an optical density at 600 nm (OD600) of 0.4, which corresponds to approximately 4 × 107 colony-forming units (cfu)/mL.

Table 1 Sugar content of core region of “S” form and rough “R” mutant strains2.3.3 Determination of minimum inhibitory concentration (MIC)

Macrotube dilution methods were used to detect the minimum inhibitory concentrations (MICs) of the test strains to synthetic antibiotics (cephalexin, gentamicin, tobramycin, and erythromycin). The MIC value is the lowest concentration of antibiotics could kill the 90% of test bacteria [15]. The data of results are presented in Table 2.

Table 2 Minimum inhibitory concentration (MIC) values of smooth (“S”) and rough (“R”) Gram-negative strains depend on the cell wall structure of bacteria and chemical composition of antimicrobials2.3.4 Thin-layer chromatography

For bioautography 5 cm × 10 cm silica gel 60 F254 aluminum sheet TLC plates (Merck, Darmstadt, Germany) were used. The positive control antibiotics, cephalexin and gentamicin (see in chapter 2.3.5), were tested with DB. After this experiment, the antibacterial effect of thyme (Thymus vulgaris L., Lamiaceae), clove (Syzygium aromaticum (L.) Merr. & L. M. Perry, Myrtaceae), and peppermint (Mentha × piperita L., Lamiaceae) EOs was investigated against the most sensitive rough bacterium S. minnesota Re595 and against the least sensitive rough mutant E. coli D31m4. Ethanol solutions (100 µL oil in 500 mL absolute ethanol) of EOs were prepared. From these solutions, 0.2, 0.4, 0.5, and 1.0 µL were applied to the TLC plate. The antibacterial activity of the main EO components (thymol, eugenol, and menthol) were also investigated by TLC‒DB. Ethanol solutions of thymol, eugenol, and menthol (20 mg/mL) were made, and an aliquot of 0.5 µL (10 µg) of the stock solution was applied to the TLC plates. The position of the starting line was 1.5 cm from the bottom and 1.5 cm from the left side. The mobile phase toluene‒ethyl acetate (95:5, V/V) was used for chromatographic separation [16]. Ascendant development in a saturated twin-trough chamber (CAMAG, Muttenz, Switzerland) was performed at room temperature (20 °C). Then, the TLC plates were dried at 90 °C for 5 min to remove the solvent completely. The visualization of the separated compounds was detected by ethanolic vanillin‒sulfuric acid reagent [5]. The identification of the separated compounds was done based on retardation factor (RF) values, color of the standards, and under ultraviolet (UV) light at 254 nm. The photos of the plates were done with digital camera (Lumix DMC-LX100, Panasonic, Osaka, Japan). It should be highlighted that the TLC plates prepared for bioautography were not treated with vanillin‒sulfuric acid, because this step interferes with the microbiological steps in bioautography.

2.3.5 Direct bioautography

The positive controls were cephalexin (0.5 mg/mL, Hungaro-Gal Kft. Kaposvár, Hungary) and gentamicin (Gentamicin 40 mg/mL, Sandoz Hungary Kft., Hungary) antibiotics. These antibiotics were dissolved in sterile water. From the cephalexin solution, 0.2, 0.4, 0.8, and 1.6 µL were applied to the 5 cm × 10 cm silica gel adsorbent with automatic pipette (Merck, Darmstadt, Germany). An aliquot of 0.2 µL from gentamicin was applied to the TLC plate. Motic Images Plus 2.0 software (Motic, Hong Kong, China) was used to measure the inhibitory zones (expressed in mm) produced by cephalexin and gentamicin. In this case, chromatographic separation was not done. In the case of DB with antibiotics or with EO samples, the TLC plates were dipped into a 100 mL of bacterial suspension to assure a homogeneous distribution and adhesion of bacteria onto the surface of the layers. Immersion time was 10 s, and then, the layers were placed into a low-wall horizontal chamber (chamber dimension: 20 cm × 14.5 cm × 5 cm) and incubated at 37 °C for 2 h. Then, the TLC plates were immersed into the aqueous solution of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, 50 mg/85 mL) (Sigma Aldrich Ltd., Darmstadt, Germany) using CAMAG immersion device for 5 s to visualize the antibacterial spots on the silica plates. Incubation of the layers (37 °C for 4 h) was the next step. The metabolically active bacteria can convert the tetrazolium salt, MTT, into formazan dye. Inhibition zones as white spots will appear against the bluish-violet background if there is no dehydrogenase activity on the plate due to the antibacterial activity of the tested EOs or their main compounds [17, 18]. The inhibition zones (expressed in mm) of the tested antibiotics without separation were measured with Motic Images Plus 2.0 program (ver. 2.0., Motic, Hong Kong, China). The experiments were prepared in six parallel repetitions.