Determination of antibacterial activity

In order to study the antimicrobial properties of S. montana hydrolate and its main volatile compound, carvacrol, the minimum inhibitory concentrations (MICs) were obtained, using the broth microdilution method in round bottom 96-well plates, according to the Clinical and Laboratory Standards Institute guideline (SCLI, M07-A9 2018) and ISO 207776-1 (2019) for bacteria, and (SCLI, M27-A3, 2017) for C. albicans, as follows.

As carvacrol is a water-insoluble compound, ethanol, DMSO and polysorbate 80 were used to prepare the stock solutions for the subsequent antimicrobial tests.

The toxicity of the solvents on bacteria was examined by the microdilution method in a 96-well plate, obtaining the maximum innocuous concentration (MInC) to ensure that the concentrations used for the tests were completely harmless to the microorganisms. Carvacrol was dissolved depending on the bacterial strain used in each experiment, to achieve maximum solubility without affecting the bacteria. The table in Support Information ¹ indicates which solvent was used to dissolve carvacrol in each test, depending on the type of bacteria. Prior to the antifungal and antibacterial analysis, all stock solutions were sonicated using an Ultrasons, J.P. SELECTA® analogue ultrasonic bath until a homogeneous and stable phase was obtained. The hydrolate (completely water soluble) was diluted directly in the corresponding culture broth.

For the tests, 100 µL of carvacrol stock solution or 100 µL of the hydrolate and 100 µL of culture broth were added to each well of the first column of the 96-well plate. Serial twofold dilutions were then applied from column 1 to 10, giving a final volume of 100 µL in each well and resulting in a concentration range between 4 and 2000 µg/mL for carvacrol and between 0.1 and 50% for the hydrolate. A positive control (wells with broth and inoculum but without testing product) and negative control (wells without inoculum and without testing product) were also included in each experiment, in columns 11 and 12, respectively. Bacteria were revived from the cryovials 24 h before the experiment, using the broth and conditions specified in Support Information¹. Fifteen minutes before inoculating the plates, the bacterial cultures were adjusted to the McFarland standard (SLCI, 2018), by spectrophotometry at 625 nm wavelength, to achieve an approximate bacterial concentration of 5 × 10⁸ CFU/mL. The cultures were then diluted 1:20 with broth, and 10 µL were inoculated into each well, except in those designated as negative controls, to reach an approximate working concentration of 2.5 × 10⁵ CFU/mL.

The C. albicans inoculum was also adjusted to the 0.5 Mcfarland standard by spectrophotometry at 520 nm wavelength, to achieve a concentration of approximately 1 × 10⁶ CFU/mL (according SCLI M27-A3). It was then diluted 1:100 with Saboraud broth, and 10 µL from this last dilution were inoculated into each well, to reach an assay concentration of 1 × 10³ CFU/mL.

The plates were incubated for 18–20 h at the optimal growth temperature for each organism in an Incuterm, Trade Raypa®, bacteriological culture Incubator and an ERI 180 DBO, Equitec®, refrigerated incubator. The pH of the wells was measured before and after incubation to ensure that they remained in the optimal range for each microorganism, according ATCC specifications (see Support Information ¹). All processes relating to the culturing, handling and inoculation of bacterial strains and the preparation of reagents were carried out under sterile conditions in a class II laminar flow biological safety hood (Model MSC Advantage 1.2).

After incubation, the MIC was considered as the lowest concentration that inhibited visible microbial growth according to SCLI, M07-A9 2018⁵⁹. To achieve a more accurate quantification of the growth, the optical density of each well was also measured at 625 nm with a BioTek™ Synergy H1 Hybrid Multi-Mode Microplate Reader.

The minimum bactericidal concentration (MBC) and minimum fungicidal concentration (MFC) were evaluated^60,61 considering that both values indicated the lowest concentration at which all microorganisms (bacteria and fungus, respectively) were killed. For their determination, an aliquot of 10 µL from each column in which there was no growth of the incubated 96-well plates was taken and inoculated onto an agar plate. The cultures were incubated for 24 h at the optimal growth temperature for each bacterial strain (see Support Information ¹) and observed to see whether any growth had occurred.

The MBC/MIC (Minimum Bactericidal Concentration/Minimum Inhibitory Concentration) ratio determines the bactericidal or bacteriostatic effect of the product on the strains. In concordance with Adrar et al.⁶², a natural product was considered to have bactericidal activity when MBC/MIC ≤ 4.

The values from the inhibition curve obtained from the micro-broth dilution test were used to calculate the LC₅₀ and LC₁₀ values (the concentration that causes the death of 50% or 10%, respectively, of a group of tested organisms).