Mueller hinton agar (mha)

In disc-diffusion method, Mueller-Hinton agar (VWR Chemicals, Radnor, PA, USA) was prepared and placed in Petri dishes. For E. coli and S. aureus inoculum of 1.5 × 10⁸ CFU (0.5 McFarland standard, BioMérieux SA, Marcy-l’Etoile, France) of bacteria/mL was used for completed seeding of the agar, and inoculum of 0.75 × 10⁸ and 3 × 10⁸ CFU of bacteria/mL was used for B. cereus and L. plantarum, respectively. Filter paper discs of 6.0 mm in diameter (Liofilchem, Roseto degli Abruzzi, Italy) were impregnated with several concentrations of infusion, decoction and essential oil, placed in the seeded agar and incubated at 37 °C for 24 h. For solvent control, DMSO 15% (solvent used for essential oil dissolution) was used. The diameter of the inhibition zone around the filter paper disc was measured in millimetres. All the determinations were performed in triplicate, and the results were expressed as mean ± standard deviation (SD).

The chemical compounds used for the antioxidant methods, the reagents for LOX and AChE inhibition assays and reference antibacterial and antifungal compounds were purchased from Sigma-Aldrich (Madrid, Spain). All compounds were of analytical grade (purity higher than 95%). All culture media were acquired from VWR Chemicals (Barcelona, Spain): Mueller-Hinton agar (MHA), Mueller-Hinton broth (MHB), Roswell Park Memorial Institute medium (RPMI-1640), Sabouraud dextrose agar (SDA), tryptic soy broth (TSB) and yeast peptone dextrose (YPD). Solvents of analytic grade and buffers were purchased from Merck (Madrid, Spain). Type I (18 MΩ·cm) deionized water (MilliQ-Reference, Millipore, Madrid, Spain) was used in this work.

The chemical compounds used for the antioxidant assays, the reagents for the LOX and AChE inhibition assays and reference antibacterial and antifungal compounds were purchased from Sigma-Aldrich Spain. All compounds were of analytical grade (purity higher than 95%). All culture media were acquired from VWR Chemicals Spain: Mueller-Hinton agar (MHA), Mueller-Hinton broth (MHB), Roswell Park Memorial Institute medium (RPMI-1640), Sabouraud dextrose agar (SDA), tryptic soy broth (TSB) and yeast peptone dextrose (YPD).
Solvents of analytic grade and buffers were purchased from Merck (Madrid, Spain). Type I (18 MΩ cm) deionized water (MilliQ-Reference, Millipore, Madrid, Spain) was used in this work.

Fifty-one strains from 40 patients plus 1 environmental sample were included in this study, for a total of 52 strains. The samples were collected from an Hospital Centre in northern Portugal, August–December 2020, using standard clinical operating procedures. Identification was performed by microbiology laboratories using conventional methods or automated systems such as MicroScan WalkAway (Beckman Coulter, Lisboa, Portugal)). Rectal swabs were seeded in a chromogenic culture medium CHROMagar™ mSuperCARBA™ (CHROMagar, Frilabo, Portugal). For further molecular characterisation, all strains were maintained frozen at −80 °C in BHI broth (VWR Prolabo, Lisboa, Portugal) plus 15% glycerol. For analysis purposes, the strains were grown overnight in BHI broth (18 h at 37 °C) and seeded on Mueller-Hinton agar (VWR, Lisboa, Portugal).

Rosmarinic acid, the 7-O-glucoside derivatives of apigenin, luteolin and eriodictyol, caffeic acid, 5-O-caffeoylquinic acid, 4-O-hydroxybenzoic acid, salvianolic acid B, ferulic acid, and 4-O-coumaric acid were obtained from Extrasynthese (Genay Cedex, France). Gallic acid, nisin, ascorbic acid, trolox, sulforhodamine B (SRB), DPPH^• (2,2-diphenyl-1-picrylhydrazyl) radical, acetic acid, ellipticine, trypan blue, trichloroacetic acid (TCA), Tris, lipopolysaccharide (LPS), nisin, and butylated hydroxyanisole (BHA) were obtained from Sigma Chemical Co (St Louis, MO, USA). Folin-Ciocalteu reagent, Na₂CO₃, formic acid and ethanol were purchased from Panreac (Barcelona, Spain). n-hexane, methanol and acetonitrile with high-performance liquid chromatography (HPLC) purity were purchased from Lab-Scan (Lisbon, Portugal). Fetal bovine serum (FBS), l-glutamine, trypsin-ethylenediaminetetraacetic acid (EDTA), penicillin/streptomycin solution (100 U/mL and 100 mg/mL, respectively), RPMI 1640 and DMEM media were obtained from Hyclone (Logan, Utah, UT, USA). The Griess reagent system was purchased from Promega Corporation (Madison, WI, USA). Mueller-Hinton agar was purchased from VWR (Prolabo Chemicals, West Chester, PA, USA). Purified water was obtained from a Direct-Q^® water purification system (Merck Life Science, Darmstadt, Germany).

For biological experiments, we used Mueller–Hinton Agar (VWR Chemicals, Prolabo, Ref.: 84686.0500), Staphylococcus aureus (Sa, CIP: ATCC 25923), vancomycin (Alfa Aesar, Ref.: J62790), DPPH (Sigma-Aldrich, Ref.: D9132), quercetin (Sigma-Aldrich, Ref.: Q4951), Artemia brine shrimp salt (JBL, Ref. 3090600), and Artemia brine shrimp eggs (Artemio Mix, JBL, Ref.: 3090600).
The cell lines were maintained with Dulbecco’s Modified Eagle’s Medium-high glucose (DMEM-HG) (Biowest, Nuaillé, France) supplemented with 10% (v/v) of heat-inactivated fetal bovine serum (FBS) (Sigma, St. Louis, MO, USA) and 1% (v/v) of penicillin-streptomycin (Sigma, St. Louis, MO, USA), stock solution of Resazurin salt dye at a concentration of 0.1 mg/mL.

Candida albicans ATCC 14,053 and C. glabrata ATCC 15,126 reference strains were employed to assess the antifungal activity. Clotrimazole (10 μg) antifungal effects of EO and EO-b-CD were evaluated as reported in the Clinical and Laboratory Standards Institute Standard M44-A. Mueller–Hinton Agar (VWR chemicals, Milan, Italy) added with 2% Glucose and 0.5 μg/mL Methylene Blue Dye (GMB) was used as medium. Briefly, strain suspensions (10⁶ CFU ml⁻¹) were swabbed on the medium surface, filter paper discs (diameter of 6 mm) were placed on the surface and added with 10 μL of the EO. The positive control was clotrimazole (10 μg). Negative controls were 1,4 Dioxane (Sigma-Aldrich, St. Louis, MO, USA; 10 μL) and organic linseed oil (10 μL) discs. Triplicate experiments were performed by incubating plates at 37 °C for 48 h. The sensitivity test for the extract is considered positive if the inhibition halo is higher than that induced by clotrimazole (positive control ≥ 100%).