Erythromycin

Teicoplanin, vancomycin, oritavancin, and chloramphenicol (Sigma-Aldrich, Germany); dalbavancin (MedChemExpress, Sweden); MA79 (Csávás et al., 2015 (link)), ERJ390 (Pintér et al., 2009 (link)), and SZZS-12 (Szucs et al., 2017 (link)); carbenicillin, gentamicin and erythromycin (Duchefa Biochemie, Netherland); vancomycin BODIPY-FL conjugate (Thermo Fisher Scientific, Germany) and fluorescently labeled Teicoplanin (Vimberg et al., 2019 (link)).

The disc diffusion method was implemented as described by The European Committee on Antimicrobial Susceptibility Testing [23 ] with some modifications, to test the susceptibility of L. monocytogenes to the EOs. The test was performed by applying a 100 µL bacterial inoculum of approximately 1–2 × 10⁸ CFU mL⁻¹ to the surface of a Mueller–Hinton agar plate (M.H, Scharlab, Barcelona, Spain). The inoculum was allowed to dry for 15 min, then three sterile disks of 6 mm (BDsensi-disc, Becton Dickinson GMBH, Heidelberg, Germany) were placed on the inoculated agar surface. One was impregnated with 10 µL of one of the EOs, which represents a concentration of 50 mg mL⁻¹, another with 10 µL of ethanol, which represented the negative control, and the last one with erythromycin (Duchefa Biochemie, Haarlem, The Netherlands), which was dissolved in 95% ethanol and then added (15 µg/disc) and represented the positive control. The plates were left 15 min at room temperature to allow the diffusion of the compounds, and then they were incubated for 24 h at 35 ± 1 °C. After incubation, the zones of growth inhibition around each of the disks were measured in millimeters. The diameter of the zone was related to the susceptibility of the strain to the EO. The experiments were carried out in triplicate.

The minimum inhibitory concentration (MIC) was evaluated using the broth dilution technique for strains used in the assessment of antibiotic efficiency. Microdilution plates containing 200 µL BHI broth medium (Oxoid) with decreasing concentration of antibiotics (erythromycin, Duchefa Biochemie; ampicillin, Melford Biolaboratories Ltd; tetracycline, Sigma; 256–0.125 µg/mL) were inoculated from overnight cultures. After overnight incubation, cultures were checked for growth, with the MIC being the lowest concentration of antibiotics that prevents visible growth.