Bdsensi disc

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 antimicrobial susceptibility profiles were determined by disk diffusion and agar dilution methods [10 ]. Nine antibiotics were used as follows: ampicillin (10 µg), vancomycin (30 µg), teicoplanin (30 µg), erythromycin (15 µg), ciprofloxacin (5 µg), chloramphenicol (30 µg), linezolid (30 µg), gentamicin (120 µg), and streptomycin (300 µg) (BD Sensi-disc, Becton–Dickinson, Mannheim, Germany). Tetracycline was diluted to minimum inhibitory concentrations (MICs) of 0.06–256 μg/ml to determine the degree of the tetracycline resistance in the isolates, and the data were interpreted according to CLSI guidelines [10 ]. Staphylococcus aureus ATCC 25923, E. faecalis ATCC 29212, and E. faecalis ATCC 51299 were used as control strains for the disk diffusion and MIC tests, respectively.