2.2.2. Determination of the Antibacterial Susceptibility by Agar Diffusion

The agar diffusion test was performed following the method of Fratianni et al. [18] with some modifications. Microbial suspensions (1 <sup>×</sup> 107 colony-forming units (cfu)/mL) were spread on LB agar plates in sterile conditions. Different amounts of extracts (2.5 and 4.9 μg) were spotted on the inoculated plates. After 10 min in sterile conditions, plates were incubated at 37 ◦C for 24 h. The diameter of the clear zone shown on plates (inhibition zone) was accurately measured ("Extra steel Caliper mod 0289", mm/inch reading scale, precision 0.05 mm, Mario De Maio, Milan, Italy). Sterile dimethylsulfoxide (DMSO, Sigma Aldrich Italy, Milano, Italy) and tetracycline (7 μg; Sigma Aldrich Italy) served as the negative and positive control, respectively. The experiments were performed in triplicate and averaged.
