The screening of inhibitory activity was studied by the agar spot test described by Fleming et al. [16], with modifications, that involved culture of the strain SMN 33.6 on nutritive agar, in a tip shaped form until the growth reached 2–4 mm, then a second layer of LB agar (10 ml) with 50% agar was added, inoculated with 100 μl of the pathogenic bacterial strain to test, in exponential growth phase. The plates were incubated at 37°C for 24 h. The antibacterial activity was evaluated by the presence of an inhibition zone around the colony of the strain SMN 33.6.
The minimum inhibitory concentration (MIC) was evaluated with a standard method of serial microdilution in Mueller–Hinton broth (MH) using microtiter plates. Each assay was performed in triplicate and the value repeated at least two times was considered as the MIC. The bacterial inoculum used was 5 × 105 UFC ml- 1, obtained from a 0.5 McFarland turbidity standard (1.5 × 108 UFC ml- 1). Microtiter plates were incubated at 35°C for 24 h and MIC was registered as the lowest concentration without turbidity. The minimum bactericidal concentration (MBC) was determined, transferring broth from wells without growth to a MH agar plate without extract and incubating at 35°C for 24 h.