3.7. Determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC)

The antimicrobial activity of free and encapsulated peptide was evaluated by the broth micro dilution method described in previous works [18,49]. Briefly, a pre-inoculum of E. coli O157:H7 in LB and MRSA in MH were grown at 37 °C for 12 h at 200 rpm; then, the culture of each strain was set at 0.5 in a McFarland scale (10⁸ CFU ml⁻¹); 100 μL aliquots of these cell suspensions were mixed with 100 μL of AMP-NPs or empty NP solutions at different concentrations in a 96 well microplate and incubated at 37 °C in an orbital shaker (200 rpm for 8 h). The bacterial growth kinetics of these microbial cultures was performed by measuring changes of absorbance at 595 nm over time in an Elisa reader for these cultures (Bio-Rad, iMark). The controls were: (1) culture media, (2) culture media plus bacteria, (3) culture media, bacteria and ofloxacin and (4) culture media, bacteria and empty NPs. MIC₅₀ was defined as the lowest concentration of AMP-NPs inhibiting 50% of the bacterial growth of these bacterial strains. After incubation for 8 h, 100 μL of these bacterial cultures were poured in 900 μL of BHI, incubated at 37 °C for 24 h; then, 10 μL of these cultures were seeded over BHI-agar petri dishes, incubated for 1 day at 37 °C and the appearance of colonies were determined. The MBC was the lowest nanoparticle concentration producing >99.9% reduction of colony-forming units (CFU).