4.4. Minimum Inhibitory Concentration (MIC) Determination

The minimum inhibitory concentration was determined by the broth microdilution method according to the NCCLS protocol. Briefly, an overnight bacterial or fungal culture was diluted in fresh MH or Sabouraud broth, respectively (300 µL O/N culture in 10 mL MHB/Sabouraud) and the culture was let grow at 37 °C or 30 °C to reach OD600 between 0.3 and 0.5. Serial two-fold dilutions of peptides were then performed in culture medium (Sabouraud, MHB or 20% MHB in PBS), to a final volume of 50 µL in the wells of a round bottom microtiter plate (Sarstedt, Milan, Italy), and then the bacterial suspension with a load of 5 × 10⁵ CFU/mL was added to the plate, halving the final concentration of bacteria and peptides. The culture was grown at 37 °C or 30 °C and the MIC was determined after 18h by visual inspection as the first clear well. The assessment was performed taking into consideration at least three independent experiments performed in duplicate.

Before the permeabilization assays, the MIC of PrAMPs and colistin against E. coli ML35p was determined with the same method but using a bacterial suspension with a load of ~2 × 10⁷ CFU/mL (=1 × 10⁶ CFU/well), so as to match the bacterial density of the permeabilization assays.