Minimal Inhibitory Concentration Determination

Minimum inhibitory concentrations (MIC) were determined using a broth microdilution assay as previously described (Wiegand et al., 2008). Bacteria from an overnight culture grown at 37°C were diluted in fresh MHb to achieve a concentration of 1 x 10⁶ CFU/ml. A bacterial suspension (100 µl) was added to wells in a 96-well polypropylene microtiter plate that had been preloaded with serial dilutions of antimicrobial peptides in MHb (100 µl) giving a final bacterial concentration of 5 x 10⁵ CFU/ml. Microtiter plates were incubated at 37°C for 18–20 h before the MIC was determined as the lowest concentration of antimicrobial able to inhibit visible growth.

To determine the MIC toward 10⁸ CFU/ml (MIC_10^8), which was the bacterial concentration used in BioSAXS experiments, bacteria from an overnight culture were diluted 1:100 in fresh MHb and incubated in a shaking incubator at 37°C and 250 RPM until an OD₆₀₀ of 0.25 was reached, which equated to approximately 2 x 10⁸ logarithmically growing CFU/ml. The MIC was then performed as above without a further dilution of the culture. After 18–20-h incubations, 10 µl of a 500 µM resazurin solution (Sigma–Aldrich) were added to each well of the microtiter plate, and the cell viability was determined after a further 1-h incubation by the colorimetric reaction that occurs in the presence of viable cells.