MICs were determined by the microwell dilution method, as described by Wiegand et al. (62) (Protocol E: Broth microdilution for AMPs that do not require the presence of acetic acid/bovine serum albumin). This method assays growth using optical density measurements (λ = 625 nm). This method was used to determine the efficacy of our peptides against a panel of 30 type cultures (table S2).

Most of our clinical isolates displayed mucoid or plaque morphologies, and growth could not be assayed using optical density readings. Instead, a modified protocol using resazurin was used to determine the efficacy of our peptides against 64 clinical isolates (Table 2). Resazurin, a weakly fluorescent dye, is reduced to fluorescent resorufin in direct proportion to microbial aerobic respiration. Microbial cultures were incubated at 37°C for 12 hours in 96-well polypropylene plates. Thirty microliters of a 0.02% (w/v) aqueous resazurin solution was then pipetted into each well. Further, incubation at 37°C was performed for 12 hours. Growth estimation was then performed on the basis of fluorescence (λexcitation, 530 nm; λemission, 590 nm). Because aerobic respiration also occurs in bacteriostatic cultures, the lack of aerobic respiration indicates bactericidal activity. Therefore, the resazurin assay measures the MBC.

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