Outer-membrane permeability test

The integrity of the outer-membranes was analyzed measuring the 1-N-phenylnaphthylamine (NPN) uptake after the treatment with the peptide. The outer-membrane acts as a permeability barrier that excludes hydrophobic compounds as NPN. If the outer-membrane is altered, the dye can enter reaching the phospholipid layer which results in a prominent fluorescence⁶⁰. Briefly, the peptides and/or antibiotics were placed in a 96-well plate at 2× the desired final concentration in HEPES buffer plus glucose (GHEPES) (5 mM HEPES, 5 mM glucose). Meantime, the Gram-negative bacteria were grown in LB until an OD₆₀₀ of 1. After that, they were washed twice in GHEPES buffer and 2-fold diluted in the same buffer. NPN was dissolved in acetone and added to the cells at 30 µM final concentration, and after that, the bacteria were distributed in the 96 wells plates previously prepared with the antimicrobials at a final OD₆₀₀ of ~0.25. Immediately the fluorescence for the dye was measured at an excitation/emission range of 350/420 nm every 10 min during 1 h. Polymyxin B was used as a positive control and non-treated cells as negative. All the tests were performed in triplicate.