S2 Table. Numerical values of Minimum Inhibitory Concentration.

The Ib-M1 peptide exhibits bactericidal activity against Escherichia coli O157:H7 and low toxicity in mammalian cells. The present study aimed to evaluate the effect of Ib-M1 on E. coli O157:H7 membrane permeabilization. To this end, the minimum inhibitory concentrations of Ib-M1 for E. coli O157:H7 and ML35 were measured. The permeability of the E. coli outer and inner membranes was determined by measuring N-phenyl-1-naphthylamine and O-nitrophenyl-β-galactosidase hydrolysis, respectively after bacterial exposure to antimicrobial peptides and control antibiotics. Morphological changes in antimicrobial-exposed E. coli O157:H7 were evaluated by scanning electron microscopy following treatment with antimicrobial peptides. Ib-M1 demonstrated inhibitory activity against E. coli O157:H7 and ML35 at MICs of 2.9±1.7 and 6.3±0 μM, respectively. The peptide induced permeabilization of the outer membrane of E. coli O157:H7 at all concentrations evaluated, and the permeabilization of the inner membrane after 50 minutes at concentrations between 1xMIC and 8xMIC. The morphological changes induced by Ib-M1 led to significant alterations in bacterial shape including collapsed cells, and pronounced surface roughness and invaginations. In conclusion, physiological and morphological evidence indicate that the Ib-M1 antimicrobial effect against E. coli O157:H7 is mediated by its permeabilizing action on the outer and inner bacterial membranes.