Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to examine the ultrastructural changes in bacteria induced by antimicrobial peptides (AMPs). Both the beta-stranded gramicidin S and the alpha-helical peptidyl-glycylleucine-carboxyamide (PGLa) are cationic amphiphilic AMPs known to interact with bacterial membranes. One representative Gram-negative strain, Escherichia coli ATCC 25922, and one representative Gram-positive strain, Staphylococcus aureus ATCC 25923, were exposed to the AMPs at sub-MICs and supra-MICs in salt-free medium. SEM revealed a shortening and swelling of the E. coli cells, and multiple blisters and bubbles formed on their surface. The S. aureus cells seemed to burst upon AMP exposure, showing open holes and deep craters in their envelope. TEM revealed the formation of intracellular membranous structures in both strains, which is attributed to a lateral expansion of the lipid membrane upon peptide insertion. Also, some morphological alterations in the DNA region were detected for S. aureus. After E. coli was incubated with AMPs in medium with low ionic strength, the cells appeared highly turgid compared to untreated controls. This observation suggests that the AMPs enhance osmosis through the inner membrane, before they eventually cause excessive leakage of the cellular contents. The adverse effect on the osmoregulatory capacity of the bacteria is attributed to the membrane-permeabilizing action of the amphiphilic peptides, even at low (sub-MIC) AMP concentrations. Altogether, the results demonstrate that both TEM and SEM, as well as appropriate sample preparation protocols, are needed to obtain detailed mechanistic insights into peptide function.
