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 β-stranded gramicidin S and the α-helical peptidyl-glycylleucine-carboxyamide (PGLa) are cationic amphiphilic AMPs known to interact with bacterial membranes. One representative Gram-negative strain,Escherichia coliATCC 25922, and one representative Gram-positive strain,Staphylococcus aureusATCC 25923, were exposed to the AMPs at sub-MICs and supra-MICs in salt-free medium. SEM revealed a shortening and swelling of theE. colicells, and multiple blisters and bubbles formed on their surface. TheS. aureuscells 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 forS. aureus. AfterE. coliwas 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.

Silver nanoparticles, as well as antimicrobial peptides (AMPs), can be used to fight infectious diseases. Since AMPs are known to permeabilize bacterial membranes and might therefore help silver nanoparticles to access internal target sites, we investigated their combined activities and showed synergistic effects between polymyxin B and silver nanoparticles for gram-negative bacteria.

Multidrug-resistant Pseudomonas aeruginosa is a major cause of severe hospital-acquired infections. Currently, polymyxin B (PMB) is a last-resort antibiotic for the treatment of infections caused by Gram-negative bacteria, despite its undesirable side effects. The delivery of drug combinations has been shown to reduce the required therapeutic doses of antibacterial agents and thereby their toxicity if a synergistic effect is present. In this study, we investigated the synergy between two cyclic antimicrobial peptides, PMB and gramicidin S (GS), against different P. aeruginosa isolates, using a quantitative checkerboard assay with resazurin as a growth indicator. Among the 28 strains that we studied, 20 strains showed a distinct synergistic effect, represented by a fractional inhibitory concentration index (FICI) of ≤0.5. Remarkably, several clinical P. aeruginosa isolates that grew as small-colony variants revealed a nonsynergistic effect, as indicated by FICIs between >0.5 and ≤0.70. In addition to inhibiting the growth of planktonic bacteria, the peptide combinations significantly decreased static biofilm growth compared with treatment with the individual peptides. There was also a faster and more prolonged effect when the combination of PMB and GS was used compared with single-peptide treatments on the metabolic activity of pregrown biofilms. The results of the present study define a synergistic interaction between two cyclic membrane-active peptides toward 17 multidrug-resistant P. aeruginosa and biofilms of P. aeruginosa strain PAO1. Thus, the application of PMB and GS in combination is a promising option for a topical medication and in the prevention of acute and chronic infections caused by multidrug-resistant or biofilm-forming P. aeruginosa .

Phenotype instability of bacterial strains can cause significant problems in biotechnological applications, since industrially useful properties may be lost. Here we report such degenerative dissociation forAneurinibacillus migulanus(formerly known asBacillus brevis) an established producer of the antimicrobial peptide gramicidin S (GS). Phenotypic variations within and between various strains maintained in different culture collections are demonstrated. The type strain, ATCC 9999, consists of six colony morphology variants, R, RC, RP, RT, SC, and SP, which were isolated and characterized as pure cultures. Correlations between colony morphology, growth, GS production, spore formation, and resistance to their own antimicrobial peptide were established in this study. We found the original R form to be the best producer, followed by RC, RP, and RT, while SC and SP yielded no GS at all. Currently available ATCC 9999^Tcontains only 2% of the original R producer and is dominated by the newly described phenotypes RC and RP. No original R form is detected in the nominally equivalent strain DSM 2895^T(=ATCC 9999^T), which grows only as SC and SP phenotypes and has thus completely lost its value as a peptide producer. Two other strains from the same collection, DSM 5668 and DSM 5759, contain the unproductive SC variant and the GS-producing RC form, respectively. We describe the growth and maintenance conditions that stabilize certain colony phenotypes and reduce the degree of degenerative dissociation, thus providing a recommendation for how to revert the nonproducing smooth phenotypes to the valuable GS-producing rough ones.