In situ and real time investigation of the evolution of a Pseudomonas fluorescens nascent biofilm in the presence of an antimicrobial peptide.

Against the increase of bacterial resistance to traditional antibiotics, antimicrobial peptides (AMP) are considered as promising alternatives. Bacterial biofilms are more resistant to antibiotics that their planktonic counterpart. The purpose of this study was to investigate the action of an AMP against a nascent bacterial biofilm. The activity of dermaseptin S4 derivative S4(1-16)M4Ka against 6 h-old Pseudomonas fluorescens biofilms was assessed by using a combination of Attenuated Total Reflectance-Fourier Transform InfraRed (ATR-FTIR) spectroscopy in situ and in real time, fluorescence microscopy using the Baclight™ kit, and Atomic Force Microscopy (AFM, imaging and force spectroscopy). After exposure to the peptide at three concentrations, different dramatic and fast changes over time were observed in the ATR-FTIR fingerprints reflecting a concentration-dependent action of the AMP. The ATR-FTIR spectra revealed major biochemical and physiological changes, adsorption/accumulation of the AMP on the bacteria, loss of membrane lipids, bacterial detachment, bacterial regrowth, or inhibition of biofilm growth. AFM allowed estimating at the nanoscale the effect of the AMP on the nanomechanical properties of the sessile bacteria. The bacterial membrane elasticity data measured by force spectroscopy were consistent with ATR-FTIR spectra, and they allowed suggesting a mechanism of action of this AMP on sessile P. fluorescens. The combination of these three techniques is a powerful tool for in situ and in real time monitoring the activity of AMPs against bacteria in a biofilm.

In vitro activity of dermaseptin S4 derivatives against genital infections pathogens.

OBJECTIVES: Sexually transmitted infections present a great risk to the reproductive health of women. Therefore, female-controlled vaginal products directed toward disease prevention are needed urgently. In the present study, efforts were made to evaluate the antimicrobial potency of dermaseptin DS4, an antimicrobial peptide derived from frog skin. To assess the structure-activity relationship between the native DS4 and their derivatives, a set of chemically modified peptides was synthesized and evaluated. METHODS: Different strains of bacteria and fungi were used to detect the antimicrobial activity of the new compounds. HeLa cultures were used to determine the safety of compounds towards their toxicity. RESULTS: All DS4 derivatives elicited concentration-dependent antimicrobial activity at micrograms concentrations (MIC values: 4 microg/mL-l mg/mL). K4S4(1-28) and K4S4(1-16)a were the most active peptides whereas S4(6-28) was the less one. The order was K4S4(1-28)>K4S4(1-16)a>S4a>S4>S4(6-28). In cytotoxicity assay, some compounds were found to be significantly safer than current antibiotics. Our data also show that increasing the number of positive charges of the peptide resulted in a reduced cytotoxicity without affecting the antimicrobial effect. CONCLUSIONS: This study indicates that dermaseptins are antimicrobial molecules that deserve to be tested as topical microbicide with useful associated activities that can add to their prophylaxis, safety and effectiveness.