title: In situ and real time investigation of the evolution of a Pseudomonas fluorescens nascent biofilm in the presence of an antimicrobial peptide.
abstract: 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.
authors.affiliation: CNRS, Laboratoire de Chimie Physique et Microbiologie pour l'Environnement, LCPME, UMR 7564, Villers-lès-Nancy, F-54600, France; Université de Lorraine, Laboratoire de Chimie Physique et Microbiologie pour l'Environnement, LCPME, UMR 7564, Villers-lès-Nancy, F-54600, France. Electronic address: fabienne.quiles@univ-lorraine.fr.
authors.forename: Fabienne
authors.initials: F
authors.lastname: Quilès
authors.affiliation: CNRS, Laboratoire de Chimie Physique et Microbiologie pour l'Environnement, LCPME, UMR 7564, Villers-lès-Nancy, F-54600, France; Université de Lorraine, Laboratoire de Chimie Physique et Microbiologie pour l'Environnement, LCPME, UMR 7564, Villers-lès-Nancy, F-54600, France; Laboratoire de Biochimie Faculté de Médecine de Sousse, Avenue Mohamed Karoui, 4002 Sousse, Tunisia.
authors.forename: Souhir
authors.initials: S
authors.lastname: Saadi
authors.affiliation: CNRS, Laboratoire de Chimie Physique et Microbiologie pour l'Environnement, LCPME, UMR 7564, Villers-lès-Nancy, F-54600, France; Université de Lorraine, Laboratoire de Chimie Physique et Microbiologie pour l'Environnement, LCPME, UMR 7564, Villers-lès-Nancy, F-54600, France.
authors.forename: Grégory
authors.initials: G
authors.lastname: Francius
authors.affiliation: CNRS, Laboratoire de Chimie Physique et Microbiologie pour l'Environnement, LCPME, UMR 7564, Villers-lès-Nancy, F-54600, France; Université de Lorraine, Laboratoire de Chimie Physique et Microbiologie pour l'Environnement, LCPME, UMR 7564, Villers-lès-Nancy, F-54600, France.
authors.forename: Jalal
authors.initials: J
authors.lastname: Bacharouche
authors.affiliation: CNRS, Laboratoire de Chimie Physique et Microbiologie pour l'Environnement, LCPME, UMR 7564, Villers-lès-Nancy, F-54600, France; Université de Lorraine, Laboratoire de Chimie Physique et Microbiologie pour l'Environnement, LCPME, UMR 7564, Villers-lès-Nancy, F-54600, France.
authors.forename: François
authors.initials: F
authors.lastname: Humbert
mesh_terms: D029845:Amphibian Proteins / Q000138:chemical synthesis / Q000494:pharmacology*; D000900:Anti-Bacterial Agents / Q000138:chemical synthesis / Q000494:pharmacology*; D023181:Antimicrobial Cationic Peptides / Q000138:chemical synthesis / Q000494:pharmacology*; D001422:Bacterial Adhesion / Q000187:drug effects; D018441:Biofilms / Q000187:drug effects* / Q000254:growth & development; D002462:Cell Membrane / Q000737:chemistry / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D055119:Elastic Modulus / Q000187:drug effects; D008563:Membrane Lipids / Q000737:chemistry; D008826:Microbial Sensitivity Tests; D018625:Microscopy, Atomic Force; D008856:Microscopy, Fluorescence; D011551:Pseudomonas fluorescens / Q000737:chemistry / Q000187:drug effects* / Q000254:growth & development / Q000648:ultrastructure; D017550:Spectroscopy, Fourier Transform Infrared
chemical_list: D029845:Amphibian Proteins; D000900:Anti-Bacterial Agents; D023181:Antimicrobial Cationic Peptides; D008563:Membrane Lipids; C070905:dermaseptin
keywords: AFM; ATR–FTIR spectroscopy; Antimicrobial peptide; Bacterial biofilm; Fluorescence images