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.

Mechanisms of Action for Antimicrobial Peptides With Antibacterial and Antibiofilm Functions

“…The above-mentioned techniques are simple and reproducible, but do not fully capture in vivo conditions (Macià et al, 2014). The flow cell system is a more sophisticated method for evaluating antibiofilm activity (de la Quilès et al, 2016). In this technique, biofilms are grown in flow cell chambers in the presence of HDPs at specific concentrations and imaged non-destructively using confocal laser scanning microscopy (CLSM) or Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) spectroscopy (Quilès et al, 2016).…”

Natural-Product Antibiotics: Cues for Modulating Bacterial Biofilm Formation

“…To address this challenge, we need experimental approaches that allow us to directly probe how metabolites affect microbial cells, ideally in situ and in real time. One such method is Attenuated Total Reflectance-Fourier Transform InfraRed (ATR-FTIR) spectroscopy, which was recently applied to gain insight into the molecular mechanism of action of an antimicrobial peptide (dermaseptin) against Pseudomonas fluorescens biofilms [67]. This study revealed that dermaseptin led to a loss of membrane lipids and a rapid inhibition of nucleic acid biosynthesis in young P. fluorescens biofilms [67].…”

“…One such method is Attenuated Total Reflectance-Fourier Transform InfraRed (ATR-FTIR) spectroscopy, which was recently applied to gain insight into the molecular mechanism of action of an antimicrobial peptide (dermaseptin) against Pseudomonas fluorescens biofilms [67]. This study revealed that dermaseptin led to a loss of membrane lipids and a rapid inhibition of nucleic acid biosynthesis in young P. fluorescens biofilms [67]. ATRFTIR thus can be used to probe and monitor, in situ and in real time, the biochemical changes induced by antibiotics in bacteria cells at the molecular scale during early biofilm formation.…”

Influence of carbon source complexity on porosity, water retention and extracellular matrix composition ofNeurospora discretabiofilms

“…Based on these results, carbon source can be used as a process lever to tune the porosity and composition of the biofilms, which would also have a direct bearing on the mechanical properties. These biofilms are natural, biodegradable and sustainable materials that can find applications as membranes for wastewater treatment (Shukla et al ; Quilès et al ), and wound care materials (Smith et al ; Suarato et al ), which require flexible ‘housing’ and a highly porous structures for exchange of fluids.…”

“…These biofilms are natural, biodegradable and sustainable materials that can find applications as membranes for wastewater treatment (Shukla et al 2014;Quil es et al 2016), and wound care materials (Smith et al 2016;Suarato et al 2018), which require flexible 'housing' and a highly porous structures for exchange of fluids.…”