Antibiofilm Properties of Temporin-L on Pseudomonas fluorescens in Static and In-Flow Conditions.

Angela Di Somma,Arianna Cirillo,Federica Recupido,Stefano Guido,Alessandra Romanelli,Sergio Caserta,Alessia Romano,Angela Duilio

doi:10.3390/ijms21228526

Abstract

Biofilms consist of a complex microbial community adhering to biotic or abiotic surfaces and enclosed within a protein/polysaccharide self-produced matrix. The formation of this structure represents the most important adaptive mechanism that leads to antibacterial resistance, and therefore, closely connected to pathogenicity. Antimicrobial peptides (AMPs) could represent attractive candidates for the design of new antibiotics because of their specific characteristics. AMPs show a broad activity spectrum, a relative selectivity towards their targets (microbial membranes), the ability to act on both proliferative and quiescent cells, a rapid mechanism of action, and above all, a low propensity for developing resistance. This article investigates the effect at subMIC concentrations of Temporin-L (TL) on biofilm formation in Pseudomonas fluorescens (P. fluorescens) both in static and dynamic conditions, showing that TL displays antibiofilm properties. Biofilm formation in static conditions was analyzed by the Crystal Violet assay. Investigation of biofilms in dynamic conditions was performed in a commercial microfluidic device consisting of a microflow chamber to simulate real flow conditions in the human body. Biofilm morphology was examined using Confocal Laser Scanning Microscopy and quantified via image analysis. The investigation of TL effects on P. fluorescens showed that when subMIC concentrations of this peptide were added during bacterial growth, TL exerted antibiofilm activity, impairing biofilm formation both in static and dynamic conditions. Moreover, TL also affects mature biofilm as confocal microscopy analyses showed that a large portion of preformed biofilm architecture was clearly perturbed by the peptide addition with a significative decrease of all the biofilm surface properties and the overall biomass. Finally, in these conditions, TL did not affect bacterial cells as the live/dead cell ratio remained unchanged without any increase in damaged cells, confirming an actual antibiofilm activity of the peptide.

Highlights

Biofilm consists of a complex self-produced matrix of polysaccharides, DNA, lipids, and proteins that protect bacteria from the environment, including the host immune system [1]
Bacteria cells were grown in the presence of serial dilution of TL from 512 μM to 0.5 μM, and the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values were evaluated
The antibiofilm properties of TL were examined on P. fluorescens biofilm cultivated under two defined conditions: Static and well-controlled flow conditions using subMIC concentrations of the peptide

Summary

Introduction

Biofilm consists of a complex self-produced matrix of polysaccharides, DNA, lipids, and proteins that protect bacteria from the environment, including the host immune system [1]. Biofilms are more resistant to hostile environmental conditions, the clearance operated by the host immune system, and the flowing action of body fluids as it occurs in catheters and other medical devices [2,3]. Studies aimed to unveil molecular and cellular mechanisms underlying biofilm formation are usually performed in static fluid conditions to standardize experimental procedures in the absence of environmental complexities. In the biomedical field, medical devices like catheters, prostheses, and heart valves exposed to organic fluids flow are often attacked by biofilm originating severe infections [5]

Methods

Results

Conclusion