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.

Biofilms prevention and removal are crucial in many industrial and medical applications. Their complex and cohesive structure provides resistance to cleaning even to strong disinfectants. A key factor for their behavior is the wetting properties of their surfaces. The main goal of this work is to study the wetting properties of biofilms produced by bacteria Pseudomonas fluorescens. Biofilms are obtained on glass coupons under well controlled flow conditions, using custom-made flow cell devices. Different nutrient concentration and shear flow conditions are investigated. Biofilm wetting properties are examined under imposed external body forces (forced wetting) through a specialized device, named Kerberos®. Kerberos® is capable of subjecting sessile droplets to varying tilting angles and centrifugal forces while monitoring the variation of the droplet shape in X, Y and Z-directions through three Wi-Fi cameras. Wetting experiments are carried out using water-based solution (dye solution) droplets on biofilm-coated glass coupons. In this work, spreading/sliding behaviour of droplets are investigated only on horizontal substrates (no tilting) under the action of centrifugal forces. Apart from wetting properties, biofilm growth kinetics and surface morphology at different nutrient and shear flow conditions are also assessed. Results show that, according to the different growth conditions, biofilms present different wetting properties. At lower nutrient concentration and shear flow conditions, spreading and sliding behaviour are similar to that observed in glass coupons in the absence of biofilm. At higher nutrient and shear flow conditions, spontaneous wicking of the biofilm occurs the moment of droplet deposition on the biofilm leading to irregular and jagged shapes of droplets, while on the contrary water droplets look like smooth spherical sections on pure glass. The spontaneous wicking affects the droplet initial shape and so the wetting behaviour during the subsequent rotation tests. In each examined condition, biofilms show hydrophilic properties.