Antibiofilm effect of Temporin-L on Pseudomonas fluorescens, in static and dynamic conditions.
Authors: Cirillo Arianna , Di Somma Angela , Romano Alessia , Recupido Federica , Caserta Sergio , Guido Stefano , Romanelli Alessandra , Duilio Angela 
Published: July 2020

&lt;p&gt;Introduction&lt;/p&gt;
&lt;p&gt;Biofilm consists of a complex self-produced matrix of polysaccharides, DNA and proteins that&amp;#160;&lt;/p&gt;
&lt;p&gt;protects bacteria from the environment including the host immune system and constitutes the main&lt;/p&gt;
&lt;p&gt;cause of bacterial resistance against antibiotics. Research is then focused on finding alternative&amp;#160;&lt;/p&gt;
&lt;p&gt;antimicrobial substances able to either hamper biofilm formation or to prevent bacterial growth.&amp;#160;&lt;/p&gt;
&lt;p&gt;Recently, we showed that the antimicrobial peptide Temporin-L impairs E.coli growth by inhibiting&amp;#160;&lt;/p&gt;
&lt;p&gt;cell division (Di Somma et al.; 2020; BBA). Here we investigate the effect of Temporin-L (TL) on&amp;#160;&lt;/p&gt;
&lt;p&gt;biofilm formation in Pseudomonas fluorescens (P. fluorescens) both in static and dynamic conditions,&amp;#160;&lt;/p&gt;
&lt;p&gt;showing that TL displays antibiofilm properties.&amp;#160;&lt;/p&gt;
&lt;p&gt;Materials and methods&lt;/p&gt;
&lt;p&gt;Biofilm formation in static conditions was performed on coverslips and analyzed by the Crystal Violet&amp;#160;&lt;/p&gt;
&lt;p&gt;assay. Biofilm morphology was assessed using imaging techniques. Investigation of biofilms in&amp;#160;&lt;/p&gt;
&lt;p&gt;dynamic conditions was performed in a flow chamber using a microfluidic system and images were&amp;#160;&lt;/p&gt;
&lt;p&gt;recorded by confocal microscopy.&lt;/p&gt;
&lt;p&gt;Results&lt;/p&gt;
&lt;p&gt;The P. fluorescens cells were either grown in the presence of TL or incubated with the antimicrobial&amp;#160;&lt;/p&gt;
&lt;p&gt;peptide after biofilm formation both in static and dynamic conditions using different concentrations&amp;#160;&lt;/p&gt;
&lt;p&gt;of the peptide. When TL was added during cell growth, the peptide affected biofilm formation at 25&amp;#160;&lt;/p&gt;
&lt;p&gt;&amp;#181;M. Confocal microscopy demonstrated that at this concentration P. fluorescens cells were still alive&lt;/p&gt;
&lt;p&gt;but a clear disruption of the biofilm architecture was observed. These results had to be ascribed to a&amp;#160;&lt;/p&gt;
&lt;p&gt;specific antibiofilm effect of TL. At 100 &amp;#181;M TL antibiofilm activity biofilm thickness was nearly&amp;#160;&lt;/p&gt;
&lt;p&gt;negligible.&amp;#160;&lt;/p&gt;
&lt;p&gt;When P. fluorescens cells were treated with TL following biofilm formation, confocal images&amp;#160;&lt;/p&gt;
&lt;p&gt;demonstrated that the peptide exerted a strong antibiofilm effect leading to cell detachment and&amp;#160;&lt;/p&gt;
&lt;p&gt;disruption the biofilm architecture.&amp;#160;&lt;/p&gt;
&lt;p&gt;Discussion and Conclusions&amp;#160;&lt;/p&gt;
&lt;p&gt;Investigation of TL effect on P. fluorescens showed that when added during bacterial growth this&amp;#160;&lt;/p&gt;
&lt;p&gt;peptide exerted antibiofilm activity at low concentration impairing biofilm formation both in static&amp;#160;&lt;/p&gt;
&lt;p&gt;and dynamic conditions, leaving most of bacterial cells still alive. However, confocal microscopy&amp;#160;&lt;/p&gt;
&lt;p&gt;measurements could not detect the long necklace-like structures observed in E.coli indicating a&amp;#160;&lt;/p&gt;
&lt;p&gt;different mechanism of action of TL on P. fluorescens. Furthermore, when TL was added to a&amp;#160;&lt;/p&gt;
&lt;p&gt;preformed P. fluorescens biofilm, the peptide showed a strong antibiofilm activity both in static and&amp;#160;&lt;/p&gt;
&lt;p&gt;dynamic conditions, suggesting that TL might penetrate biofilm architecture with a still unknown&amp;#160;&lt;/p&gt;
&lt;p&gt;mechanism leading to disruption of P. fluorescens biofilm.&lt;/p&gt;