Characterization of Anti-Biofilm Peptide Activity: A Biophysical Approach

The increasing number of multidrug resistant bacteria to available antibiotics is a growing problem worldwide. One major strategy for resistance and an important reason for failure of therapy in the clinic is biofilm formation. To cope with unfavorable surroundings many bacteria live as biofilms, sessile micro-colonies adherent to surfaces that secrete an extracellular polymeric substance. An attractive alternative to conventional antibiotics are antimicrobial peptides (AMPs), innate immune system molecules that target the bacterial cytoplasmic membrane, causing membrane disruption and cell death. AMPs biophysical properties are extensively studied regarding planktonic bacteria but much less so regarding biofilm formation. By designing and synthesizing a series of model peptides that share the same amino acids composition but differ in their biophysical properties, we investigated how different steps of biofilm formation are affected by the AMP's features. We modified the peptides characteristics using different approaches of charge segregation and amino acids D enantiomers. We used Pseudomonas aeruginosa, an opportunistic Gram-negative bacteria, which is a leading cause of severe pulmonary infections in cystic fibrosis patients and medical device contamination. Our work demonstrates that the peptides combat biofilm at different stages of its formation and maintenance: (1) killing bacteria at their planktonic stage (2) preventing bacterial adhesion to biomaterials and (3) degrading pre-formed biofilm. We show that substitution of L-to-D amino acids alters the peptides biophysical properties and improves their activity against each stage in the biofilm life cycle. By investigating which biophysical properties are essential for anti-biofilm activity we also discovered new mechanisms of peptides activity.