Design and optimization of antimicrobial peptides combating gram-negative biofilm-forming bacteria

Abstract

The accumulation of undesirable microorganisms in form of biofilms, the so-called biofouling, represents a significant challenge in numerous fields, including industry, where the presence of biocorrosive bacteria can cause in a wide range of material damage. Antimicrobial peptides (AMPs) have demonstrated potential as an alternative for the usage of antibiotics in the medical technology and pharmaceutical sectors, and increase in importance as new drug candidates. The objective of this thesis is to expand the application range of AMPs to the industrial sector, with a focus on water-bearing systems. Here, AMPs are intended to replace the current biocide treatment. For this aim, membranolytic peptides were initially selected from peptide databases based on their characteristics (charge, hydrophobicity, and amphiphilicity), and then tested for their planktonic activity against various biocorrosive and other biofilm-forming bacteria. Inhibitory concentrations of 15 μM were observed against Desulfovibrio vulgaris, the primary representative of biocorrosive bacteria. Subsequent optimization of the peptide sequences by modifying the amino acid composition and modifying the peptide termini by amidation and lipidation led to an eight-fold increase in planktonic activity. An increase in activity through the use of two AMPs could only be demonstrated in combination with one peptide (P5) due to different modes of action. The development of a modified Calgary biofilm assay enables simple and rapid screening of the antibiofilm activity of the peptides, both in terms of (long-term) biofilm inhibition and removal. The analysis is based on the determination of microbial growth and the characterization of the biofilm mass by crystal violet staining. By extending the growth surfaces to polished steel surfaces, this test could also be carried out under realistic conditions. The microbiological tests show advantages of an early AMP treatment, preferably already in the planktonic stage. Comparative experiments with two common biocides, glutaraldehyde and tetrakis hydroxymethylphosphonium sulfate, in terms of activity, toxicity and resistance rate demonstrate the huge potential of AMPs, as they are more active, less toxic and less resistant against Desulfovibrio vulgaris at the same concentration. On this basis, it is possible to extend the scope of application of AMPs from the medical to the industrial sector for the control of biocorrosive bacteria and thus represents a promising alternative to the current biocide treatment.