Characterization and investigation of intragenic antimicrobial peptide derivatives against mono and dual-species biofilm formation

Most types of infections, especially chronic infections, are of polymicrobial origin, and multiple microorganisms play a role in such infections. Staphylococcus aureus and Pseudomonas aeruginosa are two important bacterial pathogens whose interactions are well known in chronic polymicrobial infections. Studies show that the coexistence of these two bacteria in vivo has an effect on the course of the disease. In this thesis study, a suitable medium was determined for S. aureus and P. aeruginosa to form a dual-species biofilm together, and the formation of the dual-species biofilm was confirmed by microscopic methods such as gram staining, scanning electron microscopy (SEM) and fluorescence microscopy. New derivatives (VR-10 and GV-13) were designed based on the Hs02 peptide selected from the antimicrobial peptides in the APD3 database using database filtering technology. These antibiofilm peptides were purified by HPLC and confirmed by MS. The characterization of the purified peptides was performed by CD spectroscopy and FTIR. After characterization, the antimicrobial and antibiofilm activities of the peptides against both single and double biofilm models were evaluated, and their biofilm structures and cell morphologies were examined by SEM and fluorescence microscopy. In addition, the effects of these peptides on bacterial motility and cell viability were also analyzed. Changes in the expression levels of virulence-associated genes (icaA, hla, agrA, blaZ, pbp2a, lasB, pilA, algD and oprL) were determined by quantitative real-time PCR (qRT-PCR) method. In addition, the cytotoxic effects of the peptides were evaluated by MTT test and their hemolytic activities were evaluated by hemolysis test; both peptides were found to be safe. As a result, it has been revealed that the peptides VR-10 and GV-13, which are predicted to show potential antibiofilm activity as a result of bioinformatic analyzes, are new candidates that can be effective against single and double type biofilm models with their antimicrobial properties.