In Vitro Evaluation of Antimicrobial Amyloidogenic Peptides for the Treatment of Early and Mature Bacterial Biofilms

Biofilm formation by pathogenic bacteria, including methicillin-resistant <i>Staphylococcus aureus</i> (MRSA), <i>Pseudomonas aeruginosa</i>, and <i>Escherichia coli</i>, represents a major clinical challenge due to the high resistance of biofilms to conventional antimicrobial therapy. In this in vitro study, we investigated the antimicrobial and antibiofilm activity of synthetic peptides R23I^T, R23L^P, V31K^T, R44K^S, R44K^P, V31K^S, and I31K^P against methicillin-resistant <i>S. aureus</i> (MRSA, SA180-F strain), <i>S. aureus</i> (129B), <i>P. aeruginosa</i> (2943), and <i>E. coli</i> (MG1655). In liquid medium, peptides R23L^P and R44K^S exhibited the broadest and most potent antimicrobial activity against all tested strains. On solid agar, these peptides demonstrated comparable activity, with notable effects particularly against <i>E. coli</i>. We further assessed the peptides' impact on both early-stage and mature biofilms using crystal violet staining for total biomass and the MTT assay for cellular metabolic activity. Peptide R44K^S showed a strong dose-dependent inhibitory effect on early MRSA biofilm formation, while most peptides unexpectedly enhanced biofilm formation by <i>S. aureus</i> and <i>E. coli</i>. Peptides R44K^P and V31K^S at 10 mg/mL significantly reduced both biomass and metabolic activity of early <i>P. aeruginosa</i> biofilms. None of the peptides inhibited mature biofilm biomass across species; however, several, particularly I31K^P, significantly reduced the metabolic activity of MRSA within mature biofilms. These findings underscore the strain- and stage-specific effects of antimicrobial peptides and highlight R23L^P, R44K^S, R44K^P, V31K^S, and I31K^P as promising candidates for targeted biofilm control in vitro, especially against MRSA.