Evaluating the Antimicrobial Efficacy of a Designed Synthetic peptide against Pathogenic Bacteria

Recent research has focused on discovering peptides that effectively target multidrug-resistant bacteria while leaving healthy cells unharmed. In this work, we describe the antimicrobial properties of RK8, a peptide composed of eight amino acid residues. Its activity was tested against multidrugresistant Gram-negative and Gram-positive bacteria. RK8's efficacy in eradicating mature biofilm and increasing membrane permeability was assessed using Sytox Green. Cytotoxicity assays were conducted both in vitro and in vivo models. Circular dichroism analysis revealed that RK8 adopted an extended structure in water and sodium dodecyl sulfate (SDS). RK8 exhibited MICs of 8-64 pM and MBCs of 4-64 pM against various bacteria, with higher effectiveness observed in Methicillin-resistant Staphylococcus aureus (MRSA) and E. coli KPC+ strains than others. Ciprofloxacin and Vancomycin showed varying MIC and MBC values lower than RK8 for Gram-positive bacteria, but competitive for Gram-negative bacteria. The combination of RK8 and ciprofloxacin showed a synergistic effect. The RK8 peptides could reduce 38% of the mature Acinetobacter baumannii biofilm. Sytox Green reagent achieved 100% membrane permeation of Gram-positive and Gram-negative bacteria. The RK8 peptide did not show cytotoxic effects against murine macrophages (64 pM), erythrocytes (100 pM) or Galleria mellanella larvae (960 pM). In the stability test against peptidases, the RK8 peptide was stable, maintaining around 60% of the molecule intact after 120 min of incubation. These results highlight the potential of RK8 to be a promising strategy for developing a new antimicrobial and antibiofilm agent, inspiring and motivating further research in antimicrobial peptides.