RK-14: A Novel Antimicrobial Peptide Targeting Multidrug-Resistant Bacteria and Catheter-Associated Biofilms

Antimicrobial resistance (AMR) and biofilm-associated infections caused by multidrug-resistant (MDR) bacteria pose major challenges to effective antimicrobial therapy. In this study, the short cationic antimicrobial peptide RK-14 (RIWKIWKKIWKIWK-NH₂) was designed using an integrated artificial intelligence (AI)- and machine learning (ML)-assisted approach and subsequently evaluated using computational predictions and in vitro analyses. RK-14 exhibited bactericidal activity against methicillin-resistant Staphylococcus aureus and ESBL-positive Escherichia coli, with MICs of 16-64 µM and MBCs of 32-128 µM. RK-14 showed strong concentration-dependent antibiofilm activity (80-99% inhibition), outperforming LL-37, vancomycin, and colistin. Against mature catheter-associated biofilms, RK-14 achieved 87-91% eradication at 1 × MIC, compared with 77-87% for LL-37 and markedly lower activity of vancomycin and colistin. Consistently, catheter-associated CFU counts were significantly reduced following RK-14 treatment compared with the comparator treatments. Fluorescence microscopy revealed pronounced reductions in bacterial numbers and marked morphological alterations in both E. coli and S. aureus. Membrane disruption was further supported by strong membrane-binding affinity and increased propidium iodide uptake. RK-14 also suppressed key biofilm- and virulence-associated genes, including luxS, csgD, icaD, and spa. In ESBL-positive E. coli, RK-14 reduced luxS and csgD expression to approximately 0.36-0.48-fold and 0.17-0.43-fold, respectively, relative to the untreated control. In methicillin-resistant S. aureus, RK-14 reduced icaD and spa expression to approximately 0.47-fold and 0.64-fold, respectively. These effects were generally greater than or comparable to those observed following LL-37 and conventional antibiotic treatment. The peptide retained activity under various temperature and ionic conditions and showed low cytotoxicity at effective concentrations, although hemolysis increased at higher concentrations. Overall, RK-14 demonstrates potent bactericidal and antibiofilm activity, effective disruption of mature catheter-associated biofilms, and a favorable preliminary selectivity profile, supporting its further investigation as a promising lead peptide against MDR bacterial infections.