Design and Efficacy of an Ultrashort Antimicrobial Peptide-levofloxacin Conjugate Against Resistant Bacteria: A Novel Approach to Combat Antimicrobial Resistance

INTRODUCTION: The rise of Multidrug-Resistant (MDR) bacterial infections and the limited efficacy of conventional antibiotics have underscored the urgent need for innovative antimicrobial therapies. Antimicrobial Peptides (AMPs) represent a promising class of agents due to their broad-spectrum activity; however, their clinical application is often hindered by cytotoxicity and poor stability. This study aimed to develop and evaluate a novel ultrashort AMP-antibiotic conjugate, UP5-C-Levo, with improved antimicrobial efficacy and safety. METHODS: UP5-C-Levo was synthesized by covalently linking the ultrashort peptide UP5 to the antibiotic levofloxacin. The antimicrobial activity of the conjugate was assessed against MDR Gram-positive and Gram-negative bacteria using Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) assays. Antibiofilm efficacy was tested on Staphylococcus aureus biofilms. Cytotoxicity was evaluated via hemolysis of human erythrocytes and MTT assays on MDCK cells. Scanning Electron Microscopy (SEM) was employed to investigate bacterial membrane disruption. RESULTS: UP5-C-Levo exhibited potent antimicrobial activity, with MIC values ranging from 12.5 to 25 μM across all tested MDR strains. It significantly reduced biofilm biomass, achieving nearcomplete eradication of S. aureus biofilms. Hemolysis and cytotoxicity assays indicated minimal toxicity to human cells. SEM imaging revealed extensive bacterial membrane disruption, suggesting a dual mechanism of action. DISCUSSION: The conjugation of UP5 with levofloxacin resulted in a synergistic antimicrobial agent with enhanced efficacy and low cytotoxicity. The ability of UP5-C-Levo to disrupt bacterial membranes and eradicate biofilms addresses two major challenges in infection treatment. These findings align with current strategies aimed at overcoming antibiotic resistance through AMPbased drug design. CONCLUSION: UP5-C-Levo is a promising therapeutic candidate for the treatment of MDR and biofilm-associated infections. Its strong antibacterial and antibiofilm activity, combined with a favorable safety profile, supports further preclinical development and potential clinical translation.