Development of a novel ultrashort antimicrobial peptide-levofloxacin conjugate with enhanced synergistic activity against multidrug and levofloxacin-resistant bacterial isolates.

Background and purpose: Antimicrobial resistance poses a significant global health threat. A previously developed penta-amino acid ultrashort antimicrobial peptide (UP5), with alternating arginine and biphenylalanine units, showed strong antimicrobial activity, particularly in combination with levofloxacin. However, differences in pharmacokinetics between UP5 and levofloxacin may hinder their optimal clinical use. This study aimed to develop a covalent UP5-levofloxacin conjugate that retains the synergistic antimicrobial properties of both UP5 and levofloxacin. Experimental approach: The UP5-levofloxacin conjugate was synthesized and tested for antimicrobial activity against multidrug-resistant gram-positive (Enterococcus faecium, Staphylococcus aureus, Staphylococcus epidermidis) and gram-negative bacteria (Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa). Hemolytic activity was assessed on human erythrocytes, and selectivity against MDCK cells was determined. Comparative analysis was performed with individual components. Findings/Results: The conjugate exhibited synergistic antimicrobial activity with minimum inhibitory concentration (MIC) values between 2.5 and 20 μM, overcoming levofloxacin resistance. It showed minimal hemolytic activity < 1% and a favorable selectivity index of 4.4-35.2. Cytotoxicity studies indicated selective toxicity towards MDCK cells, with an IC50 of 88.34 μM, significantly higher than its MIC values. Conclusion and implications: The UP5-levofloxacin conjugate demonstrated enhanced antimicrobial efficacy against resistant bacteria, with minimal hemolytic activity and favorable selectivity toward normal cells. This conjugate presents a promising approach to combating antimicrobial resistance, offering potential for improved therapeutic strategies.