Synergistic Ciprofloxacin-RWn Peptide Therapy Overcomes Drug Resistance in Gram-Negative Bacteria

The rise of multidrug-resistant (MDR) Gram-negative bacteria necessitates novel therapeutic approaches. This study demonstrates the <i>in vitro</i> synergistic activity of ciprofloxacin (CIP), a fluoroquinolone antibiotic, combined with short arginine-tryptophan-rich antimicrobial peptides (AMPs), RWn (<i>n</i> = 4, 6, 8), against (ATCC 25922), (ATCC 700603), (ATCC 27853), and (ATCC 19606). While CIP alone showed limited efficacy against and (MICs > 32 μg/mL), coadministration with RWn peptides significantly reduced CIP minimum inhibitory concentrations (MICs) against all tested pathogens. The combination of CIP and RW4, the shortest peptide, exhibited the most potent synergy. Specifically, the MIC of CIP against decreased from 32.00 ± 0 to 3.00 ± 1.00 μg/mL with RW4, a >10-fold reduction. Fractional inhibitory concentration index (FICI) values were ≤0.5 for the CIP-RW4 combination against all strains, confirming synergy. Proline incorporation into RWn generally reduced this synergistic effect. Importantly, the combinations showed no cytotoxicity against human embryonic kidney (HEK293) or human umbilical vein endothelial (HUVEC) cells at effective concentrations (MICs >32 μg/mL). These results indicate that RWn AMPs, particularly RW4, can restore the effectiveness of CIP against MDR Gram-negative bacteria, providing a promising therapeutic strategy to combat antibiotic resistance. The MICs were obtained following the broth microdilution method.