<i>In Vitro</i> Properties of WMRK-gH625, a Novel Hybrid Peptide against Multidrug-Resistant Pathogens

Antimicrobial resistance (AMR) poses a critical global health threat, driven largely by the misuse of antibiotics and the emergence of multidrug-resistant (MDR) pathogens. Antimicrobial peptides (AMPs) have emerged as promising alternatives to traditional antibiotics due to their broad-spectrum activity and low propensity for inducing resistance. This study presents the design, synthesis, and characterization of a novel fusion peptide, WMRK-gH625, which combines the antimicrobial activity of peptide WMR-K with the membrane-translocating capability of cell-penetrating peptide gH625. The hybrid peptide exhibited a helical conformation, enhanced membrane fusion and destabilization capabilities, and selective action against bacterial membranes without significant effects on eukaryotic cells. WMRK-gH625 demonstrated potent antimicrobial activity against both Gram-positive bacteria, including <i>Staphylococcus aureus</i> and MRSA strains, and Gram-negative bacteria, including <i>Escherichia coli</i>, <i>Salmonella enterica</i> group B reference and clinical strains, with variable MIC and MBC values depending on the strain. Time-kill assays confirmed concentration-dependent bactericidal effects, particularly at ≥ 2 × MIC. Interestingly, the peptide displayed minimal cytotoxicity, low hemolytic activity, and induced minimal oxidative stress in human cells, supporting its safety profile. These findings indicate that WMRK-gH625 synergistically enhances antibacterial efficacy through targeted membrane interaction and translocation, offering a promising strategy for combating AMR and developing next-generation peptide-based antimicrobials.