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Antimicrobial peptides (AMPs) are being explored as a potential strategy to combat antibiotic resistance due to their ability to reduce susceptibility to antibiotics. This study explored whether the R4W4 peptide mode of action is bacteriostatic or bactericidal using modified two-fold serial dilution and evaluating the synergism between gentamicin and R4W4 against Escherichia coli (E. coli) and methicillin-resistant Staphylococcus aureus (MRSA) by a checkered board assay. R4W4 exhibited bactericidal activity against bacterial isolates (MBC/MIC ≤ 4), with a synergistic effect with gentamicin against E. coli (FICI = 0.3) but not against MRSA (FICI = 0.75). Moreover, we investigated the mechanism of action of R4W4 against MRSA by applying biophysical assays to evaluate zeta potential, cytoplasmic membrane depolarization, and lipoteichoic acid (LTA) binding affinity. R4W4 at a 16 mg/mL concentration stabilized the zeta potential of MRSA −31 ± 0.88 mV to −8.37 mV. Also, R4W4 at 2 × MIC and 16 × MIC revealed a membrane perturbation process associated with concentration-dependent effects. Lastly, in the presence of BODIPY-TR-cadaverine (BC) fluorescence dyes, R4W4 exhibited binding affinity to LTA comparable with melittin, the positive control. In addition, the antibacterial activity of R4W4 against MRSA remained unchanged in the absence and presence of LTA, with an MIC of 8 µg/mL. Therefore, the R4W4 mechanism of action is deemed bactericidal, involving interaction with bacterial cell membranes, causing concentration-dependent membrane perturbation. Additionally, after 30 serial passages, there was a modest increment of MRSA strains resistant to R4W4 and a change in antibacterial effectiveness MIC R4W4 and vancomycin by 8 and 4 folds with a slight change in Levofloxacin MIC 1 to 2 µg/mL. These data suggest that R4W4 warrants further consideration as a potential AMP.
Akinwale et al. (Thu,) studied this question.