Mechanistic Study of Antimicrobial Effectiveness of Cyclic Amphipathic Peptide [RW] against Methicillin-Resistant Clinical Isolates.

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 [RW] peptide mode of action is bacteriostatic or bactericidal using modified two-fold serial dilution and evaluating the synergism between gentamicin and [RW] against () and methicillin-resistant (MRSA) by a checkered board assay. [RW] exhibited bactericidal activity against bacterial isolates (MBC/MIC ≤ 4), with a synergistic effect with gentamicin against (FICI = 0.3) but not against MRSA (FICI = 0.75). Moreover, we investigated the mechanism of action of [RW] against MRSA by applying biophysical assays to evaluate zeta potential, cytoplasmic membrane depolarization, and lipoteichoic acid (LTA) binding affinity. [RW] at a 16 mg/mL concentration stabilized the zeta potential of MRSA -31 ± 0.88 mV to -8.37 mV. Also, [RW] 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, [RW] exhibited binding affinity to LTA comparable with melittin, the positive control. In addition, the antibacterial activity of [RW] against MRSA remained unchanged in the absence and presence of LTA, with an MIC of 8 µg/mL. Therefore, the [RW] 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 [RW] and a change in antibacterial effectiveness MIC [RW] and vancomycin by 8 and 4 folds with a slight change in Levofloxacin MIC 1 to 2 µg/mL. These data suggest that [RW] warrants further consideration as a potential AMP.