Mechanistic Study of Antimicrobial Effectiveness of Cyclic Amphipathic Peptide [R4W4] against Methicillin-Resistant Staphylococcus aureus 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 [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.

amphipathic; antimicrobial; cyclic peptide; mechanism of action; minimum bactericidal concentration (MBC); minimum inhibitory concentration (MIC).