Synergistic activity of mupirocin in combination with protocatechuic acid ethyl ester against mupirocin-resistant MRSA

The prevalence of mupirocin resistance in MRSA severely limits therapeutic options for skin and soft tissue infections. This study aimed to evaluate the potential synergistic activity between mupirocin and protocatechuic acid ethyl ester (EDHB) through <i>in vitro</i> and <i>in vivo</i> investigations. Clinical <i>S. aureus</i> isolates were characterized for antibiotic resistance profiles and molecular features via antimicrobial susceptibility testing, MLST, and <i>spa</i> typing. For MRSA isolates, checkerboard and time-kill assays were performed to assess <i>in vitro</i> synergy. The potential interference of EDHB with bacterial membrane integrity and efflux pumps was investigated using propidium iodide and ethidium bromide, respectively. The disk diffusion method was applied to test the retained antimicrobial activity of mupirocin and EDHB in ointment formulations. A murine dermal wound model was established to evaluate <i>in vivo</i> efficacy by topical application of mupirocin and EDHB, alone or in combination, on infected wounds. EDHB alone exhibited limited activity but synergistically reduced mupirocin MICs by 4-8-fold in most strains. Checkerboard analysis revealed synergistic or partial synergistic interactions against MRSA. Time-kill curves further indicated that combining these two drugs can effectively inhibit the planktonic <i>S. aureus</i>. EDHB rapidly disrupts cytoplasmic membrane integrity via concentration-dependent propidium iodide influx, independent of <i>norA</i>/<i>mepA</i> efflux pump modulation. The enhanced antibacterial activity of mupirocin and EDHB was sustained in ointment formulations, resulting in superior therapeutic outcomes with combination therapy compared to monotherapy. EDHB acts as a membrane-disrupting adjuvant that synergizes with mupirocin against MuR-MRSA, offering a promising strategy to combat recalcitrant <i>S. aureus</i> infections through localized combination therapy.