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Objectives and questions: Staphylococcus aureus is one of the most frequent identified bacteria in implant-associated infections and remains particularly difficult to treat due to its high prevalence of antibiotic resistance and its ability to form biofilms on implant surfaces. Most current antibiotic-based treatment options limited efficacy against S. aureus biofilm-associated infections. In this context, rifampicin is commonly recommended, however, its clinical use is constrained by a high risk of resistance development and its incompatibility with monotherapy. The study aims to investigate the potential of three selected antimicrobial peptides (AMPs) as promising non-antibiotic agents to potentiate the antimicrobial activity of rifampicin. The efficacy of the combined treatments was evaluated using both in vitro and in vivo infection models.

Material and methods: The antimicrobial activity of the AMPs Tet-20, Indolicidin, and KWI18 was evaluated alone and in combination with rifampicin using checkerboard assay to determine minimum inhibitory concentrations (MICs), minimum bactericidal concentrations (MBCs), and fractional inhibitory concentration indices (FICIs). Experiments were conducted against three clinical orthopaedic S. aureus isolates grown under planktonic conditions as well as in biofilms formed on implant surfaces. Galleria mellonella larvae infection models were used to assess the treatment efficacy in systemic, haematogenous, and biofilm-related infections. At 24 h post-infection, larvae were collected to (i) quantify bacterial burden in larval tissues and on implant surfaces using culture-based methods and (ii) evaluate host immune responses through gene expression analysis of innate immune markers.

Results: The MICs of the selected AMPs ranged from 1.8 to 7.5 µM when used alone, whereas rifampicin MICs ranged from 0.008 to 0.016 µg/mL. When combined, the treatments demonstrated synergistic activity under both planktonic and biofilm conditions, with FICI values ≤0.5 in 2 of the 3 clinical strains. In addition, combination therapy reduced the emergence of rifampicin resistance. In the haematogenous and biofilm infection models, monotherapies - either rifampicin (one-eighth of the clinical dose) or AMP alone – did not significantly improve larval survival compared with infected control groups, regardless of the strain tested. In contrast, combination treatments significantly enhanced larval survival, reduced bacterial burden, and improved host innate immune responses.

Discussion and conclusions: These findings demonstrate that rifampicin-AMP combinations enhance antimicrobial efficiency compared with single-agent treatments in both in vitro and in vivo experimental models. Further studies are ongoing to better characterize the mechanisms underlying these synergistic interactions.