Halicin‑Rifampicin Combinations Are More Effective Than Monotherapy Against Biofilm‑Residing Staphylococcus Aureus and Prevent Emergence of Rifampicin Resistance

Abstract

Background: Biofilms shield bacteria from immune defenses and antibiotics, contributing to implant-associated infections, particularly those caused by Staphylococcus aureus (S. aureus). Halicin, a repurposed antimicrobial with broad-spectrum activity, has shown promise against planktonic and biofilm-residing bacteria. Building on our previous work, we investigated whether halicin enhances the efficacy of conventional antibiotics when used in combination against S. aureus. Methods: The treatment efficacies of combination therapies of Halicin and conventional antibiotics were tested against planktonic S. aureus and S. aureus biofilms grown on titanium substrates of varying maturities. The emergence of antibiotic resistance in treated biofilm bacteria was also characterized. Results: In checkerboard assays against planktonic bacteria, halicin synergized with gentamicin, tobramycin, and cefazolin but not with rifampicin or vancomycin. In biofilms grown on titanium substrates, halicin combined most effectively with rifampicin, reducing viable bacteria by >3 logs in less-mature and >5 logs in more-mature biofilms compared to rifampicin alone. Compared to untreated biofilms, combination therapy reduced viable bacteria by >5 logs in all cases. This combination also prevented rifampicin resistance seen with rifampicin monotherapy. Halicin only modestly enhanced aminoglycoside activity against less-mature biofilms and had no significant effect when combined with vancomycin or cefazolin. Conclusions: These findings highlight that antibiotic-halicin synergy differs between planktonic and biofilm states, underscoring the importance of biofilm-specific studies. Highlighting the translatability of our results, the doses of conventional antibiotics combined with halicin required to affect planktonic and biofilm-residing S. aureus are all achievable systemically and in bone.