Enzymes-enhanced antibiotic therapy reduces biofilms to undetectable levels in an implant-associated infection model

Implant-associated infections caused by biofilm-forming bacteria, such as Staphylococcus aureus, remain a major clinical challenge due to their high tolerance to conventional antibiotic therapies. We report a dual-targeted therapeutic strategy that combines a tri-enzymatic cocktail designed to degrade key components of the biofilm matrix (TEC; comprising a DNA/RNA endonuclease, an endo-1,4-β-D-glucanase, and a β-N-acetylhexosaminidase), with vancomycin, both delivered via a thermosensitive poloxamer 407 hydrogel, for localized treatment of S. aureus biofilms. The formulation was evaluated both in vitro, on titanium-adherent biofilms, and in vivo, using a model of tissue cages containing titanium beads implanted in the back of guinea pigs. Animals additionally received intraperitoneal administration of vancomycin alone or combined with rifampicin. In vitro, this formulation enabled sequential drug release, with TEC delivered within the first 24 h and vancomycin for up to 96 h, and achieved >5 Log₁₀ reductions in CFU counts after two applications at 48 h interval. In vivo, biofilm-associated bacterial counts reached the detection limit (100 CFU; >5 Log₁₀ decrease from the initial inoculum) in 75% of implants 1 day post-treatment and remained undetectable in 37.5% of them 5 days post-treatment, with no emergence of resistance. Treatment efficacy was reduced if TEC or vancomycin were omitted in the hydrogel or if rifampicin was not included in the intraperitoneal treatment. Vancomycin in the hydrogel also prevented the emergence of rifampicin resistance. These findings underscore the therapeutic potential of a dual-targeted approach, combining biofilm disruption with local sustained antibiotic release, to improve the management of implant-associated infections.