Targeting Staphylococcus aureus biofilm-related infections on implanted material with a novel dual-action thermosensitive hydrogel containing vancomycin and a tri-enzymatic cocktail: in vitro and in vivo studies

Implant-associated infections remain a critical challenge due to the presence of biofilm-forming bacteria, which enhance tolerance to conventional treatments. This study investigates the efficacy of a tri-enzymatic cocktail (TEC; DNA/RNA endonuclease, endo-14-beta-D-glucanase, beta-N-acetylhexosaminidase) targeting biofilm matrix components combined with supratherapeutic doses of antibiotics encapsulated in a thermosensitive hydrogel (poloxamer P407) for local administration. In vitro, the hydrogel formulation enabled controlled release of active agents over 12 h. Vancomycin and TEC co-formulated in hydrogel achieved up to 3.8 Log10 CFU count reduction and 80 % biofilm biomass reduction on MRSA biofilms grown on titanium coupons, demonstrating enhanced efficacy as compared to individual active agents, with 1.3-3.2 log10 additional killing. Fluoroquinolone efficacy remained unchanged by enzyme addition. In vivo, in a model of tissue cages containing titanium beads implanted in the back of guinea pigs, hydrogel-delivered vancomycin maintained therapeutic levels for seven days. Coupled with an intraperitoneal administration of vancomycin for 4 days, a single local administration of hydrogel containing both vancomycin and TEC was more effective than hydrogels containing either vancomycin or TEC, achieving an additional 2.1 Log10 CFU reduction compared to local vancomycin, 2.3 Log10 compared to local TEC, and 4.3 Log10 compared to systemic vancomycin treatment alone. However, partial regrowth occurred at later stages, indicating room for further optimization. Nevertheless, these findings already underscore the potential of combining a high dose of antibiotic with an enzymatic cocktail in a sustained-release hydrogel delivery system as a promising strategy for improving the management of biofilm-associated implant infections.