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-β-d-glucanase, β-<i>N</i>-acetylhexosaminidase) targeting biofilm matrix components combined with supratherapeutic doses of antibiotics encapsulated in a thermosensitive hydrogel (poloxamer P407) for local administration. <i>In vitro,</i> 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. <i>In vivo,</i> 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.