Destruction of Staphylococcus aureus biofilms by combining an antibiotic with subtilisin A or calcium gluconate

In S. aureus biofilms, bacteria are embedded in a matrix of extracellular polymeric substances (EPS) and are highly tolerant to antimicrobial drugs. We thus sought to identify non-antibiotic substances with broad-spectrum activity able to destroy the EPS matrix and enhance the effect of antibiotics on embedded biofilm bacteria. Among eight substances tested, subtilisin A (0.01 U/mL) and calcium gluconate (CaG, Ca²⁺ 1.25 mmol/L) significantly reduced the biomass of biofilms formed by at least 21/24 S. aureus isolates. Confocal laser scanning microscopy confirmed that they both eliminated nearly all the proteins and PNAG from the matrix. By contrast, antibiotics alone had nearly no effect on biofilm biomass and the selected one (oxytetracycline-OTC) could only slightly reduce biofilm bacteria. The combination of OTC with CaG or subtilisin A led to an additive reduction (average of 2 log₁₀ CFU/mL) of embedded biofilm bacteria on the isolates susceptible to OTC (MBC < 10 μg/mL, 11/24). Moreover, these two combinations led to a reduction of the embedded biofilm bacteria higher than 3 log₁₀ CFU/mL for 20-25% of the isolates. Further studies are now required to better understand the factors that cause the biofilm produced by specific isolates (20-25%) to be susceptible to the combinations.