Study of the effect of pulsed washing on antibiotic susceptibility of Staphylococcus aureus biofilms

Background: Pulsed washing (PW) is a major step during debridement surgery in the context of orthopaedic device-related infections (ODRI), but its effects on biofilms are poorly characterized. The present study aims at evaluating the effect of PW alone or followed by antibiotics on MSSA and MRSA biofilms grown on titanium Ti6Al4V in-vitro. Materials/methods: Strains: 1 reference (MSSA: ATCC25923; MRSA: ATCC33591) and 2 clinical MSSA and MRSA isolated from ODRI. Biofilm culture: Incubation for 24h at 37°C with bacteria (starting inoculum ~6.6Log10CFU/mL in TGN [TSB+1%glucose+2%NaCl]), under continuous agitation. Treatment: Half the coupons received PW (50mL physiological serum, Stryker Interpulse); the coupons were then either analysed or reincubated for 24h in TGN or TGN + flucloxacillin (MSSA) or vancomycin (MRSA) at two concentrations (MIC and 20mg/L). Analysis: Biomass was determined by crystal violet (CV) staining. CFUs were counted after recovering bacteria from coupons using sonication and TSA plating. Results: See Figure. Antibiotics alone: Flucloxacillin significantly reduced both CFU and biomass for ATCC25923 but not for clinical isolates of MSSA. Vancomycin had no statistically significant effects on CFUs for MRSAs and a weak effect on biomass for one clinical isolate. PW alone markedly reduced CFUs and biomass for all strains but had no persistent effect. PW + antibiotics: Antibiotics prevented bacterial regrowth after PW when used at their MIC and further decreased CFUs when used at 20 mg/L. Conclusions: PW alone only has a transient effect on S. aureus biofilms. Vancomycin alone at therapeutic concentrations is ineffective and flucloxacillin has a strain-dependent effect. Yet, these treatments show synergistic effects, indicating the importance of PW followed by high antibiotic doses. However, PW in-vivo is limited by poor access to parts of the implants, providing arguments for the development of alternative biofilm-disrupting treatments