The limitations of mono‐ and combination antibiotic therapies on immature biofilms in a murine model of implant‐associated osteomyelitis

Treatment of implant‐associated orthopedic infections remains challenging, partly because antimicrobial treatment is ineffective after a mature biofilm covers the implant surface. Currently, the relative efficacy of systemic mono‐ and combination standard‐of‐care (SOC) antibiotic therapies over the course of mature biofilm formation is unknown. Thus, we assessed the effects of cefazoline (CEZ), gentamicin (GM), and vancomycin, with or without rifampin (RFP), on Staphylococcus aureus biofilm formation during the establishment of implant‐associated osteomyelitis in a murine tibia model. Quantitative scanning electron microscopy of the implants harvested on Days 0, 3, and 7 revealed that all treatments except CEZ monotherapy significantly reduced biofilm formation when antibiotics started at Day 0 (0.46‐ to 0.25‐fold; p < 0.05). When antibiotics commenced 3 days after the infection, only GM monotherapy significantly inhibited biofilm growth (0.63‐fold; p < 0.05), while all antibiotics inhibited biofilm formation in combination with RFP (0.56‐ to 0.44‐fold; p < 0.05). However, no treatment was effective when antibiotics commenced on Day 7. To confirm these findings, we assessed bacterial load via colony‐forming unit and histology. The results showed that GM monotherapy and all combination therapies reduced the colony‐forming unit in the implant (0.41‐ to 0.23‐fold; p < 0.05); all treatments except CEZ monotherapy reduced the colony‐forming unit and staphylococcus abscess communities in the tibiae (0.40‐ to 0.10‐fold; p < 0.05). Collectively, these findings demonstrate that systemic SOC antibiotics can inhibit biofilm formation within 3 days but not after 7 days of infection. The efficacy of SOC monotherapies, CEZ particularly, is very limited. Thus, combination treatment with RFP may be necessary to inhibit implant‐associated osteomyelitis.