Longitudinal intravital imaging of the bone marrow for analysis of the race for the surface in a murine osteomyelitis model

Abstract Critical knowledge gaps of orthopedic infections pertain to bacterial colonization. The established dogma termed the Race for the Surface posits that contaminating bacteria compete with host cells for the implant post‐op, which remains unproven without real‐time in vivo evidence. Thus, we modified the murine longitudinal intravital imaging of the bone marrow (LIMB) system to allow real‐time quantification of green fluorescent protein (GFP+) host cells and enhanced cyan fluorescent protein (ECFP+) or red fluorescent protein (RFP+) methicillin‐resistant Staphylococcus aureus (MRSA) proximal to a transfemoral implant. Following inoculation with ~10 5 CFU, an L‐shaped metal implant was press‐fit through the lateral cortex at a 90° angle ~0.150 mm below a gradient refractive index (GRIN) lens. We empirically derived a volume of interest (VOI) = 0.0161 ± 0.000675 mm 3 during each imaging session by aggregating the Z‐stacks between the first (superior) and last (inferior) in‐focus LIMB slice. LIMB postimplantation revealed very limited bacteria detection at 1 h, but by 3 h, 56.8% of the implant surface was covered by ECFP+ bacteria, and the rest were covered by GFP+ host cells. 3D volumetric rendering of the GFP+ and ECFP+ or RFP+ voxels demonstrated exponential MRSA growth between 3 and 6 h in the Z‐plane, which was validated with cross‐sectional ex vivo bacterial burden analyses demonstrating significant growth by ~2 × 10 4 CFU/h on the implant from 2 to 12 h post‐op ( p < 0.05; r 2 > 0.98). Collectively, these results show the competition at the surface is completed by 3 h in this model and demonstrate the potential of LIMB to elucidate mechanisms of bacterial colonization, the host immune response, and the efficacy of antimicrobials.