LONGITUDINAL INTRAVITAL IMAGING TO QUANTIFY THE “RACE FOR THE SURFACE” BETWEEN HOST IMMUNE CELL AND BACTERIA FOR ORTHOPAEDIC IMPLANTS WITH S. AUREUS COLONIZATION IN A MURINE MODEL

TitleLongitudinal Intravital Imaging to Quantify the “Race for the Surface” Between Host Immune Cell and Bacteria for Orthopaedic Implants with S. aureus Colonization in a Murine ModelAimTo assess S. aureus vs. host cell colonization of contaminated implants vis intravital multiphoton laser scanning microscopy (IV-MLSM) in a murine model.MethodAll animal experiments were approved by IACUC. A flat stainless steel or titanium L-shaped pin was contaminated with 105 CFU of a red fluorescent protein (RFP) expressing strain of USA300LAC, and surgically implanted through the femur of global GFP-transgenic mice. IV-MLSM was performed at 2, 4, and 6 hours post-op. Parallel cross-sectional CFU studies were performed to quantify the bacteria load on the implant at 2,4,6,12,18 and 24 hours.Results1) We developed a high-fidelity reproducible IV-MLSM system to quantify S. aureus and host cell colonization of a bone implant in the mouse femur. Proper placement of all implants were confirmed with in vivo X-rays, and ex vivo photos. We empirically derive the ROI during each imaging session by aggregating the imaged volume which ranges from (636.4um × 636.4um × 151um) = 0.625 +/- 0.014 mm3 of bone marrow in a global GFP-transgenic mouse.2) IV-MLSM imaging acquisition of the “race for the surface”.In vitro MPLSM images of implants partially coated with USA300LAC (RFP-MRSA) were verified by SEM image. Results from IV-MLSM of RFP-MRSA and GFP+ host cell colonization of the contaminated implants illustrated the mutually exclusive surface coating at 3hrs, which to our knowledge is the first demonstration of “the race for the surface” between bacteria and host cells via intravital microscopy.3) Quantifying the “race for the surface” with CFU verification of S. aureus on the implant. 3D volumetric rendering of the GFP+ voxels and RFP+ voxels within the ROI were generated in Imaris. The voxel numbers suggeste that the fight for the surface concludes ∼3hrs post-infection, and then transitions to an aggressive MRSA proliferation phase. The results of WT control demonstrate a significant increase in CFU by 12hrs post-op for both stainless steel (P<0.01) and titanium (P<0.01).ConclusionsWe developed IV-MLSM to quantify the “Race for the Surface” between host cells and contaminating S. aureus in a murine femur implant model. This race is remarkably fast, as the implant surface is completely covered with 3hrs, peak bacterial growth on the implant occurs between 2 and 12 hours and is complete by 12hrs.