Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research
Article Title: Evidence of Staphylococcus aureus deformation, proliferation and migration in canaliculi of live cortical bone in murine models of osteomyelitis
doi: 10.1002/jbmr.3055
Figure Lengend Snippet: Mice (n = 5) received a femoral osteotomy that was infected with UAMS-1 ΔSpA, and were fed BrdU in their drinking water continuously post-op until sacrifice on day 14. (A) Light microscopy image (x 4) of the toluidine blue stained cortical bone section containing a defect (yellow bracket) leading to a canaliculus (black bracket) colonized with S. aureus, which was interrogated by immunoelectron microscopy (IEM). (B) Low (x 15,000 boxed region in A), (C) high (x 40,000 boxed region in B), and (D) ultrahigh (boxed region in C enlarged from x 150,000 original) magnification of IEM images of BrdU positive S. aureus. Note the rod-shaped bacterium with a diameter of 0.36 µm at the leading edge of bacterial infiltration (C), and the immunogold labeled chromosome (12 nm black dots in D) confirming BrdU incorporation. IEM on S. aureus infected femurs from mice that were not fed BrdU were all negative for immunogold labeling (data not shown). (E) Our in vitro transwell culture system loads via an open chamber on the topside (white arrow), through which GFP+ UAMS-1 in TSB medium is placed onto a 0.4µm thick silicon nitride submicron porous (0.5µm in diameter) membrane. Note left and right access channels (black arrows) for loading TSB medium into the underside reservoir, which is a sealed chamber that is physically separated from the topside of the submicron porous membrane. (F) SEM (x15,000) of the static biofilm that forms on the top surface of the membrane at 3hrs. Validation of GFP+ UAMS-1 migration through the submicron pores is shown by confocal fluorescent microscopy of the bacteria (G-top view) and a 3D reconstructed image of 5 confocal slices (H). S. aureus migration through the submicron pores via proliferation is evidenced by low (I x 2,000) and high (J x 12,000) magnification SEM imaging of the underside of the membrane demonstrating extrusion of the bacteria at 6.5hrs. (K) The increase in GFP+ S. aureus occupancy of the submicron pores was quantified via real time fluorescent confocal microcopy from the topside of the membrane, and the data are presented as the mean of 3 independent experiments +/− SEM in which the plateau (saturation time) is estimated at 35.6 minutes (95% CI: 25.8 – 45.4 minutes; *p<0.05 vs. 5 min). (L-P) Representative fluorescent images (x 60) from the topside obtained at 15min intervals from 0 to 60 min are shown to illustrate the increase in pore occupation over the first 30 min, and subsequent saturation thereafter.
Article Snippet: Briefly, 0.4μm thick silicon nitride membrane chips containing pores 0.5μm in diameter were fabricated by SiMPore Inc. (West Henrietta, NY) as previously described. ( 14 ) Individual chips were housed within a circular culture well system made of a firm silicon gel.
Techniques: Infection, Light Microscopy, Staining, Immuno-Electron Microscopy, Labeling, BrdU Incorporation Assay, In Vitro, Membrane, Biomarker Discovery, Migration, Microscopy, Bacteria, Imaging
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