PMC6681818_01
Preoperative radiographs. Fig. 1-A Anteroposterior view of the right foot and the infected fourth toe tip (arrow) with clawing. Fig. 1-B Lateral view of the right foot and the infected fourth toe. Note the osteolysis at the distal phalanx of the fourth toe (arrow).
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Radiographs of the right foot demonstrated general osteopenia and clawing of the fourth toe (Fig. 1-A), with osteolysis at the tip of the distal phalanx (Fig. 1-B).
Figs. 2-A
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2-H TEM evidence of bacterial deformation and invasion of the submicron osteocytic-canalicular system. MC = marrow cavity, OC = osteocyte, and N = nucleus. Fig. 2-A Light microscopy image demonstrating positive Gram- stained bacteria in a section from the paraffin-embedded bone tissue of the fourth toe (x60). Fig. 2-B From the TEM epoxy-embedded portion of bone, a 1-mum epoxy section stained with toluidine blue reveals dark-blue bacteria scalloping the live bone tissue (red arrow) (note the OCs and black arrows adjacent to the MC) (x60). Fig. 2-C TEM image of a dead OC and its canaliculus (red arrow), colonized by deformed rod-shaped bacteria (x6,000). Fig. 2-D Higher magnification of the area adjacent to the red arrow in Figure 2-C reveals the septal plane of the dividing bacteria within the submicron space of the canaliculus (x30,000). Note the asymmetric plane of the 2 daughter cells. Fig. 2-E High magnification of the osteocyte in Figure 2-C displaying condensed nuclear debris and round bacteria occupying its lacunar space (x15,000). Fig. 2-F Another dead osteocyte's lacuna containing remnants of condensed chromatin bodies and round bacteria (x8,000). Fig. 2-G A TEM image from thin sections deeper into the bone tissue reveals rod-shaped bacteria within a canaliculus (x6,000). Fig. 2-H Higher-magnification TEM image of the boxed area in Figure 2-G measuring submicron-sized deformed and elongated bacteria (x15,000).
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Gram staining of paraffin-embedded bone sections confirmed Gram-positive bacteria scalloping the bone tissue adjacent to the bone marrow (Fig. 2-A). Epoxy-embedded bone tissue surveyed by light microscopy using 1-mum epoxy sections stained with toluidine blue (the stain osmicated the bacteria blue) revealed areas of dark-blue stained bacterial biofilm also scalloping the inner margin of the bone next to the marrow space (Fig. 2-B). Additional thin sectioning of these areas that we examined by TEM revealed bacterial invasion of an osteocyte's lacunar space and into its submicron canaliculus (Fig. 2-C, red arrow). A remarkable finding at the end of this canaliculus was 2 deformed submicron-sized bacteria displaying evidence of septal plane binary fission (Fig. 2-D, red arrow). A higher magnification of the osteocyte displayed in Figure 2-C (Fig. 2-E) and a neighboring osteocyte (Fig. 2-F) display apoptosis of their nuclei, as well as document bacterial invasion into their lacunar spaces. Additional scrutiny of the same epoxy-embedded block of bone and additional thin sections also revealed deformed rod-shaped bacteria within a submicron canaliculus near the marrow cavity (Fig. 2-G). The higher magnification of the boxed area in Figure 2-G displays the submicron measurement of these rod-shaped bacteria (Fig. 2-H).