Evidence of Staphylococcus Aureus Deformation, Proliferation, and Migration in Canaliculi of Live Cortical Bone in Murine Models of Osteomyelitis

Abstract

Although Staphylococcus aureus osteomyelitis is considered to be incurable, the major bacterial reservoir in live cortical bone has remained unknown. In addition to biofilm bacteria on necrotic tissue and implants, studies have implicated intracellular infection of osteoblasts and osteocytes as a mechanism of chronic osteomyelitis. Thus, we performed the first systematic transmission electron microscopy (TEM) studies to formally define major reservoirs of S. aureus in chronically infected mouse (Balb/c J) long bone tissue. Although rare, evidence of colonized osteoblasts was found. In contrast, we readily observed S. aureus within canaliculi of live cortical bone, which existed as chains of individual cocci and submicron rod-shaped bacteria leading to biofilm formation in osteocyte lacunae. As these observations do not conform to the expectations of S. aureus as non-motile cocci 1.0 to 1.5 μm in diameter, we also performed immunoelectron microscopy (IEM) following in vivo BrdU labeling to assess the role of bacterial proliferation in canalicular invasion. The results suggest that the deformed bacteria: (1) enter canaliculi via asymmetric binary fission; and (2) migrate toward osteocyte lacunae via proliferation at the leading edge. Additional in vitro studies confirmed S. aureus migration through a 0.5-μm porous membrane. Collectively, these findings define a novel mechanism of bone infection, and provide possible new insight as to why S. aureus implant-related infections of bone tissue are so challenging to treat. © 2016 American Society for Bone and Mineral Research.

Keywords: CANALICULAR SYSTEM; CORTICAL BONE; DUROTAXIS; ELECTRON MICROSCOPY; HAPTOTAXIS; OSTEOMYELITIS; STAPHYLOCOCCUS AUREUS.

Figure 1. TEM evidence of submicron-elongated S. aureus in the osteocytic lacunar-canaliculi network of infected live bone tissue

Long bones from mice (n = 5) infected with a UAMS-1 contaminated tibial pin (A-F, H, I), or a USA300 infected femoral osteotomy (G), were harvested on day 14 post-infection for TEM. (A) Low magnification TEM image (x 4,000) of UAMS-1 invasion of live bone tissue (note osteocyte OC) in a canaliculus (green arrow) communicating with the marrow cavity (MC). Also note the proximal neutrophils (yellow arrow) within the marrow. (B) Low magnification TEM image (x 4,000) of UAMS-1 invasion of an osteocytic lacunar-canaliculus adjacent to a channel infected with S. aureus (arrows) containing necrotic cells (*). Higher magnification TEM images (C x 8,000; D x10,000) of UAMS-1 colonization of osteocytic lacunae. (E) Low magnification TEM image of three parallel canaliculi in various states of colonization (1-severely infected, 2-moderately infected, and 3-uninfected) by the invading UAMS-1 within the live cortical bone (x 3,500). (F) Higher magnification TEM image measuring submicron-elongated UAMS-1 (x 15,000). (G) Similar bacterial invasion of canaliculi adjacent to the osteotomy (red arrow), and neutrophils in the marrow cavity (*) were observed in USA300 infected femurs (x 4,000), but not in long bones that received sterile implants (data not shown). (H) Low magnification TEM image (x 4,000) documenting cortical bone damage adjacent to the infected tibia pin (red arrows), and a cavity filled with UAMS-1 (yellow bracket) that leads to a canaliculus (black arrow). (I) High magnification TEM of the infected cavity in H demonstrating mitotic S. aureus in the live cortical bone (x 25,000). Note that only the bacterium entering the canaliculus has an asymmetric septal plane (red arrows), which is aligned perpendicularly with the canaliculus orifice, perhaps to anchor and propel the emerging daughter cell into the submicron channel in the cortical bone during binary fission.

Figure 2. In vivo and in vitro evidence of proliferating S. aureus through canaliculi and submicron pores

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.