Evolving concepts in bone infection: redefining "biofilm", "acute vs. chronic osteomyelitis", "the immune proteome" and "local antibiotic therapy"

Abstract

Osteomyelitis is a devastating disease caused by microbial infection of bone. While the frequency of infection following elective orthopedic surgery is low, rates of reinfection are disturbingly high. Staphylococcus aureus is responsible for the majority of chronic osteomyelitis cases and is often considered to be incurable due to bacterial persistence deep within bone. Unfortunately, there is no consensus on clinical classifications of osteomyelitis and the ensuing treatment algorithm. Given the high patient morbidity, mortality, and economic burden caused by osteomyelitis, it is important to elucidate mechanisms of bone infection to inform novel strategies for prevention and curative treatment. Recent discoveries in this field have identified three distinct reservoirs of bacterial biofilm including: Staphylococcal abscess communities in the local soft tissue and bone marrow, glycocalyx formation on implant hardware and necrotic tissue, and colonization of the osteocyte-lacuno canalicular network (OLCN) of cortical bone. In contrast, S. aureus intracellular persistence in bone cells has not been substantiated in vivo, which challenges this mode of chronic osteomyelitis. There have also been major advances in our understanding of the immune proteome against S. aureus, from clinical studies of serum antibodies and media enriched for newly synthesized antibodies (MENSA), which may provide new opportunities for osteomyelitis diagnosis, prognosis, and vaccine development. Finally, novel therapies such as antimicrobial implant coatings and antibiotic impregnated 3D-printed scaffolds represent promising strategies for preventing and managing this devastating disease. Here, we review these recent advances and highlight translational opportunities towards a cure.

Keywords: Bone; Diseases.

Fig. 1

Removal of necrotic bone and biofilm contaminated components during revision surgery for MRSA-infected total joint replacements (TJR). a–c The indications for this single-stage revision for a MRSA-infected total hip replacement is shown. a Radiographic evidence of the septic TJR in the pre-op X-ray are periosteal reaction and a non-united femoral fracture (yellow arrows). b The open infected thigh requires removal of necrotic soft tissue and white (dead) bone, adjacent to live (red) bone that needs to be retained for successful limb salvage. Complete removal of the dead bone, cement, and necrotic tissues creates a healthier environment for the new prosthesis. c Post-op X-ray of the femoral defect with modular hip prosthesis. d–g Bacterial biofilm on explanted hardware components. Photographs of the surface of a femoral total knee replacement component before (d) and after (e) osmium tetroxide staining identifying bacterial biofilm on the bone cement. f SEM of the explanted hardware reveals biofilm bacteria (yellow arrow) on the surface of the implant (x10 000) and g bacteria attached to fibrin on the explanted hardware (x10 000)

Fig. 2

Three distinct reservoirs of bacteria in chronic osteomyelitis. Chronic implant-associated osteomyelitis was established in mice with S. aureus as previously described,^,,, and the bacterial burden: (1) in Staphylococcus abscess communities (SACs) assessed by histology (a–e), (2) on the implant assessed by SEM (f–j), and (3) in cortical bone assessed by TEM (k–m) is shown. Micrographs of orange G/alcian blue-stained histology of tibiae 7 days (a) and 14 days (c) post-infection are shown highlighting SACs (arrows) in the bone marrow and adjacent soft tissues. The boxed regions in (a), (c) are shown in Brown and Brenn-stained parallel section (b, d) to highlight the Gram+ bacteria (dark blue) surrounded by dead and dying neutrophils following NETosis (red cells), which are surrounded by a ring of macrophages (white layer). Chronic infection is clearly established by day 14, as evidenced by the complete replacement of hematopoietic bone marrow (BM) with inflammatory tissue, and the presence of M2 macrophages (brown cells) surrounding the SAC, as seen by immunostaining with antibody against arginase-1 (e). Biofilm formation on the implant commences with planktonic bacterial adhesion (f), as illustrated in this case of in vitro S. aureus attachment onto a stainless-steel wire incubated in a flow chamber system (×10 000). Following transtibial implantation, the planktonic bacteria rapidly transition to biofilm (g), seen as uniform glycocalyx coating the stainless-steel pin 14 days post-op (×200). High power images of the biofilm on the implant reveal cocci adhering to fibrin strands (h, ×2 500), and clusters of S. aureus forming bacterial pods (i, ×5 000). By day 14 post-infection, bacterial emigration from the pod is complete, as evidenced by the empty lacunae (j, ×30 000). S. aureus colonization of cortical bone commences with eradication of bone lining cells to expose canaliculi (blue arrows) leading to an embedded osteocyte (OC) (k, ×6 000). Subsequently, S. aureus invasion and propagation through osteocyte lacuno-canalicular network (OLCN) renders the biofilm bacteria (*) inaccessible to activated neutrophils outside the bone (blue arrows) (l, ×1 800). The uninhibited bacteria demineralize and consume the cortical bone to expands a canaliculus, and propagate into neighboring canaliculi (yellow arrow), to reach a distant osteocyte (red arrow). m High power TEM (×12 000) of the osteocyte in (l) killed by S. aureus bacterial occupation of its lacunar space

Fig. 3

The role of S. aureus intracellular infection as a virulence mechanism in chronic osteomyelitis. Extensive TEM analyses of S. aureus-infected human bone samples (n > X) failed to identify significant evidence of viable bone cells (osteoblasts, osteoclasts, osteocytes) containing intracellular bacteria, while all S. aureus colonized OLCN contain necrotic osteocytes (OC) with extracellular bacteria (red arrows) within osteocyte lacunae (a, TEM ×15 000). In contrast, an acridine orange-stained smears of blood, harvested post-mortem from a patient that died from septic multiorgan failure, demonstrates both extracellular bacteria (orange) and colonized leukocytes (yellow cells) via fluorescent microscopy (b, ×20). c A higher power fluorescent image of the blood smear reveals a “Trojan horse” macrophage with cytoplasmic S. aureus, and acentric nucleus (fluorescent green). d TEM (×20 000) of this Trojan horse macrophage was performed via a “pop-off” technique, which confirmed intracellular S. aureus cocci within the cytoplasm, adjacent to the nucleus (yellow arrow)

Fig. 4

Histologic features of “acute” and “chronic” osteomyelitis exist in the same lesion. Hematoxylin and eosin-stained, paraffin-embedded, decalcified section of an infected metatarsal bone resected from a patient with a diabetic foot ulcer is shown, illustrating salient features of both acute and chronic osteomyelitis in the same bone. a Low power micrograph of the lesion in which most of the trabecular bone in this part of the metatarsus has been destroyed and replaced by an acute inflammatory reaction, consisting of neutrophils (*) and fibrovascular granulation tissue (black arrow) (scale bar = 1 mm). The inflammation extends to the bone beneath the articular cartilage (yellow arrow) and has destroyed much of the cortical bone (white arrow). Reactive new bone has formed in the lower part of the image, along with a chronic inflammatory and fibrovascular reaction. b A region of interest of acute inflammation (white box in a) is shown highlighting a fragment of dead cortical bone surround by neutrophils (black arrow), with an associated fibrinous exudate, which are hallmarks of acute osteomyelitis (scale bar = 25 mm). c A region of interest of chronic inflammation (black box in a) showing new bone formation (black arrow), and replacement of normal bone marrow with fibrovascular inflammatory tissue (boxed region) (scale bar = 50 mm). d This region of interest (boxed area in c) is presented at high power, showing blood vessels, osteoblasts rimming newly formed woven bone (bottom right), and collections of lymphocytes and plasma cells (arrows), which are characteristic of chronic osteomyelitis (scale bar = 25 mm)

Fig. 5

A diagnostic and prognostic immunoassay for the measurement of anti-S. aureus antibody levels in patients with osteomyelitis. a Schematic illustration of production of serum and isolation of medium enriched for newly synthesized anti-S. aureus antibodies (MENSA) from peripheral mononuclear cells of patients with osteomyelitis. b Anti-S. aureus antibody levels in serum and MENSA were determined using a custom bead-based multiantigen Luminex immunoassay developed by our group. Here, we examined anti-S. aureus IgG responses in serum and MENSA of diabetic foot infections (DFI) patients undergoing foot salvage antimicrobial therapy (FST). The change in antibody titers over the course of FST of a representative patient whose DFI was negative for S. aureus, a patient with S. aureus infection that responded to FST, and a patient with S. aureus DFI who failed FST is presented here. Remarkably, MENSA levels faithfully reflected the S. aureus infection over time while serum levels remained unchanged (see Oh et al. for more details)