Distinct vasculotropic versus osteotropic features ofS. agalactiaeversusS. aureusimplant‐associated bone infection in mice

Abstract

Osteomyelitis is a devastating complication of orthopaedic surgery and commonly caused by Staphylococcus aureus (S. aureus) and Group B Streptococcus (GBS, S. agalactiae). Clinically, S. aureus osteomyelitis is associated with local inflammation, abscesses, aggressive osteolysis, and septic implant loosening. In contrast, S. agalactiae orthopaedic infections generally involve soft tissue, with acute lifethreatening vascular spread. While preclinical models that recapitulate the clinical features of S. aureus bone infection have proven useful for research, no animal models of S. agalactiae osteomyelitis exist. Here, we compared the pathology caused by these bacteria in an established murine model of implant-associated osteomyelitis. In vitro scanning electron microscopy and CFU quantification confirmed similar implant inocula for both pathogens (~10 5 CFU/pin). Assessment of mice at 14 days post-infection demonstrated increased S. aureus virulence, as S. agalactiae infected mice had significantly greater body weight, and fewer CFU on the implant and in bone and adjacent soft tissue (p < 0.05). X-ray, µCT, and histologic analyses showed that S. agalactiae induced significantly less osteolysis and implant loosening, and fewer large TRAP + osteoclasts than S. aureus without inducing intraosseous abscess formation. Most notably, transmission electron microscopy revealed that although both bacteria are capable of digesting cortical bone, S. agalactiae have a predilection for colonizing blood vessels embedded within cortical bone while S. aureus primarily colonizes the osteocyte lacuno-canalicular network. This study establishes the first quantitative animal model of S. agalactiae osteomyelitis, and demonstrates a vasculotropic mode of S. agalactiae infection, in contrast to the osteotropic behavior of S. aureus osteomyelitis.

Staphylococcus aureus Cell Wall Biosynthesis Modulates Bone Invasion and Osteomyelitis Pathogenesis

“…All animal studies were performed in accordance with protocols approved by the University Committee on Animal Resources at the University of Rochester Medical Center and in accordance with the Animal Welfare Act. Surgeries were performed as previously described (Li et al, 2008;Varrone et al, 2014;Masters et al, 2020Masters et al, , 2021. Six-week-old, female Balb/C mice were purchased from Jackson Laboratories (Bar Harbor, ME, United States) and were acclimated for 1 week prior to surgery.…”

“…On day 14 postinfection, mice were sacrificed, and X-ray images were obtained to evaluate postinfection septic implant loosening. Pins were determined to be stably intact within the tibia as they were originally implanted or entirely dislodged from the tibia, as previously described (Masters et al, 2020(Masters et al, , 2021. Tibia, implant, and soft tissue were harvested and placed in sterile phosphatebuffered saline (PBS) on ice for immediate colony-forming unit (CFU) quantification and subsequent cytokine quantification or placed in neutral buffered formalin (NBF) for subsequent microcomputed tomography (µCT) imaging, followed by histology and transmission electron microscopy (TEM).…”

“…Tissue and implant CFUs were quantified as previously described (Masters et al, 2020(Masters et al, , 2021. Infected tibia, implant, and adjacent soft tissue were harvested following animal sacrifice and placed in sterile PBS on ice.…”

“…It is not only the most prevalent pathogen in implant-associated osteomyelitis (Arciola et al, 2005;Pulido et al, 2008) but also the most destructive. Moreover, S. aureus infection of the bone is considered very difficult to cure (Tomizawa et al, 2020;Masters et al, 2021), due to specific mechanisms that enable bacterial survival within the implant and bone microenvironment following revision surgery and antibiotic therapy [reviewed in Masters et al (2019b) and Muthukrishnan et al (2019)].…”

Biphasic effects of IL-27 during Staphylococcus aureus implant-associated osteomyelitis in mice

“…Having established an association between IL-27 and S. aureus osteomyelitis in patients and mice, we next examined if IL-27 mediates bacterial clearance during S. aureus osteomyelitis using our wellestablished murine model of osteomyelitis [32][33][34][35][36]. Mice were challenged with bioluminescent MRSA (USA300 LAC::lux ) via transtibial implantation of a contaminated stainless-steel implant following intramuscular injection of rAAV-IL-27 or adeno-associated virus expressing recombinant GFP (rAAV-GFP, control).…”