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.

Some individuals with autism spectrum disorder (ASD) carry functional mutations rarely observed in the general population. We explored the genes disrupted by these variants from joint analysis of protein-truncating (PTV), missense, and copy number variants (CNVs) in a cohort of 63,237 individuals. We discovered 72 ASD risk genes at false discovery rate (FDR)≤0.001 (185 at FDR≤0.05). De novo PTVs, damaging missense variants, and CNVs represented 57.5%, 21.1%, and 8.44% of association evidence, while CNVs conferred greatest relative risk. Meta-analysis with cohorts ascertained for developmental delay (DD, N=91,605) yielded 373 ASD/DD risk genes at FDR≤0.001 (664 at FDR≤0.05), some of which differed in relative frequency of mutation between ASD and DD. The DD-associated genes were enriched in transcriptomes of progenitor and immature neuronal cells whereas genes displaying stronger evidence in ASD were more enriched in maturing neurons and overlapped with schizophreniaassociated genes, emphasizing that these neuropsychiatric disorders share common pathways to risk.

Staphylococcus aureus infection of bone is challenging to treat because it colonizes the osteocyte lacuno-canalicular network (OLCN) of cortical bone. To elucidate factors involved in OLCN invasion and identify novel drug targets, we completed a hypothesis-driven screen of 24 S. aureus transposon insertion mutant strains for their ability to propagate through 0.5 μm-sized pores in the Microfluidic Silicon Membrane Canalicular Arrays (μSiM-CA), developed to model S. aureus invasion of the OLCN. This screen identified the uncanonical S. aureus transpeptidase, penicillin binding protein 4 (PBP4), as a necessary gene for S. aureus deformation and propagation through nanopores. In vivo studies revealed that Δpbp4 infected tibiae treated with vancomycin showed a significant 12-fold reduction in bacterial load compared to WT infected tibiae treated with vancomycin (p<0.05). Additionally, Δpbp4 infected tibiae displayed a remarkable decrease in pathogenic bone-loss at the implant site with and without vancomycin therapy. Most importantly, Δpbp4 S. aureus failed to invade and colonize the OLCN despite high bacterial loads on the implant and in adjacent tissues. Together, these results demonstrate that PBP4 is required for S. aureus colonization of the OLCN and suggest that inhibitors may be synergistic with standard of care antibiotics ineffective against bacteria within the OLCN.

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.