Biofilm Producing Staphylococcus epidermidis (RP62A Strain) Inhibits Osseous Integration Without Osteolysis and Histopathology in a Murine Septic Implant Model.

Despite its presence in orthopaedic infections, Staphylococcus epidermidis's ability to directly induce inflammation and bone destruction is unknown. Thus, we compared a clinical strain of methicillin-resistant biofilm-producing S. epidermidis (RP62A) to a highly virulent and osteolytic strain of methicillin-resistant Staphylococcus aureus (USA300) in an established murine implant-associated osteomyelitis model. Bacterial burden was assessed by colony forming units (CFUs), tissue damage was assessed by histology and micro-computed tomography, biofilm was assessed by scanning electron microscopy (SEM), host gene expression was assessed by quantitative polymerase chain reaction, and osseous integration was assessed via biomechanical push-out test. While CFUs were recovered from RP62A-contaminated implants and surrounding tissues after 14 days, the bacterial burden was significantly less than USA300-infected tibiae (p < 0.001). In addition, RP62A failed to produce any of the gross pathologies induced by USA300 (osteolysis, reactive bone formation, Staphylococcus abscess communities, marrow necrosis, and biofilm). However, fibrous tissue was present at the implant-host interface, and rigorous SEM confirmed the rare presence of cocci on RP62A-contaminated implants. Gene expression studies revealed that IL-1ß, IL-6, RANKL, and TLR-2 mRNA levels in RP62A-infected bone were increased versus Sterile controls. Ex vivo push-out testing showed that RP62A-infected implants required significantly less force compared with the Sterile group (7.5 ± 3.4 vs. 17.3 ± 4.1 N; p < 0.001), but required 10-fold greater force than USA300-infected implants (0.7 ± 0.3 N; p < 0.001). Taken together, these findings demonstrate that S. epidermidis is a commensal pathogen whose mechanisms to inhibit osseous integration are limited to minimal biofilm formation on the implant, and low-grade inflammation. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:852-860, 2020.

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

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.

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

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.

Quantifying the natural history of biofilm formation in vivo during the establishment of chronic implant-associated Staphylococcus aureus osteomyelitis in mice to identify critical pathogen and host factors.

While it is well known that Staphylococcus aureus establishes chronic implant-associated osteomyelitis by generating and persisting in biofilm, research to elucidate pathogen, and host specific factors controlling this process has been limited due to the absence of a quantitative in vivo model. To address this, we developed a murine tibia implant model with ex vivo region of interest (ROI) imaging analysis by scanning electron microscopy (SEM). Implants were coated with Staphylococcus aureus strains (SH1000, UAMS-1, USA300LAC) with distinct in vitro biofilm phenotypes, were used to infect C57BL/6 or Balb/c mice. In contrast to their in vitro biofilm phenotype, results from all bacteria strains in vivo were similar, and demonstrated that biofilm on the implant is established within the first day, followed by a robust proliferation phase peaking on Day 3 in Balb/c mice, and persisting until Day 7 in C57BL/6 mice, as detected by SEM and bioluminescent imaging. Biofilm formation peaked at Day 14, covering ∼40% of the ROI coincident with massive agr-dependent bacterial emigration, as evidenced by large numbers of empty lacunae with few residual bacteria, which were largely culture negative (80%) and PCR positive (87.5%), supporting the clinical relevance of this implant model.

Passive immunization with anti-glucosaminidase monoclonal antibodies protects mice from implant-associated osteomyelitis by mediating opsonophagocytosis of Staphylococcus aureus megaclusters.

Towards the development of a methicillin-resistant Staphylococcus aureus (MRSA) vaccine we evaluated a neutralizing anti-glucosaminidase (Gmd) monoclonal antibody (1C11) in a murine model of implant-associated osteomyelitis, and compared its effects on LAC USA300 MRSA versus a placebo and a Gmd-deficient isogenic strain (ΔGmd). 1C11 significantly reduced infection severity, as determined by bioluminescent imaging of bacteria, micro-CT assessment of osteolysis, and histomorphometry of abscess numbers (p < 0.05). Histology also revealed infiltrating macrophages, and the complete lack of staphylococcal abscess communities (SAC), in marrow abscesses of 1C11 treated mice. In vitro, 1C11 had no direct effects on proliferation, but electron microscopy demonstrated that 1C11 treatment phenocopies ΔGmd defects in binary fission. Moreover, addition of 1C11 to MRSA cultures induced the formation of large bacterial aggregates (megaclusters) that sedimented out of solution, which was not observed in ΔGmd cultures or 1C11 treated cultures of a protein A-deficient strain (ΔSpa), suggesting that the combined effects of Gmd inhibition and antibody-mediated agglutination are required. Finally, we demonstrated that macrophage opsonophagocytosis of MRSA and megaclusters is significantly increased by 1C11 (p < 0.01). Collectively, these results suggest that the primary mechanism of anti-Gmd humoral immunity against MRSA osteomyelitis is macrophage invasion of Staphylococcal abscess communities (SAC) and opsonophagocytosis of megaclusters. .

MyD88 signaling regulates both host defense and immunopathogenesis during pneumocystis infection.

The immune response protects against Pneumocystis infection but is also a key component of Pneumocystis pneumonia (PcP)-related immunopathogenesis. Signaling through myeloid differentiation factor 88 (MyD88) is critical for activation of immune pathways downstream of TLRs and IL-1R. To determine whether MyD88 regulates normal host defense against Pneumocystis, nonimmunosuppressed wild-type (WT) and MyD88-deficient mice were infected. MyD88(-/-) mice had higher early Pneumocystis burdens than did WT mice but mounted an effective adaptive immune response and cleared Pneumocystis similarly to WT. However, MyD88(-/-) mice displayed a more intense and prolonged pulmonary immune response than did WT mice. To determine the role of MyD88 in the development of PcP-related immunopathogenesis, WT and MyD88(-/-) mice were rendered susceptible to PcP by depletion of CD4(+) T cells. At 4 wk postinfection, CD4-depleted WT and MyD88(-/-) mice harbored similar organism burdens, but MyD88(-/-) mice were protected from the PcP-related respiratory impairment observed in WT mice. Improved pulmonary physiology in MyD88(-/-) mice correlated with lower lung CCL2 levels and reduced cell recruitment. However, by 5 wk postinfection, the overall health of MyD88(-/-) mice began to deteriorate rapidly relative to WT, with accelerated weight loss, impaired lung function, and exacerbated alveolar inflammation. This physiological decline of MyD88(-/-) mice was associated with increased TNF-α and IFN-γ in the lung, and by the inability to control Pneumocystis burden. Thus, MyD88 is not required for resistance to Pneumocystis infection, but limits the adaptive immune response in immunocompetent mice. In the setting of active PcP, MyD88 signaling contributes to both immunopathogenesis and control of fungal burden.

The alveolar epithelial cell chemokine response to pneumocystis requires adaptor molecule MyD88 and interleukin-1 receptor but not toll-like receptor 2 or 4.

Pneumocystis is an opportunistic fungal pathogen that causes pneumonia in a variety of clinical settings. An early step in Pneumocystis infection involves the attachment of organisms to alveolar epithelial cells (AECs). AECs produce chemokines in response to Pneumocystis stimulation, but the upstream host-pathogen interactions that activate AEC signaling cascades are not well-defined. MyD88 is an adaptor molecule required for activation of proinflammatory signaling cascades following Toll-like receptor (TLR)-dependent recognition of conserved molecular patterns on pathogens. To determine whether the TLR/MyD88 pathway is required for the AEC chemokine response to Pneumocystis, wild-type (WT) and MyD88-deficient AECs were incubated with Pneumocystis. As expected, WT AECs produced CCL2 and CXCL2 following Pneumocystis stimulation. In contrast, MyD88-deficient AECs were severely impaired in their ability to respond to Pneumocystis. MyD88-deficient AECs did not display Pneumocystis-induced Jun N-terminal protein kinase activation and produced much less chemokine than Pneumocystis-stimulated WT AECs. Using a panel of TLR agonists, primary murine AECs were found to respond vigorously to TLR2 and TLR4 agonists. However, the AEC chemokine response to Pneumocystis did not require TLR2 or TLR4. Surprisingly, the interleukin-1 receptor (IL-1R) was required for an AEC chemokine response to Pneumocystis. The role of MyD88 in early responses during Pneumocystis infection was supported by in vivo studies demonstrating that MyD88-deficient mice showed impaired Pneumocystis-stimulated chemokine production and impaired inflammatory cell recruitment. These data indicate an important role for MyD88 in the AEC inflammatory response to Pneumocystis.

Sustained high-titer antibody responses induced by conjugating a malarial vaccine candidate to outer-membrane protein complex.

The development of protein subunit vaccines to combat some of the world's deadliest pathogens such as a malaria parasite, Plasmodium falciparum, is stalled, due in part to the inability to induce and sustain high-titer antibody responses. Here, we show the induction of persistent, high-titer antibody responses to recombinant Pfs25H, a human malarial transmission-blocking protein vaccine candidate, after chemical conjugation to the outer-membrane protein complex (OMPC) of Neisseria meningitidis serogroup B and adsorption to aluminum hydroxyphosphate. In mice, the Pfs25H-OMPC conjugate vaccine was >1,000 times more potent in generating anti-Pfs25H ELISA reactivity than a similar 0.5-microg dose of Pfs25H alone in Montanide ISA720, a water-in-oil adjuvant. The immune enhancement requires covalent conjugation between Pfs25H and the OMPC, given that physically mixed Pfs25H and OMPC on aluminum hydroxyphosphate failed to induce greater activity than the nonconjugated Pfs25H on aluminum hydroxyphosphate. The conjugate vaccine Pfs25H-OMPC also was highly immunogenic in rabbits and rhesus monkeys. In rhesus monkeys, the antibody responses were sustained over 18 months, at which time another vaccination with nonconjugated Pfs25H induced strong anamnestic responses. The vaccine-induced anti-Pfs25-specific antibodies in all animal species blocked the transmission of parasites to mosquitoes. Protein antigen conjugation to OMPC or other protein carrier may have general application to a spectrum of protein subunit vaccines to increase immunogenicity without the need for potentially reactogenic adjuvants.