More Is Not Always Better-the Double-Headed Role of Fibronectin in Staphylococcus aureus Host Cell Invasion.

While Staphylococcus aureus has classically been considered an extracellular pathogen, these bacteria are also capable of being taken up by host cells, including nonprofessional phagocytes such as endothelial cells, epithelial cells, or osteoblasts. The intracellular S. aureus lifestyle contributes to infection development. The predominant recognition and internalization pathway appears to be the binding of the bacteria via a fibronectin bridge to the α5ß1-integrin on the host cell membrane, followed by phagocytosis. Although osteoblasts showed high expression of α5ß1-integrin and fibronectin, and bacteria adhered to osteoblasts to a high proportion, here we demonstrate by internalization assays and immunofluorescence microscopy that S. aureus was less engulfed in osteoblasts than in epithelial cells. The addition of exogenous fibronectin during the infection of cells with S. aureus resulted in an increased uptake by epithelial cells but not by osteoblasts. This contrasts with the previous conception of the uptake mechanism, where high expression of integrin and fibronectin would promote the bacterial uptake into host cells. Extracellular fibronectin surrounding osteoblasts, but not epithelial cells, is organized in a fibrillary network. The inhibition of fibril formation, the short interfering RNA-mediated reduction of fibronectin expression, and the disruption of the fibronectin-fibril meshwork all resulted in a significant increase in S. aureus uptake by osteoblasts. Thus, the network of fibronectin fibrils appears to strongly reduce the uptake of S. aureus into a given host cell, indicating that the supramolecular structure of fibronectin determines the capacity of particular host cells to internalize the pathogen. IMPORTANCE Traditionally, Staphylococcus aureus has been considered an extracellular pathogen. However, among other factors, the frequent failure of antimicrobial therapy and the ability of the pathogen to cause recurrent disease have established the concept of eukaryotic invasion of the pathogen, thereby evading the host's immune system. In the current model of host cell invasion, bacteria initially bind to α5ß1 integrin on the host cell side via a fibronectin bridge, which eventually leads to phagocytosis of S. aureus by host cells. However, in this study, we demonstrate that not the crude amount but the supramolecular structure of fibronectin molecules deposited on the eukaryotic cell surface plays an essential role in bacterial uptake by host cells. Our findings explain the large differences of S. aureus uptake efficacy in different host cell types as well as in vivo differences between courses of bacterial infections and the localization of bacteria in different clinical settings.

Staphylococcus aureus internalization impairs osteoblastic activity and early differentiation process.

Staphylococcus aureus is the most frequent aetiology of bone and joint infections (BJI) and can cause relapsing and chronic infections. One of the main factors involved in the chronicization of staphylococcal BJIs is the internalization of S. aureus into osteoblasts, the bone-forming cells. Previous studies have shown that S. aureus triggers an impairment of osteoblasts function that could contribute to bone loss. However, these studies focused mainly on the extracellular effects of S. aureus. Our study aimed at understanding the intracellular effects of S. aureus on the early osteoblast differentiation process. In our in vitro model of osteoblast lineage infection, we first observed that internalized S. aureus 8325-4 (a reference lab strain) significantly impacted RUNX2 and COL1A1 expression compared to its non-internalized counterpart 8325-4∆fnbAB (with deletion of fnbA and fnbB). Then, in a murine model of osteomyelitis, we reported no significant effect for S. aureus 8325-4 and 8325-4∆fnbAB on bone parameters at 7 days post-infection whereas S. aureus 8325-4 significantly decreased trabecular bone thickness at 14 days post-infection compared to 8325-4∆fnbAB. When challenged with two clinical isogenic strains isolated from initial and relapse phase of the same BJI, significant impairments of bone parameters were observed for both initial and relapse strain, without differences between the two strains. Finally, in our in vitro osteoblast infection model, both clinical strains impacted alkaline phosphatase activity whereas the expression of bone differentiation genes was significantly decreased only after infection with the relapse strain. Globally, we highlighted that S. aureus internalization into osteoblasts is responsible for an impairment of the early differentiation in vitro and that S. aureus impaired bone parameters in vivo in a strain-dependent manner.

Patient-specific effects of soluble factors from Staphylococcus aureus and Staphylococcus epidermidis biofilms on osteogenic differentiation of primary human osteoblasts.

Due to the frequency of biofilm-forming Staphylococcus aureus and Staphylococcus epidermidis in orthopedics, it is crucial to understand the interaction between the soluble factors produced by prokaryotes and their effects on eukaryotes. Our knowledge concerning the effect of soluble biofilm factors (SBF) and their virulence potential on osteogenic differentiation is limited to few studies, particularly when there is no direct contact between prokaryotic and eukaryotic cells. SBF were produced by incubating biofilm from S. aureus and S. epidermidis in osteogenic media. Osteoblasts of seven donors were included in this study. Our results demonstrate that the detrimental effects of these pathogens do not require direct contact between prokaryotic and eukaryotic cells. SBF produced by S. aureus and S. epidermidis affect the metabolic activity of osteoblasts. However, the effect of SBF derived from S. aureus seems to be more pronounced compared to that of S. epidermidis. The influence of SBF of S. aureus and S. epidermidis on gene expression of COL1A1, ALPL, BGLAP, SPP1, RUNX2 is bacteria-, patient-, concentration-, and incubation time dependent. Mineralization was monitored by staining the calcium and phosphate deposition and revealed that the SBF of S. epidermidis markedly inhibits calcium deposition; however, S. aureus shows a less inhibitory effect. Therefore, these new findings support the hypotheses that soluble biofilm factors affect the osteogenic processes substantially, particularly when there is no direct interaction between bacteria and osteoblast.

Staphylococcus aureus ST228 and ST239 as models for expression studies of diverse markers during osteoblast infection and persistence.

The ability of S. aureus to infect bone and osteoblasts is correlated with its incredible virulence armamentarium that can mediate the invasion/internalization process, cytotoxicity, membrane damage, and intracellular persistence. We comparatively analyzed the interaction, persistence, and modulation of expression of selected genes and cell viability in an ex vivo model using human MG-63 osteoblasts of two previously studied and well-characterized S. aureus clinical strains belonging to the ST239-SCCmecIII-t037 and ST228-SCCmecI-t041 clones at 3 h and 24 h post-infection (p.i). S. aureus ATCC12598 ST30-t076 was used as a control strain. Using imaging flow cytometry (IFC), we found that these strains invaded and persisted in MG-63 osteoblasts to different extents. The invasion was evaluated at 3 h p.i and persistence at 24 h p.i., in particular: ATCC12598 internalized in 70% and persisted in 50% of MG-63 cells; ST239-SCCmecIII internalized in 50% and persisted in 45% of MG-63 cells; and ST228-SCCmecI internalized in 30% and persisted in 20% of MG-63 cells. During the infection period, ST239-III exerted significant cytotoxic activity resulting from overexpression of hla and psmA and increased expression of the genes involved in adhesion, probably due to the release and re-entry of bacteria inside MG-63 cells at 24 h p.i. The lower invasiveness of ST228-I was also associated with non-cytotoxic activity inside osteoblasts. This clone was unable to activate sufficient cellular reaction and succumbed inside MG-63 cells. Our findings support the idea of considering new strategies, based on a translational approach-eukaryotic host-pathogen interaction (EHPI)-and to be applied on a large scale, to predict S. aureus /osteoblast interaction and treat bone infections. Such strategies rely on the study of the genetic and biochemical basis of both pathogen and host.

Phagocytosis and macrophage polarization on bacterially contaminated dental implant materials and effects on tissue integration.

Bacterial contamination is hard to avoid during dental implant surgery. Macrophages and their polarisation play a decisive role in bacterial colonisation and tissue integration on bacterially contaminated dental implants. The present study investigated the role of macrophages in stimulating tissue coverage overgrowth of contaminating oral bacteria on polished titanium (Ti-P) and acid-etched zirconium dioxide (ZrO2-MA) dental implant materials. Different co-culture models were employed to determine phagocytosis rates of Streptococcus mitis or Staphylococcus aureus contaminating a dental implant surface and the influence of contaminating bacteria and osteoblasts (U2OS) on macrophage polarisation. S. aureus was phagocytized in higher numbers than S. mitis in bi-cultures on smooth Ti-P surfaces. Contaminating S. mitis stimulated near full polarisation of macrophages from a non-Ym1-expressing- to a Ym1-expressing-phenotype on smooth Ti-P, but on ZrO2-MA both phenotypes occurred. In tri-cultures with U2OS-cells on smooth Ti-P, a larger percentage of macrophages remained in their non-Ym1-expressing, "fighting" M1-like phenotype to clear Ti-P surfaces from contaminating bacteria. On ZrO2-MA surfaces, more macrophages tended towards their "fix- and-repair" M2-like phenotype than on Ti-P surfaces. Surface coverage of smooth, bacterially contaminated Ti-P surfaces by U2OS-cells was more effectively stimulated by fighting, M1-like macrophages than on ZrO2-MA surfaces. Comprehensive guidelines are provided for the development of infection-resistant, dental implant materials, including bacteria, tissue and immune cells. These guidelines point to more promising results for clinical application of Ti-P as compared with ZrO2-MA.

Different Modulatory Effects of Four Methicillin-Resistant Staphylococcus aureus Clones on MG-63 Osteoblast-Like Cells.

Staphylococcus aureus is a Gram-positive bacterium responsible for a variety of mild to life-threatening infections including bone infections such as osteomyelitis. This bacterium is able to invade and persist within non-professional phagocytic cells such as osteoblasts. In the present study, four different S. aureus strains, namely, 2SA-ST239-III (ST239), 5SA-ST5-II (ST5), 10SA-ST228-I (ST228), and 14SA-ST22-IVh (ST22), were tested for their ability to modulate cell viability in MG-63 osteoblast-like cells following successful invasion and persistence. Methicillin-sensitive S. aureus (MSSA) ATCC-12598-ST30 (ST30) was used as control strain. Despite being proven that ST30, ST239, and ST22 have a similar ability to internalize and persist in MG-63 osteoblast-like cells under our experimental conditions, we demonstrated that the observed decrease in cell viability was due to the different behavior of the considered strains, rather than the number of intracellular bacteria. We focused our attention on different biochemical cell functions related to inflammation, cell metabolism, and oxidative stress during osteoblast infections. We were able to show the following: (1) ST30 and ST239 were the only two clones able to persist and maintain their number in the hostile environment of the cell during the entire period of infection; (2) ST239 was the only clone able to significantly increase gene expression (3 and 24 h post-infection (p.i.)) and protein secretion (24 h p.i.) of both interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) in MG-63 osteoblast-like cells; (3) the same clone determined a significant up-regulation of the transforming growth factorbeta 1 (TGF-ß1) and of the metabolic marker glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNAs at 24 h p.i.; and (4) neither the MSSA nor the four methicillin-resistant S. aureus (MRSA) strains induced oxidative stress phenomena in MG-63 cells, although a high degree of variability was observed for the different clones with regard to the expression pattern of nuclear factor E2-related factor 2 (Nrf2) and its downstream gene heme oxygenase 1 (HO-1) activation. Our results may pave the way for an approach to S. aureus-induced damage, moving towards individualized therapeutic strategies that take into account the differences between MSSA and MRSA as well as the distinctive features of the different clones. This approach is based on a change of paradigm in antibiotic therapy involving a case-based use of molecules able to counteract pro-inflammatory cytokines activity such as selective cytokine signaling inhibitors (IL-6, TNF-α).

Investigation of a Staphylococcus argenteus Strain Involved in a Chronic Prosthetic-Joint Infection.

Staphylococcus argenteus is an emerging species responsible for infections comparable to those induced by Staphylococcus aureus. It has been involved in few chronic or persistent infections so far. In this study, we described a case of a persistent prosthetic-joint infection (PJI) affecting a young woman. We investigated in vitro the virulence traits of the incriminated S. argenteus strain (bone cell invasion, biofilm formation and induction of inflammation) and analyzed its genome, in comparison with two other strains of S. argenteus and two S. aureus isolates. It appeared that this S. argenteus PJI strain combined biofilm formation, osteoblast invasion and intracellular persistence abilities together with genes potentially involved in the escape of the host immune defenses, which might explain the chronicization of the infection.

Injectable Chitosan-Based Thermosensitive Hydrogel/Nanoparticle-Loaded System for Local Delivery of Vancomycin in the Treatment of Osteomyelitis.

PURPOSE: Osteomyelitis, particularly chronic osteomyelitis, remains a major challenge for orthopedic surgeons. The traditional treatment for osteomyelitis, which involves antibiotics and debridement, does not provide a complete solution for infection and bone repair. Antibiotics such as vancomycin (VCM) are commonly used to treat osteomyelitis in clinical settings. VCM use is limited by a lack of effective delivery methods that provide sustained, high doses to entirely fill irregular bone tissue to treat infections. METHODS: We engineered a chitosan (CS)-based thermosensitive hydrogel to produce a VCM-nanoparticle (NPs)/Gel local drug delivery system. The VCM-NPs were formed with quaternary ammonium chitosan and carboxylated chitosan nanoparticles (VCM-NPs) by positive and negative charge adsorption to enhance the encapsulation efficiency and drug loading of VCM, with the aim of simultaneously preventing infection and repairing broken bones. This hydrogel was evaluated in a rabbit osteomyelitis model. RESULTS: The VCM-NPs had high encapsulation efficiency and drug loading, with values of 60.1±2.1% and 24.1±0.84%, respectively. When embedded in CS-Gel, the VCM-NPs maintained their particle size and morphology, and the injectability and thermosensitivity of the hydrogel, which were evaluated by injectability test and rheological measurement, were retained. The VCM-NPs/Gel exhibited sustained release of VCM over 26 days. In vitro tests revealed that the VCM-NPs/Gel promoted osteoblast proliferation and activity against Staphylococcus aureus. In vivo, VCM-NPs/Gel (with 10 mg vancomycin per rabbit) was used to treat rabbits with osteomyelitis. The VCM-NPs/Gel showed excellent anti-infection properties and accelerating bone repair under osteomyelitis conditions. CONCLUSION: The reported multifunctional NPs hydrogel system for local antibiotic delivery (VCM-NPs/Gel) showed bone regeneration promotion and anti-infection properties, demonstrating significant potential as a scaffold for effective treatment of osteomyelitis.

The Process of Osteoblastic Infection by Staphylococcus Aureus.

Bone infection is difficult to cure, and relapse frequently occurs, which is a major treatment problem. One of the main reasons for the refractory and recurrent nature of bone infection is that bacteria, such as Staphylococcus aureus (S. aureus), can be internalized into osteoblasts after infecting bone tissue, thereby avoiding attack by the immune system and antibiotics. Understanding how bacteria (such as S. aureus) are internalized into osteoblasts is key to effective treatment. S. aureus is the most common pathogenic bacterium that causes bone infection. This paper reviews the literature, analyzes the specific process of osteoblastic S. aureus infection, and summarizes specific treatment strategies to improve bone infection treatment.

EGFR/FAK and c-Src signalling pathways mediate the internalisation of Staphylococcus aureus by osteoblasts.

Internalisation of Staphylococcus aureus in osteoblasts plays a critical role in the persistence and recurrence of osteomyelitis, the mechanisms involved in this process remain largely unknown. In the present study, evidence of internalised S. aureus in osteoblasts was found in long bone of haematogenous osteomyelitis in mice after 2 weeks of infection. Meanwhile, eliminating extracellular S. aureus by gentamicin can partially rescue bone loss, whereas the remaining intracellular S. aureus in osteoblasts may be associated with continuous bone destruction. In osteoblastic MC3T3 cells, intracellular S. aureus was detectable as early as 15 min after infection, and the internalisation rates increased with the extension of infection time. Additionally, S. aureus invasion stimulated the expression of phosphor-focal adhesion kinase (FAK), phosphor-epidermal growth factor receptor (EGFR) and phosphor-c-Src in a time-dependent way, and blocking EGFR/FAK or c-Src signalling significantly reduced the internalisation rate of S. aureus in osteoblasts. Our findings provide new insights into the mechanism of S. aureus internalisation in osteoblast and raise the potential of targeting EGFR/FAK and c-Src as adjunctive therapeutics for treating chronic S. aureus osteomyelitis.

Boron doped silver-copper alloy nanoparticle targeting intracellular S. aureus in bone cells.

OBJECTIVES: Alloyed metallic nanoparticles of silver and copper are effective against intracellular infection. However, systemic toxicity may arise due to the non-specific delivery of the nanoparticles. In addressing the issue, this study deals with the targeting of silver-copper-boron (ACB) nanoparticles to infected osteoblasts, which could decrease systemic toxicity and form the basis of targeting specific markers expressed in bone infections. METHODS: ACB nanoparticles were synthesized and conjugated to the Cadherin-11 antibody (OBAb). The effect of targeting nanoparticles against extracellular and intracellular S. aureus was determined by enumeration of bacterial growth. The binding of the targeting nanoparticles to infected osteoblasts as well as the visualization of live/dead bacteria due to treatment was carried out using fluorescence microscopy. MTT assay was used to determine the viability of osteoblasts with different concentrations of the nanoparticles. RESULTS: The ACB nanoparticles conjugated to OBAb (ACB-OBAb) were effective against extracellular S. aureus. The ACB-OBAb nanoparticles showed a 1.32 log reduction of intracellular S. aureus at a concentration of 1mg/L. The ACB-OBAb nanoparticles were able to bind to the infected osteoblast and showed toxicity to osteoblasts at levels ≥20mg/L. Also, the percentage of silver, copper, and boron in the nanoparticles determined the effectiveness of their antibacterial activity. CONCLUSION: The ACB-OBAb nanoparticles were able to target the osteoblasts and demonstrated significant antibacterial activity against intracellular S. aureus. Targeting shows promise as a strategy to target specific markers expressed on infected osteoblasts for efficient nanoparticle delivery, and further animal studies are recommended to test its efficacy in vivo.

Detection of methicillin-resistant Staphylococcus aureus persistence in osteoblasts using imaging flow cytometry.

Methicillin-resistant S. aureus has been reported as the main pathogen involved in chronic infections, osteomyelitis, and prosthetic joint infections. The host/pathogen interaction is dynamic and requires several changes to promote bacterial survival. Here, we focused on the internalization and persistence behavior of well-characterized Staphylococcus aureus invasive strains belonging to the main ST-MRSA-SCCmec clones. To overcome the limitations of the cell culture method, we comparatively analyzed the ability of internalization within human MG-63 osteoblasts with imaging flow cytometry (IFC). After evaluation by cell culture assay, the MRSA clones in the study were all able to readily internalize at 3h postinfection, the persistence of intracellular bacteria was evaluated at 24h both by routine cell culture and IFC assay, after vancomycin-BODIPY staining. A statistical difference of persistence was found in ST5-SCCmecII (26.59%), ST228-SCCmecI (20.25%), ST8-SCCmecIV (19.52%), ST239-SCCmecIII (47.82%), and ST22-SCCmecIVh (50.55%) showing the same ability to internalize as ATCC12598 (51%), the invasive isolate used as control strain for invasion and persistence assays. We demonstrated that the intracellular persistence process depends on the total number of infected cells. Comparing our data obtained by IFC with those of the cell culture assay, we obtained greater reproducibility rates and a number of intracellular bacteria, with the advantage of analyzing live host cells. Moreover, with some limitations related to the lack of whole-genome sequencing analysis, we validated the different proclivities to persist in the main Italian HA-MRSA invasive isolates and our results highlighted the heterogeneity of the different clones to persist during cell infection.

Involvement of caspase-1 in inflammasomes activation and bacterial clearance in S. aureus-infected osteoblast-like MG-63 cells.

Staphylococcus aureus, a versatile Gram-positive bacterium, is the main cause of bone and joint infections (BJI), which are prone to recurrence. The inflammasome is an immune signaling platform that assembles after pathogen recognition. It activates proteases, most notably caspase-1 that proteolytically matures and promotes the secretion of mature IL-1ß and IL-18. The role of inflammasomes and caspase-1 in the secretion of mature IL-1ß and in the defence of S. aureus-infected osteoblasts has not yet been fully investigated. We show here that S. aureus-infected osteoblast-like MG-63 but not caspase-1 knock-out CASP1 -/- MG-63 cells, which were generated using CRISPR-Cas9 technology, activate the inflammasome as monitored by the release of mature IL-1ß. The effect was strain-dependent. The use of S. aureus deletion and complemented phenole soluble modulins (PSMs) mutants demonstrated a key role of PSMs in inflammasomes-related IL-1ß production. Furthermore, we found that the lack of caspase-1 in CASP1 -/- MG-63 cells impairs their defense functions, as bacterial clearance was drastically decreased in CASP1 -/- MG-63 compared to wild-type cells. Our results demonstrate that osteoblast-like MG-63 cells play an important role in the immune response against S. aureus infection through inflammasomes activation and establish a crucial role of caspase-1 in bacterial clearance.

Application of bacterial cellulose experimental dressings saturated with gentamycin for management of bone biofilm in vitro and ex vivo.

Bacterial cellulose is one of the most promising polymers of recent years. Herein, we present a possibility of BC application as a carrier of gentamycin antibiotic for the treatment and prevention of bone infections. We have shown that BC saturated with gentamycin significantly reduces the level of biofilm-forming bone pathogens, namely Staphylococcus aureus and Pseudomonas aeruginosa, and displays very low cytotoxicity in vitro against osteoblast cell cultures. Another beneficial feature of our prototype dressing is prolonged release of gentamycin, which provides efficient protection from microbial contamination and subsequent infection. Moreover, it seems that bacterial cellulose (BC) alone without any antimicrobial added, may serve as a barrier by significantly hampering the ability of the pathogen to penetrate to the bone structure. Therefore, a gentamycin-saturated BC dressing may be considered as a possible alternative for gentamycin collagen sponge broadly used in clinical setting. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 108B:30-37, 2020.

Melt electrohydrodynamic 3D printed poly (ε-caprolactone)/polyethylene glycol/roxithromycin scaffold as a potential anti-infective implant in bone repair.

Implanted scaffold or bone substitute is a common method to treat bone defects. However, the possible bone infection caused by orthopaedic surgery has created a challenging clinical problem and generally invalidate bone repair and regeneration. In this study, a poly (ε-caprolactone) (PCL)/polyethylene glycol (PEG)/roxithromycin (ROX) composite scaffold was prepared via melt electrohydrodynamic (EHD) 3D printing. Fourier transform infrared spectroscopy (FTIR) spectroscopy was performed to verify the existence of PEG and ROX in the scaffolds. By water contact angle measurement, the addition of both PEG and ROX was found to improve the hydrophilicity of the scaffolds. By in vitro drug release assay, the PCL/PEG/ROX scaffolds showed an initial burst drug release and subsequent long-term sustained release behaviour, which is favourable for the prevention and treatment of bone infections. The antibacterial assays against E. coli and S. aureus demonstrated that the composite scaffold with ROX possessed effective antibacterial activity, especially for S. aureus, the main cause of bone infection. The immunostaining and MTT assay with human osteoblast-like cells (MG63) indicated that cells showed good viability and growth on the scaffolds. Therefore, the melt EHD 3D printed PCL/PEG/ROX scaffold could be a promising anti-infective implant for bone tissue engineering.

Evaluation of the Activity of a Combination of Three Bacteriophages Alone or in Association with Antibiotics on Staphylococcus aureus Embedded in Biofilm or Internalized in Osteoblasts.

Staphylococcus aureus is responsible for difficult-to-treat bone and joint infections (BJIs). This is related to its ability to form biofilm and to be internalized and persist inside osteoblasts. Recently, bacteriophage therapy has emerged as a promising option to improve treatment of such infections, but data on its activity against the specific bacterial lifestyles presented above remain scarce. We evaluated the activity of a combination of three bacteriophages, recently used for compassionate treatment in France, against S. aureus HG001 in a model of staphylococcal biofilm and a model of osteoblasts infection, alone or in association with vancomycin or rifampin. The activity of bacteriophages against biofilm-embedded S. aureus was dose dependent. In addition, synergistic effects were observed when bacteriophages were combined with antibiotics used at the lowest concentrations. Phage penetration into osteoblasts was observed only when the cells were infected, suggesting a S. aureus-dependent Trojan horse mechanism for internalization. The intracellular bacterial count of bacteria in infected osteoblasts treated with bacteriophages as well as with vancomycin was significantly higher than in cells treated with lysostaphin, used as a control condition, owing to the absence of intracellular activity and the rapid killing of bacteria released after the death of infected cells. These results suggest that bacteriophages are both inactive in the intracellular compartment after being internalized in infected osteoblasts and present a delayed killing effect on bacteria released after cell lysis into the extracellular compartment, which avoids preventing them from infecting other osteoblasts. The combination of bacteriophages tested was highly active against S. aureus embedded in biofilm but showed no activity against intracellular bacteria in the cell model used.

Mechanisms of Immune Evasion and Bone Tissue Colonization That Make Staphylococcus aureus the Primary Pathogen in Osteomyelitis.

PURPOSE OF REVIEW: Staphylococcus aureus is the primary pathogen responsible for osteomyelitis, which remains a major healthcare burden. To understand its dominance, here we review the unique pathogenic mechanisms utilized by S. aureus that enable it to cause incurable osteomyelitis. RECENT FINDINGS: Using an arsenal of toxins and virulence proteins, S. aureus kills and usurps immune cells during infection, to produce non-neutralizing pathogenic antibodies that thwart adaptive immunity. S. aureus also has specific mechanisms for distinct biofilm formation on implants, necrotic bone tissue, bone marrow, and within the osteocyte lacuno-canicular networks (OLCN) of live bone. In vitro studies have also demonstrated potential for intracellular colonization of osteocytes, osteoblasts, and osteoclasts. S. aureus has evolved a multitude of virulence mechanisms to achieve life-long infection of the bone, most notably colonization of OLCN. Targeting S. aureus proteins involved in these pathways could provide new targets for antibiotics and immunotherapies.

Staphylococcus aureus Infects Osteoclasts and Replicates Intracellularly.

Osteomyelitis (OM), or inflammation of bone tissue, occurs most frequently as a result of bacterial infection and severely perturbs bone structure. OM is predominantly caused by Staphylococcus aureus, and even with proper treatment, OM has a high rate of recurrence and chronicity. While S. aureus has been shown to infect osteoblasts, it remains unclear whether osteoclasts (OCs) are also a target of intracellular infection. Here, we demonstrate the ability of S. aureus to intracellularly infect and divide within OCs. OCs were differentiated from bone marrow macrophages (BMMs) by exposure to receptor activator of nuclear factor kappa-B ligand (RANKL). By utilizing an intracellular survival assay and flow cytometry, we found that at 18 h postinfection the intracellular burden of S. aureus increased dramatically in cells with at least 2 days of RANKL exposure, while the bacterial burden decreased in BMMs. To further explore the signals downstream of RANKL, we manipulated factors controlling OC differentiation, NFATc1 and alternative NF-κB, and found that intracellular bacterial growth correlates with NFATc1 levels in RANKL-treated cells. Confocal and time-lapse microscopy in mature OCs showed a range of intracellular infection that correlated inversely with S. aureus-phagolysosome colocalization. The propensity of OCs to become infected, paired with their diminished bactericidal capacity compared to BMMs, could promote OM progression by allowing S. aureus to evade initial immune regulation and proliferate at the periphery of lesions where OCs are most abundant.IMPORTANCE The inflammation of bone tissue is called osteomyelitis, and most cases are caused by an infection with the bacterium Staphylococcus aureus To date, the bone-building cells, osteoblasts, have been implicated in the progression of these infections, but not much is known about how the bone-resorbing cells, osteoclasts, participate. In this study, we show that S. aureus can infect osteoclasts and proliferate inside these cells, whereas bone-residing macrophages, immune cells related to osteoclasts, destroy the bacteria. These findings elucidate a unique role for osteoclasts to harbor bacteria during infection, providing a possible mechanism by which bacteria could evade destruction by the immune system.

Double-Stranded RNA Is a Novel Molecular Target in Osteomyelitis Pathogenesis: A Translational Avian Model for Human Bacterial Chondronecrosis with Osteomyelitis.

Osteomyelitis remains a serious inflammatory bone disease that affects millions of individuals worldwide and for which there is no effective treatment. Despite scientific evidence that Staphylococcus bacteria are the most common causative species for human bacterial chondronecrosis with osteomyelitis (BCO), much remains to be understood about the underlying virulence mechanisms. Herein, we show increased levels of double-stranded RNA (dsRNA) in infected bone in a Staphylococcus-induced chicken BCO model and in human osteomyelitis samples. Administration of synthetic [poly(I:C)] or genetic (Alu) dsRNA induces human osteoblast cell death. Similarly, infection with Staphylococcus isolated from chicken BCO induces dsRNA accumulation and cell death in human osteoblast cell cultures. Both dsRNA administration and Staphylococcus infection activate NACHT, LRR and PYD domains-containing protein (NLRP)3 inflammasome and increase IL18 and IL1B gene expression in human osteoblasts. Pharmacologic inhibition with Ac-YVAD-cmk of caspase 1, a critical component of the NLRP3 inflammasome, prevents DICER1 dysregulation- and dsRNA-induced osteoblast cell death. NLRP3 inflammasome and its components are also activated in bone from BCO chickens and humans with osteomyelitis, compared with their healthy counterparts. These findings provide a rationale for the use of chicken BCO as a human-relevant spontaneous animal model for osteomyelitis and identify dsRNA as a new treatment target for this debilitating bone pathogenesis.