Staphylococcal Osteomyelitis: Disease Progression, Treatment Challenges, and Future Directions.

Osteomyelitis is an inflammatory bone disease that is caused by an infecting microorganism and leads to progressive bone destruction and loss. The most common causative species are the usually commensal staphylococci, with Staphylococcus aureus and Staphylococcus epidermidis responsible for the majority of cases. Staphylococcal infections are becoming an increasing global concern, partially due to the resistance mechanisms developed by staphylococci to evade the host immune system and antibiotic treatment. In addition to the ability of staphylococci to withstand treatment, surgical intervention in an effort to remove necrotic and infected bone further exacerbates patient impairment. Despite the advances in current health care, osteomyelitis is now a major clinical challenge, with recurrent and persistent infections occurring in approximately 40% of patients. This review aims to provide information about staphylococcus-induced bone infection, covering the clinical presentation and diagnosis of osteomyelitis, pathophysiology and complications of osteomyelitis, and future avenues that are being explored to treat osteomyelitis.

Staphylococcus aureus-mediated blood-brain barrier injury: an in vitro human brain microvascular endothelial cell model.

Blood-brain barrier (BBB) disruption constitutes a hallmark event during pathogen-mediated neurological disorders such as bacterial meningitis. As a prevalent opportunistic pathogen, Staphylococcus aureus (SA) is of particular interest in this context, although our fundamental understanding of how SA disrupts the BBB is very limited. This paper employs in vitro infection models to address this. Human brain microvascular endothelial cells (HBMvECs) were infected with formaldehyde-fixed (multiplicity of infection [MOI] 0-250, 0-48 hr) and live (MOI 0-100, 0-3 hr) SA cultures. Both Fixed-SA and Live-SA could adhere to HBMvECs with equal efficacy and cause elevated paracellular permeability. In further studies employing Fixed-SA, infection of HBMvECs caused dose-dependent release of cytokines/chemokines (TNF-α, IL-6, MCP-1, IP-10, and thrombomodulin), reduced expression of interendothelial junction proteins (VE-Cadherin, claudin-5, and ZO-1), and activation of both canonical and non-canonical NF-κB pathways. Using N-acetylcysteine, we determined that these events were coupled to the SA-mediated induction of reactive oxygen species (ROS) within HBMvECs. Finally, treatment of HBMvECs with Fixed-ΔSpA (MOI 0-250, 48 hr), a gene deletion mutant of Staphylococcal protein A associated with bacterial infectivity, had relatively similar effects to Newman WT Fixed-SA. In conclusion, these findings provide insight into how SA infection may activate proinflammatory mechanisms within the brain microvascular endothelium to elicit BBB failure.

A novel and essential role for FcγRIIa in cancer cell-induced platelet activation.

Platelets play a role in cancer by acting as a dynamic reservoir of effectors that facilitate tumor vascularization, growth, and metastasis. However, little information is available about the mechanism of tumor cell-induced platelet secretion (TCIPS) or the molecular machinery by which effector molecules are released from platelets. Here we demonstrate that tumor cells directly induce platelet secretion. Preincubation of platelets with human colon cancer (Caco-2), prostate cancer (PC3M-luc), or breast cancer cells (MDA-MB-231;MCF-7) resulted in a marked dose-dependent secretion of dense granules. Importantly, TCIPS preceded aggregation which always displayed a characteristic lag time. We investigated the role of platelet receptors and downstream molecules in TCIPS. The most potent modulators of TCIPS were the pharmacologic antagonists of Syk kinase, phospholipase C and protein kinase C, all downstream mediators of the immunoreceptor tyrosine-based activation motif (ITAM) cascade in platelets. Supporting this, we demonstrated a central role for the immune Fcγ receptor IIa (FcγRIIa) in mediating platelet-tumor cell cross-talk. In conclusion, we demonstrate that cancer cells can promote platelet dense-granule secretion, which is required to augment platelet aggregation. In addition, we show a novel essential role for FcγRIIa in prostate cancer cell-induced platelet activation opening the opportunity to develop novel antimetastatic therapies.

Amplification of bacteria-induced platelet activation is triggered by FcγRIIA, integrin αIIbß3, and platelet factor 4.

Bacterial adhesion to platelets is mediated via a range of strain-specific bacterial surface proteins that bind to a variety of platelet receptors. It is unclear how these interactions lead to platelet activation. We demonstrate a critical role for the immune receptor FcγRIIA, αIIbß3, and Src and Syk tyrosine kinases in platelet activation by Staphylococcus aureus, Streptococcus sanguinis, Streptococcus gordonii, Streptococcus oralis, and Streptococcus pneumoniae. FcγRIIA activation is dependent on immunoglobulin G (IgG) and αIIbß3 engagement. Moreover, feedback agonists adenosine 5'-diphosphate and thromboxane A2 are mandatory for platelet aggregation. Additionally, platelet factor 4 (PF4) binds to bacteria and reduces the lag time for aggregation, and gray platelet syndrome α-granule-deficient platelets do not aggregate to 4 of 5 bacterial strains. We propose that FcγRIIA-mediated activation is a common response mechanism used against a wide range of bacteria, and that release of secondary mediators and PF4 serve as a positive feedback mechanism for activation through an IgG-dependent pathway.

Human platelet activation by Escherichia coli: roles for FcγRIIA and integrin αIIbß3.

Gram-negative Escherichia coli cause diseases such as sepsis and hemolytic uremic syndrome in which thrombotic disorders can be found. Direct platelet-bacterium interactions might contribute to some of these conditions; however, mechanisms of human platelet activation by E. coli leading to thrombus formation are poorly understood. While the IgG receptor FcγRIIA has a key role in platelet response to various Gram-positive species, its role in activation to Gram-negative bacteria is poorly defined. This study aimed to investigate the molecular mechanisms of human platelet activation by E. coli, including the potential role of FcγRIIA. Using light-transmission aggregometry, measurements of ATP release and tyrosine-phosphorylation, we investigated the ability of two E. coli clinical isolates to activate platelets in plasma, in the presence or absence of specific receptors and signaling inhibitors. Aggregation assays with washed platelets supplemented with IgGs were performed to evaluate the requirement of this plasma component in activation. We found a critical role for the immune receptor FcγRIIA, αIIbß3, and Src and Syk tyrosine kinases in platelet activation in response to E. coli. IgG and αIIbß3 engagement was required for FcγRIIA activation. Moreover, feedback mediators adenosine 5'-diphosphate (ADP) and thromboxane A2 (TxA2) were essential for platelet aggregation. These findings suggest that human platelet responses to E. coli isolates are similar to those induced by Gram-positive organisms. Our observations support the existence of a central FcγRIIA-mediated pathway by which human platelets respond to both Gram-negative and Gram-positive bacteria.

Dysregulation of the endothelium following Staphylococcus aureus infection.

The cardiovascular system is typically a sterile environment; however entry of a microorganism into the circulation can cause potentially life threatening cardiac and/or vascular disease. Staphylococcus aureus endothelial cell interactions are arguably the most important interactions in the pathogenesis of cardiovascular infection. These interactions can trigger cardiac valve destruction in the case of endocarditis, multi-organ dysfunction in the case of sepsis and coagulopathy. Here, we review the interactions between S. aureus and endothelial cells and discuss the implications of these interactions in the progression of cardiovascular infection.

The expanding field of platelet-bacterial interconnections.

Given their small size, platelets are emerging as being one of the most important entities in the bloodstream. Not only do they play a key role in maintaining thrombosis and haemostasis, platelets also play a critical role in orchestrating the immune response. Being the first cell at the site of injury, they are perfectly placed to assess the extent of the damage and recruit immune cells as is necessary. As a first line of defence, platelets can act as primitive immune cells themselves by interacting with invading pathogens. A number of platelet receptors have been shown to interact with bacteria either directly or indirectly, involving a plasma protein bridge. This review will discuss the molecular mechanisms that exist between platelets and bacteria and the functional response to the interaction. We will also discuss the importance of considering animal models of disease and the use of physiological shear when studying platelet-bacterial interactions.

Staphylococcus aureus protein A binding to osteoblast tumour necrosis factor receptor 1 results in activation of nuclear factor kappa B and release of interleukin-6 in bone infection.

Staphylococcus aureus is the major pathogen among the staphylococci and the most common cause of bone infections. These infections are mainly characterized by bone destruction and inflammation, and are often debilitating and very difficult to treat. Previously we demonstrated that S. aureus protein A (SpA) can bind to osteoblasts, which results in inhibition of osteoblast proliferation and mineralization, apoptosis, and activation of osteoclasts. In this study we used small interfering RNA (siRNA) to demonstrate that osteoblast tumour necrosis factor receptor-1 (TNFR-1) is responsible for the recognition of and binding to SpA. TNFR-1 binding to SpA results in the activation of nuclear factor kappa B (NFκB). In turn, NFκB translocates to the nucleus of the osteoblast, which leads to release of interleukin 6 (IL-6). Silencing TNFR-1 in osteoblasts or disruption of the spa gene in S. aureus prevented both NFκB activation and IL-6 release. As well as playing a key role in proinflammatory reactions, IL-6 is also an important osteotropic factor. Release of IL-6 from osteoblasts results in the activation of the bone-resorbing cells, the osteoclasts. Consistent with our results described above, both silencing TNFR-1 in osteoblasts and disruption of spa in S. aureus prevented osteoclast activation. These studies are the first to demonstrate the importance of the TNFR-1-SpA interaction in bone infection, and may help explain the mechanism through which osteoclasts become overactivated, leading to bone destruction. Anti-inflammatory drug therapy could be used either alone or in conjunction with antibiotics to treat osteomyelitis or for prophylaxis in high-risk patients.

Human endothelial cell-derived exosomal microRNA-99a/b drives a sustained inflammatory response during sepsis by inhibiting mTOR expression.

The pathophysiology of sepsis and its accompanying hyper-inflammatory response are key events that lead to multi-organ failure and death. A growing body of literature now suggests that the vascular endothelium plays a critical role in driving early events of sepsis progression. In this study, we demonstrate how endothelial-derived exosomes contribute to a successive pro-inflammatory phenotype of monocytes. Exosomes isolated from S. aureus infected endothelial cells drive both CD11b and MHCII expression in monocytes and contribute dysregulated cytokine production. Conversely, healthy endothelial exosomes had no major effect. microRNA (miRNA) profiling of exosomes identified miR-99 upregulation which we hypothesised as driving this phenotypic change through mechanistic target of rapamycin (mTOR). Knockdown of mTOR with miR-99a and miR-99b mimetics in S. aureus infected monocytes increased IL-6 and decreased IL-10 production. Interestingly, inhibition of miRNAs with antagomirs has the opposing effect. Collectively, endothelial exosomes are driving a pro-inflammatory phenotype in monocytes through dysregulated expression of miR-99a and miR-99b.

Educating pharmacy students through a pandemic: Reflecting on our COVID-19 experience.

The impact of the COVID-19 pandemic on pharmacy education worldwide has been immense, affecting students, educators and regulatory agencies. Pharmacy programmes have had to rapidly adapt in their delivery of education, maintaining standards while also ensuring the safety of all stakeholders. In this commentary, we describe the challenges, compromises and solutions adopted by our institution throughout the pandemic, the lessons learnt, adaptive measures taken, and strategies to develop and future-proof our curricula.

The Sbi protein is a multifunctional immune evasion factor of Staphylococcus aureus.

The second immunoglobulin-binding protein (Sbi) of Staphylococcus aureus has two N-terminal domains that bind the Fc region of IgG in a fashion similar to that of protein A and two domains that can bind to the complement protein C3 and promote its futile consumption in the fluid phase. It has been proposed that Sbi helps bacteria to avoid innate immune defenses. By comparing a mutant defective in Sbi with mutants defective in protein A, clumping factor A, iron-regulated surface determinant H, and capsular polysaccharide, it was shown that Sbi is indeed an immune evasion factor that promotes bacterial survival in whole human blood and the avoidance of neutrophil-mediated opsonophagocytosis. Sbi is present in the culture supernatant and is also associated with the cell envelope. S. aureus strains that expressed truncates of Sbi lacking N-terminal domains D1 and D2 (D1D2) or D3 and D4 (D3D4) or a C-terminal truncate that was no longer retained in the cell envelope were analyzed. Both the secreted and envelope-associated forms of Sbi contributed to immune evasion. The IgG-binding domains contributed only when Sbi was attached to the cell, while only the secreted C3-binding domains were biologically active.

Mechanism of outside-in {alpha}IIb{beta}3-mediated activation of human platelets by the colonizing Bacterium, Streptococcus gordonii.

OBJECTIVE: To better understand the mechanism of platelet recruitment and activation by Streptococcus gordonii. The oral bacterium Streptococcus gordonii, is amongst the most common pathogens isolated from infective endocarditis patients, and has the property of being able to activate platelets, leading to thrombotic complications. The mechanism of platelet recruitment and activation by S. gordonii is poorly understood. METHODS AND RESULTS: Infective endocarditis is a bacterial infection of the heart valves that carries a high risk of morbidity and mortality. The oral bacterium, S gordonii, is among the most common pathogens isolated from patients with infective endocarditis and is able to activate platelets, leading to thrombotic complications. Platelets interact with S gordonii via glycoprotein Ibα- and α(IIb)ß(3)-recognizing S gordonii surface proteins haemaglutitin salivary antigen (Hsa) and platelet adherence protein A, respectively. The inhibition of glycoprotein Ibα or α(IIb)ß(3) using blocking antibodies or deletion of S gordonii Hsa or platelet adherence protein A significantly reduces platelet adhesion. Immunoreceptor tyrosine-based activation motif (ITAM)-containing proteins have recently played a role in transmitting activating signals into platelets. Platelet adhesion to immobilized S gordonii resulted in tyrosine phosphorylation of the ITAM-bearing receptor, FcγRIIa, and phosphorylation of downstream effectors (ie, spleen tyrosine kinase [Syk] and phospholipase C [PLC]-γ2). Tyrosine phosphorylation of FcγRIIa resulted in platelet-dense granule secretion, filopodial and lamellipodial extension, and platelet spreading. Inhibition of FcγRIIa ablated both dense granule release and platelet spreading. CONCLUSIONS: Streptococcus gordonii binding to the α(IIb)ß(3)/FcγRIIa integrin/ITAM signaling complex results in platelet activation that likely contributes to the thrombotic complications of infective endocarditis.

Platelet-bacterial interactions.

Many bacteria are capable of interacting with platelets and inducing platelet aggregation. This interaction may be a direct interaction between a bacterial surface protein and a platelet receptor or may be an indirect interaction where plasma proteins bind to the bacterial surface and subsequently bind to a platelet receptor. However, these interactions usually do not trigger platelet activation as a secondary co-signal is also required. This is usually due to specific antibody bound to the bacteria interacting with FcgammaRIIa on the platelet surface. Secreted bacterial products such as gingipains and lipopolysaccharide may also be capable of triggering platelet activation.

SARS-CoV-2 uses major endothelial integrin αvß3 to cause vascular dysregulation in-vitro during COVID-19.

The unprecedented global COVID-19 pandemic has prompted a desperate international effort to accelerate the development of anti-viral candidates. For unknown reasons, COVID-19 infections are associated with adverse cardiovascular complications, implicating that vascular endothelial cells are essential in viral propagation. The etiological pathogen, SARS-CoV-2, has a higher reproductive number and infection rate than its predecessors, indicating it possesses novel characteristics that infers enhanced transmissibility. A unique K403R spike protein substitution encodes an Arg-Gly-Asp (RGD) motif, introducing a potential role for RGD-binding host integrins. Integrin αVß3 is widely expressed across the host, particularly in the endothelium, which acts as the final barrier before microbial entry into the bloodstream. This mutagenesis creates an additional binding site, which may be sufficient to increase SARS-CoV-2 pathogenicity. Here, we investigate how SARS-CoV-2 passes from the epithelium to endothelium, the effects of αVß3 antagonist, Cilengitide, on viral adhesion, vasculature permeability and leakage, and also report on a simulated interaction between the viral and host protein in-silico.

Targeting Internalized Staphylococcus aureus Using Vancomycin-Loaded Nanoparticles to Treat Recurrent Bloodstream Infections.

The bacterial pathogen Staphylococcus aureus is a leading cause of bloodstream infections, where patients often suffer from relapse despite antibiotic therapy. Traditional anti-staphylococcal drugs display reduced effectivity against internalised bacteria, but nanoparticles conjugated with antibiotics can overcome these challenges. In the present study, we aimed to characterise the internalisation and re-emergence of S. aureus from human endothelial cells and construct a new formulation of nanoparticles that target intracellular bacteria. Using an in vitro infection model, we demonstrated that S. aureus invades and persists within endothelial cells, mediated through bacterial extracellular surface adhesion, Fibronectin-binding protein A/B. After internalising, S. aureus localises to vacuoles as determined by transmission electron microscopy. Viable S. aureus emerges from endothelial cells after 48 h, supporting the notion that intracellular persistence contributes to infection relapses during bloodstream infections. Poly lactic-co-glycolic acid nanoparticles were formulated using a water-in-oil double emulsion method, which loaded 10% vancomycin HCl with 82.85% ± 12 encapsulation efficiency. These non-toxic nanoparticles were successfully taken up by cells and demonstrated a biphasic controlled release of 91 ± 4% vancomycin. They significantly reduced S. aureus intracellular growth within infected endothelial cells, which suggests future potential applications for targeting internalised bacteria and reducing mortality associated with bacteraemia.

In vitro flow-based assay: From simple toward more sophisticated models for mimicking hemostasis and thrombosis.

In vitro flow-based assays are widely used to investigate the role of platelets and coagulation in hemostasis and thrombosis. Their main advantage over other assays relies on the fact that they integrate blood flow that regulates many aspects of platelet function, including adhesion, activation, and aggregation. Blood flow is also central in the regulation of coagulation through its ability to modulate the local concentrations of coagulation factors within and around thrombi. The most broadly used assay to study thrombus formation consists in perfusing whole blood over immobilized fibrillar collagen through a single channel, which helps to reproduce thrombus formation as it occurs in vivo after vascular injury, with platelets adhering, becoming activated, and forming a mural thrombus. This process can also be studied under conditions of thrombin generation, notably by recalcifying blood collected in sodium citrate. In this manuscript, we briefly discuss the advantages and limits of this broadly used "in vitro thrombus formation model." The main emphasis is on the description of the most recent developments regarding design of new flow models and new techniques, and how these may advance the landscape of in vitro studies into the formation of physiological or pathophysiological thrombi. Challenges linked to mimicking the formation of a hemostatic plug in a healthy vessel or a thrombus in diseased arteries and the complexity of reproducing the various aspects of venous thrombosis are discussed. Future directions are proposed to improve the physiological or pathophysiological relevance of current flow-based assays.

Intra-vital imaging of mesenchymal stromal cell kinetics in the pulmonary vasculature during infection.

Mesenchymal stem/stromal cells (MSCs) have demonstrated efficacy in pre-clinical models of inflammation and tissue injury, including in models of lung injury and infection. Rolling, adhesion and transmigration of MSCs appears to play a role during MSC kinetics in the systemic vasculature. However, a large proportion of MSCs become entrapped within the lungs after intravenous administration, while the initial kinetics and the site of arrest of MSCs in the pulmonary vasculature are unknown. We examined the kinetics of intravascularly administered MSCs in the pulmonary vasculature using a microfluidic system in vitro and intra-vital microscopy of intact mouse lung. In vitro, MSCs bound to endothelium under static conditions but not under laminar flow. VCAM-1 antibodies did not affect MSC binding. Intravital microscopy demonstrated MSC arrest at pulmonary micro-vessel bifurcations due to size obstruction. Retention of MSCs in the pulmonary microvasculature was increased in Escherichia coli-infected animals. Trapped MSCs deformed over time and appeared to release microvesicles. Labelled MSCs retained therapeutic efficacy against pneumonia. Our results suggest that MSCs are physically obstructed in pulmonary vasculature and do not display properties of rolling/adhesion, while retention of MSCs in the infected lung may require receptor interaction.

Human platelets recognize a novel surface protein, PadA, on Streptococcus gordonii through a unique interaction involving fibrinogen receptor GPIIbIIIa.

The concept of an infectious agent playing a role in cardiovascular disease is slowly gaining attention. Among several pathogens identified, the oral bacterium Streptococcus gordonii has been implicated as a plausible agent. Platelet adhesion and subsequent aggregation are critical events in the pathogenesis and dissemination of the infective process. Here we describe the identification and characterization of a novel cell wall-anchored surface protein, PadA (397 kDa), of S. gordonii DL1 that binds to the platelet fibrinogen receptor GPIIbIIIa. Wild-type S. gordonii cells induced platelet aggregation and supported platelet adhesion in a GPIIbIIIa-dependent manner. Deletion of the padA gene had no effect on platelet aggregation by S. gordonii but significantly reduced (>75%) platelet adhesion to S. gordonii. Purified N-terminal PadA recombinant polypeptide adhered to platelets. The padA mutant was unaffected in production of other platelet-interactive surface proteins (Hsa, SspA, and SspB), and levels of adherence of the mutant to fetuin or platelet receptor GPIb were unaffected. Wild-type S. gordonii, but not the padA mutant, bound to Chinese hamster ovary cells stably transfected with GPIIbIIIa, and this interaction was ablated by addition of GPIIbIIIa inhibitor Abciximab. These results highlight the growing complexity of interactions between S. gordonii and platelets and demonstrate a new mechanism by which the bacterium could contribute to unwanted thrombosis.

Direct interaction of iron-regulated surface determinant IsdB of Staphylococcus aureus with the GPIIb/IIIa receptor on platelets.

The interaction of bacteria with platelets is implicated in the pathogenesis of endovascular infections, including infective endocarditis, of which Staphylococcus aureus is the leading cause. Several S. aureus surface proteins mediate aggregation of platelets by fibrinogen- or fibronectin-dependent processes, which also requires specific antibodies. In this study S. aureus was grown in iron-limited medium to mimic in vivo conditions in which iron is unavailable to pathogens. Under such conditions, a S. aureus mutant lacking the known platelet-activating surface proteins adhered directly to platelets in the absence of plasma proteins and triggered aggregation. Platelet adhesion and aggregation was prevented by inhibiting expression of iron-regulated surface determinant (Isd) proteins. Mutants defective in IsdB, but not IsdA or IsdH, were unable to adhere to or aggregate platelets. Antibodies to the platelet integrin GPIIb/IIIa inhibited platelet adhesion by IsdB-expressing strains, as did antagonists of GPIIb/IIIa. Surface plasmon resonance demonstrated that recombinant IsdB interacts directly with GPIIb/IIIa.

A new perspective in sepsis treatment: could RGD-dependent integrins be novel targets?

Sepsis is a life-threatening condition caused by the response of the body to an infection, and has recently been regarded as a global health priority because of the lack of effective treatments available. Vascular endothelial cells have a crucial role in sepsis and are believed to be a major target of pathogens during the early stages of infection. Accumulating evidence suggests that common sepsis pathogens, including bacteria, fungi, and viruses, all contain a critical integrin recognition motif, Arg-Gly-Asp (RGD), in their major cell wall-exposed proteins that might act as ligands to crosslink to vascular endothelial cells, triggering systemic dysregulation resulting in sepsis. In this review, we discuss the potential of anti-integrin therapy in the treatment of sepsis and septic shock.