New Weapons to Fight against Staphylococcus aureus Skin Infections.

The treatment of Staphylococcus aureus skin and soft tissue infections faces several challenges, such as the increased incidence of antibiotic-resistant strains and the fact that the antibiotics available to treat methicillin-resistant S. aureus present low bioavailability, are not easily metabolized, and cause severe secondary effects. Moreover, besides the susceptibility pattern of the S. aureus isolates detected in vitro, during patient treatment, the antibiotics may never encounter the bacteria because S. aureus hides within biofilms or inside eukaryotic cells. In addition, vascular compromise as well as other comorbidities of the patient may impede proper arrival to the skin when the antibiotic is given parenterally. In this manuscript, we revise some of the more promising strategies to improve antibiotic sensitivity, bioavailability, and delivery, including the combination of antibiotics with bactericidal nanomaterials, chemical inhibitors, antisense oligonucleotides, and lytic enzymes, among others. In addition, alternative non-antibiotic-based experimental therapies, including the delivery of antimicrobial peptides, bioactive glass nanoparticles or nanocrystalline cellulose, phototherapies, and hyperthermia, are also reviewed.

Staphylococcus aureus Adaptation to the Skin in Health and Persistent/Recurrent Infections.

Staphylococcus aureus is a microorganism with an incredible capability to adapt to different niches within the human body. Approximately between 20 and 30% of the population is permanently but asymptomatically colonized with S. aureus in the nose, and another 30% may carry S. aureus intermittently. It has been established that nasal colonization is a risk factor for infection in other body sites, including mild to severe skin and soft tissue infections. The skin has distinct features that make it a hostile niche for many bacteria, therefore acting as a strong barrier against invading microorganisms. Healthy skin is desiccated; it has a low pH at the surface; the upper layer is constantly shed to remove attached bacteria; and several host antimicrobial peptides are produced. However, S. aureus is able to overcome these defenses and colonize this microenvironment. Moreover, this bacterium can very efficiently adapt to the stressors present in the skin under pathological conditions, as it occurs in patients with atopic dermatitis or suffering chronic wounds associated with diabetes. The focus of this manuscript is to revise the current knowledge concerning how S. aureus adapts to such diverse skin conditions causing persistent and recurrent infections.

Neutralization of Staphylococcus aureus Protein A Prevents Exacerbated Osteoclast Activity and Bone Loss during Osteomyelitis.

Osteomyelitis caused by Staphylococcus aureus is an important and current health care problem worldwide. Treatment of this infection frequently fails not only due to the increasing incidence of antimicrobial-resistant isolates but also because of the ability of S. aureus to evade the immune system, adapt to the bone microenvironment, and persist within this tissue for decades. We have previously demonstrated the role of staphylococcal protein A (SpA) in the induction of exacerbated osteoclastogenesis and increased bone matrix degradation during osteomyelitis. The aim of this study was to evaluate the potential of using anti-SpA antibodies as an adjunctive therapy to control inflammation and bone damage. By using an experimental in vivo model of osteomyelitis, we demonstrated that the administration of an anti-SpA antibody by the intraperitoneal route prevented excessive inflammatory responses in the bone upon challenge with S. aureus. Ex vivo assays indicated that blocking SpA reduced the priming of osteoclast precursors and their response to RANKL. Moreover, the neutralization of SpA was able to prevent the differentiation and activation of osteoclasts in vivo, leading to reduced expression levels of cathepsin K, reduced expression of markers associated with abnormal bone formation, and decreased trabecular bone loss during osteomyelitis. Taken together, these results demonstrate the feasibility of using anti-SpA antibodies as an antivirulence adjunctive therapy that may prevent the development of pathological conditions that not only damage the bone but also favor bacterial escape from antimicrobials and the immune system.

Acapsular Staphylococcus aureus with a non-functional agr regains capsule expression after passage through the bloodstream in a bacteremia mouse model.

Selection pressures exerted on Staphylococcus aureus by host factors during infection may lead to the emergence of regulatory phenotypes better adapted to the infection site. Traits convenient for persistence may be fixed by mutation thus turning these mutants into microevolution endpoints. The feasibility that stable, non-encapsulated S. aureus mutants can regain expression of key virulence factors for survival in the bloodstream was investigated. S. aureus agr mutant HU-14 (IS256 insertion in agrC) from a patient with chronic osteomyelitis was passed through the bloodstream using a bacteriemia mouse model and derivative P3.1 was obtained. Although IS256 remained inserted in agrC, P3.1 regained production of capsular polysaccharide type 5 (CP5) and staphyloxanthin. Furthermore, P3.1 expressed higher levels of asp23/SigB when compared with parental strain HU-14. Strain P3.1 displayed decreased osteoclastogenesis capacity, thus indicating decreased adaptability to bone compared with strain HU-14 and exhibited a trend to be more virulent than parental strain HU-14. Strain P3.1 exhibited the loss of one IS256 copy, which was originally located in the HU-14 noncoding region between dnaG (DNA primase) and rpoD (sigA). This loss may be associated with the observed phenotype change but the mechanism remains unknown. In conclusion, S. aureus organisms that escape the infected bone may recover the expression of key virulence factors through a rapid microevolution pathway involving SigB regulation of key virulence factors.

Protein A Modulates Neutrophil and Keratinocyte Signaling and Survival in Response to Staphylococcus aureus.

The type 1 TNF-α receptor (TNFR1) has a central role in initiating both pro-inflammatory and pro-apoptotic signaling cascades in neutrophils. Considering that TNFR1 signals Staphylococcus aureus protein A (SpA), the aim of this study was to explore the interaction of this bacterial surface protein with neutrophils and keratinocytes to underscore the signaling pathways that may determine the fate of these innate immune cells in the infected tissue during staphylococcal skin infections. Using human neutrophils cultured in vitro and isogenic staphylococcal strains expressing or not protein A, we demonstrated that SpA is a potent inducer of IL-8 in neutrophils and that the induction of this chemokine is dependent on the SpA-TNFR1 interaction and p38 activation. In addition to IL-8, protein A induced the expression of TNF-α and MIP-1α highlighting the importance of SpA in the amplification of the inflammatory response. Protein A contributed to reduce neutrophil mortality prolonging their lifespan upon the encounter with S. aureus. Signaling initiated by SpA modulated the type of neutrophil cell death in vitro and during skin and soft tissue infections (SSTI) in vivo triggering the apoptotic pathway instead of necrosis. Moreover, SpA induced pro-inflammatory cytokines in keratinocytes, modulating their survival in vitro and preventing the exacerbated necrosis and ulceration of the epithelium during SSTI in vivo. Taken together, these results highlight the importance of the inflammatory signaling induced by protein A in neutrophils and skin epithelial cells. The ability of protein A to modulate the neutrophil/epithelial cell death program in the skin is of clinical relevance considering that lysis of neutrophils and epithelial cells will promote an intense inflammatory response and contribute to tissue damage, a non-desirable feature of complicated SSTI.

Short-Term Fever-Range Hyperthermia Accelerates NETosis and Reduces Pro-inflammatory Cytokine Secretion by Human Neutrophils.

Fever is a hallmark of infections and inflammatory diseases, represented by an increase of 1-4°C in core body temperature. Fever-range hyperthermia (FRH) has been shown to increase neutrophil recruitment to local sites of infection. Here, we evaluated the impact of a short period (1 h) of FRH (STFRH) on pro-inflammatory and bactericidal human neutrophil functions. STFRH did not affect neutrophil spontaneous apoptosis but reverted the lipopolysaccharide (LPS)-induced anti-apoptotic effect compared with that under normothermic conditions. Furthermore, STFRH accelerated phorbol myristate acetate (PMA)-induced NETosis evaluated either by the nuclear DNA decondensation at 2 h post-stimulation or by the increase in extracellular DNA that colocalized with myeloperoxidase (MPO) at 4 h post-stimulation. Increased NETosis upon STFRH was associated with an increase in reactive oxygen species (ROS) production but not in autophagy levels. STFRH also increased NETosis in response to Pseudomonas aeruginosa challenge but moderately reduced its phagocytosis. However, these STFRH-induced effects did not influence the ability of neutrophils to kill bacteria after 4 h of co-culture. STFRH also significantly reduced neutrophil capacity to release the pro-inflammatory cytokines chemokine (C-X-C motif) ligand 8/interleukin 8 (CXCL8/IL-8) and IL-1ß in response to LPS and P. aeruginosa challenge. Altogether, these results indicate that a short and mild hyperthermal period is enough to modulate neutrophil responses to bacterial encounter. They also suggest that fever spikes during bacterial infections might lead neutrophils to trigger an emergency response promoting neutrophil extracellular trap (NET) formation to ensnare bacteria in order to wall off the infection and to reduce their release of pro-inflammatory cytokines in order to limit the inflammatory response.

The good side of inflammation: Staphylococcus aureus proteins SpA and Sbi contribute to proper abscess formation and wound healing during skin and soft tissue infections.

Staphylococcus aureus is the most prominent cause of skin and soft tissue infections (SSTI) worldwide. Mortality associated with invasive SSTI is a major threat to public health considering the incidence of antibiotic resistant isolates in particular methicillin resistant S. aureus both in the hospital (HA-MRSA) and in the community (CA-MRSA). To overcome the increasing difficulties in the clinical management of SSTI due to MRSA, new prophylactic and therapeutic approaches are urgently needed and a preventive vaccine would be welcome. The rational design of an anti-S. aureus vaccine requires a deep knowledge of the role that the different bacterial virulence factors play according to the type of infection. In the present study, using a set of isogenic deficient mutants and their complemented strains we determined that the staphylococcal surface proteins SpA and Sbi play an important role in the induction of inflammatory cytokines and chemokines in the skin during SSTI. SpA and Sbi initiate signaling cascades that lead to the early recruitment of neutrophils, modulate their lifespan in the skin milieu and contribute to proper abscess formation and bacterial eradication. Moreover, the expression of SpA and Sbi appear critical for skin repair and wound healing. Thus, these results indicate that SpA and Sbi can promote immune responses in the skin that are beneficial for the host and therefore, should not be neutralized with vaccine formulations designed to prevent SSTI.

TNFR1 Signaling Contributes to T Cell Anergy During Staphylococcus aureus Sepsis.

Early research on sepsis has focused on the initial hyper-inflammatory, cytokine mediated phase of the disorder whereas the events that govern the concomitant and subsequent anti-inflammatory compensatory response are not completely understood. In this context, the putative participation of TNFR1-mediated signaling in the immunosuppressive phase of Staphylococcus aureus sepsis has not been elucidated. The aim of this study was to determine the role of TNFR1 in directing the immune dysfunction during S. aureus sepsis and the potential contribution of MDSC to this process. Using a model of sepsis of peritoneal origin and tnfr1-/- mice, we demonstrated that during staphylococcal sepsis CD4+ T cell anergy is significantly dependent on TNFR1 expression and that signaling through this receptor has an impact on bacterial clearance in the spleen. MDSC played a major role in the generation of anergic CD4+ T cells and their accumulation in the spleen during S. aureus sepsis correlated with IL-6 induction. Although TNFR1 signaling was not required for MDSC accumulation and expansion in the spleen, it determined the in vivo expression of Arginase 1 and iNOS, enzymes known to participate in the suppressive function of this population. Moreover, our data indicate that TNFR1-mediated IL-10 production may modulate MDSC function during staphylococcal sepsis. Taken together these results indicate that TNFR1 plays a critical role on T cell dysfunction during S. aureus sepsis by regulating immunomodulatory mediators in MDSC. The role of TNFR1-mediated signaling during the immunosuppressive phase of staphylococcal sepsis should be considered when designing novel alternative therapeutic approaches.

Mutation of Agr Is Associated with the Adaptation of Staphylococcus aureus to the Host during Chronic Osteomyelitis.

Selection pressures exerted on Staphylococcus aureus by host factors may lead to the emergence of mutants better adapted to the evolving conditions at the infection site. This study was aimed at identifying the changes that occur in S. aureus exposed to the host defense mechanisms during chronic osteomyelitis and evaluating whether these changes affect the virulence of the organism. Genome assessment of two S. aureus isolates collected 13 months apart (HU-85a and HU-85c) from a host with chronic osteomyelitis was made by whole genome sequencing. Agr functionality was assessed by qRT-PCR. Isolates were tested in a rat model of osteomyelitis and the bacterial load (CFU/tibia) and the morphometric osteomyelitic index (OI) were determined. The ability of the isolates to trigger the release of proinflammatory cytokines was determined on macrophages in culture. Persistence of S. aureus within the host resulted in an agrC frameshift mutation that likely led to the observed phenotype. The capacity to cause bone tissue damage and trigger proinflammatory cytokines by macrophages of the agr-deficient, unencapsulated derivative (HU-85c) was decreased when compared with those of the isogenic CP8-capsulated parental strain (HU-85a). By comparison, no significant differences were found in the bacterial load or the OI from rats challenged with isogenic Reynolds strains [CP5, CP8, and non-typeable (NT)], indicating that lack of CP expression alone was not likely responsible for the reduced capacity to cause tissue damage in HU-85c compared with HU-85a. The production of biofilm was significantly increased in the isogenic derivative HU-85c. Lack of agr-dependent factors makes S. aureus less virulent during chronic osteomyelitis and alteration of the agr functionality seems to permit better adaptation of S. aureus to the chronically infected host.

High virulence of methicillin resistant Staphylococcus aureus ST30-SCCmecIVc-spat019, the dominant community-associated clone in Argentina.

Community-acquired methicillin resistant Staphylococcus aureus emerged as a worldwide health problem in the last few years. In Argentina, it is found in 70% of skin and skin structure infections in previously healthy adult patients and causes severe invasive diseases. The ST30-SCCmecIVc-spat019 clone is predominant in adult infections and has displaced the previously prevalent ST5-SCCmecIVa-spat311 clone in community settings. In the present work we compared the virulence of both clones in order to explain the displacement, and found that ST30-IVc is associated with invasive infections in adult patients from Argentina and possesses a different virulence-associated genes profile compared to ST5-IVa. A representative strain of ST30 lineage has a more aggressive behavior in animal models of infection and expresses higher level of Fibronectin binding protein A coding gene, which could enhance the bacterial invasion capacity.

Staphylococcus aureus Induces Shedding of IL-1RII in Monocytes and Neutrophils.

Interleukin 1 (IL-1) ß is a critical cytokine that orchestrates host defenses against Staphylococcus aureus and is crucial for the eradication of bacteria. The production and action of IL-1ß are regulated by multiple control pathways. Among them, IL-1RII (the type II IL-1 receptor) acts as a decoy receptor and has been shown to regulate the biological effects of IL-1ß. High levels of soluble IL-1RII are present in septic patients; however, the stimuli that regulate the expression and release of IL-1RII in pathological conditions are incompletely elucidated. In the present study, we demonstrated the ability of S. aureus and protein A to induce IL-1RII shedding in myeloid cells. The positive modulation of IL-1RII expression and cleavage was associated with the failure to detect IL-1ß in response to S. aureus both in vitro and in vivo, suggesting that the soluble form of the receptor could be masking the availability of IL-1ß. The absence of detectable IL-1ß was associated with low levels of inflammatory cytokines and chemokines known to be regulated by IL-1ß and with increased bacterial persistence. Modulation of decoy receptors during systemic S. aureus infection is proposed as a new strategy used by this bacterium to evade the immune response.

The Sbi Protein Contributes to Staphylococcus aureus Inflammatory Response during Systemic Infection.

Staphylococcus aureus is an important human pathogen that causes infections that may present high morbidity and mortality. Among its many virulence factors protein A (SpA) and Staphylococcal binding immunoglobulin protein (Sbi) bind the Fc portion of IgG interfering with opsonophagocytosis. We have previously demonstrated that SpA interacts with the TNF-α receptor (TNFR) 1 through each of the five IgG binding domains and induces the production of pro-inflammatory cytokines and chemokines. The IgG binding domains of Sbi are homologous to those of SpA, which allow us to hypothesize that Sbi might also have a role in the inflammatory response induced by S. aureus. We demonstrate that Sbi is a novel factor that participates in the induction of the inflammatory response during staphylococcal infections via TNFR1 and EGFR mediated signaling as well as downstream MAPKs. The expression of Sbi significantly contributed to IL-6 production and modulated CXCL-1 expression as well as neutrophil recruitment to the site of infection, thus demonstrating for the first time its relevance as a pro-inflammatory staphylococcal antigen in an in vivo model.

In Vitro infection of human dura-mater fibroblasts with Staphylococcus aureus: colonization and reactive production of IL-1beta.

OBJECTIVE: Post-operative meningitis, caused mainly by Staphylococcus aureus and Gram-negative rods, is a life-threatening complication after neurosurgery, and its pathogenesis is far from clear. The purpose of this work was to study the experimental infection of human dura-mater fibroblasts and whole human dura by S. aureus. METHODS: In vitro cultures of human dura-mater fibroblasts and organotypic cultures of small pieces of human dura mater were inoculated with a human-derived S. aureus strain. The pattern of bacterial infection as well as cytokines secretion by the infected fibroblasts was studied. RESULTS: Our results suggest that colonisation of human dura-mater fibroblasts in culture and whole dura-mater tissue by S. aureus includes bacterial growth on the cell surface, fibroblast intracellular invasion by bacteria and a significant synthesis of interleukin 1beta (IL-1beta) by the infected cells. CONCLUSION: This is the first report of human dura-mater fibroblast infection by S. aureus. Hopefully, these results can lead to a better understanding of the pathogenesis of meningitis caused by this bacterial species and to a more rational therapeutic approach.

Treatment in vitro with PPARα and PPARγ ligands drives M1-to-M2 polarization of macrophages from T. cruzi-infected mice.

Trypanosoma cruzi, the etiological agent of Chagas' disease, induces a persistent inflammatory response. Macrophages are a first line cell phenotype involved in the clearance of infection. Upon parasite uptake, these cells increase inflammatory mediators like NO, TNF-α, IL-1ß and IL-6, leading to parasite killing. Although desired, inflammatory response perpetuation and exacerbation may lead to tissue damage. Peroxisome proliferator-activated receptors (PPARs) are ligand-dependent nuclear transcription factors that, besides regulating lipid and carbohydrate metabolism, have a significant anti-inflammatory effect. This is mediated through the interaction of the receptors with their ligands. PPARγ, one of the PPAR isoforms, has been implicated in macrophage polarization from M1, the classically activated phenotype, to M2, the alternatively activated phenotype, in different models of metabolic disorders and infection. In this study, we show for the first time that, besides PPARγ, PPARα is also involved in the in vitro polarization of macrophages isolated from T. cruzi-infected mice. Polarization was evidenced by a decrease in the expression of NOS2 and proinflammatory cytokines and the increase in M2 markers like Arginase I, Ym1, mannose receptor and TGF-ß. Besides, macrophage phagocytic activity was significantly enhanced, leading to increased parasite load. We suggest that modulation of the inflammatory response by both PPARs might be due, at least in part, to a change in the profile of inflammatory macrophages. The potential use of PPAR agonists as modulators of overt inflammatory response during the course of Chagas' disease deserves further investigation.

Shedding of tumor necrosis factor receptor 1 induced by protein A decreases tumor necrosis factor alpha availability and inflammation during systemic Staphylococcus aureus infection.

Staphylococcus aureus infections are an important public health concern due to their increasing incidence and high rates of mortality. The success of S. aureus as a pathogen is highly related to its enormous capacity to evade the host immune response. The critical role of tumor necrosis factor alpha (TNF-α) in the initial host defense against systemic staphylococcal infection has been demonstrated in experimental models and may partially explain the lack of significant benefits observed in clinical trials attempting to neutralize this cytokine in septic patients. S. aureus protein A plays a key role in regulating inflammation through its ability to bind and signal through the TNF-α receptor 1 (TNFR1). In this study, we demonstrate that S. aureus, via protein A-mediated signaling, induces early shedding of TNFR1, which precedes the secretion of TNF-α in vitro and in vivo. The results obtained using a protein A-deficient mutant and tnfr1(-/-) mice strongly suggest that the increased levels of soluble TNFR1 present during experimental S. aureus infection may neutralize circulating TNF-α and impair the host inflammatory response. Early shedding of TNFR1 induced by protein A may constitute a novel mechanism by which S. aureus subverts the host immune response.

The length of the Staphylococcus aureus protein A polymorphic region regulates inflammation: impact on acute and chronic infection.

Staphylococcus aureus protein A (SpA) plays a critical role in the induction of inflammation. This study was aimed to determine whether the number of short sequence repeats (SSRs) present in the polymorphic region modulates the inflammatory response induced by SpA. We demonstrated that there is a dose-response effect in the activation of interferon (IFN)-ß signaling in airway epithelial and immune cells, depending on the number of SSRs, which leads to differences in neutrophil recruitment. We also determined that a significant proportion of isolates from patients with chronic infections such as osteomyelitis and cystic fibrosis carry fewer SSRs than do isolates from patients with acute infections or healthy carriers and that there was an inverse correlation between the number of SSRs and the length of disease course. Given the importance of IFN signaling in eradication of S. aureus, loss of SSRs may represent an advantageous mechanism to adapt to and persist in the host.

Staphylococcus aureus activates type I IFN signaling in mice and humans through the Xr repeated sequences of protein A.

The activation of type I IFN signaling is a major component of host defense against viral infection, but it is not typically associated with immune responses to extracellular bacterial pathogens. Using mouse and human airway epithelial cells, we have demonstrated that Staphylococcus aureus activates type I IFN signaling, which contributes to its virulence as a respiratory pathogen. This response was dependent on the expression of protein A and, more specifically, the Xr domain, a short sequence-repeat region encoded by DNA that consists of repeated 24-bp sequences that are the basis of an internationally used epidemiological typing scheme. Protein A was endocytosed by airway epithelial cells and subsequently induced IFN-beta expression, JAK-STAT signaling, and IL-6 production. Mice lacking IFN-alpha/beta receptor 1 (IFNAR-deficient mice), which are incapable of responding to type I IFNs, were substantially protected against lethal S. aureus pneumonia compared with wild-type control mice. The profound immunological consequences of IFN-beta signaling, particularly in the lung, may help to explain the conservation of multiple copies of the Xr domain of protein A in S. aureus strains and the importance of protein A as a virulence factor in the pathogenesis of staphylococcal pneumonia.