Hyperglycemia Increases Severity of Staphylococcus aureus Osteomyelitis and Influences Bacterial Genes Required for Survival in Bone.

Hyperglycemia, or elevated blood glucose, renders individuals more prone to developing severe Staphylococcus aureus infections. S. aureus is the most common etiological agent of musculoskeletal infection, which is a common manifestation of disease in hyperglycemic patients. However, the mechanisms by which S. aureus causes severe musculoskeletal infection during hyperglycemia are incompletely characterized. To examine the influence of hyperglycemia on S. aureus virulence during invasive infection, we used a murine model of osteomyelitis and induced hyperglycemia with streptozotocin. We discovered that hyperglycemic mice exhibited increased bacterial burdens in bone and enhanced dissemination compared to control mice. Furthermore, infected hyperglycemic mice sustained increased bone destruction relative to euglycemic controls, suggesting that hyperglycemia exacerbates infection-associated bone loss. To identify genes contributing to S. aureus pathogenesis during osteomyelitis in hyperglycemic animals relative to euglycemic controls, we used transposon sequencing (TnSeq). We identified 71 genes uniquely essential for S. aureus survival in osteomyelitis in hyperglycemic mice and another 61 mutants with compromised fitness. Among the genes essential for S. aureus survival in hyperglycemic mice was the gene encoding superoxide dismutase A (sodA), one of two S. aureus superoxide dismutases involved in detoxifying reactive oxygen species (ROS). We determined that a sodA mutant exhibits attenuated survival in vitro in high glucose and in vivo during osteomyelitis in hyperglycemic mice. SodA therefore plays an important role during growth in high glucose and promotes S. aureus survival in bone. Collectively, these studies demonstrate that hyperglycemia increases the severity of osteomyelitis and identify genes contributing to S. aureus survival during hyperglycemic infection.

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.

Host nutrient milieu drives an essential role for aspartate biosynthesis during invasive Staphylococcus aureus infection.

The bacterial pathogen Staphylococcus aureus is capable of infecting a broad spectrum of host tissues, in part due to flexibility of metabolic programs. S. aureus, like all organisms, requires essential biosynthetic intermediates to synthesize macromolecules. We therefore sought to determine the metabolic pathways contributing to synthesis of essential precursors during invasive S. aureus infection. We focused specifically on staphylococcal infection of bone, one of the most common sites of invasive S. aureus infection and a unique environment characterized by dynamic substrate accessibility, infection-induced hypoxia, and a metabolic profile skewed toward aerobic glycolysis. Using a murine model of osteomyelitis, we examined survival of S. aureus mutants deficient in central metabolic pathways, including glycolysis, gluconeogenesis, the tricarboxylic acid (TCA) cycle, and amino acid synthesis/catabolism. Despite the high glycolytic demand of skeletal cells, we discovered that S. aureus requires glycolysis for survival in bone. Furthermore, the TCA cycle is dispensable for survival during osteomyelitis, and S. aureus instead has a critical need for anaplerosis. Bacterial synthesis of aspartate in particular is absolutely essential for staphylococcal survival in bone, despite the presence of an aspartate transporter, which we identified as GltT and confirmed biochemically. This dependence on endogenous aspartate synthesis derives from the presence of excess glutamate in infected tissue, which inhibits aspartate acquisition by S. aureus Together, these data elucidate the metabolic pathways required for staphylococcal infection within bone and demonstrate that the host nutrient milieu can determine essentiality of bacterial nutrient biosynthesis pathways despite the presence of dedicated transporters.

Context-Dependent Roles for Toll-Like Receptors 2 and 9 in the Pathogenesis of Staphylococcus aureus Osteomyelitis.

Staphylococcus aureus is the major causative agent of bacterial osteomyelitis, an invasive infection of bone. Inflammation generated by the immune response to S. aureus contributes to bone damage by altering bone homeostasis. Increases in the differentiation of monocyte lineage cells into bone-resorbing osteoclasts (osteoclastogenesis) promote bone loss in the setting of osteomyelitis. In this study, we sought to define the role of Toll-like receptor (TLR) signaling in the pathogenesis of S. aureus osteomyelitis. We hypothesized that S. aureus-sensing TLRs 2 and 9, both of which are known to alter osteoclastogenesis in vitro, promote pathological changes to bone, including increased osteoclast abundance, bone loss, and altered callus formation during osteomyelitis. Stimulation of osteoclast precursors with S. aureus supernatant increased osteoclastogenesis in a TLR2-dependent, but not a TLR9-dependent, manner. However, in vivo studies using a posttraumatic murine model of osteomyelitis revealed that TLR2-null mice experienced similar bone damage and increased osteoclastogenesis compared to wild type (WT) mice. Therefore, we tested the hypothesis that compensation between TLR2 and TLR9 contributes to osteomyelitis pathogenesis. We found that mice deficient in both TLR2 and TLR9 (Tlr2/9-/-) have decreased trabecular bone loss in response to infection compared to WT mice. However, osteoclastogenesis is comparable between WT and Tlr2/9-/- mice, suggesting that alternative mechanisms enhance osteoclastogenesis in vivo during osteomyelitis. Indeed, we discovered that osteoclast precursors intracellularly infected with S. aureus undergo significantly increased osteoclast formation, even in the absence of TLR2 and TLR9. These results suggest that TLR2 and TLR9 have context-dependent roles in the alteration of bone homeostasis during osteomyelitis.

High Spatial Resolution MALDI Imaging Mass Spectrometry of Fresh-Frozen Bone.

Bone and bone marrow are vital to mammalian structure, movement, and immunity. These tissues are also commonly subjected to molecular alterations giving rise to debilitating diseases like rheumatoid arthritis and osteomyelitis. Technologies such as matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) facilitate the discovery of spatially resolved chemical information in biological tissue samples to help elucidate the complex molecular processes underlying pathology. Traditionally, preparation of osseous tissue for MALDI IMS has been difficult due to its mineralized composition and heterogeneous morphology, and compensation for these challenges with decalcification and fixation protocols can remove or delocalize molecular species. Here, sample preparation methods were advanced to enable multimodal MALDI IMS of undecalcified, fresh-frozen murine femurs, allowing the distribution of endogenous lipids to be linked to tissue structures and cell types. Adhesive-bound bone sections were mounted onto conductive glass slides with microscopy-compatible glue and freeze-dried to minimize artificial bone marrow damage. High spatial resolution (10 µm) MALDI IMS was employed to characterize lipid distributions, and use of complementary microscopy modalities aided tissue and cell assignments. For example, various phosphatidylcholines localize to the bone marrow, adipose tissue, marrow adipose tissue, and muscle. Further, sphingomyelin(42:1) was abundant in megakaryocytes, whereas sphingomyelin(42:2) was diminished in this cell type. These data reflect the vast molecular and cellular heterogeneity indicative of the bone marrow and the soft tissue surrounding the femur. Multimodal MALDI IMS has the potential to advance bone-related biomedical research by offering deep molecular coverage with spatial relevance in a preserved native bone microenvironment.

Staphylococcus aureus exhibits heterogeneous siderophore production within the vertebrate host.

Siderophores, iron-scavenging small molecules, are fundamental to bacterial nutrient metal acquisition and enable pathogens to overcome challenges imposed by nutritional immunity. Multimodal imaging mass spectrometry allows visualization of host-pathogen iron competition, by mapping siderophores within infected tissue. We have observed heterogeneous distributions of Staphylococcus aureus siderophores across infectious foci, challenging the paradigm that the vertebrate host is a uniformly iron-depleted environment to invading microbes.

Contemporary Clinical Isolates of Staphylococcus aureus from Pediatric Osteomyelitis Patients Display Unique Characteristics in a Mouse Model of Hematogenous Osteomyelitis.

Osteomyelitis can result from the direct inoculation of pathogens into bone during injury or surgery or from spread via the bloodstream, a condition called hematogenous osteomyelitis (HOM). HOM disproportionally affects children, and more than half of cases are caused by Staphylococcus aureus. Laboratory models of osteomyelitis mostly utilize direct injection of bacteria into the bone or implantation of foreign material and therefore do not directly interrogate the pathogenesis of pediatric hematogenous osteomyelitis. In this study, we inoculated mice intravenously and characterized the resultant musculoskeletal infections using two strains isolated from adults (USA300-LAC and NRS384) and five new methicillin-resistant S. aureus isolates from pediatric osteomyelitis patients. All strains were capable of creating stable infections over 5 weeks, although the incidence varied. Micro-computed tomography (microCT) analysis demonstrated decreases in the trabecular bone volume fraction but little effect on bone cortices. Histological assessment revealed differences in the precise focus of musculoskeletal infection, with various mixtures of bone-centered osteomyelitis and joint-centered septic arthritis. Whole-genome sequencing of three new isolates demonstrated distinct strains, two within the USA300 lineage and one USA100 isolate. Interestingly, this USA100 isolate showed a distinct predilection for septic arthritis compared to the other isolates tested, including NRS384 and LAC, which more frequently led to osteomyelitis or mixed bone and joint infections. Collectively, these data outline the feasibility of using pediatric osteomyelitis clinical isolates to study the pathogenesis of HOM in murine models and lay the groundwork for future studies investigating strain-dependent differences in musculoskeletal infection.

Staphylococcus aureus infections in children.

PURPOSE OF REVIEW: Staphylococcus aureus is the most common invasive bacterial pathogen infecting children in the U.S. and many parts of the world. This major human pathogen continues to evolve, and recognition of recent trends in epidemiology, therapeutics and future horizons is of high importance. RECENT FINDINGS: Over the past decade, a relative rise of methicillin-susceptible S. aureus (MSSA) has occurred, such that methicillin-resistant S. aureus (MRSA) no longer dominates the landscape of invasive disease. Antimicrobial resistance continues to develop, however, and novel therapeutics or preventive modalities are urgently needed. Unfortunately, several recent vaccine attempts proved unsuccessful in humans. SUMMARY: Recent scientific breakthroughs highlight the opportunity for novel interventions against S. aureus by interfering with virulence rather than by traditional antimicrobial mechanisms. A S. aureus vaccine remains elusive; the reasons for this are multifactorial, and lessons learned from prior unsuccessful attempts may create a path toward an effective preventive. Finally, new diagnostic modalities have the potential to greatly enhance clinical care for invasive S. aureus disease in children.

MyD88 and IL-1R signaling drive antibacterial immunity and osteoclast-driven bone loss during Staphylococcus aureus osteomyelitis.

Staphylococcus aureus is able to infect virtually all organ systems and is a frequently isolated etiologic agent of osteomyelitis, a common and debilitating invasive infection of bone. Treatment of osteomyelitis requires invasive surgical procedures and prolonged antibiotic therapy, yet is frequently unsuccessful due to extensive pathogen-induced bone damage that can limit antibiotic penetration and immune cell influx to the infectious focus. We previously established that S. aureus triggers profound alterations in bone remodeling in a murine model of osteomyelitis, in part through the production of osteolytic toxins. However, staphylococcal strains lacking osteolytic toxins still incite significant bone destruction, suggesting that host immune responses are also major drivers of pathologic bone remodeling during osteomyelitis. The objective of this study was to identify host immune pathways that contribute to antibacterial immunity during S. aureus osteomyelitis, and to define how these immune responses alter bone homeostasis and contribute to bone destruction. We specifically focused on the interleukin-1 receptor (IL-1R) and downstream adapter protein MyD88 given the prominent role of this signaling pathway in both antibacterial immunity and osteo-immunologic crosstalk. We discovered that while IL-1R signaling is necessary for local control of bacterial replication during osteomyelitis, it also contributes to bone loss during infection. Mechanistically, we demonstrate that S. aureus enhances osteoclastogenesis of myeloid precursors in vitro, and increases the abundance of osteoclasts residing on bone surfaces in vivo. This enhanced osteoclast abundance translates to trabecular bone loss, and is dependent on intact IL-1R signaling. Collectively, these data define IL-1R signaling as a critical component of the host response to S. aureus osteomyelitis, but also demonstrate that IL-1R-dependent immune responses trigger collateral bone damage through activation of osteoclast-mediated bone resorption.

Staphylococcus aureus Osteomyelitis: Bone, Bugs, and Surgery.

Osteomyelitis, or inflammation of bone, is most commonly caused by invasion of bacterial pathogens into the skeleton. Bacterial osteomyelitis is notoriously difficult to treat, in part because of the widespread antimicrobial resistance in the preeminent etiologic agent, the Gram-positive bacterium Staphylococcus aureus Bacterial osteomyelitis triggers pathological bone remodeling, which in turn leads to sequestration of infectious foci from innate immune effectors and systemically delivered antimicrobials. Treatment of osteomyelitis therefore typically consists of long courses of antibiotics in conjunction with surgical debridement of necrotic infected tissues. Even with these extreme measures, many patients go on to develop chronic infection or sustain disease comorbidities. A better mechanistic understanding of how bacteria invade, survive within, and trigger pathological remodeling of bone could therefore lead to new therapies aimed at prevention or treatment of osteomyelitis as well as amelioration of disease morbidity. In this minireview, we highlight recent developments in our understanding of how pathogens invade and survive within bone, how bacterial infection or resulting innate immune responses trigger changes in bone remodeling, and how model systems can be leveraged to identify new therapeutic targets. We review the current state of osteomyelitis epidemiology, diagnostics, and therapeutic guidelines to help direct future research in bacterial pathogenesis.

Concurrent Local Delivery of Diflunisal Limits Bone Destruction but Fails To Improve Systemic Vancomycin Efficacy during Staphylococcus aureus Osteomyelitis.

Staphylococcus aureus osteomyelitis is a debilitating infection of bone. Treatment of osteomyelitis is impaired by the propensity of invading bacteria to induce pathological bone remodeling that may limit antibiotic penetration to the infectious focus. The nonsteroidal anti-inflammatory drug diflunisal was previously identified as an osteoprotective adjunctive therapy for osteomyelitis, based on the ability of this compound to inhibit S. aureus quorum sensing and subsequent quorum-dependent toxin production. When delivered locally during experimental osteomyelitis, diflunisal significantly limits bone destruction without affecting bacterial burdens. However, because diflunisal's "quorum-quenching" activity could theoretically increase antibiotic recalcitrance, it is critically important to evaluate this adjunctive therapy in the context of standard-of-care antibiotics. The objective of this study is to evaluate the efficacy of vancomycin to treat osteomyelitis during local diflunisal treatment. We first determined that systemic vancomycin effectively reduces bacterial burdens in a murine model of osteomyelitis and identified a dosing regimen that decreases bacterial burdens without eradicating infection. Using this dosing scheme, we found that vancomycin activity is unaffected by the presence of diflunisal in vitro and in vivo Similarly, locally delivered diflunisal still potently inhibits osteoblast cytotoxicity in vitro and bone destruction in vivo in the presence of subtherapeutic vancomycin. However, we also found that the resorbable polyester urethane (PUR) foams used to deliver diflunisal serve as a nidus for infection. Taken together, these data demonstrate that diflunisal does not significantly impact standard-of-care antibiotic therapy for S. aureus osteomyelitis, but they also highlight potential pitfalls encountered with local drug delivery.

Innate Immunity to Staphylococcus aureus: Evolving Paradigms in Soft Tissue and Invasive Infections.

Staphylococcus aureus causes a wide range of diseases that together embody a significant public health burden. Aided by metabolic flexibility and a large virulence repertoire, S. aureus has the remarkable ability to hematogenously disseminate and infect various tissues, including skin, lung, heart, and bone, among others. The hallmark lesions of invasive staphylococcal infections, abscesses, simultaneously denote the powerful innate immune responses to tissue invasion as well as the ability of staphylococci to persist within these lesions. In this article, we review the innate immune responses to S. aureus during infection of skin and bone, which serve as paradigms for soft tissue and bone disease, respectively.

MicroLESA: Integrating Autofluorescence Microscopy, In Situ Micro-Digestions, and Liquid Extraction Surface Analysis for High Spatial Resolution Targeted Proteomic Studies.

The ability to target discrete features within tissue using liquid surface extractions enables the identification of proteins while maintaining the spatial integrity of the sample. Here, we present a liquid extraction surface analysis (LESA) workflow, termed microLESA, that allows proteomic profiling from discrete tissue features of ∼110 µm in diameter by integrating nondestructive autofluorescence microscopy and spatially targeted liquid droplet micro-digestion. Autofluorescence microscopy provides the visualization of tissue foci without the need for chemical stains or the use of serial tissue sections. Tryptic peptides are generated from tissue foci by applying small volume droplets (∼250 pL) of enzyme onto the surface prior to LESA. The microLESA workflow reduced the diameter of the sampled area almost 5-fold compared to previous LESA approaches. Experimental parameters, such as tissue thickness, trypsin concentration, and enzyme incubation duration, were tested to maximize proteomics analysis. The microLESA workflow was applied to the study of fluorescently labeled Staphylococcus aureus infected murine kidney to identify unique proteins related to host defense and bacterial pathogenesis. Proteins related to nutritional immunity and host immune response were identified by performing microLESA at the infectious foci and surrounding abscess. These identifications were then used to annotate specific proteins observed in infected kidney tissue by MALDI FT-ICR IMS through accurate mass matching.

Monoclonal Antibodies Against the Staphylococcus aureus Bicomponent Leukotoxin AB Isolated Following Invasive Human Infection Reveal Diverse Binding and Modes of Action.

The 2-component leukotoxin LukAB is critical for Staphylococcus aureus targeting and killing of human neutrophils ex vivo and is produced in the setting of human infection. We report 3 LukAB-specific human monoclonal antibodies (mAbs) with distinct mechanisms of toxin neutralization and in vivo efficacy. Three hybridomas secreting mAbs with anti-LukAB activity (designated SA-13, -15, and -17) were generated from B cells obtained from a 12-year-old boy with S. aureus osteomyelitis. Each of the 3 mAbs neutralized LukAB-mediated neutrophil toxicity, exhibited differing levels of potency, recognized different antigenic sites on the toxin, and displayed at least 2 distinct mechanisms for cytotoxic inhibition. SA-15 bound exclusively to the dimeric form of the toxin, suggesting that human B cells recognize epitopes on the dimerized form of LukAB during natural infection. Both SA-13 and SA-17 bound the LukA monomer and the LukAB dimer. Although all 3 mAbs potently neutralized cytotoxicity, only SA-15 and SA-17 significantly inhibited toxin association with the cell surface. Treatment with a 1:1 mixture of mAbs SA-15 and SA-17 resulted in significantly lower bacterial colony counts in heart, liver, and kidneys in a murine model of S. aureus sepsis. These data describe the isolation of diverse and efficacious antitoxin mAbs.

Bacterial Hypoxic Responses Revealed as Critical Determinants of the Host-Pathogen Outcome by TnSeq Analysis of Staphylococcus aureus Invasive Infection.

Staphylococcus aureus is capable of infecting nearly every organ in the human body. In order to infiltrate and thrive in such diverse host tissues, staphylococci must possess remarkable flexibility in both metabolic and virulence programs. To investigate the genetic requirements for bacterial survival during invasive infection, we performed a transposon sequencing (TnSeq) analysis of S. aureus during experimental osteomyelitis. TnSeq identified 65 genes essential for staphylococcal survival in infected bone and an additional 148 mutants with compromised fitness in vivo. Among the loci essential for in vivo survival was SrrAB, a staphylococcal two-component system previously reported to coordinate hypoxic and nitrosative stress responses in vitro. Healthy bone is intrinsically hypoxic, and intravital oxygen monitoring revealed further decreases in skeletal oxygen concentrations upon S. aureus infection. The fitness of an srrAB mutant during osteomyelitis was significantly increased by depletion of neutrophils, suggesting that neutrophils impose hypoxic and/or nitrosative stresses on invading bacteria. To more globally evaluate staphylococcal responses to changing oxygenation, we examined quorum sensing and virulence factor production in staphylococci grown under aerobic or hypoxic conditions. Hypoxic growth resulted in a profound increase in quorum sensing-dependent toxin production, and a concomitant increase in cytotoxicity toward mammalian cells. Moreover, aerobic growth limited quorum sensing and cytotoxicity in an SrrAB-dependent manner, suggesting a mechanism by which S. aureus modulates quorum sensing and toxin production in response to environmental oxygenation. Collectively, our results demonstrate that bacterial hypoxic responses are key determinants of the staphylococcal-host interaction.

Impact of sarA and Phenol-Soluble Modulins on the Pathogenesis of Osteomyelitis in Diverse Clinical Isolates of Staphylococcus aureus.

We used a murine model of acute, posttraumatic osteomyelitis to evaluate the virulence of two divergent Staphylococcus aureus clinical isolates (the USA300 strain LAC and the USA200 strain UAMS-1) and their isogenic sarA mutants. The results confirmed that both strains caused comparable degrees of osteolysis and reactive new bone formation in the acute phase of osteomyelitis. Conditioned medium (CM) from stationary-phase cultures of both strains was cytotoxic to cells of established cell lines (MC3TC-E1 and RAW 264.7 cells), primary murine calvarial osteoblasts, and bone marrow-derived osteoclasts. Both the cytotoxicity of CM and the reactive changes in bone were significantly reduced in the isogenic sarA mutants. These results confirm that sarA is required for the production and/or accumulation of extracellular virulence factors that limit osteoblast and osteoclast viability and that thereby promote bone destruction and reactive bone formation during the acute phase of S. aureus osteomyelitis. Proteomic analysis confirmed the reduced accumulation of multiple extracellular proteins in the LAC and UAMS-1 sarA mutants. Included among these were the alpha class of phenol-soluble modulins (PSMs), which were previously implicated as important determinants of osteoblast cytotoxicity and bone destruction and repair processes in osteomyelitis. Mutation of the corresponding operon reduced the cytotoxicity of CM from both UAMS-1 and LAC cultures for osteoblasts and osteoclasts. It also significantly reduced both reactive bone formation and cortical bone destruction by CM from LAC cultures. However, this was not true for CM from cultures of a UAMS-1 psmα mutant, thereby suggesting the involvement of additional virulence factors in such strains that remain to be identified.

Repurposing the Nonsteroidal Anti-inflammatory Drug Diflunisal as an Osteoprotective, Antivirulence Therapy for Staphylococcus aureus Osteomyelitis.

Staphylococcus aureus osteomyelitis is a common and debilitating invasive infection of bone. Treatment of osteomyelitis is confounded by widespread antimicrobial resistance and the propensity of bacteria to trigger pathological changes in bone remodeling that limit antimicrobial penetration to the infectious focus. Adjunctive therapies that limit pathogen-induced bone destruction could therefore limit morbidity and enhance traditional antimicrobial therapies. In this study, we evaluate the efficacy of the U.S. Food and Drug Administration-approved, nonsteroidal anti-inflammatory (NSAID) compound diflunisal in limiting S. aureus cytotoxicity toward skeletal cells and in preventing bone destruction during staphylococcal osteomyelitis. Diflunisal is known to inhibit S. aureus virulence factor production by the accessory gene regulator (agr) locus, and we have previously demonstrated that the Agr system plays a substantial role in pathological bone remodeling during staphylococcal osteomyelitis. Consistent with these observations, we find that diflunisal potently inhibits osteoblast cytotoxicity caused by S. aureus secreted toxins independently of effects on bacterial growth. Compared to commonly used NSAIDs, diflunisal is uniquely potent in the inhibition of skeletal cell death in vitro Moreover, local delivery of diflunisal by means of a drug-eluting, bioresorbable foam significantly limits bone destruction during S. aureus osteomyelitis in vivo Collectively, these data demonstrate that diflunisal potently inhibits skeletal cell death and bone destruction associated with S. aureus infection and may therefore be a useful adjunctive therapy for osteomyelitis.

A fungal metabolic regulator underlies infectious synergism during Candida albicans - Staphylococcus aureus intra-abdominal co-infection.

Candida albicans and Staphylococcus aureus are two commonly associated pathogens that cause nosocomial infections with high morbidity and mortality. Our prior and current work using a murine model of polymicrobial intra-abdominal infection (IAI) uncovered synergistic lethality that was driven by Candida -induced upregulation of functional S. aureus ⍺-toxin leading to polymicrobial sepsis and organ damage. In order to determine the candidal effector(s) mediating enhanced virulence, an unbiased screen of C. albicans transcription factor mutants was undertaken and revealed that zcf13 Δ/Δ failed to drive augmented ⍺-toxin or lethal synergism during co-infection. Using a combination of transcriptional and phenotypic profiling approaches, ZCF13 was shown to regulate genes involved in pentose metabolism, including RBK1 and HGT7 that contribute to fungal ribose catabolism and uptake, respectively. Subsequent experiments revealed that ribose inhibited the staphylococcal agr quorum sensing system and concomitantly repressed toxicity. Unlike wild-type C. albicans , zcf13 Δ/Δ was unable to effectively utilize ribose during co-culture or co-infection leading to exogenous ribose accumulation and agr repression. Forced expression of RBK1 and HGT7 in the zcf13 Δ/Δ mutant fully restored pathogenicity during co-infection. Collectively, our results detail the interwoven complexities of cross-kingdom interactions and highlight how intermicrobial metabolism impacts polymicrobial disease pathogenesis with devastating consequences for the host.

The 2023 Orthopaedic Research Society's International Consensus Meeting on musculoskeletal infection: Summary from the host immunity section.

Musculoskeletal infections (MSKI), which are a major problem in orthopedics, occur when the pathogen eludes or overwhelms the host immune system. While effective vaccines and immunotherapies to prevent and treat MSKI should be possible, fundamental knowledge gaps in our understanding of protective, nonprotective, and pathogenic host immunity are prohibitive. We also lack critical knowledge of how host immunity is affected by the microbiome, implants, prior infection, nutrition, antibiotics, and concomitant therapies, autoimmunity, and other comorbidities. To define our current knowledge of these critical topics, a Host Immunity Section of the 2023 Orthopaedic Research Society MSKI International Consensus Meeting (ICM) proposed 78 questions. Systematic reviews were performed on 15 of these questions, upon which recommendations with level of evidence were voted on by the 72 ICM delegates, and another 12 questions were voted on with a recommendation of "Unknown" without systematic reviews. Two questions were transferred to another ICM Section, and the other 45 were tabled for future consideration due to limitations of available human resources. Here we report the results of the voting with internet access to the questions, recommendations, and rationale from the systematic reviews. Eighteen questions received a consensus vote of ≥90%, while nine recommendations failed to achieve this threshold. Commentary on why consensus was not achieved on these questions and potential ways forward are provided to stimulate specific funding mechanisms and research on these critical MSKI host defense questions.