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Streptococcus pyogenes (group A Streptococcus ) is a globally disseminated and human-adapted bacterial pathogen that causes a wide range of infections, including scarlet fever. Scarlet fever is a toxin-mediated disease characterized by the formation of an erythematous, sandpaper-like rash that typically occurs in children aged 5 to 15. This infectious disease is caused by toxins called superantigens, a family of highly potent immunomodulators. Although scarlet fever had largely declined in both prevalence and severity since the late 19th century, outbreaks have now reemerged in multiple geographical regions over the past decade. Here, we review recent findings that address the role of superantigens in promoting a fitness advantage for S . pyogenes within human populations and discuss how superantigens may be suitable targets for vaccination strategies.

Superantigens (SAgs) are potent exotoxins secreted by Staphylococcus aureus and Streptococcus pyogenes. They target a large fraction of T cell pools to set in motion a \"cytokine storm\" with severe and sometimes life-threatening consequences typically encountered in toxic shock syndrome (TSS). Given the rapidity with which TSS develops, designing timely and truly targeted therapies for this syndrome requires identification of key mediators of the cytokine storm's initial wave. Equally important, early host responses to SAgs can be accompanied or followed by a state of immunosuppression, which in turn jeopardizes the host's ability to combat and clear infections. Unlike in mouse models, the mechanisms underlying SAg-associated immunosuppression in humans are ill-defined. In this work, we have identified a population of innate-like T cells, called mucosa-associated invariant T (MAIT) cells, as the most powerful source of pro-inflammatory cytokines after exposure to SAgs. We have utilized primary human peripheral blood and hepatic mononuclear cells, mouse MAIT hybridoma lines, HLA-DR4-transgenic mice, MAIThighHLA-DR4+ bone marrow chimeras, and humanized NOD-scid IL-2Rγnull mice to demonstrate for the first time that: i) mouse and human MAIT cells are hyperresponsive to SAgs, typified by staphylococcal enterotoxin B (SEB); ii) the human MAIT cell response to SEB is rapid and far greater in magnitude than that launched by unfractionated conventional T, invariant natural killer T (iNKT) or γδ T cells, and is characterized by production of interferon (IFN)-γ, tumor necrosis factor (TNF)-α and interleukin (IL)-2, but not IL-17A; iii) high-affinity MHC class II interaction with SAgs, but not MHC-related protein 1 (MR1) participation, is required for MAIT cell activation; iv) MAIT cell responses to SEB can occur in a T cell receptor (TCR) Vβ-specific manner but are largely contributed by IL-12 and IL-18; v) as MAIT cells are primed by SAgs, they also begin to develop a molecular signature consistent with exhaustion and failure to participate in antimicrobial defense. Accordingly, they upregulate lymphocyte-activation gene 3 (LAG-3), T cell immunoglobulin and mucin-3 (TIM-3), and/or programmed cell death-1 (PD-1), and acquire an anergic phenotype that interferes with their cognate function against Klebsiella pneumoniae and Escherichia coli; vi) MAIT cell hyperactivation and anergy co-utilize a signaling pathway that is governed by p38 and MEK1/2. Collectively, our findings demonstrate a pathogenic, rather than protective, role for MAIT cells during infection. Furthermore, we propose a novel mechanism of SAg-associated immunosuppression in humans. MAIT cells may therefore provide an attractive therapeutic target for the management of both early and late phases of severe SAg-mediated illnesses.

Background The lack of a reliable scoring system that predicts the development of septic shock and death precludes comparison of disease and/or treatment outcomes in animal models of sepsis. We developed a murine sepsis score (MSS) that evaluates seven clinical variables, and sought to assess its validity and reliability in an experimental mouse model of polymicrobial sepsis. Methods Stool collected from the cecum of C57BL/6 (B6) mice was dissolved in 0.9% normal saline (NS) and filtered, resulting in a fecal solution (FS) which was injected intraperitoneally into B6 mice. Disease severity was monitored by MSS during the experimental timeline. Blood and tissue samples were harvested for the evaluation of inflammatory changes after sepsis induction. The correlation between pro-inflammatory markers and MSS was assessed by the Spearman rank correlation coefficient. Results Mice injected with FS at a concentration of 90 mg/mL developed polymicrobial sepsis with a 75% mortality rate at 24 hours. The MSS was highly predictive of sepsis progression and mortality, with excellent discriminatory power, high internal consistency (Cronbach alpha coefficient = 0.92), and excellent inter-rater reliability (intra-class coefficient = 0.96). An MSS of 3 had a specificity of 100% for predicting onset of septic shock and death within 24 hours. Hepatic dysfunction and systemic pro-inflammatory responses were confirmed by biochemical and cytokine analyses where the latter correlated well with the MSS. Significant bacterial dissemination was noted in multiple organs. Furthermore, the liver, spleen, and intestine demonstrated histopathological evidence of injury. Conclusions The MSS reliably predicts disease progression and mortality in an animal model of polymicrobial sepsis. More importantly, it may be used to assess and compare outcomes among various experimental models of sepsis, and serve as an ethically acceptable alternative to death as an endpoint.

Streptococcus pyogenes is a globally prominent human-specific pathogen responsible for an enormous burden of human illnesses, including >600 million pharyngeal and >100 million skin infections each year. Despite intensive efforts that focus on invasive indications, much remains unknown about this bacterium in its natural state during colonization of the nasopharynx and skin. Using acute experimental infection models in HLA-transgenic mice, we evaluated how the hyaluronic acid (HA) capsule contributes to S . pyogenes MGAS8232 infection within these limited biological niches. Herein, we demonstrate that HA capsule expression promotes bacterial burden in murine nasal turbinates and skin lesions by resisting neutrophil-mediated killing. HA capsule production is encoded by the hasABC operon and compared to wildtype S . pyogenes infections, mice infected with a Δ hasA mutant exhibited over a 1000-fold CFU reduction at 48-hours post-nasal challenge, and a 10,000-fold CFU reduction from skin lesions 72-hours post-skin challenge. HA capsule expression contributed substantially to skin lesion size development following subdermal inoculations. In the absence of capsule expression, S . pyogenes revealed drastically impeded growth in whole human blood and increased susceptibility to killing by isolated neutrophils ex vivo , highlighting its important role in resisting phagocytosis. Furthermore, we establish that neutrophil depletion in mice recovered the reduced burden by the Δ hasA mutant in both the nasopharynx and skin. Together, this work confirms that the HA capsule is a key virulence determinant during acute infections by S . pyogenes and demonstrates that its predominant function is to protect S . pyogenes against neutrophil-mediated killing.

The production of the staphylococcal exotoxin toxic shock syndrome toxin-1 (TSST-1) by Staphylococcus aureus has been associated with essentially all cases of menstruation-associated toxic shock syndrome (TSS). In this work, we show that the human vaginal isolate Lactobacillus reuteri RC-14 produces small signaling molecules that are able to interfere with the staphylococcal quorum-sensing system agr, a key regulator of virulence genes, and repress the expression of TSST-1 in S. aureus MN8, a prototype of menstrual TSS S. aureus strains. Quantitative real-time PCR data showed that transcription from the Ptst promoter, as well as the P2 and P3 promoters of the agr system from all four agr subgroups of S. aureus, was strongly inhibited in response to growth with L. reuteri RC-14 cultural supernatant. Alterations in the transcriptional levels of two other virulence-associated regulators sarA and saeRS were also observed, indicating a potential overall influence of L. reuteri RC-14 signals on the production of virulence factors in S. aureus. S. aureus promoter-lux reporter strains were used to screen biochemically fractionated L. reuteri RC-14 supernatant, and the cyclic dipeptides cyclo(L-Phe-L-Pro) and cyclo(L-Tyr-L-Pro) were identified as the signaling molecules. The results from this work contribute to a better understanding of interspecies cell-to-cell communication between Lactobacillus and Staphylococcus, and provide a unique mechanism by which endogenous or probiotic strains may attenuate virulence factor production by bacterial pathogens.

The re-emergence of scarlet fever poses a new global public health threat. The capacity of North-East Asian serotype M12 ( emm 12) Streptococcus pyogenes (group A Streptococcus , GAS) to cause scarlet fever has been linked epidemiologically to the presence of novel prophages, including prophage ΦHKU.vir encoding the secreted superantigens SSA and SpeC and the DNase Spd1. Here, we report the molecular characterization of ΦHKU.vir-encoded exotoxins. We demonstrate that streptolysin O (SLO)-induced glutathione efflux from host cellular stores is a previously unappreciated GAS virulence mechanism that promotes SSA release and activity, representing the first description of a thiol-activated bacterial superantigen. Spd1 is required for resistance to neutrophil killing. Investigating single, double and triple isogenic knockout mutants of the ΦHKU.vir-encoded exotoxins, we find that SpeC and Spd1 act synergistically to facilitate nasopharyngeal colonization in a mouse model. These results offer insight into the pathogenesis of scarlet fever-causing GAS mediated by prophage ΦHKU.vir exotoxins. The pathogenesis of Streptococcus pyogenes (GAS) causing scarlet fever has been associated with the presence of prophages, such as ΦHKU.vir, and their products. Here, the authors characterize the exotoxins SpeC and Spd1 of ΦHKU.vir and show these to act synergistically to facilitate nasopharyngeal colonization in mice.

Streptococcus pyogenes is a human-specific pathogen that commonly colonizes the upper respiratory tract and skin, causing a wide variety of diseases ranging from pharyngitis to necrotizing fasciitis and toxic shock syndrome. S . pyogenes has a repertoire of secreted virulence factors that promote infection and evasion of the host immune system including the cytolysins streptolysin O (SLO) and streptolysin S (SLS). S . pyogenes does not naturally infect the upper respiratory tract of mice although mice transgenic for MHC class II human leukocyte antigens (HLA) become highly susceptible. Here we used HLA-transgenic mice to assess the role of both SLO and SLS during both nasopharyngeal and skin infection. Using S . pyogenes MGAS8232 as a model strain, we found that an SLS-deficient strain exhibited a 100-fold reduction in bacterial recovery from the nasopharynx and a 10-fold reduction in bacterial burden in the skin, whereas an SLO-deficient strain did not exhibit any infection defects in these models. Furthermore, depletion of neutrophils significantly restored the bacterial burden of the SLS-deficient bacteria in skin, but not in the nasopharynx. In mice nasally infected with the wildtype S . pyogenes , there was a marked change in localization of the tight junction protein ZO-1 at the site of infection, demonstrating damage to the nasal epithelia that was absent in mice infected with the SLS-deficient strain. Overall, we conclude that SLS is required for the establishment of nasopharyngeal infection and skin infection in HLA-transgenic mice by S . pyogenes MGAS8232 and provide evidence that SLS contributes to nasopharyngeal infection through the localized destruction of nasal epithelia.

INTRODUCTION:Infective endocarditis is associated with high morbidity and mortality. Currently, there is concern that the incidence of infective endocarditis associated with people who inject drugs (PWID) is increasing. However, it is difficult to monitor population-wide trends in PWID-associated infective endocarditis, as there is no International Statistical Classification of Diseases, 10th Revision (ICD-10) code for injection drug use. To address this barrier, we sought to develop a validated algorithm using ICD-10 discharge diagnosis codes. MATERIALS AND METHODS:We constructed a cohort of patients whose hospital discharge diagnosis included infective endocarditis. We reviewed 100 patients with incident infective endocarditis from 2014 to 2016 for their infective endocarditis and injection drug use status. We calculated the operating characteristics for algorithms constructed using permutations of ICD-10 codes associated with injection drug use. We repeated this analysis in a cohort of 100 patients with incident infective endocarditis from 2009 to 2011 to examine the temporal stability of the operating characteristics of each algorithm. RESULTS:We found that a combination of hepatitis C virus, drug use, and mental/behavioral disorder codes yielded the highest sensitivity (93%) and positive predictive value (83%) of the algorithms analyzed. DISCUSSION:We have described the first algorithm, validated against chart review data, for identifying PWID-associated infective endocarditis cases using ICD-10 codes. The high sensitivity and positive predictive value indicate that this algorithm can be used for surveillance and research with confidence. CONCLUSIONS:This algorithm will enable researchers to examine epidemiological trends in PWID-associated infective endocarditis.

Injection drug use-associated endocarditis (IDUaIE) incidence in Ontario has recently been associated with hydromorphone prescribing rates. Staphylococcus aureus causes the majority of cases of IDUaIE in Ontario and across North America. Hydromorphone controlled-release (Hydromorphone-CR) requires a complex technique for injection and therefore provides multiple opportunities for contamination. Hydromorphone-CR contains several excipients, which could enhance staphylococcal survival and increase risk of contaminating the injectate. Used injection drug preparation equipment (cookers/filters) was collected from persons who inject drugs (PWID), rinsed with water, and plated on Mannitol salt agar. Bacterial isolates from bacteremic PWID were used to assess the survival of S. aureus and Streptococcus pyogenes on cookers/filters with Hydromorphone-CR, hydromorphone immediate-release (Hydromorphone-IR) or oxycodone controlled-release (Oxycodone-CR). The solutions spiked with S. aureus were heated and the remaining viable bacteria enumerated. S. aureus was detected in 12/87 (14%, 95%CI 8-23%) cookers/filters samples used for injection of Hydromorphone-CR. Hydromorphone-CR was the only opioid associated with greater survival of methicillin-sensitive S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) on cookers/filters when compared to sterile water vehicle control. There was a ~2 log reduction in the number of S. aureus that survived when cookers/filters were heated. 14% of all cookers/filters used in the preparation of Hydromorphone-CR were contaminated with S. aureus. Hydromorphone-CR prolongs the survival of MRSA and MSSA in cookers/filters. Heating cookers/filters may be a harm-reduction strategy.

Superantigens (SAgs) are a family of potent immunostimulatory exotoxins known to be produced by only a few bacterial pathogens, including Staphylococcus aureus. More than 20 distinct SAgs have been characterized from different S. aureus strains and at least 80% of clinical strains harbor at least one SAg gene, although most strains encode many. SAgs have been classically associated with food poisoning and toxic shock syndrome (TSS), for which these toxins are the causative agent. TSS is a potentially fatal disease whereby SAg-mediated activation of T cells results in overproduction of cytokines and results in systemic inflammation and shock. Numerous studies have also shown a possible role for SAgs in other diseases such as Kawasaki disease (KD), atopic dermatitis (AD), and chronic rhinosinusitis (CRS). There is also now a rich understanding of the mechanisms of action of SAgs, as well as their structures and function. However, we have yet to discover what purpose SAgs play in the life cycle of S. aureus, and why such a wide array of these toxins exists. This review will focus on recent developments within the SAg field in terms of the molecular biology of these toxins and their role in both colonization and disease.