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Purpose of Review: This review summarizes the general concepts of innate and acquired immunity, including vaccine use and hesitancy, as they relate to reduction of the global burden of highly communicable infectious diseases., Recent Findings: Vaccination to increase herd immunity remains the cornerstone of disease prevention worldwide yet global vaccination goals are not being met. Modern obstacles to vaccine acceptance include hesitancy, reduced altruistic intentions, impact of COVID-19, distrust of science and governmental agencies as well as recent geopolitical and environmental disasters. Together, such barriers have negatively impacted immunization rates worldwide, resulting in epidemics and pandemics of serious life-threatening infections from vaccine-preventable diseases, especially those affecting children. In addition, pathogens thought to be controlled or eradicated are reemerging with new genetic traits, making them more able to evade natural and acquired immunity, including that induced by available vaccines. Lastly, many serious and widespread infectious diseases await development and utilization of efficacious vaccines., Summary: The global burden of communicable diseases remains high, necessitating continued pathogen surveillance as well as vaccine development, deployment and continued efficacy testing. Equally important is the need to educate aggressively the people and their leaders on the benefits of vaccination to the individual, local community and the human population as a whole., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

Background: Apart from their antimicrobial activities, some antibiotics have immunomodulatory effects on host cells, particularly monocytes. Because hyperactivation of the pro-inflammatory cytokine response contributes to acute lung injury in patients with bacterial pneumonia and other lung diseases, antimicrobial agents with immunomodulatory activity can reduce cytokine-mediated tissue injury and improve outcomes., Objectives: Omadacycline has been recently FDA-approved for community-acquired bacterial pneumonia and acute bacterial skin and skin-structure infections. The present study investigated omadacycline's ability to modulate LPS-induced production of pro-inflammatory cytokines (TNF-α, IL-1β), acute-phase reactants (IL-6) and anti-inflammatory cytokines (IL-4, IL-10) by human monocytes in vitro., Methods: Isolated human monocytes from healthy consenting adults were cultured in RPMI with 1% pooled human serum. Cells were pre-exposed to omadacycline (0.5-64 μg/mL), minocycline (25, 50 or 25 μg/mL) or azithromycin (20, 40 or 80 μg/mL) for 2 h, followed by stimulation with Escherichia coli LPS for 24 h. Cytokines elaborated in the culture supernatant were quantitated by multiplex immunoassay., Results: Omadacycline dose-dependently suppressed LPS-induced production of all cytokines tested. Only high-dose minocycline (100 μg/mL) modestly suppressed TNF-α whereas minocycline significantly increased LPS-induced IL-1β production. Lower concentrations of minocycline were also stimulatory for IFN-γ, IL-6 and IL-4. Except for suppression of IL-6, azithromycin was largely without effect., Conclusions: Omadacycline has unique and broad immunomodulatory properties. Such activity supports its use in settings where hyperactivation of the immune response contributes to tissue injury and poor outcomes, especially at sites where pro-inflammatory M-type 1 macrophages dominate the cellular immune response., (© The Author(s) 2022. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy.)

Necrotizing soft tissue infections caused by Streptococcus pyogenes (group A streptococcus [GAS]) are characterized by rapid and extensive necrosis of fascia and muscle. Molecular epidemiological studies have demonstrated a positive correlation between GAS isolates that cause invasive infections and the production of S. pyogenes NAD+-glycohydrolase (SPN), an NADase secreted by GAS, but the effect of SPN on muscle cells has not been described. Thus, using standard βNAD+ and ATP quantification assays, we investigated the effects of SPN on cultured human skeletal muscle cell (SkMC) βNAD+ and ATP with and without streptolysin O (SLO)-a secreted cholesterol-dependent cytolysin known to act synergistically with SPN. We found that culture supernatants from GAS strains producing SLO and SPN depleted intracellular βNAD+ and ATP, while exotoxins from a GAS strain producing SLO and an enzymatically-inactive form of SPN had no effect on βNAD+ or ATP. Addition of purified, enzymatically-active SPN to NADase-negative culture supernatants or sterile media reconstituted βNAD+ depletion but had no effect ATP levels. Further, SPN-mediated βNAD+ depletion could be augmented by SLO or the homologous cholesterol-dependent cytolysin, perfringolysin O (PFO). Remarkably, SPN-mediated βNAD+ depletion was SkMC-specific, as purified SPN had minimal effect on epithelial cell βNAD+. Taken together, this study identifies a previously unrecognized role for SPN as a major disruptor of skeletal muscle βNAD+. Such activity could contribute to the rapid and widespread myonecrosis characteristic of severe GAS soft tissue infections.

Necrotizing soft tissue infections occur after traumatic injuries, minor skin lesions, nonpenetrating injuries, natural childbirth, and in postsurgical and immunocompromised patients. Infections can be severe, rapidly progressive, and life threatening. Survivors often endure multiple surgeries and prolonged hospitalization and rehabilitation. Despite subtle nuances that may distinguish one entity from another, clinical approaches to diagnosis and treatment are highly similar. This review describes the clinical and laboratory features of necrotizing soft tissue infections and addresses recommended diagnostic and treatment modalities. It discusses the impact of delays in surgical debridement, antibiotic use, and resuscitation on mortality, and summarizes key pathogenic mechanisms., Competing Interests: Disclosure The authors have nothing to disclose., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Objectives: Clindamycin (CLI) and erythromycin (ERY) resistance is increasing among group A streptococci (GAS) causing invasive disease and alternative treatments are urgently required. In this study, the efficacy of the newer oxazolidinone tedizolid (TZD) was compared with the first drug in this class, linezolid (LNZ), in experimental murine myonecrosis caused by ERY-susceptible/CLI-susceptible (ERY S /CLI S ) or ERY- resistant/CLI-resistant (ERY R /CLI R ) GAS., Methods: Normal adult outbred Swiss Webster female mice (10 per group) were infected intramuscularly with ERY S /CLI S (ATCC 12384) or ERY R /CLI R (15-003) GAS. Treatments began 4 h post-infection and continued for 72 h. TZD and LNZ (10, 20 and 40 mg/kg) were given intraperitoneally every 12 h. Saline, penicillin (PEN), CLI and ERY were given every 6 h. Survival and infection severity signs and symptoms were followed for 12 days., Results: Both GAS strains were susceptible to LNZ, TZD and PEN; strain 15-003 was confirmed as constitutively resistant to ERY and CLI. Blood levels following a 40 mg/kg dose of LZD and TZD were 30.9 ± 4.0 μg/mL and 21.9 ± 5.3 μg/mL, respectively. Both TZD and LNZ were highly efficacious for the treatment of severe experimental myonecrosis caused by ERY S /CLI S and, importantly, ERY R /CLI R GAS., Conclusion: In the current era of emerging macrolide/lincosamide resistance among GAS, these data support the use of TZD and LNZ as first-line antibiotics for the treatment of life-threatening GAS infections in humans., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Purpose: Extracellular protein toxins contribute to the pathogenesis of Staphylococcus aureus infections. The present study compared the effects of iclaprim and trimethoprim - two folic acid synthesis inhibitors - with nafcillin and vancomycin on production of Panton-Valentine leukocidin (PVL), alpha haemolysin (AH) and toxic-shock syndrome toxin I (TSST-1) in methicillin-resistant and vancomycin-intermediate S. aureus (MRSA and VISA, respectively)., Methodology: Northern blotting and RT-PCR were used to assess gene transcription; toxin-specific bioassays were used to measure protein toxin production., Results: As shown previously, sub-inhibitory concentrations (sub-MIC) of nafcillin increased and prolonged MRSA toxin gene transcription and enhanced PVL, TSST-1 and AH production. Sub-inhibitory doses of iclaprim and trimethoprim delayed maximal AH gene (hla) transcription and suppressed AH production; both drugs delayed, but neither reduced, maximal TSST-1 production. Trimethoprim significantly increased lukF-PV expression and PVL production compared to both untreated and iclaprim-treated cultures. Higher concentrations of iclaprim and trimethoprim markedly suppressed MRSA growth, mRNA synthesis and toxin production. In VISA, iclaprim, vancomycin and nafcillin variably increased tst and hla expression, but only nafcillin increased toxin production. Despite its ability to increase hla expression, iclaprim was the most potent inhibitor of AH production., Conclusions: We conclude that, due to its ability to suppress toxin production, iclaprim should be effective against severe staphylococcal infections caused by toxin-producing MRSA and VISA strains, especially given its ability to concentrate at sites of infection such as skin and skin structures and the lung.

Purpose: Clostridium difficile is an anaerobic spore-forming bacterial pathogen that causes a spectrum of illness severity ranging from mild diarrhoea to severe life-threatening pseudomembranous colitis. C. difficile infection (CDI) is antibiotic-associated and primarily mediated by two exotoxins, Toxins A and B. We and others have shown that some antibiotics stimulate Toxin A and B production by C. difficile in a strain-specific manner. Still, the effects of newer anti-C. difficile antibiotics on this process and spore formation remain to be investigated., Methodology: Surotomycin (formally CB-183,315) is a novel, minimally absorbed, narrow-spectrum antibiotic. We determined the effects of surotomycin on C. difficile growth, toxin production and sporulation in historical and BI/NAP1/027 epidemic strains of C. difficile.Results/Key findings. While antibiotic free controls showed toxin production during the stationary phase growth, all strains exposed to sub-inhibitory concentrations of surotomycin and vancomycin demonstrated increased TcdA and TcdB production during early (log phase) growth by all strains. However, this effect was not observed at 24 or 48 h post-treatment by any of the C. difficile strains exposed to either antibiotic. Additionally, all doses of surotomycin and vancomycin suppressed spore formation in all tested strains., Conclusion: In summary, these findings demonstrate that surotomycin and vancomycin have similar effects on exotoxin production and sporulation by C. difficile in vitro. Furthermore, since spores contribute to recurrent infection, the ability of surotomycin to suppress spore formation may explain its ability to disrupt the reinfection cycle in the clinical setting.

Clostridium septicum is a highly pathogenic microbe that causes gas gangrene in humans, and is the principal cause of spontaneous gas gangrene in patients with gastrointestinal maladies, including adenocarcinoma of the colon. Despite modern approaches to manage C. septicum infection, morbidity and mortality remain high (>60%). At present, no objective in-vivo data exist supporting the current antibiotic treatment recommendations for C. septicum infection. Utilizing an established murine model of clostridial myonecrosis, this study investigated the efficacy of standard antibiotics for anaerobic Gram-positive soft tissue infections (penicillin, clindamycin, tetracycline and vancomycin) in treating C. septicum gas gangrene. Following intramuscular challenge with 1 × 10 6 colony-forming units of C. septicum, antibiotics were administered by intraperitoneal injection every 4 h for a total of four doses. At 30 h, all animals in all treatment groups survived the C. septicum challenge, compared with no survivors in the untreated controls (100% mortality by 10 h). However, by 60 h, mice treated with vancomycin exhibited 40% mortality, with no mortality observed in any other antibiotic treatment group. Microbroth dilution minimum inhibitory concentration analyses for three strains of C. septicum also demonstrated high susceptibility to penicillin, clindamycin and tetracycline, but considerably lower susceptibility to vancomycin. This study suggests that penicillin, clindamycin and tetracycline are suitable alternatives for the treatment of C. septicum infection in humans., (Copyright © 2018 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.)

As the infectious disease paradigm undergoes a subtle shift, unusual infections associated with malignancy and immunosuppression are being increasingly reported. Spontaneous or non-traumatic Clostridium septicum infection is one such unusual infection which has gained prominence. This article aims to understand the pathophysiology, clinical manifestations and current trends in diagnosing and treating this rare but deadly infection. To understand the multifactorial causation of this infection a review of published cases of spontaneous C. septicum gas gangrene was performed and a total of 94 such cases were identified. Several factors were analyzed for each case: age, infection location and underlying illness, presenting signs and symptoms, neutropenia, gross pathology of the colon, antibiotic use, surgical intervention, and survival. A known or occult malignancy was present in 71% patients and an overall mortality of 67% was observed., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

This study investigated the effects of subinhibitory doses of the lipoglycopeptide antibiotic dalbavancin on Staphylococcus aureus toxin production in vitro S. aureus toxin production levels were compared to those seen with the natural glycopeptide antibiotic vancomycin and with representative beta-lactam and oxazolidinone antibiotics. While neither dalbavancin nor vancomycin adversely affected toxin production, of these glycopeptide antibiotics, only dalbavancin significantly attenuated toxin production at subinhibitory concentrations. These findings support the recent success of dalbavancin for treatment of staphylococcal infections., (Copyright © 2017 American Society for Microbiology.)

Purpose: Fidaxomicin, a macrocyclic antibiotic, has been approved for the treatment of Clostridium difficile infection (CDI). Previous work by our group has demonstrated that some antibiotics at sub-inhibitory concentrations stimulate early toxin production and sporulation by C. difficile. Prior studies revealed that fidaxomicin, when added to late stationary-phase organisms, reduced exotoxin production and spore formation by C. difficile. However, the ability of fidaxomicin to trigger early virulence factor production and spore formation has never been investigated., Methodology: Sub-inhibitory concentrations of the RNA synthesis inhibitor fidaxomicin (1/4×, 1/8×, 1/16× MIC) were added immediately to lag-phase cultures of historical (strain 9689) and epidemic BI/NAP1/027 (strain 5325) strains of C. difficile, and their effects on sporulation and toxin A (TcdA) and toxin B (TcdB) production were compared.Results/Key findings. Even at sub-inhibitory concentrations, all doses of fidaxomicin reduced both TcdA and TcdB gene expression and protein production in the historical and epidemic C. difficile strains. Fidaxomicin also dose-dependently reduced viable spore production by the 9689 and 5325 strains. Reductions in spore formation were also observed in both strains treated with tigecycline and vancomycin. However, all concentrations of metronidazole stimulated a ~2 log increase in spore production by the 5325 isolate., Conclusion: The ability of fidaxomicin to suppress early exotoxin production and endospore formation by historical and epidemic strains of C. difficile may explain its clinical success in treating severe and recurrent cases of CDI disease.

Background: Acute muscle injuries are exceedingly common and non-steroidal anti-inflammatory drugs (NSAIDs) are widely consumed to reduce the associated inflammation, swelling and pain that peak 1-2 days post-injury. While prophylactic use or early administration of NSAIDs has been shown to delay muscle regeneration and contribute to loss of muscle strength after healing, little is known about the effects of delayed NSAID use. Further, NSAID use following non-penetrating injury has been associated with increased risk and severity of infection, including that due to group A streptococcus, though the mechanisms remain to be elucidated. The present study investigated the effects of delayed NSAID administration on muscle repair and sought mechanisms supporting an injury/NSAID/infection axis., Methods: A murine model of eccentric contraction (EC)-induced injury of the tibialis anterior muscle was used to profile the cellular and molecular changes induced by ketorolac tromethamine administered 47 hr post injury., Results: NSAID administration inhibited several important muscle regeneration processes and down-regulated multiple cytoprotective proteins known to inhibit the intrinsic pathway of programmed cell death. These activities were associated with increased caspase activity in injured muscles but were independent of any NSAID effect on macrophage influx or phenotype switching., Conclusions: These findings provide new molecular evidence supporting the notion that NSAIDs have a direct negative influence on muscle repair after acute strain injury in mice and thus add to renewed concern about the safety and benefits of NSAIDS in both children and adults, in those with progressive loss of muscle mass such as the elderly or patients with cancer or AIDS, and those at risk of secondary infection after trauma or surgery.

Clostridium sordellii infections have been reported in women following natural childbirth and spontaneous or medically-induced abortion, injection drug users and patients with trauma. Death is rapid and mortality ranges from 70 to 100%. Clinical features include an extreme leukemoid reaction, the absence of fever, and only minimal pain or erythema at the infected site. In the current study, we developed a murine model of C. sordellii soft tissue infection to elucidate the pathogenic mechanisms. Mice received 0.5, 1.0 or 2.0 × 10(6) CFU C. sordellii (ATCC 9714 type strain) in the right thigh muscle. All doses caused fatal infection characterized by intense swelling of the infected limb but no erythema or visible perfusion deficits. Survival rates and time to death were inoculum dose-dependent. Mice developed a granulocytic leukocytosis with left shift, the onset of which directly correlated with disease severity. Histopathology of infected tissue showed widespread edema, moderate muscle damage and minimal neutrophil infiltration. Circulating levels of granulocyte colony-stimulating factor (G-CSF), soluble tumor necrosis factor receptor I (sTNF-RI) and interlukin-6 (IL-6) were significantly increased in infected animals, while TNF-α, and IL-1β levels were only mildly elevated, suggesting these host factors likely mediate the leukocytosis and innate immune dysfunction characteristic of this infection. Thus, this model mimics many of the salient features of this infection in humans and has allowed us to identify novel targets for intervention., (Published by Elsevier Ltd.)

Purpose of Review: This review summarizes clinical and basic science evidence linking trauma and nonsteroidal anti-inflammatory drug (NSAID) use to initiation and progression of severe group A streptococcal (GAS) soft tissue infection., Recent Findings: New evidence includes recent clinical series and controlled studies that lend support to an NSAID/GAS association, basic science studies that demonstrate unique roles for nonpenetrating injury and NSAID administration in initiation of cryptogenic GAS infection and experimental studies showing that nonselective NSAIDs accelerate disease progression and limit antibiotic efficacy in established GAS soft tissue infections. Potential mechanisms for these processes are discussed., Summary: NSAIDs are important anti-inflammatory and analgesic drugs; however, new experimental data suggest that nonselective NSAIDs do more than simply mask the signs and symptoms of developing GAS infection. A more thorough understanding of the triadic interplay of injury-triggered immune signaling, GAS soft tissue infection and NSAIDs is of significant clinical importance and could shift the current paradigm of pain management to avert the consequences of such devastating infections.

Drylands comprise 40% of Earth's land mass and are critical to food security, carbon sequestration, and threatened and endangered wildlife. Exotic weed invasions, overgrazing, energy extraction, and other factors have degraded many drylands, and this has placed an increased emphasis on dryland restoration. The increased restoration focus has generated a wealth of experience, innovations and empirical data, yet the goal of restoring diverse, native, dryland plant assemblages composed of grasses, forbs, and shrubs has generally proven beyond reach. Of particular concern are shrubs, which often fail to establish or establish at trivially low densities. We used data from two Great Plains, USA coal mines to explore factors regulating shrub establishment. Our predictor data related to weather and restoration (e.g., seed rates, rock cover) variables, and our response data described shrub abundances on fields of the mines. We found that seeded non-shrubs, especially grasses, formed an important competitive barrier to shrub establishment: With every one standard deviation increase in non-shrub seed rate, the probability shrubs were present decreased ~0.1 and shrub cover decreased ~35%. Since new fields were seeded almost every year for > 20 years, the data also provided a unique opportunity to explore effects of stochastic drivers (i.e., precipitation, year effects). With every one standard deviation increase in precipitation the first growing season following seeding, the probability shrubs were present decreased ~0.07 and shrub cover decreased ~47%. High precipitation appeared to harm shrubs by increasing grass growth/competition. Also, weak evidence suggested shrub establishment was better in rockier fields where grass abundance/competition was lower. Multiple lines of evidence suggest reducing grass seed rates below levels typically used in Great Plains restoration would benefit shrubs without substantially impacting grass stand development over the long term. We used Bayesian statistics to estimate effects of seed rates and other restoration predictors probabilistically to allow knowledge of the predictors' effects to be refined through time in an adaptive management framework. We believe this framework could improve restoration planning in a variety of systems where restoration outcomes remain highly uncertain and ongoing restoration efforts are continually providing new data of value for reducing the uncertainty.

Septic cardiomyopathy is a severe complication among some patients who develop group A streptococcal toxic shock syndrome. Despite the importance of cardiac dysfunction in determining prognosis, very little is known about mechanisms that reduce cardiac output in association with streptococcal infection. Here, we investigated the effects of streptococcal extracellular toxins on mechanical contractility of electrically paced primary murine cardiomyocytes. Our data demonstrate that streptolysin O (SLO) is the major streptococcal toxin responsible for cardiomyocyte contractile dysfunction. Streptolysin O dose-dependently affected cardiac myocyte function in discrete stages. Exposure to SLO caused a failure of cardiac cells to respond to electrical pacing, followed by spontaneous dysregulated contractions and augmented strength of contraction. Central to these SLO-mediated effects is a marked influx of calcium into the cytosol through SLO-mediated pores in the cytoplasmic membrane. Such calcium mobilization in response to SLO correlated temporally with hypercontractility and unpaced contractions. During continued exposure to SLO, cardiomyocytes exhibited periods of reversion to normal electrical pacing suggestive of membrane lesion repair and restoration of calcium handling. Together, these observations are consistent with the clinical observation that septic cardiomyopathy is a reversible condition in patients who survive streptococcal toxic shock syndrome. These data provide strong evidence that streptococcal exotoxins, specifically SLO, can directly impact cardiac mechanical function.

Background: Clostridium difficile infection (CDI) is mediated by potent extracellular toxins and is spread largely via bacterial spores. We and others have shown that some antibiotics stimulate C. difficile toxin production in a strain-specific manner; however, the effects of newer anti-C. difficile antibiotics on this process remain to be investigated., Methods: The effects of the protein synthesis inhibitor tigecycline on sporulation and toxin A and toxin B production were compared in historical (strain 9689) and hypervirulent BI/NAP1/027 (strain 5325) isolates of C. difficile in vitro., Results: Tigecycline at 1/4× MIC stimulated an increased and earlier toxin A and/or B gene expression in both the historical and the hypervirulent strains, although a commensurate increase in toxin protein production was observed only in the 9689 strain. In fact, in the hypervirulent 5325 strain, toxin production was dramatically suppressed. By comparison, subinhibitory concentrations of vancomycin and metronidazole also stimulated increased protein toxin production by the historical, but not the hypervirulent, strain. In addition, tigecycline dose-dependently reduced viable spore production by both the 9689 and 5325 strains. Vancomycin treatment also suppressed spore formation in both C. difficile strains; however, metronidazole, while reducing spore formation in the 9689 strain, stimulated a near 2 log increase in spore production by the 5325 isolate., Conclusions: In summary, these findings suggest that the treatment of CDI patients with tigecycline could effectively both control disease progression and limit its spread by disrupting sporulation., (© The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

A large number of individuals are acquitted of criminal charges after being found "not guilty by reason of insanity." Most of these individuals are hospitalized and later seek hospital discharge under a court-ordered provision called conditional release ("CR"). Courts rely on opinions from forensic evaluators to determine acquittees' readiness for CR. However, how evaluators make these decisions are unknown. Eighty-nine CR readiness evaluators from nine states were surveyed to understand which factors evaluators prioritize and to understand evaluators' assessment methodologies and their beliefs about the CR process itself. Little uniformity was found among evaluators on any aspect of the decision-making process. Evaluators utilized a wide variety of methodologies when making their decisions on readiness for CR. Moreover, evaluators' conceptualizations of the CR process itself varied widely. The results highlight the difficulty and confusion evaluators face when conducting CR readiness evaluations, and demonstrate the need for enhanced training, statutory guidance, and standardized evaluation protocols for these evaluations., (Copyright © 2014 John Wiley & Sons, Ltd.)

Background:  Epidemiologic evidence suggests that nonsteroidal anti-inflammatory drugs (NSAIDs) contribute to more severe group A streptococcal (GAS) infections, yet a beneficial role for NSAIDs has been demonstrated in other experimental bacterial infections., Methods:  Nonselective (ketorolac tromethamine, ibuprofen, indomethacin), COX-1-selective (SC-560), or COX-2-selective (SC-236) NSAIDs ± antibiotics (penicillin, clindamycin) were given to mice challenged intramuscularly with M-type 3 GAS and disease course was followed for 14 days. RESULTS. All nonselective NSAIDs significantly accelerated mortality and reduced antibiotic efficacy; COX-selective NSAIDs had no significant effects., Conclusions:  Use of nonselective NSAIDs, either alone or as adjuncts to antibiotic therapy, for GAS soft tissue infection may contribute to worse outcomes.

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) causes severe hemorrhagic necrotizing pneumonia associated with high mortality. Exotoxins have been implicated in the pathogenesis of this infection; however, the cellular mechanisms responsible remain largely undefined. Because platelet-neutrophil aggregates (PNAs) can dysregulate inflammatory responses and contribute to tissue destruction, we investigated whether exotoxins from MRSA could stimulate formation of PNAs in human whole blood. Strong PNA formation was stimulated by toxins from stationary phase but not log phase CA-MRSA, and α-hemolysin was singularly identified as the mediator of this activity. MRSA exotoxins also caused neutrophil (polymorphonuclear leukocyte) activation, as measured by increased CD11b expression, although platelet binding was not driven by this mechanism; rather, α-hemolysin-induced PNA formation was solely platelet P-selectin dependent. These findings suggest a role for S. aureus α-hemolysin-induced PNA formation in alveolar capillary destruction in hemorrhagic/necrotizing pneumonia caused by CA-MRSA and offer novel targets for intervention.

Puerperal sepsis caused by group A Streptococcus (GAS) remains an important cause of maternal and infant mortality worldwide, including countries with modern antibiotic regimens, intensive care measures and infection control practices. To provide insights into the genesis of modern GAS puerperal sepsis, we reviewed the published cases and case series from 1974 to 2009, specifically seeking relationships between the likely source of pathogen acquisition, clinical signs, and symptoms at infection onset and patient outcomes that could provide clues for early diagnosis. Results suggest that the pathogenesis of pregnancy-related GAS infections in modern times is complex and not simply the result of exposure to GAS in the hospital setting. Additional research is needed to further explore the source of GAS, the specific M types involved, and the pathogenesis of these pregnancy-related infections to generate novel preventative and therapeutic strategies.

Hypervirulent BI/NAP1/027 strains of Clostridium difficile have been associated with increased mortality of C. difficile infection (CDI). The emergence of highly fluoroquinolone (FLQ)-resistant BI/NAP1/027 strains suggests that FLQ exposure may be a risk factor for CDI development. However, the mechanism for this is not clear. We compared the effects of subinhibitory concentrations of ciprofloxacin on Toxin A and B gene expression and protein production in recent (strain 039) and historical (strain 5325) BI/NAP1/027 clinical isolates with high- and low-level ciprofloxacin resistance, respectively. In the highly ciprofloxacin-resistant isolate (strain 039), ciprofloxacin significantly and dose-dependently increased Toxin A gene expression and shifted its expression to earlier in its growth cycle; TcdB gene expression also increased but was less sensitive to low-dose ciprofloxacin. Maximal Toxin A/B production (4 ng ml(-1)) was increased twofold and occurred significantly earlier than in the untreated control. In strain 5325, ciprofloxacin at 0.25×MIC markedly increased both tcdA and tcdB expression but their temporal dynamics were unchanged. Maximal toxin production (250 ng ml(-1)) was reduced approximately threefold compared with that of the untreated control. These results demonstrate significant differences in ciprofloxacin-induced toxin gene expression and protein production among BI/NAP1/027 isolates, and offer a new paradigm for FLQ-associated CDI caused by recent, highly antibiotic-resistant strains.

Myocardial dysfunction in group A streptococcal (GAS) toxic shock syndrome (StrepTSS) is characterized by severe biventricular dilatation and a striking reduction in ventricular performance; however, the mechanisms have not been fully elucidated. We have previously shown that pro-inflammatory cytokines are upregulated in the hearts of experimental animals with GAS bacteremia and that cardiomyocytes themselves as well as macrophages are the principal cytokine sources. Although macrophage-derived cytokines can clearly affect cardiac contractility, we questioned whether soluble cardiomyocyte-derived mediators might in turn affect macrophage function. Thus, we sought evidence of cardiomyocyte-to-macrophage directional cross-talk under resting versus GAS-stimulated conditions, using production of matrix metalloproteinase-9 (MMP-9) as an indicator of such signaling. Our results demonstrate that unstimulated cardiomyocytes produce a soluble inhibitor/s that maintains macrophage functional quiescence. Further, viable GAS induced production of cardiomyocyte-derived stimulator/s that overcomes quiescence and boosts macrophages production of MMP-9 and expression of pro-inflammatory cytokines (IL-1β, IL-6) and cardiodepressant factors (iNOS). Understanding the role of these cardiomyocyte-derived effectors of macrophage function (herein termed "cardiokines") in sepsis-associated cardiomyopathy may suggest new targets for therapeutic intervention., (Published by Elsevier Ltd.)

Life-threatening soft tissue infections caused by Clostridium species have been described in the medical literature for hundreds of years largely because of their fulminant nature, distinctive clinical presentations and complex management issues. The Clostridium species perfringens, septicum and histolyticum are the principal causes of trauma-associated gas gangrene and their incidence increases dramatically in times of war, hurricanes, earthquakes and other mass casualty conditions. Recently, there has also been an increased incidence of spontaneous gas gangrene caused by Clostridium septicum in association with gastrointestinal abnormalities and neutropenia. Similarly, over the last 15 years there has been increased recognition of a toxic shock-like syndrome associated with Clostridium sordellii in individuals skin-popping black tar heroin, in women undergoing childbirth or other gynecologic procedures including medically-induced abortion. Like their cousins Clostridium tetanus and Clostridium botulinum, the pathogenesis of these clostridial infections is largely the consequence of potent exotoxin production. Strategies to inhibit toxin production, neutralize circulating toxins and prevent their interaction with cells of the innate immune response are sorely needed. Recent studies have elucidated novel targets that may hold promise for newer therapeutic modalities., (Published by Elsevier Ltd.)

Some patients with Group A Streptococcal toxic shock syndrome (StrepTSS) develop a unique form of cardiomyopathy characterized by global hypokinesia and reduced cardiac index. Here we investigated the immune responses of cardiomyocytes to Group A Streptococcus both in vivo and in vitro. Our data demonstrate that cardiomyocyte-derived cytokines are produced following both direct GAS stimulation and after exposure to GAS-activated inflammatory cells. These locally produced, cardiomyocyte-derived cytokines may mediate cardiac contractile dysfunction observed in patients with StrepTSS-associated cardiomyopathy and may hold the key to our ability to attenuate this severe complication., (Published by Elsevier Ltd.)

In 2008, an unusual strain of methicillin-sensitive Staphylococcus aureus (MSSA68111), producing both Panton-Valentine leukocidin (PVL) and toxic shock syndrome toxin-1 (TSST-1), was isolated from a fatal case of necrotizing pneumonia. Because PVL/TSST-1 co-production in S. aureus is rare, we characterized the molecular organization of these toxin genes in strain 68111. MSSA68111 carries the PVL genes within a novel temperate prophage we call ФPVLv68111 that is most similar, though not identical, to phage ФPVL--a phage type that is relatively rare worldwide. The TSST-1 gene (tst) in MSSA68111 is carried on a unique staphylococcal pathogenicity island (SaPI) we call SaPI68111. Features of SaPI68111 suggest it likely arose through multiple major recombination events with other known SaPIs. Both ФPVLv68111 and SaPI68111 are fully mobilizable and therefore transmissible to other strains. Taken together, these findings suggest that hypervirulent S. aureus have the potential to emerge worldwide.

Clostridial myonecrosis remains an important cause of human morbidity and mortality worldwide. Although traumatic gas gangrene can be readily diagnosed from clinical findings and widely available technologies, spontaneous gas gangrene is more insidious, and gynecologic infections due to Clostridium sordellii progress so rapidly that death often precedes diagnosis. In each case, extensive tissue destruction and the subsequent systemic manifestations are mediated directly and indirectly by potent bacterial exotoxins. The management triumvirate of timely diagnosis, thorough surgical removal of necrotic tissue, and treatment with antibiotics that inhibit toxin synthesis remains the gold standard of care. Yet, despite these measures, mortality remains 30% to 100% and survivors often must cope with life-altering amputations. Recent insights regarding the genetic regulation of toxin production, the molecular mechanisms of toxin-induced host cell dysfunction, and the roles of newly described toxins in pathogenesis suggest that novel prevention, diagnostic, and treatment modalities may be on the horizon for these devastating infections.

Clostridium sordellii, an anaerobic pathogen, has recently been associated with rapidly fatal infections following medically induced abortions and injecting drug use. Patients with C. sordellii infection display few signs of inflammation such as fever, or redness and pain at the site of infection. We hypothesized that this could be due to reduced recognition of the organism by Toll-like receptors (TLRs) of the innate immune system. An ELAM-NF-kappaB luciferase reporter system in TLR-transfected HEK cells was used to measure TLR-dependent recognition of washed, heat-killed C. sordellii and other pathogenic clostridial species. Results demonstrated that all clostridia were well recognized by TLR2 alone and that responses were greatest when TLR2 was co-expressed with TLR6. Further, isolated human monocytes produced the pro-inflammatory cytokine TNFalpha and the immunoregulator IL-10 in response to C. sordellii. In addition, C. sordellii-stimulated monocytes produced 30% less TNFalpha following treatment with an anti-TLR2 blocking antibody. These data demonstrate that innate immune recognition of, and response to, cell-associated components of C. sordellii and other clostridial pathogens are mediated by TLR2 in combination with TLR6. We conclude that the characteristic absence of inflammatory signs and symptoms in C. sordellii infection is not related to inadequate immune detection of the organism, but rather is attributable to a species-specific immune system dysfunction that remains to be elucidated., (Published by Elsevier Ltd.)

The role of Panton-Valentine leukocidin (PVL) in Staphylococcus aureus infections is controversial. We used a mouse model of skin infection to compare the virulence of methicillin-resistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) strains with different levels of PVL production. Differences in PVL production were not associated with mutations in the genes lukS-PV and lukF-PV. However, MSSA and MRSA strains that produced high levels of PVL caused larger skin abscesses, higher bacterial burdens, and more tissue inflammation than did low-PVL-producing strains. Together, these data suggest that (1) the effect of PVL on the pathogenesis of staphylococcal infection may depend on the level of toxin produced and (2) many strains of MSSA that cause soft-tissue infections produce higher levels of PVL than do MRSA strains.

A gene unique to Streptococcus pyogenes, called vfr, that negatively regulates speB, an important extracellular proteinase, has been identified. Disruption of vfr markedly increased SpeB production in a clinical strain of S. pyogenes and relieved its growth phase dependency. These findings may provide important insights into the pathogenesis of invasive S. pyogenes infections.

Background: Myonecrosis due to group A streptococci (GAS) often develops at sites of nonpenetrating muscle injury, and nonsteroidal anti-inflammatory drugs (NSAIDs) may increase the severity of these cryptic infections. We have previously shown that GAS bind to vimentin on injured skeletal muscles in vitro. The present study investigated whether vimentin up-regulation in injured muscles in vivo is associated with homing of circulating GAS to the injured site and whether NSAIDs facilitate this process., Methods: M type 3 GAS were delivered intravenously 48 h after eccentric contraction (EC)-induced injury of murine hind-limb muscles. Vimentin gene expression and homing of GAS were followed by real-time reverse-transcriptase polymerase chain reaction and quantitative bacteriology of muscle homogenates, respectively. In separate experiments, ketorolac tromethamine (Toradol) was given 1 h before GAS infusion., Results: Vimentin was up-regulated approximately 8-fold 48 h after EC. Significantly more GAS were found in moderately injured muscles than in noninjured controls. NSAIDs greatly augmented the number of GAS in injured muscles., Conclusions: Vimentin may tether circulating GAS to injured muscle, and NSAIDs enhance this process. Strategies targeting the vimentin-GAS interaction may prevent or attenuate GAS myonecrosis. Use of NSAIDs should increase suspicion of cryptic GAS infection in patients with increasing pain at sites of nonpenetrating muscle injury.

Clostridium perfringens-induced gas gangrene is mediated by potent extracellular toxins, especially alpha toxin (a phospholipase C [PLC]) and theta toxin (perfringolysin O [PFO], a thiol-activated cytolysin); and antibiotic-induced suppression of toxin synthesis is an important clinical goal. The production of PLC and PFO by a gatifloxacin-induced, fluoroquinolone-resistant mutant strain of C. perfringens, strain 10G, carrying a stable mutation in DNA gyrase was compared with that of the wild-type (WT) parent strain. Zymography (with sheep red blood cell and egg yolk overlays) and time course analysis [with hydrolysis of egg yolk lecithin and O-(4 nitrophenyl-phosphoryl)choline] demonstrated that strain 10G produced more PLC and PFO than the WT strain. Increased toxin production in strain 10G was not related either to differences in growth characteristics between the wild-type and the mutant strain or to nonsynonymous polymorphisms in PLC, PFO, or their known regulatory proteins. Increased PLC and PFO production by strain 10G was associated with increased cytotoxic activity for HT-29 human adenocarcinoma cells and with increased platelet-neutrophil aggregate formation. Four other gatifloxacin-induced gyrase mutants did not show increased toxin production, suggesting that gatifloxacin resistance was not always associated with increased toxin production in all strains of C. perfringens. This is the first report of increased toxin production in a fluoroquinolone-resistant strain of C. perfringens.

Background: Community-acquired methicillin-resistant Staphylococcus aureus strains have recently been associated with severe necrotizing infections. Greater than 75% of these strains carry the genes for Panton-Valentine leukocidin (PVL), suggesting that this toxin may mediate these severe infections. However, to date, studies have not provided evidence of toxin production., Methods: Twenty-nine community-acquired methicillin-resistant Staphylococcus aureus and 2 community-acquired methicillin-susceptible S. aureus strains were collected from patients with infections of varying severity. Strains were analyzed for the presence of lukF-PV and SCCmecA type. PVL production in lukF-PV gene-positive strains was measured by ELISA, and the amount produced was analyzed relative to severity of infection., Results: Only 2 of the 31 strains tested, 1 methicillin-resistant Staphylococcus aureus abscess isolate and 1 nasal carriage methicillin-susceptible S. aureus isolate, were lukF-PV negative. All methicillin-resistant Staphylococcus aureus strains were SCCmec type IV. PVL was produced by all strains harboring lukF-PV, although a marked strain-to-strain variation was observed. Twenty-six (90%) of 29 strains produced 50-350 ng/mL of PVL; the remaining strains produced PVL in excess of 500 ng/mL. The quantity of PVL produced in vitro did not correlate with severity of infection., Conclusions: Although PVL likely plays an important role in the pathogenesis of these infections, its mere presence is not solely responsible for the increased severity. Factors that up-regulate toxin synthesis in vivo could contribute to more-severe disease and worse outcomes in patients with community-acquired methicillin-resistant Staphylococcus aureus infection.

Objectives: To prevent perinatal HIV transmission, providers must identify HIV status for all women in labor and newborns, and provide timely antiretroviral therapy if necessary. The objective of this study is to evaluate the availability and accessibility of zidovudine (AZT) in Illinois birthing hospitals., Methods: We surveyed all Illinois birthing hospital pharmacies by telephone in February 2005 regarding availability, accessibility, and protocols surrounding AZT use in the perinatal period., Results: All 137 pharmacies participated. Only 43.1% reported having syrup and IV AZT available and only 37.2% indicated the ability to have AZT available on labor and delivery within 30 min during off hours. Protocols for treating HIV positive women in labor and exposed newborns were available in only 37.2% of pharmacies while 72.4% had protocols for antiretroviral therapy for occupational post-exposure prophylaxis. Variables associated with pharmacies having AZT available included being a major academic hospital and serving a predominately (greater than 96%) white patient population. Timely provision of AZT was more likely to occur in urban, major academic hospitals serving a predominately white population., Conclusions: In order to further reduce perinatal HIV transmission, availability and timely access to both syrup and IV AZT must be improved in Illinois birthing hospitals.

Life-threatening Clostridium sordellii infections have recently been reported in women undergoing therapeutic abortion, during natural childbirth, and in injection drug users. Shock, diffuse capillary leak, and a leukemoid reaction (LR) are cardinal features of these infections. The magnitude of the LR is highly correlated with mortality. We have isolated a 42-kDa extractable protein from C. sordellii culture supernatant that stimulates proliferation of promyelocytic HL-60 cells in vitro. Using mass spectrometry, we have identified this protein as the C. sordellii neuraminidase, NanS. Recombinant NanS (rNanS) dose dependently stimulated HL-60 cell proliferation. Increased proliferation was observed when HL-60 cells were cocultured with both rNanS and granulocyte-macrophage colony stimulating factor. In addition, NanS also modified vascular cell adhesion molecule 1, which orchestrates the release of mature and immature granulocytes from bone marrow stromal cells. Thus, neuraminidase likely plays an important role in the characteristic LR in C. sordellii infection.

Objective: To estimate whether there is an association between chronic psychosocial stress and low birth weight neonates in low-income women., Methods: Between 1999 and 2004, a random sample of women receiving welfare in nine Illinois counties was selected. The women were then interviewed annually. Women who delivered during this period were identified. Self-reported stress that occurred in temporal proximity to the delivery was assessed by 1) external stressors, 2) enhancers of stress, 3) buffers against stress, and 4) perceived stress and was compared between women who delivered low birth weight neonates and women who delivered normal birth weight neonates., Results: Of the 1,363 women in the study, 294 women (21.6%) became pregnant and delivered during the study period. Of the 294 deliveries, 39 (13.3%) were low birth weight. The only demographic factor associated with a low birth weight delivery was increasing maternal age. However, multiple psychosocial factors, including food insecurity (odds ratio [OR] 3.2, 95% confidence interval [CI] 1.4-7.2), a child with chronic illness in the home (OR 3.4, 95% CI 1.5-7.9), increased crowding in the home (OR 2.7, 95% CI 1.3-5.6), unemployment (OR 3.1, 95% CI 1.2-7.9), and poor coping skills (OR 3.8, 95% CI 1.7-8.7), were significantly associated with low birth weight delivery (P < .01 for all comparisons). These significant associations persisted after adjusting for maternal age in multivariable analysis., Conclusion: This study provides evidence that chronic psychosocial stress may be associated with low birth weight neonates in a low-income population of women., Level of Evidence: II.

Extracellular protein toxins contribute to the pathogenesis of a wide variety of Staphylococcus aureus infections. The present study investigated the effects that cell-wall active antibiotics and protein-synthesis inhibitors have on transcription and translation of genes for Panton-Valentine leukocidin, alpha-hemolysin, and toxic-shock syndrome toxin 1, in both methicillin-sensitive and methicillin-resistant S. aureus. Subinhibitory concentrations of nafcillin induced and prolonged mRNA for Panton-Valentine leukocidin, alpha-toxin, and toxic-shock syndrome toxin 1 and increased toxin production. In contrast, clindamycin and linezolid markedly suppressed translation, but not transcription, of toxin genes. These results suggest (1) that protein-synthesis inhibition is an important consideration in the selection of antimicrobial agents to treat serious infections caused by toxin-producing gram-positive pathogens and (2) that, by inducing and enhancing toxin production, inadvertent use of beta-lactam antibiotics to treat methicillin-resistant S. aureus infections may contribute to worse outcomes.

Clostridium sordellii infections pose difficult clinical challenges and are usually fatal. Most commonly, these infections occur after trauma, childbirth, and routine gynecological procedures, but they have recently been associated with medically induced abortions and injection drug use. We report 2 fatal cases, one of which was associated with minor trauma, and the other of which was associated with normal childbirth, and we summarize the clinical features of 43 additional cases of reported C. sordellii infection. Of these 45 cases, 8 (18%) were associated with normal childbirth, 5 (11%) were associated with medically induced abortion, and 2 (0.4%) were associated with spontaneous abortion. The case-fatality rate was 100% in these groups. Ten (22%) of the C. sordellii infections occurred in injection drug users, and 50% of these patients died. Other cases of C. sordellii infection (in 19 patients [43%]) occurred after trauma or surgery, mostly in healthy persons, and 53% these patients died. Overall, the mortality rate was 69% (31 of 45 patients). Eighty-five percent of all patients with fatal cases died within 2-6 days of initial infection, and nearly 80% of fatal cases developed leukemoid reactions. Rapid diagnostic tests and improved treatments are needed to reduced the morbidity and mortality associated with this devastating infection.

Severe, invasive group A streptococcal infections have reemerged worldwide, and extracellular toxins, including streptococcal pyrogenic exotoxin B (SpeB), have been implicated in pathogenesis. The genetic regulation of SpeB is not fully understood, and the mechanisms involved in the processing of the protoxin to its enzymatically active form have not been definitively established. The present work demonstrated that the genes encoding SpeB (speB) and a peptidyl-prolyl isomerase (prsA) constitute an operon with transcription initiated from two promoters upstream of speB. Further, the speB-prsA operon was transcribed as a bicistronic mRNA. This finding is in contrast to the generally accepted notion that speB is transcribed only as a monocistronic gene. In addition, prsA has its own promoter, and transcription from this promoter starts in early log phase, prior to the transcription of speB. Genomic disruption of prsA decreased the production of enzymatically active SpeB but not the level of the pro-SpeB zymogen. Taken together, these results demonstrate that prsA is required for production of fully mature, enzymatically active SpeB.

Objective: Women who have undergone type III female genital cutting may suffer long-term complications. Defibulation (reconstructive surgery of the infibulated scar) can alleviate some of these complications. We studied the physical and sexual outcomes after defibulation and evaluated both patient and husband satisfaction., Methods: The medical records of 40 consecutive women with a history of type III female genital cutting who underwent defibulation between 1995 and 2003 were reviewed. Data collected included demographics, indications for the procedure, closure type, intraoperative and postoperative complications. Telephone surveys were conducted between 6 months and 2 years postprocedure to evaluate the long-term health and sexual satisfaction outcomes., Results: Of 40 women identified as having undergone defibulation, 95% were Somali, 65% were married, and 73% were between the ages of 19 and 30. Primary indications for defibulation were being pregnant (30%), dysmenorrhea (30%), apareunia (20%), and dyspareunia (15%). Secondary indications were apareunia (20%), difficulty urinating (12.5%), and dyspareunia (10%). Sixty-five percent had a subcuticular repair. Forty-eight percent had an intact clitoris buried beneath the scar. None had intraoperative or postoperative complications. Of the 32 patients reached by telephone, 94% stated they would highly recommend it to others. One hundred percent of patients and their husbands were satisfied with the results, felt their appearance had improved, and were sexually satisfied., Conclusion: Defibulation is recommended for all infibulated women who suffer long-term complications. The complication rates are minimal, with high satisfaction rates among patients and their husbands., Level of Evidence: II-3.

Necrotizing fasciitis and myonecrosis caused by invasive infection with group A streptococci (GAS) are life-threatening conditions that have reemerged worldwide. Half of all GAS myonecrosis cases have no known portal of entry; yet, for unknown reasons, infection becomes established precisely at the site of a prior, nonpenetrating minor injury, such as a muscle strain. We hypothesized that GAS establishes infection by binding to surface molecules that are up-regulated on injured skeletal-muscle cells. Here, we isolated and identified vimentin as the major skeletal-muscle GAS-binding protein. Furthermore, we found that vimentin expression was up-regulated on injured skeletal-muscle cells in vitro and was expressed in muscle tissues from a patient with GAS myonecrosis who died of streptococcal toxic shock syndrome. These findings provide a molecular mechanism to explain the development of severe GAS soft-tissue infections at the sites of prior minor muscle trauma. This understanding may provide a basis for novel preventive strategies or therapies for patients with this devastating infection.

Clostridium perfringens gas gangrene is a fulminant necrotizing infection in which inflammatory cells are notably absent from infected tissues but are often massed within adjacent vessels. It has been shown that C. perfringens phospholipase C (PLC) stimulates formation of large intravascular platelet/leukocyte complexes and that PLC-induced activation of platelet gpIIbIIIa plays a major role. In vivo, such aggregates contribute to microvascular thrombosis and ischaemic necrosis of tissue. However, the effects of adherent platelets on neutrophil diapedesis have not been established. The present work investigated (1) the contribution of platelet P-selectin (CD62P) to PLC-induced cellular complex formation and (2) the effects of platelet adhesion on neutrophil diapedesis. The effects of anti-gpIIbIIIa and anti-CD62P strategies on PLC-induced complex formation were measured by flow cytometry and followed by light microscopy. Both platelet gpIIbIIIa and CD62P contributed to the formation of platelet/leukocyte complexes. Specifically, gpIIbIIIa mediated the formation of large platelet/platelet aggregates that were tethered to the leukocyte principally via CD62P. Neutrophil diapedesis, quantified by a transendothelial cell migration assay and visualized by electron microscopy, was significantly reduced (>60%) by the adherence of large platelet aggregates. It was concluded that the absence of a tissue inflammatory response in C. perfringens gas gangrene is due, in part, to impaired neutrophil mobility caused by large aggregates of adherent platelets induced by PLC. Further, an adjunctive immunotherapeutic strategy targeting both gpIIbIIIa and CD62P may improve the tissue inflammatory response, prevent vascular occlusion, maintain tissue viability, and reduce the need for radical amputation in patients with clostridial gas gangrene.

Rapid tissue destruction in group A streptococcal (GAS) necrotizing fasciitis/myonecrosis often necessitates extensive debridement to ensure survival. The mechanisms responsible for this fulminant process remain unknown; we hypothesized that toxin-induced ischemia contributes to necrosis. In a rat model, Doppler flowmetry was used to measure local blood flow at the site of the intramuscular injection of exotoxins from an invasive M-type 1 GAS, which caused a rapid, dose-dependent decrease in perfusion that was irreversible at the highest toxin concentration tested. Videomicroscopic results revealed that blood flow was impeded by occlusive intravascular cellular aggregates. Flow-cytometric results confirmed that GAS toxins induced the coaggregation of platelets and neutrophils, that this activity was attributable to streptolysin O, and that platelet/neutrophil complex formation was largely mediated by platelet P-selectin (CD62P). Strategies that target platelet adherence molecules may prevent vascular occlusion, maintain tissue viability, and reduce the need for amputation in necrotizing GAS infections.