Bacteroides fragilis toxin expression enables lamina propria niche acquisition in the developing mouse gut.

Bacterial toxins are well-studied virulence factors; however, recent studies have revealed their importance in bacterial niche adaptation. Enterotoxigenic Bacteroides fragilis (ETBF) expresses B. fragilis toxin (BFT) that we hypothesized may contribute to both colonic epithelial injury and niche acquisition. We developed a vertical transmission model for ETBF in mice that showed that BFT enabled ETBF to access a lamina propria (LP) niche during colonic microbiome development that was inaccessible to non-toxigenic B. fragilis. LP entry by ETBF required BFT metalloprotease activity, and showed temporal restriction to the pre-weaning period, dependent on goblet-cell-associated passages. In situ single-cell analysis showed bft expression at the apical epithelial surface and within the LP. BFT expression increased goblet cell number and goblet-cell-associated passage formation. These findings define a paradigm by which bacterial toxin expression specifies developmental niche acquisition, suggesting that a selective advantage conferred by a toxin may impact long-term host health.

Endothelial ADAM10 utilization defines a molecular pathway of vascular injury in mice with bacterial sepsis.

The endothelium plays a critical role in the host response to infection and has been a focus of investigation in sepsis. While it is appreciated that intravascular thrombus formation, severe inflammation, and loss of endothelial integrity impair tissue oxygenation during sepsis, the precise molecular mechanisms that lead to endothelial injury remain poorly understood. We demonstrate here that endothelial ADAM10 was essential for the pathogenesis of Staphylococcus aureus sepsis, contributing to α-toxin-mediated (Hla-mediated) microvascular thrombus formation and lethality. As ADAM10 is essential for endothelial development and homeostasis, we examined whether other major human sepsis pathogens also rely on ADAM10-dependent pathways in pathogenesis. Mice harboring an endothelium-specific knockout of ADAM10 were protected against lethal Pseudomonas aeruginosa and Streptococcus pneumoniae sepsis, yet remained fully susceptible to group B streptococci and Candida albicans sepsis. These studies illustrate a previously unknown role for ADAM10 in sepsis-associated endothelial injury and suggest that understanding pathogen-specific divergent host pathways in sepsis may enable more precise targeting of disease.

Derivation, Validation, and Clinical Relevance of a Pediatric Sepsis Phenotype With Persistent Hypoxemia, Encephalopathy, and Shock.

OBJECTIVES: Untangling the heterogeneity of sepsis in children and identifying clinically relevant phenotypes could lead to the development of targeted therapies. Our aim was to analyze the organ dysfunction trajectories of children with sepsis-associated multiple organ dysfunction syndrome (MODS) to identify reproducible and clinically relevant sepsis phenotypes and determine if they are associated with heterogeneity of treatment effect (HTE) to common therapies. DESIGN: Multicenter observational cohort study. SETTING: Thirteen PICUs in the United States. PATIENTS: Patients admitted with suspected infections to the PICU between 2012 and 2018. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: We used subgraph-augmented nonnegative matrix factorization to identify candidate trajectory-based phenotypes based on the type, severity, and progression of organ dysfunction in the first 72 hours. We analyzed the candidate phenotypes to determine reproducibility as well as prognostic, therapeutic, and biological relevance. Overall, 38,732 children had suspected infection, of which 15,246 (39.4%) had sepsis-associated MODS with an in-hospital mortality of 10.1%. We identified an organ dysfunction trajectory-based phenotype (which we termed persistent hypoxemia, encephalopathy, and shock) that was highly reproducible, had features of systemic inflammation and coagulopathy, and was independently associated with higher mortality. In a propensity score-matched analysis, patients with persistent hypoxemia, encephalopathy, and shock phenotype appeared to have HTE and benefit from adjuvant therapy with hydrocortisone and albumin. When compared with other high-risk clinical syndromes, the persistent hypoxemia, encephalopathy, and shock phenotype only overlapped with 50%-60% of patients with septic shock, moderate-to-severe pediatric acute respiratory distress syndrome, or those in the top tier of organ dysfunction burden, suggesting that it represents a nonsynonymous clinical phenotype of sepsis-associated MODS. CONCLUSIONS: We derived and validated the persistent hypoxemia, encephalopathy, and shock phenotype, which is highly reproducible, clinically relevant, and associated with HTE to common adjuvant therapies in children with sepsis.

Prognostic significance of early platelet dynamics in Staphylococcus aureus bacteremia.

BACKGROUND: Platelets are recognized as key immune effectors, but they are targets of bacterial virulence factors. In the present study, we aimed to examine the relationship between early platelet dynamics and the outcome of Staphylococcus aureus bacteremia (SAB). METHOD: Electronic medical records of adult patients hospitalized for SAB between July 2012 and November 2020 were retrospectively reviewed for relevant demographic, laboratory, and clinical data. The outcome endpoints were mortality and microbial persistence. RESULTS: Among the 811 patients evaluated, 29% experienced thrombocytopenia on Day 1. Platelet count nadir occurred on Days 2-3 following SAB onset, and Day 4 was a determining point of platelet count trajectory and mortality. Mortality risk was 6% or less for those with normal platelet count by Day 4 regardless of whether they experienced thrombocytopenia on Day 1, but the risk increased to 16-21% for those who experienced thrombocytopenia on Day 4 regardless of whether they had normal platelet count on Day 1 or sustained thrombocytopenia. The duration of bacteremia was prolonged by one day (median 3 d vs. 2 d) for those with sustained thrombocytopenia compared to those without. CONCLUSION: Early platelet dynamics during SAB have prognostic significance and represent an early window for potential platelet-directed therapeutic interventions to improve outcome.

A colon cancer "prequel".

The development of colorectal cancer has been predicted to inolve the enteric microbiome. In this issue of Cell Host & Microbe, Kordahi et al. (2021) address this predisposition by identifying not just pathobiont micro-organisms but also distinct microbial signatures within those bacteria that predict the presence of pre-cancerous polyps.

HOME2 Study: Household Versus Personalized Decolonization in Households of Children With Methicillin-Resistant Staphylococcus aureus Skin and Soft Tissue Infection-A Randomized Clinical Trial.

BACKGROUND: A household approach to decolonization decreases skin and soft tissue infection (SSTI) incidence, though this is burdensome and costly. As prior SSTI increases risk for SSTI, we hypothesized that the effectiveness of decolonization measures to prevent SSTI when targeted to household members with prior year SSTI would be noninferior to decolonizing all household members. METHODS: Upon completion of our 12-month observational Household Observation of Methicillin-resistant Staphylococcus aureus in the Environment (HOME) study, 102 households were enrolled in HOME2, a 12-month, randomized noninferiority trial. Pediatric index patients with community-associated methicillin-resistant Staphylococcus aureus (MRSA) SSTI, their household contacts, and pets were enrolled. Households were randomized 1:1 to the personalized (decolonization performed only by household members who experienced SSTI during the HOME study) or household (decolonization performed by all household members) approaches. The 5-day regimen included hygiene education, twice-daily intranasal mupirocin, and daily bleach-water baths. At 5 follow-up visits in participants' homes, swabs to detect S. aureus were collected from participants, environmental surfaces, and pets; incident SSTIs were ascertained. RESULTS: Noninferiority of the personalized approach was established for the primary outcome 3-month cumulative SSTI: 23 of 212 (10.8%) participants reported SSTI in household approach households, while 23 of 236 (9.7%) participants reported SSTI in personalized approach households (difference in proportions, -1.1% [95% confidence interval, -6.7% to 4.5%]). In multivariable analyses, prior year SSTI and baseline MRSA colonization were associated with cumulative SSTI. CONCLUSIONS: The personalized approach was noninferior to the household approach in preventing SSTI. Future studies should interrogate longer durations of decolonization and/or decontamination of the household environment to reduce household MRSA burden. CLINICAL TRIALS REGISTRATION: NCT01814371.

Longitudinal, strain-specific Staphylococcus aureus introduction and transmission events in households of children with community-associated meticillin-resistant S aureus skin and soft tissue infection: a prospective cohort study.

BACKGROUND: Devising effective, targeted approaches to prevent recurrent meticillin-resistant Staphylococcus aureus (MRSA) skin and soft tissue infection requires an understanding of factors driving MRSA acquisition. We comprehensively defined household longitudinal, strain-level S aureus transmission dynamics in households of children with community-associated MRSA skin and soft tissue infection. METHODS: From 2012-15, otherwise healthy paediatric patients with culture-confirmed, community-onset MRSA infections were recruited for the Household Observation of MRSA in the Environment (HOME) prospective cohort study from hospitals and community practices in metropolitan St Louis (MO, USA). Children with health-care-related risk factors were excluded, as determined by evidence of recent hospital admission, an invasive medical device, or residence in a long-term care facility. Household contacts (individuals sleeping in the home ≥four nights per week) and indoor dogs and cats were also enrolled. A baseline visit took place at the index patient's primary home, followed by four quarterly visits over 12 months. At each visit, interviews were done and serial cultures were collected, to detect S aureus from three anatomic sites of household members, two anatomic sites on dogs and cats, and 21 environmental surfaces. Molecular typing was done by repetitive-sequence PCR to define distinct S aureus strains within each household. Longitudinal, multivariable generalised mixed-effects logistic regression models identified factors associated with S aureus acquisition. FINDINGS: Across household members, pets, and environmental surfaces, 1267 strain acquisition events were observed. Acquisitions were driven equally by 510 introductions of novel strains into households and 602 transmissions within households, each associated with distinct factors. Frequent handwashing decreased the likelihood of novel strain introduction into the household (odds ratio [OR] 0·86, credible interval [CrI] 0·74-1·01). Transmission recipients were less likely to own their homes (OR 0·77, CrI 0·63-0·94) and were more likely to share bedrooms with strain-colonised individuals (OR 1·33, CrI 1·12-1·58), live in homes with higher environmental S aureus contamination burden (OR 3·97, CrI 1·96-8·20), and report interval skin and soft tissue infection (OR 1·32, CrI 1·07-1·64). Transmission sources were more likely to share bath towels (OR 1·25, CrI 1·01-1·57). Pets were often transmission recipients, but rarely the sole transmission source. INTERPRETATION: The household environment plays a key role in transmission, a factor associated with skin and soft tissue infection. Future interventions should inclusively target household members and the environment, focusing on straightforward changes in hand hygiene and household sharing behaviours. FUNDING: National Institutes of Health, Agency for Healthcare Research and Quality, Children's Discovery Institute, Burroughs Wellcome Foundation, Defense Advanced Research Projects Agency.

Another Score for the Pore: S. aureus Leukocidins Take a Shot on the Endothelium.

Sepsis is a complex disease characterized by severe endothelial injury. In this issue of Cell Host & Microbe, Lubkin et al. (2019) reveal that an interaction between Staphylococcus aureus leukocidins and their cellular receptor DARC on endothelial cells leads to vascular injury, shedding light on pathogen-driven contributions to sepsis.

Vancomycin Monotherapy May Be Insufficient to Treat Methicillin-resistant Staphylococcus aureus Coinfection in Children With Influenza-related Critical Illness.

Background: Coinfection with influenza virus and methicillin-resistant Staphylococcus aureus (MRSA) causes life-threatening necrotizing pneumonia in children. Sporadic incidence precludes evaluation of antimicrobial efficacy. We assessed the clinical characteristics and outcomes of critically ill children with influenza-MRSA pneumonia and evaluated antibiotic use. Methods: We enrolled children (<18 years) with influenza infection and respiratory failure across 34 pediatric intensive care units 11/2008-5/2016. We compared baseline characteristics, clinical courses, and therapies in children with MRSA coinfection, non-MRSA bacterial coinfection, and no bacterial coinfection. Results: We enrolled 170 children (127 influenza A, 43 influenza B). Children with influenza-MRSA pneumonia (N = 30, 87% previously healthy) were older than those with non-MRSA (N = 61) or no (N = 79) bacterial coinfections. Influenza-MRSA was associated with increased leukopenia, acute lung injury, vasopressor use, extracorporeal life support, and mortality than either group (P ≤ .0001). Influenza-related mortality was 40% with MRSA compared to 4.3% without (relative risk [RR], 9.3; 95% confidence interval [CI], 3.8-22.9). Of 29/30 children with MRSA who received vancomycin within the first 24 hours of hospitalization, mortality was 12.5% (N = 2/16) if treatment also included a second anti-MRSA antibiotic compared to 69.2% (N = 9/13) with vancomycin monotherapy (RR, 5.5; 95% CI, 1.4, 21.3; P = .003). Vancomycin dosing did not influence initial trough levels; 78% were <10 µg/mL. Conclusions: Influenza-MRSA coinfection is associated with high fatality in critically ill children. These data support early addition of a second anti-MRSA antibiotic to vancomycin in suspected severe cases.

Staphylococcus aureus alpha toxin activates Notch in vascular cells.

Staphylococcus aureus infection is one of the leading causes of morbidity in hospitalized patients in the United States, an effect compounded by increasing antibiotic resistance. The secreted agent hemolysin alpha toxin (Hla) requires the receptor A Disintegrin And Metalloproteinase domain-containing protein 10 (ADAM10) to mediate its toxic effects. We hypothesized that these effects are in part regulated by Notch signaling, for which ADAM10 activation is essential. Notch proteins function in developmental and pathological angiogenesis via the modulation of key pathways in endothelial and perivascular cells. Thus, we hypothesized that Hla would activate Notch in vascular cells. Human umbilical vein endothelial cells were treated with recombinant Hla (rHla), Hla-H35L (genetically inactivated Hla), or Hank's solution (HBSS), and probed by different methods. Luciferase assays showed that Hla (0.01 µg/mL) increased Notch activation by 1.75 ± 0.5-fold as compared to HBSS controls (p < 0.05), whereas Hla-H35L had no effect. Immunocytochemistry and Western blotting confirmed these findings and revealed that ADAM10 and γ-secretase are required for Notch activation after inhibitor and siRNA assays. Retinal EC in mice engineered to express yellow fluorescent protein (YFP) upon Notch activation demonstrated significantly greater YFP intensity after Hla injection than controls. Aortic rings from Notch reporter mice embedded in matrix and incubated with rHla or Hla-H35L demonstrate increased Notch activation occurs at tip cells during sprouting. These mice also had higher skin YFP intensity and area of expression after subcutaneous inoculation of S. aureus expressing Hla than a strain lacking Hla in both EC and pericytes assessed by microscopy. Human liver displayed strikingly higher Notch expression in EC and pericytes during S. aureus infection by immunohistochemistry than tissues from uninfected patients. In sum, our results demonstrate that the S. aureus toxin Hla can potently activate Notch in vascular cells, an effect which may contribute to the pathobiology of infection with this microorganism.

Comprehensive modeling reveals proximity, seasonality, and hygiene practices as key determinants of MRSA colonization in exposed households.

BACKGROUND: Staphylococcus aureus is the leading cause of skin and soft tissue infections (SSTIs). To develop interventions to prevent recurrent infections, household attributes and individual practices influencing S. aureus colonization must be discerned. METHODS: Households of healthy children with methicillin-resistant S. aureus (MRSA) SSTI (n = 150; 671 participants) were interviewed regarding health history, activities, and hygiene practices. S. aureus colonization was assessed in household members, and recovered isolates were typed by repetitive sequence-based PCR. RESULTS: The number of unique strain types in a household (median 1, range 0-7) correlated with the number of colonized individuals (p < 0.001). The MRSA infecting strain type colonized a household member in 57% of 91 households with an available infecting strain, and was the most common strain type recovered in 45% of these households. In multivariable models, household MRSA colonization burden (p < 0.001), sharing a bedroom with MRSA-colonized individuals (p = 0.03), renting dwelling (p = 0.048), and warmer seasons (p = 0.02) were associated with increased MRSA colonization. Increasing age (p = 0.02), bathing at least daily (p = 0.01), and antibacterial soap use (p = 0.03) correlated with reduced MRSA colonization. CONCLUSIONS: This study identified practices that correlate with MRSA colonization, which will inform physician counseling and multifaceted interventions among MRSA-affected households to mitigate MRSA in the community.

α-Toxin Induces Platelet Aggregation and Liver Injury during Staphylococcus aureus Sepsis.

During sepsis, small blood vessels can become occluded by large platelet aggregates of poorly understood etiology. During Staphylococcal aureus infection, sepsis severity is linked to the bacterial α-toxin (α-hemolysin, AT) through unclear mechanisms. In this study, we visualized intravascular events in the microcirculation and found that intravenous AT injection induces rapid platelet aggregation, forming dynamic micro-thrombi in the microcirculation. These aggregates are retained in the liver sinusoids and kidney glomeruli, causing multi-organ dysfunction. Acute staphylococcal infection results in sequestration of most bacteria by liver macrophages. Platelets are initially recruited to these macrophages and help eradicate S. aureus. However, at later time points, AT causes aberrant and damaging thrombosis throughout the liver. Treatment with an AT neutralizing antibody (MEDI4893∗) prevents platelet aggregation and subsequent liver damage, without affecting the initial and beneficial platelet recruitment. Thus, AT neutralization may represent a promising approach to combat staphylococcal-induced intravascular coagulation and organ dysfunction.

A Two-Component System Regulates Bacteroides fragilis Toxin to Maintain Intestinal Homeostasis and Prevent Lethal Disease.

Intestinal microbes are recognized for their role in human disease. Enterotoxigenic Bacteroides fragilis (ETBF) has been implicated in inflammatory bowel disease and colorectal cancer; however, colonization alone is insufficient to cause these illnesses. We hypothesized that homeostasis in healthy carriers is maintained by colonic mucus, the major constituent of which is the glycoprotein Muc2. We found that Muc2-deficient mice succumb to lethal disease from ETBF colonization in a B. fragilis toxin (BFT)-dependent manner. We identify a toxin regulator, the two-component system RprXY, which suppresses BFT expression in vitro and in vivo. Overexpression of either component was sufficient to prevent lethal disease in Muc2-deficient mice. Our studies demonstrate that homeostasis in the context of ETBF colonization is dependent on a dynamic interaction between intestinal mucus, a bacterial toxin, and a toxin regulatory system. Regulation of virulence may offer a therapeutic target to maintain intestinal homeostasis in susceptible patients.

The Bacteroides fragilis pathogenicity island links virulence and strain competition.

The mature microbiome is a stable ecosystem that resists perturbation despite constant host exposure to exogenous microbes. However, the microbial mechanisms determining microbiome development and composition are poorly understood. We recently demonstrated that a non-toxigenic B. fragilis (NTBF) strain restricts enteric colonization by an enterotoxigenic (ETBF) strain dependent on a type VI secretion system (T6SS). We show here that a second enterotoxigenic strain is competent to colonize, dependent on the Bacteroides fragilis pathogenicity island (BFPAI). Additional data showing complex environmental regulation of the Bacteroides fragilis toxin (BFT) suggest that virulence factors may be adapted to modify the colonic niche to provide a strain-specific colonization advantage. We conclude that more complex models of host-microbe-microbiome interactions are needed to investigate this hypothesis.

Staphylococcus aureus pore-forming toxins: The interface of pathogen and host complexity.

Staphylococcus aureus is a prominent human pathogen capable of infecting a variety of host species and tissue sites. This versatility stems from the pathogen's ability to secrete diverse host-damaging virulence factors. Among these factors, the S. aureus pore-forming toxins (PFTs) α-toxin and the bicomponent leukocidins, have garnered much attention for their ability to lyse cells at low concentrations and modulate disease severity. Although many of these toxins were discovered nearly a century ago, their host cell specificities have only been elucidated over the past five to six years, starting with the discovery of the eukaryotic receptor for α-toxin and rapidly followed by identification of the leukocidin receptors. The identification of these receptors has revealed the species- and cell type-specificity of toxin binding, and provided insight into non-lytic effects of PFT intoxication that contribute to disease pathogenesis.

Pediatric Multiple Organ Dysfunction Syndrome: Promising Therapies.

OBJECTIVE: To describe the state of the science, identify knowledge gaps, and offer potential future research questions regarding promising therapies for children with multiple organ dysfunction syndrome presented during the Eunice Kennedy Shriver National Institute of Child Health and Human Development Workshop on Pediatric Multiple Organ Dysfunction Syndrome (March 26-27, 2015). DATA SOURCES: Literature review, research data, and expert opinion. STUDY SELECTION: Not applicable. DATA EXTRACTION: Moderated by an expert from the field, issues relevant to the association of multiple organ dysfunction syndrome with a variety of conditions were presented, discussed, and debated with a focus on identifying knowledge gaps and research priorities. DATA SYNTHESIS: Summary of presentations and discussion supported and supplemented by relevant literature. CONCLUSIONS: Among critically ill children, multiple organ dysfunction syndrome is relatively common and associated with significant morbidity and mortality. For outcomes to improve, effective therapies aimed at preventing and treating this condition must be discovered and rigorously evaluated. In this article, a number of potential opportunities to enhance current care are highlighted including the need for a better understanding of the pharmacokinetics and pharmacodynamics of medications, the effect of early and optimized nutrition, and the impact of effective glucose control in the setting of multiple organ dysfunction syndrome. Additionally, a handful of the promising therapies either currently being implemented or developed are described. These include extracorporeal therapies, anticytokine therapies, antitoxin treatments, antioxidant approaches, and multiple forms of exogenous steroids. For the field to advance, promising therapies and other therapies must be assessed in rigorous manner and implemented accordingly.

Staphylococcus aureus in the Intensive Care Unit: Are These Golden Grapes Ripe for a New Approach?

Staphylococcus aureus is the leading cause of infection in the setting of critical illness and injury. This pathogen causes life-threatening infection in otherwise healthy individuals and also complicates the clinical course of patients requiring intensive care as a result of their primary medical or surgical disease processes. S. aureus infection in the intensive care unit (ICU) most commonly manifests as sepsis, ventilator-associated pneumonia, and infection of surgical sites and indwelling medical devices. With the epidemic spread of methicillin-resistant S. aureus, many cases of staphylococcal infection in the ICU are now classified as drug resistant, prompting hospital-based screening for methicillin-resistant S. aureus and implementation of both isolation practices and decolonization strategies in ICU patients. The genetic adaptability of S. aureus, heterogeneity of disease presentation, clinical course, and outcome between individual S. aureus-infected ICU patients remains enigmatic, suggesting a need to define disease classification subtypes that inform disease progression and therapy. We propose that S. aureus infection in the ICU now presents a unique opportunity for individualized risk stratification coupled with the investigation of novel approaches to mitigate disease. Given our increasing knowledge of the molecular pathogenesis of S. aureus disease, we suggest that the application of molecular pathological epidemiology to S. aureus infection can usher in a new era of highly focused personalized therapy that may be particularly beneficial in the setting of critical illness and injury.

Staphylococcus aureus α-Toxin Response Distinguishes Respiratory Virus-Methicillin-Resistant S. aureus Coinfection in Children.

BACKGROUND: Development of methicillin-resistant Staphylococcus aureus (MRSA) pneumonia after a respiratory viral infection is frequently fatal in children. In mice, S. aureus α-toxin directly injures pneumocytes and increases mortality, whereas α-toxin blockade mitigates disease. The role of α-toxin in pediatric staphylococcal-viral coinfection is unclear. METHODS: We enrolled children across 34 North American pediatric intensive care units with acute respiratory failure and suspected influenza virus infection. Serial serum anti-α-toxin antibody titers and functional α-toxin neutralization capacity were compared across children coinfected with MRSA or methicillin-susceptible S. aureus (MSSA) and control children infected with influenza virus only. MRSA isolates were tested for α-toxin production and lethality in a murine pneumonia model. RESULTS: Influenza virus was identified in 22 of 25 children with MRSA coinfection (9 died) and 22 patients with MSSA coinfection (all survived). Initial α-toxin-specific antibody titers were similar, compared with those in the 13 controls. In patients with serial samples, only MRSA-coinfected patients showed time-dependent increases in anti-α-toxin titer and functional neutralization capacity. MRSA α-toxin production from patient isolates correlated with initial serologic titers and with mortality in murine pneumonia. CONCLUSIONS: These data implicate α-toxin as a relevant antigen in severe pediatric MRSA pneumonia associated with respiratory viral infection, supporting a potential role for toxin-neutralizing therapy.