Intestinal Inflammation and Altered Gut Microbiota Associated with Inflammatory Bowel Disease Render Mice Susceptible to Clostridioides difficile Colonization and Infection.

Clostridioides difficile is a noteworthy pathogen in patients with inflammatory bowel disease (IBD). Patients with IBD who develop concurrent C. difficile infection (CDI) experience increased morbidity and mortality. IBD is associated with intestinal inflammation and alterations of the gut microbiota, both of which can diminish colonization resistance to C. difficile. Here, we describe the development of a mouse model to explore the role that IBD-induced changes of the gut microbiome play in susceptibility to C. difficile. Helicobacter hepaticus, a normal member of the mouse gut microbiota, triggers pathological inflammation in the distal intestine akin to human IBD in mice that lack intact interleukin 10 (IL-10) signaling. We demonstrate that mice with H. hepaticus-induced IBD were susceptible to C. difficile colonization in the absence of other perturbations, such as antibiotic treatment. Concomitant IBD and CDI were associated with significantly worse disease than observed in animals with colitis alone. Development of IBD resulted in a distinct intestinal microbiota community compared to that of non-IBD controls. Inflammation played a critical role in the susceptibility of animals with IBD to C. difficile colonization, as mice colonized with an isogenic mutant of H. hepaticus that triggers an attenuated intestinal inflammation maintained full colonization resistance. These studies with a novel mouse model of IBD and CDI emphasize the importance of host responses and alterations of the gut microbiota in susceptibility to C. difficile colonization and infection in the setting of IBD. IMPORTANCE The incidence of C. difficile infection (CDI) has increased significantly among patients with IBD, independently of antibiotic use, yet the relationship between IBD and increased risk for CDI remains to be understood. Our study sought to describe and utilize an antibiotic-independent mouse model to specifically explore the relationship between the IBD-associated gut and susceptibility to C. difficile colonization and CDI development. We demonstrate that the development of IBD is sufficient to render mice susceptible to C. difficile colonization and results in significantly worse disease than IBD alone. Furthermore, this model requires IBD-induced inflammation to overcome colonization resistance to C. difficile. This model recapitulates human IBD and CDI comorbidity and will aid in developing new clinical approaches to predict, diagnose, and treat C. difficile infection in the IBD population.

Lung and gut microbiota are altered by hyperoxia and contribute to oxygen-induced lung injury in mice.

Inhaled oxygen, although commonly administered to patients with respiratory disease, causes severe lung injury in animals and is associated with poor clinical outcomes in humans. The relationship between hyperoxia, lung and gut microbiota, and lung injury is unknown. Here, we show that hyperoxia conferred a selective relative growth advantage on oxygen-tolerant respiratory microbial species (e.g., Staphylococcus aureus) as demonstrated by an observational study of critically ill patients receiving mechanical ventilation and experiments using neonatal and adult mouse models. During exposure of mice to hyperoxia, both lung and gut bacterial communities were altered, and these communities contributed to oxygen-induced lung injury. Disruption of lung and gut microbiota preceded lung injury, and variation in microbial communities correlated with variation in lung inflammation. Germ-free mice were protected from oxygen-induced lung injury, and systemic antibiotic treatment selectively modulated the severity of oxygen-induced lung injury in conventionally housed animals. These results suggest that inhaled oxygen may alter lung and gut microbial communities and that these communities could contribute to lung injury.

Systemic Inflammatory Mediators Are Effective Biomarkers for Predicting Adverse Outcomes in Clostridioides difficile Infection.

Clostridioides difficile infection (CDI) can result in severe disease and death, with no accurate models that allow for early prediction of adverse outcomes. To address this need, we sought to develop serum-based biomarker models to predict CDI outcomes. We prospectively collected sera ≤48 h after diagnosis of CDI in two cohorts. Biomarkers were measured with a custom multiplex bead array assay. Patients were classified using IDSA severity criteria and the development of disease-related complications (DRCs), which were defined as ICU admission, colectomy, and/or death attributed to CDI. Unadjusted and adjusted models were built using logistic and elastic net modeling. The best model for severity included procalcitonin (PCT) and hepatocyte growth factor (HGF) with an area (AUC) under the receiver operating characteristic (ROC) curve of 0.74 (95% confidence interval, 0.67 to 0.81). The best model for 30-day mortality included interleukin-8 (IL-8), PCT, CXCL-5, IP-10, and IL-2Rα with an AUC of 0.89 (0.84 to 0.95). The best model for DRCs included IL-8, procalcitonin, HGF, and IL-2Rα with an AUC of 0.84 (0.73 to 0.94). To validate our models, we employed experimental infection of mice with C. difficile Antibiotic-treated mice were challenged with C. difficile and a similar panel of serum biomarkers was measured. Applying each model to the mouse cohort of severe and nonsevere CDI revealed AUCs of 0.59 (0.44 to 0.74), 0.96 (0.90 to 1.0), and 0.89 (0.81 to 0.97). In both human and murine CDI, models based on serum biomarkers predicted adverse CDI outcomes. Our results support the use of serum-based biomarker panels to inform Clostridioides difficile infection treatment.IMPORTANCE Each year in the United States, Clostridioides difficile causes nearly 500,000 gastrointestinal infections that range from mild diarrhea to severe colitis and death. The ability to identify patients at increased risk for severe disease or mortality at the time of diagnosis of C. difficile infection (CDI) would allow clinicians to effectively allocate disease modifying therapies. In this study, we developed models consisting of only a small number of serum biomarkers that are capable of predicting both 30-day all-cause mortality and adverse outcomes of patients at time of CDI diagnosis. We were able to validate these models through experimental mouse infection. This provides evidence that the biomarkers reflect the underlying pathophysiology and that our mouse model of CDI reflects the pathogenesis of human infection. Predictive models can not only assist clinicians in identifying patients at risk for severe CDI but also be utilized for targeted enrollment in clinical trials aimed at reduction of adverse outcomes from severe CDI.

Aging Dampens the Intestinal Innate Immune Response during Severe Clostridioides difficile Infection and Is Associated with Altered Cytokine Levels and Granulocyte Mobilization.

Clostridioides (formerly Clostridium) difficile is the most common cause of hospital-acquired infection, and advanced age is a risk factor for C. difficile infection. Disruption of the intestinal microbiota and immune responses contribute to host susceptibility and severity of C. difficile infection. However, the specific impact of aging on immune responses during C. difficile infection remains to be well described. This study explores the effect of age on cellular and cytokine immune responses during C. difficile infection. Young mice (2 to 3 months old) and aged mice (22 to 28 months old) were rendered susceptible to C. difficile infection with the antibiotic cefoperazone and then infected with C. difficile strains with varied disease-causing potentials. We observe that the host age and the infecting C. difficile strain influenced the severity of disease associated with infection. Tissue-specific CD45+ immune cell responses occurred at the time of peak disease severity in the ceca and colons of all mice infected with a high-virulence strain of C. difficile; however, significant deficits in intestinal neutrophils and eosinophils were detected in aged mice, with a corresponding decrease in circulating CXCL1, an important neutrophil recruiter and activator. Interestingly, this lack of intestinal granulocyte response in aged mice during severe C. difficile infection was accompanied by a simultaneous increase in circulating white blood cells, granulocytes, and interleukin 17A (IL-17A). These findings demonstrate that age-related alterations in neutrophils and eosinophils and systemic cytokine and chemokine responses are associated with severe C. difficile infection and support a key role for intestinal eosinophils in mitigating C. difficile-mediated disease severity.

Novel therapies and preventative strategies for primary and recurrent Clostridium difficile infections.

Clostridium difficile is the leading infectious cause of antibiotic-associated diarrhea and colitis. C. difficile infection (CDI) places a heavy burden on the healthcare system, with nearly half a million infections yearly and an approximate 20% recurrence risk after successful initial therapy. The high incidence has driven new research on improved prevention such as the emerging use of probiotics, intestinal microbiome manipulation during antibiotic therapies, vaccinations, and newer antibiotics that reduce the disruption of the intestinal microbiome. While the treatment of acute C. difficile is effective in most patients, it can be further optimized by adjuvant therapies that improve the initial treatment success and decrease the risk of subsequent recurrence. Finally, the high risk of recurrence has led to multiple emerging therapies that target toxin activity, recovery of the intestinal microbial community, and elimination of latent C. difficile in the intestine. In summary, CDIs illustrate the complex interaction among host physiology, microbial community, and pathogen that requires specific therapies to address each of the factors leading to primary infection and recurrence.

Reducing Recurrence of C. difficile Infection.

Clostridium difficile infection (CDI) is facilitated by alteration of the microbiome following antibiotic administration. Antimicrobial therapy directed against the pathogen can treat CDI. Unfortunately, ∼20% of successfully treated patients will suffer recurrence. Bezlotoxumab, a human monoclonal antibody, binds to C. difficile toxin B (TcdB), reducing recurrence presumably by limiting epithelial damage and facilitating microbiome recovery.

Polyphosphate granule biogenesis is temporally and functionally tied to cell cycle exit during starvation in Pseudomonas aeruginosa.

Polyphosphate (polyP) granule biogenesis is an ancient and ubiquitous starvation response in bacteria. Although the ability to make polyP is important for survival during quiescence and resistance to diverse environmental stresses, granule genesis is poorly understood. Using quantitative microscopy at high spatial and temporal resolution, we show that granule genesis in Pseudomonas aeruginosa is tightly organized under nitrogen starvation. Following nucleation as many microgranules throughout the nucleoid, polyP granules consolidate and become transiently spatially organized during cell cycle exit. Between 1 and 3 h after nitrogen starvation, a minority of cells have divided, yet the total granule number per cell decreases, total granule volume per cell dramatically increases, and individual granules grow to occupy diameters as large as ∼200 nm. At their peak, mature granules constitute ∼2% of the total cell volume and are evenly spaced along the long cell axis. Following cell cycle exit, granules initially retain a tight spatial organization, yet their size distribution and spacing relax deeper into starvation. Mutant cells lacking polyP elongate during starvation and contain more than one origin. PolyP promotes cell cycle exit by functioning at a step after DNA replication initiation. Together with the universal starvation alarmone (p)ppGpp, polyP has an additive effect on nucleoid dynamics and organization during starvation. Notably, cell cycle exit is temporally coupled to a net increase in polyP granule biomass, suggesting that net synthesis, rather than consumption of the polymer, is important for the mechanism by which polyP promotes completion of cell cycle exit during starvation.

Probing the subcellular localization of hopanoid lipids in bacteria using NanoSIMS.

The organization of lipids within biological membranes is poorly understood. Some studies have suggested lipids group into microdomains within cells, but the evidence remains controversial due to non-native imaging techniques. A recently developed NanoSIMS technique indicated that sphingolipids group into microdomains within membranes of human fibroblast cells. We extended this NanoSIMS approach to study the localization of hopanoid lipids in bacterial cells by developing a stable isotope labeling method to directly detect subcellular localization of specific lipids in bacteria with ca. 60 nm resolution. Because of the relatively small size of bacterial cells and the relative abundance of hopanoid lipids in membranes, we employed a primary (2)H-label to maximize our limit of detection. This approach permitted the analysis of multiple stable isotope labels within the same sample, enabling visualization of subcellular lipid microdomains within different cell types using a secondary label to mark the growing end of the cell. Using this technique, we demonstrate subcellular localization of hopanoid lipids within alpha-proteobacterial and cyanobacterial cells. Further, we provide evidence of hopanoid lipid domains in between cells of the filamentous cyanobacterium Nostoc punctiforme. More broadly, our method provides a means to image lipid microdomains in a wide range of cell types and test hypotheses for their functions in membranes.

Characterization of the temperature-sensitive mutations un-7 and png-1 in Neurospora crassa.

The model filamentous fungus Neurospora crassa has been studied for over fifty years and many temperature-sensitive mutants have been generated. While most of these have been mapped genetically, many remain anonymous. The mutation in the N. crassa temperature-sensitive lethal mutant un-7 was identified by a complementation based approach as being in the open reading frame designated NCU00651 on linkage group I. Other mutations in this gene have been identified that lead to a temperature-sensitive morphological phenotype called png-1. The mutations underlying un-7 result in a serine to phenylalanine change at position 273 and an isoleucine to valine change at position 390, while the mutation in png-1 was found to result in a serine to leucine change at position 279 although there were other conservative changes in this allele. The overall morphology of the strain carrying the un-7 mutation is compared to strains carrying the png-1 mutation and these mutations are evaluated in the context of other temperature-sensitive mutants in Neurospora.

Microcalorimetric study of adsorption of human monoclonal antibodies on cation exchange chromatographic materials.

Adsorption of two human monoclonal antibodies on two different strong cation exchange resins is studied by isothermal titration microcalorimetry and independent adsorption isotherm measurements. The pH value is varied between 4.5 and 7.0, using different buffer systems, the temperature is always 25 degrees C. The adsorption isotherm data is fitted using two different Langmuir type models. Combining the calorimetric and the adsorption data, the specific enthalpy of adsorption of the protein Deltah(p)(ads) is determined. At pH values near 7.0, where the antibodies are only weakly charged, the adsorption is exothermal. At small loadings the absolute number of Deltah(p)(ads) is then large and almost constant but it significantly decreases at higher loadings. This shows that the arrangement of antibody molecules on the absorber material depends on the loading and is less favourable at higher loadings. Despite the high positive charge of the antibody at pH values of about 5 the value of Deltah(p)(ads) is almost zero along the entire isotherm. Furthermore, at pH 4.5 even endothermal effects are observed, although high binding capacities are found. At these conditions the adsorption process seems to be strongly influenced by the ions bound to the antibody. Their release upon absorption explains the endothermal caloric effect. The adsorption equilibrium constant K(eq) is calculated from the isotherms. From Deltag(p)(ads) and the calorimetric results for Deltah(p)(ads), Deltas(p)(ads), the entropy change upon adsorption of the protein is found for the different studied conditions.