Rational Design of Live Biotherapeutic Products for the Prevention of Clostridioides difficile Infection.

Clostridioides difficile infection (CDI) is one of the leading causes of healthcare- and antibiotic-associated diarrhea. While fecal microbiota transplantation (FMT) has emerged as a promising therapy for recurrent CDI, its exact mechanisms of action and long-term safety are not fully understood. Defined consortia of clonal bacterial isolates, known as live biotherapeutic products (LBPs), have been proposed as an alternative therapeutic option. However, the rational design of LBPs remains challenging. Here, we employ a computational pipeline and three independent metagenomic datasets to systematically identify microbial strains that have the potential to inhibit CDI. We first constructed the CDI-related microbial genome catalog, comprising 3,741 non-redundant metagenome-assembled genomes (nrMAGs) at the strain level. We then identified multiple potential protective nrMAGs that can be candidates for the design of microbial consortia targeting CDI, including strains from Dorea formicigenerans , Oscillibacter welbionis , and Faecalibacterium prausnitzii . Importantly, some of these potential protective nrMAGs were found to play an important role in the success of FMT, and the majority of the top protective nrMAGs can be validated by various previously reported findings. Our results demonstrate a computational framework for the rational selection of microbial strains targeting CDI, paving the way for the computational design of microbial consortia against other enteric infections.

How Future Pharmacologic Therapies for Celiac Disease Will Complement the Gluten-Free Diet.

The only proven treatment for celiac disease is adherence to a strict, lifelong, gluten-free diet. However, complete dietary gluten avoidance is challenging and a substantial number of patients do not respond fully, clinically, or histologically, despite their best efforts. As celiac disease is common and its central pathophysiology is well elucidated, it has become attractive for drug development to address the limitations of dietary treatment. Most efforts address nonresponsive celiac disease, defined as continued symptoms and/or signs of disease activity despite a gluten-free diet, including the more severe forms of refractory celiac disease, types I and II. An increasing spectrum of therapeutic approaches target defined mechanisms in celiac disease pathogenesis and some have advanced to current phase 2 and 3 clinical studies. We discuss these approaches in terms of potential efficiency, practicability, safety, and need, as defined by patients, regulatory authorities, health care providers, and payors.

A computational method to dissect colonization resistance of the gut microbiota against pathogens.

The mammalian gut microbiome protects the host through colonization resistance (CR) against the incursion of exogenous and often harmful microorganisms, but identifying the exact microbes responsible for the gut microbiota-mediated CR against a particular pathogen remains a challenge. To address this limitation, we developed a computational method: generalized microbe-phenotype triangulation (GMPT). We first systematically validated GMPT using a classical population dynamics model in community ecology and demonstrated its superiority over baseline methods. We then tested GMPT on simulated data generated from the ecological network inferred from a real community (GnotoComplex microflora) and real microbiome data on two mouse studies on Clostridioides difficile infection. We demonstrated GMPT's ability to streamline the discovery of microbes that are potentially responsible for microbiota-mediated CR against pathogens. GMPT holds promise to advance our understanding of CR mechanisms and facilitate the rational design of microbiome-based therapies for preventing and treating enteric infections.

Budesonide, an anti-inflammatory drug, exacerbate clostridioides difficile colitis in mice.

BACKGROUND AND AIMS: Clostridioides difficile infection (CDI) induces intense acute inflammatory responses through toxin release. A combination of antibiotic and anti-inflammatory agents is sometimes recommended in severe, non-responsive cases, although clinical trials have been inconclusive, raising concerns about potential complications. This study aims to investigate the effect of budesonide and mesalamine in the treatment of CDI in a murine model, by evaluating the combination of fidaxomicin and these anti-inflammatory drugs. METHOD: C57BL/6 J female mice pretreated with an antimicrobial mixture were challenged with C. difficile VPI 10463 or culture media by gavage. After the challenge, mice received placebo, fidaxomicin alone (20 mg/kg), or fidaxomicin combined with mesalamine (200, 400 mg/kg) or budesonide (0.2, 1, 10 mg/kg) for 5 days. The mice were monitored for 7 days with weight and survival. Colon and cecum tissues were harvested for histological assessment. RESULTS: CDI of mice caused 80% mortality. Fidaxomicin completely protected against CDI in all parameters (weight, survival and pathscores). Mortality rates were up to 90%, 70% in budesonide(10 mg/kg) and mesalamine (400 mg/kg) treatment group, respectively. Budesonide (0.02,0.1 and 1 mg/kg) adjunction to fidaxomicin worsened the disease outcome according to all tested parameters. While mesalamine in combination with fidaxomicin (200, 400 mg/kg) did not lead to any deaths during CDI treatment, it did not provide additional benefits. CONCLUSIONS: Anti-inflammatory drugs including corticosteroid therapy may worsen the incidence and severity of CDI in this mouse model. These studies may have important clinical implications for understanding the role of anti-inflammatory/ corticosteroid therapy in CDI and inflammatory bowel disease management.

Characteristics and Outcomes of Young Patients With ST-Elevation Myocardial Infarction Without Standard Modifiable Risk Factors.

Patients with ST-elevation myocardial infarction (STEMI) with no standard modifiable risk factors (SMuRFs: hypertension, diabetes mellitus, hypercholesterolemia, and smoking) have worse short-term mortality than those with SMuRFs. Whether this association extends to younger patients is unclear. A retrospective cohort study was performed of patients aged 18 to 45 years with STEMI at 3 Australian hospitals between 2010 and 2020. Nonatherosclerotic causes of STEMI were excluded. The primary outcome was 30-day all-cause mortality. Secondary outcomes included 1 and 2-year mortality. Cox proportional hazards analysis was used. Of 597 patients, the median age was 42 (interquartile range 38 to 44) years, 85.1% were men and 8.4% were SMuRF-less. Patients who are SMuRF-less were >2 times more likely to have cardiac arrest (28.0% vs 12.6%, p = 0.003); require vasopressors (16.0% vs 6.8%, p = 0.018), mechanical support (10.0% vs 2.3%, p = 0.046), or intensive care admission (20.0% vs 5.7%, p <0.001); and have higher rate of left anterior descending artery infarcts than those with SMuRFs (62.0% vs 47.2%, p = 0.045). No significant differences in thrombolysis or percutaneous intervention were observed. Guideline-directed medical therapy at discharge was high (>90%), and not different in the SMuRF-less. 30-day mortality was almost fivefold higher in the SMuRF-less (hazard ratio 4.70, 95% confidence interval 1.66 to 13.35, p = 0.004), remaining significant at 1 and 2 years. In conclusion, young patients who are SMuRF-less have a higher 30-day mortality after STEMI than their counterparts with SMuRFs. This may be partially mediated by higher rates of cardiac arrest and left anterior descending artery territory events. These findings further highlight the need for improved prevention and management of SMuRF-less STEMI.

Redefining Clostridioides difficile infection antibiotic response and clinical outcomes.

With the approval and development of narrow-spectrum antibiotics for the treatment of Clostridioides difficile infection (CDI), the primary endpoint for treatment success of CDI antibiotic treatment trials has shifted from treatment response at end of therapy to sustained response 30 days after completed therapy. The current definition of a successful response to treatment (three or fewer unformed bowel movements [UBMs] per day for 1-2 days) has not been validated, does not reflect CDI management, and could impair assessments for successful treatment at 30 days. We propose new definitions to optimise trial design to assess sustained response. Primarily, we suggest that the initial response at the end of treatment be defined as (1) three or fewer UBMs per day, (2) a reduction in UBMs of more than 50% per day, (3) a decrease in stool volume of more than 75% for those with ostomy, or (4) attainment of bowel movements of Bristol Stool Form Scale types 1-4, on average, by day 2 after completion of primary CDI therapy (ie, assessed on day 11 and day 12 of a 10-day treatment course) and following an investigator determination that CDI treatment can be ceased.

Preservation of the Innate Immune Response to Clostridioides difficile Infection in Hospitalized Immunocompromised Patients.

Background: Clostridioides difficile infection (CDI) immune response is influenced by the innate and adaptive (humoral) immune systems. Our prior research found attenuated humoral responses to C difficile in immunocompromised hosts (ICHs) with CDI. We sought to evaluate whether the innate immune response to CDI was influenced by ICH status. Methods: We conducted a prospective study of hospitalized adults with CDI (acute diarrhea, positive C difficile stool nucleic acid amplification testing [NAAT], and decision to treat), with and without immunosuppression and measured a panel of cytokines (granulocyte colony-stimulating factor [G-CSF], interleukin [IL]-10, IL-15, IL-1ß, IL-4, IL-6, IL-8, and tumor necrosis factor-α) in blood and stool at CDI diagnosis. Results were compared with measurements from a cohort of asymptomatic carrier patients (ASCs) (NAAT positive, without diarrhea) with and without immunocompromise. Results: One hundred twenty-three subjects (42 ICHs, 50 non-ICHs, 31 ASCs) were included. Median values for blood and stool cytokines were similar in ICH versus non-ICH CDI subjects. In blood, G-CSF, IL-10, IL-15, IL-6, and IL-8 were higher in both groups of CDI subjects versus the ASC cohort (P < .05). In stool, IL-1ß and IL-8 were higher in both groups of CDI subjects versus the ASC cohort (P < .05). Median stool concentrations of IL-1ß demonstrated significant differences between the groups (ICHs, 10.97 pg/mL; non-ICHs, 9.71 pg/mL; and ASCs, 0.56 pg/mL) (P < .0001). Conclusions: In this small exploratory analysis, ICH status did not significantly impact blood and fecal patterns of cytokines in humans at the diagnosis of CDI, suggesting that the innate immune response to C difficile may be conserved in immunocompromised patients.

Baseline stool toxin concentration is associated with risk of recurrence in children with Clostridioides difficile infection.

BACKGROUND: In adults with Clostridioides difficile infection (CDI), higher stool concentrations of toxins A and B are associated with severe baseline disease, CDI-attributable severe outcomes, and recurrence. We evaluated whether toxin concentration predicts these presentations in children with CDI. METHODS: We conducted a prospective cohort study of inpatients aged 2-17 years with CDI who received treatment. Patients were followed for 40 days after diagnosis for severe outcomes (intensive care unit admission, colectomy, or death, categorized as CDI primarily attributable, CDI contributed, or CDI not contributing) and recurrence. Baseline stool toxin A and B concentrations were measured using ultrasensitive single-molecule array assay, and 12 plasma cytokines were measured when blood was available. RESULTS: We enrolled 187 pediatric patients (median age, 9.6 years). Patients with severe baseline disease by IDSA-SHEA criteria (n = 34) had nonsignificantly higher median stool toxin A+B concentration than those without severe disease (n = 122; 3,217.2 vs 473.3 pg/mL; P = .08). Median toxin A+B concentration was nonsignificantly higher in children with a primarily attributed severe outcome (n = 4) versus no severe outcome (n = 148; 19,472.6 vs 429.1 pg/mL; P = .301). Recurrence occurred in 17 (9.4%) of 180 patients. Baseline toxin A+B concentration was significantly higher in patients with versus without recurrence: 4,398.8 versus 280.8 pg/mL (P = .024). Plasma granulocyte colony-stimulating factor concentration was significantly higher in CDI patients versus non-CDI diarrhea controls: 165.5 versus 28.5 pg/mL (P < .001). CONCLUSIONS: Higher baseline stool toxin concentrations are present in children with CDI recurrence. Toxin quantification should be included in CDI treatment trials to evaluate its use in severity assessment and outcome prediction.

American College of Gastroenterology Guidelines Update: Diagnosis and Management of Celiac Disease.

This guideline presents an update to the 2013 American College of Gastroenterology Guideline on the Diagnosis and Management of Celiac Disease with updated recommendations for the evaluation and management of patients with celiac disease (CD). CD is defined as a permanent immune-mediated response to gluten present in wheat, barley, and rye. CD has a wide spectrum of clinical manifestations that resemble a multisystemic disorder rather than an isolated intestinal disease, and is characterized by small bowel injury and the presence of specific antibodies. Detection of CD-specific antibodies (e.g., tissue transglutaminase) in the serum is very helpful for the initial screening of patients with suspicion of CD. Intestinal biopsy is required in most patients to confirm the diagnosis. A nonbiopsy strategy for the diagnosis of CD in selected children is suggested and discussed in detail. Current treatment for CD requires strict adherence to a gluten-free diet (GFD) and lifelong medical follow-up. Most patients have excellent clinical response to a GFD. Nonresponsive CD is defined by persistent or recurrent symptoms despite being on a GFD. These patients require a systematic workup to rule out specific conditions that may cause persistent or recurrent symptoms, especially unintentional gluten contamination. Refractory CD is a rare cause of nonresponsive CD often associated with poor prognosis.

Unique features of regulation of sulfate assimilation in monocots.

Sulfate assimilation is an essential pathway of plant primary metabolism, regulated by the demand for reduced sulfur (S). The S-containing tripeptide glutathione (GSH) is the key signal for such regulation in Arabidopsis, but little is known about the conservation of these regulatory mechanisms beyond this model species. Using two model monocot species, C3 rice (Oryza sativa) and C4Setaria viridis, and feeding of cysteine or GSH, we aimed to find out how conserved are the regulatory mechanisms described for Arabidopsis in these species. We showed that while in principle the regulation is similar, there are many species-specific differences. For example, thiols supplied by the roots are translocated to the shoots in rice but remain in the roots of Setaria. Cysteine and GSH concentrations are highly correlated in Setaria, but not in rice. In both rice and Setaria, GSH seems to be the signal for demand-driven regulation of sulfate assimilation. Unexpectedly, we observed cysteine oxidation to sulfate in both species, a reaction that does not occur in Arabidopsis. This reaction is dependent on sulfite oxidase, but the enzyme(s) releasing sulfite from cysteine still need to be identified. Altogether our data reveal a number of unique features in the regulation of S metabolism in the monocot species and indicate the need for using multiple taxonomically distinct models to better understand the control of nutrient homeostasis, which is important for generating low-input crop varieties.

Stool Interleukin-1ß Differentiates Clostridioides difficile Infection (CDI) From Asymptomatic Carriage and Non-CDI Diarrhea.

BACKGROUND: Despite advances in the understanding and diagnosis of Clostridioides difficile infection (CDI), clinical distinction within the colonization-infection continuum remains an unmet need. METHODS: By measuring stool cytokines and antitoxin antibodies in well-characterized cohorts of CDI (diarrhea, nucleic acid amplification test [NAAT] positive), non-CDI diarrhea (NCD; diarrhea, NAAT negative), asymptomatic carriers (ASC; no diarrhea, NAAT positive) and hospital controls (CON; no diarrhea, NAAT negative), we aim to discover novel biological markers to distinguish between these cohorts. We also explore the relationship of these stool cytokines and antitoxin antibody with stool toxin concentrations and disease severity. RESULTS: Stool interleukin (IL) 1ß, stool immunoglobulin A (IgA), and immunoglobulin G (IgG) anti-toxin A had higher (P < .0001) concentrations in CDI (n = 120) vs ASC (n = 43), whereas toxins A, B, and fecal calprotectin did not. Areas under the receiver operating characteristic curve (ROC-AUCs) for IL-1ß, IgA, and IgG anti-toxin A were 0.88, 0.83, and 0.83, respectively. A multipredictor model including IL-1ß and IgA anti-toxin A achieved an ROC-AUC of 0.93. Stool IL-1ß concentrations were higher in CDI compared to NCD (n = 75) (P < .0001) and NCD + ASC+ CON (CON, n = 75) (P < .0001), with ROC-AUCs of 0.83 and 0.86, respectively. Stool IL-1ß had positive correlations with toxins A (ρA = +0.55) and B (ρB = +0.49) in CDI (P < .0001) but not in ASC (P > .05). CONCLUSIONS: Stool concentrations of the inflammasome pathway, proinflammatory cytokine IL-1ß, can accurately differentiate CDI from asymptomatic carriage and NCD, making it a promising biomarker for CDI diagnosis. Significant positive correlations exist between stool toxins and stool IL-1ß in CDI but not in asymptomatic carriers.

Anemia Etiology and the Response to a Gluten-Free Diet in Untreated Patients With Celiac Disease: A 2-Year Follow-Up.

INTRODUCTION: Anemia and micronutrient deficiencies are common in newly diagnosed patients with celiac disease (CeD). We aim to determine the prevalence and etiology of anemia in a cohort of patients with CeD in the United States and examine the effect of a gluten-free diet (GFD) on the laboratory parameters related to anemia in CeD. METHODS: We analyzed a prospectively collected cohort of adults with biopsy-proven CeD followed in a specialized CeD center between January 2000 and June 2016. We used the level of hemoglobin (Hb) and micronutrients suggested by the World Health Organization to establish the diagnosis of anemia or deficiencies. Demographic data and laboratory parameters related to anemia and micronutrients were recorded at the time of diagnosis and on a GFD. A celiac expert nutritionist or gastroenterologist evaluated all patients. RESULTS: In 572 patients with laboratory evaluation before starting a GFD, approximately 25% presented with anemia at the time of diagnosis of CeD. Iron deficiency was present in 50.8% of the cohort and in 78.8% of the patients with anemia. Within the anemic population, 84.4% of female patients as compared with 58.3% of male patients ( P = 0.02) showed iron deficiency. Folate deficiency (23.2%), vitamin B12 deficiency (11%), and anemia of chronic diseases (7.8%) were also part of both sexes' anemia etiology. Of the initially anemic patients, 81% and 89% normalized their Hb levels within 1 year and 2 years of beginning a GFD, respectively. All patients received appropriate supplementation when needed. DISCUSSION: Approximately 25% of individuals have anemia at CeD diagnosis. The anemia etiology included iron deficiency, vitamin deficiencies, and anemia of chronic diseases. Most of the patients will normalize their Hb levels and the anemia laboratory parameters 1 year after starting a strict GFD.

Stool Toxin Concentration Does Not Distinguish Clostridioides difficile Infection from Colonization in Children Less Than 3 Years of Age.

In a prospective cohort study, stools from children <3 years with and without diarrhea who were Clostridioides difficile nucleic acid amplification test-positive underwent ultrasensitive and quantitative toxin measurement. Among 37 cases and 46 controls, toxin concentration distributions overlapped substantially. Toxin concentration alone does not distinguish C. difficile infection from colonization in young children.

Analysis of Intestinal Mycobiota of Patients with Clostridioides difficile Infection among a Prospective Inpatient Cohort.

Clostridioides difficile infection (CDI) is a burden to health care systems worldwide. Gut microbiota dysbiosis associated with CDI has been well accepted. However, contribution of fungal mycobiota to CDI has recently gained research interest. Here, we report the gut mycobiota composition of 149 uniquely well characterized participants from a prospective clinical cohort and evaluate the discriminating ability of gut mycobiota to classify CDI and non-CDI patients. Fecal samples were divided into two groups: (i) CDI (inpatients who had clinically significant diarrhea and positive nucleic acid amplification testing [NAAT] and received subsequent CDI therapy, n = 58) and (ii) non-CDI, which can be further divided into three subgroups: (a) carrier (inpatients with positive stool NAAT but without diarrhea; n = 28); (b) diarrhea (inpatients with negative stool NAAT; n = 31); and (c) control (inpatients with negative stool NAAT and without diarrhea; n = 32). Fecal mycobiota composition was analyzed by internal transcribed spacer 2 (ITS2) sequencing. In comparison to non-CDI patients, CDI patients tend to have gut mycobiota with lower biodiversity, weaker fungi correlations, and weaker correlations between fungi and host immune factors. Notably, 11 genera (Saccharomyces, Penicillium, Aspergillus, Cystobasidium, Cladosporium, and so on) were significantly enriched in non-CDI patients, and Pichia and Suhomyces were enriched in patients with CDI, while 1 two genera, Cystobasidium and Exophiala, had higher abundance in patients with diarrhea compared with CDI (linear discriminant analysis [LDA] > 3.0; P < 0.05). Ascomycota and Basidiomycota (or Candida and Saccharomyces) exhibited a strong negative correlation (r ≤ -0.714 or r ≤ -0.387; P < 0.05), and the ratios of Ascomycota to Basidiomycota or genera Candida to Saccharomyces were dramatically higher in CDI patients than in non-CDI patients (P < 0.05). A disease-specific pattern with much weaker fungal abundance correlations was observed in the CDI group compared to that in the non-CDI and diarrhea groups, suggesting that these correlations may contribute to the development of CDI. Our findings provided specific markers of stool fungi that distinguish CDI from all non-CDI hospitalized patients. This study's potential clinical utility for better CDI diagnosis warrants further investigation. IMPORTANCE Clostridioides difficile is an opportunistic bacterial pathogen that causes a serious and potentially life-threatening infection of the human gut. It remains an existing challenge to distinguish active infection of CDI from diarrhea with non-CDI causes. A few large prospective studies from recent years suggest that there is no single optimal test for the diagnosis of CDI. Previous research has concentrated on the relationship between bacteria and CDI, while the roles of fungi, as a significant proportion of the gut microbial ecosystem, remain understudied. In this study, we report a series of fungal markers that may add diagnostic values for the development of a more systematic approach to accurate CDI diagnosis. These results help open the door for better understanding of the relationship between host immune factors and the fungal community in the context of CDI pathogenesis.

Higher In Vivo Fecal Concentrations of Clostridioides difficile Toxins A and B in Patients With North American Pulsed-Field Gel Electrophoresis Type 1/Ribotype 027 Strain Infection.

Ultrasensitive, quantitative Clostridioides difficile stool toxin measurement demonstrated significantly higher concentrations of toxins A and B in patients infected with the North American pulsed-field gel electrophoresis type 1/ribotype 027 (NAP-1/027) strain compared with other strains, providing in vivo confirmation of the in vitro association between NAP-1/027 and elevated toxin production.