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Although the role of gut microbiota in Clostridioides difficile infection (CDI) has been well established, little is known about the role of mycobiota in CDI. Here, we performed mycobiome data analysis in a well-characterized human cohort to evaluate the potential of using gut mycobiota features for CDI diagnosis.Stool samples were collected from 118 hospital patients, divided into 3 groups: CDI (n = 58), asymptomatic carriers (Carrier, n = 28), and Control (n = 32). The nuclear ribosomal DNA internal transcribed spacer 2 was sequenced using the Illumina HiSeq platform to assess the fungal composition. Downstream statistical analyses (including Alpha diversity analysis, ordination analysis, differential abundance analysis, fungal correlation network analysis, and classification analysis) were then performed.Significant differences were observed in alpha and beta diversity between patients with CDI and Carrier (P.05). Differential abundance analysis identified 2 genera (Cladosporium and Aspergillus) enriched in Carrier. The ratio of Ascomycota to Basidiomycota was dramatically higher in patients with CDI than in Carrier and Control (P.05). Correlations between host immune factors and mycobiota features were weaker in patients with CDI than in Carrier. Using 4 fungal operational taxonomic units combined with 6 host immune markers in the random forest classifier can achieve very high performance (area under the curve ∼92.38%) in distinguishing patients with CDI from Carrier.Our study provides specific markers of stool fungi combined with host immune factors to distinguish patients with CDI from Carrier. It highlights the importance of gut mycobiome in CDI, which may have been underestimated. Further studies on the diagnostic applications and therapeutic potentials of these findings are warranted.

Background & Aims Clostridium difficile induces intestinal inflammation by releasing toxins A and B. The antimicrobial compound cationic steroid antimicrobial 13 (CSA13) has been developed for treating gastrointestinal infections. The CSA13-Eudragit formulation can be given orally and releases CSA13 in the terminal ileum and colon. We investigated whether this form of CSA13 reduces C difficile infection (CDI) in mice. Methods C57BL/6J mice were infected with C difficile on day 0, followed by subcutaneous administration of pure CSA13 or oral administration of CSA13-Eudragit (10 mg/kg/d for 10 days). Some mice were given intraperitoneal vancomycin (50 mg/kg daily) on days 0–4 and relapse was measured after antibiotic withdrawal. The mice were monitored until day 20; colon and fecal samples were collected on day 3 for analysis. Blood samples were collected for flow cytometry analyses. Fecal pellets were collected each day from mice injected with CSA13 and analyzed by high-performance liquid chromatography or 16S sequencing; feces were also homogenized in phosphate-buffered saline and fed to mice with CDI via gavage. Results CDI of mice caused 60% mortality, significant bodyweight loss, and colonic damage 3 days after infection; these events were prevented by subcutaneous injection of CSA13 or oral administration CSA13-Eudragit. There was reduced relapse of CDI after administration of CSA13 was stopped. Levels of CSA13 in feces from mice given CSA13-Eudragit were significantly higher than those of mice given subcutaneous CSA13. Subcutaneous and oral CSA13 each significantly increased the abundance of Peptostreptococcaceae bacteria and reduced the abundance of C difficile in fecal samples of mice. When feces from mice with CDI and given CSA13 were fed to mice with CDI that had not received CSA13, the recipient mice had significantly increased rates of survival. CSA13 reduced fecal levels of inflammatory metabolites (endocannabinoids) and increased fecal levels of 4 protective metabolites (ie, citrulline, 3-aminoisobutyric acid, retinol, and ursodeoxycholic acid) in mice with CDI. Oral administration of these CSA13-dependent protective metabolites reduced the severity of CDI. Conclusions In studies of mice, we found the CSA13-Eudragit formulation to be effective in eradicating CDI by modulating the intestinal microbiota and metabolites.

Background & Aims Saccharomyces boulardii (Sb) is a probiotic yeast with anti-inflammatory and anti-microbial activities and has been used for decades in the prevention and treatment of a variety of human gastrointestinal disorders. We reported previously that Sb modulates host inflammatory responses through down-regulation of extracellular signal-regulated kinase (Erk)1/2 activities both in vitro and in vivo. The aim of this study was to identify upstream mediators responsible for extracellular signal-regulated kinase (Erk)1/2 inactivation and to examine the effects of Sb on tumor development in Apc Min mice. Methods Signaling studies of colon cancer cells were done by western blot. Cell proliferation was measured by MTS and BrdU assay. Apoptosis was examined by flow cytometry, tunel assay and caspase assay. Apc Min mice were orally given Sb for 9 weeks before sacrifice for tumor analysis. Results We found that the epidermal growth factor receptor (EGFR) was deactivated upon exposure to Sb , leading to inactivation of both the EGFR-Erk and EGFR-Akt pathways. In human colonic cancer cells, Sb prevented EGF-induced proliferation, reduced cell colony formation, and promoted apoptosis. HER-2, HER-3, and insulin-like growth factor-1 receptor were also found to be inactivated by Sb . Oral intake of Sb reduced intestinal tumor growth and dysplasia in C57BL/6J Min/+ ( Apc Min ) mice. Conclusions Thus, in addition to its anti-inflammatory effects, Sb inhibits EGFR and other receptor tyrosine kinase signaling and thereby may also serve a novel therapeutic or prophylactic role in intestinal neoplasia.

Background & Aims Infection with Clostridium difficile causes nosocomial antibiotic–associated diarrhea and colitis. Hamsters historically have been used to investigate disease pathogenesis and treatment, but are not ideal models because of the lack of hamster-specific reagents and genetically modified animals, and because they develop fulminant disease. The aim of this study was to establish a mouse model of antibiotic-induced C difficile–associated disease (CDAD) that more closely resembles human disease. Methods C57BL/6 mice were exposed to a mixture of antibiotics (kanamycin, gentamicin, colistin, metronidazole, and vancomycin) for 3 days. Two days later, they were given injections of clindamycin and then challenged 1 day later with different doses of C difficile. Results Mice that were exposed to antibiotics and then challenged with C difficile developed diarrhea and lost weight. Disease severity varied from fulminant to minimal in accordance with the challenge dose. Typical histologic features of CDAD were evident. Oral vancomycin prevented CDAD in all mice, but 68% died from colitis after treatment was discontinued. All animals that survived an initial episode of CDAD showed no evidence of diarrhea or colitis after subsequent rechallenge with C difficile. Different strains of C difficile tested in the model showed different levels of virulence in mice. Conclusions We have developed a mouse model of CDAD that closely represents the human disease. In light of the recent substantial increases in CDAD incidence and severity, this model will be valuable in testing new treatments, examining disease pathogenesis, and elucidating mechanisms of protective immunity.

G A A b st ra ct s factor. The serum concentrations for all the studied cytokines were compared according to these outcomes through rank sum tests. Whenever a statistically significant difference with a p

Background and Aims: Clostridium difficile infection is the major cause of nosocomial antibiotic-associated colitis. C. difficile produces toxin A, a proinflammatory enterotoxin involved in the pathophysiology of C. difficile infection. Cathelicidin is an endogenous antimicrobial peptide that protects the host from infections. Recent findings also support the ability of cathelicidin to modulate innate immunity and alter the development of intestinal inflammation. Here we explored whether toxin Amodifies the expression of cathelicidin inmouse ileum and cultured human colonocytes. We also determined the anti-inflammatory mechanism of cathelicidin in toxin A-mediated intestinal inflammation using a mouse model of enteritis and human primary peripheral blood monocytes. Methods: Ileal loops were prepared in anesthetized mice and injected with either toxin A (10 microg/20g mouse) or saline (control) (n= 6 loops per group). A separate group was treated with cathelicidin (200 microg/mouse) administered into the ileal loops. After 4 hrs, levels of cathelicidin and TNFalpha and mucosal histological changes of ileal loops were evaluated. Results: Toxin A significantly increased cathelicidin mRNA and protein expression (up to 5 fold, P=0.005) in mouse ileum. In human colonic epithelial NCM460 cells, toxin A induced cathelicidin protein (by ~2 fold, P 50%, P 50%, P 80%, P 50%, P

Background: Intravenous immunoglobulin (IVIG) has been used for many years as an adjunct to treat severe and recurrent Clostridium difficile infection. The concentration of serum antibodies to toxins A and B have been shown to correlate with an asymptomatic carrier state in patients with C. difficile colonization. Furthermore, the lack of development of an effective humoral response to C. difficile toxins may result in increased risk of development of recurrent infection. Objective: Studies of IVIG treatment for C. difficile infection have been inconclusive. We therefore sought to examine whether the effectiveness of IVIG therapy could be altered by variability in anti-toxin antibody content in commercial preparations of IVIG. Methods: Antibodies against C. difficile toxins A and B were measured by ELISA in eight commercially-available immunoglobulin preparations. These preparations represent those that are commonly available for IVIG therapy and included two products that have high titers of antibodies against cytomegalovirus (CMV) and respiratory syncytial virus (RSV). Results: Different preparations of IVIG demonstrated approximately a fourfold variability in antibody content against toxin A and toxin B. Preparations that are formulated to contain high-titers of antibodies against CMV and RSV did not contain higher titers of antibody against C. difficle toxins over standard IVIG preparations. Conclusions: Currently-utilized preparations of immunoglobulin, used as IVIG in the treatment of C. difficile colitis show substantial variability in their anti-C. difficile toxin A and B titers. The ability of these preparations to neutralize toxin effect may be affected by anti-toxin titers and this variability may contribute to variations in the published literature with regard to efficacy of IVIG in severe and recurrent C. difficile colitis.