Teaching old mice new tricks: the utility of aged mouse models of C. difficile infection to study pathogenesis and rejuvenate immune response.

BACKGROUND: Clostridioides difficile is a serious problem for the aging population. Aged mouse model of C. difficile infection (CDI) has emerged as a valuable tool to evaluate the mechanism of aging in CDI. METHODS: We reviewed five published studies utilizing aged mice (7-28 months) for CDI model for findings that may advance our understanding of how aging influences outcome from CDI. RESULTS: Aged mouse models of CDI uniformly demonstrated more severe disease in the old compared to young mice. Diminished neutrophil recruitment to intestinal tissue in aged mice is the most consistent finding. Differences in innate and humoral immune responses were also observed. The effects of aging on the outcome of infection were reversed by pharmacologic or microbiota-targeted interventions. CONCLUSION: The aged mouse presents an important in vivo model to study CDI and elucidate the mechanisms underlying advanced age as an important risk factor for severe disease.

Nasal Immunization with the C-Terminal Domain of Bcla3 Induced Specific IgG Production and Attenuated Disease Symptoms in Mice Infected with Clostridioides difficile Spores.

Clostridioides difficile is a Gram-positive, spore-forming bacterium that causes a severe intestinal infection. Spores of this pathogen enter in the human body through the oral route, interact with intestinal epithelial cells and persist in the gut. Once germinated, the vegetative cells colonize the intestine and produce toxins that enhance an immune response that perpetuate the disease. Therefore, spores are major players of the infection and ideal targets for new therapies. In this context, spore surface proteins of C. difficile, are potential antigens for the development of vaccines targeting C. difficile spores. Here, we report that the C-terminal domain of the spore surface protein BclA3, BclA3CTD, was identified as an antigenic epitope, over-produced in Escherichia coli and tested as an immunogen in mice. To increase antigen stability and efficiency, BclA3CTD was also exposed on the surface of B. subtilis spores, a mucosal vaccine delivery system. In the experimental conditions used in this study, free BclA3CTD induced antibody production in mice and attenuated some C. difficile infection symptoms after a challenge with the pathogen, while the spore-displayed antigen resulted less effective. Although dose regimen and immunization routes need to be optimized, our results suggest BclA3CTD as a potentially effective antigen to develop a new vaccination strategy targeting C. difficile spores.

Interleukin-22-mediated host glycosylation prevents Clostridioides difficile infection by modulating the metabolic activity of the gut microbiota.

The involvement of host immunity in the gut microbiota-mediated colonization resistance to Clostridioides difficile infection (CDI) is incompletely understood. Here, we show that interleukin (IL)-22, induced by colonization of the gut microbiota, is crucial for the prevention of CDI in human microbiota-associated (HMA) mice. IL-22 signaling in HMA mice regulated host glycosylation, which enabled the growth of succinate-consuming bacteria Phascolarctobacterium spp. within the gut microbiome. Phascolarctobacterium reduced the availability of luminal succinate, a crucial metabolite for the growth of C. difficile, and therefore prevented the growth of C. difficile. IL-22-mediated host N-glycosylation is likely impaired in patients with ulcerative colitis (UC) and renders UC-HMA mice more susceptible to CDI. Transplantation of healthy human-derived microbiota or Phascolarctobacterium reduced luminal succinate levels and restored colonization resistance in UC-HMA mice. IL-22-mediated host glycosylation thus fosters the growth of commensal bacteria that compete with C. difficile for the nutritional niche.

IL-17-producing γδ T cells protect against Clostridium difficile infection.

Colitis caused by Clostridium difficile infection is a growing cause of human morbidity and mortality, especially after antibiotic use in health care settings. The natural immunity of newborn infants and protective host immune mediators against C. difficile infection are not fully understood, with data suggesting that inflammation can be either protective or pathogenic. Here, we show an essential role for IL-17A produced by γδ T cells in host defense against C. difficile infection. Fecal extracts from children with C. difficile infection showed increased IL-17A and T cell receptor γ chain expression, and IL-17 production by intestinal γδ T cells was efficiently induced after infection in mice. C. difficile-induced tissue inflammation and mortality were markedly increased in mice deficient in IL-17A or γδ T cells. Neonatal mice, with naturally expanded RORγt+ γδ T cells poised for IL-17 production were resistant to C. difficile infection, whereas elimination of γδ T cells or IL-17A each efficiently overturned neonatal resistance against infection. These results reveal an expanded role for IL-17-producing γδ T cells in neonatal host defense against infection and provide a mechanistic explanation for the clinically observed resistance of infants to C. difficile colitis.

Acetate coordinates neutrophil and ILC3 responses against C. difficile through FFAR2.

Antibiotic-induced dysbiosis is a key predisposing factor for Clostridium difficile infections (CDIs), which cause intestinal disease ranging from mild diarrhea to pseudomembranous colitis. Here, we examined the impact of a microbiota-derived metabolite, short-chain fatty acid acetate, on an acute mouse model of CDI. We found that administration of acetate is remarkably beneficial in ameliorating disease. Mechanistically, we show that acetate enhances innate immune responses by acting on both neutrophils and ILC3s through its cognate receptor free fatty acid receptor 2 (FFAR2). In neutrophils, acetate-FFAR2 signaling accelerates their recruitment to the inflammatory sites, facilitates inflammasome activation, and promotes the release of IL-1ß; in ILC3s, acetate-FFAR2 augments expression of the IL-1 receptor, which boosts IL-22 secretion in response to IL-1ß. We conclude that microbiota-derived acetate promotes host innate responses to C. difficile through coordinate action on neutrophils and ILC3s.

Evaluation of a Gastrointestinal Pathogen Panel Immunoassay in Stool Testing of Patients with Suspected Clostridioides (Clostridium) difficile Infection.

Clostridioides (Clostridium) difficile infection (CDI) is the most common causative pathogen of health care-associated gastrointestinal infections; however, due to the overlap of clinical symptoms with those of other causes of acute gastroenteritis, the selection of the most appropriate laboratory test is difficult. From April to October 2018, 640 stool samples requested for CDI testing were examined using the mariPOC CDI and Gastro test (ArcDia), which allows the detection of C. difficile glutamate dehydrogenase (GDH) and toxin A/B, norovirus genogroups GI and GII.4, rotavirus, adenovirus, and Campylobacter spp. In parallel, the C. Diff Quik Chek Complete test (Alere) was used as a routine diagnostic assay, and C. difficile toxigenic culture was used as a reference method. The sensitivity of the mariPOC CDI and Gastro test was comparable to that of C. Diff Quik Chek Complete for the detection of GDH (96.40% [95% confidence interval {CI}, 91.81% to 98.82%] versus 95.68% [95% CI, 90.84 to 98.40%]; P = 1.00) and was higher for the detection of toxin A/B (66.67% [95% CI, 57.36 to 75.11%] versus 55.56% [95% CI, 46.08 to 64.74%]; P = 0.00). The specificity of the mariPOC CDI and Gastro test was lower than that of C. Diff Quik Chek Complete for GDH detection (95.21% [95% CI, 92.96% to 96.91%] versus 97.60% [95% CI, 95.85% to 98.76%]; P = 0.04) and comparable to that of C. Diff Quik Chek Complete for toxin A/B detection (99.24 [95% CI, 98.05% to 99.79%] versus 99.81% [95% CI, 98.94% to 100.0%]; P = 0.37). In 29 cases (4.53%), other causative agents of diarrhea were detected (Campylobacter spp. [n = 17], rotavirus [n = 7], and norovirus genogroup GII.4 [n = 5]).

IL-33 drives group 2 innate lymphoid cell-mediated protection during Clostridium difficile infection.

Clostridium difficile (C. difficile) incidence has tripled over the past 15 years and is attributed to the emergence of hypervirulent strains. While it is clear that C. difficile toxins cause damaging colonic inflammation, the immune mechanisms protecting from tissue damage require further investigation. Through a transcriptome analysis, we identify IL-33 as an immune target upregulated in response to hypervirulent C. difficile. We demonstrate that IL-33 prevents C. difficile-associated mortality and epithelial disruption independently of bacterial burden or toxin expression. IL-33 drives colonic group 2 innate lymphoid cell (ILC2) activation during infection and IL-33 activated ILC2s are sufficient to prevent disease. Furthermore, intestinal IL-33 expression is regulated by the microbiota as fecal microbiota transplantation (FMT) rescues antibiotic-associated depletion of IL-33. Lastly, dysregulated IL-33 signaling via the decoy receptor, sST2, predicts C. difficile-associated mortality in human patients. Thus, IL-33 signaling to ILC2s is an important mechanism of defense from C. difficile colitis.

Epithelium-specific MyD88 signaling, but not DCs or macrophages, control acute intestinal infection with Clostridium difficile.

Infection with Clostridium difficile is one of the major causes of health care acquired diarrhea and colitis. Signaling though MyD88 downstream of TLRs is critical for initiating the early protective host response in mouse models of C. difficile infection (CDI). In the intestine, MyD88 is expressed in various tissues and cell types, such as the intestinal epithelium and mononuclear phagocytes (MNP), including DC or macrophages. Using a genetic gain-of-function system, we demonstrate here that restricting functional MyD88 signaling to the intestinal epithelium, but also to MNPs is sufficient to protect mice during acute CDI by upregulation of the intestinal barrier function and recruitment of neutrophils. Nevertheless, we also show that mice depleted for CD11c-expressing MNPs in the intestine display no major defects in mounting an effective inflammatory response, indicating that the absence of these cells is irrelevant for inducing host protection during acute infection. Together, our results highlight the importance of epithelial-specific MyD88 signaling and demonstrate that although functional MyD88 signaling in DC and macrophages alone is sufficient to correct the phenotype of MyD88-deficiency, these cells do not seem to be essential for host protection in MyD88-sufficient animals during acute infection with C. difficile.

Clostridioides difficile Colonization Is Differentially Associated With Gut Microbiome Profiles by Infant Feeding Modality at 3-4 Months of Age.

Colonization with Clostridioides difficile occurs in up to half of infants under the age of 3 months, is strongly influenced by feeding modality and is largely asymptomatic. In spite of this, C. difficile's presence has been associated with susceptibility to chronic disease later in childhood, perhaps by promoting or benefiting from changes in infant gut microbiome development, including colonization with pathogenic bacteria and disrupted production of microbial bioactive metabolites and proteins. In this study, the microbiomes of 1554 infants from the CHILD Cohort Study were described according to C. difficile colonization status and feeding mode at 3-4 months of age. C. difficile colonization was associated with a different gut microbiome profile in exclusively breastfed (EBF) vs. exclusively formula fed (EFF) infants. EBF infants colonized with C. difficile had an increased relative abundance of Firmicutes and Proteobacteria, decreased relative abundance of Bifidobacteriaceae, greater microbiota alpha-diversity, greater detectable fecal short chain fatty acids (SCFA), and lower detectable fecal secretory Immunoglobulin A (sIgA) than those not colonized. Similar but less pronounced differences were seen among partially breastfed infants (PBF) but EFF infants did not possess these differences in the gut microbiome according to colonization status. Thus, breastfed infants colonized with C. difficile appear to possess a gut microbiome that differs from non-colonized infants and resembles that of EFF infants, but the driving force and direction of this association remains unknown. Understanding these compositional differences as drivers of C. difficile colonization may be important to ensure future childhood health.

Novel Chimeric Protein Vaccines Against Clostridium difficile Infection.

Clostridium difficile infection (CDI) is the leading cause of world-wide nosocomial acquired diarrhea in adults. Active vaccination is generally accepted as a logical and cost-effective approach to prevent CDI. In this paper, we have generated two novel chimeric proteins; one designated Tcd169, comprised of the glucosyltransferase domain (GT), the cysteine proteinase domain (CPD), and receptor binding domain (RBD) of TcdB, and the RBD of TcdA; the other designated Tcd169FI, which contains Salmonella typhimurium flagellin (sFliC) and Tcd169. Both proteins were expressed in and purified from Bacillus megaterium. Point mutations were made in the GT (W102A, D288N) and CPD (C698) of TcdB to ensure that Tcd169 and Tcd169FI were atoxic. Immunization with Tcd169 or Tcd169Fl induced protective immunity against TcdA/TcdB challenge through intraperitoneal injection, also provided mice full protection against infection with a hyper-virulent C. difficile strain (BI/NAP1/027). In addition, inclusion of sFlic in the fusion protein (Tcd169Fl) enhanced its protective immunity against toxin challenge, reduced C. difficile numbers in feces from Tcd169Fl-immunized mice infected C. difficile. Our data show that Tcd169 and Tcd169FI fusion proteins may represent alternative vaccine candidates against CDI.

Apoptosis of intestinal epithelial cells restricts Clostridium difficile infection in a model of pseudomembranous colitis.

Clostridium difficile is the leading cause of pseudomembranous colitis in hospitalized patients. C. difficile enterotoxins TcdA and TcdB promote this inflammatory condition via a cytotoxic response on intestinal epithelial cells (IECs), but the underlying mechanisms are incompletely understood. Additionally, TcdA and TcdB engage the Pyrin inflammasome in macrophages, but whether Pyrin modulates CDI pathophysiology is unknown. Here we show that the Pyrin inflammasome is not functional in IECs and that Pyrin signaling is dispensable for CDI-associated IEC death and for in vivo pathogenesis. Instead, our studies establish that C. difficile enterotoxins induce activation of executioner caspases 3/7 via the intrinsic apoptosis pathway, and demonstrate that caspase-3/7-mediated IEC apoptosis is critical for in vivo host defense during early stages of CDI. In conclusion, our findings dismiss a critical role for inflammasomes in CDI pathogenesis, and identify IEC apoptosis as a host defense mechanism that restricts C. difficile infection in vivo.

Development of vaccine for Clostridium difficile infection using membrane fraction of nontoxigenic Clostridium difficile.

Although standard antibiotic therapy is performed for diarrhea and pseudomembranous colitis caused by Clostridium difficile, a high recurrence rate of C. difficile infection (CDI) remains a major problem. We previously showed that a membrane fraction of nontoxigenic C. difficile (ntCDMF) was effective as a vaccine antigen by in vitro experiments. In this study, we examined whether ntCDMF had an in vivo effect in animal challenge experiments. By intrarectal immunization with ntCDMF, the number of C. difficile cells in feces of mice was decreased approximately 99% compared to the control mice. In addition, survival rate of C. difficile-challenged hamsters was increased almost 30% by immunization with ntCDMF. These results showed that ntCDMF could be a practical vaccine candidate.

The pyrin inflammasome: from sensing RhoA GTPases-inhibiting toxins to triggering autoinflammatory syndromes.

Numerous pathogens including Clostridium difficile and Yersinia pestis have evolved toxins or effectors targeting GTPases from the RhoA subfamily (RhoA/B/C) to inhibit or hijack the host cytoskeleton dynamics. The resulting impairment of RhoA GTPases activity is sensed by the host via an innate immune complex termed the pyrin inflammasome in which caspase-1 is activated. The cascade leading to activation of the pyrin inflammasome has been recently uncovered. In this review, following a brief presentation of RhoA GTPases-modulating toxins, we present the pyrin inflammasome and its regulatory mechanisms. Furthermore, we discuss how some pathogens have developed strategies to escape detection by the pyrin inflammasome. Finally, we present five monogenic autoinflammatory diseases associated with pyrin inflammasome deregulation. The molecular insights provided by the study of these diseases and the corresponding mutations on pyrin inflammasome regulation and activation are presented.

Detection of chicken interleukin-10 production in intestinal epithelial cells and necrotic enteritis induced by Clostridium perfringens using capture ELISA.

AIMS: To assess the production level of interleukin (IL)-10 inClostridium perfringens (CP)-stimulated intestinal epithelial cells (IECs) and CP-infected chickens using an antigen capture ELISA developed by mouse monoclonal antibodies against chicken IL-10. Also, to investigate which CP toxins induced IL-10 in chicken IECs stimulated with CP. METHODS AND RESULTS: Chicken IECs were stimulated with CP toxin in the absence or presence of antibodies (α-toxin or NetB) for 18 h. Expression of chicken IL-10 and IL-6 was monitored by quantitative real-time polymerase chain reaction. Serum and jejunum samples were collected from CP-infected chickens at 9 days post-infection and expression of IL-10 and IL-6 was analyzed. IL-10 production was detected by antigen capture ELISA in chicken IECs stimulated with CP and in serum samples collected from CP-infected birds. The mRNA levels were consistent with the results of antigen capture ELISA. CONCLUSION: CP induced IL-10 production in chicken IECs. Increased IL-10 production was detected in CP-stimulated chicken IECs and in serum collected from CP-infected birds, using antigen capture ELISA. Antigen capture ELISA could be a useful tool to monitor IL-10 production in chicken disease.

Faecal microbiota transplantation in patients with Clostridium difficile and significant comorbidities as well as in patients with new indications: A case series.

Fecal microbiota transplantation (FMT) is effective in recurrent Clostridium difficile infection (rCDI). Knowledge of the safety and efficacy of FMT treatment in immune deficient patients is scarce. FMT has been suggested as a potential method for an increasing number of new indications besides rCDI. Among our FMT-treated rCDI patients, we reviewed those with major comorbidities: two human immunodeficiency virus patients, six haemodialysis patients, two kidney transplant patients, two liver transplant patients and a patient with chronic lymphatic leukaemia. We also reviewed those treated with FMT for indications other than rCDI: Salmonella carriage (two patients), trimethylaminuria (two patients), small intestinal bacterial overgrowth (SIBO; one patient), and lymphocytic colitis (one patient), as well as a common variable immunodeficiency patient with chronic norovirus infection and ESBL-producing Escherichia coli (E. coli) carriage. Of the thirteen rCDI patients treated with FMT, eleven cleared the CDI. The observed adverse events were not directly attributable to FMT. Concerning the special indications, both Salmonellas and ESBL-producing E. coli were eradicated. One trimethylaminuria patient and one SIBO-patient reported a reduction of symptoms. Three patients did not experience a benefit from FMT: chronic norovirus, lymphocytic colitis and the other fish malodour syndrome. There were no reported side effects in this group. FMT appeared to be safe and effective for immunocompromised patients with rCDI. FMT showed promise for the eradication of antibiotic-resistant bacteria, but further research is warranted.

Clostridium difficile flagellin FliC: Evaluation as adjuvant and use in a mucosal vaccine against Clostridium difficile.

The immunogenicity of bacterial flagellin has been reported in different studies. By its close interaction with the immune system, the flagellin represents an interesting adjuvant and vaccine candidate. Salmonella Typhimurium flagellin has already been tested as adjuvant to stimulate mucosal immunity. Here, we assessed the ability of Clostridium difficile flagellin FliC to act as a mucosal adjuvant, first combined with ovalbumin as antigen and second with a C. difficile surface protein, the precursor of the S-layer proteins SlpA. Using ovalbumin as antigen, we compared the gut mucosal adjuvanticity of FliC to Salmonella Typhimurium flagellin and cholera toxin. Two routes of immunization were tested in a mouse model: intra-rectal and intra-peritoneal, following which, gut mucosal and systemic antibody responses against ovalbumin (Immunoglobulins G and Immunoglobulins A) were analyzed by Enzyme-Linked Immuno Assay in intestinal contents and in sera. In addition, ovalbumin-specific immunoglobulin producing cells were detected in the intestinal lamina propria by Enzyme-Linked Immunospot. Results showed that FliC as adjuvant for immunization targeting ovalbumin was able to stimulate a gut mucosal and systemic antibody response independently of the immunization route. In order to develop a mucosal vaccine to prevent C. difficile intestinal colonization, we assessed in a mouse model the efficacy of FliC as adjuvant compared with cholera toxin co-administrated with the C. difficile S-layer precursor SlpA as antigen. After challenge, a significant decrease of C. difficile intestinal colonization was observed in immunized groups compared to the control group. Our results showed that C. difficile FliC could be used as adjuvant in mucosal vaccination strategy against C. difficile infections.

Clostridium perfringens beta toxin DNA prime-protein boost elicits enhanced protective immune response in mice.

Clostridium perfringens beta toxin (CPB) is the primary pathogenic factor responsible for necrotic enteritis in sheep, cattle and humans. Owing to rapid progression of the disease, vaccination is the only possible recourse to avoid high mortality in animal farms and huge economic losses. The present study reports evaluation of a cpb gene-based DNA vaccine encoding the beta toxin of C. perfringens with homologous as well as heterologous booster strategy. Immunization strategy employing heterologous booster with heat-inactivated rCPB mounted stronger immune response when compared to that generated by homologous booster. Antibody isotyping and cytokine ELISA demonstrated the immune response to be Th1-biased mixed immune response. While moderate protection of immunized BALB/c and C57BL/6 mice against rCPB challenge was observed with homologous booster strategy, heterologous booster strategy led to complete protection. Thus, beta toxin-based DNA vaccine using the heterologous prime-boosting strategy was able to generate better immune response and conferred greater degree of protection against high of dose rCPB challenge than homologous booster regimen, making it an effective vaccination approach against C. perfringens beta toxin.

Outcomes of Fecal Microbiota Transplantation for Clostridium difficile Infection in Patients with Inflammatory Bowel Disease.

BACKGROUND AND AIMS: Fecal microbiota transplantation (FMT) has recently been shown to be a promising therapy for recurrent and refractory Clostridium difficile infections (CDI) despite lack of protocol standardization. Patients with inflammatory bowel disease (IBD) present a particular challenge to CDI therapy as they are reported to have worse clinical outcomes, including higher colectomy rates and increased mortality. We aimed to assess the outcomes of FMT for recurrent CDI in patients with IBD at our healthcare system. METHODS: We constructed a retrospective cohort of all patients who underwent FMT at our healthcare system between December 2012 and May 2014. Patients with concurrent IBD were identified. We evaluated the differences in demographic and clinical characteristics, along with the outcomes to FMT between patients with IBD as compared to the general population. RESULTS: Over the study period, 201 patients underwent FMT of which 20 patients had concurrent IBD. Patients with IBD were younger but did not differ from the general population in terms of CDI risk factors or disease severity. The response to FMT and rate of CDI relapse in the IBD group were not statistically different compared to the rest of the cohort. The overall response rate in the IBD population was 75% at 12 weeks. Of the patients who failed FMT 4 of 5 patients had active or untreated IBD. CONCLUSION: Fecal microbiota transplantation provides a good alternative treatment option with high success rates for recurrent or refractory Clostridium difficile infection in patients with well-controlled IBD who fail standard antimicrobial therapy.

Immunotropic aspect of the Bacillus coagulans probiotic action.

OBJECTIVES: Currently, probiotics are increasingly used as the alternative to antibiotics as well as the preventive measures in humans. In particular, probiotics occupy a key position in the treatment of antibiotics-associated intestinal dysbiosis. A spore-forming microorganism lactobacillus Bacillus coagulans is one of the most promising probiotics. However, some of its pharmacological effects remain poorly understood. This study was aimed at investigation of the effect of B. coagulans (Laktovit Forte) on the intestinal dysbiosis syndrome in mice caused by streptomycin against the background of cyclophosphamide-induced cellular immunodeficiency. METHODS: Pharmacological method: mouse model in vivo with immunodeficiency caused by cyclophosphamide. KEY FINDINGS: In mice with colitis caused by streptomycin treatment, the administration of B. coagulans (Laktovit Forte medicinal product) resulted in an antidiarrhoeal effect, normalisation of gastrointestinal motility and prevention of the animals' weight loss. Given the cyclophosphamide-induced immunosuppression and streptomycin-associated diarrhoea, the immunity was completely restored only under the action of B. coagulans. CONCLUSIONS: According to all parameters, B. coagulans has been proved to be more effective as compared to the Linex Forte reference product containing lacto- and bifidobacteria.