Variants at the MHC Region Associate With Susceptibility to Clostridioides difficile Infection: A Genome-Wide Association Study Using Comprehensive Electronic Health Records.

Background: Clostridioides difficile is a major cause of healthcare-associated and community-acquired diarrhea. Host genetic susceptibility to Clostridioides difficile infection has not been studied on a large-scale. Methods: A total of 1,160 Clostridioides difficile infection cases and 15,304 controls were identified by applying the eMERGE Clostridioides difficile infection algorithm to electronic health record data. A genome-wide association study was performed using a linear mixed model, adjusted for significant covariates in the full dataset and the antibiotic subgroup. Colocalization and MetaXcan were performed to identify potential target genes in Clostridioides difficile infection - relevant tissue types. Results: No significant genome-wide association was found in the meta-analyses of the full Clostridioides difficile infection dataset. One genome-wide significant variant, rs114751021, was identified (OR = 2.42; 95%CI = 1.84-3.11; p=4.50 x 10-8) at the major histocompatibility complex region associated with Clostridioides difficile infection in the antibiotic group. Colocalization and MetaXcan identified MICA, C4A/C4B, and NOTCH4 as potential target genes. Down-regulation of MICA, upregulation of C4A and NOTCH4 was associated with a higher risk for Clostridioides difficile infection. Conclusions: Leveraging the EHR and genetic data, genome-wide association, and fine-mapping techniques, this study identified variants and genes associated with Clostridioides difficile infection, provided insights into host immune mechanisms, and described the potential for novel treatment strategies for Clostridioides difficile infection. Future replication and functional validation are needed.

The glucosyltransferase activity of C. difficile Toxin B is required for disease pathogenesis.

Enzymatic inactivation of Rho-family GTPases by the glucosyltransferase domain of Clostridioides difficile Toxin B (TcdB) gives rise to various pathogenic effects in cells that are classically thought to be responsible for the disease symptoms associated with C. difficile infection (CDI). Recent in vitro studies have shown that TcdB can, under certain circumstances, induce cellular toxicities that are independent of glucosyltransferase (GT) activity, calling into question the precise role of GT activity. Here, to establish the importance of GT activity in CDI disease pathogenesis, we generated the first described mutant strain of C. difficile producing glucosyltransferase-defective (GT-defective) toxin. Using allelic exchange (AE) technology, we first deleted tcdA in C. difficile 630Δerm and subsequently introduced a deactivating D270N substitution in the GT domain of TcdB. To examine the role of GT activity in vivo, we tested each strain in two different animal models of CDI pathogenesis. In the non-lethal murine model of infection, the GT-defective mutant induced minimal pathology in host tissues as compared to the profound caecal inflammation seen in the wild-type and 630ΔermΔtcdA (ΔtcdA) strains. In the more sensitive hamster model of CDI, whereas hamsters in the wild-type or ΔtcdA groups succumbed to fulminant infection within 4 days, all hamsters infected with the GT-defective mutant survived the 10-day infection period without primary symptoms of CDI or evidence of caecal inflammation. These data demonstrate that GT activity is indispensable for disease pathogenesis and reaffirm its central role in disease and its importance as a therapeutic target for small-molecule inhibition.

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.

The xenobiotic sensing pregnane X receptor regulates tissue damage and inflammation triggered by C difficile toxins.

Clostridioides difficile (formerly Clostridium difficile; C difficile), the leading cause of nosocomial antibiotic-associated colitis and diarrhea in the industrialized world, triggers colonic disease through the release two toxins, toxin A (TcdA) and toxin B (TcdB), glucosyltransferases that modulate monomeric G-protein function and alter cytoskeletal function. The initial degree of the host immune response to C difficile and its pathogenic toxins is a common indicator of disease severity and infection recurrence. Thus, targeting the intestinal inflammatory response during infection could significantly decrease disease morbidity and mortality. In the current study, we sought to interrogate the influence of the pregnane X receptor (PXR), a modulator of xenobiotic and detoxification responses, which can sense and respond to microbial metabolites and modulates inflammatory activity, during exposure to TcdA and TcdB. Following intrarectal exposure to TcdA/B, PXR-deficient mice (Nr1i2-/- ) exhibited reduced survival, an effect that was associated with increased levels of innate immune cell influx. This exacerbated response was associated with a twofold increase in the expression of Tlr4. Furthermore, while broad-spectrum antibiotic treatment (to deplete the intestinal microbiota) did not alter the responses in Nr1i2-/- mice, blocking TLR4 signaling significantly reduced TcdA/B-induced disease severity and immune responses in these mice. Lastly, to assess the therapeutic potential of targeting the PXR, we activated the PXR with pregnenolone 16α-carbonitrile (PCN) in wild-type mice, which greatly reduced the severity of TcdA/B-induced damage and intestinal inflammation. Taken together, these data suggest that the PXR plays a role in the host's response to TcdA/B and may provide a novel target to dampen the inflammatory tissue damage in C difficile infections.

Klebsiella oxytoca enterotoxins tilimycin and tilivalline have distinct host DNA-damaging and microtubule-stabilizing activities.

Establishing causal links between bacterial metabolites and human intestinal disease is a significant challenge. This study reveals the molecular basis of antibiotic-associated hemorrhagic colitis (AAHC) caused by intestinal resident Klebsiella oxytoca Colitogenic strains produce the nonribosomal peptides tilivalline and tilimycin. Here, we verify that these enterotoxins are present in the human intestine during active colitis and determine their concentrations in a murine disease model. Although both toxins share a pyrrolobenzodiazepine structure, they have distinct molecular targets. Tilimycin acts as a genotoxin. Its interaction with DNA activates damage repair mechanisms in cultured cells and causes DNA strand breakage and an increased lesion burden in cecal enterocytes of colonized mice. In contrast, tilivalline binds tubulin and stabilizes microtubules leading to mitotic arrest. To our knowledge, this activity is unique for microbiota-derived metabolites of the human intestine. The capacity of both toxins to induce apoptosis in intestinal epithelial cells-a hallmark feature of AAHC-by independent modes of action, strengthens our proposal that these metabolites act collectively in the pathogenicity of colitis.

The predictive value of quantitative nucleic acid amplification detection of Clostridium difficile toxin gene for faecal sample toxin status and patient outcome.

BACKGROUND: Laboratory diagnosis of Clostridium difficile infection (CDI) remains unsettled, despite updated guidelines. We investigated the potential utility of quantitative data from a nucleic acid amplification test (NAAT) for C. difficile toxin gene (tg) for patient management. METHODS: Using data from the largest ever C. difficile diagnostic study (8853 diarrhoeal samples from 7335 patients), we determined the predicative value of C. difficile tgNAAT (Cepheid Xpert C.diff) low cycle threshold (CT) value for patient toxin positive status, CDI severity, mortality and CDI recurrence. Reference methods for CDI diagnosis were cytotoxicity assay (CTA) and cytotoxigenic culture (CTC). RESULTS: Of 1281 tgNAAT positive faecal samples, 713 and 917 were CTA and CTC positive, respectively. The median tgNAAT CT for patients who died was 25.5 vs 27.5 for survivors (p = 0.021); for toxin-positivity was 24.9 vs 31.6 for toxin-negative samples (p<0.001) and for patients with a recurrence episode was 25.6 vs 27.3 for those who did not have a recurrent episode (p = 0.111). Following optimal cut-off determination, low CT was defined as ≤25 and was significantly associated with a toxin-positive result (P<0.001, positive predictive value 83.9%), presence of PCR-ribotype 027 (P = 0.025), and mortality (P = 0.032). Recurrence was not associated with low CT (p 0.111). CONCLUSIONS: Low tgNAAT CT could indicate CTA positive patients, have more severe infection, increased risk of mortality and possibly recurrence. Although, the limited specificity of tgNAAT means it cannot be used as a standalone test, it could augment a more timely diagnosis, and optimise management of these at-risk patients.

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.

Identification of Clostridium difficile Immunoreactive Spore Proteins of the Epidemic Strain R20291.

PURPOSE: Clostridium difficile infections are the leading cause of diarrhea associated with the use of antibiotics. During infection, C. difficile initiates a sporulation cycle leading to the persistence of C. difficile spores in the host and disease dissemination. The development of vaccine and passive immunization therapies against C. difficile has focused on toxins A and B. In this study, an immunoproteome-based approach to identify immunogenic proteins located on the outer layers of C. difficile spores as potential candidates for the development of immunotherapy and/or diagnostic methods against this devastating infection is used. EXPERIMENTAL DESIGN: To identify potential immunogenic proteins on the surface of C. difficile R20291, spore coat/exosporium extracts are separated by 2D electrophoresis (2-DE) and analyzed for reactivity against C. difficile spore-specific goat sera. Finally, the selected spots are in-gel digested with chymotrypsin, peptides generated are separated by nanoUPLC followed by MS/MS using Quad-TOF-MS, corroborated by Ultimate 3000RS-nano-UHPLC coupled to Q-Exactive-Plus-Orbitrap MS. RESULTS: The analysis identify five immunoreactive proteins: spore coat proteins CotE, CotA, and CotCB; exosporium protein CdeC; and a cytosolic methyltransferase. CONCLUSION: This data provides a list of spore surface protein candidates as antigens for vaccine development against C. difficile infections.

More than 50% of Clostridium difficile Isolates from Pet Dogs in Flagstaff, USA, Carry Toxigenic Genotypes.

Nosocomial acquisition of Clostridium difficile is well documented, yet recent studies have highlighted the importance of community acquired infections and identified community associated reservoirs for this pathogen. Multiple studies have implicated companion pets and farm animals as possible sources of community acquired C. difficile infections in humans. To explore the potential role of pet dogs in human C. difficile infections we systematically collected canine fecal samples (n = 197) in Flagstaff, AZ. Additionally, nineteen fecal samples were collected at a local veterinary clinic from diarrheic dogs. We used these combined samples to investigate important questions regarding C. difficile colonization in pet canines: 1) What is the prevalence and diversity of C. difficile in this companion pet population, and 2) Do C. difficile isolates collected from canines genetically overlap with isolates that cause disease in humans? We used a two-step sequence typing approach, including multilocus sequence typing to determine the overall genetic diversity of C. difficile present in Flagstaff canines, and whole-genome sequencing to assess the fine-scale diversity patterns within identical multilocus sequence types from isolates obtained within and among multiple canine hosts. We detected C. difficile in 17% of the canine fecal samples with 10% containing toxigenic strains that are known to cause human disease. Sequencing analyses revealed similar genotypes in dogs and humans. These findings suggest that companion pets are a potential source of community acquired C. difficile infections in humans.

[A NOVEL APPROACH TO GENOTYPING OF HOSPITAL ISOLATES OF CLOSTRIDIUM DIFFICILE].

AIM: Development of a novel approach in genotyping of Clostridium difficile and its testing on the example of 140 hospital isolates. MATERIALS AND METHODS: The approach is based on an idea of double digest and selective label (DDSL), used previously during genotyping of other bacterial pathogens. Selection of optimal enzymes for restriction of MluI and Mph1103I was carried out, condition of DDSL reaction execution were optimized. RESULTS: Genotyping of C. difficile hospital isolates was carried out, index of strain discrimination was calculated, conclusions regarding possibilities of the method in elucidation of spread pathways and identification of infection sources were made. CONCLUSION: The developed method of genotyping has a number of advantages over the existing method and can be used to'address issues in epidemiology of infections caused by C. difficile.

The Clostridium difficile Dlt Pathway Is Controlled by the Extracytoplasmic Function Sigma Factor σV in Response to Lysozyme.

Clostridium difficile (also known as Peptoclostridium difficile) is a major nosocomial pathogen and a leading cause of antibiotic-associated diarrhea throughout the world. Colonization of the intestinal tract is necessary for C. difficile to cause disease. Host-produced antimicrobial proteins (AMPs), such as lysozyme, are present in the intestinal tract and can deter colonization by many bacterial pathogens, and yet C. difficile is able to survive in the colon in the presence of these AMPs. Our prior studies established that the Dlt pathway, which increases the surface charge of the bacterium by addition of d-alanine to teichoic acids, is important for C. difficile resistance to a variety of AMPs. We sought to determine what genetic mechanisms regulate expression of the Dlt pathway. In this study, we show that a dlt null mutant is severely attenuated for growth in lysozyme and that expression of the dltDABC operon is induced in response to lysozyme. Moreover, we found that a mutant lacking the extracytoplasmic function (ECF) sigma factor σ(V) does not induce dlt expression in response to lysozyme, indicating that σ(V) is required for regulation of lysozyme-dependent d-alanylation of the cell wall. Using reporter gene fusions and 5' RACE (rapid amplification of cDNA ends) analysis, we identified promoter elements necessary for lysozyme-dependent and lysozyme-independent dlt expression. In addition, we observed that both a sigV mutant and a dlt mutant are more virulent in a hamster model of infection. These findings demonstrate that cell wall d-alanylation in C. difficile is induced by lysozyme in a σ(V)-dependent manner and that this pathway impacts virulence in vivo.

Analysis of TcdB Proteins within the Hypervirulent Clade 2 Reveals an Impact of RhoA Glucosylation on Clostridium difficile Proinflammatory Activities.

Clostridium difficile strains within the hypervirulent clade 2 are responsible for nosocomial outbreaks worldwide. The increased pathogenic potential of these strains has been attributed to several factors but is still poorly understood. During a C. difficile outbreak, a strain from this clade was found to induce a variant cytopathic effect (CPE), different from the canonical arborizing CPE. This strain (NAP1V) belongs to the NAP1 genotype but to a ribotype different from the epidemic NAP1/RT027 strain. NAP1V and NAP1 share some properties, including the overproduction of toxins, the binary toxin, and mutations in tcdC. NAP1V is not resistant to fluoroquinolones, however. A comparative analysis of TcdB proteins from NAP1/RT027 and NAP1V strains indicated that both target Rac, Cdc42, Rap, and R-Ras but only the former glucosylates RhoA. Thus, TcdB from hypervirulent clade 2 strains possesses an extended substrate profile, and RhoA is crucial for the type of CPE induced. Sequence comparison and structural modeling revealed that TcdBNAP1 and TcdBNAP1V share the receptor-binding and autoprocessing activities but vary in the glucosyltransferase domain, consistent with the different substrate profile. Whereas the two toxins displayed identical cytotoxic potencies, TcdBNAP1 induced a stronger proinflammatory response than TcdBNAP1V as determined in ex vivo experiments and animal models. Since immune activation at the level of intestinal mucosa is a hallmark of C. difficile-induced infections, we propose that the panel of substrates targeted by TcdB is a determining factor in the pathogenesis of this pathogen and in the differential virulence potential seen among C. difficile strains.

Clostridium Difficile Colonization in Hematopoietic Stem Cell Transplant Recipients: A Prospective Study of the Epidemiology and Outcomes Involving Toxigenic and Nontoxigenic Strains.

Clostridium difficile is a leading cause of infectious diarrhea in hematopoietic stem cell transplant (HSCT) recipients. Asymptomatic colonization of the gastrointestinal tract occurs before development of C. difficile infection (CDI). This prospective study examines the rates, risk factors, and outcomes of colonization with toxigenic and nontoxigenic strains of C. difficile in HSCT patients. This 18-month study was conducted in the HSCT unit at the Karmanos Cancer Center and Wayne State University in Detroit. Stool samples from the patients who consented for the study were taken at admission and weekly until discharge. Anaerobic culture for C. difficile and identification of toxigenic strains by PCR were performed on the stool samples. Demographic information and clinical and laboratory data were collected. Of the 150 patients included in the study, 29% were colonized with C. difficile at admission; 12% with a toxigenic strain and 17% with a nontoxigenic strain. Over a 90-day follow-up, 12 of 44 (26%) patients colonized with any C. difficile strain at admission developed CDI compared with 13 of 106 (12%) of patients not colonized (odds ratio [OR], 2.70; 95% confidence interval [95% CI], 1.11 to 6.48; P = .025). Eleven of 18 (61%) patients colonized with the toxigenic strain and 1 of 26 (4%) of those colonized with nontoxigenic strain developed CDI (OR, 39.30; 95% CI, 4.30 to 359.0; P < .001) at a median of 12 days. On univariate and multivariate analyses, none of the traditional factors associated with high risk for C. difficile colonization or CDI were found to be significant. Recurrent CDI occurred in 28% of cases. Asymptomatic colonization with C. difficile at admission was high in our HSCT population. Colonization with toxigenic C. difficile was predictive of CDI, whereas colonization with a nontoxigenic C. difficile appeared protective. These findings may have implications for infection control strategies and for novel approaches for the prevention and preemptive treatment of CDI in the HSCT patient population.

Regulation of Clostridium difficile Spore Formation by the SpoIIQ and SpoIIIA Proteins.

Sporulation is an ancient developmental process that involves the formation of a highly resistant endospore within a larger mother cell. In the model organism Bacillus subtilis, sporulation-specific sigma factors activate compartment-specific transcriptional programs that drive spore morphogenesis. σG activity in the forespore depends on the formation of a secretion complex, known as the "feeding tube," that bridges the mother cell and forespore and maintains forespore integrity. Even though these channel components are conserved in all spore formers, recent studies in the major nosocomial pathogen Clostridium difficile suggested that these components are dispensable for σG activity. In this study, we investigated the requirements of the SpoIIQ and SpoIIIA proteins during C. difficile sporulation. C. difficile spoIIQ, spoIIIA, and spoIIIAH mutants exhibited defects in engulfment, tethering of coat to the forespore, and heat-resistant spore formation, even though they activate σG at wildtype levels. Although the spoIIQ, spoIIIA, and spoIIIAH mutants were defective in engulfment, metabolic labeling studies revealed that they nevertheless actively transformed the peptidoglycan at the leading edge of engulfment. In vitro pull-down assays further demonstrated that C. difficile SpoIIQ directly interacts with SpoIIIAH. Interestingly, mutation of the conserved Walker A ATP binding motif, but not the Walker B ATP hydrolysis motif, disrupted SpoIIIAA function during C. difficile spore formation. This finding contrasts with B. subtilis, which requires both Walker A and B motifs for SpoIIIAA function. Taken together, our findings suggest that inhibiting SpoIIQ, SpoIIIAA, or SpoIIIAH function could prevent the formation of infectious C. difficile spores and thus disease transmission.

Recombinant lipoprotein-based vaccine candidates against C. difficile infections.

BACKGROUND: Opportunistically nosocomial infections in hospitalized patients are often related to Clostridium difficile infections (CDI) due to disruption of the intestinal micro-flora by antibiotic therapies during hospitalization. Clostridial exotoxins A and B (TcdA and TcdB) specifically bind to unknown glycoprotein(s) in the host intestine, disrupt the intestinal barrier leading to acute inflammation and diarrhea. The C-terminal receptor binding domain of TcdA (A-rRBD) has been shown to elicit antibody responses that neutralize TcdA toxicity in Vero cell cytotoxicity assays, but not effectively protect hamsters against a lethal dose challenge of C. difficile spores. To develop an effective recombinant subunit vaccine against CDI, A-rRBD was lipidated (rlipoA-RBD) as a rational design to contain an intrinsic adjuvant, a toll-like receptor 2 agonist and expressed in Escherichia coli. RESULTS: The purified rlipoA-RBD was characterized immunologically and found to have the following properties: (a) mice, hamsters and rabbits vaccinated with 3 µg of rlipoA-RBD produced strong antibody responses that neutralized TcdA toxicity in Vero cell cytotoxicity assays; furthermore, the neutralization titer was comparable to those obtained from antisera immunized either with 10 µg of TcdA toxoid or 30 µg of A-rRBD; (b) rlipoA-RBD elicited immune responses and protected mice from TcdA challenge, but offered insignificant protection (10 to 20 %) against C. difficile spores challenge in hamster models; (c) only rlipoA-RBD formulated with B-rRBD consistently confers protection (90 to 100 %) in the hamster challenge model; and (d) rlipoA-RBD was found to be 10-fold more potent than A-rRBD as an adjuvant to enhancing immune responses against a poor antigen such as ovalbumin. CONCLUSION: These results indicate that rlipoA-RBD formulated with B-rRBD could be an excellent vaccine candidate for preclinical studies and future clinical trials.

The use of pulsed-field gel electrophoresis for genotyping of Clostridium difficile.

Genotyping approaches are important for tracking infectious agents and can be used for various purposes. Pulsed-Field Gel Electrophoresis (PFGE) is among the highly discriminatory genotyping approaches commonly used for characterizing Clostridium difficile. Other genotyping methods used for C. difficile include Ribotyping, Restriction Endonuclease Assay (REA), Multilocus Variable Number Tandem Repeats (VNTR) Assay, and others. PFGE has a high discriminatory power, high reproducibility, and typeability. We utilized PFGE for typing C. difficile isolates of porcine and human origin. We used a macrorestriction fragment analysis of an intact genomic DNA using SmaI, a rare cutting restriction endonuclease. Using a Contour-Clamped Homogeneous Electric Field (CHEF) system with running conditions of 120° angle; initial switch time of 5 s; final switch time of 40 s with a run time of 18 h in a low-melting temperature agarose (Seakem Gold); and 0.5× TBE circulated in the CHEF system at 6 V/cm [CDC (2014) Pulsenet. http://www.cdc.gov/pulsenet/index.html . Accessed 22 Aug 2014] supported by 14 °C cooling module, we were able to separate very large DNA fragments (up to 2 Mb).

Complete genome sequence of the Clostridium difficile laboratory strain 630Δerm reveals differences from strain 630, including translocation of the mobile element CTn5.

BACKGROUND: Clostridium difficile strain 630Δerm is a spontaneous erythromycin sensitive derivative of the reference strain 630 obtained by serial passaging in antibiotic-free media. It is widely used as a defined and tractable C. difficile strain. Though largely similar to the ancestral strain, it demonstrates phenotypic differences that might be the result of underlying genetic changes. Here, we performed a de novo assembly based on single-molecule real-time sequencing and an analysis of major methylation patterns. RESULTS: In addition to single nucleotide polymorphisms and various indels, we found that the mobile element CTn5 is present in the gene encoding the methyltransferase rumA rather than adhesin CD1844 where it is located in the reference strain. CONCLUSIONS: Together, the genetic features identified in this study may help to explain at least part of the phenotypic differences. The annotated genome sequence of this lab strain, including the first analysis of major methylation patterns, will be a valuable resource for genetic research on C. difficile.

The role of Gr-1(+) cells and tumour necrosis factor-α signalling during Clostridium difficile colitis in mice.

The host response to Clostridium difficile infection in antibiotic-treated mice is characterized by robust recruitment of Gr-1(+) cells, increased expression of inflammatory cytokines including tumour necrosis factor-α (TNF-α), and the development of severe epithelial damage. To investigate the role of Gr-1(+) cells and TNF-α during C. difficile colitis, we treated infected mice with monoclonal antibodies against Gr-1 or TNF-α. Mice were challenged with vegetative cells of C. difficile strain VPI 10463 following treatment with the third-generation cephalosporin ceftriaxone. Ceftriaxone treatment alone was associated with significant changes in cytokine expression within the colonic mucosa but not overt inflammatory histopathological changes. In comparison, C. difficile infection following ceftriaxone treatment was associated with increased expression of inflammatory cytokines and chemokines including Cxcl1, Cxcl2, Il1b, Il17f and Tnfa, as well as robust recruitment of Ly6C(Mid)  Gr-1(High) neutrophils and Ly6C(High) Gr-1(Mid) monocytes and the development of severe colonic histopathology. Anti-Gr-1 antibody treatment resulted in effective depletion of both Ly6C(Mid) Gr-1(High) neutrophils and Ly6C(High) Gr-1(Mid) monocytes: however, we observed no protection from the development of severe pathology or reduction in expression of the pro-inflammatory cytokines Il1b, Il6, Il33 and Tnfa following anti-Gr-1 treatment. By contrast, anti-TNF-α treatment did not affect Gr-1(+) cell recruitment, but was associated with increased expression of Il6 and Il1b. Additionally, Ffar2, Ffar3, Tslp, Tff and Ang4 expression was significantly reduced in anti-TNF-α-treated animals, in association with marked intestinal histopathology. These studies raise the possibility that TNF-α may play a role in restraining inflammation and protecting the epithelium during C. difficile infection.

The molecular basis of Clostridium difficile disease and host response.

PURPOSE OF REVIEW: Clostridium difficile infection (CDI) ranges from asymptomatic colonization to severe colitis and death. The physiologic and molecular mechanisms determining disease outcome are thus far poorly understood. Here, we review recent advances in the relationship between host response to infection and disease outcome. Furthermore, we review recent studies on the relationship between intestinal microbial ecology and pathogenesis of CDI. RECENT FINDINGS: Severe CDI is characterized by toxin-induced epithelial injury and marked intestinal inflammation. Recent studies demonstrate that systemic markers of inflammation correlate with disease outcome. Peripheral neutrophil count, C-reactive protein, and proinflammatory cytokines are elevated in patients with severe disease as compared with asymptomatic controls. Furthermore, fecal inflammatory biomarkers are better predictors of disease severity and diarrhea persistence than C. difficile abundance. A landmark study reported higher than 80% success rate of fecal microbiota transplantation for treatment of recurrent CDI. The commensal microbes responsible for C. difficile protection, and the molecular basis by which microbial ecology impacts disease outcome, are under active investigation. SUMMARY: Under conditions of altered microbial ecology, C. difficile incites epithelial injury and marked intestinal inflammation, the primary determinant of disease outcome. Restoration of a diverse intestinal microbial population by fecal microbiota transplantation attenuates disease and prevents recurrence by mechanisms that are yet to be fully elucidated.

Interleukin-22 and CD160 play additive roles in the host mucosal response to Clostridium difficile infection in mice.

Our previous work has shown the significant up-regulation of Il22 and increased phosphorylation of signal transducer and activator of transcription 3 (STAT3) as part of the mucosal inflammatory response to Clostridium difficile infection in mice. Others have shown that phosphorylation of STAT3 at mucosal surfaces includes interleukin-22 (IL-22) and CD160-mediated components. The current study sought to determine the potential role(s) of IL-22 and/or CD160 in the mucosal response to C. difficile infection. Clostridium difficile-infected mice treated with anti-IL-22, anti-CD160 or a combination of the two showed significantly reduced STAT3 phosphorylation in comparison to C. difficile-infected mice that had not received either antibody. In addition, C. difficile-infected mice treated with anti-IL-22/CD160 induced a smaller set of genes, and at significantly lower levels than the untreated C. difficile-infected mice. The affected genes included pro-inflammatory chemokines and cytokines, and anti-microbial peptides. Furthermore, histopathological and flow cytometric assessments both showed a significantly reduced influx of neutrophils in C. difficile-infected mice treated with anti-IL-22/CD160. These data demonstrate that IL-22 and CD160 are together responsible for a significant fraction of the colonic STAT3 phosphorylation in C. difficile infection. They also underscore the additive effects of IL-22 and CD160 in mediating both the pro-inflammatory and pro-survival aspects of the host mucosal response in this infection.