Toxigenic C. difficile induced inflammatory marker expression by human intestinal epithelial cells is asymmetrical

Clostridium difficile infection of the intestinal epithelium and consequent pseudomembranous colitis is an important cause of morbidity and mortality. Pathogenesis has been ascribed exclusively to toxin production. Using in vitro models of human intestinal epithelial layers, we show that exposure to toxigenic C. difficile upregulates epithelial expression of IL-8 and ICAM-1, two molecules important in neutrophil chemoattraction and adhesion and subsequent inflammation. IL-8 production was also stimulated by toxin-containing supernatants. C difficile induced IL-8 release was inhibited by specific antiserum. Increased ICAM-1 expression only occurred after basolateral exposure to C. difficile while apical exposure had no effect on ICAM-1 expression. However, transepithelial electrical resistance was impaired by apical exposure to bacterial suspensions. We suggest that apical exposure to C. difficile induces changes in epithelial layer integrity which allows the bacteria and/or the toxin access to the basolateral compartment where pathogenic inflammatory mechanisms are activated.     

Protective Role of Commensals against Clostridium difficile Infection via an IL-1β-Mediated Positive-Feedback Loop

Clostridium difficile is a Gram-positive obligate anaerobic pathogen that causes pseudomembranous colitis in antibiotic-treated individuals. Commensal bacteria are known to have a significant role in the intestinal accumulation of C. difficile after antibiotic treatment, but little is known about how they affect host immunity during C. difficile infection. In this article, we report that C. difficile infection results in translocation of commensals across the intestinal epithelial barrier that is critical for neutrophil recruitment through the induction of an IL-1β-mediated positive-feedback loop. Mice lacking ASC, an essential mediator of IL-1β and IL-18 processing and secretion, were highly susceptible to C. difficile infection. ASC(-/-) mice exhibited enhanced translocation of commensals to multiple organs after C. difficile infection. Notably, ASC(-/-) mice exhibited impaired CXCL1 production and neutrophil influx into intestinal tissues in response to C. difficile infection. The impairment in neutrophil recruitment resulted in reduced production of IL-1β and CXCL1 but not IL-18. Importantly, translocated commensals were required for ASC/Nlrp3-dependent IL-1β secretion by neutrophils. Mice lacking IL-1β were deficient in inducing CXCL1 secretion, suggesting that IL-1β is the dominant inducer of ASC-mediated CXCL1 production during C. difficile infection. These results indicate that translocated commensals play a crucial role in CXCL1-dependent recruitment of neutrophils to the intestine through an IL-1β/NLRP3/ASC-mediated positive-feedback mechanism that is important for host survival and clearance of translocated commensals during C. difficile infection.     

Induction of cytokines in a macrophage cell line by proteins of Clostridium difficile

Clostridium difficile is a major cause of nosocomial diarrhoea. The toxins produced by C. difficile are responsible for the characteristic pathology observed in C. difficile disease, but several surface-associated proteins of C. difficile are also recognized by the immune system and could modulate the immune response in infection. The aim of this study was to assess the induction of cytokines in a macrophage cell line in response to different antigens prepared from five C. difficile strains: the hypervirulent ribotype 027, ribotypes 001 and 106 and reference strains VPI 10463 and 630 (ribotype 012). PMA-activated THP-1 cells were challenged with surface-layer proteins, flagella, heat-shock proteins induced at 42 and 60 °C and culture supernatants of the five C. difficile strains. The production of the pro-inflammatory cytokines such as TNF-α, IL-1β, IL-6, IL-8 and IL-12p70 was observed in response to the surface-associated proteins, and high levels of TNF-α, IL-1β and IL-8 were detected in response to challenge with culture supernatants. The immune response triggered by the surface-associated proteins was independent of the strain from which the antigens were derived, suggesting that these proteins might not be related to the varying virulence of the hypervirulent ribotype 027 or ribotypes 001 and 106. There was no interstrain difference observed in response to the culture supernatants of the tested C. difficile strains, but this was perhaps due to toxicity induced in the macrophages by large amounts of toxin A and toxin B.     

Clostridium difficile in emergency room

Clostridium difficile strains are known as etiological agents of pseudomembranous colitis (PMC), antibiotic-associated diarrhea (AAC) and colitis (AAC) and hospital-acquired infections. The aim of this study was to determine the frequency of C. difficile infection among patients in the emergency room and to compare isolated strains by phenotypic and genotypic characteristics. During a period of 11 months, 56 stool samples taken from diarrheic patients hospitalized in the emergency room of the Medical Center UC Davis and 14 environmental samples were cultured for isolation of C. difficile strains. Eighteen C. difficile strains were isolated from stool samples cultured on selective TCCCA plates and 5 strains from environmental samples using Rodac plates. Eleven toxigenic (TcdA+/TcdB+), 6 non-toxigenic (TcdA-/TcdB-) and unique toxin A-negative/toxin B-positive (TcdA-/TcdB+) C. difficile strains were detected among patients' isolates and 3 toxigenic and 2 non-toxigenic strains-among environmental samples. The majority of C. difficile-positive patients were treated previously by antibiotics. Four strains isolated from patients' fecal samples and one strain isolated from the environment demonstrated high-level resistance to erythromycin and clindamycin (MIC >256mug/mL). The results obtained by AP-PCR and PCR-ribotyping revealed genetic heterogeneity among the strains isolated from patients' fecal samples. However, similarity was observed among environmental strains and strains isolated from patients' fecal samples. Considering the importance of emergency room patients as a potential source of C. difficile strains, it appears to be important examine these patients for C. difficile before transfer to the other hospital units.     

Evaluation of the effect of medicines on biological properties of Clostridium difficile

The presence of the persistence factors (anti-lysozyme and anti-complement activity) in the vegetative forms of C. difficile was experimentally proved. The effect of different medicines (vitamins B1, B6 and C, prebiotic inulin, probiotics Bifidumbacterin and Enterol) on the persistence factors of C. difficile and microbial resistance to vancomycin, thienam, lincomycin, clindamycin was evaluated. The anti-lysozyme and anti-complement activity of C. difficile was found to decrease under the influence of vitamins B1, B6, C, inulin, exometabolites of bifidobacteria. Under the impact of the preparations used in this study changes in the sensitivity of C. difficile to antibiotics of the lincoamide, carbapenem, glycopeptide groups were found to occur. The data obtained reveal one of the possible mechanisms of the corrective action of the medicines under study on the intestinal microbiocenosis in patients with antibiotic-associated colitis.     

Nucleotide-binding oligomerization domain 1 mediates recognition of Clostridium difficile and induces neutrophil recruitment and protection against the pathogen

Clostridium difficile is a Gram-positive obligate anaerobic pathogen that causes pseudomembranous colitis in antibiotics-treated individuals. However, host immune protective mechanisms against C. difficile are largely unknown. In this study, we show that C. difficile possesses potent stimulatory activity for nucleotide-binding oligomerization domain 1 (Nod1), an intracellular pattern recognition molecule that senses bacterial peptidoglycan-related molecules. Nod1(-/-), but not Nod2(-/-), mice exhibited increased lethality in response to C. difficile intestinal infection despite comparable levels of intestinal damage and epithelial permeability in Nod1(-/-) and control mice. The enhanced lethality was accompanied by impaired C. difficile clearance, increased bacterial translocation, and elevated levels of endotoxin and IL-1β in the serum of Nod1(-/-) mice. Histological and flow cytometric analyses revealed that Nod1(-/-) mice had defective recruitment of neutrophils, but not macrophages, to the intestine after C. difficile infection. The reduced recruitment of neutrophils correlated with impaired production of CXCL1, but not CCL2, XCL1, and other cytokines/chemokines, in infected Nod1(-/-) mice. The influx of neutrophils also was reduced when C. difficile was administered i.p., suggesting that Nod1 directly recognizes C. difficile to induce the recruitment of neutrophils to the infected site. These results indicate that Nod1 regulates host susceptibility to C. difficile and suggest that Nod1-mediated neutrophil recruitment is an important immune response against the enteric pathogen.     

In vivo lysogenization of a Clostridium difficile bacteriophage ФCD119

Clostridium difficile is a nosocomial pathogen identified as the cause of antibiotic-associated diarrhea and colitis. In this study, we have documented the lysogeny of a C.?difficile bacteriophage in hamsters during C.?difficile infection. The lysogens isolated from the hamsters were toxin typed and their phage integration site was confirmed by PCR. Through toxin ELISA it was found that the toxin production in the in?vivo isolated lysogens was affected due to ФCD119 lysogenization as in the case of in?vitro isolated ФCD119 lysogens. Together our findings indicate that a baceriophage can lysogenize its C.?difficile host even during the infection process and highlights the importance of lysogeny of C.?difficile phages as an evolutionary adaptation for survival.     

Passive immunisation of hamsters against Clostridium difficile infection using antibodies to surface layer proteins

Clostridium difficile is a major cause of antibiotic-associated diarrhoea and the primary cause of pseudomembraneous colitis in hospitalised patients. We assessed the protective effect of anti-surface layer protein (SLP) antibodies on C. difficile infection in a lethal hamster challenge model. Post-challenge survival was significantly prolonged in the anti-SLP treated group compared with control groups (P=0.0281 and P=0.0283). The potential mechanism of action of the antiserum was shown to be through enhancement of C. difficile phagocytosis. This report indicates that anti-SLP antibodies can modulate the course of C. difficile infection and may therefore merit closer investigation for use as constituents of multi-component vaccines against C. difficile associated diarrhoea.     

Pseudomembranous colitis: isolation of two species of cytotoxic clostridia and successful treatment with vancomycin.

Lincomycin-resistant Clostridium sporogenes obtained from the stools of a patient with lincomycin-associated pseudomembranous colitis produced a heat-stable cytotoxin in low titre when grown in chopped meat medium. Vancomycin eradicated this strain and all other clostridia, and controlled the symptoms. When diarrhea recurred 7 days after treatment with vancomycin was stopped, clostridia including C. sporogenes and C. difficile were again isolated. The C. difficile produced a heat-labile cytotoxin in high titre that was unaffected by growth in various media and induced colitis in hamsters. Treatment with vancomycin, to which all the clostridia were sensitive, eradicated both toxic species and controlled the diarrhea. Antibiotic-induced pseudomembranous colitis may be associated with more than one species of toxin-producing clostridia. Vancomycin therapy should be continued for 10 days or more in patients with severe disease to eradicate the responsible organism.     

Prevalence of Clostridium spp. and Clostridium difficile in children with acute diarrhea in São Paulo city,Brazil

Species of Clostridium are widely distributed in the environment, inhabiting both human and animal gastrointestinal tracts. Clostridium difficile is an important pathogen associated with outbreaks of pseudomembranous colitis and other intestinal disorders, such as diarrhea. In this study, the prevalence of Clostridium spp. and C. difficile, from hospitalized children with acute diarrhea, was examined. These children were admitted to 3 different hospitals for over 12 months. Eighteen (20%) and 19 (21%) stool specimens from children with (90) and without (91) diarrhea respectively, were positive to clostridia. Only 10 C. difficile strains were detected in 5.5% of the stool samples of children with diarrhea. None healthy children (without diarrhea) harbored C. difficile. From these 10 C. difficile, 9 were considered as toxigenic and genotyped as tcdA+/tcdB+ or tcdA-/tcdB+, and 1 strain as nontoxigenic (tcdA-/tdcB-). They were detected by the citotoxicity on VERO cells and by the multiplex-polymerase chain reaction. Thirty clinical fecal extracts produced minor alterations on VERO cells. The presence of C. difficile as a probable agent of acute diarrhea is suggested in several countries, but in this study, the presence of these organisms was not significant. More studies will be necessary to evaluate the role of clostridia or C. difficile in diarrhoeal processes in children.     

Human mucosa/submucosa interactions during intestinal inflammation: involvement of the enteric nervous system in interleukin-8 secretion

Interleukin-8 (IL-8) is a key chemokine upregulated in various forms of intestinal inflammation, especially those induced by bacteria such as Clostridium difficile (C. difficile). Although interactions between different mucosal and submucosal cellular components have been reported, whether such interactions are involved in the regulation of IL-8 secretion during C. difficile infection is unknown. Moreover, whether the enteric nervous system, a major component of the submucosa, is involved in IL-8 secretion during an inflammatory challenge remains to be determined. In order to investigate mucosa/submucosa interactions that regulate IL-8 secretion, we co-cultured human intestinal mucosa and submucosa. In control condition, IL-8 secretion in co-culture was lower than the sum of the IL-8 secretion of both tissue layers cultured alone. Contrastingly, IL-8 secretion increased in co-culture after mucosal challenge with toxin B of C. difficile through an IL-1 beta-dependent pathway. Moreover, we observed that toxin B of C. difficile increased IL-8 immunoreactivity in submucosal enteric neurones in co-culture and in intact preparations of mucosa/submucosa, through an IL-1 beta-dependent pathway. IL-1 beta also increased IL-8 secretion and IL-8 mRNA expression in human neuronal cell lines (NT2-N and SH-SY5Y), through p38 and ERK1/2 MAP kinase-dependent pathways. Our results demonstrate that mucosa/submucosa interactions regulate IL-8 secretion during inflammatory processes in human through IL-1 beta-dependent pathways. Finally we observed that human submucosal neurones synthesize IL-8, whose production in neurones is induced by IL-1 beta via MAPK-dependent pathways.     

抗生素在体内外对厌氧菌和艰难梭菌细胞毒素生成的作用

氯林霉素、灭滴灵和甲砜霉素对大多数肠道厌氧菌的生长具抑制作用。氯林霉素还会破坏肠道菌群平衡,使原来受抑制的艰难梭菌得以定植,并在艰难梭菌浓度达10~8/g盲肠内含物时,检测到艰难梭菌细胞毒素。培养基中亚抑菌浓度的氯林霉素和灭滴灵会推迟艰难梭菌细胞霉素的生成。灭滴灵还可保护无菌小鼠及受氯林霉素处理的悉生小鼠免遭艰难梭菌细胞毒素的致死作用,从而证实了灭滴灵在伪膜性结肠炎临床治疗中的可用性。     

Separate isolation of Clostridium difficile spores and vegetative cells from the feces of newborn infants.

A modified taurocholate-cefoxitin-cycloserine-fructose agar medium, pH 5.5, on which vegetative cells alone could grow, was newly devised for separate isolation of Clostridium difficile vegetative cells and spores from feces. The ratio of C. difficile-positive feces from healthy newborn infants younger than 10 days of the age was 30.8%, and 93.3% of feces from healthy infants older than 20 days were positive for C. difficile. C. difficile spores alone were detected in twenty-one samples (75%) of C. difficile-positive Twenty-eight specimens. Only 10.7% (3/28) C. difficile vegetative cells alone were detected. C. difficile spores alone were detected in one of nine healthy adults. These collective results offer potential explanations for high frequent isolations of C. difficile from newborn infants without occurrence of pseudomembranous colitis.     

Cytotoxicity and antimicrobial susceptibility of Clostridium difficile isolated from hospitalized children with acute diarrhea

Clostridium difficile is an important pathogen associated with outbreaks of pseudomembranous colitis and other intestinal disorders such as diarrhea. In this study, 181 stool samples from children with and without acute diarrhea were analysed. Eighteen children with acute diarrhea were positive to C. ramosum, C. difficile, C. limosum, C. clostridioforme, C. septicum, C. butyricum, C. innocuum and Clostridium sp. Nineteen children without diarrhea harbored C. ramosum, C. septicum, C. barattii, C. butyricum, C. innocuum, C. sphenoides, C. bifermentans, C. clostridioforme and C. paraputrificum. No patient with diarrhea harbored C. barattii, C. bifermentans, C. paraputrificum and C. sphenoides. In addition, ten C. difficile strains were detected in 5 (5.5%) of the children with diarrhea. Also, no children from control group harbored C. difficile, C. limosum and Clostridium sp. Most of the tested strains were resistant to all the used antimicrobial. Nine C. difficile were toxigenic on VERO cells and by multiplex PCR, six strains showed both toxin A and B genes and three strains showed only toxin B gene. In this study, the presence of C. difficile was not significant, and it is suggested the need of more studies to evaluate the role of clostridia or C. difficile play in the childhood diarrhea and these organisms must be looked for routinely and a periodic evaluation of antimicrobial susceptibility should be performed.     

Clostridium difficile cell-surface polysaccharides composed of pentaglycosyl and hexaglycosyl phosphate repeating units

Clostridium difficile is a Gram-positive bacterium that is known to be a cause of enteric diseases in humans. It is the leading cause of antibiotic-associated diarrhea and pseudomembranous colitis. Recently, large outbreaks of C. difficile-associated diarrhea have been reported internationally, and there have been reports of increases in severe disease, mortality and relapse rates. At the moment, there is no vaccine against C. difficile, and the medical prevention of C. difficile infection is mostly based on the prophylactic use of antibiotics; however, this has led to an increase in the incidence of the disease. Here, we describe the chemical structure of C. difficile cell-surface polysaccharides. The polysaccharides of three C. difficile strains were structurally analyzed; ribotype 027 (North American pulsotype 1) strain was observed to express two polysaccharides, one was composed of a branched pentaglycosyl phosphate repeating unit: [-->4)-alpha-l-Rhap-(1-->3)-beta-D-Glcp-(1-->4)-[alpha-l-Rhap-(1-->3]-alpha-D-Glcp-(1-->2)-alpha-D-Glcp-(1-->P] and the other was composed of a hexaglycosyl phosphate repeating unit: [-->6)-beta-D-Glcp-(1-->3)-beta-D-GalpNAc-(1-->4)-alpha-D-Glcp-(1-->4)-[beta-D-Glcp-(1-->]-beta-D-GalpNAc-(1-->3)-alpha-D-Manp-(1-->P]. The latter polysaccharide was also observed to be produced by strains MOH900 and MOH718. The results described here represent the first literature report describing the covalent chemical structures of C. difficile cell-surface polysaccharides, of which PS-II appears to be a regular C. difficile antigen. These C. difficile teichoic-acid-like polysaccharides will be tested as immunogens in vaccine preparations in a rat and horse model.     

Immunization of hamsters against Clostridium difficile infection using the Cwp84 protease as an antigen

Clostridium difficile is a pathogen responsible for diarrhoea and colitis, particularly after antibiotic treatment. We evaluated the C. difficile protease Cwp84, found to be associated with the S-layer proteins, as a vaccine antigen to limit the C. difficile intestinal colonization and therefore the development of the infection in a clindamycin-treated hamster model. First, we evaluated the immune response and the animal protection against death induced by several immunization routes: rectal, intragastric and subcutaneous. Antibody production was variable according to the immunization routes. In addition, serum Cwp84 antibody titres did not always correlate with animal protection after challenge with a toxigenic C. difficile strain. The best survival rate was observed with the rectal route of immunization. Then, in a second assay, we selected this immunization route to perform a larger immunization assay including a Cwp84 immunized group and a control group. Clostridium difficile intestinal colonization and survival rate, as well as the immune response were examined. Clostridium difficile hamster challenge resulted in a 26% weaker and slower C. difficile intestinal colonization in the immunized group. Furthermore, hamster survival in the Cwp84 immunized group was 33% greater than that of the control group, with a significant statistical difference.     

Acute colitis induced by dextran sulfate sodium progresses to chronicity in C57BL/6 but not in BALB/c mice: correlation between symptoms and inflammation

Exposure to dextran sulfate sodium (DSS) induces acute colitis, which is normally resolved after DSS removal. To study chronicity, mice are typically subjected to three to five cycles of weekly DSS exposures, each followed by a 1- to 2-wk rest period. Here, we describe a novel and convenient way of inducing chronic, progressive colitis by a single exposure to DSS. C57BL/6 mice exposed to DSS for 5 days developed acute colitis that progressed to severe chronic inflammation. The plasma haptoglobin levels remained high during the chronic phase, showing that the inflammation was active. Surprisingly, the mice regained their original weight along with the progression of colitis, and the only apparent symptom was loose feces. Histopathological changes 4 wk after DSS removal were dense infiltrates of mononuclear cells, irregular epithelial structure, and persistent deposits of collagen. A progressive production of the cytokines IL-1beta, IL-12 p70, and IL-17 correlated with the extensive cellular infiltration, whereas high IFN-gamma production was mainly found late in the chronic phase. Similar to C57BL/6 mice, BALB/c mice exposed to 5 days of DSS developed acute colitis as previously described. The acute colitis was accompanied by elevated plasma levels of haptoglobin and increased colonic levels of IL-1alpha/beta, IL-6, IL-18, and granulocyte colony-stimulating factor. However, soon after DSS removal, BALB/c mice recovered and were symptom free within 2 wk and completely recovered 4 wk after DSS removal in terms of histopathology, haptoglobin levels, and local cytokine production. In summary, these data stress the effect of genetic background on the outcome of DSS provocation. We believe that the present protocol to induce chronic colitis in C57BL/6 mice offers a robust model for validating future therapies for treatment of inflammatory bowel disease.