Modulation of cytotoxin production by Clostridium difficile in the intestinal tracts of gnotobiotic mice inoculated with various human intestinal bacteria

Gnotobiotic mice died 2 days after inoculation of a cytotoxigenic Clostridium difficile strain. Protection occurred when mice were previously inoculated with a strain of Escherichia coli or Bifidobacterium bifidum. Intestinal cytotoxin production was highly reduced in the surviving mice, whereas the C. difficile population level did not decrease to a great extent.     

Inhibitory effect of a copper-dipeptide complex on the establishment of a Clostridium perenne strain in the intestinal tract of gnotobiotic mice.

A semisynthetic diet fed to axenic mice was found to prevent the establishment of a Clostridium perenne strain in their intestinal tract. This inhibitory effect did not occur when axenic mice were preinoculated with a strain of Clostridium difficile. The inhibitory effect was related to the presence in the intestinal contents of axenic mice of both dietary copper and a dipeptide, aspartic-epsilon-lysine. When C. difficile was inoculated into axenic mice, the dipeptide disappeared from the digesta, and C. perenne became established even in the presence of high concentrations of copper.     

Inhibitory effect of a copper-dipeptide complex on the establishment of a Clostridium perenne strain in the intestinal tract of gnotobiotic mice

A semisynthetic diet fed to axenic mice was found to prevent the establishment of a Clostridium perenne strain in their intestinal tract. This inhibitory effect did not occur when axenic mice were preinoculated with a strain of Clostridium difficile. The inhibitory effect was related to the presence in the intestinal contents of axenic mice of both dietary copper and a dipeptide, aspartic-epsilon-lysine. When C. difficile was inoculated into axenic mice, the dipeptide disappeared from the digesta, and C. perenne became established even in the presence of high concentrations of copper.     

Effect of toxins produced by various Clostridium difficile strains on cecum size reduction in gnotobiotic mice

Inoculation of axenic mice with Clostridium difficile strains induced a significant reduction in ceca weight (dry or wet), whereas a nontoxinogenic strain led to a partial reduction. A strain, which produces cytotoxin and no enterotoxin in vivo, caused a reduction similar to that observed with a nontoxinogenic strain. Simultaneous cytotoxin and enterotoxin production by various C. difficile strains caused the cecum size to diminish to that observed for conventional control mice.     

Presence and molecular characterization of Clostridium difficile and Clostridium perfringens in intestinal compartments of healthy horses

ABSTRACT: BACKGROUND: Clostridium difficile and Clostridium perfringens are commonly associated with colitis in equids, but healthy carriers exist. Scarce information is available on the prevalence of Clostridium spp. in gastrointestinal compartments other than faeces in healthy horses, and it is unknown whether faecal samples are representative of proximal compartments. The objectives were to investigate the prevalence of C. difficile and C. perfringens in different intestinal compartments of healthy adult horses and to determine whether faecal samples are representative of colonization in proximal sites and overall carrier status. RESULTS: Toxigenic C. difficile was isolated from 14/135 (10.3%) samples from 8/15 (53.3%) horses. Between zero and three sites were positive per horse, and multiple sites were positive in four horses. Isolates were recovered from duodenum, jejunum, ileum, right dorsal colon, small colon and rectum. When multiple compartments were positive in a single horse, two different C. difficile ribotypes were always present. Clostridium perfringens Type A (CPE, beta2 toxin gene negative) was recovered from the left ventral colon of one horse (0.74%, 1/135 samples). Agreement between faeces and overall C. difficile carrier status was good. CONCLUSIONS: Clostridium difficile can be found in different compartments of the gastrointestinal tract of healthy horses, and multiple strains can be present in an individual horse. The prevalence of C. perfringens in healthy adult hoses was low, consistent with previous reports. Faecal samples were representative for presence of C. difficile in proximal compartments in 5/8 horses (63%) but were not representative for the specific strain.     

Intestinal bacteria antagonistic to Clostridium difficile in mice

Overgrowth by Clostridium difficile has been reported in conventional mice injected intraperitoneally with ampicillin. In this study, we aimed to determine which types of indigenous intestinal bacteria were eliminated by ampicillin to allow overgrowth by C. difficile. C. difficile overgrowth was associated with a decrease in the numbers of lactobacilli, an increase in bacteroidaceae and a slight decrease in the frequency of isolation of fusiform-shaped bacteria (clostridia). C. difficile cytotoxin was detected in caeca from mice in which the numbers of C. difficile were greater than 10(5) per gram of faeces. Gnotobiotic mice were inoculated with various groups of intestinal anaerobes to determine which members of the indigenous flora would antagonize C. difficile. Gnotobiotic mice inoculated with three strains of lactobacilli, 37 strains of bacteroides or 46 strains of clostridia isolated from limited-flora mice were unable to eliminate C. difficile. C. difficile was eliminated, however, from the gastrointestinal tracts of gnotobiotic mice inoculated with whole faeces or chloroform-treated faeces from conventional mice or whole faeces from limited-flora mice containing only clostridia.     

酪酸梭菌对艰难梭菌感染的防治研究

目的:观察酪酸梭菌对艰难梭菌感染的防治效果.方法:用艰难梭菌产毒株人工感染BALB/C小鼠,感染前后分别用酪酸梭菌进行预防与治疗,并检测盲肠内容物细胞毒性和进行肠黏膜病理观察.结果:酪酸梭菌不能预防艰难梭菌的感染,但在艰难梭菌感染后则能明显降低艰难梭菌的产毒力和盲肠黏膜的病理损伤.结论:酪酸梭菌对小鼠艰难梭菌感染有明显的治疗作用.     

Prevention of Clostridium difficile-induced experimental pseudomembranous colitis by Saccharomyces boulardii: a scanning electron microscopic and microbiological study

The ability of Saccharomyces boulardii to protect mice against intestinal pathology caused by toxinogenic Clostridium difficile was studied. Different regions of the intestine of experimental mice were prepared for observation by scanning electron microscopy or homogenized for C. difficile enumeration and quantification of toxin A by enzyme immunoassay and toxin B by cytotoxicity. The test group was treated for 6 d with an S. boulardii suspension in drinking water and challenged with C. difficule on day 4. The three control groups were: axenic mice, mice treated with only S. boulardii and mice only challenged with C. difficile. The results showed that: (i) 70% of the mice infected by C. difficile survived when treated with S. boulardii; (ii) the C. difficile-induced lesions on the small and large intestinal mucosa were absent or markedly less severe in S. boulardii-treated mice; and (iii) there was no decrease in the number of C. difficile but rather a reduction in the amount of toxins A and B in S. boulardii-treated mice.     

Isolation of Clostridium difficile from various colonies of laboratory mice

An attempt was made to isolate Clostridium difficile from a total of 565 mice from nine different conventional mouse colonies and six different specified-pathogen-free mouse colonies. C. difficile was isolated from all the conventional colonies but from none of the specified-pathogen-free colonies. Ampicillin injected intraperitoneally increased the isolation rate of C. difficile from mouse faeces to 63.6% compared with 19.4% from untreated mice.     

Draft genome sequence of the nontoxigenic Clostridium difficile strain CD37

Here we report the draft genome sequence of Clostridium difficile strain CD37, the first nontoxigenic strain sequenced. Every sequenced strain of Clostridium difficile has been shown to contain multiple different mobile genetic elements. The draft genome sequence of strain CD37 reveals the presence of two putative conjugative transposons.     

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.     

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。     

悉生小鼠体内大肠杆菌和青春型双歧杆菌对艰难梭菌的拮抗作用

本文应用悉生小鼠做模型,研究了大肠杆菌(E.coli)和青春型双歧杆菌(Bifidobacterium adolescentis)对艰难梭菌(Clostridium diffi-cile)的拮抗作用。E.coli和B.adolescentis预先接种无菌SSB小鼠,再用C.difficile攻击。结果表明,E.coli和E.coli B.adolescentis对小鼠均有保护作用,保护平分别为87.5%(7/8)和100%(8/8)。B.adolescentis定值后数量达10~(10.28)CFU/g,且对E.coli数量和小鼠本身无影响。E.coli和B.adolescentis联合比E.coli单独抑制C.difficile在肠道中繁殖的作用更强(0.02>P>0.01),但对其毒素产生和粘附力的作用无明显差异。C.difficile攻击后的1～14天,小鼠粪便中C.difficile菌数在10~4至10~8CFU/g内变化,细胞毒素为10~3CFU/g,A毒素滴度为10~2/g,B.adolescentis也一度下降10~2CFU/g。接种C.difficile后,小鼠虽无明显的腹泻症状,但组织学仍可观察到肠粘膜有充血和分泌增加等轻度损害。扫描电镜和普通光镜均发现E.coli单独或与B.adolescentis共同吸附在肠粘膜微绒毛表面,未见有C.difficile吸附。     

Modulation of Clostridium difficile induced mortality as a function of the dose and the viability of the Saccharomyces boulardii used as a preventative agent in gnotobiotic mice

The ability of viable and nonviable Saccharomyces boulardii to protect gnotobiotic mice from Clostridium difficile induced mortality was tested. With the exception of irradiated S. boulardii, which retained some activity, only viable yeast protected the mice from lethality. The survival of C. difficile infected mice was dependent on the dose of the yeast provided in the drinking water.     

Isolation and characterization of temperate bacteriophages of Clostridium difficile

The lack of information on bacteriophages of Clostridium difficile prompted this study. Three of 56 clinical C. difficile isolates yielded double-stranded DNA phages phiC2, phiC5, phiC6, and phiC8 upon induction. Superinfection and DNA analyses revealed relatedness between the phages, while partial sequencing of phiC2 showed nucleotide homology to the sequenced C. difficile strain CD630.     

Effect of Bacterial Contamination on Cecal Size and Cecal Contents of Gnotobiotic Rodents

In the present investigation the effect of various bacterial contaminations of gnotobiotic mice and rats on cecal size is presented. Of the species tested, Bacteroides oralis and Fusobacterium nucleatum did not establish in germ-free mice. Streptococcus mutans, Clostridium difficile, a Neisseria strain and two recent cecal isolates established, but failed to exert an effect upon the cecum of mice. A group K streptococcus and B. fragilis increased the cecal size apparently by increasing the levels of water-soluble protein, peptides, and carbohydrates in the cecal contents. Mixed ileal bacteria decreased the cecal size by preventing accumulation of soluble proteins and carbohydrates in the cecum. A Peptococcus strain caused a reduction by lowering the levels of insoluble material in the cecum. When this strain was combined with two Clostridium isolates and introduced into gnotobiotic rats, 50 to 65% cecal reduction was observed. This polycontamination did not decrease the per cent water of the cecal contents but caused lower levels of both soluble and insoluble material to accumulate in the cecum. No net nitrogen absorption from the distal small intestine occurred in either the germ-free or polycontaminated rats.     

酪酸梭菌与婴双歧杆菌对艰难梭菌体外生物拮抗作用的作用

用酪酸梭菌（Clostridium butyricum)和婴双歧杆菌（Bifidobacterium infantis)单独或联合艰难菌（Clostridium difficile)进行试管内的生物拮抗作用,将酪酸梭菌、婴儿双歧杆菌单独或酪酸梭菌和婴儿双歧杆菌联合分别与艰难梭菌以一定的比例等量混合后,接种于GAM液体培养基中进行厌氧培养。证明酪酸梭菌和婴儿双歧杆菌能明显抑制艰难梭菌的生长,并且两菌联合比各自单独培养时显示出更强的生物拮抗作用。     

Motility as an intestinal colonization factor for Campylobacter jejuni

The colonization of the intestinal tract of suckling mice by Campylobacter jejuni was examined by orally challenging the mice with a wild-type strain and several nonmotile mutant strains which were isolated after treating the wild-type strain with mutagens. The wild-type strain had colonized the lower portion of the small intestine, the caecum and the colon 2 d after inoculation. Two nonmotile strains, one of which (M8) had lost all the flagellar structure including the filament, the hook and the basal structure, and the other (M1) which had lost only the filament region, were both cleared from the intestinal tract 2 d after challenge. Another nonmotile strain (M14), which had a complete flagellar structure like that of the wild-type strain, did not colonize and was cleared from the intestinal tract like the other nonmotile and nonflagellated strains. One atypically motile strain (M5), which had a shorter flagellar filament than that of the wild-type strain, colonized the intestinal tract only when mice were challenged with a large inoculum. None of the mice challenged with either the wild-type or any of the mutant strains showed signs of illness. We concluded that motility is an important factor in the colonization of the intestinal tract of suckling mice by C. jejuni.     

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.     

Prevention of Clostridium difficile induced mortality in gnotobiotic mice by Saccharomyces boulardii

Oral preventive treatment of gnotobiotic mice by Saccharomyces boulardii significantly decreased mortality following Clostridium difficile infection. A single S. boulardii ingestion protected 16% of mice, whereas 56% were protected when S. boulardii was given continuously in the drinking water. No direct antagonistic effect of the yeast on C. difficile numbers was detected, whereas a modulation of fecal cytotoxin production was demonstrated.