Molecular analysis of the digestive microbiota in a gnotobiotic mouse model during antibiotic treatment: Influence of Saccharomyces boulardii

The probiotic Saccharomyces boulardii is a non-pathogenic yeast that has been proven efficient in the prevention of antimicrobial-associated diarrhea and of Clostridium difficile associated colitis. We evaluated the influence of the administration of S. boulardii on the composition of the fecal microbiota in a human microbiota-associated mouse model. This evaluation was run before, during and after a 7-day oral treatment with amoxicillin clavulanic acid. Predominant groups of bacteria were quantified with fluorescence in situ hybridization combined with flow cytometry using group-specific 16S rRNA targeted oligonucleotide probes designed for the Eubacteria, Bacteroides-Porphyromonas-Prevotella, Clostridium coccoides-Eubacterium rectale, Faecalibacterium prausnitzii, Clostridium histolyticum, Lactobacillus-Enterococcus and Enterobacteriaceae groups and Bifidobacterium species. S. boulardii did not quantitatively alter the total anaerobic microbiota nor the dominant bacterial groups. During the antibiotic treatment in the two groups of mice receiving the yeast or not, the level of Enterobacteriaceae and Bacteroides groups increased when the C. coccoides-E. rectale group decreased dramatically. After the antibiotic treatment was discontinued, the return to the initial level was reached more rapidly in the S. boulardii-treated mice than in the control mice (p<0.05) for the C. coccoides-E. rectale and Bacteroides-Porphyromonas-Prevotella groups. This quicker recovery of normal intestinal microbiota equilibrium after antibiotic therapy could be a mechanism for S. boulardii preventive effect on antibiotic-associated diarrhea in humans.     

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。     

Saccharomyces boulardii inhibits ERK1/2 mitogen-activated protein kinase activation both in vitro and in vivo and protects against Clostridium difficile toxin A-induced enteritis

Saccharomyces boulardii (Sb), a probiotic yeast, protects against intestinal injury and inflammation caused by a wide variety of enteric pathogens, including Clostridium difficile. Given the broad range of protective effects of Sb in multiple gastrointestinal disorders, we hypothesize that Sb modulates host signaling pathways involved in intestinal inflammatory responses. In this study, we found that Sb culture supernatant (SbS) inhibits interleukin-8 production induced by C. difficile toxin A or IL-1beta in human colonocyte NCM460 cells in a dose-dependent fashion. Furthermore, SbS inhibited IL-1beta and toxin A induced Erk1/2 and JNK/SAPK but not p38 activation in NCM460 cells. To test whether this inhibition also occurs in vivo, we used a previously established mouse ileal loop model. On its own, SbS had no significant effect on basal fluid secretion or intestinal histology. However, Erk1/2 activation was significantly inhibited by SbS in toxin A exposed mouse ileal mucosa. In control loops, toxin A increased fluid secretion (2.2-fold), histological score (3.3-fold), and levels of the chemokine KC (4.5-fold). SbS pretreatment completely normalized toxin A mediated fluid secretion (p < 0.01), and histopathologic changes (p < 0.01) and substantially inhibited toxin A-associated KC increases (p < 0.001). In summary, the probiotic yeast S. boulardii inhibits C. difficile toxin A-associated enteritis by blocking the activation of Erk1/2 MAP kinases. This study indicates a new mechanism whereby Sb protects against intestinal inflammation and supports the hypothesis that Sb modulates host inflammatory signaling pathways to exert its beneficial effects.     

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.     

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.     

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.     

Comparative role of antibiotic and proton pump inhibitor in experimental Clostridium difficile infection in mice

Clostridium difficile inoculated BALB/c mice were investigated to assess the comparative role of antibiotic and proton pump inhibitor. They were examined for colonization and toxin production by C. difficile as well as myeloperoxidase activity and histopathological changes in the intestinal tract. The C. difficile count, toxin A and B titres and myeloperoxidase activity were significantly higher (P>0.05) in ampicillin and lansoprazole receiving groups as compared to the control and the C. difficile receiving groups. Similarly they showed significant difference (P >0.05) for epithelial damage, oedema and neutrophilic infiltrate in colons. In addition to antibiotic, PPI also acts as an independent risk factor for C. difficile infection in experimental studies.     

Effect of Saccharomyces boulardii against experimental oral infection with Salmonella typhimurium and Shigella flexneri in conventional and gnotobiotic mice

A.C.P. RODRIGUES, R.M. NARDI, E.A. BAMBIRRA, E.C. VIEIRA AND J.R. NICOLI. 1996. Saccharomyces boulardii was shown to be capable of inhibiting multiplication of enteropathogenic bacteria in vitro and is currently used for its anti-diarrhoea properties. We studied the capacity of this yeast to antagonize Salmonella typhimurium and Shigella flexneri in the intestinal tract of conventional or gnotobiotic NMRI mice. Conventional animals were given daily 10 mg doses of S. boulardii , whereas germ-free animals were given a single 10 mg dose. Both groups were challenged orally 5 d later with the pathogenic bacteria (10⁸ or 10² viable cells, respectively). Control groups were treated with saline instead of S. boulardii. Mortality and/or histopathological data showed a protective effect against the pathogenic bacteria in yeast-treated mice. Saccharomyces boulardii colonized the digestive tract of gnotobiotic mice and the number of viable cells ranged around 10¹⁰ g^-1 of faeces. In experimental and control gnotobiotic animals, Salm. typhimurium and Sh. flexneri became rapidly established at a level of about 10¹⁰ viable cells g^-1 of faeces and remained at high levels until the animals died or were sacrificed. The protection against Salm. typhimurium and Sh. flexneri obtained in conventional and/or gnotobiotic mice previously associated with S. boulardii is not due to the reduction of the bacterial populations in the intestines.     

Yeast, beef and pork extracts counteract Clostridium difficile toxin A enterotoxicity

Clostridium difficile is responsible for a large proportion of nosocomial cases of antibiotic-associated diarrhoea and pseudomembranous colitis. The present study provides evidence that yeast, beef and pork extracts, ingredients commonly used to grow bacteria, can counteract C. difficile toxin A enterotoxicity in vitro and in vivo . In model intestinal epithelial cells the individual extracts could prevent the toxin A-induced decrease in epithelial barrier function and partially prevented actin disaggregation and cell rounding. Mice with ad libitum access to individual extracts for 1 week had almost complete reduction in toxin A-induced fluid secretion in intestinal loops. Concomitantly, the toxin A-induced expression of the essential proinflammatory mediator Cox-2 was normalized. Moreover this protective effect was also seen when mice received only two doses of extract by intragastric gavage within 1 week. These results show that yeast, beef and pork extracts have the potential to counteract the intestinal pathogenesis triggered by C. difficile toxin A.     

Saccharomyces boulardii stimulates sIgA production and the phagocytic system of gnotobiotic mice

The effect of Saccharomyces boulardii on the immune system was evaluated, comparing germ-free Swiss/NIH mice monoassociated with the probiotic with germ-free mice. Saccharomyces boulardii colonized the gut of germ-free mice and survived the gastrointestinal conditions. An increase in sIgA production, both total and anti-S. boulardii, was observed in the intestinal contents of monoassociated mice when compared with germ-free controls. The number of Kupffer cells was significantly higher in monoassociated mice than in germ-free controls. In S. boulardii-monoassociated mice, clearance of Escherichia coli B41 was higher than in germ-free controls. TNF-alpha, IFN-gamma and IL-12 serum levels were higher at earlier time points in monoassociated mice when compared with germ-free mice. These results show that the yeast S. boulardii modulates the host immune responses. This effect may be of interest for improving the resistance to enteropathogenic bacterial infections.     

Isolation of toxigenic strains of Clostridium difficile and enterotoxin producing Bacteroides fragilis from fecal specimens of patients suspected of antibiotic associated diarrhoea

Fifty faecal samples from patients suspected of AAD (antibiotic associated diarrhoea) were studied for Clostridium difficile and enterotoxin producing Bacteroides fragilis (ETBF). Using TCD (Becton-Dickinson) and C. difficile Toxin A test (Oxoid) in 34% of specimens the presence of toxin A was detected. From all specimens 25 C. difficile strains were isolated. All isolated strains produced toxin B in vitro which was shown in Mc Coy cytotoxicity test. Eighteen strains only were toxin A positive in vitro. From all isolated C. difficile strains 28% were tox A (-) tox B (+). By means of PCR presence of toxin A and toxin B genes was tested directly in faecal samples and in strains. From the same 50 faecal samples 17 B. fragilis strains were isolated. Four of them produced the enterotoxin (fragilisin) which was detected on the HT 29/C1 cell line. Genes of fragilisin were found in strains and directly in faecal samples. Toxin producing C. difficile and B. fragilis (ETBF) together were found in 3 samples. From one faecal sample only ETBF was cultured.     

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.     

Evaluation of different methods for detection of Clostridium difficile toxins in Poland

The aim of this study was to compare different methods for C. difficile toxins detection. Fifty three stool samples taken from patients with antibiotic-associated diarrhoea were studied. TCD toxin A EIA (Becton Dickinson, USA), Tox A/B ELISA test (TechLab, USA), cytotoxicity and neutralization assay on McCoy cells and PCR for detection of both toxin A and B genes were performed in vivo (in stool samples) and in vitro (in isolated strains). Reference toxigenic and nontoxigenic and two Japanese toxin A-negative and toxin B-positive C. difficile strains were used as a controls. TCD toxin A EIA detected in vivo only 19 positive samples. Tox A/B test detected 52 positive samples out of 53 studied. All 53 stool samples were C. difficile culture positive (53 strains were cultured). Toxin B was detected in 52 strain-supernatants and in all controls (except the nontoxigenic one). Both toxin A and B genes were detected by PCR in all 53 isolated strains, Japanese and reference strain (except the nontoxigenic one). In vitro toxin A was detected by TCD toxin A EIA in 42 strains. These results were compared with those obtained in Tox A/B ELISA test. We observed 52 positive strains. Toxigenic reference strain and two Japanese toxA(-)/toxB(+) strains were also positive. Only 2 negative results were obtained with the nontoxigenic reference strain and unique nontoxigenic isolated strain. Tox A/B ELISA test seems to be the best for detection of C. difficile toxins in vivo and in vitro. Test avoids the false-negative results in the case of presence of toxin A-negative and toxin B-positive strain.     

Profile of toxigenicity of Clostridium difficile strains isolated from paediatric patients with clinical diagnosis of antibiotic associated diarrhea (AAD)

This study was performed to determine profile of toxigenicity of 18 Clostridium difficile strains isolated from paeditric patients suffering from antibiotic associated diarrhea (AAD). Toxigenicity of C. difficile strains was tested for detection toxin A and toxin B by phenotypic methods and for detection of the tcdA and tcdB genes using of PCR. Changes in the repeating regions of the tcdA genes were detected with the NK9/NKV011 primer pairs. For detection of binary toxin (CDT) cdtA and cdtB genes, cdtApos/cdtArev i cdtBpos/cdtBrev two pair primers in PCR was used. Among C. difficile strains was detected three profiles of toxigenicity: C. difficile strains possesing of tcdA and tcdB genes but not possesing cdtA and cdtB genes of binary toxin (A+B+CDT-), strains possesing tcdA and tcdB and cdtA and cdtB genes (A+B+CDT+), strains with deletion of toxin A gene (A-B+CDT-). This is the first report on the occurence of binary positive C. difficile strains isolated from paediatric patients.     

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.     

Influence of selected Lactobacillus sp. on Clostridium difficile strains with different toxigenicity profile

This study was performed for determination of antagonistic activity of Lactobacillus spp. (L. plantarum 2017405, L. rhamnosus GG, L. acidophilus DSM 21007 and L. fernmentumn 353) on Clostridiunl difficile strains belonging to different toxigenicity profiles. Forty strains C. difficile isolated from patients suffering from antibiotic associated diarrhea (AAD) were used. Among C. difficile strains 13 produced toxin A and B (A+B+CDT-), 14 produced only toxin B (AB'CDT), 9 produced toxins A and B and possessing of binary toxin genes (A+B+CDT-) and 4 were non-toxigenic (A-B-CDT-). We did not observe relationship between degree of antagonistic activity Lactobacillus spp. and profile of toxigenicity of C. difficile strains.     

Effect of Streptococcus parvulus and Peptostreptococcus magnus on cytotoxin levels of Clostridium difficile in anaerobic continuous flow culture

An anaerobic continuous flow (CF) culture method was used in order to study the effect of Peptostreptococcus magnus and Streptococcus parvulus, anaerobic gram-positive cocci which are members of intestinal bacterial flora, on growth and cytotoxin-activity of Clostridium difficile. The growth- and the cytotoxin activity-patterns of C. difficile in an established CF culture of P. magnus were similar to those of C. difficile alone. On the other hand, in the mixed culture system of C. difficile and S. parvulus, the cytotoxin levels were significantly lower as compared with C. difficile alone in spite of the fact that no differences existed between growth of C. difficile in mixed and single culture systems. The culture filtrate of P. magnus did not influence the growth and cytotoxin production of C. difficile, nor did that of S. parvulus have any effect on growth of C. difficile in static culture. The cytotoxin activity of C. difficile was, however, suppressed by the culture filtrate of S. parvulus. Furthermore, when P. magnus or S. parvulus was statically cultured in a medium containing cytotoxic culture filtrate of C. difficile, the toxin in the medium was not inactivated.     

Toxin B of Clostridium difficile activates human VIP submucosal neurons,in part via an IL-1beta-dependent pathway

This study investigated whether toxin B of Clostridium difficile can activate human submucosal neurons and the involved pathways. Isolated segments of human colon were placed in organ culture for 3 h in the presence of toxin B or IL-1beta. Whole mounts of internal submucosal plexus were stained with antibodies against c-Fos, neuron-specific enolase (NSE), vasoactive intestinal polypeptide (VIP), and substance P (SP). The membrane potential (Vm) response of submucosal neurons to local application of toxin B and IL-1beta was determined by a multisite optical recording technique. Toxin B (0.1 to 10 ng/ml) increased the proportion of c-Fos-positive neurons dose dependently compared with the control. In the presence of toxin B (10 ng/ml), most c-Fos-positive neurons were immunoreactive for VIP (79.8 +/- 22.5%) but only 19.4 +/- 14.0% for SP. Toxin B induced a rapid rise in IL-1beta mRNA level and a sixfold increase in IL-1beta protein in supernatant after 3 h of incubation. c-Fos expression induced by toxin B was reduced dose dependently by IL-1 receptor antagonist (0.1-10 ng/ml). IL-1beta significantly increased c-Fos expression in submucosal neurons compared with the control (34.2 +/- 10.1 vs. 5.1 +/- 1.3% of NSE neurons). Microejection of toxin B had no effect on the Vm of enteric neurons. Evidence of a direct excitatory effect of IL-1beta on Vm was detected in a minority of enteric neurons. Therefore, toxin B of C. difficile activates VIP-positive submucosal neurons, at least in part, via an indirect IL-1beta-dependent pathway.     

ADP-ribosylation in Clostridium difficile toxin-treated cells is not related to cytopathogenicity of toxin B

ADP-ribosylation of a protein in human fibroblasts treated with partially purified Clostridium difficile toxin B was previously reported. Here we show that the same protein was ADP-ribosylated also in human fibroblasts exposed to supernatant from a C. difficile strain producing neither toxin A nor toxin B. Furthermore, in Chinese hamster ovary and in Vero cells, showing toxin B-induced cytopathogenic effect, the protein was not significantly ADP-ribosylated. The results indicate that the ADP-ribosylation is unrelated to the cytopathogenic effect of toxin B. It appears to be caused by another unidentified factor from C. difficile, and the substrate may correspond to a protein modified endogenously in cells exposed to stressful situations. Cellular actin was not ADP-ribosylated by toxin B.