Intestinal Microbiota Increases Cell Proliferation of Colonic Mucosa in Human-Flora-Associated (HFA) Mice.

Intestinal epithelium renewal strictly depends on fine regulation between cell proliferation, differentiation, and apoptosis. While murine intestinal microbiota has been shown to modify some epithelial cell kinetics parameters, less is known about the role of the human intestinal microbiota. Here, we investigated the rate of intestinal cell proliferation in C3H/HeN germ-free mice associated with human flora (HFA, n = 8), and in germ-free (n = 15) and holoxenic mice (n = 16). One hour before sacrifice, all mice were intraperitoneally inoculated with 5-bromodeoxyuridine (BrdU), and the number of BrdU-positive cells/total cells (labelling index, LI), both in the jejunum and the colon, was evaluated by immunohistochemistry. Samples were also observed by scanning electron microscopy (SEM). Moreover, the microbiota composition in the large bowel of the HFA mice was compared to that of of human donor's fecal sample. No differences in LI were found in the small bowels of the HFA, holoxenic, and germ-free mice. Conversely, the LI in the large bowel of the HFA mice was significantly higher than that in the germ-free and holoxenic counterparts (p = 0.017 and p = 0.048, respectively). In the holoxenic and HFA mice, the SEM analysis disclosed different types of bacteria in close contact with the intestinal epithelium. Finally, the colonic microbiota composition of the HFA mice widely overlapped with that of the human donor in terms of dominant populations, although Bifidobacteria and Lactobacilli disappeared. Despite the small sample size analyzed in this study, these preliminary findings suggest that human intestinal microbiota may promote a high proliferation rate of colonic mucosa. In light of the well-known role of uncontrolled proliferation in colorectal carcinogenesis, these results may deserve further investigation in a larger population study.

Intestinal microflora and digestive toxicity of irinotecan in mice.

PURPOSE: Delayed diarrhea is the most important side effect of irinotecan. The aim of this study was to investigate the role of intestinal microflora on the induction of systemic and intestinal toxicity and diarrhea, studying germ-free and holoxenic mice i.p. injected with irinotecan. EXPERIMENTAL DESIGN: To evaluate the lethal dose, starting with 100 mg/kg/4 d, we treated the holoxenic mice with 100, 80, and 60 mg/kg/4 d and germ-free mice with 60, 80, 100, and 150 mg/kg/4 d. We recorded the percentage of dead animals, diarrhea, and the epithelial damage to the jejunum, ileum, cecum, and colon at optical and scanning electron microscopy. RESULTS: Germ-free mice were more resistant to irinotecan than the holoxenic group. The lethal dose was between 60 and 80 mg of irinotecan for holoxenic mice and > or =150 mg for the germ-free. The intestinal damage score was higher in holoxenic than germ-free mice at 100 mg and equally diffuse in the small and large bowel. The damage in germ-free mice was less severe (8 of 40 samples) prevailing in the ileum. The differences were significant for all sites (jejunum, P < 0.001; ileum, P = 0.012; cecum, P = 0.001; colon, P < 0.001). No damage was found in germ-free mice at 60 mg. Diarrhea was present in all 100 and 80 mg holoxenic mice and in 19 of 20 cases at 60 mg whereas it was absent in 60 mg or sporadic in 80 and 100 mg germ-free mice. CONCLUSIONS: The intestinal microflora plays a key role in the intestinal toxicity of irinotecan.

Epimerization of chenodeoxycholic acid to ursodeoxycholic acid by Clostridium baratii isolated from human feces.

Ursodeoxycholic acid-producing bacteria are of clinical and industrial interest due to the multiple beneficial effects of this bile acid on human health. This work reports the first isolation of 7-epimerizing bacteria from feces of a healthy volunteer, on the basis of their capacity to epimerize the primary bile acid, chenodeoxycholic acid, to ursodeoxycholic acid. Five isolates were found to be active starting from unconjugated chenodeoxycholic acid and its tauro-conjugated homologue, but none of these strains could epimerize the glyco-conjugated form. Biochemical testing and 16S ribosomal DNA sequencing converged to show that all five isolates were closely related to Clostridium baratii (99% sequence similarity), suggesting that this bacterial species could be responsible at least partially, for this bioconversion in the human gut.

Bacteroides sp. strain D8, the first cholesterol-reducing bacterium isolated from human feces.

The microbial community in the human colon contains bacteria that reduce cholesterol to coprostanol, but the species responsible for this conversion are still unknown. We describe here the first isolation and characterization of a cholesterol-reducing bacterium of human intestinal origin. Strain D8 was isolated from a 10(-8) dilution of a fresh stool sample provided by a senior male volunteer with a high capacity to reduce luminal cholesterol to coprostanol. Cholesterol-to-coprostanol conversion by strain D8 started on the third day, while cells were in stationary phase, and was almost complete after 7 days. Intermediate products (4-cholesten-3-one and coprostanone) were occasionally observed, suggesting an indirect pathway for cholesterol-to-coprostanol conversion. Resting-cell assays showed that strain D8 could reduce 1.5 mumol of cholesterol/mg bacterial protein/h. Strain D8 was a gram-negative, non-spore-forming, rod-shaped organism identified as a member of the genus Bacteroides closely related to Bacteroides vulgatus, based on its morphological and biochemical characteristics. The 16S rRNA gene sequence of strain D8 was most similar (>99.5%) to those of two isolates of the recently described species Bacteroides dorei. Phylogenetic tree construction confirmed that Bacteroides sp. strain D8 clustered within an independent clade together with these B. dorei strains. Nevertheless, no cholesterol-reducing activity could be detected in cultures of the B. dorei type strain. Based on Bacteroides group-specific PCR-temporal temperature gradient gel electrophoresis, there was no correlation between the presence of a band comigrating with the band of Bacteroides sp. strain D8 and cholesterol conversion in 11 human fecal samples, indicating that this strain is unlikely to be mainly responsible for cholesterol conversion in the human population.

Colonization of gnotobiotic mice with human gut microflora at birth protects against Escherichia coli heat-labile enterotoxin-mediated abrogation of oral tolerance.

Previous work has shown that the indigenous gut microflora in mice plays a protective role against Escherichia coli heat-labile enterotoxin (LT)-mediated abrogation of oral tolerance to an unrelated co-ingested protein. To assess potential protection by human gut microflora, we studied the effect of human gut microflora in a murine model. Oral tolerance was studied in adult gnotobiotic mice (i.e. ex-germ-free mice) colonized with the entire human fecal microflora and orally administered once with LT and ovalbumin. Systemic suppression of IgG, IgG1, IgG2a, and IgE antibody responses was assessed by ELISA. Both specific IgG subclasses and IgE hyporesponsiveness was induced in LT + ovalbumin-fed gnotobiotic mice, indicating that the human gut microflora can protect against the LT-mediated abrogation of oral tolerance. However, as confirmed with mouse gut microflora, this protective effect only occurs when the gut microflora is associated from birth on. Colonization of germ-free mice with a single bacterial strain, E. coli, predominant in the human and mouse gut microflora in the neonatal period, showed that this strain alone did not induce protection. These results supported the hypothesis that the natural establishment of the gut microflora in neonates crucially influenced resistance to LT-mediated abrogation of oral tolerance by reinforcing suppression of both T helper type 1- and T helper type 2-controlled responses, and suggested that sequential bacterial colonization of the gut rather than a single bacterial species may be involved in this phenomenon.

Antagonismo exercido por bactérias anaeróbias estritas da flora fecal humana contra Clostrdium perfringens, estudado em camundongos gnotobióticos / antagonism exerted by an association of strict anaerobic bacteria from human fecal flora against clostridium perfringens in gnotobiotic mice

Resumo: O antagonismo entre um número limitado de bactérias anaeróbicas estritas isoladas de uma flora fecal humana e Clostridium perfrigens tipo A (CpA) foi estudado no trato intestinal de camundongos gnotobióticos. Uma associaçäo de 18 bactérias (três bacteroides, cinco peptostreptococcus, cinco eubacterium e cinco clostridium) eliminou CpA dos intestinos de todos os camundongos gnotobióticos em três dias. Durante o processo de simplificaçäo dessa associaçäo de 18 para 7 bactérias houve uma perda na capacidade de eliminar a cepa alvo. Uma eliminaçäo definitiva de CpA foi observada em somente 23 (por cento) dos camundongos inoculados intragastricamente com as 7 bactérias. esses resultados sugerem uma relaçäo complexa entre bactérias diretamente envolvidas no efeito antagonista com cepas auxiliares (au)

Efeito antagonista contra clostridium perfrigens de uma substância produzido por Peptostreptocucus sp. em camundongo gnotobiótico / Anatagonistic effect against clostridium perfrigens of a diffusible copound produced by a peptostreptococcus sp from human intestinal flora in mice

Resumo: Um Peptrostreptocuccus so foi isolado de uma flora intestinal fecal humana. Esse microorganismo produz uma substância difusível apresentando um efeito antagonista contra Clostridium perfringens em camundongo gnotobiótico. Esse composto diluível foi ativo contra a cepa alvo tanto na presença como na ausência de uma microflora intestinal omplexa de origem humana quando foi testado em camundongo gnotobiótico (au)