The microbial community resident in the human gastrointestinal tract has a major impact on host physiology. Manipulating this complex ecosystem by dietary intervention requires knowledge of the parameters that influence its composition and the activity. More recent developments have taken advantage of culture-independent molecular methods for bacterial identification on the basis of the highly discriminatory sequence database of 16S rRNA. This culture-independent approach was applied to asses the fecal microbiota of human subjects from different European countries in relation to nutrition. In one of two human studies, a positive correlation was observed between the intake of fruits and vegetables and the proportion of bacteria belonging to the Clostridium leptum group. In the second study, the relative proportion of bifidobacteria was much higher in the Italian study group than in any other study group. This finding correlated positively with a consumption of water-soluble fiber. Intestinal bacteria also play a role in the activation and inactivation of plant-derived polyphenolic substances such as lignans and flavonoids. To assess the influence of bacterial metabolism on the bioavailability of flavonoids in the human intestinal tract, two model organisms were used to characterize the transformation of flavones in comparison to that of flavonols. Lignans require activation by intestinal bacteria to exert estrogenic and antioxidant activities. Several bacterial species involved in lignan activation were isolated and the catalytic steps involved were characterized. The results indicate that the activation of dietary lignans involves phylogenetically diverse bacteria, most of which are common members of the dominant human intestinal microbiota.
Systemic treatment with infliximab, a monoclonal antibody that binds TNFα is among the most potent therapies for Crohn's disease. Despite initial successes with intravenous addition of IL-10, this treatment was abandoned in later trials. Both treatments suffered a serious drawback from the fact that they had to be administered systemically and thus caused potentially serious side effects. We used two mouse models for IBD to evaluate the efficacy of L. lactis mediated topical delivery of IL-10. In the Dextran Sulphate Sodium (DSS)-induced chronic colitis intragastric administration of mIL-10-secreting L. lactis led to a 50% reduction in inflammation. Also, daily administration of the engineered strain prevented the onset of colitis, normally associated with the IL-10 knockout genotype. In both models, the effect was strictly dependent on delivery of live bacteria. We engineered a strain exhibiting biological containment. To this end, the thymidilate synthase (thyA) gene of L. lactis was replaced with a synthetic human IL-10 gene. ThyA-deficient bacteria are suicidal in the absence of thymine or thymidine and therefore cannot accumulate in the environment. A limited clinical trail in Crohn's patients under physical containment proved that the treatment was safe, the biological containment strategy was effective and the results obtained suggested a clinical effect. In contrast to oral administration of purified protein, intragastric administration of Trefoil factor-secreting L. lactis was very effective in prevention and healing of acute DSS colitis. In addition this approach was successful in improving established chronic colitis in IL-10 knockout mice.
We have developed a terminal restriction fragment length polymorphism (T-RFLP) method for determining the structure and dynamics of the microbial gut community. In this paper, the improved T-RFLP method in combination with an analysis of the fecal 16S rDNA (ribosomal RNA gene) clone libraries from six individuals is described. A total of 418 different partial sequences of 16S rDNA were determined and subjected to a phylogenetic analysis and homology examination. We found that the sequences were roughly divided into six phylogenetic groups containing seven subgroups and were related to 71 known species with over 90% similarity. With the exception of a few cases, we found that in silico BslI-digestion of the sequences belonging to the same bacterial group or subgroup generated terminal restriction fragments of similar lengths. We concluded that human intestinal microbiota predominantly consists of the members of approximately ten phylogenetic bacterial groups and that these bacterial groups are effectively distinguished by our T-RFLP system.
A synthetic mixture of fructo-oligosaccharides (mFOS), consisting largely of nystose (GF3) and a lesser amount of 1-kestose (GF2) has been reported to be selectively utilized by bifidobacteria. In the present study, we tried to identify which fructo-oligosaccharide molecule in mFOS is really involved in the stimulation of bifidobacteria in the gut, using both the gnotobiotic murine model and in vitro culture. 1-Kestose administration to gnotobiotic mice that were associated with human fecal microbiota significantly increased the number of bifidobacteria while mFOS administration was unable to sustain bifidobacteria in these hosts. Moreover a simultaneous decrease in the number of clostridia was found in host mice administered 1-kestose but not in those administered mFOS. The acetate/propionate ratio in the feces was far higher in host mice administered 1-kestose than in those administered mFOS, suggesting the selective growth activation of bifidobacteria by 1-kestose. The culture study demonstrated that 1-kestose exerts a strong growth-stimulating activity on bifidobacteria but a negligible one on clostridia. On the other hand, nystose was able to stimulate clostridia if the clostridia were exposed to nystose for some time. These results suggest the superiority of 1-kestose to mFOS, which consists largely of nystose, in the selective stimulating activity on bifidobacteria.
A polyclonal antibody of Salmonella enterica serovar Enteritidis (SE), designated RY542, inhibited SE from attaching to and invading the human intestinal epithelial cell line, Caco-2, in a dose-dependent manner. A major immunoreactive band of 55 kDa obtained in western blotting using RY542 was identified as FliC. An affinity-purified anti-flagellin antibody from RY542 similarly inhibited the infection. These results suggest that RY542 is a neutralizing antibody that blocks the infection of SE in Caco-2 cells by interacting with flagella, and that flagellin may potentially be useful as a component of a Salmonella vaccine.