Evaluation of inter-individual differences in gut bacterial isoflavone bioactivation in humans by PCR-based targeting of genes involved in equol formation.

AIM: To identify human subjects harbouring intestinal bacteria that bioactivate daidzein to equol using a targeted PCR-based approach. METHODS AND RESULTS: In a pilot study including 17 human subjects, equol formation was determined in faecal slurries. In parallel, faecal DNA was amplified by PCR using degenerate primers that target highly conserved regions of dihydrodaidzein reductase and tetrahydrodaidzein reductase genes. PCR products of the expected size were observed for six of the eight subjects identified as equol producers. Analysis of clone libraries revealed the amplification of sequences exclusively related to Adlercreutzia equolifaciens in four of the subjects tested positive for equol formation, whereas in three of the equol producers, only sequences related to Slackia isoflavoniconvertens were observed. No amplicons were obtained for one equol-forming subject, thus suggesting the presence of nontargeted alternative genes. Amplicons were only sporadically observed in the nonequol producers. CONCLUSION: The majority of human subjects who produced equol were also detected with the developed PCR-based approach. SIGNIFICANCE AND IMPACT OF THE STUDY: The obtained results shed light on the distribution and the diversity of known equol-forming bacterial species in the study group and indicate the presence of as yet unknown equol-forming bacteria.

Intestinal alkaline phosphatase at the crossroad of intestinal health and disease - a putative role in type 1 diabetes.

BACKGROUND: Patients with type 1 diabetes have shown an increase in circulating cytokines, altered lipoprotein metabolism and signs of vascular dysfunction in response to high-fat meals. Intestinal alkaline phosphatase (IAP) regulates lipid transport and inflammatory responses in the gastrointestinal tract. We therefore hypothesized that changes in IAP activity could have profound effects on gut metabolic homeostasis in patients with type 1 diabetes. METHODS: Faecal samples of 41 nondiabetic controls and 46 patients with type 1 diabetes were analysed for IAP activity, calprotectin, immunoglobulins and short-chain fatty acids (SCFAs). The impact of oral IAP supplementation on intestinal immunoglobulin levels was evaluated in C57BL/6 mice exposed to high-fat diet for 11 weeks. RESULTS: Patients with type 1 diabetes exhibited signs of intestinal inflammation. Compared to controls, patients with diabetes had higher faecal calprotectin levels, lower faecal IAP activities accompanied by lower propionate and butyrate concentrations. Moreover, the amount of faecal IgA and the level of antibodies binding to oxidized LDL were decreased in patients with type 1 diabetes. In mice, oral IAP supplementation increased intestinal IgA levels markedly. CONCLUSION: Deprivation of protective intestinal factors may increase the risk of inflammation in the gut - a phenomenon that seems to be present already in patients with uncomplicated type 1 diabetes. Low levels of intestinal IgA and antibodies to oxidized lipid epitopes may predispose such patients to inflammation-driven complications such as cardiovascular disease and diabetic nephropathy. Importantly, oral IAP supplementation could have beneficial therapeutic effects on gut metabolic homeostasis, possibly through stimulation of intestinal IgA secretion.

Increased bacterial putrescine has no impact on gut morphology and physiology in gnotobiotic adolescent mice.

Gut bacteria influence host anatomy and physiology. It has been proposed that bacterial metabolites including polyamines are responsible for intestinal maturation and mucosal growth. We have hypothesised that bacterially produced polyamines act as trophic factors and thereby influence large intestinal crypt depth and thickness of the different gut layers. For that purpose, germ-free mice were associated with two different microbial consortia. One group was colonised with a simplified human microbiota (SIHUMI). The second group was associated with SIHUMI + Fusobacterium varium (SIHUMI + Fv), which is known to produce high amounts of polyamines. Polyamine concentrations were measured by HPLC and morphological parameters were determined microscopically. Germ-free and conventional mice served as controls. The caecal putrescine concentration of the SIHUMI + Fv was 61.8 µM (47.6-75.5 µM), whereas that of conventional and SIHUMI mice was 28.8 µM (1.3-41.7 µM) and 24.5 µM (16.8-29.1 µM), respectively. The caecal putrescine concentration of germ-free mice was only 0.6 µM (0-1.0 µM). Caecal crypt depth and thickness of the different caecal layers revealed no significant differences between SIHUMI and SIHUMI + Fv mice. However, the crypt depth in the caeca of conventional, SIHUMI and SIHUMI + Fv mice was increased by 48.6% (P<0.001), 39.7% (P<0.001) and 28.5% (P<0.05), respectively, compared to germ-free mice. These findings indicate that increased intestinal putrescine concentrations do not influence gut morphology in our gnotobiotic adolescent mice.

Evolution of the gut microbiota and the influence of diet.

Diet is a major force that shapes the composition and activity of the gut microbiota. This is evident from alterations in gut microbiota composition after weaning or drastic dietary changes. Owing to the complexity of the microbiota, interactions of intestinal bacteria with the host are difficult to study. Gnotobiotic animal models offer the opportunity to reduce the complexity and the interindividual variability of the intestinal microbiota. Germ-free animals were associated with a simplified microbial community consisting of eight bacterial species, that are found in the human gut. These microbes were selected because their genome sequences are available, and they mimic to some extent the metabolic activity of the human gut microbiota. The microbiota responded to dietary modifications by changes in the relative proportions of the community members. This model offers the chance to better define the role of intestinal bacteria in obesity development, but little is known on how diet affects intestinal bacteria at the cellular level. Mice monoassociated with Escherichia coli were used as a simplified model to investigate the influence of dietary factors on bacterial protein expression in the intestine. The mice were fed three different diets: a carbohydrate (lactose)-rich diet, a protein-rich diet and a diet rich in starch. The lactose-rich diet led to an induction of proteins involved in E. coli's oxidative stress response (Fur, AhpF, Dps). The corresponding genes are under control of the OxyR transcriptional regulator which is activated by oxidative stress. Further experiments demonstrated that osmotic stress exerted by various carbohydrates leads to an upregulation of proteins belonging to the oxyR regulon. The data suggest that the upregulated proteins enable intestinal E. coli to better cope with diet-induced osmotic stress. These examples demonstrate that gnotobiotic animal models are a valuable tool for studying diet-induced changes at the community and the cell level.

Enterococcus faecium NCIMB 10415 does not protect interleukin-10 knock-out mice from chronic gut inflammation.

Enterococcus faecium NCIMB 10415 reduces diarrhoea incidence and duration in animals and human study subjects. We tested whether the strain is also capable of reducing chronic gut inflammation and aimed to identify mechanisms that are involved in possible probiotic effects. To identify health-promoting mechanisms of the strain, we used interleukin-10-deficient mice that spontaneously develop gut inflammation and fed these mice a diet containing NCIMB 10415 for 3, 8 and 24 weeks, respectively. Control mice were fed a diet which was identically composed but did not contain the strain. After 3 weeks of intervention the experimental animals were less inflamed in the caecum than the control animals. This effect was not observed in the colon and there were no differences between experimental and control mice at any other time point. The application of the strain was associated with higher expression levels of interferon gamma and interferon gamma-induced protein 10 after 3 and 24 but not after 8 weeks of feeding. No differences between the animals were observed in intestinal barrier function or intestinal microbiota composition. However, we observed a low abundance of the mucin-degrading bacterium Akkermansia muciniphila in the mice that were fed NCIMB 10415 for 8 weeks. These low cell numbers were associated with a significantly lower caecal inflammation score and improved paracellular permeability as compared to the NCIMB-treated mice that were killed after 3 and 24 weeks of intervention. In conclusion, NCIMB 10415 is not capable of reducing gut inflammation in the IL-10-/- mouse model. The exact role of A. muciniphila and of a possible interaction between this bacterium, NCIMB 10415 and the host in gut inflammation requires further investigation.

Expression of heparan sulfate proteoglycans in murine models of experimental colitis.

BACKGROUND: Heparan sulfate proteoglycans (HSPGs) are considered important in maintaining physiological homeostasis in many systems. Their expression is altered greatly in several pathophysiological conditions. Herein, we assess the expression and cellular localization of HSPGs in two murine models of human inflammatory bowel disease (IBD). METHODS: Expression and localization of HSPGs, syndecans, and HS epitopes were examined in the colon of 129SvEv interleukin 10 knockout (IL10(-/-)), C3Bir IL10(-/-), and their genetic control (IL10(+/+)) counterparts (129SvEv; C3H/HeJ). mRNA expression of syndecans and heparan sulfate biosynthesis enzymes were evaluated by real-time polymerase chain reaction (PCR). Localization of HSPGs was determined by immunofluorescence. RESULTS: mRNA for all syndecans was detected and expression in colonic tissues altered in IL10(-/-) mice. Syndecan-1 protein was expressed in the intestinal epithelium and on lamina propria cells of IL10(-/-) and control mice but was significantly reduced on the intestinal epithelial cells of IL10(-/-), mice particularly with severe colitis. Syndecan-2 was not detected, whereas syndecan-3 immunoreactivity was localized in the lamina propria but did not differ between control and IL10(-/-) mice. Syndecan-4 was present on epithelial cells of all mice but was significantly reduced in IL10(-/-) mice. Differences in the expression of HS epitopes between control and IL10(-/-) mice were also confirmed. CONCLUSIONS: The study has revealed altered expression of syndecan-1 and -4 and HS epitopes in the gut of mice with an IBD-like gut disorder. The IL10(-/-) mouse is a useful model for further study of the functional role of HSPGs in chronic inflammation and in maintaining healthy gut barrier.

Isolation of catechin-converting human intestinal bacteria.

AIMS: To isolate and characterize bacteria from the human intestine that are involved in the conversion of catechins, a class of bioactive polyphenols abundant in the human diet. METHODS AND RESULTS: Two bacterial strains, rK3 and aK2, were isolated from an epicatechin-converting human faecal suspension. The isolates catalysed individual steps in the degradation of ⁻-epicatechin and ⁺-catechin. Based on their phenotypic characteristics and 16S rRNA gene sequences, the isolates were identified as Eggerthella lenta and Flavonifractor plautii (formerly Clostridium orbiscindens). Eggerthella lenta rK3 reductively cleaved the heterocyclic C-ring of both ⁻-epicatechin and ⁺-catechin giving rise to 1-(3,4-dihydroxyphenyl)-3-(2,4,6-trihydroxyphenyl)propan-2-ol. The conversion of catechin proceeded five times faster than that of epicatechin. Higher (epi)catechin concentrations led to an accelerated formation of the ring fission product without affecting the growth of Eg. lenta rK3. Flavonifractor plautii aK2 further converted 1-(3,4-dihydroxyphenyl)-3-(2,4,6-trihydroxyphenyl)propan-2-ol to 5-(3,4-dihydroxyphenyl)-γ-valerolactone and 4-hydroxy-5-(3,4-dihydroxyphenyl)valeric acid. Flavonifractor plautii DSM 6740 catalysed the identical reaction indicating it is not strain specific. CONCLUSIONS: The conversion of dietary catechins by the isolated Eg. lenta and F. plautii strains in the human intestine may affect their bioavailability. SIGNIFICANCE AND IMPACT OF THE STUDY: The majority of catechin metabolites are generated by the intestinal microbiota. The identification of catechin-converting gut bacteria therefore contributes to the elucidation of the bioactivation and the health effects of catechins.

Balancing inflammatory, lipid, and xenobiotic signaling pathways by VSL#3, a biotherapeutic agent, in the treatment of inflammatory bowel disease.

BACKGROUND: The interleukin 10 knockout mouse (IL10-KO) is a model of human inflammatory bowel disease (IBD) used to study host microbial interactions and the action of potential therapeutics. Using Affymetrix data analysis, important signaling pathways and transcription factors relevant to gut inflammation and antiinflammatory probiotics were identified. METHODS: Affymetrix microarray analysis on both wildtype (WT) and IL10-KO mice orally administered with and without the probiotic VSL#3 was performed and the results validated by real-time polymerase chain reaction (PCR), immunocytochemistry, proteomics, and histopathology. Changes in metabolically active bacteria were assessed with denaturing gradient gel electrophoresis (DGGE). RESULTS: Inflammation in IL10-KO mice was characterized by differential regulation of inflammatory, nuclear receptor, lipid, and xenobiotic signaling pathways. Probiotic intervention resulted in downregulation of CXCL9 (fold change [FC] = -3.98, false discovery rate [FDR] = 0.019), CXCL10 (FC = -4.83, FDR = 0.0008), CCL5 (FC = -3.47, FDR = 0.017), T-cell activation (Itgal [FC = -4.72, FDR = 0.00009], Itgae [FC = -2.54 FDR = 0.0044]) and the autophagy gene IRGM (FC = -1.94, FDR = 0.01), a recently identified susceptibility gene in human IBD. Consistent with a marked reduction in integrins, probiotic treatment decreased the number of CCL5+ CD3+ double-positive T cells and upregulated galectin2, which triggers apoptosis of activated T cells. Importantly, genes associated with lipid and PPAR signaling (PPARalpha [FC = 2.36, FDR = 0.043], PPARGC1alpha [FC = 2.58, FDR = 0.016], Nr1d2 [FC = 3.11, FDR = 0.0067]) were also upregulated. Altered microbial diversity was noted in probiotic-treated mice. CONCLUSIONS: Bioinformatics analysis revealed important immune response, phagocytic and inflammatory pathways dominated by elevation of T-helper cell 1 type (TH1) transcription factors in IL10-KO mice. Probiotic intervention resulted in a site-specific reduction of these pathways but importantly upregulated PPAR, xenobiotic, and lipid signaling genes, potential antagonists of NF-kappaB inflammatory pathways.

Exacerbation of murine ileitis by Toll-like receptor 4 mediated sensing of lipopolysaccharide from commensal Escherichia coli.

BACKGROUND: In the course of inflammatory bowel diseases (IBD) and acute murine ileitis following peroral Toxoplasma gondii infection, commensal Escherichia coli accumulate at inflamed mucosal sites and aggravate small intestinal immunopathology. AIM: To unravel the molecular mechanisms by which commensal E coli exacerbate ileitis. METHODS: Ileitis was investigated in mice that lack Toll-like receptors (TLR) 2 or 4, specific for bacterial lipoproteins (LP) or lipopolysaccharide (LPS), respectively. Gnotobiotic mice, in which any cultivable gut bacteria were eradicated by antibiotic treatment, were used to study the role of LPS in ileitis. RESULTS: Microbiological analyses revealed that E coli increase in the inflamed ileum. TLR4(-/-), but not TLR2(-/-), mice displayed reduced mortality and small intestinal immunopathology. Decreased interferon (IFN)-gamma and nitric oxide (NO) levels in the inflamed terminal ileum of TLR4(-/-) mice indicated that TLR4 signalling aggravates ileitis via local mediator release from immune cells. E coli strains isolated from the inflamed ileum activated cultured mouse macrophages and induced TLR4-dependent nuclear factor kappaB activation and NO production in human embryonic kidney 293 cells and in peritoneal macrophages, respectively. Most strikingly, in contrast with wild-type mice, gnotobiotic TLR4(-/-) mice were protected from induction of ileitis by treatment with purified E coli lipid A or colonisation with live E coli. Finally, prophylactic treatment with the LPS scavenger polymyxin B ameliorated T gondii-induced ileitis. CONCLUSION: These findings highlight the innate immune system as a key player in T gondii-induced ileal immunopathology. Treatment with LPS or TLR4 antagonists may represent a novel strategy for prophylaxis and/or therapy of small intestinal inflammation in IBD.

Bacterial and fungal microbiota in relation to probiotic therapy (VSL#3) in pouchitis.

BACKGROUND: The intestinal microbiota plays a critical role in the pathophysiology of pouchitis, a major complication after ileal pouch anal anastomosis in patients with ulcerative colitis. Recently, controlled trials have demonstrated that probiotics are effective in maintenance of remission in pouchitis patients. However, the mechanism by which therapy with probiotics works remains elusive. This study explores the role of the bacterial and fungal flora in a controlled trial for maintenance of remission in pouchitis patients with the probiotic VSL#3 compound. METHODS: The mucosa associated pouch microbiota was investigated before and after therapy with VSL#3 by analysis of endoscopic biopsies using ribosomal DNA/RNA based community fingerprint analysis, clone libraries, real time polymerase chain reaction (PCR), and fluorescence in situ hybridisation. Patients were recruited from a placebo controlled remission maintenance trial with VSL#3. RESULTS: Patients who developed pouchitis while treated with placebo had low bacterial and high fungal diversity. Bacterial diversity was increased and fungal diversity was reduced in patients in remission maintained with VSL#3 (p = 0.001). Real time PCR experiments demonstrated that VSL#3 increased the total number of bacterial cells (p = 0.002) and modified the spectrum of bacteria towards anaerobic species. Taxa specific clone libraries for Lactobacilli and Bifidobacteria showed that the richness and spectrum of these bacteria were altered under probiotic therapy. CONCLUSIONS: Probiotic therapy with VSL#3 increases the total number of intestinal bacterial cells as well as the richness and diversity of the bacterial microbiota, especially the anaerobic flora. The diversity of the fungal flora is repressed. Restoration of the integrity of a "protective" intestinal mucosa related microbiota could therefore be a potential mechanism of probiotic bacteria in inflammatory barrier diseases of the lower gastrointestinal tract.

Effect of isomalt consumption on faecal microflora and colonic metabolism in healthy volunteers.

Due to its low digestibility in the small intestine, a major fraction of the polyol isomalt reaches the colon. However, little is known about effects on the intestinal microflora. During two 4-week periods in a double-blind, placebo-controlled, cross-over design, nineteen healthy volunteers consumed a controlled basal diet enriched with either 30 g isomalt or 30 g sucrose daily. Stools were collected at the end of each test phase and various microbiological and luminal markers were analysed. Fermentation characteristics of isomalt were also investigated in vitro. Microbiological analyses of faecal samples indicated a shift of the gut flora towards an increase of bifidobacteria following consumption of the isomalt diet compared with the sucrose diet (P<0.05). During the isomalt phase, the activity of bacterial beta-glucosidase decreased (P<0.05) whereas beta-glucuronidase, sulfatase, nitroreductase and urease remained unchanged. Faecal polyamines were not different between test periods with the exception of cadaverine, which showed a trend towards a lower concentration following isomalt (P=0.055). Faecal SCFA, lactate, bile acids, neutral sterols, N, NH3, phenol and p-cresol were not affected by isomalt consumption. In vitro, isomalt was metabolized in several bifidobacteria strains and yielded high butyrate concentrations. Isomalt, which is used widely as a low-glycaemic and low-energy sweetener, has to be considered a prebiotic carbohydrate that might contribute to a healthy luminal environment of the colonic mucosa.

Bacterial response to eukaryotic cells. Analysis of differentially expressed proteins using nano liquid chromatography-electrospray ionization tandem mass spectrometry.

Host-bacteria interactions have mostly been investigated with regard to the host response or to activities of pathogenic bacteria. In contrast, we aim to identify reactions of non-pathogenic bacteria that result from their contact with host cells of the gastrointestinal tract. In a proteomic approach, the response of non-pathogenic human Escherichia coli bacteria on gut epithelial cells (rat IEC-6) was investigated in an in vitro co-culture model. For this purpose, a sensitive analytical procedure was developed based on the identification of two-dimensional polyacrylamide gel electrophoresis separated proteins by online nanoLC-electrospray ionization MS/MS using a quadrupole time-of-flight tandem mass spectrometer for accurate mass determination. We demonstrate here the efficiency of this technique by the identification of a total of 43 differentially expressed proteins, out of which 25 were up-regulated and 18 were down-regulated. They represent a wide range of molecular weight and different metabolic and physiological functions.

An improved method for the automated enumeration of fluorescently labelled bacteria in human faeces.

The study aimed to improve microscopy-based automated recognition of faecal bacterial cells labelled with 16S rRNA-targeted oligonucleotides and 4',6-diamidino-2-phenylindole (DAPI). Based on the software KS400 (Carl Zeiss Vision, Hallbergmoos, Germany), designed for automising microscopy-based image capture and image analysis, a routine was developed that affords the recognition of doubly stained bacteria and the rejection of artefacts. The accuracy of the automated enumeration was investigated by comparing the resulting data with those obtained by manual counting. The newly developed method was subsequently used to compare the total bacterial counts in human faecal samples using the domain specific probe Eub338 alone and a mixture of 5 domain-specific probes, respectively. Faecal samples from 90 healthy volunteers were analysed. The cell counts obtained with Eub338 were 10% lower than those obtained with the probe mixture. Since the cells detected with the probe mixture covered a wide range of signal intensities, a dynamic analysis routine was developed to effectively detect the whole range of bright to weak signals within the same image, while at the same time reliably rejecting artefacts.

Mucosal and invading bacteria in patients with inflammatory bowel disease compared with controls.

BACKGROUND: Endogenous intestinal bacteria and/or specific bacterial pathogens are suspected of being involved in the pathogenesis of inflammatory bowel diseases (IBD). The aim of this study was to investigate IBD tissues for different bacterial population groups harbouring the mucosal surface and/or invading the mucosa. METHODS: Tissue sections from surgical resections from the terminal ileum and/or the colon from 24 IBD patients (12 active ulcerative colitis (UC), 12 active Crohn disease (CD)) and 14 non-IBD controls were studied by fluorescent in situ hybridization on a quantifiable basis. RESULTS: More bacteria were detected on the mucosal surface of IBD patients than on those of non-IBD controls (P < 0.05). Bacterial invasion of the mucosa was evident in 83.3% of colonic specimens from the UC patients, in 55.6% of the ileal and in 25% of the colonic specimens from the CD patients, but no bacteria were detected in the tissues of the controls. Colonic UC specimens were colonized by a variety of organisms, such as bacteria belonging to the gamma subdivision of Proteobacteria, the Enterobacteriaceae, the Bacteroides/Prevotella cluster, the Clostridium histolyticum/Clostridium lituseburense group, the Clostridium coccoides/Eubacterium rectale group, high G + C Gram-positive bacteria, or sulphate-reducing bacteria, while CD samples harboured mainly bacteria belonging to the former three groups. CONCLUSION: Pathogenic events in CD and UC may be associated with different alterations in the mucosal flora of the ileum and colon.

Influence of resistant starch on the SCFA production and cell counts of butyrate-producing Eubacterium spp. in the human intestine.

AIMS: The genus Eubacterium, which is the second most common genus in the human intestine, includes several known butyrate producers. We hypothesized that Eubacterium species play a role in the intestinal butyrate production and are inducible by resistant starch. METHODS AND RESULTS: In a human pilot study species-specific and group-specific 16S rRNA-targeted, Cy3 (indocarbocyanine)-labelled oligonucleotide probes were used to quantify butyrogenic species of the genera Eubacterium, Clostridium and Ruminococcus. Following the intake of RS type III a significant increase in faecal butyrate but not in total SCFA was observed. However, increase in butyrate was not accompanied by a proliferation in the targeted bacteria. CONCLUSIONS: The tested Eubacterium species have the capacity to produce butyrate but do not appear to play a major role for butyric acid production in the human intestine. SIGNIFICANCE AND IMPACT OF THE STUDY: In view of the fact that the bacteria responsible for butyrate production are largely unknown, it is still difficult to devise a dietary intervention to stimulate butyrogenic bacteria in a targeted way.

Molecular biological methods for studying the gut microbiota: the EU human gut flora project.

Seven European laboratories co-operated in a joint project (FAIR CT97-3035) to develop, refine and apply molecular methods towards facilitating elucidation of the complex composition of the human intestinal microflora and to devise robust methodologies for monitoring the gut flora in response to diet. An extensive database of 16S rRNA sequences for tracking intestinal bacteria was generated by sequencing the 16S rRNA genes of new faecal isolates and of clones obtained by amplification with polymerase chain reaction (PCR) on faecal DNA from subjects belonging to different age groups. The analyses indicated that the number of different species (diversity) present in the human gut increased with age. The sequence information generated, provided the basis for design of 16S rRNA-directed oligonucleotide probes to specifically detect bacteria at various levels of phylogenetic hierarchy. The probes were tested for their specificity and used in whole-cell and dot-blot hybridisations. The applicability of the developed methods was demonstrated in several studies and the major outcomes are described.

[Bifidobacterium adolescentis suppresses the humoral immune response to an autochthonous intestinal bacterium--experiments with gnotobiotic rats]. / Bifidobacterium adolescentis supprimiert die humorale Immunantwort gegen ein autochthones Darmbakterium--Versuche an gnotobiotischen Ratten.

On the basis of association-experiments with gnotobiotic rats, we described the immunogenicity of two selected bacterial species (Bifidobacterium adolescentis and Bacteroides thetaiotaomicron). B. adolescentis is a gram-positive lactic acid producing bacterium, strains of which are claimed to have probiotic properties. B. thetaiotaomicron is a gram-negative rod, autochthonous to the human as well as to the rats' intestinal tract. Colonization of the gut was monitored by determination of bacterial cell counts in the animals' feces. In order to investigate the systemic immune reaction, the amounts of specific serum-IgG and -IgA against both bacterial species were measured in the serum. The intestinal immune reaction was examined by measuring the specific IgA in the rats' feces. Knowing about the antibody levels in gnotobiotic rats induced by monoassociation we subsequently disassociated two groups of rats in order to investigate the impact of B. adolescentis on the immune reaction against B. thetaiotaomicron. One group was disassociated simultaneously with B. adolescentis and B. thetaiotaomicron, the second group was disassociated with these bacteria in sequence. B. adolescentis was merely able to induce a mucosal immune reaction, while B. thetaiotaomicron challenged the mucosal as well as the systemic immune system. Furthermore B. adolescentis obviously suppressed the systemic and mucosal immune reaction against the autochthonous B. thetaiotaomicron.

A fluorescence quenching test for the detection of flavonoid transformation.

A novel fluorescence quenching test for the detection of flavonoid degradation by microorganisms was developed. The test is based on the ability of the flavonoids to quench the fluorescence of 1,6-diphenyl-1,3,5-hexatriene (DPH). Several members of the anthocyanidins, flavones, isoflavones, flavonols, flavanones, dihydroflavanones, chalcones, dihydrochalcones and catechins were tested with regard to their quenching properties. The anthocyanidins were the most potent quenchers of DPH fluorescence, while the flavanones, dihydroflavanones and dihydrochalcones, quenched the fluorescence only weakly. The catechins had no visible impact on DPH fluorescence. The developed test allows a quick and easy differentiation between flavonoid-degrading and flavonoid-non-degrading bacteria. The investigation of individual reactions of flavonoid transformation with the developed test system is also possible.

Degradation of quercetin and luteolin by Eubacterium ramulus.

The degradation of the flavonol quercetin and the flavone luteolin by Eubacterium ramulus, a strict anaerobe of the human intestinal tract, was studied. Resting cells converted these flavonoids to 3,4-dihydroxyphenylacetic acid and 3-(3,4-dihydroxyphenyl)propionic acid, respectively. The conversion of quercetin was accompanied by the transient formation of two intermediates, one of which was identified as taxifolin based on its specific retention time and UV and mass spectra. The structure of the second intermediate, alphitonin, was additionally elucidated by (1)H and (13)C nuclear magnetic resonance analysis. In resting-cell experiments, taxifolin in turn was converted via alphitonin to 3,4-dihydroxyphenylacetic acid. Alphitonin, which was prepared by enzymatic conversion of taxifolin and subsequent purification, was also transformed to 3,4-dihydroxyphenylacetic acid. The coenzyme-independent isomerization of taxifolin to alphitonin was catalyzed by cell extract or a partially purified enzyme preparation of E. ramulus. The degradation of luteolin by resting cells of E. ramulus resulted in the formation of the intermediate eriodictyol, which was identified by high-performance liquid chromatography and mass spectrometry analysis. The observed intermediates of quercetin and luteolin conversion suggest that the degradation pathways in E. ramulus start with an analogous reduction step followed by different enzymatic reactions depending on the additional 3-hydroxyl group present in the flavonol structure.