Isolated fecal microorganisms capable of 7-alpha-dehydroxylating bile acids.

Strains of microorganisms capable of 7alpha-dehydroxylation of chenodeoxycholate were isolated from rat and human feces. All the strains were strictly anaerobic, non-motile, moderately themioresistant Gram-positive rods. They showed some saccharolytic properties with the production of both acid and gas. They were H(2)S-positive but indole-, skatole-, citrate-, catalase-, and oxidase-negative. The isolated strains capable of 7alpha-dehydroxylation of chenodeoxycholate were also able to oxidize the hydroxyl groups at C-3 and C-7 to keto groups. The following metabolites were isolated: 3-keto-7alpha-hydroxy-5beta-cholanoic acid, 3alpha-hydroxy-7-keto-5beta-cholanoic acid, 3alpha-hydroxy-5beta-cholanoic acid, and 3-keto-5beta-cholanoic acid. The isolated strains did not have the enzymes necessary for hydrolyzing conjugated bile acids. In mixed anaerobic cultures of fecal microorganisms, extensive reduction of the 3-keto group to the 3beta-hydroxyl group occurred. The microorganism(s) responsible for this reaction have as yet not been isolated.

A model of host-microbial interactions in an open mammalian ecosystem.

The maintenance and significance of the complex populations of microbes present in the mammalian intestine are poorly understood. Comparison of conventionally housed and germ-free NMRI mice revealed that production of fucosylated glycoconjugates and an alpha1, 2-fucosyltransferase messenger RNA in the small-intestinal epithelium requires the normal microflora. Colonization of germ-free mice with Bacteroides thetaiotaomicron, a component of this flora, restored the fucosylation program, whereas an isogenic strain carrying a transposon insertion that disrupts its ability to use L-fucose as a carbon source did not. Simplified models such as this should aid the study of open microbial ecosystems.

Secreted enteric antimicrobial activity localises to the mucus surface layer.

OBJECTIVES: The intestinal mucosa is constantly exposed to a dense and highly dynamic microbial flora and challenged by a variety of enteropathogenic bacteria. Antibacterial protection is provided in part by Paneth cell-derived antibacterial peptides such as the alpha-defensins. The mechanism of peptide-mediated antibacterial control and its functional importance for gut homeostasis has recently been appreciated in patients with Crohn's ileitis. In the present study, the spatial distribution of antimicrobial peptides was analysed within the small intestinal anatomical compartments such as the intestinal crypts, the overlaying mucus and the luminal content. METHODS: Preparations from the different intestinal locations as well as whole mouse small intestine were extracted and separated by reversed-phase high-performance liquid chromatography. Antibacterial activity was determined in extracts, and the presence of antimicrobial peptides/proteins was confirmed by N-terminal sequencing, mass spectrometry analysis and immunodetection. RESULTS: The secreted antibacterial activity was largely confined to the layer of mucus, whereas only minute amounts of activity were noted in the luminal content. The extractable activity originating from either crypt/mucus/lumen compartments respectively (given as a percentage) was for Listeria monocytogenes, 48 (4)/44 (4)/8 (8); Enterococcus faecalis, 44 (10)/49 (3)/7 (7); Bacterium megaterium, 56 (4)/42 (3)/2 (1); Streptococcus pyogenes, 48 (4)/46 (3)/6 (6); Escherichia coli, 46 (4)/47 (3)/7 (7); and Salmonella enterica sv. Typhimurium, 38 (3)/43 (7)/19 (10). A spectrum of antimicrobial peptides was identified in isolated mucus, which exhibited strong and contact-dependent antibacterial activity against both commensal and pathogenic bacteria. CONCLUSION: These findings show that secreted antimicrobial peptides are retained by the surface-overlaying mucus and thereby provide a combined physical and antibacterial barrier to prevent bacterial attachment and invasion. This distribution facilitates high local peptide concentration on vulnerable mucosal surfaces, while still allowing the presence of an enteric microbiota.

Therapeutic potential of an anaerobic cultured human intestinal microbiota, ACHIM, for treatment of IBS.

By administering an anaerobic cultivated human intestinal microbiota (ACHIM) via upper gastrointestinal route using endoscopy we aimed to rectify intestinal dysbiosis and simultaneously achieve a treatment response in IBS patients. The study population fulfilled the Rome III IBS criteria and comprised 50 patients. During 10 days, patients recorded the irritable bowel syndrome symptom severity scale (IBS-SSS) along with the Bristol stool scale and number of stools/day. The enrolled patients were categorized as follows: 37 with diarrhea, 5 with constipation and 8 with mixed symptoms. The treatment response showed reduction in a majority of patients, 32 of which with 50-point reduction of IBS-SSS and 21 with a 100-point IBS-SSS reduction. The percentage improvement was 36 (23-49) and 28 (18-38) for women and men respectively. Short-chain fatty acids were not changed. We consider fecal microbiota transplantation in the form of ACHIM as an option for the future therapeutic armamentarium in IBS. REGISTERED TRIAL: www.clinicaltrials.gov NCT02857257.

Creating and maintaining the gastrointestinal ecosystem: what we know and need to know from gnotobiology.

Studying the cross talk between nonpathogenic organisms and their mammalian hosts represents an experimental challenge because these interactions are typically subtle and the microbial societies that associate with mammalian hosts are very complex and dynamic. A large, functionally stable, climax community of microbes is maintained in the murine and human gastrointestinal tracts. This open ecosystem exhibits not only regional differences in the composition of its microbiota but also regional differences in the differentiation programs of its epithelial cells and in the spatial distribution of its component immune cells. A key experimental strategy for determining whether "nonpathogenic" microorganisms actively create their own regional habitats in this ecosystem is to define cellular function in germ-free animals and then evaluate the effects of adding single or several microbial species. This review focuses on how gnotobiotics-the study of germ-free animals-has been and needs to be used to examine how the gastrointestinal ecosystem is created and maintained. Areas discussed include the generation of simplified ecosystems by using genetically manipulatable microbes and hosts to determine whether components of the microbiota actively regulate epithelial differentiation to create niches for themselves and for other organisms; the ways in which gnotobiology can help reveal collaborative interactions among the microbiota, epithelium, and mucosal immune system; and the ways in which gnotobiology is and will be useful for identifying host and microbial factors that define the continuum between nonpathogenic and pathogenic. A series of tests of microbial contributions to several pathologic states, using germ-free and ex-germ-free mice, are proposed.

Alteration in human defensin-5 expression following gastric bypass surgery.

BACKGROUND: Roux-en-Y gastric bypass surgery provides a novel human model to investigate small bowel mucosal innate immunity, in which there is loss of gastric acid-mediated protection against orally-acquired microorganisms. AIM: To study changes in jejunal mucosal immunoreactivity of human defensin (HD)-5, an antimicrobial peptide normally produced by Paneth cells. METHODS: Mucosal samples were obtained from 18 female patients (24-54 years), from the same segment of jejunum during and after gastric bypass surgery. Samples were used for bacterial culture and immunohistochemistry using anti-HD-5 antibody. The number of immunoreactive cells per crypt and villus were determined and expressed as mean (SD). RESULTS: No bacteria were cultured from any of the perioperative jejunal samples but colonies of bacteria normally present in the pharynx were identified during culture of all postoperative jejunal biopsy specimens (1->100 colonies). Paneth cell numbers per crypt were unchanged after gastric bypass (4.16 (0.71) vs 4.24 (0.78)). However, following surgery, there was an increase in HD-5-positive intermediate cells per crypt (0.25 (0.41) vs 1.12 (0.66), p<0.01), HD-5 staining enterocytes per crypt (0.03 (0.09) vs 1.38 (1.10), p<0.01), HD-5 staining material in the crypt lumen (crypt lumens: 5.0% (10.9%) vs 68.1% (27.9%), p<0.01) and HD-5 immunoreactivity coating the luminal surface of villus enterocytes (villi sampled: 15.0% (31.0%) vs 67.5% (42.0%), p<0.01). CONCLUSIONS: Bacteria normally resident in the pharynx were present in the proximal jejunal mucosa following Roux-en-Y gastric bypass surgery. After gastric bypass, there was increased secretion of HD-5 and an increase in HD-5 expressing intermediate cells and enterocytes in the crypt. The increase in HD-5 expression in the jejunal mucosa following gastric bypass surgery is likely to be secondary to exposure to orally-acquired microorganisms.

A glycoprotein of high molecular weight in the urine of germ-free rats.

A carbohydrate-rich glycoprotein fraction was obtained by purification of the non-dialysable material of germ-free rat urine. This procedure involved proteolytic digestion with pronase, presipitation of the acidic glycoprotein with cetyltrimethyl-ammonium bromide (CTAB), and chromatography on DEAE-Sephadex and finally on Sepharose 4B from which it was partly excluded. The purified glycoprotein contained fucose (10.1%), galactose (12.2%).N-acetylglucosamine (16.6%), N-acetylgalactosamine (19.8%), sialic acid (11.9%), and sulphate ester groups (0.7%). The amino acid content was 19%, threonine, serine and proline being the predominating amino acids. Gel electrophoresis and gel chromatography revealed a pronounced poly-dispersity of the material. Treatment of the glycoprotein with alkali in the presence of borohydride led to partial degradation of the polymer with the formation of fragments containing N-acetylgalactosaminitol. The chemical properties of the purified glycoprotein are characteristic of a mucin. The presence of this glycoprotein could not be demonstrated in conventional rat urine.

Influence of ofloxacin on the faecal flora.

In a continuing, Norwegian-Swedish collaborative study, the effects of several antimicrobials upon intestinal biochemical microflora-associated characteristics are being investigated. In the present study, 7 healthy volunteers were given ofloxacin 200mg bid for 6 days. Faecal sampling was performed twice before, on days 3 and 6 during treatment, and weekly to monthly afterwards. Blood sampling was performed on day 6 during treatment. The concentrations of ofloxacin were measured in faeces and serum on day 6. Additionally, the following microflora-associated characteristics were studied in all the faecal samples: presence of short chain fatty acids, absence of beta-aspartylglycine, production of coprostanol, formation of urobilinogen, inactivation of tryptic activity and breakdown of mucin. The mean concentrations of ofloxacin in faeces and serum were 44.0 mg/kg and 2.1 mg/L, respectively. In contrast to most of the other antimicrobials so far tested, ofloxacin exerted almost no influence upon the microflora-associated characteristics investigated.

Sulphasalazine, olsalazine and sulphapyridine induce mitogenic actions in the rat intestinal epithelium.

Our aim was to study the influence of sulphasalazine (SASP), olsalazine (ADS) and sulphapyridine (SP) on the cell kinetics of the intestinal epithelium in conventional rats. Groups of rats were treated with SASP, ADS or SP for 9 days. After an intraperitoneal injection of a metaphase blocker, the rats were killed and the jejunum, ileum and colon were examined in histological sections by means of the cumulative mitotic index (MI), growth fraction and number of cells in crypts and villi. SP increased both the MI in the jejunum, ileum and colon and the number of crypt cells (p < 0.05 vs controls). In contrast, SASP and ADS increased the MI only in the colonic epithelium (p < 0.05 vs controls). The growth fraction was essentially unaffected. Our results suggest that SASP, SP and ADS have a selective compartment-dependent proliferative action on the epithelium of the intestinal tract.

Intestinal microbial conversion of cholesterol to coprostanol in man. Influence of antibiotics.

The intestinal microbial conversion of cholesterol to coprostanol has been measured in groups of healthy subjects before, during and after they received the antibiotics ampicillin, bacitracin, clindamycin, co-trimoxazole, doxycycline, erythromycin, metronidazole, nalidixic acid, ofloxacin or vancomycin orally for 6 days. Before they received antibiotics, the subjects demonstrated two distinct patterns of cholesterol conversion. One pattern was characterised by extensive conversion of cholesterol, the other by little or no conversion. Intake of bacitracin, clindamycin, erythromycin, metronidazole and vancomycin significantly reduced the conversion to coprostanol. In the groups receiving ampicillin or doxycycline, marked reductions were found in most of the subjects. No alterations were found in the groups receiving co-trimoxazole, nalidixic acid or ofloxacin. In 6 subjects no conversion of cholesterol to coprostanol was found up to 5 weeks after the end of the antibiotic intake. We conclude that orally given antibiotics may cause alterations in the intestinal conversion of cholesterol, reflecting changes in the anaerobic, Gram-positive component of the gut flora.

Gas supersaturation in the cecal wall of mice due to bacterial CO2 production.

PCO2 in the lumen and serosa of cecum and jejunum was measured in mice. The anesthetic used was a fentanyl-fluanisone-midazolam mixture. PCO2 was recorded in vivo and postmortem. PCO2 was 409 +/- 32 Torr (55 +/- 4 kPa) in the cecal lumen and 199 +/- 22 Torr (27 +/- 3 kPa) on the serosa in normal mice. Irrigation of the cecum resulted in serosal and luminal PCO2 levels of 65-75 Torr. Cecal PCO2 was significantly lower in germ-free mice (65 +/- 5 Torr). Cecal PCO2 increased significantly after introduction of normal bacterial flora into germ-free mice. Introduction of bacterial monocultures into germ-free mice had no effect. After the deaths of the mice, cecal PCO2 increased rapidly in normal mice. The intestinal bacteria produced the majority of the cecal PCO2, and the use of tonometry in intestinal segments with a high bacterial activity should be interpreted with caution. We propose that serosal PCO2 levels >150-190 Torr (20-25 kPa) in the cecum of mice with a normal circulation may represent a state of gas supersaturation in the cecal wall.

Ca(2+)-dependent and Ca(2+)-independent exhaled nitric oxide, presence in germ-free animals, and inhibition by arginine analogues.

Nitric oxide (NO) was detected by chemiluminescence in exhaled air from awake humans, anaesthetized rabbits, guinea pigs, germ-free rats and conventional rats. Rabbits exhibited the highest concentrations, followed by guinea pigs, humans and rats. There was no significant difference between germ-free rats and control rats. The authenticity of NO was confirmed in cold-trap experiments. Intravenous administration of inhibitors of NO synthase (0.01-300 mg kg-1) to guinea pigs dose dependently reduced NO concentrations in exhaled air with the following potency order: L-N omega-nitro-arginine-methylester > asymmetric NG,NG-dimethyl-L-arginine-dihydrochloride = L-NG-mono-methyl -arginine = L-N5- (1-iminoethyl)-ornithine = aminoguanidine > L-canavanine. The effect of the NO synthase inhibitors was partly or fully reversed by L-arginine (1 g kg-1 i.v.), and L-arginine per se induced a significant increment of NO in exhaled air. In rats, L-N omega-nitro-arginine-methylester was considerably less potent than in guinea pigs. The concentration of NO in exhaled air increased 3-fold when changing from in situ blood auto-perfusion of rabbit lungs to in situ perfusion with saline medium. Addition of L-N omega-nitro-arginine-methylester to the saline perfusion medium evoked a reduction of NO concentrations in the air from the ventilated perfused lungs. Perfusion of lungs with Ca(2+)-free medium induced significant decrements in NO concentrations in exhaled air, an effect partly reversed upon reintroducing Ca2+ into the medium. In conclusion, NO was detected in exhaled air from humans and animals by chemiluminescence.(ABSTRACT TRUNCATED AT 250 WORDS)

Probiotic mixture decreases DNA adduct formation in colonic epithelium induced by the food mutagen 2-amino-9H-pyrido[2,3-b]indole in a human-flora associated mouse model.

Consumption of probiotic bacteria such as bifidobacteria has been shown to reduce the risk of colon cancer in animal models. However, the composition and metabolic activities of the intestinal flora of experimental animals are significantly different from those of humans. The aim of the study was to examine whether the probiotic mixture, which consisted of Streptococcus faecalis, Clostridium butyricum and Bacillus mesentericus, could decrease DNA adduct formation induced by 2-amino-9H-pyrido[2,3-b]indole (2-amino-alpha-carboline; AAC) in the colonic epithelium of a human-flora-associated (HFA) mouse model. Ten HFA mice were divided into a control group (n=4) and a probiotic group (n=6). The control group was administered AAC for 3 days and sacrificed 24 h after the last dose. The probiotic group was administered the probiotic mixture for 2 weeks prior to the administration of AAC. Analysis of DNA adducts with the 32P-high-performance liquid chromatography method was performed on stomach, jejunum and colonic epithelium, representing direct exposure sites of AAC, and colon wall, liver and kidney, representing indirect exposure sites. The mean level of the DNA adducts in the colonic epithelium of the probiotic group was significantly lower than that of control group, while the mean levels at the other sites did not differ significantly between the groups. The results indicated that the probiotic mixture could decrease the DNA adduct formation in the colonic epithelium induced by AAC.

Epoxydicarboxylic aciduria resulting from the ingestion of castor oil.

A woman who was investigated because of suspected malabsorption was found to excrete large amounts of 3,6-epoxyoctanedioic, 3,6-epoxydecanedioic and 3,6-epoxydodecanedioic acids. The origin of these compounds could be traced to the factitious ingestion of castor oil. Healthy subjects and conventional as well as germ-free rats also excrete these substances after ingestion of castor oil. The epoxydicarboxylic acids are thus endogenous metabolites from ricinoleic acid. The shortest of the three homologues is present in small amounts in normal urine. It may be formed by cyclization of a hydroxylated beta-oxidation intermediate.

Enteral nutrition and the function of the intestinal microflora in healthy adults.

An oral feeding formula was given to 5 healthy volunteers for 8 days. Faecal samples were collected before, during and after the feeding period. The effect of enteral nutrition (EN) on the following seven intestinal microflora-associated characteristics (MACs) was studied: formation of urobilinogen, coprostanol and deoxycholic acid, degradation of mucin and beta-aspartylglycine, faecal tryptic activity, and production of short-chain fatty acids (SCFAs). None of the microbial functions studied were lost during the study. The urobilinogen level increased during EN (P < 0.05) but it seems reasonable to assume that this was a concentration effect due to a decrease in stool mass. The concentration of SCFAs decreased during EN (P < 0.05) and this reflects the absence of dietary fibre in the feed used.

The characteristic cellular organization and CEACAM1 expression in the junctional epithelium of rats and mice are genetically programmed and not influenced by the bacterial microflora.

BACKGROUND: The epithelial cell adhesion molecule CEACAM1 exhibits an interesting dynamic expression during tooth development. It is first expressed in the reduced enamel epithelium, its expression then increases in the orally faced reduced epithelium and the overlying oral epithelium that then fuse to give rise to the junctional epithelium. The expression of CEACAM1 remains at high levels in the junctional epithelium, in contrast to the surrounding oral sulcular epithelium which shows much lower expression levels. We investigated if the high expression levels of CEACAM1 and the loosely organized cells characteristic of the junctional epithelium are genetically programmed or result from bacterial infiltration. METHODS: Oral tissues from germ-free rats and mice and animals with conventional bacterial flora were analyzed by transmission electron microscopy and immunohistochemical staining for CEACAM1. RESULTS: The junctional epithelium of both germ-free and conventional animals was identical with respect to both CEACAM1 expression and morphology. Also the presence of leukocytes was the same in both types of animals. CONCLUSIONS: The results indicate that the characteristic morphology and the high expression levels of CEACAM1 in the junctional epithelium are genetically programmed and not a result of bacterial infiltration. This suggests that CEACAM1 has an important role for the structural integrity of the junctional epithelium. This conclusion was supported by the observation that the junctional epithelium does not express any E-cadherin, which is another abundant epithelial cell adhesion molecule.

Intestinal mucin of germ-free rats. Biochemical and electron-microscopic characterization.

Purified germ-free rat intestinal mucin was found by chemical analysis to contain 25% protein, enriched in serine, threonine, and proline, 75% carbohydrate, and no nucleic acid. It was analyzed by darkfield electron microscopy and found to consist of long filamentous molecules with a maximum length of approximately 740 nm, a mean length of 456 nm, and a mean width of 7 nm. Given reasonable assumptions derived from earlier work on other well-characterized mucins, the molecular weight of the peptide, calculated by the length from electron microscopy, was 200,000, and, given the chemical composition, the molecular weight of the entire mucin molecule was calculated to be approximately 800,000.

Mutagen excretion and cytochrome P-450-dependent activity in germfree and conventional rats fed a diet containing fried meat.

To evaluate a possible role of the intestinal microflora in the metabolism of the highly mutagenic compounds formed in fried meat, conventional and germfree male AGUS rats were fed a semi-synthetic diet containing fried meat. Changes in mutagen excretion in urine and faeces over time were studied using the Ames Salmonella assay. The faecal and urinary extracts were separated by means of high-performance liquid chromatography (HPLC), and the mutagenicity of the collected fractions was determined. Cytochrome P-450 IA (IA1 and/or IA2) were detected by the use of antibodies with the Western blot technique, and the corresponding enzyme activities were measured in microsomes from the small intestine and the liver. A quantitative as well as qualitative difference in excretion of mutagens between germfree and conventional rats was observed. The total excreted of mutagenicity was significantly higher for the conventional than for the germfree rats, as a result of a higher faecal excretion of mutagens in the conventional animals. The HPLC separations of urinary and faecal extracts showed a different mutagenic metabolite pattern between the germfree and conventional rats. An increased activity of the cytochrome P-450-dependent enzyme ethoxyresorufin-O-deethylase was observed in the small intestine of conventional rats on the fried meat diet, whereas no effect of this diet was observed in the germfree rats. Similar results were obtained in immunoblotting experiments using a P-450 IA antiserum. The present study indicates that the excretion pattern and thus also the metabolism of compounds present in fried meat are affected by the germfree status.

The relevance of gene transfer to the safety of food and feed derived from genetically modified (GM) plants.

In 2000, the thematic network ENTRANSFOOD was launched to assess four different topics that are all related to the testing or assessment of food containing or produced from genetically modified organisms (GMOs). Each of the topics was linked to a European Commission (EC)-funded large shared cost action (see http://www.entransfood.com). Since the exchange of genetic information through horizontal (lateral) gene transfer (HGT) might play a more important role, in quantity and quality, than hitherto imagined, a working group dealing with HGT in the context of food and feed safety was established. This working group was linked to the GMOBILITY project (GMOBILITY, 2003) and the results of the deliberations are laid down in this review paper. HGT is reviewed in relation to the potential risks of consuming food or feed derived from transgenic crops. First, the mechanisms for obtaining transgenic crops are described. Next, HGT mechanisms and its possible evolutionary role are described. The use of marker genes is presented in detail as a special case for genes that may pose a risk. Furthermore, the exposure to GMOs and in particular to genetically modified (GM) deoxyribonucleic acid (DNA) is discussed as part of the total risk assessment. The review finishes off with a number of conclusions related to GM food and feed safety. The aim of this paper is to provide a comprehensive overview to assist risk assessors as well as regulators and the general public in understanding the safety issues related to these mechanisms.

Lack of differences in blood and tissue concentrations of endogenous ethanol in conventional and germfree rats.

Headspace gas chromatography was used to determine the concentrations of endogenous ethanol in blood and tissue of conventional and germfree rats. In all biological specimens analysed, the four principal volatile endogenous substances were identified as methanol, acetaldehyde, ethanol and acetone. No statistically significant differences in the concentrations of endogenous ethanol were noted between conventional and germfree animals. In whole blood, liver, kidney, and brain of germfree rats the concentrations of endogenous ethanol were 4.2 +/- 0.19 microM, 5.1 +/- 0.55 microM, 8.2 +/- 0.59 microM and 4.4 +/- 0.17 microM (means +/- SE), respectively. The higher concentration in kidney was also observed in conventional rats. Our results suggest that ethanol is a normal metabolic intermediate in rats and does not exclusively arise from microbial fermentation reactions in the gastrointestinal tract.