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.

The effects of oats on the function of gut microflora in children with coeliac disease.

BACKGROUND: Faecal short chain fatty acids (SCFAs) are produced by the gut microflora. We have previously reported high faecal SCFA levels in children with coeliac disease (CD), indicating alteration in gut microfloral metabolism. Data accumulated over recent decades by us and others suggest that wheat-free oats can safely be included in a gluten-free diet (GFD). However, concerns have been raised with respect to the safety of oats in a subset of coeliacs. AIM: To describe faecal SCFA patterns in children with newly diagnosed CD treated for 1 year with a GFD with or without oats. METHODS: This report is part of a randomised, double-blind study on the effect of a GFD containing oats (GFD-oats) vs. a standard GFD (GFD-std). Faecal samples were received from 34 children in the GFD-oats group and 37 in the GFD-std group at initial diagnosis and/or after 1 year on a GFD. Faecal SCFAs were analysed. RESULTS: The GFD-std group had a significantly lower total faecal SCFA concentration at 12 months compared with 0 months (P < 0.05). In contrast, total SCFA in the GFD-oats group remained high after 1 year on the GFD. The children in the GFD-oats group had significantly higher acetic acid (P < 0.05), n-butyric acid (P < 0.05) and total SCFA concentration (P < 0.01) after 1-year diet treatment compared to the GFD-std group. CONCLUSIONS: Our results indicate that oats do affect the gut microflora function, and that some coeliac children receiving oats may develop gut mucosal inflammation, that may present a risk for future complications.

Family relationship of female breeders reduce the systematic inter-individual variation in the gut microbiota of inbred laboratory mice.

The gut microbiota (GM) may influence disease expression in several animal models for inflammatory diseases. It may therefore seem reasonable to pursue reduction in the number of animals used for individual studies by reducing the variation in the GM. Previous studies have shown that the composition of the GM is related to genetics to a certain extent. We hypothesized that the GM similarity in a group of mice born by mothers not being sisters would be lower than that in a group born by mothers being sisters. The lower similarity could lead to clustering of the GM of mice born by non-sisters according to their mothers, while such clustering would not be visible if the mothers were sisters. We used 16S rRNA gene (V3 region) polymerase chain reaction-derived amplicon profiling by denaturing gradient gel electrophoresis (DGGE) to study the GM composition in caecum samples of 33 eight-week-old C57BL/6Sca mice from a breeding set-up with dam breeders that were sisters, as well as caecum samples of 35 eight-week-old C57BL/6Sca mice from a breeding set-up with dam breeders that were not sisters. Principal component analysis revealed a significant difference between the litters from the breeding set-up with dam breeders that were not sisters, whereas no significant difference between the litters based on the breeding set-up with dam breeders that were sisters was observed. The results obtained indicate that the systematic variation in the GM of inbred mice can be reduced by increasing the family relatedness of the breeding pairs.

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.

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.

Gastrointestinal bacteria generate nitric oxide from nitrate and nitrite.

Denitrifying bacteria in soil generate nitric oxide (NO) from nitrite as a part of the nitrogen cycle, but little is known about NO production by commensal bacteria. We used a chemiluminescence assay to explore if human faeces and different representative gut bacteria are able to generate NO. Bacteria were incubated anaerobically in gas-tight bags, with or without nitrate or nitrite in the growth medium. In addition, luminal NO levels were measured in vivo in the intestines in germ-free and conventional rats, and in rats mono-associated with lactobacilli. We show that human faeces can generate NO after nitrate or nitrite supplementation. Lactobacilli and bifidobacteria generated much NO from nitrite, but only a few of the tested strains produced NO from nitrate and at much lower levels. In contrast, Escherichia coli, Bacteroides thetaiotaomicron, and Clostridium difficile did not produce significant amounts of NO either with nitrate or nitrite. NO generation in the gut lumen was also observed in vivo in conventional rats but not in germ-free rats or in rats mono-associated with lactobacilli. We conclude that NO can be generated by the anaerobic gut flora in the presence of nitrate or nitrite. Future studies will reveal its biological significance in regulation of gastrointestinal integrity.

Biochemical intestinal parameters in germ-free minipigs and rats and in ex-germ-free minipigs and rats monoassociated with Escherichia coli.

Intestinal contents of newborn and young germ-free minipigs and germ-free rats were investigated for the following biochemical parameters - conversion of cholesterol to coprostanol, degradation of beta-aspartylglycine, level of tryptic activity, formation of urobilinogen and the profile of short-chain fatty acids. Additionally, germ-free minipigs and germ-free rats were monoassociated with non-pathogenic strains of Escherichia coli and were investigated for the same biochemical parameters. The conversion of cholesterol to coprostanol, degradation of beta-aspartylglycine, tryptic activity and the short-chain fatty acid profile were similar to those found in previous studies in germ-free animals. Slightly higher amounts of urobilinogen than in the other species investigated so far were found in samples from germ-free and monoassociated minipigs. Except for the total amount of short-chain fatty acids in rats, monoassociation with E. coli did not alter any of the parameters either in the minipigs or in the rats.

Gut microflora associated characteristics in children with celiac disease.

OBJECTIVES: The aim of the study was to investigate the metabolic function of intestinal microflora in children with celiac disease (CD) in order to find out if there is a deviant gut flora in CD patients compared to healthy controls. METHODS: The study group comprised children with CD, consecutively diagnosed according to current criteria given by the European Society for Paediatric Gastroenterology, Hepatology, and Nutrition. Thirty-six children were studied at presentation, i.e., on a normal gluten-containing diet, with clinical symptoms and signs indicative of CD, positive celiac serology markers, and a small bowel biopsy showing severe enteropathy. Forty-seven patients were studied when they had been on a gluten-free diet (GFD) for at least 3 months. For comparison, a group of 42 healthy controls (HC) were studied. The functional status of the intestinal microflora was evaluated by gas-liquid chromatography of short chain fatty acids (SCFAs) in fecal samples. RESULTS: There was a significant difference between untreated CD children and HC as well as between treated CD children and HC regarding acetic, i-butyric, i-valeric acid, and total SCFAs. The propionic and n-valeric acids differed significantly between CD children on GFD and HC. Moreover, there was a strong correlation between i-butyric and i-valeric acids in all study groups. CONCLUSIONS: This is the first study of the SCFA pattern in fecal samples from children with CD. The results indicate that there is a difference in the metabolic activity of intestinal microbial flora in children with CD compared to that in HC. The finding of a different pattern of some SCFAs in celiacs both at presentation and during treatment with GFD indicates that it is a genuine phenomenon of CD not affected by either the diet, the inflammation, or the autoimmune status of the patient.

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.

Influence of zinc bacitracin and Bacillus licheniformis on microbial intestinal functions in weaned piglets.

The influence of zinc bacitracin (ZB) and of Bacillus licheniformis on host microbial-related functions in young piglets was investigated by applying the concept of microflora-associated characteristics. Six biochemical parameters were determined before and after weaning in faecal samples from piglets in four litters having access to a diet containing ZB, to a diet containing B. licheniformis, to a diet with both additives, or to a diet with no additives, from 3 weeks of age. Statistically significant differences were found in three of the intestinal functions investigated: formation of short-chain fatty acids (at 7 and 10 weeks of age). degradation of mucin (at 7 and 10 weeks of age) and conversion of bilirubin to urobilins (at 7 weeks of age). We also found age-dependent influences on the formation of short-chain fatty acids, on conversion of cholesterol to coprostanol and on conversion of bilirubin to urobilins. We conclude that a functional approach is appropriate for measuring exogenous influence(s) on the microbial intestinal metabolisms in weaned piglets.

A human flora-associated rat model of the breast-fed infant gut.

OBJECTIVES: Bacterial colonization of the infant gut may have important influences on the development of gastrointestinal, respiratory, and allergic disease. Early diet is a major determinant of the gut microflora. It is very difficult to carry out studies in human infants that can investigate the interaction of diet, flora, and mucosa. In this study we have developed an infant human flora-associated (IHFA) rat model to allow such investigation. METHODS: Germ-free infant rats were infected with fecal bacteria from exclusively breast-fed infants and were maintained on a modified infant formula for 8 weeks. The fecal and cecal contents were collected and compared with feces of breast-fed infants for bacterial populations, bacterial metabolites, and enzymes and for the ability to inhibit adhesion of pathogenic bacteria to human mucosal cells. RESULTS: The IHFA cecum and feces were dominated by lactic acid bacteria, Bifidobacterium, and lactobacilli, which were representative of the infant feces. The fecal short-chain fatty acid profile was dominated by acetic and lactic acid in a similar manner to human infant feces. Other bacterial metabolites were similar to those of the human infant. Rat intestinal samples were able to inhibit the adhesion of pathogens to mucosal cells, but to a lesser extent than the human samples. CONCLUSIONS: This IHFA infant model of the intestinal flora of the breast-fed infant is considered valid for studying the effect of diet on bacterial colonization and metabolism.

Etiology of cecal and hepatic lesions in mice after administration of gas-carrier contrast agents used in ultrasound imaging.

The aim of the study was to investigate the etiology of cecal and hepatic lesions in mice and rats after intravenous administration of gas-carrier contrast agents (GCAs). A modified fluorescein flowmetry technique and 24 h necropsy were used in mice (conventional and germ free), rats, and guinea pigs after GCA administration. Different diets and oral nonabsorbable antibiotics were used. Nonfluorescence, edema, congestion, hemorrhage, and mucosal erosion in cecum and colon and nonfluorescent areas in the liver were observed from 16 min after GCA administration in conventional mice on standard diet. Numerous gas bubbles (>50 microm) were observed in the vasculature around the nonfluorescent areas of cecum and colon and in mesenteric vessels draining to the portal vein. Acute inflammation, edema, hemorrhage, and ulceration of the cecum and colon and liver necrosis were seen 24 h after GCA administration in conventional mice on standard diet. When mice were maintained on either a diet with glucose as the only carbohydrate source or on a standard diet supplemented with antibiotics, uniform fluorescence and no organ lesions were observed after GCA administration. Uniform fluorescence and no organ lesions were observed in germ-free mice, rats, and guinea pigs dosed with GCAs and in control animals (mice, rats, and guinea pigs) dosed with sucrose. The results indicate that intravascular growth of GCA microbubbles occurs in the cecal and colonic wall of mice, leading to occlusive ischemia and necrosis in these intestinal segments and secondary gas embolisation in the liver. Transmural gas supersaturation in the cecal wall may explain the intravascular bubble growth in mice.

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.

Orally delivered antibiotics which lower bacterial numbers decrease experimental intra-abdominal adhesions.

BACKGROUND: Postsurgical adhesion formation is a common occurrence after most surgical procedures and is still a major cause of postoperative morbidity because no satisfactory treatment or prophylaxis has yet been developed. Further elucidation of the basic mechanisms of postsurgical adhesion formation is needed. Recent studies using germfree rats have found the indigenous bowel flora to be important in the adhesive response. The present study examined whether antibiotic treatment affects intra-abdominal adhesion formation. METHODS: Using the cecal crush model to inflict adhesions, groups of rats ( n=12) were treated with placebo or amoxicillin/clavulanic acid in the drinking water. Treatment started 3 days before operation and continued until evaluation. Adhesion scores were recorded after 7 days. Bacterial counts were made from cultures of fecal samples on operation day and at termination. RESULTS: Amoxicillin/clavulanic acid decreased adhesion score compared to placebo. Adhesion incidence was 50% in the treatment group and 92% in the placebo group. Bacterial numbers were lower in the treatment group. CONCLUSIONS: Antibiotic treatment which lowers bacterial numbers can decrease adhesions.

Biochemical intestinal parameters in pigs reared outdoors and indoors, and in germ-free pigs.

Intestinal microbial functions reflect cross-talk between a host and its flora, and external factors may influence these functions. The aim of this investigation was to follow the development of six biochemical microbial-related functions of piglets, raised outdoors (OPs) or indoors (IPs), from birth to slaughter age. The following parameters (microflora-associated characteristic; MAC) were consecutively measured at five different ages: production of short-chain fatty acids (SCFA), conversion of cholesterol to coprostanol and of bilirubin to urobilinogens, inactivation of trypsin, degradation of beta-aspartylglycine and of mucin. Additionally, four parameters (production of SCFA. conversion of cholesterol to coprostanol, inactivation of trypsin, degradation of beta-aspartylglycine) were investigated in faecal samples from germ-free minipigs. The differences in MAC patterns between OPs and IPs were most pronounced at 20 days of age. Differences were found in the total amount of SCFAs, proportions of the acetic, propionic and butyric acids, conversion of bilirubin to urobilinogens, degradation of faecal tryptic activity and degradation of mucin. The values found in the minipigs were within the range of a germ-free animal characteristic (GAC) pattern. Our results show that environmental factors influence the development of some intestinal microbial functions in pigs.

Increased enterocyte production in gnotobiotic rats mono-associated with Lactobacillus rhamnosus GG.

There is increasing scientific and commercial interest in using beneficial microorganisms (i.e., probiotics) to enhance intestinal health. Of the numerous microbial strains examined, Lactobacillus rhamnosus GG has been most extensively studied. Daily intake of L. rhamnosus GG shortens the course of rotavirus infection by mechanisms that have not been fully elucidated. Comparative studies with germfree and conventional rats have shown that the microbial status of an animal influences the intestinal cell kinetics and morphology. The present study was undertaken to study whether establishment of L. rhamnosus GG as a mono-associate in germfree rats influences intestinal cell kinetics and morphology. L. rhamnosus GG was easily established in germfree rats. After 3 days of mono-association, the rate of mitoses in the upper part of the small intestine (jejunum 1) increased as much as 14 and 22% compared to the rates in germfree and conventional counterparts, respectively. The most striking alteration in morphology was an increase in the number of cells in the villi. We hypothesis that the compartmentalized effects of L. rhamnosus GG may represent a reparative event for the mucosa.

Escherichia coli S fimbriae do not contribute to intestinal colonization or translocation in the gnotobiotic rat.

Escherichia coli S fimbriae, which bind to sialic acid residues, are a virulence factor for extraintestinal infection, but also promote binding to intestinal epithelial cells. In this study, we investigated whether S fimbriae would enhance intestinal colonization by E. coli or promote translocation to extraintestinal sites. A mixture of two E. coli isogenic strains both expressing type-1 fimbriae but differing in the carriage of S fimbriae (Sfim+ and Sfim-) were given perorally to germfree neonatal, infant or adult rats. The Sfim+ bound better to rat intestinal mucus and epithelial cells. However, both strains colonized equally well in both the small and large intestine and their rate of translocation to the mesenteric lymph nodes was similar. Infant rats had higher E. coli levels in the small intestine than adult rats, but their translocation rates were lower. This was at least partly due to their milk diet, since weaned infant rats had more translocating bacteria than infant rats that continued suckling their mother. The results suggest that S fimbriae, despite binding to intestinal epithelial cells and mucus, do not contribute to either colonization or translocation in the gnotobiotic rat.

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.