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The human gut microbiota develops soon after birth and can acquire inter-individual variation upon exposure to intrinsic and environmental cues. However, inter-individual variation has not been comprehensively assessed in a multi-ethnic study. We studied a longitudinal birth cohort of 106 infants of three Asian ethnicities (Chinese, Malay, and Indian) that resided in the same geographical location (Singapore). Specific and temporal influences of ethnicity, mode of delivery, breastfeeding status, gestational age, birthweight, gender, and maternal education on the development of the gut microbiota in the first 2 years of life were studied. Mode of delivery, breastfeeding status, and ethnicity were identified as the main factors influencing the compositional development of the gut microbiota. Effects of delivery mode and breastfeeding status lasted until 6M and 3M, respectively, with the primary impact on the diversity and temporal colonization of the genera Bacteroides and Bifidobacterium. The effect of ethnicity was apparent at 3M post-birth, even before the introduction of weaning (complementary) foods, and remained significant after adjusting for delivery mode and breastfeeding status. Ethnic influences remained significant until 12M in the Indian and Chinese infants. The microbiota of Indian infants was characterized by higher abundances of Bifidobacterium and Lactobacillus, while Chinese infants had higher abundances of Bacteroides and Akkermansia. These findings provide a detailed insight into the specific and temporal influences of early life factors and ethnicity in the development of the human gut microbiota. Trial Registration: Clinicaltrials.gov registration no. NCT01174875.

The neonatal body provides a range of potential habitats, such as the gut, for microbes. These sites eventually harbor microbial communities (microbiotas). A "complete" (adult) gut microbiota is not acquired by the neonate immediately after birth. Rather, the exclusive, milk-based nutrition of the infant encourages the assemblage of a gut microbiota of low diversity, usually dominated by bifidobacterial species. The maternal fecal microbiota is an important source of bacterial species that colonize the gut of infants, at least in the short-term. However, development of the microbiota is influenced by the use of human milk (breast feeding), infant formula, preterm delivery of infants, caesarean delivery, antibiotic administration, family details and other environmental factors. Following the introduction of weaning (complementary) foods, the gut microbiota develops in complexity due to the availability of a diversity of plant glycans in fruits and vegetables. These glycans provide growth substrates for the bacterial families (such as members of the Ruminococcaceae and Lachnospiraceae) that, in due course, will dominate the gut microbiota of the adult. Although current data are often fragmentary and observational, it can be concluded that the nutrition that a child receives in early life is likely to impinge not only on the development of the microbiota at that time but also on the subsequent lifelong, functional relationships between the microbiota and the human host. The purpose of this review, therefore, is to discuss the importance of promoting the assemblage of functionally robust gut microbiotas at appropriate times in early life.

Background Gut microbiota data obtained by DNA sequencing are complex and compositional because of large numbers of detectable taxa, and because microbiota characteristics are described in relative terms. Nutrition researchers use principal component analysis (PCA) to derive dietary patterns from food data. Although compositional PCA methods are not commonly used to describe patterns from complex microbiota data, this approach would be useful for identifying gut microbiota patterns associated with diet and body composition. Objectives To use compositional PCA to describe the principal components (PCs) of gut microbiota in 5-y-old children and explore associations between microbiota components, diet, and BMI z-score. Methods A fecal sample was provided by 319 children aged 5 y. Their primary caregiver completed a validated 123-item quantitative FFQ. Body composition was determined using DXA, and a BMI z-score was calculated. Compositional PCA identified characterizing taxa and weightings for calculation of gut microbiota PC scores at the genus level, and was examined in relation to diet and body size. Results Three gut microbiota PCs were found. PC1 (negative loadings on uncultured Christensenellaceae and Ruminococcaceae) was related to lower BMI z-scores and longer duration of breastfeeding (per month) (β = -0.14; 95% CI: -0.26, -0.02; and β = 0.02; 95% CI: 0.003, 0.34, respectively). PC2 (positive loadings on Fusicatenibacter and Bifidobacterium; negative loadings on Bacteroides) was associated with a lower intake of nuts, seeds, and legumes (β = -0.05 per gram; 95% CI: -0.09, -0.01). When adjusted for fiber intake, PC2 was also associated with higher BMI z-scores (β = 0.12; 95% CI: 0.01, 0.24). PC3 (positive loadings on Faecalibacterium, Eubacterium, and Roseburia) was associated with higher intakes of fiber (β = 0.02 per gram; 95% CI: 0.003, 0.04) and total nonstarch polysaccharides (β = 0.02 per gram; 95% CI: 0.003, 0.04). Conclusions Our results suggest that specific gut microbiota components determined using compositional PCA are associated with diet and BMI z-score.This trial was registered at clinicaltrials.gov as NCT00892983.

The human colon contains a complex microbial community (the microbiota), composed mostly of bacteria, that degrades and ferments indigestible polysaccharides known collectively as dietary fibre. Ac...

International audience; Pectin is abundant in modern day diets, as it comprises the middle lamellae and one-third of the dry carbohydrate weight of fruit and vegetable cell walls. Currently there is no specialized model organism for studying pectin fermentation in the human colon, as our collective understanding is informed by versatile glycan-degrading bacteria rather than by specialist pectin degraders. Here we show that the genome of Monoglobus pectinilyticus possesses a highly specialized glycobiome for pectin degradation, unique amongst Firmicutes known to be in the human gut. Its genome encodes a simple set of metabolic pathways relevant to pectin sugar utilization, and its predicted glycobiome comprises an unusual distribution of carbohydrate-active enzymes (CAZymes) with numerous extracellular methyl/acetyl esterases and pectate lyases. We predict the M. pectinilyticus degradative process is facilitated by cell-surface S-layer homology (SLH) domain-containing proteins, which proteomics analysis shows are differentially expressed in response to pectin. Some of these abundant cell surface proteins of M. pectinilyticus share unique modular organizations rarely observed in human gut bacteria, featuring pectin-specific CAZyme domains and the cell wall-anchoring SLH motifs. We observed M. pectinilyticus degrades various pectins, RG-I, and galactan to produce polysaccharide degradation products (PDPs) which are presumably shared with other inhabitants of the human gut microbiome (HGM). This strain occupies a new ecological niche for a primary degrader specialized in foraging a habitually consumed plant glycan, thereby enriching our understanding of the diverse community profile of the HGM.

Background Our Prevention of Overweight in Infancy (POI) study suggested that a brief sleep intervention in infancy reduced the risk of obesity at age 2 y. In contrast, we observed no benefit from the nutrition and activity intervention. Objective The objective of the study was to determine how these interventions influenced growth at ages 3.5 and 5 y compared with usual care (Control). Design A follow-up of a parallel, 4-arm, single-blind, 2-y, randomized controlled trial in 802 women (86% European, 48% primiparous) recruited in pregnancy (58% response rate) was undertaken. All groups received standard Well-Child care with additional support for 3 intervention groups: FAB (promotion of breastfeeding, healthy eating, physical activity: 8 contacts, antenatal, 18 mo); Sleep (prevention of sleep problems: antenatal, 3 wk); Combination (both interventions). Follow-up measures were collected by staff blinded to group allocation. The primary outcome was child body mass index (BMI) z score, and secondary outcomes were prevalence of obesity (BMI ≥95th percentile), self-regulation (psychological measures), sleep, physical activity (accelerometry, questionnaires), and dietary intake (food-frequency questionnaire). Analyses were conducted through the use of multiple imputation. Results Retention was 77% at age 3.5 y and 69% at age 5 y. Children in the FAB group had significantly higher BMI z scores than did Controls at age 5 y (adjusted difference: 0.25; 95% CI: 0.04, 0.47) but not at age 3.5 y (0.15; 95% CI: -0.04, 0.34). Children who received the Sleep intervention (Sleep and Combination groups) had significantly lower BMI z scores at age 3.5 y (-0.24; 95% CI: -0.38, -0.10) and at age 5 y (-0.23; 95% CI: -0.38, -0.07) than children who did not (Control and FAB groups). Conclusions A conventional intervention had unexpected adverse long-term weight outcomes, whereas positive outcomes from a less conventional sleep intervention remained promising at age 5 y. More intensive or extended sleep intervention might have larger or longer-lasting effects and should be investigated. This trial was registered at clinicaltrials.gov as NCT00892983.

Kiwifruit are a nutrient dense food and an excellent source of vitamin C. Supplementation of the diet with kiwifruit enhances plasma vitamin C status and epidemiological studies have shown an association between vitamin C status and reduced insulin resistance and improved blood glucose control. In vitro experiments suggest that eating kiwifruit might induce changes to microbiota composition and function, however, human studies to confirm these findings are lacking. The aim of this study was to investigate the effect of consuming two SunGold kiwifruit per day over 12 weeks on vitamin C status, clinical and anthropometric measures and faecal microbiota composition in people with prediabetes. This pilot intervention trial compared baseline measurements with those following the intervention. Participants completed a physical activity questionnaire and a three-day estimated food diary at baseline and on completion of the trial. Venous blood samples were collected at each study visit (baseline, 6, 12 weeks) for determination of glycaemic indices, plasma vitamin C concentrations, hormones, lipid profiles and high-sensitivity C-reactive protein. Participants provided a faecal sample at each study visit. DNA was extracted from the faecal samples and a region of the 16S ribosomal RNA gene was amplified and sequenced to determine faecal microbiota composition. When week 12 measures were compared to baseline, results showed a significant increase in plasma vitamin C (14 &micro, mol/L, p &lt, 0.001). There was a significant reduction in both diastolic (4 mmHg, p = 0.029) and systolic (6 mmHg, p = 0.003) blood pressure and a significant reduction in waist circumference (3.1 cm, p = 0.001) and waist-to-hip ratio (0.01, p = 0.032). Results also showed a decrease in HbA1c (1 mmol/mol, p = 0.005) and an increase in fasting glucose (0.1 mmol/L, p = 0.046), however, these changes were small and were not clinically significant. Analysis of faecal microbiota composition showed an increase in the relative abundance of as yet uncultivated and therefore uncharacterised members of the bacterial family Coriobacteriaceae. Novel bacteriological investigations of Coriobacteriaceae are required to explain their functional relationship to kiwifruit polysaccharides and polyphenols.

Summary The composition and function of the mammalian gut microbiota has been the subject of much research in recent years, but the principles underlying the assembly and structure of this complex community remain incompletely understood. Processes that shape the gut microbiota are thought to be mostly niche‐driven, with environmental factors such as the composition of available nutrients largely determining whether or not an organism can establish. The concept that the nutrient landscape dictates which organisms can successfully colonize and persist in the gut was first proposed in Rolf Freter's nutrient niche theory. In a situation where nutrients are perfectly mixed and there is balanced microbial growth, Freter postulated that an organism can only survive if it is able to utilize one or a few limiting nutrients more efficiently than its competitors. Recent experimental work indicates, however, that nutrients in the gut vary in space and time. We propose that in such a scenario, Freter's nutrient niche theory must be expanded to account for the co‐existence of microorganisms utilizing the same nutrients but in distinct sites or at different times, and that metabolic flexibility and mixed‐substrate utilization are common strategies for survival in the face of ever‐present nutrient fluctuations.

Lactobacillus rhamnosus HN001 is a probiotic strain reported to increase resistance to epithelium-adherent and -invasive intestinal pathogens in experimental animals. To increase understanding of the relationship between strain HN001 and the bowel, transcription of selected genes in the mucosa of the murine small bowel was measured. Mice previously naive to lactobacilli ( Lactobacillus -free mice) were examined after daily exposure to HN001 in drinking water. Comparisons were made to results from matched Lactobacillus -free mice. Infant and adult mice were investigated to provide a temporal view of gene expression in response to exposure to HN001. Genes sgk1 , angptl4 , and hspa1b , associated with the apoptosis pathway, were selected for investigation by reverse transcription-quantitative PCR on the basis of a preliminary duodenal DNA microarray screen. Normalized to gapdh gene transcription, these three genes were upregulated after 6 to 10 days exposure of adult mice to HN001. Angptl4 was shown by immunofluorescence to be upregulated in duodenal epithelial cells of mucosal samples. Epithelial cell migration was faster in HN001-exposed mice than in the Lactobacillus -free controls. Transcriptional responses in infant mice differed according to bowel region and age. For example, sgk1 was upregulated in duodenal, jejunal, and ileal mucosa of mice less than 25 days old, whereas angptl4 and hspa1b were upregulated at 10 days in the duodenum but downregulated in the jejunal mucosa until mice were 25 days old. Overall, the results provide links between a probiotic strain, mucosal gene expression, and host phenotype, which may be useful in delineating mechanisms of probiotic action.

Lactobacillus ruminis is an inhabitant of human bowels and bovine rumens. None of 10 isolates (three from bovine rumen, seven from human feces) of L. ruminis that were tested could utilize barley β-glucan for growth. Seven of the strains of L. ruminis were, however, able to utilize tetrasaccharides (3- O -β-cellotriosyl- d -glucose [LDP4] or 4- O -β-laminaribiosyl- d -cellobiose [CDP4]) present in β-glucan hydrolysates for growth. The tetrasaccharides were generated by the use of lichenase or cellulase, respectively. To learn more about the utilization of tetrasaccharides by L. ruminis , whole-transcriptome shotgun sequencing (RNA-seq) was tested as a transcriptional screen to detect altered gene expression when an autochthonous human strain (L5) was grown in medium containing CDP4. RNA-seq results were confirmed and extended by reverse transcription-quantitative PCR assays of selected genes in two upregulated operons when cells were grown as batch cultures in medium containing either CDP4 or LDP4. The cellobiose utilization operon had increased transcription, particularly in early growth phase, whereas the chemotaxis/motility operon was upregulated in late growth phase. Phenotypic changes were seen in relation to upregulation of chemotaxis/flagellar operons: flagella were rarely seen by electron microscopy on glucose-grown cells but cells cultured in tetrasaccharide medium were commonly flagellated. Chemotactic movement toward tetrasaccharides was demonstrated in capillary cultures. L. ruminis utilized 3- O -β-cellotriosyl- d -glucose released by β-glucan hydrolysis due to bowel commensal Coprococcus sp., indicating that cross feeding of tetrasaccharide between bacteria could occur. Therefore, the RNA-seq screen and subsequent experiments had utility in revealing foraging attributes of gut commensal Lactobacillus ruminis .

Aim The composition of faecal microbiota of babies is known to be influenced by diet. Faecal calprotectin and α1-antitrypsin concentrations may be associated with mucosal permeability and inflammation. We aimed to assess whether there was any difference after consumption of a probiotic/prebiotic formula on faecal microbiota composition, calprotectin and α1-antitrypsin levels, and diarrhoea in comparison with breast milk-fed Indonesian infants. Methods One hundred sixty infants, 2 to 6 weeks old, were recruited to the study. They were either breastfed or formula fed (80 per group). Faecal samples were collected at recruitment and 3 months later. Bacterial groups characteristic of the human faecal microbiota were quantified in faeces by quantitative polymerase chain reaction. Calprotectin and α1-antitrypsin concentrations were measured using commercial kits. Details of diarrhoeal morbidity were documented and rated for severity. Results The compositions of the faecal microbiota of formula-fed compared with breast milk-fed children were similar except that the probiotic strain Bifidobacterium animalis subsp. lactisâ€...DR10 was more abundant after 3 months consumption of the formula. Alpha1-antitrypsin levels were higher in breastfed compared with formula-fed infants. The occurrence of diarrhoea did not differ between the groups of babies. Conclusion Feeding Indonesian babies with a probiotic/prebiotic formula did not produce marked differences in the composition of the faecal microbiota in comparison with breast milk. Detrimental effects of formula feeding on biomarkers of mucosal health were not observed.

The ability to predictably engineer the composition of bowel microbial communities (microbiota) using dietary components is important because of the reported associations of altered microbiota composition with medical conditions. In a synecological study, weanling conventional Sprague-Dawley rats (21 days old) were fed a basal diet (BD) or a diet supplemented with resistant starch (RS) at 5%, 2.5%, or 1.25% for 28 days. Pyrosequencing of 16S rRNA genes and temporal temperature gradient electrophoresis (TTGE) profiles in the colonic digesta showed that rats fed RS had altered microbiota compositions due to blooms of Bacteroidetes and Actinobacteria . The altered microbiota was associated with changes in colonic short-chain fatty acid (SCFA) concentrations, colonic-tissue gene expression ( Gsta2 and Ela1 ), and host physiology (serum metabolite profiles and colonic goblet cell numbers). Comparisons between germ-free and conventional rats showed that transcriptional and serum metabolite differences were mediated by the microbiota and were not the direct result of diet composition. Altered transcriptomic and physiological responses may reflect the young host's attempts to maintain homeostasis as a consequence of exposure to a new collection of bacteria and their associated biochemistry.

Autoinducer-2 (AI-2)-mediated quorum sensing has been extensively studied in relation to the regulation of microbial behavior. There are, however, two potential roles for the AI-2 synthase (LuxS). The first is in the production of AI-2 and the second is as an enzyme in the activated methyl cycle, where it catalyzes the conversion of S-ribosylhomocysteine to homocysteine. The by-product of the reaction catalyzed by LuxS is ( S )-4,5-dihydroxy-2,3-pentanedione, which spontaneously forms the furanones known collectively as AI-2. The mammalian gut contains a complex collection of bacterial species so a method of interspecies communication might influence community structure and function. Lactobacillus reuteri 100-23 is an autochthonous inhabitant of the rodent forestomach, where it adheres to the nonsecretory epithelium, forming a biofilm. Microarray comparisons of gene expression profiles of the L. reuteri 100-23 wild type and a luxS mutant under different culture conditions revealed altered transcription of genes encoding proteins associated with cysteine biosynthesis/oxidative stress response, urease activity, and sortase-dependent proteins. Metabolomic analysis showed that the luxS mutation affected cellular levels of fermentation products, fatty acids and amino acids. Cell density-dependent changes (log phase versus stationary phase growth) in gene transcription were not detected, indicating that AI-2 was unlikely to be involved in gene regulation mediated by quorum sensing in L. reuteri 100-23.

Lactobacillus reuteri strain 100-23 together with a Lactobacillus-free mouse model, provides a system with which the molecular traits underpinning bacterial commensalism in vertebrates can be studied. A polysaccharide was extracted from sucrose-containing liquid cultures of strain 100-23. Chemical analysis showed that this exopolysaccharide was a levan (β-2, 6-linked fructan). Mutation of the fructosyl transferase (ftf) gene resulted in loss of exopolysaccharide production. The ftf mutant was able to colonise the murine gastrointestinal tract in the absence of competition, but colonisation was impaired in competition with the wild type. Biofilm formation by the mutant on the forestomach epithelial surface was not impaired and the matrix between cells was indistinguishable from that of the wild type in electron micrographs. Colonisation of the mouse gut by the wild-type strain led to increased proportions of regulatory T cells (Foxp3+) in the spleen, whereas colonisation by the ftf mutant did not. Survival of the mutant in sucrose-containing medium was markedly reduced relative to the wild type. Comparison of the genomic ftf loci of strain 100-23 with other L. reuteri strains suggested that the ftf gene was acquired by lateral gene transfer early in the evolution of the species and subsequently diversified at accelerated rates. Levan production by L. reuteri 100-23 may represent a function acquired by the bacterial species for life in moderate to high-sucrose extra-gastrointestinal environments that has subsequently been diverted to novel uses, including immunomodulation, that aid in colonisation of the murine gut.

Given the relatively immature state of the neonatal gut and gut-associated immune system, the safety of probiotic strains for use as ingredients in infant milk formulae must be demonstrated in infant populations. As part of a double-blind placebo-controlled clinical trial of two commercially available probiotic strains in the reduction of risk for infant eczema, a number of safety outcomes were measured. Infants received daily doses of Lactobacillus rhamnosus HN001 (6 × 10 9 cfu day −1 ) or Bifidobacterium animalis subsp. lactis HN019 (9 × 10 9 cfu day −1 ), or placebo from birth to 24 months. Mothers received the same treatment from 35 weeks gestation, for up to 6 months postnatally while breastfeeding. No statistically significant differences were observed between the treatment groups for study withdrawal, incidence of adverse events, morphometric data, wheeze, and antibiotic use over the treatment period. We conclude that probiotics strains HN001 and HN019 were safe and well tolerated in infants, and did not affect normal growth.

Objective.Ileocolitis is a recognized feature of ankylosing spondylitis (AS) and is likely to play a role in the pathogenesis of AS, in conjunction with the normal intestinal microbiota. In order to investigate the host immune response in AS, we measured cytokines in tissue culture following exposure of peripheral blood mononuclear cells (PBMC) to autologous colonic bacteria.Methods.Twenty-one patients with AS and 21 matched controls were recruited. Subjects in the AS group were assessed clinically.Bacteroidesspecies belonging to theB. fragilisgroup were selectively cultured from stool samples and paired with blood samples from each participant. Ten cultures of autologousBacteroideswere randomly selected from cultures grown from the fecal specimens of each of the 21 patients with AS and 21 controls. These were then tested for reactivity with PBMC and the cytokines produced by proliferating lymphocytes [interleukin 10 (IL-10), IL-17, interferon-γ, tumor necrosis factor-α] were measured in cell culture supernatants. Differences between groups were analyzed using censored normal regression analysis.Results.The patients with AS had severe active AS with Bath AS Disease Activity Index 5.5 (± 1.6) and C-reactive protein (mg/l) 13.8 (± 12.2) (mean ± standard deviation). IL-10 concentrations inex vivoassay supernatants were lower in the AS group compared with controls (p = 0.047). There were no statistically significant differences between the groups for other cytokines.Conclusion.In AS, reduced IL-10 production in response to stimulation with autologousBacteroidescultures may represent a mechanism by which intestinal inflammation develops and persists, a situation analogous to inflammatory bowel disease.

An undisturbed sediment core taken from Round Lake, Hawke's Bay, New Zealand, using a specially designed sterile sampler, has been dated by tephra analysis and radiocarbon and analysed for pollen, charcoal and DNA. It appears to cover the last ca. 3600 years. There are clear indications of Māori effects on vegetation from ca. 800 BP. DNA was extracted from the core and amplified using bacterial and a range of other PCR primers targeting human and other animal mtDNA sequences. Cloned bacterial DNA sequences from just above the Taupo tephra (dated to AD 217-247) produced 98% and 99% similarity matches with human faecal bacteria. The 98% match was to the human gut bacterial genus Prevotella. This genus can, however, be found in a range of animal (including insect) guts. Extracted DNA was matched closely to the New Zealand native fish species Gobiomorphus cotidianus. Further work is needed to establish unequivocally that no DNA movement through the profile is occurring, but these results suggest a new technique of potentially revolutionary importance for Quaternary research. © 2007 Elsevier Ltd and INQUA.

Members of the genus Lactobacillus are common inhabitants of the proximal gastrointestinal tract of animals such as mice, rats, chickens and pigs, where they form epithelial biofilms. Little is known about the traits that facilitate biofilm formation and gut colonization. This study investigated the ecological role of a glucosyltransferase (GtfA) and inulosucrase (Inu) of Lactobacillus reuteri TMW1.106 and a fructosyltransferase (FtfA) of L. reuteri LTH5448. In vitro experiments using isogenic mutants revealed that GtfA was essential for sucrose-dependent autoaggregation of L. reuteri TMW1.106 cells under acidic conditions, while inactivation of Inu slowed the formation of cell aggregates. Experiments using an in vitro biofilm assay showed that GtfA and Inu contributed to biofilm formation of L. reuteri TMW1.106. Experiments using ex-Lactobacillus-free mice revealed that the ecological performance of the inu mutant, but not of the gtfA or ftfA mutant, was reduced in the gastrointestinal tract when in competition with the parental strain. In the absence of competition, the gtfA mutant showed delayed colonization of the murine gut relative to the wild-type. In addition, the gtfA mutant showed reduced ecological performance in competition experiments with Lactobacillus johnsonii #21. From the evidence provided in this study we conclude that GtfA and Inu confer important ecological attributes of L. reuteri TMW1.106 and contribute to colonization of the mouse gastrointestinal tract.

Summary The dlt operon of Gram-positive bacteria encodes proteins required for the incorporation of d-alanine esters into cell wall-associated teichoic acids (TA). d-Alanylation of TA has been shown to be important for acid tolerance, resistance to antimicrobial peptides, adhesion, biofilm formation, and virulence of a variety of pathogenic organisms. The aim of this study was to determine the importance of d-alanylation for colonization of the gastrointestinal tract by Lactobacillus reuteri 100-23. Insertional inactivation of the dltA gene resulted in complete depletion of d-alanine substitution of lipoteichoic acids. The dlt mutant had similar growth characteristics as the wild type under standard in vitro conditions, but formed lower population sizes in the gastrointestinal tract of ex-Lactobacillus-free mice, and was almost eliminated from the habitat in competition experiments with the parental strain. In contrast to the wild type, the dlt mutant was unable to form a biofilm on the forestomach epithelium during gut colonization. Transmission electron microscope observations showed evidence of cell wall damage of mutant bacteria present in the forestomach. The dlt mutant had impaired growth under acidic culture conditions and increased susceptibility to the cationic peptide nisin relative to the wild type. Ex vivo adherence of the dlt mutant to the forestomach epithelium was not impaired. This study showed that d-alanylation is an important cell function of L. reuteri that seems to protect this commensal organism against the hostile conditions prevailing in the murine forestomach.

Straight-run broiler chickens were raised either in floor pens or wire-floored cages (trial 1) or in floor pens only (trials 2, 3, and 4). Birds raised in floor pens had lower BW and feed intakes than those raised in cages. The administration of bacitracin in the feed increased feed intake from d 12 to d 35, decreased the feed conversion ratio during the same period in trial 2, and improved the weight gain of broilers from d 0 to 10 in trial 3. The concentrations of conjugated bile salts (taurocholic and taurochenodeoxycholic acids) were higher in the ileal contents of broilers administered the antimicrobials compared with untreated birds. Supplementation of the feed with monensin increased fat digestibility in the ileum of the birds. Although total numbers of bacteria in ileal contents were the same regardless of whether antimicrobials were administered or not, the bacterial community differed qualitatively. Populations of Lactobacillus salivarius were reduced in birds fed antimicrobials relative to untreated broilers. A representative ileal isolate of L. salivarius deconjugated bile salts in pure culture in the laboratory and in the ileal contents of ex-Lactobacillus-free chickens maintained in a protective environment and colonized by the Lactobacillus isolate. These observations provide a link between bile salt deconjugation in the ileum by L. salivarius and decreased weight gain of broilers. Lactobacillus salivarius populations could be targeted in future studies aimed at modification of the ileal bacterial community to achieve growth promotion of broilers without the administration of antimicrobial drugs.

Gerald W Tannock University of Otago, Department of Microbiology & Immunology, PO Box 56, 720 Cumberland Street, Dunedin, New Zealand Tel.: +643 479 7713; Fax: +643 479 8540; gerald.tannock@stonebow. otago.ac.nz ‘Medical knowledge focuses on the pathogenesis of diseases and the derivation of intervention strategies, but outside of public health medicine, where fundamental improvements have been driven by hygiene, Hygeia has been neglected’

A study was undertaken to test the effect of 2 commercially available probiotics on the production efficiency of broiler chickens hatched from the same breeder flock at 3 different ages (28, 43, and 57 wk). At each of the 3 breeder flock ages, 1,600 broiler chickens were hatched and randomly allocated to 1 of 4 treatments: 1) no probiotics (control), 2) probiotic 1 administered in the drinking water, 3) probiotic 1 administered as a spray, and 4) probiotic 2 administered in the feed. A coccidiostat was included in the feed, but no other antimicrobial agents were given. Broilers were then reared on straw litter in identical floor pens for a period of 6 wk. There were no significant differences in broiler BW, feed conversion, or mortality between the probiotic treatments and the control group in any of the trials. The 43-wk-old breeder flock had the highest fertility and hatchability and the lowest percentage of chicks culled at hatching. Throughout the broiler production period, the broilers from the 43- and 57-wk-old breeder flocks had higher BW and weight gains than the broilers produced at 28 wk of breeder flock age. Broiler feed conversion over the 6-wk production period decreased as the breeder flock aged. Probiotics had no effect on chick quality or production efficiency in broilers produced by the breeder flock ages examined.

HLA-B27 transgenic rats develop spontaneous colitis under specific pathogen-free conditions (SPF) but germ-free rats remain disease-free, emphasizing a role for intestinal bacteria in the pathogenesis of chronic intestinal inflammation. Prebiotics are dietary substances that affect the host by stimulating growth and/or activity of potentially health promoting bacteria. The aims of this study were to investigate whether prebiotics can prevent colitis in SPF HLA-B27 rats, and secondly, to explore mechanisms of protection. SPF HLA-B27 transgenic rats received orally the prebiotic combination long-chain inulin and oligofructose (Synergy 1), or not, prior to the development of clinically detectable colitis. After seven weeks, cecal and colonic tissues were collected for gross cecal scores (GCS), histologic inflammatory scores (scale 0-4), and mucosal cytokine measurement. Cecal and colonic contents were collected for analysis of the gut microbiota by PCR-denaturing gradient gel electrophoresis (PCR-DGGE) and fluorescent in-situ hybridization (FISH), and analysis of short-chain fatty acid composition. Prebiotic treatment significantly decreased GCS and inflammatory histologic scores in the cecum and colon. Prebiotic treatment also decreased cecal IL-1beta, but increased cecal TGF-beta concentrations. Inulin/oligofructose altered the cecal and colonic PCR-DGGE profiles, and FISH analysis showed significant increases in cecal Lactobacillus and Bifidobacterium populations after prebiotic treatment compared with water-treated rats. In conclusion, the prebiotic combination Synergy 1 reduced colitis in HLA-B27 transgenic rats, which effect was associated with alterations to the gut microbiota, decreased tissue proinflammatory cytokines and increased immunomodulatory molecules. These results show promise for prebiotics as primary or adjuvant maintenance therapy for chronic inflammatory bowel diseases.

Aim: To test combined polymerase chain reaction amplification of 16S rRNA gene sequences and denaturing gradient gel electrophoresis (PCR/DGGE) as an analytical method to investigate the composition of the large bowel microbiota of mice during the development of colitis. Methods and Results: The colonic microbiota of formerly germfree interleukin 10 (IL-10)-deficient mice that had been exposed to the faecal microbiota of specific pathogen-free animals was screened using PCR/DGGE. The composition of the large bowel microbiota of IL-10-deficient mice changed as colitis progressed. DNA fragments originating from four bacterial populations (‘Bacteroides sp.’, Bifidobacterium animalis, Clostridium cocleatum, enterococci) were more apparent in PCR/DGGE profiles of colitic mice relative to non-colitic animals, whereas two populations were less apparent (Eubacterium ventriosum, Acidophilus group lactobacilli). Specific DNA:RNA dot blot analysis showed that bifidobacterial ribosomal RNA (rRNA) abundance increased as colitis developed. Conclusions: PCR/DGGE was shown to be an effective method to demonstrate changes in the composition of the large bowel microbiota of mice in relation to progression of inflammatory disease. The intensity of staining of DNA fragments in DGGE profiles reflected increased abundance of bifidobacterial rRNA in the microbiota of colitic animals. As bifidobacterial fragments in PCR/DGGE profiles generated from microbiota DNA showed increased intensity of fragment staining, an increase in bifidobacterial numbers in colitic mice was indicated. Significance and Impact of the Study: PCR/DGGE analysis demonstrated an altered composition of the large bowel microbiota of colitic mice. This work will allow specific groups of bacteria to be targeted in future research concerning the pathogenesis of colitis.

A metagenomic (community genomic) library consisting of 5,760 bacterial artificial chromosome clones was prepared in Escherichia coli DH10B from DNA extracted from the large-bowel microbiota of BALB/c mice. DNA inserts detected in 61 randomly chosen clones averaged 55 kbp (range, 8 to 150 kbp) in size. A functional screen of the library forβ -glucanase activity was conducted using lichenin agar plates and Congo red solution. Three clones with β-glucanase activity were detected. The inserts of these three clones were sequenced and annotated. Open reading frames (ORF) that encoded putative proteins with identity to glucanolytic enzymes (lichenases and laminarinases) were detected by reference to databases. Other putative genes were detected, some of which might have a role in environmental sensing, nutrient acquisition, or coaggregation. The insert DNA from two clones probably originated from uncultivated bacteria because the ORF had low sequence identity with database entries, but the genes associated with the remaining clone resembled sequences reported in Bacteroides species.

The consumption of phytoestrogens has been related to a reduced risk of hormone-dependent cancer types as well as coronary heart diseases. Plant lignans that represent one class of phytoestrogens are found in relatively high concentrations in rye bran. When consumed, they are metabolized into mammalian lignans (enterodiol and enterolactone) by gut bacteria. In order to investigate whether an increased intake of plant lignans had an impact on the composition of the predominant bacterial community of the human gut a 2x6-week intervention study, with a crossover design, was conducted. Eighteen healthy postmenopausal women (age 55-71 years) were recruited and randomly assigned to two groups. The intervention comprised bread products with either rye bran or an inert wheat cellulose (Vitacel† ). Faecal samples were collected before and at the end of each intervention period and analysed for their predominant bacterial species composition by a cultivationindependent approach - polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). The rye bran diet had no impact on the composition of gut microflora. However, the DGGE profiles demonstrated unique bacterial communities for each individual, which implied a differing efficiency among individuals to metabolize lignans.Key words: rye bran, lignans, human gut microflora, PCR-DGGE

Members of the genus Lactobacillus are common inhabitants of the gut, yet little is known about the traits that contribute to their ecological performance in gastrointestinal ecosystems. Lactobacillus reuteri 100-23 persists in the gut of the reconstituted Lactobacillus -free mouse after a single oral inoculation. Recently, three genes of this strain that were specifically induced (in vivo induced) in the murine gut were identified (38). We report here the detection of a gene of L. reuteri 100-23 that encodes a high-molecular-mass surface protein (Lsp) that shows homology to proteins involved in the adherence of other bacteria to epithelial cells and in biofilm formation. The three in vivo-induced genes and lsp of L. reuteri 100-23 were inactivated by insertional mutagenesis in order to study their biological importance in the murine gastrointestinal tract. Competition experiments showed that mutation of lsp and a gene encoding methionine sulfoxide reductase (MsrB) reduced ecological performance. Mutation of lsp impaired the adherence of the bacteria to the epithelium of the mouse forestomach and altered colonization dynamics. Homologues of lsp and msrB are present in the genomes of several strains of Lactobacillus and may play an important role in the maintenance of these bacteria in gut ecosystems.

John B. S. Haldane (1892-1964), noted British geneticist, physiologist, and popularizer of science, established new paths of research in population genetics and evolution. Emphasizing the immensity of the Milky Way in the night sky and the fact that there were 400,000 species of beetles but only 8

Human subjects consumed biscuits containing either galacto-oligosaccharides or fructo-oligosaccharides in a double-blinded, crossover study. The impact of supplementing the diet with three biscuits per day on the fecal microbiota was evaluated by selective culture of particular bacterial groups, measurement of β-galactosidase activity, and nucleic acid-based analytical methods (PCR-denaturing gradient gel electrophoresis [PCR-DGGE] and fluorescent in situ hybridization). The composition of the bifidobacterial populations was monitored at the level of species (PCR-DGGE) and strains (pulsed-field gel electrophoresis of DNA digests), and representative cultures were tested quantitatively for their ability to use galacto-oligosaccharides. Technical improvements to DGGE analysis of the microbiota were made by the use of an internal standard that allowed valid comparisons of fragment staining intensities to be made between profiles, the use of S1 nuclease digestion to remove single-stranded DNA to facilitate cloning of DNA sequences cut from gels, and the extraction of RNA to be used as the template in reverse transcription-PCR-DGGE. RNA-DGGE profiles were markedly different (Dice's similarity coefficient, 58.5%) from those generated by DNA-DGGE. Neither the sizes of the bacterial populations nor the DNA-DGGE profiles of the microbiota were altered by the consumption of the biscuits, but the RNA-DGGE profiles were altered by the detection or increased staining intensity of 16S rRNA gene sequences originating from Bifidobacterium adolescentis and/or Colinsella aerofaciens in the feces of 11 of 15 subjects. β-Galactosidase activity was elevated in the feces of some subjects as a result of biscuit consumption. Subjects differed in the ability of the bifidobacterial strains harbored in their feces to use galacto-oligosaccharides. Our observations suggest that a phylogenetic approach to analysis of the gut ecosystem may not always be optimal and that a more physiological (biochemical) method might be more informative.

Lactobacilli are common inhabitants of the gastrointestinal tracts of mammals and have received considerable attention due to their putative health-promoting properties. Little is known about the traits that enhance the ability of these bacteria to inhabit the gastrointestinal tract. In this paper we describe the development and application of a strategy based on in vivo expression technology (IVET) that enables detection of Lactobacillus reuteri genes specifically induced in the murine gut. A plasmid-based system was constructed containing ′ ermGT (which confers lincomycin resistance) as the primary reporter gene for selection of promoters active in the gastrointestinal tract of mice treated with lincomycin. A second reporter gene, ′ bglM (β-glucanase), allowed differentiation between constitutive and in vivo inducible promoters. The system was successfully tested in vitro and in vivo by using a constitutive promoter. Application of the IVET system with chromosomal DNA of L. reuteri 100-23 and reconstituted lactobacillus-free mice revealed three genes induced specifically during colonization. Two of the sequences showed homology to genes encoding xylose isomerase ( xylA ) and peptide methionine sulfoxide reductase ( msrB ), which are involved in nutrient acquisition and stress responses, respectively. The third locus showed homology to the gene encoding a protein whose function is not known. Our IVET system has the potential to identify genes of lactobacilli that have not previously been functionally characterized but which may be essential for growth of these bacteria in the gastrointestinal ecosystem.

A large and complex bacterial community inhabits the distal intestinal tract of humans. This collection, known as the intestinal microflora, is dominated numerically by obligately anaerobic bacterial species. Many of these species have never been cultivated under laboratory conditions. Nucleic acid-based techniques now permit, however, the analysis of even the non-cultivable members of the bacterial community. Polymerase chain reaction (PCR) coupled with denaturing gradient gel electrophoresis (DGGE) provides a useful technique for comparisons of the composition of faecal or intestinal microfloras. PCR/DGGE has been shown to be useful in demonstrating changes that occur in the composition of the faecal microflora of infants administered antibacterial drugs. This research is important because treatment with oral antibiotics during the first 2 y of life has been identified as a predictor of subsequent atopic disease. The treatment of young children with broad spectrum oral antibiotics might produce perturbations in the composition of the intestinal microflora such that bacteria important in promoting Th1 mechanisms are depleted at a crucial age. This could result in Th2 dominance over Th1 immune responses to environmental antigens and an increased incidence of atopic disorders. PCR/DGGE provides a useful screening method to determine the impact of antibiotic treatment on the composition of the intestinal microflora of children and to identify the bacterial groups that are most affected.

The large bowel is home to a complex microbial community that is present throughout the life of the human host. Relatively few microbial species detected in faeces in relatively low numbers have been implicated as major aetiological agents of urinary tract infections. The impact of these few species on human health is considerable, especially when recurrent urinary tract infections are considered, and ways must be found to reduce their pathogenic activities. One approach may be to learn about the ecology of the bowel ecosystem and devise ways by which the numbers of enterobacteria, in particular, can be restricted. This, in turn, would decrease the dose of potential urinary tract pathogens present in the faeces.

Lactobacillus isolates were identified by PCR amplification and sequencing of the region between the 16S and 23S rRNA genes (spacer region). The sequences obtained from the isolates were compared to those of reference strains held in GenBank. A similarity of 97.5% or greater was considered to provide identification. To check the reliability of the method, the V2-V3 region of the 16S rRNA gene was amplified and sequenced in the case of isolates whose spacer region sequences were less than 99% similar to that of a reference strain. Confirmation of identity was obtained in all instances. Spacer region sequencing provided rapid and accurate identification of Lactobacillus isolates obtained from gastrointestinal, yoghurt, and silage samples. It had an advantage over 16S V2-V3 sequence comparisons because it distinguished between isolates of Lactobacillus casei and Lactobacillus rhamnosus .

The effect on growth of a conjugated bile salt (sodium taurocholate) at physiological concentration was determined using cultures of Lactobacillus strains of murine origin. The bile salt stimulated the growth of one strain, did not affect the growth of another, but inhibited the growth of strains that produced relatively large amounts of the enzyme bile salt hydrolase. Comparison of the growth of isogenic strains that differed in the ability to produce bile salt hydrolase demonstrated that inhibition of growth was due to the accumulation of cholic acid in the culture medium as a result of the enzyme activity.

Diets rich in complex carbohydrates that resist digestion in the small bowel can alter large bowel ecology and microbiota biochemistry because the carbohydrates become substrates for bacterial growth and metabolism. Conventional or germ-free weanling rats were fed a control diet or diets containing 1.25, 2.5, or 5% konjac (KJ), a commonly used ingredient in Asian foods, for 28 d. In the absence of bowel microbiota, 5% KJ elicited a significant increase in colonic goblet cell numbers and increased expression of mast cell protease genes and of genes that were overrepresented in the KEGG pathway "Metabolism of xenobiotics by cytochrome P450" relative to the control diet. In contrast, feeding 5% KJ caused few changes in mucosal gene expression in conventional rats. Analysis of the colonic microbiota of conventional rats fed KJ showed modest increases in the proportions of Actinobacteria and Bacteroidetes relative to rats fed the control diet, with a concomitant reduction in Firmicutes, which included a 50% reduction in Lactobacillus abundance. Colonic concentrations of short-chain fatty acids and colonic crypt lengths were increased by feeding KJ. Goblet cell numbers were greater in conventional rats fed KJ relative to the control diet but were lower compared with germ-free animals. Serum metabolite profiles were different in germ-free and conventional rats. Metabolites that differed in concentration included several phospholipids, a bile acid metabolite, and an intermediate product of tryptophan metabolism. Overall, KJ in the diet was potentially damaging to the bowel mucosa and produced a protective response from the host. This response was reduced by the presence of the bowel microbiota, which therefore ameliorated potentially detrimental effects of dietary KJ.

The composition of faecal microbiota of babies is known to be influenced by diet. Faecal calprotectin and α1-antitrypsin concentrations may be associated with mucosal permeability and inflammation. We aimed to assess whether there was any difference after consumption of a probiotic/prebiotic formula on faecal microbiota composition, calprotectin and α1-antitrypsin levels, and diarrhoea in comparison with breast milk-fed Indonesian infants.One hundred sixty infants, 2 to 6 weeks old, were recruited to the study. They were either breastfed or formula fed (80 per group). Faecal samples were collected at recruitment and 3 months later. Bacterial groups characteristic of the human faecal microbiota were quantified in faeces by quantitative polymerase chain reaction. Calprotectin and α1-antitrypsin concentrations were measured using commercial kits. Details of diarrhoeal morbidity were documented and rated for severity.The compositions of the faecal microbiota of formula-fed compared with breast milk-fed children were similar except that the probiotic strain Bifidobacterium animalis subsp. lactis DR10 was more abundant after 3 months consumption of the formula. Alpha1-antitrypsin levels were higher in breastfed compared with formula-fed infants. The occurrence of diarrhoea did not differ between the groups of babies.Feeding Indonesian babies with a probiotic/prebiotic formula did not produce marked differences in the composition of the faecal microbiota in comparison with breast milk. Detrimental effects of formula feeding on biomarkers of mucosal health were not observed.

Evidence suggests that gut microbes colonize the mammalian intestine through propagation as an adhesive microbial community. A bacterial artificial chromosome (BAC) library of murine bowel microbiota DNA in the surrogate host Escherichia coli DH10B was screened for enhanced adherence capability. Two out of 5,472 DH10B clones, 10G6 and 25G1, exhibited enhanced capabilities to adhere to inanimate surfaces in functional screens. DNA segments inserted into the 10G6 and 25G1 clones were 52 and 41 kb and included 47 and 41 protein-coding open reading frames (ORFs), respectively. DNA sequence alignments, tetranucleotide frequency, and codon usage analysis strongly suggest that these two DNA fragments are derived from species belonging to the genus Bacteroides . Consistent with this finding, a large portion of the predicted gene products were highly homologous to those of Bacteroides spp. Transposon mutagenesis and subsequent experiments that involved heterologous expression identified two operons associated with enhanced adherence. E. coli strains transformed with the 10a or 25b operon adhered to the surface of intestinal epithelium and colonized the mouse intestine more vigorously than did the control strain. This study has revealed the genetic determinants of unknown commensals (probably resembling Bacteroides species) that enhance the ability of the bacteria to colonize the murine bowel.

Four DNA probes were derived that hybridized specifically to DNA from Lactobacillus acidophilus O. The probes were constructed by randomly cloning lactobacillus DNA in plasmid vector pBR322. Two of the probes (pSR1 and pSR2) were composed of vector and plasmid DNA inserts (3.6 and 1.6 kb, respectively); the others (pSR3 and pSR4) were composed of vector and chromosomally derived inserts (6.9 and 1.4 kb, respectively). The probes were used to enumerate, by colony hybridization, strain O in digestive tract samples collected from piglets inoculated 24 hours previously with a culture of the strain. The probes did not hybridize to DNA from lactobacilli inhabiting the digestive tract of uninoculated piglets. Strain O made up about 10% of the total lactobacillus population of the pars esophagea and about 20% of the population in other digestive tract samples.

Fifty-four lactobacillus strains were differentiated by ribotyping. The stability of ribotypes characteristic of four strains of lactobacilli inhabiting the digestive tract of mice was investigated. One of four isolates of Lactobacillus delbrueckii GT21, which had been associated with mice for 22 months, had an altered ribotype.

The human bowel contains a large and biodiverse bacterial community known as the microbiota or microbiome. It seems likely that the microbiota, fractions of the microbiota, or specific species comprising the microbiota provide the antigenic fuel that drives the chronic immune inflammation of the bowel mucosa that is characteristic of Crohn's disease and ulcerative colitis. At least twenty years of microbiological research have been expended on analysis of the composition of the bowel microbiota of inflammatory bowel disease patients in comparison to that of control subjects. Despite extensive speculations about the aetiological role of dysbiosis in inflammatory bowel diseases, knowledge that can be easily translated into effective remedies for patients has not eventuated. The causes of this failure may be due to poorly defined and executed bacteriological studies, as well as the overwhelming complexity of a biome that contains hundreds of bacterial species and trillions of bacterial cells.

Nucleic acid-based (molecular) analytical methods have utility in discriminating between bowel microbiota of altered compositions. This has been demonstrated in studies involving both experimental animals and humans with inflammation of the bowel. Although alterations in the composition of the microbiota can be demonstrated, future studies need to provide functional links between the candidate proinflammatory agents and disease processes.

The bowels of humans contain resident bacterial communities, the members of which are numerous and biodiverse. Changes in the composition of bowel communities is accepted to occur in relation to antibiotic-associated colitis of the elderly, but compositional alterations could also be relevant to allergic diseases in children and inflammatory bowel diseases (i.e. Crohn's disease and ulcerative colitis). It is timely, therefore, to reflect on current knowledge of the bacterial community of the human bowel in relation to disease. Modern analytical methods provide tools by which compositional shifts in bacterial communities can be detected, but inadequate bowel-sampling procedures and poorly designed studies hamper progress. Moreover, demonstration that population shifts cause the disease and are not just reflections of a diseased state is necessary. Therefore, important challenges remain for bacteriologists in investigations of the bowel bacterial community in relation to disease.

The dlt operon of Gram-positive bacteria encodes proteins required for the incorporation of D-alanine esters into cell wall-associated teichoic acids (TA). D-alanylation of TA has been shown to be important for acid tolerance, resistance to antimicrobial peptides, adhesion, biofilm formation, and virulence of a variety of pathogenic organisms. The aim of this study was to determine the importance of D-alanylation for colonization of the gastrointestinal tract by Lactobacillus reuteri 100-23. Insertional inactivation of the dltA gene resulted in complete depletion of D-alanine substitution of lipoteichoic acids. The dlt mutant had similar growth characteristics as the wild type under standard in vitro conditions, but formed lower population sizes in the gastrointestinal tract of ex-Lactobacillus-free mice, and was almost eliminated from the habitat in competition experiments with the parental strain. In contrast to the wild type, the dlt mutant was unable to form a biofilm on the forestomach epithelium during gut colonization. Transmission electron microscope observations showed evidence of cell wall damage of mutant bacteria present in the forestomach. The dlt mutant had impaired growth under acidic culture conditions and increased susceptibility to the cationic peptide nisin relative to the wild type. Ex vivo adherence of the dlt mutant to the forestomach epithelium was not impaired. This study showed that D-alanylation is an important cell function of L. reuteri that seems to protect this commensal organism against the hostile conditions prevailing in the murine forestomach.

The significance of bile salt hydrolase production by lactobacilli in the microecology of the murine intestinal tract has not been extensively studied previously. Assays of bile salt hydrolase (sodium taurocholate as substrate) associated with cell extracts of five Lactobacillus strains of murine origin gave a range of activities (from 915 nmol of cholate released per mg of protein per 30 min to none detected). All of the strains tested colonized the murine gastrointestinal tract equally well. The growth rates of mice were not affected by colonization of their intestinal tracts by lactobacilli whether or not the bacteria produced bile salt hydrolase.

Purpose Chronic pouchitis is an important long-term complication following ileal pouch formation. Antibiotics reduce symptoms indicating bacteria play a role in pathogenesis. We analyzed and compared the bacterial content (microbiota) of stool collected from chronic pouchitis and familial adenomatous polyposis patients. Additionally, we compared the microbiota of chronic pouchitis patients on and off antibiotics. We aimed to determine which of the bacterial inhabitants of pouches had a role in the pathogenesis of pouchitis. Methods The stool microbiota of 17 patients with chronic pouchitis and of 15 familial adenomatous polyposis patients were analyzed by nucleic acid-based methods (temporal temperature gradient gel electrophoretic profiles, fluorescence in situ hybridization with flow cytometry). Results The composition of the stool of chronic pouchitis and familial adenomatous polyposis patients was markedly different. Electrophoretic profiles of the stool microbiota of familial adenomatous patients clustered at the 80% level of similarity, whereas those of chronic pouchitis patients were disparate. The results of fluorescence in situ hybridization analysis showed that bacteria not commonly present in human faeces, nor in the stool of familial adenomatous polyposis patients, comprised about 50% of the stool microbiota of untreated chronic pouchitis patients. Antibiotic treatment reduced the proportion of these unknown bacteria in the stool of every chronic pouchitis patient. Conclusion Chronic pouchitis is associated with a microbiota that contains bacteria not commonly associated with human faeces. These bacteria are reduced with antibiotics and are likely to be the pathogenic agents of chronic pouchitis.