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Over the last years, there has been increasing interest in the intestinal microbiota internationally and in New Zealand. As an illustration of this, more than 26,000 items were selected on a curren...

The gut microbiota harvests energy from indigestible plant polysaccharides, forming short-chain fatty acids (SCFAs) that are absorbed from the bowel. SCFAs provide energy—presumably after easily digested food components have been absorbed from the small intestine. Infant night waking is believed by many parents to be due to hunger. Our objective was to determine whether faecal SCFAs are associated with longer uninterrupted sleep in infants. Infants (n = 57) provided faecal samples for determining SCFAs (7 months of age), and questionnaire data for determining infant sleep (7 and 8 months). Linear regression determined associations between SCFAs—faecal acetate, propionate and butyrate—and sleep. For each 1% higher propionate at 7 months of age, the longest night sleep was 6 (95% CI: 1, 10) minutes longer at both 7 and 8 months. A higher proportion of total faecal SCFA as propionate was associated with longer uninterrupted infant sleep.

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...

The human colon contains a community of microbial species, mostly bacteria, which is often referred to as the gut microbiota. The community is considered essential to human well-being by conferring additional energy-harvesting capacity, niche exclusion of pathogens, and molecular signaling activities that are integrated into human physiological processes. Plant polysaccharides (glycans, dietary fiber) are an important source of carbon and energy that supports the maintenance and functioning of the gut microbiota. Therefore, the daily quantity and quality of plant glycans consumed by the human host have the potential to influence health. Members of the gut microbiota differ in ability to utilize different types of plant glycans. Dietary interventions with specific glycans could modulate the microbiota, counteracting ecological perturbations that disrupt the intricate relationships between microbiota and host (dysbiosis). This review considers prospects and research options for modulation of the gut microbiota by the formulation of diets that, when consumed habitually, would correct dysbiosis by building diverse consortia that boost functional resilience. Traditional “prebiotics” favor bifidobacteria and lactobacilli, whereas dietary mixtures of plant glycans that are varied in chemical complexity would promote high-diversity microbiotas. It is concluded that research should aim at improving knowledge of bacterial consortia that, through shared nourishment, degrade and ferment plant glycans. The consortia may vary in composition from person to person, but functional outputs will be consistent in a given context because of metabolic redundancy among bacteria. Thus, the individuality of gut microbiotas could be encompassed, functional resilience encouraged, and correction of dysbiosis achieved.

Preterm infants are exposed to major perinatal, post-natal, and early infancy events that could impact on the gut microbiome. These events include infection, steroid and antibiotic exposure, parenteral nutrition, necrotizing enterocolitis, and stress. Studies have shown that there are differences in the gut microbiome during the early months of life in preterm infants. We hypothesized that differences in the gut microbial composition and metabolites in children born very preterm persist into mid-childhood. Participants were healthy prepubertal children aged 5–11 years who were born very preterm (≤32 weeks of gestation; n = 51) or at term (37–41 weeks; n = 50). We recorded the gestational age, birth weight, mode of feeding, mode of birth, age, sex, and the current height and weight of our cohort. We performed a multi'omics [i.e., 16S rRNA amplicon and shotgun metagenomic sequencing, SPME-GCMS (solid-phase microextraction followed by gas chromatography-mass spectrometry)] analysis to investigate the structure and function of the fecal microbiome (as a proxy of the gut microbiota) in our cross-sectional cohort. Children born very preterm were younger (7.8 vs. 8.3 years; p = 0.034), shorter [height-standard deviation score (SDS) 0.31 vs. 0.92; p = 0.0006) and leaner [BMI (body mass index) SDS −0.20 vs. 0.29; p < 0.0001] than the term group. Children born very preterm had higher fecal calprotectin levels, decreased fecal phage richness, lower plasma arginine, lower fecal branched-chain amino acids and higher fecal volatile (i.e., 3-methyl-butanoic acid, butyrolactone, butanoic acid and pentanoic acid) profiles. The bacterial microbiomes did not differ between preterm and term groups. We speculate that the observed very preterm-specific changes were established in early infancy and may impact on the capacity of the very preterm children to respond to environmental changes.

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.

Diet is considered one of the key drivers of the world-wide obesity epidemic, and the gut microbiota may play a role in this multifaceted disease due to their mutualistic relationship. This study investigated relationships between habitual dietary intake of New Zealand European and Pacific women and their gut microbiota and body fat content. Pacific (44%) and NZ European (NZE; 56%) women (n = 287) aged 18–45 years were recruited based on body mass index (normal versus obese) and stratified as low (< 35%) or high (≥ 35%) body fat percentage (BF%). Dietary intake was assessed with a 5-day estimated food record and a semi-quantitative food frequency questionnaire, which were used to calculate habitual dietary intake using the National Cancer Institute (NCI) method. BF% was assessed by dual-energy x-ray absorptiometry (DXA). Fasting blood samples were analysed for markers of insulin sensitivity. The DNA from faecal samples was analysed following shotgun sequencing. There were no significant differences in BF% between Pacific and NZE women (p = 0.498). Significant differences in homeostasis model assessment of insulin resistance (HOMA-IR) index were observed between Pacific (3.4 [2.3, 5.9]) and NZE (2.1 [1.5, 3.1], p ≤ 0.001) women, and between; low-BF% (1.9 [1.3, 2.7]) and high-BF% (3.4 [2.5, 5.9], p ≤ 0.001) groups. The highest (27.6g/d [24.9, 30.6]) compared to the lowest tertile (16g/d [13.3, 17.6]) of habitual total dietary fibre (DF) intake was associated with a significantly lower HOMA-IR (2.1 [1.3, 3.1] versus 3.3 [2.1, 5.3] p ≤ 0.001) respectively. Higher DF intake was also associated with significantly lower BF% (β -0.35, p ≤ 0.001), and this relationship became stronger when considering the intake of other macronutrients (β -0.47, p ≤ 0.001). Alpha diversity; observed taxonomic units (OTU's; rs = -0.15, p = 0.011), Pielou's evenness (rs = -0.20, p = 0.001), and Shannon index (rs = -0.22, p ≤ 0.001), were all negatively correlated with BF%. In contrast BF% was positively correlated with the Firmicutes:Bacteroidetes ratio (rs = 0.26, p ≤ 0.001). HOMA-IR index was significantly higher in Pacific and women in the higher BF% group, indicating an increased metabolic disease risk. Higher habitual DF intake was associated with lower BF% and HOMA-IR, suggesting a potential metabolically protective effect. The positive effects of higher DF intake may be associated with microbiota diversity, as higher BF% was associated with reduced alpha diversity and an increased Firmicutes:Bacteroidetes ratio. Further analysis will explore which foods contributed to the higher DF intake, and associations with body composition, microbiota and biomarkers of metabolic health.

Dietary fiber is an important nutrient for the gut microbiota, with different fiber fractions having different effects. The aim of this study was to determine the relative validity and reproducibility of a food frequency questionnaire (EAT5 FFQ) for measuring intake of fiber, and low and high fiber foods, in studies examining diet and gut microbiota in young children. One hundred parents of 5-year old children completed the 123-item EAT5 FFQ on two occasions four weeks apart. A 3-day weighed diet record (WDR) was completed on non-consecutive days between FFQ appointments. Mean correlations between the (randomly chosen) FFQ and WDR were acceptable for nutrient and food group intakes (r = 0.34 and r = 0.41 respectively). Gross misclassification was below chance (12.5%) for quartiles of nutrient (mean 5.7%) and food group (mean 5.1%) intake. &lsquo, Absolute values for surrogate categories&rsquo, suggested the FFQ clearly differentiated between highest and lowest quartiles for all nutrients and food groups tested. Mean correlations between repeat administrations of the FFQ suggested very good reproducibility for nutrients (r = 0.83) and food groups (r = 0.80). The EAT5 FFQ appears to be an appropriate tool for investigating the intake of nutrients and food groups of relevance to the gut microbiota, and is the first FFQ validated to measure total, soluble and insoluble non-starch polysaccharide intakes in young children.

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.

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.

Background The prevalence of obesity has increased substantially over recent decades and is associated with considerable health inequalities. Although the causes of obesity are complex, key drivers include overconsumption of highly palatable, energy-dense, and nutrient-poor foods, which have a profound impact on the composition and function of the gut microbiome. Alterations to the microbiome may play a critical role in obesity by affecting energy extraction from food and subsequent energy metabolism and fat storage. Objective We report the study protocol and recruitment strategy of the PRedictors linking Obesity and the gut MIcrobiomE (PROMISE) study, which characterizes the gut microbiome in 2 populations with different metabolic disease risk (Pacific and European women) and different body fat profiles (normal and obese). It investigates (1) the role of gut microbiome composition and functionality in obesity and (2) the interactions between dietary intake; eating behavior; sweet, fat, and bitter taste perception; and sleep and physical activity; and their impact on the gut microbiome, metabolic and endocrine regulation, and body fat profiles. Methods Healthy Pacific and New Zealand (NZ) European women aged between 18 and 45 years from the Auckland region were recruited for this cross-sectional study. Participants were recruited such that half in each group had either a normal weight (body mass index [BMI] 18.5-24.9 kg/m2) or were obese (BMI ≥30.0 kg/m2). In addition to anthropometric measurements and assessment of the body fat content using dual-energy x-ray absorptiometry, participants completed sweet, fat, and bitter taste perception tests; food records; and sleep diaries; and they wore accelerometers to assess physical activity and sleep. Fasting blood samples were analyzed for metabolic and endocrine biomarkers and DNA extracted from fecal samples was analyzed by shotgun sequencing. Participants completed questionnaires on dietary intake, eating behavior, sleep, and physical activity. Data were analyzed using descriptive and multivariate regression methods to assess the associations between dietary intake, taste perception, sleep, physical activity, gut microbiome complexity and functionality, and host metabolic and body fat profiles. Results Of the initial 351 women enrolled, 142 Pacific women and 162 NZ European women completed the study protocol. A partnership with a Pacific primary health and social services provider facilitated the recruitment of Pacific women, involving direct contact methods and networking within the Pacific communities. NZ European women were primarily recruited through Web-based methods and special interest Facebook pages. Conclusions This cross-sectional study will provide a wealth of data enabling the identification of distinct roles for diet, taste perception, sleep, and physical activity in women with different body fat profiles in modifying the gut microbiome and its impact on obesity and metabolic health. It will advance our understanding of the etiology of obesity and guide future intervention studies involving specific dietary approaches and microbiota-based therapies. Trial Registration Australian New Zealand Clinical Trials Registry ACTRN12618000432213; https://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=370874 International Registered Report Identifier (IRRID) RR1-10.2196/14529

Background: New Zealand is culturally diverse and has one of the highest rates of obesity in theworld, especially among women. [...]

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.

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.

Alterations to the composition of the bowel microbiota (dysbioses) are associated with particular diseases and conditions of humans. There is a need to discover new, indigestible polysaccharides which are selective growth substrates for commensal bowel bacteria. These substrates (prebiotics) could be added to food in intervention studies to correct bowel dysbiosis. A collection of commensal bacteria was screened for growth in culture using a highly-branched xylan produced by New Zealand flax. Two, Bacteroides ovatus ATCC 8483 and Bacteroides xylanisolvens DSM 18836 grew well on this substrate. The utilisation of the xylan was studied chromatographically and by constituent sugar analysis. The two closely related species utilised the xylan in different ways, and differently from their use of wheat arabinoxylan. The growth of Bacteroides species on other plant xylans having differing chemical structures was also investigated. Novel xylans expand the choice of potential prebiotics that could be used to correct bowel dysbioses.

Background The Prevention of Overweight in Infancy (POI) study was a four-arm randomised controlled trial (RCT) in 802 families which assessed whether additional education and support on sleep (Sleep group); food, physical activity and breastfeeding (FAB group); or both (Combination group), reduced excessive weight gain from birth to 2 years of age, compared to usual care (Control group). The study had high uptake at recruitment (58 %) and retention at 2 years (86 %). Although the FAB intervention produced no significant effect on BMI or weight status at 2 years, the odds of obesity were halved in those who received the sleep intervention, despite no apparent effect on sleep duration. We speculate that enhanced self-regulatory behaviours may exist in the Sleep group. Self-regulation was not measured in our initial intervention, but extensive measures have been included in this follow-up study. Thus, the overall aim of the POI follow-up is to determine the extent to which augmented parental support and education on infant sleep, feeding, diet, and physical activity in the first 2 years of life reduces BMI at 3.5 and 5 years of age, and to determine the role of self-regulation in any such relationship. Methods/design We will contact all 802 families and seek renewed consent to participate in the follow-up study. The families have received no POI intervention since the RCT finished at 2 years of age. Follow-up data collection will occur when the children are aged 3.5 and 5 years (i.e. up to 3 years post-intervention). Outcomes of interest include child anthropometry, body composition (DXA scan), diet (validated food frequency questionnaire), physical activity (accelerometry), sleep (questionnaire and accelerometry), and self-regulation (questionnaires and neuropsychological assessment). Discussion Our follow-up study has been designed primarily to enable us to determine whether the intriguing benefit of the sleep intervention suggested at 2 years of age remains as children approach school age. However, cohort analyses will also investigate how BMI, self-regulation, and sleep consolidation develop during the early years. This information will be valuable to researchers and policy makers progressing the field of early childhood obesity prevention. Trial registration ClinicalTrials.gov number NCT00892983 .

A feeding trial was performed with broilers receiving a diet of wheat-based feed (WBF), maize-based feed (MBF), or maize-based concentrates supplemented with 15% or 30% crimped kernel maize silage (CKMS-15 or CKMS-30, respectively). The aim of the study was to investigate the bacterial community compositions of the crop, gizzard, ileum, and cecum contents in relation to the feeding strategy and age (8, 15, 22, 25, 29, or 36 days). Among the four dietary treatments, bacterial diversity was analyzed for MBF and CKMS-30 by 454 pyrosequencing of the 16S rRNA gene. Since the diets had no significant influence on bacterial diversity, data were pooled for downstream analysis. With increasing age, a clear succession of bacterial communities and increased bacterial diversity were observed. Lactobacillaceae (belonging mainly to the genus Lactobacillus ) represented most of the Firmicutes at all ages and in all segments of the gut except the cecum. The development of a “mature” microbiota in broilers occurred during the period from days 15 to 22. Striking increases in the relative abundances of Lactobacillus salivarius (17 to 36%) and clostridia (11 to 18%), and a concomitant decrease in the relative abundance of Lactobacillus reuteri , were found in the ileum after day 15. The concentration of deconjugated bile salts increased in association with the increased populations of L. salivarius and clostridia. Both L. salivarius and clostridia deconjugate bile acids, and increases in the abundances of these bacteria might be associated with growth reduction and gastrointestinal (GI) disorders occurring in the critical period of broiler life between days 20 and 30.

Background Probiotic administration has been proposed for the prevention and treatment of specific allergic manifestations such as eczema, rhinitis, gastrointestinal allergy, food allergy, and asthma. However, published statements and scientific opinions disagree about the clinical usefulness.Objective A World Allergy Organization Special Committee on Food Allergy and Nutrition review of the evidence regarding the use of probiotics for the prevention and treatment of allergy.Methods A qualitative and narrative review of the literature on probiotic treatment of allergic disease was carried out to address the diversity and variable quality of relevant studies. This variability precluded systematization, and an expert panel group discussion method was used to evaluate the literature. In the absence of systematic reviews of treatment, meta-analyses of prevention studies were used to provide data in support of probiotic applications.Results Despite the plethora of literature, probiotic research is still in its infancy. There is a need for basic microbiology research on the resident human microbiota. Mechanistic studies from biology, immunology, and genetics are needed before we can claim to harness the potential of immune modulatory effects of microbiota. Meanwhile, clinicians must take a step back and try to link disease state with alterations of the microbiota through well-controlled long-term studies to identify clinical indications.Conclusions Probiotics do not have an established role in the prevention or treatment of allergy. No single probiotic supplement or class of supplements has been demonstrated to efficiently influence the course of any allergic manifestation or long-term disease or to be sufficient to do so. Further epidemiologic, immunologic, microbiologic, genetic, and clinical studies are necessary to determine whether probiotic supplements will be useful in preventing allergy. Until then, supplementation with probiotics remains empirical in allergy medicine. In the future, basic research should focus on homoeostatic studies, and clinical research should focus on preventive medicine applications, not only in allergy. Collaborations between allergo-immunologists and microbiologists in basic research and a multidisciplinary approach in clinical research are likely to be the most fruitful. Keywords: probiotics, prevention of allergy, pediatric allergy

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.

BACKGROUND: Allergic disease has been associated with altered intestinal microbiota. Therefore, probiotics have been suggested as a potential treatment for eczema. OBJECTIVE: We investigated whether dietary supplementation of infants with eczema at age 3-6 months with Lactobacillus paracasei CNCM I-2116 or Bifidobacterium lactis CNCM I-3446 had a treatment effect or altered allergic disease progression. METHODS: Primary outcome included eczema severity (SCORing Atopic Dermatitis, SCORAD) 3 months post-randomization. Secondary: SCORAD (other visits); infant dermatitis quality of life (IDQoL); gastrointestinal permeability; urinary eosinophilic protein X; allergen-sensitization; allergic symptoms (age 12, 18, 36 months). A total of 208 infants aged 3-6 months with physician-diagnosed eczema were recruited; 137/208 (SCORAD ? 10, consuming ? 200 mL standard formula/day) were randomized to daily supplements containing L. paracasei or B. lactis or placebo for a 3-month period, while receiving extensively hydrolysed whey-formula (dairy-free diet). There were two open observational groups, one group exclusively breastfed (n = 22) and the other, standard formula-fed (n = 49). TRIAL NUMBER: ISRCTN41490500. RESULTS: Eczema severity decreased significantly over time in all groups. No significant difference was observed between randomized groups after 12-week treatment-period (SCORAD-score pre-/post-intervention: B. lactis 25.9 [95% CI: 22.8-29.2] to 12.8 [9.4-16.6]; L. paracasei 25.4 [22.1-29] to 12.5 [9.2-16.4]; placebo 26.9 [23.4-30.6] to 11.8 [9.6-14.3]; P = 0.7). Results were similar when analysis was controlled for allergen-sensitization, or when only sensitized infants were analysed. No differences were found for secondary outcomes. No difference was observed in SCORAD-score between randomized and observational groups. CONCLUSION AND CLINICAL RELEVANCE: We found no benefit from supplementation with B. lactis or L. paracasei in the treatment of eczema, when given as an adjunct to basic topical treatment, and no effect on the progression of allergic disease from age 1 to 3 years.

A high carriage rate of multidrug-resistant Escherichia coli (MDREC) was observed in elderly residents in long-term care facilities. A double-blinded, placebo-controlled trial was carried out to determine whether the probiotic product E. coli strain Nissle 1917 (Mutaflor) would compete with MDREC in the bowel and thereby reduce the prevalence of the multiresistant bacteria in faeces and urine. Sixty-nine patients excreting norfloxacin-resistant E. coli were randomized to probiotic or placebo groups and administered capsules twice daily. The daily dose of probiotic was 5×109–5×1010 bacteria. Faecal and urine samples were cultured at baseline and during and after the treatment period. A reduction in baseline carriage was not influenced by probiotic administration. The probiotic strain was detected in faecal specimens collected during the treatment period of only two out of 12 probiotic group subjects that were tested. Genotyping of norfloxacin-resistant E. coli isolates showed that 32 strains were prevalent among the patients. Thus, E. coli Nissle 1917 does not have the capacity to compete effectively with MDREC in the bowel of elderly patients.

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.

Monoassociations of germ-free animals with colitogenic and probiotic bacterial strains trigger intestinal epithelial cell (IEC) activation and host-derived feedback mechanisms. To characterize the impact of a single nonpathogenic bacterial strain on the intestinal epithelium in the presence of an established microbiota, we inoculated reconstituted Lacotobacillus-free (RLF) mice at 8 wk of age with Lactobacillus reuteri 100-23. Primary IEC from the small intestine of L. reuteri-inoculated and control RLF mice were isolated 2, 6, and 21 d after inoculation followed by gene expression analysis (real-time PCR; Affymetrix microarrays) as well as 2-dimensional-gel electrophoreses (2D SDS-PAGE) and peptide mass fingerprinting via matrix-assisted laser desorption/ionization time of flight MS. At d 6, gene expression of proinflammatory cytokines and chemokines including interleukin (IL)-1alpha, IL-6, interferon-gamma-inducible protein 10, and macrophage inflammatory protein 2 was transiently induced, whereas gene expression levels of regulatory proteins A20 and Toll-interacting protein decreased. In addition, 8 target proteins with changes in the steady-state protein expression levels were identified at d 2 and 6 of L. reuteri colonization. Consistent with the absence of histopathology, L. reuteri-induced activation of primary IEC returned to control levels by d 21 after inoculation of RLF mice. The capability of L. reuteri 100-23 to directly trigger epithelial cell activation was confirmed in small IEC cultures using the murine cell line Mode-K. These results clearly indicate that the intestinal epithelium is reactive toward environmental changes induced by the commensal bacterial strain L. reuteri even in the presence of an already-established microbiota. The induction of transient IEC activation may help to maintain mucosal homeostasis.

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.

Bowel commensals appear to be an important source of antigens that drive the chronic immune inflammation characteristic of Crohn's disease and ulcerative colitis [inflammatory bowel diseases (IBD)]. Biopsy-associated bacteria are assumed to be particularly relevant in bacteriological investigations of IBD because they are assumed to be located on the mucosal surface and hence close to immunological cells. This investigation analysed the bacterial collections associated with bowel biopsies, aspirates of residual fluid after bowel cleansing and faeces from IBD patients and non-IBD subjects in Edmonton, Canada, and Mexico City, Mexico. Temporal temperature gradient gel electrophoresis of 16S rRNA gene sequences produced profiles of the bacterial collections and their similarities were compared. Similarity analysis showed that the profiles did not cluster according to disease status, but that Canadian and Mexican profiles could be differentiated by this method. Comparison of biopsy, aspirate and faecal samples obtained from the same subject showed that, on average, the profiles were highly similar. Therefore, biopsy-associated bacteria are likely to represent, at least in part, contaminants from the fluid, which resembles a faecal solution, that pools in the bowel after cleansing prior to endoscopy.

The fecal microbiota of 37 infants with (n = 20) or without (n = 17) probiotic administration was evaluated on D 3, and at 1, 3, and 12 mo by fluorescence in situ hybridization-flow cytometry (FISH-FC), PCR, and bacteriological culture methods. They represent consecutive subjects of an ongoing double-blind, placebo-controlled trial on a probiotic formula (LGG and Bifidobacterium longum) administered during the first 6 mo of life. Despite varying composition in each baby, there was a general bacterial colonization pattern in the first year. Bifidobacteria increased markedly (p = 0.0003) with a parallel decrease in Enterobacteriaceae (p0.001) and Bacteroides-Prevotella (p = 0.005) populations. Eubacterium rectale-Clostridium coccoides (p0.001) and Atopobium (p = 0.039) groups also gradually increased. This overall pattern was unaffected by probiotic administration (p0.05). B. longum (p = 0.005) and Lactobacillus rhamnosus (p0.001) were detected more frequently in probiotic group during supplementation, but no difference after supplementation had ceased (p0.05). Cultured lactic acid bacteria were also more numerous in the probiotic-administered babies during treatment period (log CFU/g 8.4 versus 7.4; p = 0.035). Our results indicate that supplemented strains could be detected but did not persist in the bowel once probiotic administration had ceased.

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’

The bacterial community (microbiota) that inhabits the gut of humans appears to be an important source of antigens that drive the chronic immunological processes characteristic of Crohn's disease (CD) and ulcerative colitis (UC). Most of the members of the microbiota have not yet been cultured, but nucleic-acid-based methods of detection and enumeration can provide information about the community. This investigation used these methods to obtain information about the bacteria associated with mucosal surfaces in the gut of 20 CD and 15 UC patients. Biopsies were collected from inflamed and non-inflamed sites in the intestines of newly diagnosed, untreated patients. Biopsies were also collected from several intestinal sites of 14 healthy subjects. The bacterial collections associated with the biopsies were analysed by generating PCR/denaturing gradient gel electrophoresis (DGGE) profiles, the preparation of 16S rRNA gene clone libraries, and qualitative PCR to detect specific groups of bacteria. The total numbers of bacteria associated with the biopsies were determined by real-time quantitative PCR. DGGE profiles generated from the terminal ileum and various colonic regions were characteristic of each individual but differed between subjects. DGGE profiles and 16S rRNA gene libraries showed that the bacteria associated with inflamed and non-inflamed tissues did not differ. UC patients had more bacteria associated with biopsies than did CD patients (PP

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.

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 gut microbiota may be important in the postnatal development of the immune system and hence may influence the prevalence of atopic diseases. Bifidobacteria are the most numerous bacteria in the guts of infants, and the presence or absence of certain species could be important in determining the geographic incidence of atopic diseases. We compared the fecal populations of bifidobacteria from children aged 25 to 35 days in Ghana (which has a low prevalence of atopy), New Zealand, and the United Kingdom (high-prevalence countries). Natal origin influenced the detection of bifidobacterial species in that fecal samples from Ghana almost all containedBifidobacterium infantiswhereas those of the other children did not. Choosing species on the basis of our bacteriological results, we tested bifidobacterial preparations for their effects on cell surface markers and cytokine production by dendritic cells harvested from cord blood. Species-specific effects on the expression of the dendritic-cell activation marker CD83 and the production of interleukin-10 (IL-10) were observed. Whereas CD83 expression was increased and IL-10 production was induced byBifidobacterium bifidum,Bifidobacterium longum, andBifidobacterium pseudocatenulatum,B. infantisfailed to produce these effects. We concluded thatB. infantisdoes not trigger the activation of dendritic cells to the degree necessary to initiate an immune response but thatB. bifidum,B. longum, andB. pseudocatenulatuminduce a Th2-driven immune response. A hypothesis is presented to link our observations to the prevalence of atopic diseases in different countries.

An overly aggressive immune response to the intestinal microflora in a genetically susceptible host background has been implicated in the pathogenesis of inflammatory bowel diseases. We measured the impact of a probiotic preparation (SIM) containing inulin on the severity of colitis and on intestinal microflora profiles of HLA-B27-β 2 -microglobulin transgenic (TG) rats. SIM is a mixture of lactobacilli, bifidobacteria, and inulin. Two-month-old TG rats received either SIM or water. Control TG rats received metronidazole, alone or in combination with SIM, for 8 weeks. Nontransgenic rats received SIM or water. The cecal content was removed for analysis of the intestinal microflora by PCR combined with denaturing gradient gel electrophoresis. The colon was scored for histological evidence of inflammation, colonic myeloperoxidase activity and interleukin-1β RNA levels were measured photometrically or by real-time quantitative PCR. At 4 months, the colonic inflammation of TG rats treated with SIM was histologically diminished compared to that in untreated TG rats (2.2 ± 0.2 versus 2.9 ± 0.1; P ≤ 0.03). The administration of SIM altered the microflora profiles of TG rats by increasing the diversity and stimulating specifically the growth of Bifidobacterium animalis . The probiotic bacteria added to SIM were below the detection level in cecal stool samples at the end of the study period. The administration of SIM resulted in a measurable impact on the cecal microflora profiles of TG rats with attenuation of colitis. The lack of detection of any added probiotic bacteria in the cecal content suggests that prebiotic inulin is the major effective compound.

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 terminal ileum and the large bowel (cecum and colon) are hospitable places for bacterial proliferation, and a complex and numerous bacterial community resides in this site. This community is often referred to as the normal gut microbiota (microflora). Many of the numerically important members of the gut microbiota have not yet been cultivated under laboratory conditions and are known and detected on the basis of their 16S rRNA gene sequences. Comparisons of the characteristics of germfree and conventional animals have clearly demonstrated that the gut microbiota has considerable influences on host biochemistry, physiology, immunology, and low-level resistance to gut infections. Cytoskeletal rearrangements within the enterocyte form a socket by which the filament becomes permanently attached to the mucosal surface. The influence of the gut microbiota of neonates on the immune system is of especial interest because of the observed increase in the incidence of allergies in children in affluent countries over recent decades. The adherence of Lactobacillus cells to, and proliferation on, epithelial surfaces in rodents, pigs, and poultry has tempted some researchers to consider that the same phenomenon occurs in the human gut. This overlooks the differences in the anatomy and histology of the human gut relative to that of other monogastric animals. In other words, the immune systems of different humans or other animals recognize different epitopes. This is apparent from experimental-animal studies because the composition of the gut microbiota of HLA-B27 rats and interleukin-10-deficient mice is different, yet colitis results in both types of animals.

This chapter suggests ways in which knowledge of the microbial ecology of the human bowel can be obtained using modern technologies. Probiotics aimed at altering the composition of oral and vaginal ecosystems have been developed or are under development, most products target bowel health. There is little doubt that lacto-bacilli transit the digestive tract following consumption of probiotic products, but in order to evaluate the probiotics phenomenon, one must consider the microbial ecology of the human gut. Some members of the medical profession, as well as the laity, have greeted with enthusiasm the use of current probiotic products as prophylaxis for atopic diseases (allergies), inflammatory bowel diseases (Crohn’s disease [CD] and ulcerative colitis [UC]), and pouchitis. Regulatory mechanisms generated within the ecosystem (autogenic factors) and by external forces (allogenic factors) permit the episodic persistence of some bacterial populations but the elimination of others in a classical biological succession. The best evidence for the efficacy of probiotics in inflammatory conditions of the bowel comes from studies of the maintenance of remission of pouchitis. Metabolomics is the nontargeted, holistic, quantitative analysis of changes in the complete set of metabolites in the cell (the metabolome) in response to environmental or cellular changes. The study of the metabonome might contribute to a full systems biology approach to understanding and maintaining bowel health. The primary aim of the research will be to understand how all of the heterogeneous parts are integrated, with a supplementary aim of identifying biomarkers of health or disease.

Three molecular analytical approaches based on small ribosomal subunit RNA (16S rRNA) sequences have proven to be able to provide reliable knowledge about the richness (diversity of species) and evenness (population sizes) of the fecal microflora of humans. These approaches are phylogenetic analysis by PCR, cloning, sequencing; total fluorescent in situ hybridization (FISH) analysis using oligonucleotide probes that target specific 16S rRNA sequences; and oligonucleotide/total bacterial RNA hybridizations on membranes. Relatively well-described examples of the biological succession that occurs in the infant gut are provided by investigations of the acquisition of the intestinal microflora by mice and humans. Enterococci are inherently more resistant to antimicrobial drugs than other clinically important gram-positive bacteria, but the reason for this is not clear. Enterococci inhabit the gastrointestinal tract or environments contaminated by human waste and as such may be exposed to antibiotics that pass through the gastrointestinal tract. It is unlikely that enterococcal species experience selection with respect to antibacterial drugs during human infections. On the basis of these observations, it can be assumed that the intrinsic resistance of enterococci to many antimicrobials might have resulted from their need to survive and persist in highly competitive, and potentially detrimental, ecosystems such as the intestinal tract. The impact of imipenem on the composition of the intestinal microflora is, however, worthy of attention because enterococci have been isolated more frequently from pediatric cases of septicemia after the introduction of antibacterial drug.

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.

This article describes nucleic acid-based methods to assess the composition and function of the bowel microbiota. The methods range from the relatively simple (polymerase chain reaction) to the technically sophisticated (metatranscriptomics). Not all are accessible to the majority of laboratories, but a core of validated (used in more than 1 study) tools is readily available to most researchers. Reliance on a single methodology per human study is not recommended. Generally, a study could commence with a screening of samples to determine whether it will be worthwhile expending further time and money on an in-depth analysis.

The strain composition of Lactobacillus populations inhabiting the stomach and caecal contents of piglets aged 6–150 days was determined using ribotyping and pulsed field gel electrophoresis of DNA digests. These genetic fingerprinting methods proved effective in demonstrating transitions occurring in the Lactobacillus microflora of the pigs raised on a commercial farm. The composition of the Lactobacillus microflora showed periods of stability in which animals of the same age harboured similar collections of predominant strains. Periods of instability were also detected: post-weaning (stomach and caecum) and in grower accommodation (caecum). The majority of the lactobacillus isolates were resistant to erythromycin and tetracycline, presumably reflecting the use of antibiotics as prophylactic agents on the farm. A strain of Lactobacillus acidophilus , used to inoculate the piglets at 5 days old, was detected in the stomach contents of only the two pigs examined at 18 days old. Antibodies reactive with whole cells of lactobacilli were detected in serum samples obtained from the pigs. Keywords: lactobacilli, pigs, ribotyping, pulsed field gel electrophoresis, antibiotics.

Alkaline phosphatase and phosphodiesterase I activities of duodenal enterocytes harvested from mice with or without lactobacilli as intestinal inhabitants were determined. The presence of lactobacilli as members of the digestive tract microbiota did not influence the two enzyme activities. Keywords - Lactobacilli, Duodenum, Enterocytes, Enzymes, Alkaline phosphatase, Phosphodiesterase.

Clostridium difficile infection (CDI) is the most common identifiable cause of diarrhoea in hospitalized patients. Current therapies rely on the administration of metronidazole or vancomycin, which reduce vegetative populations of C. difficile in the bowel. Recurrence of the disease when treatment with these antibiotics ceases indicates that metronidazole and vancomycin affect not only C. difficile but also commensal populations that normally mediate competitive exclusion. Fidaxomicin is a new antibiotic that inhibits C. difficile. Our study shows that fidaxomicin had little effect on the composition of the faecal microbiota in terms of its major phylogenetic clusters. Notably, clostridial clusters XIVa and IV, and Bifidobacterium, were much less affected by fidaxomicin compared to vancomycin treatment. These findings help to explain the substantially reduced rates of relapse following treatment of CDI with fidaxomicin in recent clinical trials.

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.

Lactobacillus reuteri 100-23 is a bacterial commensal of the gastrointestinal tract of mice. Previous studies have shown that colonization of the murine gut by this strain stimulates small-bowel enterocytes to produce proinflammatory cytokines. This is associated with a mild, transitory inflammatory response 6 days after inoculation of formerly Lactobacillus-free animals. The inflammation subsides by 21 days after colonization, although lactobacilli continue to be present in the bowel. To determine the immunological mechanisms that underpin tolerance to bowel commensals, we investigated cytokine responses of dendritic cells and T cells after exposure to cells of L. reuteri 100-23. Interleukin-10 (IL-10), IL-2 and transforming growth factor-beta (TGF-beta) concentrations in supernatants of cultured immune cells, as well as the results of proliferative assays of mesenteric lymph node (MLN) cells and quantification of Foxp3-positive cells in MLN and spleen, indicated that L. reuteri 100-23 stimulated the development of an increased number of regulatory T cells.

Background: We recently showed that Bifidobacterium animalis is more prevalent within the colons of interleukin (IL)-10-deficient (−/−) mice than in wildtype (WT) animals colonized with the same specific pathogen-free (SPF) fecal contents. Here we tested the ability of this organism to cause T-cell-mediated intestinal inflammation by introducing it into germ-free (GF) IL-10−/− mice. Methods: GF IL-10−/− or WT mice were monoassociated with Bifidobacterium animalis subsp. animalis ATCC (American Type Culture Collection, Manassas, VA) 25527T or with B. infantis ATCC 15697T. Inflammation was measured by blinded histologic scores of the duodenum, cecum, and colon and by spontaneous secretion of IL-12/IL-23 p40 from colonic explants. Bacterial antigen-specific CD4+ mesenteric lymph node (MLN) T-cell recall responses were measured in response to antigen-presenting cells (APC) pulsed with bacterial lysates. Results:B. animalis caused marked duodenal inflammation and mild colitis in monoassociated IL-10−/− mice, whereas the intestinal tracts of WT animals remained free of inflammation. B. infantis colonization resulted in mild inflammation in the duodena of IL-10−/− mice. CD4+ MLN T cells from B. animalis monoassociated IL-10−/− mice secreted high levels of IFN-γ and IL-17 in response to B. animalis lysate. B. animalis equally colonized the different intestinal regions of WT and IL-10−/− mice. Conclusions:B. animalis, a traditional probiotic species that is expanded in experimental colitis in this model, induces marked duodenal and mild colonic inflammation and TH1/TH17 immune responses when introduced alone into GF IL-10−/− mice. This suggests a potential pathogenic role for this commensal bacterial species in a susceptible host. (Inflamm Bowel Dis 2009)

The fate of the broad-host-range, conjugative plasmid pAMβ1 in the digestive tract of mice was monitored by DNA-DNA hybridisations using a probe containing the ermAM determinant (macrolide-lincosamide-streptogramin type B resistance). Transfer of pAMβ1 from Lactobacillus reuteri to Enterococcus faecium occurred in the digestive tract of infant mice and from E. faecium to E. faecalis and to L. fermentum in the adult intestinal tract. Long-term administration of lincomycin to the animals resulted in a modified form of pAMβ1 in the L. reuteri host. The modified plasmid was present in caecal isolates of the lactobacillus at higher concentration than in the stock culture used to inoculate the mice at the start of the experiment, and was maintained stably by the lactobacilli in vivo . In contrast, pAMβ1 was not stably maintained in the stock strain in the absence of antibiotic administration. The modified plasmid was no longer self-transmissible. Long-term administration of antibiotic to the animals resulted in erythromycin-resistant strains of Escherichia coli and eubacteria. Keywords: Digestive tract; Microflora; Antibiotic; Lactobacillus; Enterococcus; pAMβ1.

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.

International audience; The bacterial community, in whole or in part, resident in the bowel of humans is considered to fuel the chronic immune inflammatory conditions characteristic of Crohn's disease and ulcerative colitis. Chronic or recurrent pouchitis in ulcerative colitis patients is responsive to antibiotic therapy, indicating that bacteria are the etiological agents. Microbiological investigations of the bacterial communities in stool or of biopsy-associated bacteria have so far failed to reveal conclusively the existence of pathogens or bacterial communities of consistently altered composition in IBD patients relative to control subjects. Confounding factors need to be eliminated from future studies by using better-defined patient populations of newly diagnosed and untreated individuals and by improved sampling procedures.