Type of intrinsic resistant starch type 3 determines in vitro fermentation by pooled adult faecal inoculum.

Resistant starch (RS) results in relatively high health-beneficial butyrate levels upon fermentation by gut microbiota. We studied how physico-chemical characteristics of RS-3 influenced butyrate production during fermentation. Six highly resistant RS-3 substrates (intrinsic RS-3, 80-95 % RS) differing in chain length (DPn 16-76), Mw distribution (PI) and crystal type (A/B) were fermented in vitro by pooled adult faecal inoculum. All intrinsic RS-3 substrates were fermented to relatively high butyrate levels (acetate/butyrate ≤ 2.5), and especially fermentation of A-type RS-3 prepared from polydisperse α-1,4 glucans resulted in the highest relative butyrate amount produced (acetate/butyrate: 1). Analysis of the microbiota composition after fermentation revealed that intrinsic RS-3 stimulated primarily Lachnospiraceae, Bifidobacterium and Ruminococcus, but the relative abundances of these taxa differed slightly depending on the RS-3 physico-chemical characteristics. Especially intrinsic RS-3 of narrow disperse Mw distribution stimulated relatively more Ruminococcus. Selected RS fractions (polydisperse Mw distribution) obtained after pre-digestion were fermented to acetate and butyrate (ratio ≤ 1.8) and stimulated Lachnospiraceae and Bifidobacterium. This study indicates that especially the α-1,4 glucan Mw distribution dependent microstructure of RS-3 influences butyrate production and microbiota composition during RS-3 fermentation.

The level and distribution of methyl-esters influence the impact of pectin on intestinal T cells, microbiota, and Ahr activation.

Pectins are dietary fibres that modulate T cell immunity, microbiota composition, and fermentation profiles, but how this is influenced by the degree of methyl-esterification (DM) and degree-of-blockiness (DB) of pectin is unknown. Here, we demonstrate that supplementation of DM19(high-DB), DM49(low-DB) and DM43(high-DB) pectins at a low dose increased the frequencies of intestinal T-helper (Th)1 and Th2 cells after 1 week of pectin supplementation in mice, whereas DM18(low-DB) did not. After 4 weeks of supplementation with those pectins, Th1 and Th2 frequencies returned to control levels, whereas Rorγt+ regulatory T-cell frequencies increased. These structure-dependent effects could derive from induced shifts in microbiota composition that differed between DM18(low-DB) pectin and the other pectins. T-cell-modulating effects were not short-chain-fatty acid-dependent, but rather through an increase in Aryl-hydrocarbon-receptor-activating components. Thus, pectins with a specific combination of DM and DB have an impact on intestinal T cell-immunity in mice, when supplemented at a low dose.

Differences in Fecal Gut Microbiota, Short-Chain Fatty Acids and Bile Acids Link Colorectal Cancer Risk to Dietary Changes Associated with Urbanization Among Zimbabweans.

The incidence of colorectal cancer (CRC) is gradually rising in sub-Saharan Africa. This may be due to dietary changes associated with urbanization, which may induce tumor-promoting gut microbiota composition and function. We compared fecal microbiota composition and activity in 10 rural and 10 urban Zimbabweans for evidence of a differential CRC risk. Dietary intake was assessed by a food frequency questionnaire. Fecal microbiota composition, metabolomic profile, functional microbial genes were analyzed, and bile acids and short chain fatty acids quantified. Animal protein intake was higher among urban volunteers, but carbohydrate and fiber intake were similar. Bacteria related to Blautia obeum, Streptococcus bovis, and Subdoligranulum variabile were higher in urban residents, whereas bacteria related to Oscillospira guillermondii and Sporobacter termitidis were higher in rural volunteers. Fecal levels of primary bile acids, cholic acid, and chenodeoxycholic acid (P < 0.05), and secondary bile acids, deoxycholic acid (P < 0.05) and ursodeoxycholic acid (P < 0.001) were higher in urban residents. Fecal levels of acetate and propionate, but not butyrate, were higher in urban residents. The gut microbiota composition and activity among rural and urban Zimbabweans retain significant homogeneity (possibly due to retention of dietary fiber), but urban residents have subtle changes, which may indicate a higher CRC risk.

Metabolic improvement in obese patients after duodenal-jejunal exclusion is associated with intestinal microbiota composition changes.

BACKGROUND: Intestinal microbiota have been suggested to play an important role in the pathogenesis of obesity and type 2 diabetes. Bariatric surgery improves both conditions and has been associated with changes in intestinal microbiota composition. We investigated the effect of a nonsurgical bariatric technique on intestinal microbiota composition in relation to metabolic improvement. METHODS: Seventeen patients with obesity and type 2 diabetes were treated with the nonsurgical duodenal-jejunal bypass liner, which excludes the proximal 60 cm small intestine from food. Fecal samples as well as metabolic parameters reflecting obesity and type 2 diabetes were obtained from the patients at baseline, after 6 months with the device in situ, and 6 months after explantation. RESULTS: After 6 months of treatment, both obesity and type 2 diabetes had improved with a decrease in weight from 106.1 [99.4-123.5] to 97.4 [89.4-114.0] kg and a decrease in HbA1c from 8.5% [7.6-9.2] to 7.2% [6.3-8.1] (both p < 0.05). This was paralleled by an increased abundance of typical small intestinal bacteria such as Proteobacteria, Veillonella, and Lactobacillus spp. in feces. After removal of the duodenal-jejunal bypass liner, fecal microbiota composition was similar to that observed at baseline, despite persistent weight loss. CONCLUSION: Improvement of obesity and type 2 diabetes after exclusion of the proximal 60 cm small intestine by treatment with a nonsurgical duodenal-jejunal bypass liner may be promoted by changes in fecal microbiota composition.

Molecular ecological tools to decipher the role of our microbial mass in obesity.

After birth, our gastrointestinal (GI) tract is colonised by a highly complex assemblage of microbes, collectively termed the GI microbiota, that develops intimate interactions with our body. Recent evidence indicates that the GI microbiota and its products may contribute to the development of obesity and related diseases. This, coupled with the current worldwide epidemic of obesity, has moved microbiome research into the spotlight of attention. Although the main cause of obesity and its associated metabolic complications is excess caloric intake compared with expenditure, differences in GI tract microbial ecology between individuals might be an important biomarker, mediator or new therapeutic target. This can be investigated using a diverse set of complementary so called -omics technologies, such as 16S ribosomal RNA gene-targeted composition profiling, metabolomics, metagenomics, metatranscriptomics and metaproteomics. This review aims to describe the different molecular approaches and their contributions to our understanding of the role of the GI microbiota in host energy homeostasis. Correspondingly, we highlight their respective strengths, but also try to create awareness for their specific limitations. However, it is currently still unclear which bacterial groups play a role in the development of obesity in humans. This might partly be explained by the heterogeneity in genotype, lifestyle, diet and the complex ethology of obesity and its associated metabolic disorders (OAMD). Nevertheless, recent research on this matter has shown a conceptual shift by focusing on more homogenous subpopulations, through the use of both anthropometric (weight, total body fat) as well as biochemical variables (insulin resistance, hyperlipidaemia) to define categories. Combined with technological advances, recent data suggests that an OAMD associated microbiota can be characterised by a potential pro-inflammatory composition, with less potential for the production of short chain fatty acids and butyrate in particular.

Transient inflammatory-like state and microbial dysbiosis are pivotal in establishment of mucosal homeostasis during colonisation of germ-free mice.

The gut microbiota is increasingly recognised as a key-player in defining the health status of the gastrointestinal tract. Recently, we demonstrated that colonisation of healthy germfree mice with a conventional microbiota (conventionalisation) elicits temporal and region specific host-microbe communication responses that lead to the establishment of a microbiota-accommodating homeostatic state within 30 days. Here, the microbiota composition profiles, mucosal transcriptomes and plasma-analytes in germ-free and conventionalised C57/BL 6 J mice were assessed to decipher the features of the distinctive and pivotal events occurring four days after initiation of the conventionalisation process. The dominance of the microbial genera Helicobacter, Sphingomonas and Mucispirillum in the gut microbiota coincided with the transient mounting of proinflammatory responses in the mucosa and the transiently elevated levels of specific (inflammatory) cytokines and amines in plasma. The overrepresented microbes have previously been associated with the potential to cause disease under certain conditions, illustrating that conventionalisation proceeds through a transient state that resembles situations associated with dysbiosis. However, no overt mucosal inflammation was observed, suggesting a pivotal role of the overrepresented bacterial groups in priming and maturation of the immune system during the process of conventionalisation. These findings imply that the transiently elevated relative overgrowth of particular microbial genera functions as pivotal adjuvants to elicit the corresponding proinflammatory cascades, which precede the full maturation of the different arms of the immune system following these events and is required to achieve a microbiota-accommodating homeostasis in healthy animals.

Obesity, non-alcoholic fatty liver disease, and atherothrombosis: a role for the intestinal microbiota?

Whereas the association between intestinal microorganisms and health has been widely accepted in the area of infectious disease, recent advances have now implied a role for the intestinal microbiota in human energy balance. In fact, numerous studies support an intricate relationship between the intestinal microbiota and obesity, as well as subsequent insulin resistance and non-alcoholic fatty liver disease. Intestinal microorganisms also seem to be involved in haemostatic tone and atherogenesis. However, as most of the findings stem from observational data, intervention studies in humans using interventions selectively aimed at altering the composition and activity of the intestinal microbiota are crucial to prove causality. If substantiated, this could open the arena for modulation of the intestinal microbiota as a future target in obesity-associated disease, both as a diagnostic test for personalized algorithms and for selective therapeutic strategies.

The therapeutic potential of manipulating gut microbiota in obesity and type 2 diabetes mellitus.

Obesity and type 2 diabetes mellitus (T2DM) are attributed to a combination of genetic susceptibility and lifestyle factors. Their increasing prevalence necessitates further studies on modifiable causative factors and novel treatment options. The gut microbiota has emerged as an important contributor to the obesity--and T2DM--epidemic proposed to act by increasing energy harvest from the diet. Although obesity is associated with substantial changes in the composition and metabolic function of the gut microbiota, the pathophysiological processes remain only partly understood. In this review we will describe the development of the adult human microbiome and discuss how the composition of the gut microbiota changes in response to modulating factors. The influence of short-chain fatty acids, bile acids, prebiotics, probiotics, antibiotics and microbial transplantation is discussed from studies using animal and human models. Ultimately, we aim to translate these findings into therapeutic pathways for obesity and T2DM in humans.

The environment within: how gut microbiota may influence metabolism and body composition.

Obesity, diabetes and consequently atherosclerotic vascular disease have become major health and public health issues worldwide. The increasing and staggering prevalence of obesity might not only be explained by nutritional habits or the reduction of energy expenditure through decreased physical activity. In addition, recent studies have focused on intestinal microbiota as environmental factors that increase energy yield from diet, regulate peripheral metabolism and thereby increase body weight. Obesity is associated with substantial changes in composition and metabolic function of gut microbiota, but the pathophysiological processes driving this bidirectional relationship have not been fully elucidated. This review discusses the relationships between the following: composition of gut microbiota, energy extracted from diet, synthesis of gut hormones involved in energy homeostasis, production of butyrate and the regulation of fat storage.

High-throughput diversity and functionality analysis of the gastrointestinal tract microbiota.

The human gastrointestinal (GI) tract microbiota plays a pivotal role in our health. For more than a decade a major input for describing the diversity of the GI tract microbiota has been derived from the application of small subunit ribosomal RNA (SSU rRNA)-based technologies. These not only provided a phylogenetic framework of the GI tract microbiota, the majority of which has not yet been cultured, but also advanced insights into the impact of host and environmental factors on the microbiota community structure and dynamics. In addition, it emerged that GI tract microbial communities are host and GI tract location-specific. This complicates establishing relevant links between the host's health and the presence or abundance of specific microbial populations and argues for the implementation of novel high-throughput technologies in studying the diversity and functionality of the GI tract microbiota. Here, we focus on the recent developments and applications of phylogenetic microarrays based on SSU rRNA sequences and metagenomics approaches exploiting rapid sequencing technologies in unravelling the secrets of our GI tract microbiota.

Clinical trial: multispecies probiotic supplementation alleviates the symptoms of irritable bowel syndrome and stabilizes intestinal microbiota.

BACKGROUND: Irritable bowel syndrome is the most common diagnosis in gastroenterology. Trials suggest certain probiotics to be beneficial. AIM: To investigate the effects of multispecies probiotic supplementation (Lactobacillus rhamnosus GG, L. rhamnosus Lc705, Propionibacterium freudenreichii ssp. shermanii JS and Bifidobacterium animalis ssp. lactis Bb12) on abdominal symptoms, quality of life, intestinal microbiota and inflammatory markers in irritable bowel syndrome. METHODS: Eighty-six irritable bowel syndrome patients (Rome II criteria) participated in this randomized, placebo-controlled 5-month intervention. Patients were randomized to receive daily either multispecies probiotic supplementation or placebo. Irritable bowel syndrome symptoms, quality of life, microarray-based intestinal microbiota stability (n = 20), serum cytokines and sensitive C-reactive protein were monitored. RESULTS: The composite irritable bowel syndrome score had at 5 months decreased 14 points (95% CI: -19 to -9) from baseline with the multispecies probiotic vs. three points (95% CI: -8 to 1) with placebo (P = 0.0083). Especially, distension and abdominal pain were affected. A stabilization of the microbiota was observed, as the microbiota similarity index increased with the probiotic supplementation (1.9 +/- 3.1), while it decreased with placebo (-2.9 +/- 1.7). No differences were seen in C-reactive protein. CONCLUSIONS: This multispecies probiotic seems to be an effective and safe option to alleviate symptoms of irritable bowel syndrome, and to stabilize the intestinal microbiota.

Diversity of the Lactobacillus group in breast milk and vagina of healthy women and potential role in the colonization of the infant gut.

AIMS: To evaluate the diversity of the Lactobacillus group in breast milk and the vagina of healthy women and understand their potential role in the infant gut colonization using the 16S rRNA gene approaches. METHODS AND RESULTS: Samples of breast milk, vaginal swabs and infant faeces were aseptically collected from five mothers whose neonates were born by vaginal delivery and another five that had their babies by caesarean section. After polymerase chain reaction (PCR) amplification using Lactobacillus group-specific primers, amplicons were analysed by denaturing gradient gel electrophoresis (DGGE). Clone libraries were constructed to describe the Lactobacillus group diversity. DGGE fingerprints were not related to the delivery method. None of the species detected in vaginal samples were found in breast milk-derived libraries and only few were detected in infant faeces. CONCLUSIONS: The bacterial composition of breast milk and infant faeces is not related to the delivery method. SIGNIFICANCE AND IMPACT OF THE STUDY: It has been suggested that neonates acquire lactobacilli by oral contamination with vaginal strains during delivery; subsequently, newborns would transmit such bacteria to the breast during breastfeeding. However, our findings confirm, at the molecular level that in contrast to the maternal vagina, breast milk seems to constitute a good source of lactobacilli to the infant gut.

DNA isolation protocols affect the detection limit of PCR approaches of bacteria in samples from the human gastrointestinal tract.

A major concern in molecular ecological studies is the lysis efficiency of different bacteria in a complex ecosystem. We used a PCR-based 16S rDNA approach to determine the effect of two DNA isolation protocols (i.e. the bead beating and Triton-X100 method) on the detection limit of seven feces-associated bacterial species of different genera. Glycogen was used in these protocols to improve the precipitation of small concentrations of DNA in ethanol without affecting the sequential procedures. The PCR detection limit of 16S rDNA amplicons on agarose gel from the seven strains tested varied between 8.0 (+/- 1.3) x 10(4) and 4.3 (+/- 1.6) x 10(6) cells for the bead beating method, and between 8.0 (+/- 1.3) x 10(4) and 5.4 (+/- 0.7) x 10(8) cells for the Triton X-100 method. These large differences are most like due to the difference in cell lysis efficiency, since a competitive PCR experiment did not indicate any preference for gram negative, low G+C gram positive or high G+C gram positive bacteria. Denaturing gradient gel electrophoresis (DGGE) analysis was performed to investigate the effect of both DNA isolation protocols on the lysis efficiency of bacteria in fecal samples. A higher diversity in fecal samples was observed with the bead beating method than with the Triton-X100 method. Bands in the bead beating method-derived DGGE profiles corresponding to bands of cloned sequences of the Clostridium coccoides-Eubacterium rectale group and uncultured Fusobacterium prausnitzii were absent or had low intensity in the Triton X-100 method-derived profiles. The applicability of the bead beating method was further investigated by analyzing biopsy samples from the human colon which contain approximately 10(6) cells.

Caloramator coolhaasii sp. nov., a glutamate-degrading, moderately thermophilic anaerobe.

An obligately anaerobic, moderately thermophilic, glutamate-degrading bacterium (strain ZT) was isolated from an enrichment culture obtained from anaerobic thermophilic granular sludge. The cells were rod-shaped to filamentous and showed no motility or spore formation. The cell wall had a Gram-positive structure, which was revealed by electron microscopy. Optimum growth of the strain was observed under neutrophilic conditions at 50-55 degrees C. The doubling time of strain ZT grown in rich medium was approximately 1 h at optimal pH and temperature. Strain ZT was able to grow on a variety of organic compounds. Most carbon sources were converted to acetate, CO2, H2, and traces of propionate and lactate. Strain ZT oxidized glutamate to acetate, CO2, NH4+, traces of propionate and H2. The doubling time on this substrate was 1-6 d. The strain fermented glutamate syntrophically in co-culture with Methanobacterium thermoautotrophicum Z-245T to the same products, but the co-culture had a fourfold higher growth rate. 16S rDNA sequence analysis revealed a relationship with Thermobrachium celere, Caloramator indicus and Caloramator proteoclasticus. The G+C content was 31.7 mol%. Based on its morphological, phylogenetic and physiological characteristics, it is proposed that strain ZT should be classified in the genus Caloramator as a new species, Caloramator coolhaasii.

A molecular view of the intestinal ecosystem.

This review describes the state of the art as well as the initial results of molecular methodologies used to study the ecology of the complex microflora of the human intestinal tract. The detection and identification of many of these organisms has largely been hampered by the incomplete knowledge of their culture conditions. Many of the molecular methodologies are rooted in the use of ribosomal RNA (rRNA) and its encoding genes to describe the relationship between the bacteria in such communities and their individual identity. This approach permits the elucidation both qualitatively as well as quantitatively of the abundance of bacterial species and how their presence interacts with diet and health. Emphasis is given to the analysis of complex communities rather than detection of individual groups of bacteria. The potential of novel advances in molecular technologies such as DNA arrays for analysis of the intestinal ecosystem are also discussed.

Temperature gradient gel electrophoresis analysis of 16S rRNA from human fecal samples reveals stable and host-specific communities of active bacteria.

The diversity of the predominant bacteria in the human gastrointestinal tract was studied by using 16S rRNA-based approaches. PCR amplicons of the V6 to V8 regions of fecal 16S rRNA and ribosomal DNA (rDNA) were analyzed by temperature gradient gel electrophoresis (TGGE). TGGE of fecal 16S rDNA amplicons from 16 individuals showed different profiles, with some bands in common. Fecal samples from two individuals were monitored over time and showed remarkably stable profiles over a period of at least 6 months. TGGE profiles derived from 16S rRNA and rDNA amplicons showed similar banding patterns. However, the intensities of bands with similar mobilities differed in some cases, indicating a different contribution to the total active fraction of the prominent fecal bacteria. Most 16S rRNA amplicons in the TGGE pattern of one subject were identified by cloning and sequence analysis. Forty-five of the 78 clones matched 15 bands, and 33 clones did not match any visible band in the TGGE pattern. Nested PCR of amplified 16S rDNA indicated preferential amplification of a sequence corresponding to 12 of the 33 nonmatching clones with similar mobilities in TGGE. The sequences matching 15 bands in the TGGE pattern showed 91.5 to 98.7% homology to sequences derived from different Clostridium clusters. Most of these were related to strains derived from the human intestine. The results indicate that the combination of cloning and TGGE analysis of 16S rDNA amplicons is a reliable approach to monitoring different microbial communities in feces.

Phylogenetic characterization of ineffective Frankia in Alnus glutinosa (L.) Gaertn. nodules from wetland soil inoculants.

Ineffective Frankia endophytes were retrieved from various wet soils by using Alnus glutinosa clones as trapping plants. No pure cultures could be isolated from these ineffective nodules. Therefore, the phylogenetic position of these endophytes was determined by sequence analysis of cloned PCR products of bacterial 16S rDNA, derived from nodules. The results showed that all nodule endophytes belong to a hitherto undescribed cluster of the Frankia phylogenetic tree. The position of these uncultured ineffective Frankia nodule endophytes is different from that of the ineffective Frankia isolates derived from A. glutinosa nodules, even when originating from the same geographical location. This suggests a bias in current isolation techniques.