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Recent years have seen the development of high-accuracy and high-throughput genetic manipulation techniques, which have greatly improved our understanding of genetically tractable microbes. However, challenges remain in establishing genetic manipulation techniques in novel organisms, owing largely to exogenous DNA defence mechanisms, lack of selectable markers, lack of efficient methods to introduce exogenous DNA and an inability of genetic vectors to replicate in their new host. In this review, we describe some of the techniques that are available for genetic manipulation of novel microorganisms. While many reviews exist that focus on the final step in genetic manipulation, the editing of recipient DNA, we particularly focus on the first step in this process, the transfer of exogenous DNA into a strain of interest. Examples illustrating the use of these techniques are provided for a selection of human gut bacteria in which genetic tractability has been established, such as Bifidobacterium, Bacteroides and Roseburia. Ultimately, this review aims to provide an information source for researchers interested in developing genetic manipulation techniques for novel bacterial strains, particularly those of the human gut microbiota.

Alex Johnstone, Alison Donaldson, Karen Scott and Phyo Myint all contributed equally to the writing and preparation of the manuscript. This study is funded by Tenovus Scotland Research Project No. G16‐08 (start 1 June 2017, end date 31 January 2019) and NHS‐Grampian Research and Development Endowment Research Grants Project No: 16/11/043 (start date 1 April 2017, end date 31 January, 2019) and the Scottish government as part of the Strategic Research Programme at the Rowett Institute (start date 1 April 2016–31 March 2021).

Microbiome Research Reports, 1 (1), ISSN:2771-5965

In May 2019, the International Scientific Association for Probiotics and Prebiotics (ISAPP) convened a panel of nutritionists, physiologists and microbiologists to review the definition and scope of synbiotics. The panel updated the definition of a synbiotic to “a mixture comprising live microorganisms and substrate(s) selectively utilized by host microorganisms that confers a health benefit on the host”. The panel concluded that defining synbiotics as simply a mixture of probiotics and prebiotics could suppress the innovation of synbiotics that are designed to function cooperatively. Requiring that each component must meet the evidence and dose requirements for probiotics and prebiotics individually could also present an obstacle. Rather, the panel clarified that a complementary synbiotic, which has not been designed so that its component parts function cooperatively, must be composed of a probiotic plus a prebiotic, whereas a synergistic synbiotic does not need to be so. A synergistic synbiotic is a synbiotic for which the substrate is designed to be selectively utilized by the co-administered microorganisms. This Consensus Statement further explores the levels of evidence (existing and required), safety, effects upon targets and implications for stakeholders of the synbiotic concept., Gut microbiota can be manipulated to benefit host health, including the use of probiotics, prebiotics and synbiotics. This Consensus Statement outlines the definition and scope of the term ‘synbiotics’ as determined by an expert panel convened by the International Scientific Association for Probiotics and Prebiotics in May 2019.

Gut microbiota influences many aspects of host health including immune, metabolic, and gut health. We examined the effect of a fermented whey concentrate (FWC) drink rich in L-(+)-Lactic acid, consumed daily, in 18 healthy men (n = 5) and women (n = 13) in free-living conditions.Objective: The aims of this 6-weeks pilot trial were to (i) identify changes in the gut microbiota composition and fecal short chain fatty acid (SCFA) profile, and (ii) to monitor changes in glucose homeostasis.Results: Total fecal SCFA (mM) concentration remained constant throughout the intervention. Proportionally, there was a significant change in the composition of different SCFAs compared to baseline. Acetate levels were significantly reduced (−6.5%; p < 0.01), coupled to a significant increase in the relative amounts of propionate (+2.2%; p < 0.01) and butyrate (+4.2%; p < 0.01), respectively. No changes in the relative abundance of any specific bacteria were detected. No significant changes were observed in glucose homeostasis in response to an oral glucose tolerance test.Conclusion: Daily consumption of a fermented whey product led to significant changes in fecal SCFA metabolite profile, indicating some potential prebiotic activity. These changes did not result in any detectable differences in microbiota composition. Post-hoc analysis indicated that baseline microbiota composition might be indicative of participants likely to see changes in SCFA levels. However, due to the lack of a control group these findings would need to be verified in a rigorously controlled trial. Future work is also required to identify the biological mechanisms underlying the observed changes in microbiota activity and to explore if these processes can be harnessed to favorably impact host health.Clinical Trial Registration: www.clinicaltrials.gov, identifier NCT03615339; retrospectively registered on 03/08/2018.

Faecal samples have often been used to characterise the gut microbiota in health and disease. There is significant debate whether faecal bacterial communities accurately reflect the mucosa associated bacterial populations, which are considered critical in the aetiopathogenesis of several gastrointestinal diseases. We simultaneously assessed faecal and mucosal microbiota from healthy volunteers to unravel the degree of concordance between the two profiles. Paired fresh rectal biopsies and faecal samples were obtained from ten healthy volunteers and processed under stringent anaerobic conditions. Composition and diversity of the microbiota were studied using next generation sequencing targeting the 16S ribosomal nucleic acid (rRNA) gene and culturomics. Bacterial richness and diversity were comparable between mucosal and faecal samples with no significant statistical differences. The relative abundance of Oxalobacteraceae, Propionibacteriaceae, Campylobacteraceae and Corynebacteriaceae were significantly increased (Corncob analysis; FDR=0.00027, 0.000046, 0.011 and 0.025 respectively) in biopsy compared to faecal samples at the family level. Conversely, there was increased abundance from the family Ruminococcaceae and Clostridiaceae (Corncob analysis; FDR=0.025 and 0.025 respectively) in faecal samples. Principal Coordinates Analysis of a Bray Curtis distance matrix generated from sequence variant tables did not show distinct clustering of biopsy and faecal samples (PERMANOVA; p=0.991). A total of 528 bacteria were isolated from a subset of 6 volunteer samples (biopsy and faeces) out of which there were 97 unique and 39 novel species identified. Our study showed good concordance between faecal and gut mucosal microbial profile, corroborating that faecal samples can act as a convenient surrogate to study gut microbiota.

Roseburia and Eubacterium species of the human gut microbiota play an important role in the maintaince of human health, partly by producing butyrate, the main energy source of our colonic epithelial cells. However, our knowledge of the biochemistry and physiology of these bacteria has been limited by a lack of genetic manipulation techniques. Conjugative transposons previously introduced into Roseburia species could not be easily modified, greatly limiting their applicability as genetic modification platforms. Modular plasmid shuttle vectors have previously been developed for Clostridium species, which share a taxonomic order with Roseburia and Eubacterium, raising the possibility that these vectors could be used in these organisms. Here, we describe an optimized conjugation protocol enabling the transfer of autonomously replicating plasmids from an E. coli donor strain into Roseburia inulinivorans and Eubacterium rectale. The modular nature of the plasmids and their ability to be maintained in the recipient bacterium by autonomous replication makes them ideal for investigating heterologous gene expression, and as a platform for other genetic tools including antisense RNA silencing or mobile group II interon gene disruption strategies.

International audience; Technical variation in metagenomic analysis must be minimized to confidently assess the contributions of microbiota to human health. Here we tested 21 representative DNA extraction protocols on the same fecal samples and quantified differences in observed microbial community composition. We compared them with differences due to library preparation and sample storage, which we contrasted with observed biological variation within the same specimen or within an individual over time. We found that DNA extraction had the largest effect on the outcome of metagenomic analysis. To rank DNA extraction protocols, we considered resulting DNA quantity and quality, and we ascertained biases in estimates of community diversity and the ratio between Gram-positive and Gram-negative bacteria. We recommend a standardized DNA extraction method for human fecal samples, for which transferability across labs was established and which was further benchmarked using a mock community of known composition. Its adoption will improve comparability of human gut microbiome studies and facilitate meta-analyses.

The concept of using specific dietary components to selectively modulate the gut microbiota to confer a health benefit, defined as prebiotics, originated in 1995. In 2018, a group of scientists met at the International Scientific Association for Probiotics and Prebiotics annual meeting in Singapore to discuss advances in the prebiotic field, focussing on issues affecting functionality, research methodology, and geographical differences. The discussion ranged from examining scientific literature supporting the efficacy of established prebiotics, to the prospects for establishing health benefits associated with novel compounds, isolated from different sources. While many promising candidate prebiotics from across the globe have been highlighted in preliminary research, there are a limited number with both demonstrated mechanism of action and defined health benefits as required to meet the prebiotic definition. Prebiotics are part of a food industry with increasing market sales, yet there are great disparities in regulations in different countries. Identification and commercialisation of new prebiotics with unique health benefits means that regulation must improve and remain up-to-date so as not to risk stifling research with potential health benefits for humans and other animals. This summary of the workshop discussions indicates potential avenues for expanding the range of prebiotic substrates, delivery methods to enhance health benefits for the end consumer, and guidance to better elucidate their activities in human studies. This article is protected by copyright. All rights reserved.

Commensal butyrate-producing bacteria in the Firmicutes phylum are abundant in the human intestine and are important for maintaining health. However, understanding of the metabolism and host interaction of these bacteria is limited by the lack of genetic modification techniques. Here we establish a protocol enabling the transfer of autonomously-replicating shuttle vectors by conjugative plasmid transfer from an Escherichia coli donor into representatives of an important sub-group of strictly anaerobic human colonic Firmicutes. Five different plasmid shuttle vectors were tested, each carrying a different origin of replication from Gram-positive bacteria. Plasmid pMTL83151 (pCB102 replicon) were successfully transferred into two strains of Eubacterium rectale, while pMTL83151 and pMTL82151 (pBP1 replicon) were transferred into Roseburia inulinivorans A2-194. Plasmids that carried a Streptococcus bovis JB1 glycoside hydrolase family 16 β-(1,3-1,4)-glucanase gene were constructed and conjugated into Roseburia inulinivorans A2-194 and Eubacterium rectale T1-815, resulting in successful heterologous expression of this introduced enzymatic activity in these two strains of butyrate-producing Firmicutes.

The diverse microbial community that inhabits the human gut has an extensive metabolic repertoire that is distinct from, but complements the activity of mammalian enzymes in the liver and gut mucosa and includes functions essential for host digestion. As such, the gut microbiota is a key factor in shaping the biochemical profile of the diet and, therefore, its impact on host health and disease. The important role that the gut microbiota appears to play in human metabolism and health has stimulated research into the identification of specific microorganisms involved in different processes, and the elucidation of metabolic pathways, particularly those associated with metabolism of dietary components and some host-generated substances. In the first part of the review, we discuss the main gut microorganisms, particularly bacteria, and microbial pathways associated with the metabolism of dietary carbohydrates (to short chain fatty acids and gases), proteins, plant polyphenols, bile acids, and vitamins. The second part of the review focuses on the methodologies, existing and novel, that can be employed to explore gut microbial pathways of metabolism. These include mathematical models, omics techniques, isolated microbes, and enzyme assays.

With the continued interest in the role of the gut microbiota in health, attention has now turned to how to harness the microbiota for the benefit of the host. This Consensus Statement outlines the definition and scope of the term 'prebiotic' as determined by an expert panel convened by the International Scientific Association for Probiotics and Prebiotics in December 2016. In December 2016, a panel of experts in microbiology, nutrition and clinical research was convened by the International Scientific Association for Probiotics and Prebiotics to review the definition and scope of prebiotics. Consistent with the original embodiment of prebiotics, but aware of the latest scientific and clinical developments, the panel updated the definition of a prebiotic: a substrate that is selectively utilized by host microorganisms conferring a health benefit. This definition expands the concept of prebiotics to possibly include non-carbohydrate substances, applications to body sites other than the gastrointestinal tract, and diverse categories other than food. The requirement for selective microbiota-mediated mechanisms was retained. Beneficial health effects must be documented for a substance to be considered a prebiotic. The consensus definition applies also to prebiotics for use by animals, in which microbiota-focused strategies to maintain health and prevent disease is as relevant as for humans. Ultimately, the goal of this Consensus Statement is to engender appropriate use of the term 'prebiotic' by relevant stakeholders so that consistency and clarity can be achieved in research reports, product marketing and regulatory oversight of the category. To this end, we have reviewed several aspects of prebiotic science including its development, health benefits and legislation.

The rapid detection of pathogenic strains in food products is essential for the prevention of disease outbreaks. It has already been demonstrated that whole-metagenome shotgun sequencing can be used to detect pathogens in food but, until recently, strain-level detection of pathogens has relied on whole-metagenome assembly, which is a computationally demanding process. Here we demonstrated that three short-read-alignment-based methods, i.e., MetaMLST, PanPhlAn, and StrainPhlAn, could accurately and rapidly identify pathogenic strains in spinach metagenomes that had been intentionally spiked with Shiga toxin-producing Escherichia coli in a previous study. Subsequently, we employed the methods, in combination with other metagenomics approaches, to assess the safety of nunu, a traditional Ghanaian fermented milk product that is produced by the spontaneous fermentation of raw cow milk. We showed that nunu samples were frequently contaminated with bacteria associated with the bovine gut and, worryingly, we detected putatively pathogenic E. coli and Klebsiella pneumoniae strains in a subset of nunu samples. Ultimately, our work establishes that short-read-alignment-based bioinformatics approaches are suitable food safety tools, and we describe a real-life example of their utilization. IMPORTANCE Foodborne pathogens are responsible for millions of illnesses each year. Here we demonstrate that short-read-alignment-based bioinformatics tools can accurately and rapidly detect pathogenic strains in food products by using shotgun metagenomics data. The methods used here are considerably faster than both traditional culturing methods and alternative bioinformatics approaches that rely on metagenome assembly; therefore, they can potentially be used for more high-throughput food safety testing. Overall, our results suggest that whole-metagenome sequencing can be used as a practical food safety tool to prevent diseases or to link outbreaks to specific food products.

Supplemental Digital Content is available in the text, The human microbiome is of considerable interest to pediatric inflammatory bowel disease (PIBD) researchers with 1 potential mechanism for disease development being aberrant immune handling of the intestinal bacteria. This study analyses the fecal microbiome through treatment in newly diagnosed PIBD patients and compares to cohabiting siblings where possible. Patients were recruited on clinical suspicion of PIBD before diagnosis. Treatment-naïve fecal samples were collected, with further samples at 2 and 6 weeks into treatment. Samples underwent 16S ribosomal ribonucleic acid (RNA) gene sequencing and short-chain fatty acids (SCFAs) analysis, results were analyzed using quantitative-insights-into-microbial-ecology. Six PIBD patients were included in the cohort: 4 Crohn disease (CD), 1 ulcerative colitis (UC), 1 inflammatory bowel disease (IBD) unclassified, and median age 12.6 (range 10–15.1 years); 3 patients had an unaffected healthy sibling recruited. Microbial diversity (observed species/Chao1/Shannon diversity) was reduced in treatment-naïve patients compared to siblings and patients in remission. Principal coordinate analysis using Bray–Curtis dissimilarity and UniFrac revealed microbial shifts in CD over the treatment course. In treatment-naïve PIBD, there was reduction in functional ability for amino acid metabolism and carbohydrate handling compared to controls (P = .038) and patients in remission (P = .027). Metabolic function returned to normal after remission was achieved. SCFA revealed consistent detection of lactate in treatment-naïve samples. This study adds in-depth 16S rRNA sequencing analysis on a small longitudinal cohort to the literature and includes sibling controls and patients with UC/IBD unclassified. It highlights the initial dysbiosis, reduced diversity, altered functional potential, and subsequent shifts in bacteria from diagnosis over time to remission.

Warmer temperatures and increasing interest in high provenance food and drink products are creating new opportunities for cereal growing in northern Europe. Nevertheless, cultivation of oats and barley in these areas for malting and milling remains a challenge, primarily because of the weather, and there are few reports of their nutritional content from this region. In this study, trials in Orkney compared agronomic characteristics and nutritional content of recommended UK oat and barley varieties with Scandinavian varieties over three years. For a subset of varieties, nutritional content was compared with samples cultivated in more southerly sites. For Orkney, barley was considered a more suitable crop than oats because varieties matured earlier. In both crops, Scandinavian varieties matured earlier than UK varieties and some produced comparable yields. The range of values for macronutrients and minerals in oats and barley in Orkney were similar to those reported previously for other locations, but there were some significant differences attributable to variety and year. Compared with grain samples from more southerly locations, oats in Orkney had a significantly lower β-glucan and higher sodium content. The lower β-glucan may have resulted from higher rainfall and lower temperatures during the months of grain filling and maturation.

The gut microbiota and its metabolic products interact with the host in many different ways, influencing gut homoeostasis and health outcomes. The species composition of the gut microbiota has been shown to respond to dietary change, determined by competition for substrates and by tolerance of gut conditions. Meanwhile, the metabolic outputs of the microbiota, such as SCFA, are influenced both by the supply of dietary components and via diet-mediated changes in microbiota composition. There has been significant progress in identifying the phylogenetic distribution of pathways responsible for formation of particular metabolites among human colonic bacteria, based on combining cultural microbiology and sequence-based approaches. Formation of butyrate and propionate from hexose sugars, for example, can be ascribed to different bacterial groups, although propionate can be formed via alternative pathways from deoxy-sugars and from lactate by a few species. Lactate, which is produced by many gut bacteria in pure culture, can also be utilised by certain Firmicutes to form butyrate, and its consumption may be important for maintaining a stable community. Predicting the impact of diet upon such a complex and interactive system as the human gut microbiota not only requires more information on the component groups involved but, increasingly, the integration of such information through modelling approaches.

Study how the dietary intake affects the fecal microbiota of a group of obese individuals after a 6-week very low-energy diet (VLED) and thereafter during a follow-up period of 5, 8, and 12 months. Additionally, we compared two different methods, fluorescent in situ hybridization (FISH) and real-time PCR (qPCR), for the quantification of fecal samples. Sixteen subjects participated in a 12-month dietary intervention which consisted of a VLED high in protein and low in carbohydrates followed by a personalized diet plan, combined with exercise and lifestyle counseling. Fecal samples were analyzed using qPCR, FISH, and denaturing gradient gel electrophoresis. The VLED affected the fecal microbiota, in particular bifidobacteria that decreased approximately two logs compared with the baseline numbers. The change in numbers of the bacterial groups studied followed the dietary intake and not the weight variations during the 12-month intervention. Methanogens were detected in 56 % of the participants at every sampling point, regardless of the dietary intake. Moreover, although absolute numbers of comparable bacterial groups were similar between FISH and qPCR measurements, relative proportions were higher according to FISH results. Changes in the fecal microbial numbers of obese individuals were primarily affected by the dietary intake rather than weight changes.

Diet is a major factor driving the composition and metabolism of the colonic microbiota. The amount, type and balance of the main dietary macronutrients (carbohydrates, proteins and fats) have a great impact on the large intestinal microbiota. The human colon contains a dense population of bacterial cells that outnumber host cells 10-fold. Bacteroidetes, Firmicutes and Actinobacteria are the three major phyla that inhabit the human large intestine and these bacteria possess a fascinating array of enzymes that can degrade complex dietary substrates. Certain colonic bacteria are able to metabolise a remarkable variety of substrates whilst other species carry out more specialised activities, including primary degradation of plant cell walls. Microbial metabolism of dietary carbohydrates results mainly in the formation of short chain fatty acids and gases. The major bacterial fermentation products are acetate, propionate and butyrate; and the production of these tends to lower the colonic pH. These weak acids influence the microbial composition and directly affect host health, with butyrate the preferred energy source for the colonocytes. Certain bacterial species in the colon survive by cross-feeding, using either the breakdown products of complex carbohydrate degradation or fermentation products such as lactic acid for growth. Microbial protein metabolism results in additional fermentation products, some of which are potentially harmful to host health. The current 'omic era promises rapid progress towards understanding how diet can be used to modulate the composition and metabolism of the gut microbiota, allowing researchers to provide informed advice, that should improve long-term health status.

Firmicutes and Bacteroidetes are the predominant bacterial phyla colonizing the healthy human large intestine. Whilst both ferment dietary fibre, genes responsible for this important activity have been analysed only in the Bacteroidetes , with very little known about the Firmicutes . This work investigates the carbohydrate-active enzymes (CAZymes) in a group of Firmicutes , Roseburia spp. and Eubacterium rectale , which play an important role in producing butyrate fr om dietary carbohydrates and in health maintenance. Genome sequences of 11 strains representing E. rectale and four Roseburia spp. were analysed for carbohydrate-active genes. Following assembly into a pan-genome, core, variable and uniqu e genes were identified. The 1840 CAZyme genes identified in the pan-genome were assigned to 538 orthologous groups, of which only 26 were present in all strains, indicating considerable inter- strain variability. This analysis was used to categorize the 11 strains into four carbohydrate utilization ecotypes (CUEs), which were shown to correspond to utilization of diffe rent carbohydrates for growth. Many glycoside hydrolase genes were found linked to genes encoding oligosaccharide transporters and regulatory elements in the genomes of Roseburia spp. and E. rectale ,forming distinct polysaccharide utilization loci (PULs). Whilst PULs are also a common feature in Bacteroidetes , key differences were noted in these Firmicutes , including the absence of close homologues of Bacteroides polysaccharide utilization genes, hence we refer to Gram-positive PULs (gpPULs). Most CAZyme genes in the Roseburia / E. rectale group are organized into gpPULs. Variation in gpPULs can explain the high degree of nutritional specialization at the species level within this group.

BACKGROUND: Metabolites released by the gut microbiota may influence host metabolism and immunity. We have tested the hypothesis that inulin-type fructans (ITF), by promoting microbial production of short-chain fatty acids (SCFA), influence cancer cell proliferation outside the gut. METHODS: Mice transplanted with Bcr-Abl-transfected BaF3 cells, received ITF in their drinking water. Gut microbiota was analysed by 16S rDNA polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) and qPCR. Serum Short-chain fatty acids were quantified by UHPLC-MS. Cell proliferation was evaluated in vivo, by molecular biology and histology, and in vitro. RESULTS: Inulin-type fructans treatment reduces hepatic BaF3 cell infiltration, lessens inflammation and increases portal propionate concentration. In vitro, propionate reduces BaF3 cell growth through a cAMP level-dependent pathway. Furthermore, the activation of free fatty acid receptor 2 (FFA2), a Gi/Gq-protein-coupled receptor also known as GPR43 and that binds propionate, lessens the proliferation of BaF3 and other human cancer cell lines. CONCLUSION: We show for the first time that the fermentation of nutrients such as ITF into propionate can counteract malignant cell proliferation in the liver tissue. Our results support the interest of FFA2 activation as a new strategy for cancer therapeutics. This study highlights the importance of research focusing on gut microbes-host interactions for managing systemic and severe diseases such as leukaemia.

Research into the microbial community within the human gastrointestinal tract (GIT) has developed from investigating micro-organisms that cause disease to trying to establish those that are important to maintain health. Thus we are now at a point where the bacterial community in healthy individuals has been well characterized, emphasizing the huge interindividual variation that exists. Molecular advances have also facilitated initial studies on establishing the composition of the viral and eukaryotic communities. In this article, we provide an overview of the current state of knowledge, illustrating the structure and function of the gut microbial community.

In November 2008, a group of scientists met at the 6th Meeting of the International Scientific Association of Probiotics and Prebiotics (ISAPP) in London, Ontario, Canada, to discuss the functionality of prebiotics. As a result of this, it was concluded that the prebiotic field is currently dominated by gastrointestinal events. However, in the future, it may be the case that other mixed microbial ecosystems may be modulated by a prebiotic approach, such as the oral cavity, skin and the urogenital tract. Therefore, a decision was taken to build upon the current prebiotic status and define a niche for ‘dietary prebiotics’. This review is co-authored by the working group of ISAPP scientists and sets the background for defining a dietary prebiotic as ‘‘a selectively fermented ingredient that results in specific changes in the composition and/or activity of the gastrointestinal microbiota, thus conferring benefit(s) upon host health’’.

The human gut harbours a wide range of bacterial communities that play key roles in supplying nutrients and energy to the host through anaerobic fermentation of dietary components and host secretions. This fermentative process involves different functional groups of microorganisms linked in a trophic chain. Although the diversity of the intestinal microbiota has been studied extensively using molecular techniques, the functional aspects of this biodiversity remain mostly unexplored. The aim of the present work was to enumerate the principal metabolic groups of microorganisms involved in the fermentative process in the gut of healthy humans. These functional groups of microorganisms were quantified by a cultural approach, while the taxonomic composition of the microbiota was assessed by in situ hybridization on the same faecal samples. The functional groups of microorganisms that predominated in the gut were the polysaccharide-degrading populations involved in the breakdown of the most readily available exogenous and endogenous substrates and the predominant butyrate-producing species. Most of the functional groups of microorganisms studied appeared to be present at rather similar levels in all healthy volunteers, suggesting that optimal numbers of these various bacterial groups are crucial for efficient gut fermentation, as well as for host nutrition and health. Significant interindividual differences were, however, confirmed with respect to the numbers of methanogenic archaea, filter paper-degrading and acetogenic bacteria and the products formed by lactate-utilizing bacteria.

Summary The complex human colonic microbiota plays a key role in gut health, and both the composition and metabolism of the gut microbiota are strongly diet related. Controlled dietary intake studies in obese individuals demonstrated that consumption of low-carbohydrate diets results in low faecal butyrate concentrations together with a low abundance of the major butyrate-producing bacteria, Roseburia spp. and Eubacterium rectale group. Resistant dietary carbohydrates, including pre-biotics, escape digestion in the upper gastrointestinal tract and are fermented to short-chain fatty acids (SCFAs) by bacteria in the colon. The main SCFAs formed are acetate, propionate and butyrate. Model colonic fermentor systems employed to determine which bacterial groups utilise specific substrates indicated that the bacterial composition and production of SCFAs is substrate dependent. The bacterial colonisers of specific substrates were identified using 16S rRNA sequence analysis. Sequences recovered from bran were most closely related to Roseburia spp., E. rectale and Clostridium hathewayi, while on resistant starch, sequences were most closely related to Ruminococcus bromii and Bifidobacterium adolescentis. Differing intakes of dietary carbohydrate also impact on other factors in the colon such as transit and pH. In continuous fermentor systems, a slightly acidic pH (5.5) representative of the proximal colon gave fourfold higher butyrate concentrations than at the higher pH of 6.5, which is more representative of the distal colon. Bacteroidetes dominated the fermentor at pH 6.5, while the major butyrate-producing bacterial groups competed more effectively for carbohydrate substrates when the pH was mildly acidic (5.5). In in vitro studies in which pH values were even lower, pH 5.2, lactate and acetate accumulated. In contrast, lactate was not detected at pH 6.4 although stable isotope studies revealed that lactate was actively produced. Lactate accumulation at low pH appears to be as a result of the loss of lactate-utilising bacteria including Eubacterium hallii. It is clear that the composition of the colonic microbiota and the balance of its metabolic products is strongly influenced by diet and, in particular, by the intake of resistant carbohydrates and dietary fibre.

Commensal gut bacteria have received a small fraction of the research effort that has been accorded to pathogens. The importance of commensal bacteria for normal gut function and health is increasingly being recognized. These bacteria play crucial roles in the metabolism of dietary components and the supply of energy to the gut mucosa and other host tissues, and influence the gut environment. They provide protection against pathogens through effective competition for growth requirements and binding sites while also participating in genetic exchange. Analyses of 16S rRNA genes amplified from gut and fecal samples have demonstrated remarkable diversity within the microbial communities of the gastrointestinal tract. Lactate is produced by a wide range of commensal gut bacteria but is not normally detected at concentrations above 2 mM in the feces of healthy individuals, although it is detected at much higher concentrations in patients with ulcerative colitis. Several low- G+C-content gram-positive species, including Ruminococcus hydrogenotrophicus, have been shown to be acetogenic and can both consume hydrogen and supply acetate when in coculture with the butyrate producer Roseburia intestinalis. Bacterial attachment must play a key role in the colonization of insoluble substrates. The impact of diet on susceptibility to pathogen infection may therefore be exerted partly through the effects of diet on the balance of commensal species and metabolic activity, as discussed in this chapter. In addition, poor diet is considered to contribute to long-term gut disorders, including inflammatory bowel disease, irritable bowel syndrome, and colorectal cancer.

We investigated the effects of oral therapy with doxycycline, a tetracycline group antibiotic, on the gastrointestinal (GI) survival and tetracycline susceptibility of probiotic strains Lactobacillus acidophilus LaCH-5 and Bifidobacterium animalis subsp. lactis Bb-12. In addition, the influence of doxycycline therapy on the diversity of the predominant faecal microbiota was evaluated by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). Faecal samples from the antibiotic group (receiving antibiotics and probiotics) and the control group (receiving probiotics only) were analysed for anaerobically and aerobically growing bacteria, bifidobacteria and lactic acid bacteria as well as for the dominant microbiota. Although doxycycline consumption did not have a large impact on GI survival of the probiotics, it had a detrimental effect on the bifidobacteria and on the diversity of the dominant faecal microbiota. A higher proportion of tetracycline-resistant anaerobically growing bacteria and bifidobacteria was detected in the antibiotic group than in the control group. Several antibiotic group subjects had faecal B. animalis subsp. lactis Bb-12-like isolates with reduced tetracycline susceptibility. This was unlikely to be due to the acquisition of novel tetracycline resistance determinants, since only tet(W), which is also present in the ingested B. animalis subsp. lactis Bb-12, was found in the resistant isolates. Thus, concomitant ingestion of probiotic L. acidophilus LaCH-5 and B. animalis subsp. lactis Bb-12 with the antibiotic did not generate a safety risk regarding the possible GI transfer of tetracycline resistance genes to the ingested strains.

Mosaic tetracycline resistance genes comprising tet (O), tet (W), and tet (32) sequences were abundant in DNA extracted from pig and human fecal samples, accounting for 78% (50/64) and 46% (37/80) of genes amplified with a tet (O) primer set, respectively, in two samples. The nonmosaic tet (32) gene was isolated from a human saliva bacterium.

Background\ud \ud Considerable effort has been made to categorise the bacterial composition of the human gut and correlate findings with gastrointestinal disease. The infant gut has long been considered sterile at birth followed by rapid colonisation; however, this view has recently been challenged. We examined first-pass meconium from healthy term infants to confirm or refute sterility.\ud \ud Methods\ud \ud Healthy mothers were approached following vaginal delivery. First-pass meconium stools within 24 hours of delivery were obtained from healthy, breastfed infants with tight inclusion/exclusion criteria including rejecting any known antibiotic exposure - mother within 7 days preceding delivery or infant after birth. Stools were processed in triplicate for fluorescent in-situ hybridisation (FISH) with 16S rRNA-targeted probes including Bifidobacterium; Bacteroides-Prevotella; Lactobacillaceae/Enterococcaceae; Enterobacteriaceae; Streptococcaceae; Staphylococcaceae and Enterococcaceae. Absolute counts of all bacteria and proportional identification of each bacterial group were calculated. Confirmation of bacterial presence by PCR was undertaken on FISH-positive samples.\ud \ud Results\ud \ud The mothers of 31 newborn infants were recruited, 15 met inclusion/exclusion criteria and provided a sample within 24 hours of birth, processed in the lab within 4 hours. All babies were 37–40 weeks gestation. 8/15 were male, mean birth weight was 3.4kg and mean maternal age was 32 years. Meconium samples from 10/15 (66%) infants had evidence of bacteria based on FISH analysis. Of these, PCR was positive in only 1. Positive FISH counts ranged from 2.2 - 41.8 x 104 cells/g with a mean of 15.4 x 104 cells/g. (The limit of detection for automated counting is 106 cells/g). Cell counts were too low to allow formal diversity analysis. Amplification by PCR was not possible despite positive spiked samples demonstrating the feasibility of reaction. One baby was dominated by Enterobacteriaceae. The others contained 2-5 genera, with Bifidobacterium, Enterobacteriaceae, Enterococcaceae and Bacteroides-Prevotella the most prevalent. There was no association between bacterial counts and rupture of membrane duration, time to passage of meconium or time to lab.\ud \ud Conclusion\ud \ud This study provides evidence that low numbers of bacteria are present in first-pass meconium samples from healthy, vaginally-delivered, breastfed term infants. Only two-thirds of meconium samples had detectable bacteria, though at levels too low for automated counting or for reliable confirmation by PCR. This study suggests that gut bacterial colonisation is extremely limited at birth and occurs rapidly thereafter.

Background : The intestinal microbiota composition varies between healthy and diseased individuals for numerous diseases. Although any cause or effect relationship between the alterations in the gut microbiota and disease is not always clear, targeting the intestinal microbiota might offer new possibilities for prevention and/or treatment of disease. Objective : Here we review some examples of manipulating the intestinal microbiota by prebiotics, probiotics, and fecal microbial transplants. Results : Prebiotics are best known for their ability to increase the number of bifidobacteria. However, specific prebiotics could potentially also stimulate other species they can also stimulate other species associated with health, like Akkermansia muciniphila , Ruminococcus bromii , the Roseburia/ Enterococcus rectale group, and Faecalibacterium prausnitzii . Probiotics have beneficial health effects for different diseases and digestive symptoms. These effects can be due to the direct effect of the probiotic bacterium or its products itself, as well as effects of the probiotic on the resident microbiota. Probiotics can influence the microbiota composition as well as the activity of the resident microbiota. Fecal microbial transplants are a drastic intervention in the gut microbiota, aiming for total replacement of one microbiota by another. With numerous successful studies related to antibiotic-associated diarrhea and Clostridium difficile infection, the potential of fecal microbial transplants to treat other diseases like inflammatory bowel disease, irritable bowel syndrome, and metabolic and cardiovascular disorders is under investigation. Conclusions : Improved knowledge on the specific role of gut microbiota in prevention and treatment of disease will help more targeted manipulation of the intestinal microbiota. Further studies are necessary to see the (long term) effects for health of these interventions. Keywords: Clostridium difficile; fecal microbial transplants; inflammatory bowel disease; irritable bowel syndrome; obesity; prebiotics; probiotics (Published: 2 February 2015) Citation: Microbial Ecology in Health & Disease 2015, 26: 25877 - http://dx.doi.org/10.3402/mehd.v26.25877 This paper is part of the Proceedings from the 2013 ENGIHR Conference in Valencia, Spain . More papers from this supplement can be found at http://www.microbecolhealthdis.net

The human gut microbiota represents a complex collection of microorganisms, which contribute considerably to host health. They occupy different ecological niches and habitats within the gastrointestinal tract and vary both in compositional make up and metabolic output at different sites along the gut. In this chapter, we describe the microbial “geography” within the human gastrointestinal tract and discuss available methods for studying the gut microbiota at both taxonomic and metabolic levels. Tremendous advances have been made in culture independent molecular microbiology over the past 20 years giving previously undreamt of insight into the architecture of the gut microbiota. Similarly, advances in “omics” technologies, especially metagenomics and metabolomics, are providing the tools necessary to give, for the first time, a real insight into both the gut microbiota metabolic potential (encoded by the genes of microbiota metagenome) and the metabolic kinetic (comprising the flux of microbially derived metabolites) and how these then interact with host physiology influencing health and disease risk.