Prebiotic effects: metabolic and health benefits

The different compartments of the gastrointestinal tract are inhabited by populations of micro-organisms. By far the most important predominant populations are in the colon where a true symbiosis with the host exists that is a key for well-being and health. For such a microbiota, 'normobiosis' characterises a composition of the gut 'ecosystem' in which micro-organisms with potential health benefits predominate in number over potentially harmful ones, in contrast to 'dysbiosis', in which one or a few potentially harmful micro-organisms are dominant, thus creating a disease-prone situation. The present document has been written by a group of both academic and industry experts (in the ILSI Europe Prebiotic Expert Group and Prebiotic Task Force, respectively). It does not aim to propose a new definition of a prebiotic nor to identify which food products are classified as prebiotic but rather to validate and expand the original idea of the prebiotic concept (that can be translated in 'prebiotic effects'), defined as: 'The selective stimulation of growth and/or activity(ies) of one or a limited number of microbial genus(era)/species in the gut microbiota that confer(s) health benefits to the host.' Thanks to the methodological and fundamental research of microbiologists, immense progress has very recently been made in our understanding of the gut microbiota. A large number of human intervention studies have been performed that have demonstrated that dietary consumption of certain food products can result in statistically significant changes in the composition of the gut microbiota in line with the prebiotic concept. Thus the prebiotic effect is now a well-established scientific fact. The more data are accumulating, the more it will be recognised that such changes in the microbiota's composition, especially increase in bifidobacteria, can be regarded as a marker of intestinal health. The review is divided in chapters that cover the major areas of nutrition research where a prebiotic effect has tentatively been investigated for potential health benefits. The prebiotic effect has been shown to associate with modulation of biomarkers and activity(ies) of the immune system. Confirming the studies in adults, it has been demonstrated that, in infant nutrition, the prebiotic effect includes a significant change of gut microbiota composition, especially an increase of faecal concentrations of bifidobacteria. This concomitantly improves stool quality (pH, SCFA, frequency and consistency), reduces the risk of gastroenteritis and infections, improves general well-being and reduces the incidence of allergic symptoms such as atopic eczema. Changes in the gut microbiota composition are classically considered as one of the many factors involved in the pathogenesis of either inflammatory bowel disease or irritable bowel syndrome. The use of particular food products with a prebiotic effect has thus been tested in clinical trials with the objective to improve the clinical activity and well-being of patients with such disorders. Promising beneficial effects have been demonstrated in some preliminary studies, including changes in gut microbiota composition (especially increase in bifidobacteria concentration). Often associated with toxic load and/or miscellaneous risk factors, colon cancer is another pathology for which a possible role of gut microbiota composition has been hypothesised. Numerous experimental studies have reported reduction in incidence of tumours and cancers after feeding specific food products with a prebiotic effect. Some of these studies (including one human trial) have also reported that, in such conditions, gut microbiota composition was modified (especially due to increased concentration of bifidobacteria). Dietary intake of particular food products with a prebiotic effect has been shown, especially in adolescents, but also tentatively in postmenopausal women, to increase Ca absorption as well as bone Ca accretion and bone mineral density. Recent data, both from experimental models and from human studies, support the beneficial effects of particular food products with prebiotic properties on energy homaeostasis, satiety regulation and body weight gain. Together, with data in obese animals and patients, these studies support the hypothesis that gut microbiota composition (especially the number of bifidobacteria) may contribute to modulate metabolic processes associated with syndrome X, especially obesity and diabetes type 2. It is plausible, even though not exclusive, that these effects are linked to the microbiota-induced changes and it is feasible to conclude that their mechanisms fit into the prebiotic effect. However, the role of such changes in these health benefits remains to be definitively proven. As a result of the research activity that followed the publication of the prebiotic concept 15 years ago, it has become clear that products that cause a selective modification in the gut microbiota's composition and/or activity(ies) and thus strengthens normobiosis could either induce beneficial physiological effects in the colon and also in extra-intestinal compartments or contribute towards reducing the risk of dysbiosis and associated intestinal and systemic pathologies.

Legumes regulate Rhizobium bacteroid development and persistence by the supply of branched-chain amino acids

One of the largest contributions to biologically available nitrogen comes from the reduction of N-2 to ammonia by rhizobia in symbiosis with legumes. Plants supply dicarboxylic acids as a carbon source to bacteroids, and in return they receive ammonia. However, metabolic exchange must be more complex, because effective N-2 fixation by Rhizobium leguminosarum bv viciae bacteroids requires either one of two broad-specificity amino acid ABC transporters (Aap and Bra). It was proposed that amino acids cycle between plant and bacteroids, but the model was unconstrained because of the broad solute specificity of Aap and Bra. Here, we constrain the specificity of Bra and ectopically express heterologous transporters to demonstrate that branched-chain amino acid (LIV) transport is essential for effective N-2 fixation. This dependence of bacteroids on the plant for LIV is not due to their known down-regulation of glutamate synthesis, because ectopic expression of glutamate dehydrogenase did not rescue effective N-2 fixation. Instead, the effect is specific to LIV and is accompanied by a major reduction in transcription and activity of LIV biosynthetic enzymes. Bacteroids become symbiotic auxotrophs for LIV and depend on the plant for their supply. Bacteroids with aap bra null mutations are reduced in number, smaller, and have a lower DNA content than wild type. Plants control LIV supply to bacteroids, regulating their development and persistence. This makes it a critical control point for regulation of symbiosis. MICROBIOLOGY

Interference competition and high temperature reduce the ‘virulence’ of fig wasps and contribute to stability of a fig-wasp mutualism

Fig trees are pollinated by fig wasps, which also oviposit in female flowers. The wasp larvae gall and eat developing seeds. Although fig trees benefit from allowing wasps to oviposit, because the wasp offspring disperse pollen, figs must prevent wasps from ovipositing in all flowers, or seed production would cease, and the mutualism would go extinct. In Ficus racemosa, we find that syconia (‘figs’) that have few foundresses (ovipositing wasps) are underexploited in the summer (few seeds, few galls, many empty ovules) and are overexploited in the winter (few seeds, many galls, few empty ovules). Conversely, syconia with many foundresses produce intermediate numbers of galls and seeds, regardless of season. We use experiments to explain these patterns, and thus, to explain how this mutualism is maintained. In the hot summer, wasps suffer short lifespans and therefore fail to oviposit in many flowers. In contrast, cooler temperatures in the winter permit longer wasp lifespans, which in turn allows most flowers to be exploited by the wasps. However, even in winter, only in syconia that happen to have few foundresses are most flowers turned into galls. In syconia with higher numbers of foundresses, interference competition reduces foundress lifespans, which reduces the proportion of flowers that are galled. We further show that syconia encourage the entry of multiple foundresses by delaying ostiole closure. Taken together, these factors allow fig trees to reduce galling in the wasp-benign winter and boost galling (and pollination) in the wasp-stressing summer. Interference competition has been shown to reduce virulence in pathogenic bacteria. Our results show that interference also maintains cooperation in a classic, cooperative symbiosis, thus linking theories of virulence and mutualism. More generally, our results reveal how frequency-dependent population regulation can occur in the fig-wasp mutualism, and how a host species can ‘set the rules of the game’ to ensure mutualistic behavior in its symbionts.

The phenology and potential for self-pollination of two Australian monoecious fig species

In this preliminary study, the reproductive phenology of two monoecious fig species, Ficus racemosa and F. rubiginosa, was examined in tropical Australia. Syconia (inflorescences) occurred on both species all year round, but pre-floral and interfloral syconia were much commoner than the wasp-receptive and wasp-emitting phases in both species. The temporal overlap of the wasp-receptive and wasp-emitting phases on a single tree indicated that self-pollination was possible in both species and that pollinators may sometimes persist through multiple generations on one tree. This sexual phase overlap was commoner in F. rubiginosa than in F racemosa. The two species also differed in their general within-tree asynchrony, with a higher diversity of phases on F. rubiginosa than on F. racemosa. The time from syconium initiation to ripening was very similar in F. rubiginosa (mean = 48.51 days) and F. racemosa (mean = 43.53 days). However, there was much more variation within and between trees for F. rubiginosa. In addition, the wasp-receptive phase was found to last up to 5 days (rnean = 4.38) in F. rubiginosa. Such longevity should contribute substantially to local pollinator population persistence. Future work should use genetic studies to determine whether self-pollination is common in these fig species.

Diversity, phylogeny and distribution of bean rhizobia in salt-affected soils of North-West Morocco

Different molecular methods: BOX-PCR fingerprinting, R-FLP-PCR and sequencing of the 16S rDNA as well as the symbiotic genes nodC and nifH, were used to study the genetic diversity within a collection of nodulating bean rhizobia isolated from five soils of North-West Morocco. BOX fingerprints analysis of 241 isolates revealed 19 different BOX profiles. According to the PFLP-PCR and sequencing of 16S rDNA carried out on 45 representative isolates, 5 genotypes were obtained corresponding to the species Rhizobium etli, R. tropici, R. gallicum, R. leguminosarum and Sinorhizobium meliloti. The most abundant species were R. etli and R. tropici (61% and 24%, respectively). A high intraspecific diversity was observed among the R. etli isolates, while the R. tropici group was homogeneous. Most of the rhizobia studied belong to species known to nodulate common bean, while 2 species were unconventional microsymbionts: R. leguminosarum biovar viciae and S. meliloti. Our results, especially the nodulation promiscuity of common bean and the relation between the predominance of some species of rhizobia in particular soils and the salt content of these soils, indicate that there is a real need for a better understanding of the distribution of common bean rhizobia species in the soils of Morocco before any inoculation attempt.

Metabolic changes of rhizobia in legume nodules

Bacteria have evolved a wide variety of metabolic strategies to cope with varied environments. Some are specialists and only able to survive in restricted environments; others are generalists and able to cope with diverse environmental conditions. Rhizolbia (e.g. Rhizobium, Sinorhizobium, Bradyrhizobium, Mesorhizobium and Azorhizobium species) can survive and compete for nutrients in soil and the plant rhizosphere but can also form a beneficial symbiosis with legumes in a highly specialized plant cell environment. Inside the legume-root nodule, the bacteria (bacteroids) reduce dinitrogen to ammonium, which is secreted to the plant in exchange for a carbon and energy source. A new and challenging aspect of nodule physiology is that nitrogen fixation requires the cycling of amino acids between the bacteroid and plant. This review aims to summarize the metabolic plasticity of rhizobia and the importance of amino acid cycling.

New scientific paradigms for probiotics and prebiotics

The inaugural meeting of the International Scientific Association for Probiotics and Prebiotics (ISAPP) was held May 3 to May 5 2002 in London, Ontario, Canada. A group of 63 academic and industrial scientists from around the world convened to discuss current issues in the science of probiotics and prebiotics. ISAPP is a non-profit organization comprised of international scientists whose intent is to strongly support and improve the levels of scientific integrity and due diligence associated with the study, use, and application of probiotics and prebiotics. In addition, ISAPP values its role in facilitating communication with the public and healthcare providers and among scientists in related fields on all topics pertinent to probiotics and prebiotics. It is anticipated that such efforts will lead to development of approaches and products that are optimally designed for the improvement of human and animal health and well being. This article is a summary of the discussions, conclusions, and recommendations made by 8 working groups convened during the first ISAPP workshop focusing on the topics of: definitions, intestinal flora, extra-intestinal sites, immune function, intestinal disease, cancer, genetics and genomics, and second generation prebiotics. Humans have evolved in symbiosis with an estimated 1014 resident microorganisms. However, as medicine has widely defined and explored the perpetrators of disease, including those of microbial origin, it has paid relatively little attention to the microbial cells that constitute the most abundant life forms associated with our body. Microbial metabolism in humans and animals constitutes an intense biochemical activity in the body, with profound repercussions for health and disease. As understanding of the human genome constantly expands, an important opportunity will arise to better determine the relationship between microbial populations within the body and host factors (including gender, genetic background, and nutrition) and the concomitant implications for health and improved quality of life. Combined human and microbial genetic studies will determine how such interactions can affect human health and longevity, which communication systems are used, and how they can be influenced to benefit the host. Probiotics are defined as live microorganisms which, when administered in adequate amounts confer a health benefit on the host.1 The probiotic concept dates back over 100 years, but only in recent times have the scientific knowledge and tools become available to properly evaluate their effects on normal health and well being, and their potential in preventing and treating disease. A similar situation exists for prebiotics, defined by this group as non-digestible substances that provide a beneficial physiological effect on the host by selectively stimulating the favorable growth or activity of a limited number of indigenous bacteria. Prebiotics function complementary to, and possibly synergistically with, probiotics. Numerous studies are providing insights into the growth and metabolic influence of these microbial nutrients on health. Today, the science behind the function of probiotics and prebiotics still requires more stringent deciphering both scientifically and mechanistically. The explosion of publications and interest in probiotics and prebiotics has resulted in a body of collective research that points toward great promise. However, this research is spread among such a diversity of organisms, delivery vehicles (foods, pills, and supplements), and potential health targets such that general conclusions cannot easily be made. Nevertheless, this situation is rapidly changing on a number of important fronts. With progress over the past decade on the genetics of lactic acid bacteria and the recent, 2,3 and pending, 4 release of complete genome sequences for major probiotic species, the field is now armed with detailed information and sophisticated microbiological and bioinformatic tools. Similarly, advances in biotechnology could yield new probiotics and prebiotics designed for enhanced or expanded functionality. The incorporation of genetic tools within a multidisciplinary scientific platform is expected to reveal the contributions of commensals, probiotics, and prebiotics to general health and well being and explicitly identify the mechanisms and corresponding host responses that provide the basis for their positive roles and associated claims. In terms of human suffering, the need for effective new approaches to prevent and treat disease is paramount. The need exists not only to alleviate the significant mortality and morbidity caused by intestinal diseases worldwide (especially diarrheal diseases in children), but also for infections at non-intestinal sites. This is especially worthy of pursuit in developing nations where mortality is too often the outcome of food and water borne infection. Inasmuch as probiotics and prebiotics are able to influence the populations or activities of commensal microflora, there is evidence that they can also play a role in mitigating some diseases. 5,6 Preliminary support that probiotics and prebiotics may be useful as intervention in conditions including inflammatory bowel disease, irritable bowel syndrome, allergy, cancer (especially colorectal cancer of which 75% are associated with diet), vaginal and urinary tract infections in women, kidney stone disease, mineral absorption, and infections caused by Helicobacter pylori is emerging. Some metabolites of microbes in the gut may also impact systemic conditions ranging from coronary heart disease to cognitive function, suggesting the possibility that exogenously applied microbes in the form of probiotics, or alteration of gut microecology with prebiotics, may be useful interventions even in these apparently disparate conditions. Beyond these direct intervention targets, probiotic cultures can also serve in expanded roles as live vehicles to deliver biologic agents (vaccines, enzymes, and proteins) to targeted locations within the body. The economic impact of these disease conditions in terms of diagnosis, treatment, doctor and hospital visits, and time off work exceeds several hundred billion dollars. The quality of life impact is also of major concern. Probiotics and prebiotics offer plausible opportunities to reduce the morbidity associated with these conditions. The following addresses issues that emerged from 8 workshops (Definitions, Intestinal Flora, Extra-Intestinal Sites, Immune Function, Intestinal Disease, Cancer, Genomics, and Second Generation Prebiotics), reflecting the current scientific state of probiotics and prebiotics. This is not a comprehensive review, however the study emphasizes pivotal knowledge gaps, and recommendations are made as to the underlying scientific and multidisciplinary studies that will be required to advance our understanding of the roles and impact of prebiotics, probiotics, and the commensal microflora upon health and disease management.

Influence of the Photorhabdus luminescens phosphomannose isomerase gene, manA, on mannose utilization, exopolysaccharide structure, and biofilm formation

Extracellular polysaccharide (EPS) is produced by diverse bacterial pathogens and fulfills assorted roles, including providing a structural matrix for biofilm formation and more specific functions in virulence, such as protection against immune defenses. We report here the first investigation of some of the genes important for biofilm formation in Photorhabdus luminescens and demonstrate the key role of the phosphomannose isomerase gene, manA, in the structure of functional EPS. Phenotypic analyses of a manA-deficient mutant showed the importance of EPS in motility, insect virulence, and biofilm formation on abiotic surfaces as well as the requirement of this gene for the use of mannose as the sole carbon source. Conversely, this defect had no apparent impact on symbiosis with the heterorhabditid nematode vector. A more detailed analysis of biofilm formation revealed that the manA mutant was able to attach to surfaces with the same efficiency as that of the wild-type strain but could not develop the more extended biofilm matrix structures. A compositional analysis of P. luminescens EPS reveals how the manA mutation has a major effect on the formation of a complete, branched EPS.

Pathogenicity and other genomic islands in plant pathogenic bacteria

Pathogenicity islands (PAIs) were first described in uropathogenic E. coli. They are now defined as regions of DNA that contain virulence genes and are present in the genome of pathogenic strains, but absent from or only rarely present in non-pathogenic variants of the same or related strains. Other features include a variable G+C content, distinct boundaries from the rest of the genome and the presence of genes related to mobile elements such as insertion sequences, integrases and transposases. Although PAIs have now been described in a wide range of both plant and animal pathogens it has become evident that the general features of PAIs are displayed by a number of regions of DNA with functions other than pathogenicity, such as symbiosis and antibiotic resistance, and the general term genomic islands has been adopted. This review will describe a range of genomic islands in plant pathogenic bacteria including those that carry effector genes, phytotoxins and the type III protein secretion cluster. The review will also consider some medically important bacteria in order to discuss the range, acquisition and stabilization of genomic islands.

Beyond the American century: Walter Lippmann and American grand strategy, 1943-1950

As the United States became a world Power, journalist and intellectual Walter Lippmann feared that it would become its own worst enemy. During and after the Second World War, he tried to steer the country towards coherent statecraft, to define the national interest and the limits of power, and give geopolitical expression to the role of the United States as the core of an Atlantic strategic system. But in response to world war, the Truman Doctrine, and the Korean War, he became pessimistic about the country's ability to conduct strategy effectively. In the prophetic tradition, he believed that a fatal symbiosis between America's growing strength and domestic politics led it towards crisis. Though at times ahistorical, Lippmann's concept of strategy deserves attention for its dialogue between power and identity, for its questioning of “ends” as well as means, and for its focus on the danger of self-defeating behaviour.

Therapeutic modulation of microbiota-host metabolic interactions

The complex metabolic relationships between the host and its microbiota change throughout life and vary extensively between individuals, affecting disease risk factors and therapeutic responses through drug metabolism. Elucidating the biochemical mechanisms underlying this human supraorganism symbiosis is yielding new therapeutic insights to improve human health, treat disease, and potentially modify human disease risk factors. Therapeutic options include targeting drugs to microbial genes or co-regulated host pathways and modifying the gut microbiota through diet, probiotic and prebiotic interventions, bariatric surgery, fecal transplants, or ecological engineering. The age-associated co-development of the host and its microbiota provides a series of windows for therapeutic intervention from early life through old age

Response kinetics in the complex chemotaxis signalling pathway of Rhodobacter sphaeroides

Chemotaxis is one of the best characterised signalling systems in biology. It is the mechanism by which bacteria move towards optimal environments and is implicated in biofilm formation, pathogenesis and symbiosis. The properties of the bacterial chemosensory response have been described in detail for the single chemosensory pathway of Escherichia coli. We have characterised the properties of the chemosensory response of Rhodobacter sphaeroides, an -proteobacterium with multiple chemotaxis pathways, under two growth conditions allowing the effects of protein expression levels and cell architecture to be investigated. Using tethered cell assays we measured the responses of the system to step changes in concentration of the attractant propionate and show that, independently of the growth conditions, R. sphaeroides is chemotactic over at least five orders of magnitude and has a sensing profile following Weber’s law. Mathematical modelling also shows that, like E. coli, R. sphaeroides is capable of showing Fold-Change Detection (FCD). Our results indicate that general features of bacterial chemotaxis such as the range and sensitivity of detection, adaptation times, adherence to Weber’s law and the presence of FCD may be integral features of chemotaxis systems in general, regardless of network complexity, protein expression levels and cellular architecture across different species.

Dual mycorrhizal associations of jarrah (Eucalyptus marginata) in a nurse-pot system

Jarrah (Eucalyptus marginata Donn ex Sm.) plants, like many other eucalypts, can form symbiotic associations with both arbuscular mycorrhizal (AM) and ectomycorrhizal (ECM) fungi. To study this tripartite relationship we developed a novel nurse-pot system to allow us to investigate the extent and temporal colonisation dynamics of jarrah by two AM species (Rhizophagus irregularis (Błaszk., Wubet, Renker & Buscot) C. Walker & A. Schüßler comb. nov. and Scutellospora calospora Nicol. & Gerd.) and two putative ECM species (Austroboletus occidentalis Watling & N.M. Greg. and Scleroderma sp.) and their potential effects on jarrah growth and nutrition. Our nurse-pot system, using jarrah as both the nurse plant and test plant, was developed to establish extraradical hyphal networks of both AM and ECM fungi that act as single or dual inoculum for test plants. Mycorrhizal colonisation was described and quantified, and growth and nutritional effects measured and analysed. Mycorrhizal colonisation increased with time for the test seedlings exposed to hyphae networks from S. calospora and Scleroderma sp. The nurse-pot system was effective at initiating colonisation of functioning AM or (putative) ECM systems separately but the ECM symbiosis was inhibited where a dual AM + ECM inoculum (R. irregularis and Scleroderma sp.) was present. The presence of S. calospora, A. occidentalis and Scleroderma sp. individually significantly increased the shoot biomass of seedlings compared with non-mycorrhizal controls. The two AM isolates had different physiological effects on jarrah plants. S. calospora improved growth and micronutrient uptake of jarrah seedlings whereas no positive response was observed with R. irregularis. In addition, as an interesting observation, the non-responsive AM fungus R. irregularis suppressed the ECM symbiosis in dually inoculated plants where ECM structures, positive growth response and nutritional effects were absent. When inoculated individually, ECM isolates dominated the growth response and uptake of P and other nutrients in this dual symbiotic plant. Despite the positive growth response in the A. occidentalis treatment, ECM structures were not observed in either nurse or test seedlings. From the effects of A. occidentalis on jarrah we hypothesise that this fungus forms a functional mycorrhizal-type partnership even without forming archetypal structures in and on the root