Movement of newly assimilated 13C carbon in the grass Lolium perenne and its incorporation into rhizosphere microbial DNA

One of the key processes that drives rhizosphere microbial activity is the exudation of soluble organic carbon (C) by plant roots. We describe an experiment designed to determine the impact of defoliation on the partitioning and movement of C in grass (Lolium perenne L.), soil and grass-sterile sand microcosms, using a (13)CO(2) pulse-labelling method. The pulse-derived (13)C in the shoots declined over time, but that of the roots remained stable throughout the experiment. There were peaks in the atom% (13)C of rhizosphere CO(2) in the first few hours after labelling probably due to root respiration, and again at around 100 h. The second peak was only seen in the soil microcosms and not in those with sterilised sand as the growth medium, indicating possible microbial activity. Incorporation of the (13)C label into the microbial biomass increased at 100 h when incorporation into replicating cells, as indicated by the amounts of the label in the microbial DNA, started to increase. These results indicate that the rhizosphere environment is conducive to bacterial growth and replication. The results also show that defoliation had no impact on the pattern of movement of (13)C from plant roots into the microbial population in the rhizosphere.

Nutrimetabonomics: applications for nutritional sciences, with specific reference to gut microbial interactions

Understanding the role of the diet in determining human health and disease is one major objective of modern nutrition. Mammalian biocomplexity necessitates the incorporation of systems biology technologies into contemporary nutritional research. Metabonomics is a powerful approach that simultaneously measures the low-molecular-weight compounds in a biological sample, enabling the metabolic status of a biological system to be characterized. Such biochemical profiles contain latent information relating to inherent parameters, such as the genotype, and environmental factors, including the diet and gut microbiota. Nutritional metabonomics, or nutrimetabonomics, is being increasingly applied to study molecular interactions between the diet and the global metabolic system. This review discusses three primary areas in which nutrimetabonomics has enjoyed successful application in nutritional research: the illumination of molecular relationships between nutrition and biochemical processes; elucidation of biomarker signatures of food components for use in dietary surveillance; and the study of complex trans-genomic interactions between the mammalian host and its resident gut microbiome. Finally, this review illustrates the potential for nutrimetabonomics in nutritional science as an indispensable tool to achieve personalized nutrition.

Contrasting effects of necrotrophic and biotrophic plant pathogens on the aphid Aphis fabae

Phytophagous insects have to contend with a wide variation in food quality brought about by a variety of factors intrinsic and extrinsic to the plant. One of the most important factors is infection by plant pathogenic fungi. Necrotrophic and biotrophic plant pathogenic fungi may have contrasting effects on insect herbivores due to their different infection mechanisms and induction of different resistance pathways, although this has been little studied and there has been no study of their combined effect. We studied the effect of the biotrophic rust fungus Uromyces viciae-fabae (Pers.) Schroet (Basidiomycota: Uredinales: Pucciniaceae) and the necrotrophic fungus Botrytis cinerea Pers. (Ascomycota: Helotiales: Sclerotiniaceae) singly and together on the performance of the aphid Aphis fabae Scop. (Hemiptera: Aphididae) on Vicia faba (L.) (Fabaceae). Alone, botrytis had an inhibitory effect on individual A. fabae development, survival and fecundity, while rust infection consistently enhanced individual aphids’ performance. These effects varied in linear relation to lesion or pustule density. However, whole-plant infection by either pathogen resulted in a smaller aphid population of smaller aphids than on uninfected plants, indicating a lowering of aphid carrying capacity with infection. When both fungi were applied simultaneously to a leaf they generally cancelled the effect of each other out, resulting in most performance parameters being similar to the controls, although fecundity was reduced. However, sequential plant infection (pathogens applied five days apart) led to a 70% decrease in fecundity and 50% reduction in intrinsic rate of increase. The application of rust before botrytis had a greater inhibitory effect on aphids than applying botrytis before rust. Rust infection increased leaf total nitrogen concentration by 30% while infection by botrytis with or without rust led to a 38% decrease. The aphids’ responses to the two plant pathogens individually is consistent with the alteration in plant nutrient content by infection and also the induction of different plant defence pathways and the possible cross-talk between them. This is the first demonstration of the complex effects of the dual infection of a plant by contrasting pathogens on insect herbivores. Key words: Vicia faba, Botrytis cinerea, Uromyces viciae-fabae, tripartite interactions, induced resistance

A defined intestinal colonization microbiota for gnotobiotic pigs

Maximising the ability of piglets to survive exposure to pathogens is essential to reduce early piglet mortality, an important factor in efficient commercial pig production. Mortality rates can be influenced by many factors, including early colonization by microbial commensals. Here we describe the development of an intestinal microbiota, the Bristol microbiota, for use in gnotobiotic pigs and its influence on synthesis of systemic immunoglobulins. Such a microbiota will be of value in studies of the consequences of early microbial colonization on development of the intestinal immune system and subsequent susceptibility to disease. Gnotobiotic pig studies lack a well-established intestinal microbiota. The use of the Altered Schaedler Flora (ASF), a murine intestinal microbiota, to colonize the intestines of Caesarean-derived, gnotobiotic pigs prior to gut closure, resulted in unreliable colonization with most (but not all) strains of the ASF. Subsequently, a novel, simpler porcine microbiota was developed. The novel microbiota reliably colonized the length of the intestinal tract when administered to gnotobiotic piglets. No health problems were observed, and the novel microbiota induced a systemic increase in serum immunoglobulins, in particular IgA and IgM. The Bristol microbiota will be of value for highly controlled, reproducible experiments of the consequences of early microbial colonization on susceptibility to disease in neonatal piglets, and as a biomedical model for the impact of microbial colonization on development of the intestinal mucosa and immune system in neonates.

Restricting microbial exposure in early life negates the immune benefits associated with gut colonization in environments of high microbial diversity

Background: Acquisition of the intestinal microbiota in early life corresponds with the development of the mucosal immune system. Recent work on caesarean-delivered infants revealed that early microbial composition is influenced by birthing method and environment. Furthermore, we have confirmed that early-life environment strongly influences both the adult gut microbiota and development of the gut immune system. Here, we address the impact of limiting microbial exposure after initial colonization on the development of adult gut immunity. Methodology/Principal Findings: Piglets were born in indoor or outdoor rearing units, allowing natural colonization in the immediate period after birth, prior to transfer to high-health status isolators. Strikingly, gut closure and morphological development were strongly affected by isolator-rearing, independent of indoor or outdoor origins of piglets. Isolator-reared animals showed extensive vacuolation and disorganization of the gut epithelium, inferring that normal gut closure requires maturation factors present in maternal milk. Although morphological maturation and gut closure were delayed in isolatorreared animals, these hard-wired events occurred later in development. Type I IFN, IL-22, IL-23 and Th17 pathways were increased in indoor-isolator compared to outdoor-isolator animals during early life, indicating greater immune activation in pigs originating from indoor environments reflecting differences in the early microbiota. This difference was less apparent later in development due to enhanced immune activation and convergence of the microbiota in all isolator-reared animals. This correlated with elevation of Type I IFN pathways in both groups, although T cell pathways were still more affected in indoor-reared animals. Conclusions/Significance: Environmental factors, in particular microbial exposure, influence expression of a large number of immune-related genes. However, the homeostatic effects of microbial colonization in outdoor environments require sustained microbial exposure throughout development. Gut development in high-hygiene environments negatively impacts on normal succession of the gut microbiota and promotes innate immune activation which may impair immune homeostasis.

Hippurate: the natural history of a mammalian-microbial co-metabolite

Hippurate, the glycine conjugate of benzoic acid, is a normal constituent of the endogenous urinary metabolite profile and has long been associated with the microbial degradation of certain dietary components, hepatic function and toluene exposure, and is also commonly used as a measure of renal clearance. Here we discuss the potential relevance of hippurate excretion with regards to normal endogenous metabolism and trends in excretion relating to gender, age, and the intestinal microbiota. Additionally, the significance of hippurate excretion with regards to disease states including obesity, diabetes, gastrointestinal diseases, impaired renal function, psychological disorders and autism, as well as toxicity and parasitic infection, are considered.

Microbial−mammalian cometabolites dominate the age-associated urinary metabolic phenotype in Taiwanese and American populations

Understanding the metabolic processes associated with aging is key to developing effective management and treatment strategies for age-related diseases. We investigated the metabolic profiles associated with age in a Taiwanese and an American population. 1H NMR spectral profiles were generated for urine specimens collected from the Taiwanese Social Environment and Biomarkers of Aging Study (SEBAS; n = 857; age 54–91 years) and the Mid-Life in the USA study (MIDUS II; n = 1148; age 35–86 years). Multivariate and univariate linear projection methods revealed some common age-related characteristics in urinary metabolite profiles in the American and Taiwanese populations, as well as some distinctive features. In both cases, two metabolites—4-cresyl sulfate (4CS) and phenylacetylglutamine (PAG)—were positively associated with age. In addition, creatine and β-hydroxy-β-methylbutyrate (HMB) were negatively correlated with age in both populations (p < 4 × 10–6). These age-associated gradients in creatine and HMB reflect decreasing muscle mass with age. The systematic increase in PAG and 4CS was confirmed using ultraperformance liquid chromatography–mass spectrometry (UPLC–MS). Both are products of concerted microbial–mammalian host cometabolism and indicate an age-related association with the balance of host–microbiome metabolism.

Environmentally-acquired bacteria influence microbial diversity and natural innate immune responses at gut surfaces

Background: Early microbial colonization of the gut reduces the incidence of infectious, inflammatory and autoimmune diseases. Recent population studies reveal that childhood hygiene is a significant risk factor for development of inflammatory bowel disease, thereby reinforcing the hygiene hypothesis and the potential importance of microbial colonization during early life. The extent to which early-life environment impacts on microbial diversity of the adult gut and subsequent immune processes has not been comprehensively investigated thus far. We addressed this important question using the pig as a model to evaluate the impact of early-life environment on microbe/host gut interactions during development. Results: Genetically-related piglets were housed in either indoor or outdoor environments or in experimental isolators. Analysis of over 3,000 16S rRNA sequences revealed major differences in mucosa-adherent microbial diversity in the ileum of adult pigs attributable to differences in earlylife environment. Pigs housed in a natural outdoor environment showed a dominance of Firmicutes, in particular Lactobacillus, whereas animals housed in a hygienic indoor environment had reduced Lactobacillus and higher numbers of potentially pathogenic phylotypes. Our analysis revealed a strong negative correlation between the abundance of Firmicutes and pathogenic bacterial populations in the gut. These differences were exaggerated in animals housed in experimental isolators. Affymetrix microarray technology and Real-time Polymerase Chain Reaction revealed significant gut-specific gene responses also related to early-life environment. Significantly, indoorhoused pigs displayed increased expression of Type 1 interferon genes, Major Histocompatibility Complex class I and several chemokines. Gene Ontology and pathway analysis further confirmed these results.

Human microbiota in health and disease

Each human body plays host to a microbial population which is both numerically vast (at around 1014 microbial cells) and phenomenally diverse (over 1,000 species). The majority of the microbial species in the gut have not been cultured but the application of culture-independent approaches for high throughput diversity and functionality analysis has allowed characterisation of the diverse microbial phylotypes present in health and disease. Studies in monozygotic twins, showing that these retain highly similar microbiota decades after birth and initial colonisation, are strongly indicative that diversity of the microbiome is host-specific and affected by the genotype. Microbial diversity in the human body is reflected in both richness and evenness. Diversity increases steeply from birth reaching its highest point in early adulthood, before declining in older age. However, in healthy subjects there appears to be a core of microbial phylotypes which remains relatively stable over time. Studies of individuals from diverse geopraphies suggest that clusters of intestinal bacterial groups tend to occur together, constituting ‘enterotypes’. So variation in intestinal microbiota is stratified rather than continuous and there may be a limited number of host/microbial states which respond differently to environmental influences. Exploration of enterotypes and functional groups may provide biomarkers for disease and insights into the potential for new treatments based on manipulation of the microbiome. In health, the microbiota interact with host defences and exist in harmonious homeostasis which can then be disturbed by invading organisms or when ‘carpet bombing’ by antibiotics occurs. In a portion of individuals with infections, the disease will resolve itself without the need for antibiotics and microbial homeostasis with the host’s defences is restored. The administration of probiotics (live microorganisms which when administered in adequate amounts confer a health benefit on the host) represents an artificial way to enhance or stimulate these natural processes. The study of innate mechanisms of antimicrobial defence on the skin, including the production of numerous antimicrobial peptides (AMPs), has shown an important role for skin commensal organisms. These organisms may produce AMPs, and also amplify the innate immune responses to pathogens by activating signalling pathways and processing host produced AMPs. Research continues into how to enhance and manipulate the role of commensal organisms on the skin. The challenges of skin infection (including diseases caused by multiply resistant organisms) and infestations remain considerable. The potential to re-colonise the skin to replace or reduce pathogens, and exploring the relationship between microbiota elsewhere and skin diseases are among a growing list of research targets. Lactobacillus species are among the best known ‘beneficial’ bacterial members of the human microbiota. Of the approximately 120 species known, about 15 are known to occur in the human vagina. These organisms have multiple properties, including the production of lactic acid, hydrogen peroxide and bacteriocins, which render the vagina inhospitable to potential pathogens. Depletion of the of the normal Lactobacillus population and overgrowth of vaginal anaerobes, accompanied by the loss of normal vaginal acidity can lead to bacterial vaginosis – the commonest cause of abnormal vaginal discharge in women. Some vaginal anaerobes are associated with the formation of vaginal biofilms which serve to act as a reservoir of organisms which persists after standard antibiotic therapy of bacterial vaginosis and may help to account for the characteristically high relapse rate in the condition. Administration of Lactobacillus species both vaginally and orally have shown beneficial effects in the treatment of bacterial vaginosis and such treatments have an excellent overall safety record. Candida albicans is a frequent coloniser of human skin and mucosal membranes, and is a normal part of the microbiota in the mouth, gut and vagina. Nevertheless Candida albicans is the most common fungal pathogen worldwide and is a leading cause of serious and often fatal nosocomial infections. What turns this organism from a commensal to a pathogen is a combination of increasing virulence in the organism and predisposing host factors that compromise immunity. There has been considerable research into the use of probiotic Lactobacillus spp. in vaginal candidiasis. Studies in reconstituted human epithelium and monolayer cell cultures have shown that L. rhamnosus GG can protect mucosa from damage caused by Candida albicans, and enhance the immune responses of mucosal surfaces. Such findings offer the promise that the use of such probiotic bacteria could provide new options for antifungal therapy. Studies of changes of the human intestinal microbiota in health and disease are complicated by its size and diversity. The Alimentary Pharmabiotic Centre in Cork (Republic of Ireland) has the mission to ‘mine microbes for mankind’ and its work illustrates the potential benefits of understanding the gut microbiota. Work undertaken at the centre includes: mapping changes in the microbiota with age; studies of the interaction between the microbiota and the gut; potential interactions between the gut microbiota and the central nervous system; the potential for probiotics to act as anti-infectives including through the production of bacteriocins; and the characterisation of interactions between gut microbiota and bile acids which have important roles as signalling molecules and in immunity. The important disease entity where the role of the gut microbiota appears to be central is the Irritable Bowel Syndrome (IBS). IBS patients show evidence of immune activation, impaired gut barrier function and abnormal gut microbiota. Studies with probiotics have shown that these organisms can exert anti-inflammatory effects in inflammatory bowel disease and may strengthen the gut barrier in IBS of the diarrhoea-predominant type. Formal randomised trials of probiotics in IBS show mixed results with limited benefit for some but not all. Studies confirm that administered probiotics can survive and temporarily colonise the gut. They can also stimulate the numbers of other lactic acid bacilli in the gut, and reduce the numbers of pathogens. However consuming live organisms is not the only way to influence gut microbiota. Dietary prebiotics are selectively fermented ingredients that can change the composition and/or activity of the gastrointestinal microbiota in beneficial ways. Dietary components that reach the colon, and are available to influence the microbiota include poorly digestible carbohydrates, such as non-starch polysaccharides, resistant starch, non-digestible oligosaccharides (NDOs) and polyphenols. Mixtures of probiotic and prebiotic ingredients that can selectively stimulate growth or activity of health promoting bacteria have been termed ‘synbiotics’. All of these approaches can influence gut microbial ecology, mainly to increase bifidobacteria and lactobacilli, but metagenomic approaches may reveal wider effects. Characterising how these changes produce physiological benefits may enable broader use of these tactics in health and disease in the future. The current status of probiotic products commercially available worldwide is less than ideal. Prevalent problems include misidentification of ingredient organisms and poor viability of probiotic microorganisms leading to inadequate shelf life. On occasions these problems mean that some commercially available products cannot be considered to meet the definition of a probiotic product. Given the potential benefits of manipulating the human microbiota for beneficial effects, there is a clear need for improved regulation of probiotics. The potential importance of the human microbiota cannot be overstated. ‘We feed our microbes, they talk to us and we benefit. We just have to understand and then exploit this.’ (Willem de Vos).

Aspects of in vitro and in vivo research approaches directed towards identifying probiotics and prebiotics for human use

The microbiota of the human gastrointestinal tract plays a key role in nutrition and health. Through the process of fermentation, gut bacteria metabolize various substrates (principally dietary components) to end products such as short-chain fatty acids and gases. This anaerobic metabolism is thought to contribute positively toward host daily energy requirements. However, under certain circumstances, the fermentative process may produce undesirable metabolites. This may cause the onset of gut disorders that can be manifest through both acute and chronic conditions. Moreover, the gut flora may become contaminated by transient pathogens that serve further to upset the normal community structure. There has been a recent increase in the use of dietary components that help to maintain, or even improve, the gut microflora "balance." Probiotics are live microbial feed supplements added to appropriate food vehicles (usually fermented milks), whereas prebiotics are dietary carbohydrates that have a selective metabolism in the colon and serve to increase numbers of bacteria seen as desirable. Because of their purported health-promoting properties, lactic acid-producing bacteria, including bifidobacteria, are the usual target organisms. The market value and biological potential of both approaches are enormous. This article will summarize how efficacious types can be identified.

Effects of plant pathogens on population dynamics and community composition in grassland ecosystems: two case studies

Grassland ecosystems comprise a major portion of the earth’s terrestrial surface, ranging from high-input cultivated monocultures or simple species mixtures to relatively unmanaged but dynamic systems. Plant pathogens are a component of these systems with their impact dependent on many interacting factors, including grassland species population dynamics and community composition, the topics covered in this paper. Plant pathogens are affected by these interactions and also act reciprocally by modifying their nature. We review these features of disease in grasslands and then introduce the 150-year long-term Park Grass Experiment (PGE) at Rothamsted Research in the UK. We then consider in detail two plant-pathogen systems present in the PGE, Tragopogon pratensis-Puccinia hysterium and Holcus lanata-Puccinia coronata. These two systems have very different life history characteristics: the first, a biennial member of the Asteraceae infected by its host-specific, systemic rust; the second, a perennial grass infected by a host-non-specific rust. We illustrate how observational, experimental and modelling studies can contribute to a better understanding of population dynamics, competitive interactions and evolutionary outcomes. With Tragopogon pratensis-Puccinia hysterium, characterised as an “outbreak” species in the PGE, we show that pathogen-induced mortality is unlikely to be involved in host population regulation; and that the presence of even a short-lived seed-bank can affect the qualitative outcomes of the host-pathogen dynamics. With Holcus lanata-Puccinia coronata, we show how nutrient conditions can affect adaptation in terms of host defence mechanisms, and that co-existence of competing species affected by a common generalist pathogen is unlikely.

Effects of single- and multi-strain probiotics on biofilm formation and in vitro adhesion to bladder cells by urinary tract pathogens

Inhibition of biofilm seems to be a major mechanism of urinary tract pathogen exclusion, related to, and possibly dependent upon, the probiotic ability to reduce environmental pH. Exclusion via competition of binding sites is a possible in vivo mechanism for these probiotics. If an additive or synergistic effect exists between strains within a mixture, it does not manifest itself in a greater effect through these two inhibitory mechanisms.