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Introduction
Few developments in biology promise as much to the advancement of medicine as the exploration of the indigenous human microbiota. Since the publication of historic experiments performed with germ-free animals, it has been evident that the microbiota is a source of trophic, metabolic and protective signals, from which the host benefits. Host–microbe interactions are now known to be bidirectional and disturbances of these interactions contribute to gastrointestinal and extraintestinal disorders. Mining host–microbe signalling has long promised much, but several pivotal discoveries and advances in molecular microbiology are now poised for translation to clinical medicine. This article focuses on the clinical implications of advances in human microbial ecology; the lessons learned extend beyond the gut and are germane to all clinical specialties.
Features of the gut microbiota
In 2007, J. Craig Venter wrote that “Without understanding the environment in which cells or species exist, life cannot be understood. An organism’s environment is
ultimately as unique as its genetic code.”1
This judgement from one so closely linked with the human genome is particularly pertinent to the human microbial environment. Gastrointestinal pathophysiology cannot be conclusively examined outside the context of the relationship with our microbial selves. Over the past decade, a convergence of research interest from disparate cognitive disciplines has greatly enhanced the understanding of the human microbiota and of host–microbe interactions. Progress has been accelerated by metagenomics, which combines high-throughput DNA sequencing and computational methods to define the composition of complex microbial communities without needing to culture the constituents. Microbial genes (the microbiome) numerically exceed those of the human genome by 100‑fold, and microbes from the three domains of life (Bacteria, Archaea and Eukarya), along with viruses, are represented within the normal human microbiota, including species that were unknown until recently.2,3 Work from various laboratories has revealed the complexity, majesty and diversity of the microbiota (Table 1).2,3
The discovery of Helicobacter pylori by clinicians refusing to accept dogma is arguably the field’s greatest success story and has yielded several lessons of continuing clinical relevance. First, it showed that the solution to some diseases cannot be found by focussing exclusively on the host. Almost certainly, other diseases, including some forms of colorectal cancer, have a microbial aetiology.3 Second, the story showed the value of traditional culture-based techniques and the wisdom of working with model organisms to understand disease mechanisms. Third, after decades of missing a transmissible cause of peptic ulceration, the discovery exposed the limitations of ‘risk factor epidemiology’ without taking into account the disease mechanisms, whilst it also highlighted the importance of thinking across the boundaries of traditional research disciplines to solve important biological problems. Finally, as the prevalence of H. pylori was in decline in developed countries long before its existence was known, the story poses important clinical questions regarding the changing nature of the human microbiota.
Microbiota change—disease risk
An abrupt rise in the frequency of immunoallergic disorders, such as IBD and asthma, occurs with socioeconomic development. This association has been attributed to reduced environmental exposure to microbes (the hygiene hypothesis), but more accurately might be related to changes in microbial colonization during the earliest stages of life, when the immune system is maturing. Microbial signalling is required, not only for mucosal homeostasis, but also for full development of extraintestinal systems, including the brain–gut axis and the immune response. Loss of ancestral organisms, such as H. pylori and helminths, is associated with socioeconomic development, and is a risk factor for certain diseases. Reduced microbial diversity accompanies many gastrointestinal and extraintestinal disorders, but reduced levels of specific organisms, such as Lactobacilli spp., Bifidobacterium spp., Akkermansia muciniphilia and Faecalibacterium prausnitzii, might confer a particular risk of developing IBD.2–5
The earlier the exposure to a modern lifestyle in a developed country, the greater is the risk of disease. This finding is consistent with the onset of many immunoallergic disorders in adolescence or early adulthood, and is confirmed by migration studies5 (Figure 1). Many of the elements of
OPINION
The gut microbiota—a clinical perspective on lessons learned
Fergus Shanahan
Abstract | Once considered obscure and largely ignored by microbiologists, the human microbiota has moved centre-stage in biology. The gut microbiota is now a focus of disparate research disciplines, with its contributions to health and disease ready for translation to clinical medicine. The changing composition of the microbiota is linked with changes in human behaviour and the rising prevalence of immunoallergic and metabolic disorders. The microbiota is both a target for drug therapy and a repository for drug discovery. Its secrets promise the realization of personalized medicine and nutrition, and will change and improve conventional dietary management.
Shanahan, F. Nat. Rev. Gastroenterol. Hepatol. 9, 609–614 (2012); published online 14 August 2012;
doi:10.1038/nrgastro.2012.145
Competing interests
The author declares associations with the following companies: Alimentary Health, GlaxoSmithKline, Procter & Gamble. See the article online for full details of the relationships. FOCUS ON GUT MICROBIOTA
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a modern lifestyle in a developed country influence the composition of the indigenous microbiota, disturbances of which have been reported in several diseases of developed society. The most obvious lifestyle or environmental modifier of the microbiota is increased antibiotic exposure, particularly in infancy, which has been linked with an increased risk of IBD in childhood in population-based studies.6,7 Early antibiotic exposure might also increase the risk of asthma, and perhaps even metabolic and obesity-related disease, in later life.
Dietary intake is another prominent modifier of the microbiota (Figure 2). For example, dietary polysaccharides and oligosaccharides, including fibres, are bifidogenic (that is, they enhance the growth of beneficial bifidobacteria), whereas microbial compositional changes have been linked with high levels of dietary fat, iron and protein (casein).8 Of note, increased consumption of dietary fat in Japan, particularly animal fat and n‑6 polyunsaturated fatty acids, has been closely correlated with increases in the incidence of both Crohn’s disease and ulcerative colitis.9
Dietary modification of the intestinal microbiota has also been linked with metabolic and cardiovascular disease. A striking example is the discovery of a microbial-dependent pathway for the metabolism of dietary phospholipids that generates metabolites that promote atherosclerosis after absorption and hepatic metabolism.10 This finding has brought personalized nutrition a step closer to reality but, as discussed below, is only one part of an unfolding story linking the microbiota with both immunoinflammatory and metabolic
signalling in the host.
Microbe–host signalling
Microbe–host signalling is reciprocal, and occurs at several levels: with the immune system; with host metabolic processes; and with the enteric nervous system and brain–gut axis. Interdependency within this network is shown by the mutual regulation of the microbiota and immune system. Microbial signalling is required for immune development and homeostasis, whereas an intact immune system is necessary for maintenance of a healthy microbiota. A depleted microbiota might result in an immune deficit, whereas defects in innate immunity lead to an altered gut microbiota, which might transfer inflammatory and metabolic disease phenotypes upon
faecal transplantation.11–13
Interactions between inflammatory and metabolic cascades are well established. Modulation of both of these processes by the microbiota has added an intriguing layer of complexity, with therapeutic implications for several diseases beyond the gut, including diabetes, obesity and related complications (Figure 3). The first intersection of microbes, immunity and metabolism arises at the intestinal epithelium. The immune and metabolic functions of the epithelium (for example, IgA release and lipid absorption, respectively) are functionally interconnected and inversely regulated.14 IgA influences the composition of the commensal microbiota and, if deficient, the commensal bacteria drive interferon-dependent expression of genes controlling immunity, at the expense of those regulating metabolism. This effect might contribute to lipid malabsorption in some forms of immune deficiency.
By contrast, disturbed host metabolism with excess fat storage might arise from defects in innate immunity. Experimental mice lacking Toll-like receptor (TLR)5, the immunosensory receptor for microbial flagellin, develop obesity and insulin resistance.12 This result seems to be attributable to alterations in the composition of the gut microbiota, which induce proinflammatory cytokines, leading to desensitization of insulin receptor signalling with consequent hyperphagia and weight gain. Defects at the level of the inflammasomes are also associated with changes in microbial composition, activation of inflammatory cascades and progression of metabolic disease.13,15,16 Inflammasomes, as discussed later, are
Table 1 | Overview of the gut microbiota*
Feature
Comment
High diversity and density
Loss of microbial diversity predisposes to pathogenic infections and is linked with several immunoallergic and metabolic disorders
Individuality
Variation arises at species and strain levels with limited variability at phylum level; members of two phyla (Firmicutes and Bacteroidetes) contribute to ~90% of the species in the distal gut
Maternal transmission
Colonization at birth is influenced by mode of delivery (vaginal versus caesarean section)
Age-dependent variability
Rapid diversification during infancy influenced by diet and environment, including antibiotics, reaching relative stability with idiosyncrasy in adulthood, and changing in the elderly depending on physiological status, diet, drugs and morbidity
Variation over long axis of gut
After the oral cavity, complexity and numbers increase distally
Variation over cross-sectional axis of gut
The aerobe:anaerobe ratio is greater at the mucosal surface than at the lumen
Resilience
The microbiota tends to return to normal after antibiotic challenge, but some strains might be eliminated, particularly after repeated or prolonged antibiotic exposure, with the greatest effect in infancy
Plasticity and adaptability
On a background of relative stability, there are continual variations in metabolic behaviour and composition depending on diet, other lifestyle variables and disease
Host–microbe interactions
Bidirectional; microbial, immunoinflammatory and metabolic cascades are interactive
Spatial segregation and compartmentalization
Microbes have restricted access to the small intestinal epithelia because of host-derived factors, such as the antibacterial lectin RegIII-γ; and in the colon, the structure of the inner layer of colonic mucin ensures that it is microbe-free; if commensal organisms penetrate the mucosa they are restricted from the systemic circulation by a gatekeeper effect of the mesenteric lymph node
Experimental transferrable microbiota
‘Colitogenic’ microbiota from animal models of colitis can transfer disease to naive genetically wild-type recipients; transplants of microbiota have similarly revealed transferrable metabolic phenotypes
*Source material reviewed, in part, in references 2 and 3.
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intracellular sensors of microbial-induced damage, but also sense metabolic disturbance in the host and might determine why some patients with obesity are metabolically normal and why others progress to multiorgan complications, including steatohepatitis and insulin resistance.15,16 The microbiota might confound host metabolism by additional mechanisms (Figure 3). However, the microbiota also intersects host metabolism and inflammatory tone by regulating fatty-acid composition within fat tissue, the bioactivity of which influences the production of inflammatory cytokines.17 Thus, the microbiota has a regulatory influence on both fat quantity and quality in the host.
As microbial, inflammatory and metabolic signalling pathways are interlinked and each limb of this triangular network is influenced by diet, it follows that identification and manipulation of the microbial signals and/or alteration of the inflammatory response offer new therapeutic adjuncts to the management of obesity-related disease. The molecular details underpinning this prospect have been addressed elsewhere,8 and proof of principle for an improved metabolic outcome by targeted manipulation of gut microbiota in diet-induced obesity has been established.18
The sensory conundrum
What defines a commensal and how does the host distinguish harmless commensals from dangerous or opportunistic pathogens? The distinction is not always clear, even at a clinical level. The simplest answer is that all commensals probably have pathogenic potential, depending on the context and host susceptibility, and some organisms might be both beneficial and hazardous. For premature babies, colonization with otherwise harmless commensals before optimal development of immunity and mucosal barrier function poses a pathogenic threat. Risk and benefit are also well represented in the H. pylori story,3 with some clinicians taking the view that ‘the only good H. pylori is a dead H. pylori’. However, the outcome of the Helicobacter–host interaction varies depending on the bacterial strain, the host susceptibility and the age of the host. Acquired in childhood, with a latent period of apparent health, H. pylori might cause peptic ulceration in adulthood in some individuals, lymphoma in others and gastric cancer at a later age. By contrast, the same organism might confer protection against asthma and possibly infections in early life, and almost certainly protects against reflux-associated complications, including metaplasia and neoplasia at the
gastroesophageal junction, in later life.
How does the host interpret the microbial environment in terms of risk and benefit or what are traditionally referred to as pathogens versus commensals? As the molecular patterns involved in recognition of pathogens are also expressed by nonpathogenic microbes, detection is only part of the process. The response decision is complex and seems to be based partly on specific inputs or symbiosis-associated molecular patterns from the microbiota19 and partly by sensing danger or damage-associated molecular patterns by epithelial and other host cells13 (Figure 4). An example of the former is the production of an immunomodulatory polysaccharide (polysaccharide A) by Bacteroides fragilis. In contrast to other TLR2 ligands that promote clearance of pathogens, polysaccharide A signals though TLR2 on regulatory T cells and suppresses T‑helper 17 effector cells, thereby avoiding an adverse immune response and favouring colonization of the host.19 Whether other commensals deploy symbiosis-associated molecular patterns is unclear, but the host has an intracellular surveillance system to detect danger within the microbiota and to respond and maintain compositional equilibrium.
Intracellular inflammasomes are multiprotein complexes, partly comprised of Nod-like receptors (NLRs), which sense exogenous or endogenous stress or damage. The epithelium mobilizes the NLRP6 inflammasome in response to pathogenic components of the microbiota and triggers a cascade of events including: activation of caspase 1; conversion of prointerleukin IL‑18 to mature IL‑18; recruitment of γ‑interferon-producing NK and T cells; and enhanced bacteriocidal activity of local macrophages.13 This inflammasome mobilization has a conditioning influence on the composition of the gut microbiota, which becomes evident when NLRP6 is deficient and the commensal bacteria become colitogenic. Intestinal macrophages and dendritic cells have also been reported to have divergent responses to commensals
Figure 1 | Migration and disease risk. Migration studies confirm that lifestyle factors exert their influence at the earliest stages of life (when the microbiota is becoming established and whilst the immune system is maturing). The risk of various immunoallergic disorders is greater the earlier a migrant moves from a region of low-risk (‘developing’ socioeconomically) to one of high-risk (developed) and is low if they migrate in later life.
Migration from developing (low-risk) regions to developed (high-risk) r
egionsAge at time of migrationRisk of acquiring disease of new worldLow-riskregionsHigh-riskregionHigh-riskregion
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versus pathogens, which are driven by the
NLRC4 inflammasome.20
Once an antimicrobial immune response is launched, the host must determine the scale of the threat and adapt accordingly to limit inflammatory collateral damage. The molecular mechanisms by which this effect is achieved are becoming clear and promise new therapeutic targets. Failure of the checkpoints for modifying the response to microbes might underpin or contribute to chronicity of inflammatory disease.21
The ‘drugable’ microbiota
Although most drugs are absorbed in the upper gastrointestinal tract with little exposure to bacteria in the distal gut, an increasing list of drugs and other xenobiotics are substrates for bacterial enzymes and might arrive at the distal gut because of delayed release formulations or after biliary excretion. This delay might result in metabolites with more or less activity, a desirable example of the former being the release of aminosalicylate from the parent prodrug, sulphasalazine, whereas a classic example of the latter is microbial action on digoxin. In other instances, toxins might be generated by microbial enzymatic action on drugs. A particularly informative example of the clinical effect of bacterial action on drugs has been shown in the case of the colon cancer chemotherapeutic agent CPT‑11.22 After parenteral administration, this drug is activated in vivo to generate the antineoplastic topoisomerase I toxin and is inactivated by glucuronidation in the liver, after which it arrives in the intestine by biliary excretion, where it is reactivated by bacterial glucuronidase. This process leads to dose-limiting diarrhoea, a problem that can be circumvented using inhibitors that are specific to the bacterial enzyme. Thus, the microbiota metabolizes some drugs and is a target for others.
Mining the microbiota
Mankind has exploited microbes with ingenuity, from cleaning up oil slicks to production of monoclonal antibodies and life-saving drugs. New therapeutic opportunities arise as the molecular basis of host–microbe interactions unfold. These include mining the microbiota for bioactive compounds that might be formulated as functional food ingredients or novel
drugs (Table 2).
The diversity of microbial metabolites and signalling molecules is testimony to the richness of the microbiota as a repository for drug discovery, but the pressing need for exploring this avenue is perhaps best illustrated by increasing bacterial resistance from overuse of antibiotics and diminished pharmaceutical research.23 Concerns about the long-term consequences of antibacterial action on the commensal microbiota also call for agents with a narrower spectrum of activity. An approach to these problems is shown by the discovery that a Bacillus thuringiensis strain, isolated from human faeces, produces thuricin CD, a potent antimicrobial peptide with narrow-spectrum efficacy against Clostridium difficile. This peptide is a naturally occurring, potential adjunct to existing antibiotics, of
Figure 3 | A signalling internet a | Diet influences each component of a triangular network of signalling among the microbiota, host immunity and host metabolism. b | Mechanisms by which the microbiota influences host metabolism include: harvest of energy from dietary nutrients, production of short-chain fatty acids (which signal via G protein-coupled receptors expressed by the epithelium), and promotion of lipid storage in adipose tissue by suppressing fasting-induced adipocyte factor, an inhibitor of lipoprotein lipase; modification of satiety and behaviour by signalling through the brain–gut–microbe axis; and influencing the host’s inflammatory tone, including the ratio of proinflammatory and anti-inflammatory cytokines.
Metabolic
signallingAntibiotics/vaccinationsUrban life Diet and nutrition Cooking and refrigerationHygiene andwater quality Smaller family size Delayed infectionsLifestyle(early life)MicrobiotaImmunepriming andin ammatorysignallingabIn ammatorytoneBioavailability & storageof dietary nutrientsSatiety,behaviourDietMicrobiotaMetabolismImmunity
Figure 2 | Lifestyle, microbiota and disease. The link between the elements of a modern lifestyle in developed countries and risk of immune and metabolic disorders in later life might be through an influence on the microbiota, particularly in infancy. Microbial, immune and metabolic signalling events are interactive.
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comparable efficacy, but with little resistance evident to date. More importantly, unlike antibiotics currently used against C. difficile, thuricin CD has a narrow spectrum of activity without collateral damage to the commensal microbiota.24
Rebooting the system
Clinicians often make a therapeutic leap before basic science catches up, the story of H. pylori and peptic ulceration being one example. Faecal microbial transplantation is an old remedy undergoing a resurgence of interest because of promising results in various conditions, particularly C. difficile-associated disease (CDAD). The problem of CDAD has escalated because of increasing antibiotic resistance, emergence of an epidemic hypervirulent strain (NAP1/B1/027) and recurrence rates of about 20–25%.25 Curiously, the appendix might act as a sanctuary for the resident microbiota that protects against C. difficile recurrence, with increased rates of recurrence reported in patients who have had an appendicectomy.26 It has been suggested that the appendix might be both a locus of mucosal lymphoid tissue and a reservoir of normal microbiota, from which the colon can be re-populated to restore homeostasis after challenge from antibiotics, disease and perhaps phage viruses.
Different centres have wide variability concerning the acquisition, storage, preparation and mode of administration of faecal material to patients, although a standardized preparation and protocol has been described.27 Critics claim that the bacterial components of the administered material should be well defined and their interactions with other microbes established prior to making this therapy a routine practice. Others raise safety concerns, which will increase as faecal microbial transplantation becomes more widespread or is applied to less serious or trivial conditions. Concern might even become crisis if reports linking specific bacteria with colorectal cancer are replicated and if the risk of cancer is shown to be transferrable.3 An alternative strategy now underway in several centres is to define the minimal microbiota, that is, the combination(s) of strains sufficient to safely confer protection against recurrence of CDAD and which can be characterized, stored and safely prepared for human administration without the risk of
human–human disease transmission.
More nuanced approaches to mimic the normal microbiota, including prebiotics, probiotics and pharmabiotics, in CDAD and other disorders, have been addressed elsewhere, the most important lesson being the need to match the selection of the probiotic
strain with the clinical indication.28
Conclusions
Helpful recommendations for filling persisting gaps in our knowledge of host–microbe interactions in health and disease have been offered by several
Bor
n too soon—premature babyCommensal bacteriaPathogens?MucosalTREG cellTLR2Microbial factorsSymbiosis-associatedmolecular patterns (SAMPS)Host factorsDamage-associatedmolecular patterns (DAMPS)ContextWrong place at wrong timeor host susceptibilityIn ammasome
Figure 4 | The sensory conundrum—friend or foe? The distinction between a harmless commensal and a potential pathogen occurs at different levels. Microbial factors: although the immune system does not express specific receptors to discriminate pathogens from commensals, some microbes produce molecules that act directly on regulatory T cells that promote colonization by the organism. Host factors: epithelial inflammasomes are multiprotein intracellular sensors of cellular stress or damage that activate an immune response, thereby modifying the composition of the microbiota. Context: the host will respond to any commensal found in the wrong place at the wrong time; for example, premature babies are colonized with commensals that have pathogenic potential because the mucosal barrier, immune function and blood–brain barrier are not yet completely developed.
Table 2 | Microbial activity translated to drug discovery or to functional foods4,24,33–37
Bacterial action
Potential drug category
Representative examples
Microbe–microbe signalling
Antimicrobial (bacteriocin)
Lactococcus lactis and Bacillus thuringiensis-derived broad and narrow-spectrum bacteriocins against Clostridium difficile
Microbe–host signalling
Anti-inflammatory: Bacteroides fragilis-derived anti-inflammatory polysaccharide antigen; Lactobacillus-derived cell wall peptide
Protective in experimental models of IBD
Microbe–host signalling
Cytoprotective
Inhibition of cytokine-induced epithelial cell apoptosis by a probiotic (Lactobacillus rhamnosus)-derived soluble protein acting as an epidermal growth factor receptor agonist
Microbe–host signalling
Analgesic
Probiotic-derived analgesic effect in experimental functional bowel disorder
Microbial metabolism
Vitamins and short-chain fatty acids
Short-chain fatty acids, conjugated linoleic acid
Genetically modified organisms
Delivery of vaccines or bioactive agents to gut
Reversal of autoimmune diabetes with L. lactis engineered to deliver proinsulin and IL‑10; treatment of colitis with Bacteroides ovatus engineered to secrete TGF‑β1 under control of dietary xylan
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investigators.3,29 The clinical benefits from exploring the microbiota should drive the research, and although an extensive list of priorities remains, the benefits will include the following. First, greater exploration of the diversity and variation of the human gut virome and how it shapes the rest of the microbiota in health and disease is needed; interactions between viruses and commensal bacteria have already been shown to influence the onset of experimental colitis and to adversely affect viral infection.30,31 Understanding the virome might also yield new phagebiotic therapies for targeting specific constituents of the microbiota. Second, interpersonal variation in enteric bacteria and viruses, which occurs even in genetically identical twins, underpins the promise of extending and realising the scope of personalized medicine.32 Third, diet is the most important influence on the microbiota in health; improved understanding of diet–microbe interactions and their influence on metabolic and inflammatory cascades promises strategies beyond conventional dietary advice for prevention of metabolic disease, and for promotion of healthy aging. Fourth, unravelling the molecular basis of commensal–host interactions will improve probiotic selection for different clinical indications and will facilitate ‘bugs-to-drugs’ discovery. Fifth, the relationship between microbes or combinations of microbes and various disorders will yield new microbial biomarkers of disease risk and response to therapy. Finally, in sounding a warning about misuse of antibiotics, their negative influence on the microbiota, particularly in the earliest stages of life, might be a stronger message for society and for clinicians than admonishment about future antibiotic resistance.
Department of Medicine and Alimentary Pharmabiotic Centre, University College Cork, National University of Ireland, Biosciences Building, College Road, Cork, Ireland.
f.shanahan@ucc.ie
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Acknowledgements
F. Shanahan is supported, in part, by Science Foundation Ireland, in the form of a centre grant: the Alimentary Pharmabiotic Centre.
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