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Keywords:

  • bacteriotherapy;
  • colonization resistance;
  • inflammation;
  • intestinal microbiota;
  • pathogens;
  • short-chain fatty acids;
  • T helper type 17/regulatory T-cell balance

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Colonization of the gastrointestinal tract
  5. Ecology of the healthy adult gastrointestinal tract
  6. Microenvironments within the gastrointestinal tract
  7. Proposed mechanisms of colonization resistance
  8. Subversion of colonization resistance by intestinal pathogens
  9. Therapeutic restoration of a healthy intestinal microbiota
  10. Concluding remarks
  11. Acknowledgements
  12. Disclosure
  13. References

Dense, complex microbial communities, collectively termed the microbiota, occupy a diverse array of niches along the length of the mammalian intestinal tract. During health and in the absence of antibiotic exposure the microbiota can effectively inhibit colonization and overgrowth by invading microbes such as pathogens. This phenomenon is called ‘colonization resistance’ and is associated with a stable and diverse microbiota in tandem with a controlled lack of inflammation, and involves specific interactions between the mucosal immune system and the microbiota. Here we overview the microbial ecology of the healthy mammalian intestinal tract and highlight the microbe–microbe and microbe–host interactions that promote colonization resistance. Emerging themes highlight immunological (T helper type 17/regulatory T-cell balance), microbiota (diverse and abundant) and metabolic (short-chain fatty acid) signatures of intestinal health and colonization resistance. Intestinal pathogens use specific virulence factors or exploit antibiotic use to subvert colonization resistance for their own benefit by triggering inflammation to disrupt the harmony of the intestinal ecosystem. A holistic view that incorporates immunological and microbiological facets of the intestinal ecosystem should facilitate the development of immunomodulatory and microbe-modulatory therapies that promote intestinal homeostasis and colonization resistance.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Colonization of the gastrointestinal tract
  5. Ecology of the healthy adult gastrointestinal tract
  6. Microenvironments within the gastrointestinal tract
  7. Proposed mechanisms of colonization resistance
  8. Subversion of colonization resistance by intestinal pathogens
  9. Therapeutic restoration of a healthy intestinal microbiota
  10. Concluding remarks
  11. Acknowledgements
  12. Disclosure
  13. References

Human beings are born into, and develop in, a microbial world. We are completely sterile at birth but are immediately colonized by microbes from the local environment. We are also born immunologically naive but, faced with this influx of foreign invaders, our immune system rapidly adapts and develops.[1] As we age the microbial communities that inhabit us (collectively termed the ‘microbiota’) become more complex and stable in the absence of external disturbances like infection or antibiotics.[2] Indeed, at maturity it is estimated that 1014 microbes are present on and in the human body. The number of colonizing bacterial cells therefore exceeds the number of human cells that make up the body by a factor of 10.[3] Furthermore, it is estimated that the microbiota-encoded ‘microbiome’ (i.e. the collective gene set of all colonizing microbes) contains 150 times more unique genes than are encoded by the human genome.[4] Many microbiome genes are complementary to those encoded by the human genome, endowing us with numerous capabilities that we have not had to evolve ourselves, impacting on our physiology during both health and disease states. Our microbiota may therefore be considered an accessory ‘organ’ that functions as a superorganism and co-exists with us in harmony during health.[5]

The mammalian intestinal microbiota plays a central role in host development and basic physiology, including immune system development, tissue integrity, digestion, vitamin and nutrient production and colonization resistance.[1] This review will focus on intestinal colonization resistance, which is defined as the resistance to colonization by ingested bacteria or inhibition of overgrowth of resident bacteria normally present at low levels within the intestinal tract. This may include either commensal or pathogenic bacteria. The importance of the indigenous intestinal microbiota to the phenomenon of colonization resistance was initially recognized in the 1950s when Bohnhoff et al.[6] demonstrated that antibiotic treatment resulted a 100 000-fold decrease in the dose of Salmonella enterica serovar Typhimurium required to infect mice. We are not aware of any element of the mammalian immune system that protects us from pathogens to this degree.

From a bacterial point of view successful colonization of the intestinal tract represents a formidable task.[7] First, a bacterium must typically survive in an environmental reservoir before finding its way to the oral cavity. Then the bacterium must pass through the oesophagus, survive the low pH of the stomach, locate to a suitable niche within the intestine and ultimately gain access to nutrients to begin replication. To establish colonization, replication to sufficient numbers is required for the invading bacteria to resist peristalsis and washout from the intestine.[7] During this journey, the invading bacterium must continuously contend and compete with the established resident microbiota for niches and nutrients as well as the immune response directed at these indigenous microbial communities.[8]

Colonization resistance therefore plays an important role during the development of our microbial communities shortly after birth and also in protecting us from invading intestinal pathogens throughout life. Pathogenic bacteria often cause disease only after a sufficient population size is achieved and virulence gene expression is induced. A sufficient population density increases the likelihood that the pathogen will express its virulence factors (i.e. toxins, effector proteins) and interact with the mucosal/epithelial surface to trigger a local inflammatory response or invade deeper tissues.[9] There is increasing evidence that intestinal pathogens exploit host immune responses to outcompete the indigenous microbiota and therefore disrupt colonization resistance.[10, 11] Furthermore, re-establishment of a diverse indigenous microbiota following intestinal perturbations such as diarrhoea[12] and antibiotic treatment[13] is associated with pathogen clearance. Therefore a fundamental understanding of the processes underlying colonization resistance will probably lead to therapies that modulate the intestinal ecosystem to restore and promote intestinal health and homeostasis.[14] This review will outline the current knowledge of the mechanisms of colonization resistance against intestinal pathogens and offer some perspective on future challenges for developing therapies to promote colonization resistance.

Colonization of the gastrointestinal tract

  1. Top of page
  2. Summary
  3. Introduction
  4. Colonization of the gastrointestinal tract
  5. Ecology of the healthy adult gastrointestinal tract
  6. Microenvironments within the gastrointestinal tract
  7. Proposed mechanisms of colonization resistance
  8. Subversion of colonization resistance by intestinal pathogens
  9. Therapeutic restoration of a healthy intestinal microbiota
  10. Concluding remarks
  11. Acknowledgements
  12. Disclosure
  13. References

Initial microbial colonization of the gastrointestinal tract (GIT) begins immediately postpartum with simple microbial communities and is directly influenced by the local environment.[15] During natural childbirth the infant is initially exposed to microbes from the mother's birth canal and intestinal tract, representing an underappreciated form of familial inheritance. In contrast, during caesarean section the infant is initially exposed to microbes present on the mother's skin as well as to those from the surrounding environment such as the hospital delivery room.[16-18] However, it remains to be determined if and how the initial colonizing communities influence the subsequent development of the microbiota and the immune system. Interestingly, 50–60% of newborns are colonized with the intestinal pathogen Clostridium difficile yet disease is rarely observed.[19] Half of the adult population, the majority of which have never had C. difficile disease, harbour serum antibodies specific for C. difficile toxins and this is associated with resistance to severe, recurrent disease.[20, 21] It has been proposed that the presence of toxin-specific antibodies in adults is linked to C. difficile exposure early in life.[19]

After childbirth the establishment of the prevailing microbiota is largely influenced by the infant's diet. For example, the intestinal microbiota of breast-fed infants is typically dominated by Bifidobacterium species.[22] Human breast milk contains a subset of oligosaccharides that are completely indigestible for the nursing infant. From a human evolutionary perspective this would appear to be an inefficient use of the mother's energy resources. However, these oligosaccharides can be preferentially used as carbon sources by health-associated bifidobacteria and promote their growth in the colon.[23] This may be an evolutionary co-adaptation, which brings mutualistic benefits.[24] In contrast to breast-fed infants, formula-fed infants tend to be colonized by a wider range of species, with bifidobacteria typically less predominant.[25]

The introduction of a wide range of complex dietary carbohydrates at weaning appears to trigger wholesale changes in microbiota composition and by 1–2 years of age a dense and diverse microbiota resembling that of an adult becomes established.[15, 16] In some respects the development of the adult intestinal microbiota represents a classical ecological succession, resulting in a stable ‘climax’ community of dominant species.[26] Indeed, longitudinal studies within individual hosts demonstrate that the species composition of an adult's microbiota is relatively stable over extended time periods.[27, 28] However, despite the stability in overall species content of the adult intestinal microbiota, the relative abundances of constituent bacterial groups fluctuate over time in response to changes in the host's diet.[29, 30] Other important drivers of changes in community structure include insults such as antibiotic treatment and diarrhoeal disease. Although the microbiota often shows a remarkable degree of resilience in recovering from these insults it is likely that complete species recovery may not occur after exposure to broad-spectrum antibiotics.[31, 32] The long-term consequences of antibiotic treatment for human health and colonization resistance remain to be determined.[2]

Ecology of the healthy adult gastrointestinal tract

  1. Top of page
  2. Summary
  3. Introduction
  4. Colonization of the gastrointestinal tract
  5. Ecology of the healthy adult gastrointestinal tract
  6. Microenvironments within the gastrointestinal tract
  7. Proposed mechanisms of colonization resistance
  8. Subversion of colonization resistance by intestinal pathogens
  9. Therapeutic restoration of a healthy intestinal microbiota
  10. Concluding remarks
  11. Acknowledgements
  12. Disclosure
  13. References

There is a broad range of physiological conditions in the GIT, creating distinct niches for colonization by microorganisms[33] (Fig. 1). Accordingly, the indigenous microbiota differs in composition along the length of the alimentary canal.[3] Saliva may contain up to 109 microbial cells/ml[34] but the stomach, with its extremely low pH, is an imposing barrier against microbial entry into the lower intestinal tract. The upper regions of the small intestine are therefore normally only colonized by relatively simple microbial communities.[35] It is not until the ileum, with decreased peristalsis and acidity, that both microbial abundance and species diversity increase significantly.[33] However, it is in the colon, where there is a slower transit time and a readily available supply of nutrients that have escaped enzymatic digestion in the small intestine, that the most abundant microbial communities reside.[36] Overall, commensal bacteria levels vary between individuals but up to 1011 bacterial cells/g of faeces are commonly detected. Indeed, the mammalian intestine is among the most densely colonized microbial habitats found in nature.[37]

image

Figure 1. Indigenous microbiota of the human gastrointestinal tract (GIT). The indigenous microbiota varies in density and composition along the length of the human GIT. The densities of colonizing microbes are shown at different locations within the GIT and some examples of distinct environmental niches available for microbial growth within the colon are highlighted (includes information from Cummings and Macfarlane[61]). Invading foreign microorganisms such as pathogens must actively compete against these indigenous microbial communities to successfully replicate and cause disease.

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Not only is the adult intestinal microbiota numerically abundant it is also highly diverse with well over 1000 species capable of colonizing the human colon.[38] However, even this impressive figure is a vast underestimate of the inherent diversity present in the microbiota because different strains of a bacterial species can have extremely variable genome content and phenotypic traits.[39] Adding further to this complexity, there is tremendous variation in intestinal microbiota species content between humans, perhaps as the result of host genetics or early colonization events,[40] such that not even a single ‘core’ microbial species is present in all human beings.[41] This inter-individual variation in microbiota composition may impact the host's degree of colonization resistance.[42] Indeed, inconsistent results in murine models of infectious disease are common, even when animals are bred from the same line, purchased from the same vendors, housed in identical conditions and exposed to the same pathogen strain. These observations may in part be explained by underlying differences in microbiota composition.[43, 44] Understanding the basic, conserved functions of these diverse, health-associated microbial communities remains a challenge to fully appreciate the role of the microbiota during intestinal homeostasis and protection from invading pathogens.

Despite the inherent variability in microbiota composition between different individuals it is clear that there are some consistent themes. First of all, the vast majority of microbial cells present within the colon (>99%) are obligate anaerobic bacteria (i.e. only grow and survive in the absence of oxygen).[33] Second, regardless of species content, the microbiota remains relatively functionally stable and generally appears to carry out a core set of biochemical reactions such as the metabolism of potentially harmful substances (i.e. bile acids, bilirubin and heterocyclic amines) and the breakdown and fermentation of polysaccharides into short-chain fatty acids (SCFAs; predominantly acetate, propionate and butyrate).[45, 46] The SCFAs produced by indigenous intestinal bacteria contribute 5–10% of the total calorie requirements of humans, and have beneficial effects at both local and systemic sites.[47]

Another striking feature of the microbiota is the limited number of bacteria phyla (this is roughly equivalent to the ‘Chordata’ taxonomic rank that human beings share with all animals that have a bilateral body plan and notochord) that reproducibly colonize the intestine. There are at least 55 currently recognized phyla of bacteria but the vast majority (approximately 99%) of intestinal microbiota species appear to belong to just four of these phyla: the Gram-positive Firmicutes and Actinobacteria and the Gram-negative Bacteroidetes and Proteobacteria.[38, 41, 48] Therefore only certain bacterial lineages are adapted to the mammalian intestine. Commonly identified groups of bacteria from each of these phyla, and some phenotypic characteristics, are shown in Table 1. Although the majority of microbial biomass is comprised of bacteria, it is important to note that eukaryotes, archaea and viruses are also important colonizers of the human GIT. It appears that the eukaryotic and archaeal microbiota are much less numerous and diverse than bacteria[38, 48, 49] but viruses, particularly bacteriophages (viruses that infect bacteria), are highly abundant.[50, 51]

Table 1. Common bacterial constituents of the human gastrointestinal tract microbiota
PhylumBrief descriptionCommonly detected constituent genera
  1. While the human intestinal microbiota is dominated by just four bacterial phyla there are many hundreds of constituent species. The most commonly identified bacterial genera within the dominant phyla are shown. Note that many traditional classifications, particularly within the Firmicutes phylum, have not been supported by subsequent DNA sequence analysis.[146] For example, many species placed in the ‘Clostridium’ genus[103] in fact appear to largely belong to two distinct taxonomic Families; the Lachnospiraceae and the Ruminococcaceae.[146, 147]

Actinobacteria Gram-positive, typically obligately anaerobic or microaerophilic. Some genera most abundant in infantsAtopobium, Bifidobacterium, Collinsella, Eggerthella
Bacteroidetes Gram-negative bacilli, typically obligately anaerobic. Often abundant in the gut microbiotaAlistipes, Bacteroides, Barnesiella, Parabacteroides, Prevotella
Firmicutes Gram-positive, typically obligately anaerobic. Often abundant in the gut microbiota, and typically highly diverse. Majority of constituent species have yet to be cultured in the laboratory

Lachnospiraceae family: Anaerostipes, Blautia, Butyrivibrio, Coprococcus, Dorea, Lachnospira, Roseburia

Ruminococcaceae family: Anaerotruncus, Coprobacillus, Faecalibacterium, Ruminococcus, Subdoligranulum

Other Firmicutes Acidaminococcus, Dialister, Enterococcus, Finegoldia, Holdemania, Lactobacillus, Megasphaera, Phascolarctobacterium, Streptococcus, Veillonella

*Segmented filamentous bacteria (note: these bacteria do not appear to inhabit the human intestine)

Proteobacteria Gram-negative, mainly facultatively anaerobic species. Includes many pathogenic speciesAlcaligenes, Bilophila, Campylobacter, Desulfovibrio, Enterobacter, Escherichia, Hafnia, Helicobacter, Klebsiella, Oxalobacter, Parasutterella, Proteus, Sutterella
OthersTypically less abundant members of the gut microbiotaAkkermansia, Fusobacterium, Victivallis

Microenvironments within the gastrointestinal tract

  1. Top of page
  2. Summary
  3. Introduction
  4. Colonization of the gastrointestinal tract
  5. Ecology of the healthy adult gastrointestinal tract
  6. Microenvironments within the gastrointestinal tract
  7. Proposed mechanisms of colonization resistance
  8. Subversion of colonization resistance by intestinal pathogens
  9. Therapeutic restoration of a healthy intestinal microbiota
  10. Concluding remarks
  11. Acknowledgements
  12. Disclosure
  13. References

The indigenous colonic microbiota has evolved to take advantage of numerous distinct microenvironments within the intestine (see Fig. 1). The microbial communities associated with the mucosal surface, for example, are distinct from those present in the luminal contents/faeces.[48, 52] This difference may be largely driven by the presence of a layer of mucus (produced by goblets cells) that covers the intestinal epithelial cells and serves as a rich source of energy for a sub-set of colonizing intestinal microbes.[36] In addition, the mucus layer serves an important physical barrier function to limit direct contact between commensal intestinal microbes and the underlying epithelial cells.[53]

Secreted mucus varies in composition and organizational structure along the length of the GIT.[54] In the small intestine the mucus is thinner than in the large intestine, probably so that the small intestine can effectively absorb dietary nutrients.[55] In contrast, in the large intestine the much thicker mucus layer is organized into two distinct layers.[55] A thin inner layer of mucin, situated adjacent to the gut epithelium, is dense, compacted and seemingly largely devoid of microbial colonization.[56] The much thicker outer layer, however, is more loosely arranged, can be effectively degraded by bacteria and is densely colonized by the indigenous microbiota.[55] The barrier function of the GIT mucus is complemented by the large-scale secretion of IgA by mucosal B cells[57] and a range of antimicrobial compounds (i.e. α-defensins, cathelicidins, lysozymes and lectins such as RegIIIα and RegIIIγ) produced by specialized cells like Paneth cells and leucocytes.[58-60]

There are also differences in microbial species content and activity within the luminal contents of the colon. The proximal colon is the site where undigested dietary substrates from the small intestine first become available for breakdown by colonic bacteria. As a result, the colonic lumen in this region is characterized by elevated levels of bacterial fermentation linked to a reduction in local luminal pH. The dietary polysaccharides become depleted as the digesta moves along the length of the colon leading to a reduction in bacterial growth/fermentation and a gradual rise in pH towards neutrality in the distal regions of the colon.[61] Furthermore, insoluble dietary components such as fibre and resistant starch promote the formation of distinct substrate-attached microbial populations that are different from those found free-living in the gut lumen/faeces or associated with the mucosa[62, 63] (Fig. 1).

It is clear, therefore, that the indigenous intestinal microbiota has evolved and adapted to fill numerous environmental niches in the mammalian GIT. This forms a formidable barrier to subsequent colonization by invading foreign microbes and underpins the phenomenon of colonization resistance.

Proposed mechanisms of colonization resistance

  1. Top of page
  2. Summary
  3. Introduction
  4. Colonization of the gastrointestinal tract
  5. Ecology of the healthy adult gastrointestinal tract
  6. Microenvironments within the gastrointestinal tract
  7. Proposed mechanisms of colonization resistance
  8. Subversion of colonization resistance by intestinal pathogens
  9. Therapeutic restoration of a healthy intestinal microbiota
  10. Concluding remarks
  11. Acknowledgements
  12. Disclosure
  13. References

Healthy, immunocompetent hosts are constantly exposed to intestinal pathogens yet disease is uncommon because pathogens fail to colonize and subsequently replicate to sufficient levels. A working model of colonization resistance proposes that there are multiple layers of host defence that are the products of direct interactions between microbes, as well as indirect mechanisms mediated by stimulation of the mucosal immune system by members of the health-associated microbiota (Fig. 2). Such interactions have probably evolved to maintain health and homeostasis and, as a result, provide survival benefits to both parties. Murine models have served as valuable surrogates to dissect these mechanisms of colonization resistance because of the availability of immune system reagents, transgenic mouse lines and, importantly, the ability of select human commensal and pathogenic bacteria to colonize and cause disease, respectively. Below we discuss some of the proposed mechanisms of colonization resistance based on bacterial culture-based, human and mouse studies.

image

Figure 2. Microbial and immunological signatures of intestinal colonization resistance. Schematic of colon illustrating the associations between the intestinal microbiota composition and the immune system status during differing degrees of colonization resistance.

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Microbe–microbe interactions

Inhibition of pathogen growth by common commensal bacteria has been demonstrated ex vivo (i.e. in the absence of host immune responses) and in vivo demonstrating that direct microbe–microbe interactions are a critical component of colonization resistance.[64-66] There are a number of ways in which these microbe–microbe interactions may inhibit pathogenic colonization of the GIT.

Competition for niches and nutrients

A health-associated intestinal microbiota is characterized by a diverse and abundant community that maximizes the carrying capacity of the entire ecosystem. The intestinal microbiota fills a wide range of available niches forming complex nutrient webs where the metabolic by-product from one microbe is the growth substrate for another.[67, 68] In effect this means that any invading foreign bacterium is in direct competition with the indigenous microbiota for niches and nutrients that the resident microbes are actively sequestering for their own growth and sustenance.[69] Nutrient depletion by the indigenous microbiota probably plays a role in suppressing resident pathogenic species such as C. difficile to low levels within the intestine such that hosts remain asymptomatic carriers.[70] In addition, the mucosa-associated component of the indigenous microbiota also acts as an extra barrier to penetration of the gut epithelium by pathogens. Invading pathogens must navigate through the heavily colonized outer mucin layer and compete with the microbiota for adhesion receptors on the gut epithelium.[54] Therefore, direct competition for niches and nutrients limits the scope for pathogenic microbes to establish and replicate within the lumen to the sufficient densities required to reach the epithelial surface or invade deeper tissues.[7]

Metabolic exclusion by SCFA production, O2 consumption and bacteriocins

Bacteria constantly sense signals from the local environment and regulate gene expression accordingly.[71] Regulation of colonization and virulence functions by intestinal pathogens is central to their ability to manipulate host cells to cause disease and subvert immune responses. Intrinsic metabolic activities of the indigenous microbiota create conditions within the intestine that can inhibit the growth and virulence gene expression of invading pathogens. The SCFAs can act as inhibitors of virulence gene expression by pathogenic Enterobacteriaceae. For example, exposure of Salmonella enterica to butyrate down-regulates expression of virulence genes (i.e. Type III secretion system) and decreases its ability to invade or induce apoptosis of host cells,[72] a key first step that Salmonella spp. use to induce intestinal inflammation and invade deeper tissues.[73] In addition to direct inhibitory effects, the production of SCFAs and other organic acids such as lactate also causes localized reductions in pH (see Fig. 1) to levels below the optimum for enteric pathogens such as Salmonella spp. and Escherichia coli O157, suppressing their replication rate in vivo.[74-76] Furthermore, a recent study using a mouse model of colitis demonstrates that commensal-derived SCFAs reduce intestinal inflammation by altering the microbiota composition and specifically suppressing colitogenic Enterobacteriaceae.[77]

The metabolic activity of the indigenous microbiota also reduces free oxygen in the gut lumen, leading to predominantly anaerobic conditions within the GIT during health.[78] Available oxygen is a key signal and resource for bacteria and, because of the metabolic networks within the intestinal microbiota, its presence can have a profound effect on the entire microbiota composition and the activity of specific bacterial groups. Some of the most important intestinal pathogens of mammals, such as the Enterobacteriaceae, are facultative anaerobes (can survive in aerobic and anaerobic environments) allowing them to readily survive in the surrounding environment (i.e. during transmission) and within the intestinal tract.[78] The resulting low oxidation-reduction potential within the colon slows the growth rate and virulence gene expression of Enterobacteriaceae pathogens.[79] For example, Shigella flexneri senses small differences in available O2 within the intestine and under specific conditions expresses its virulence factors to manipulate epithelial cells leading to inflammation.[80] Hence, the ability of the indigenous microbiota to promote a largely anaerobic environment in the intestine limits the virulence potential of many important pathogens.

The indigenous microbiota is also armed with an arsenal of compounds that can be used to directly attack microbial competitors. Of particular note are a group of metabolites known as bacteriocins. These are a broad range of peptides that are released by bacterial cells and can have either narrow-spectrum or broad-spectrum bactericidal activity against competing microbes.[81] Numerous bacteriocins produced by gut commensals have bactericidal activity against pathogens such as Listeria monocytogenes, Salmonella spp., Clostridium botulinum, Clostridium perfringens and C. difficile.[82-85] Bacteriocins are therefore attractive candidates to develop as novel, narrow-spectrum antibiotics that selectively target pathogenic microbes but leave the wider indigenous microbiota (and therefore colonization resistance) largely intact. Promising studies have demonstrated novel antibiotic compounds with potent activity against C. difficile, but not the wider microbiota, in vitro[86, 87] but further work is needed to confirm their efficacy and specificity in vivo.

Microbe–host interactions

Intestinal bacteria and their metabolic by-products are powerful stimulants of the intestinal immune system, and directly impact the general health of intestinal epithelial cells[88] and the repertoire and activity of the underlying immune system cells.[89, 90] Because of the complexity and variability of the intestinal microbiota[41] unravelling the specific microbial–host interactions that play a key role in colonization resistance will prove to be challenging. However, specific host–commensal interactions have been identified that promote immune resistance to pathogens and mucosal damage leading to the emergence of general immunological themes that are associated with colonization resistance.

The epithelial interface

The interface between the intestinal epithelium and the resident microbiota is critical for establishing intestinal homeostasis and colonization resistance.[91] Toll-like receptors (TLRs) and nucleotide-binding oligomerization domain-like receptors (NODs) are key host receptors. These receptors are strategically located on a variety of host cell surfaces, within specific organelles and cytoplasmically to recognize an array of intestinal microbes and their products, such as lipopolysaccharides, peptidoglycans, nucleotides, proteins and lipoproteins.[92] The TLR–commensal microbe interactions maintain the steady-state homeostasis of the intestinal mucosa[93] and TLR/NODs are central to recognizing pathogens and their damaging activities to initiate host defence and inflammation.[94] How the TLR system differentiates between commensal and pathogenic bacteria remains poorly understood. Remarkably, TLR recognition of specific commensal bacteria induces innate antimicrobial activity that can specifically target Salmonella enterica and limit systemic invasion.[95] The specificity of TLR/NOD-ligand recognition and the well-characterized downstream cellular responses highlight these pathways as viable targets for therapeutic interventions to establish intestinal homeostasis directly or as adjuvants to boost vaccine efficacy.[96]

The T-cell balance

The presence of specific T-cell subsets in the lamina propria is important for establishing and maintaining colonization resistance.[97] One emerging theme is that a balanced regulatory T cell/T helper type 17 (Treg/Th17) status is a signature of intestinal homeostasis.[98] Regulatory T cells (Tregs; CD4+ CD25+ Foxp3+) located within the intestinal lamina propria function to suppress pathological inflammatory responses via interleukin-10-mediated signalling cascades, particularly those mediated by excessive effector T-cell activity.[99, 100] Interestingly, evolutionarily diverse groups of commensal bacteria are now known to induce the expansion of Tregs from naive Th cells and, as a result, protect mice from exuberant inflammation caused by intestinal pathogens and mucosal injury. For example, specific bacteria such as Bacteroides fragilis[101] and Bifidobacterium infantis[102] or defined mixtures of bacteria such as a collection of Firmicutes bacteria[103] and those contained within the Altered Schaedler's Flora[104] promote Treg-cell expansion and fortify the intestinal barrier through distinct mechanisms.

Other members of the microbiota (in mice) appear to promote Th17 activation that is associated with increased colonization resistance. For example, segmented filamentous bacteria intimately interact with ileal epithelial cells and induce a strong Th17 response without intestinal pathology (Fig. 2). Colonization of mice with segmented filamentous bacteria has a moderate protective effect against Citrobacter rodentium (pathogenic E. coli-like bacteria) infection[105] but segmented filamentous bacteria also predispose mice to other extra-intestinal autoimmune diseases.[106, 107] There are more general products of intestinal microbiota activity, for example ATP, that also promote the development and recruitment of Th17 cells to the lamina propria, which is associated with resistance to experimental colitis.[89] The fact that physiologically and biochemically diverse commensal bacteria and their metabolites promote conserved, mutualistic relationships with the host highlights an evolutionary adaptation that benefits the host and indigenous microbes. The emerging ‘yin and yang’ relationship between Treg and Th17 responses may serve as a valuable biomarker of intestinal colonization resistance that may be exploited during the development of immune-modulating or microbial-modulating therapies.

Metabolic links: the SCFA paradigm

The SCFAs also play important roles in mucosal health and resolving intestinal pathology. One SFCA, butyrate, is the major energy source for enterocytes and thereby indirectly helps to fortify epithelial barrier functions. Other SCFAs, such as acetate and propionate, are readily detected in the blood, highlighting a link between the intestinal microbiota and general health and homeostasis.[108] Recent experiments indicate that acetate production by bifidobacteria protects mice from enterohaemorrhagic E. coli infection by reducing translocation of Shiga toxin from the intestine to the blood.[109] Maslowski et al.[110] demonstrated that SCFAs signal through G-proteins (GPR43) present on leucocytes, such as neutrophils, to initiate a signalling cascade resulting in stimulation of cell migration and apoptosis pathways. The authors propose that SCFAs play a role in resolving intestinal inflammation by suppressing the damaging effects of neutrophils.

Subversion of colonization resistance by intestinal pathogens

  1. Top of page
  2. Summary
  3. Introduction
  4. Colonization of the gastrointestinal tract
  5. Ecology of the healthy adult gastrointestinal tract
  6. Microenvironments within the gastrointestinal tract
  7. Proposed mechanisms of colonization resistance
  8. Subversion of colonization resistance by intestinal pathogens
  9. Therapeutic restoration of a healthy intestinal microbiota
  10. Concluding remarks
  11. Acknowledgements
  12. Disclosure
  13. References

Intestinal pathogens disrupt the harmony of a healthy intestinal microbiota by interacting with the mucosal surface to weaken the barrier function and trigger inflammation, generally resulting in diarrhoea. Diarrhoea contains high levels of the disease-inducing pathogen, which are excreted into the local environment, increasing the likelihood of infecting another susceptible host.[13, 111] Therefore the ability of intestinal pathogens to reach and manipulate the intestinal epithelial cells, leading to a strong inflammatory response, provides a strategy for dissemination. Recently it has become apparent that different pathogens exploit intestinal inflammation to achieve this goal.

Salmonella Typhimurium and Citrobacter rodentium use an array of virulence factors to colonize and directly manipulate host cells, leading to colonic inflammation.[112, 113] TLR/NOD-mediated recognition of S. Typhimurium and Citrobacter rodentium triggers a Th1/Th17 response that is characterized by macrophage and neutrophil recruitment.[114-116] Remarkably, both S. Typhimurium and Citrobacter rodentium have evolved a number of mechanisms to survive and thrive within the inflammatory environment.[10, 11] For example, S. Typhimurium replicates inside phagocytic cells,[117] resists host-derived antimicrobials such as lipocalin-2, RegIIIβ and calprotectin[118-120] and even uses host reactive oxygen species and other compounds released during inflammation for enhanced growth.[121, 122] In contrast, many commensal bacteria cannot survive within the inflamed environment and, as a result, the complexity of the indigenous microbiota is reduced.[10, 11] Therefore, S. Typhimurium and Citrobacter rodentium exploit a strong, pathological Th1/Th17-mediated inflammation response to outcompete beneficial commensals and transmit to susceptible hosts.[111, 123]

Antibiotic treatment perturbs the intestinal microbiota, leading to an immediate reduction in microbial abundance and species diversity[124] and suppression of the innate immune system.[125] Therefore, antibiotic exposure reduces colonization resistance by freeing niches and nutrients and creating an immunosuppressed host state for invading pathogens to exploit. Indeed, some pathogens have evolved to specifically exploit antibiotic use. For example, C. difficile is the leading cause of antibiotic-associated diarrhoea in the healthcare setting, colonizes patients (mainly by environmental spores) and rapidly overgrows in the intestine.[126, 127] Clostridium difficile produces two potent enterotoxins, harbouring glucosyltransferase activity, that are translocated into enterocytes, resulting in irreversible disassembly of the actin cytoskeleton.[128] Cellular intoxication can ultimately lead to cell death and disruption of the barrier function to allow bacterial translocation and induce a strong inflammatory response, characterized by neutrophil infiltration.[129] In humans and mice, C. difficile infection is associated with a simplified microbiota[13, 130, 131] containing high levels of C. difficile that are excreted as highly resistant and infectious spores.[132]

Therapeutic restoration of a healthy intestinal microbiota

  1. Top of page
  2. Summary
  3. Introduction
  4. Colonization of the gastrointestinal tract
  5. Ecology of the healthy adult gastrointestinal tract
  6. Microenvironments within the gastrointestinal tract
  7. Proposed mechanisms of colonization resistance
  8. Subversion of colonization resistance by intestinal pathogens
  9. Therapeutic restoration of a healthy intestinal microbiota
  10. Concluding remarks
  11. Acknowledgements
  12. Disclosure
  13. References

Diarrhoeal infections linked to an unbalanced intestinal microbiota remain a serious and growing problem in developed countries (i.e. C. difficile)[133] and developing countries (i.e. pathogenic Enterobacteriaceae).[134] First-line treatments are invariably antibiotics, but their effectiveness is in decline. This point is underscored by the alarming increase in antibiotic-resistant bacteria (pathogens and commensals) and the fact that the antibiotic research and development pipeline is running dry.[135] An alternative and opposite clinical response to such diseases is to re-establish harmony and make peace with our microbiota instead of wreaking havoc with antibiotics. To develop therapies that restore colonization resistance we need to formulate a holistic view of the intestinal ecosystem during health and disease. Clear aims of such therapies are to re-establish a stable and diverse microbial community, eliminate the eliciting pathogen and resolve intestinal pathologies.

Traditional probiotic approaches, including either single or a small number of microbial species, predominantly from the Lactobacillus and Bifidobacterium genera, are designed to modulate the immune system but only colonize the consumer transiently[136] and do not stably restore the microbial diversity of the intestine. Another, more radical, approach is to restore the entire community with faecal transplantation using stool from healthy donors, which is highly effective for treating intestinal ailments like C. difficile disease and ulcerative colitis.[137-139] However, bacteriotherapy is not widely used because of the time required to identify a suitable donor, the risk of introducing opportunistic pathogens as well as a general patient aversion.[140] The development of murine infection models of bacteriotherapy[141] and advances in culturing novel health-associated bacteria[142, 143] promise to lead to the rational design of more novel ‘probiotics’ and other therapeutic options.[144] The description of immunological (Th17/Treg balance), microbiological (diversity and abundance) and metabolic (SCFA) signatures that distinguish between healthy and diseased intestinal ecosystems should guide the development of such therapies.

Concluding remarks

  1. Top of page
  2. Summary
  3. Introduction
  4. Colonization of the gastrointestinal tract
  5. Ecology of the healthy adult gastrointestinal tract
  6. Microenvironments within the gastrointestinal tract
  7. Proposed mechanisms of colonization resistance
  8. Subversion of colonization resistance by intestinal pathogens
  9. Therapeutic restoration of a healthy intestinal microbiota
  10. Concluding remarks
  11. Acknowledgements
  12. Disclosure
  13. References

Our indigenous intestinal microbiota, with a collective gene repertoire 150 times greater than that of the human host, exceeds the host immune system in complexity and plays an equally important role in maintaining intestinal health and preventing infectious diseases. This is an enormously complex ecosystem and we are just beginning to understand how our intestinal microbiota is shaped by our immune system and, in turn, how our microbiota shapes our immune system. However, microbiota research is currently undergoing a considerable resurgence in interest, including major international efforts such as the Human Microbiome Project and MetaHIT.[145] Further recent advances in DNA sequencing technologies, microbial culturing, murine transgenics and mucosal immunology, coupled with the ability to humanize the murine microbiota mean that relevant and innovative studies are now possible to unravel the biology underlying our intestinal ecosystem. We believe that we are on the cusp of exciting new discoveries that will eventually allow us to bolster colonization resistance to prevent or resolve intestinal infectious diseases.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Colonization of the gastrointestinal tract
  5. Ecology of the healthy adult gastrointestinal tract
  6. Microenvironments within the gastrointestinal tract
  7. Proposed mechanisms of colonization resistance
  8. Subversion of colonization resistance by intestinal pathogens
  9. Therapeutic restoration of a healthy intestinal microbiota
  10. Concluding remarks
  11. Acknowledgements
  12. Disclosure
  13. References

Our work was funded by the Wellcome Trust (grant numbers 098051 and 076964) and the Medical Research Council New Investigator Research Grant (TDL; grant number 93614). We are grateful to Rob Kingsley and Arthur Kaser for thoughtful comments on the manuscript.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Colonization of the gastrointestinal tract
  5. Ecology of the healthy adult gastrointestinal tract
  6. Microenvironments within the gastrointestinal tract
  7. Proposed mechanisms of colonization resistance
  8. Subversion of colonization resistance by intestinal pathogens
  9. Therapeutic restoration of a healthy intestinal microbiota
  10. Concluding remarks
  11. Acknowledgements
  12. Disclosure
  13. References