2.3.5 Microbiome
Humans encounter their initial gut microbiota in very early life with most studies focusing on colonisation after birth and the microbial assembly that ensues.Some studies have led to the hypothesis that colonisation begins in utero by providing evidence of a placental microbiome in healthy pregnancies, the presence of microorganisms in the amniotic fluid and meconium.These studies have been presented and critically.The authors concluded that there is insufficient evidence to support the “in utero colonisation hypothesis”, since the studies lacked appropriate controls for contamination, the molecular approaches used were incapable of studying low-biomass microbial populations and evidence of bacterial viability was not provided.Thus, for the purpose of this review, we have focused on gut microbiota development from birth onwards [79, 80].
Although gut microbiota composition varies considerably between infants, certain patterns have been identified.Several factors have been shown to influence microbial colonisation of the gut at this stage, including gestational age, birth mode, sanitation, antibiotic exposure, feeding regime and host genetics.However, facultative anaerobes have been identified as the initial colonisers.Depletion of the available oxygen creates the necessary environment for the establishment of strict anaerobes.Anaerobes which colonise the gut in the early days and weeks of life include Bifidobacterium, Bacteroides, Clostridia and Parabacteroides.Although the diversity of the microbiota is generally low at this stage, dominated largely by members of the Actinobacteria phylum in the case of full-term spontaneously vaginally delivered infants, it has been shown to be greatest in this cohort compared to full-term infants delivered by caesarean section (dominated by Firmicutes) or pre-term infants (dominated by Proteobacteria) at one week old.Interestingly, by week 24, no significant differences in alpha diversity were recorded between any of these groups.Diversity increases with age with a gradual increase in the presence of Firmicutes and Bacteroidetes by the first year where the introduction of solid food to the diet has been identified as an important step in the succession of the microbiota (Spanish infants and Danish infants).In terms of metabolic function, genes involved in the de novo biosynthesis of folate have been shown to be enriched in the infant microbiome across three different populations (Malawian rural communities, Amerindians from the Amazonas of Venezuela, and families from the USA)relative to adults.By three years of age, obligate anaerobes have been shown to dominate the microbiota in breast-fed infants which is trending towards an adult-like composition.The establishment of a stable adult-like microbiota occurs between 2-5 years of age and is dominated by Firmicutes and Bacteroidetes [81-85].
Few studies have specifically investigated the pre-adolescent and adolescent microbiota of healthy humans.However, those which have investigated these age groups indicate that the microbiota has not yet reached the adult-state and is providing essential functions towards the developmental process of its human host.The gut microbiota of children is more stable than that of infants with composition largely influenced by dietary habits and geography.The pre-adolescent microbiota(7-12 years of age) is still in a state of immaturity and based on observations from a group of children from Houston, Texas, has been shown to be more diverse and harbors significantly greater abundances of Firmicutes and Actinobacteria than observed in healthy adults.The pre-adolescent microbiome was also found to be enriched in functions potentially involved in ongoing development, such as vitamin B12 synthesis and de novo folate synthesis relative to the adult microbiome.In terms of the adolescent microbiota, it is identified that a core microbiota of 46 species common to both adults and adolescents (11-18 years) who consumed a standard Western diet.However, the abundances of the genera Bifidobacterium and Clostridium were significantly higher in adolescents relative to adults [86].
The healthy adult gut microbiota is composed primarily of the phyla Firmicutes and Bacteroidetes,and to a lesser extent the phyla Actinobacteria, Proteobacteria, and Verrucomicrobia.As with any age-group, due to the extensive inter-individual variation, it has been virtually impossible to define the composition of the “healthy” adult gut microbiota.However, the “enterotype” concept was introduced in 2011 when faecal metagenomes of individuals from America, Europe and Japan were found to be dominated by one of three different bacterial communities, namely Bacteroides(enterotype 1), Prevotella (enterotype 2) or Ruminococcus (enterotype 3).The enterotype “concept”has since been used in other studies when evaluating the gut microbiota as we will see further on,although further analysis has resulted in the identification of only two enterotypes, one of which is dominated by Prevotella and the other by Bacteroides which have been linked to long term carbohydrate-, or animal fat and protein-rich diets, respectively.Prevotella and Bacteroides should be interpreted as “biomarkers” of diet, lifestyle and disease state given that gradients of both Bacteroides and Prevotella have been found within gut communities as opposed to distinct and consistent community taxa [87-89].
Reports on the estimated number of species and strains in an individual have varied greatly, but in a study examining the stability of the gut microbiota in 37 US adults over a five year period, an average of 101 ± 27 species and 195 ± 48 strains are reported in the faecal gut microbiota of each individual with family members sharing strains, which was not observed in unrelated individuals.However, other studies estimate the number of species to be greater than 1000.The Human Gastrointestinal Bacteria Culture Collection which consisted of 737 whole-genome-sequenced bacterial isolates, representing 273 different species, including 105 novel species from 31 families were found in the human gastrointestinal microbiota.The healthy adult microbiota has been shown to be stable over long periods of time but can be influenced by a number of factors.These including geographical location albeit diet would appear to be an important contributing factor in this regard,direct antibiotic usage, and indirectly by consumption of antibiotic containing animal derived products, such as beef and chicken as a result of their use in livestock production, non-antibiotic drugs, illness, injury and hormonal changes.The healthy gut microbiota is generally characterized by rich species diversity which has been found to be reduced/altered in individuals with certain diseases, particularly those typified by a dysregulated immune response[90, 91].
Ageing has a significant impact on the gut microbiota with dramatic compositional and functional changes observed in the elderly microbiota (in general ＞65 years).Several physiological and lifestyle changes associated with the ageing process may be contributing factors resulting in changes in dietary habits and ultimately nutrition, including a decline in dentition and salivary function, a reduction in digestion and absorption, due to gastrointestinal dysmotility, changes in appetite as a result of prescribed drugs and psychological state, or changes in living conditions,such as residential care or hospitalisation.Gastric hypochlorhydria which is associated with ageing and is prevalent in individuals experiencing or who have experienced Helicobacter pylori infection can cause malabsorption and bacterial overgrowth in the small intestine [92].
The gut microbiome is integral to the health of its host, serving a myriad of functions.It provides essential nutrients and bioactive metabolites, which can be produced directly by the microorganisms or indirectly by microbial conversion of host or environmental molecules.It is involved in energy regulation.It prevents pathogen colonisation directly or indirectly through a phenomenon referred to as colonisation resistance.It sustains the integrity of the mucosal barrier and is an essential component in the orchestration of immune functioning within the gut.The bidirectional interactions within the brain-gut-microbiome axis, in which the gut microbes communicate to the central nervous system, have been demonstrated largely by preclinical and some clinical studies.Alterations in brain-gut-microbiome communication have been implicated in various disease states, from irritable bowel syndrome to psychiatric and neurologic disorders, and this is an area of research that has the potential for identification of novel therapeutic targets and therapies [93].
Fruits, vegetables and cereals are major components of the human diet, providing essential carbohydrates and dietary fibres, although digestion of the latter is beyond the scope of the human genome.Only 17 enzymes within the human genome can breakdown carbohydrate nutrients which include starch, lactose and sucrose.Thus, plant cell wall polysaccharides and resistant starch, which constitute most dietary fibres and cannot be digested or absorbed in the small intestine, enter the large intestine and undergo microbial breakdown and subsequent fermentation.The microbiota also feeds on animal-derived dietary carbohydrates (glycosaminoglycans and N-linked glycans from cartilage and tissue), host epithelial glycome, and microbe-derived carbohydrates from resident gut microbes or foodborne microbes.Collectively, the carbohydrates consumed by the microbiota have been termed “microbiota accessible carbohydrates” (MACs) [94].
Carbohydrate active enzymes (CAZymes) breakdown MACs into fermentable monosaccharides.For example, the gut bacterium Bacteroides thetaiotaomicron was shown to metabolise the most structurally complex plant polysaccharide known, rhamnogalacturonan-II, using a highly specific enzyme system.Bifidobacterium longum strains derived from the infant gut were shown to be capable of metabolising human milk oligosaccharides.In-silico analysis of CAZyme profiles in the guts of 448 individuals from diverse geographies and age groups revealed 89 CAZyme families which were present across 85% of the gut microbiome and revealed several geography/age-specific trends in the CAZyme repertoires of individuals.The major end products of microbial fermentation of the resulting monosaccharides are the SCFAs, including butyrate, propionate, and acetate, which reach a combined concentration of 50-150 mM in the colon at a ratio of 1:1:3, respectively.They are rapidly absorbed by the intestinal epithelial cells where they are involved in a number of cellular and regulatory processes with only 5% excreted in faeces.Butyrate is mainly produced by Firmicutes, propionate by Bacteroidetes, and acetate by most gut anaerobes.Butyrate is the main energy source for the epithelial cells and plays an important role in brain function.It is also known for its anti-cancer and anti-inflammatory properties and for its role in the development of the intestinal barrier.Propionate contributes to gluconeogenesis in the liver and, along with butyrate,has been shown to activate intestinal gluconeogenesis, albeit both use different circuits.Propionate derived from the gut microbiota has also been shown to reduce cancer cell proliferation in the liver.The SCFAs are also involved in regulating immune responses.Acetate has been shown to promote intestinal antibody IgA responses to the gut microbiota via the G protein coupled receptor GPR43.Intestinal IgA is specialized in protecting the mucosa.These SCFAs also stimulate secretion of gut hormones, such as glucagon-like peptide 1 (GLP-1) and plasma peptide YY (PYY) involved in appetite regulation and satiety from enteroendocrine cells proposedly through the SCFA receptors GPR41 and GPR43, thus playing a role in energy regulation in the body.Unsurprisingly, changes in the production of these compounds as a result of disturbances to the gut microbiota can result in pathological consequences for the host.As an example, increased acetate production from an altered gut microbiota in a rodent model was shown to promote metabolic syndrome [95, 96].
The gut microbiota is also responsible for the biosynthesis of several essential vitamins, including B vitamins, such as cobalamin, folic acid, biotin, thiamine, riboflavin, nicotinic acid, pyrodixine and pantothenic acid, as well as vitamin K.Interestingly, vitamin biosynthesis pathways across the three identified enterotypes; however, enterotype 1 was enriched in the biosynthesis of riboflavin,biotin, ascorbate and pantothenic acid, while enterotype 2 was enriched in the biosynthesis of thiamine and folic acid [97].
Primary bile acids are produced in the liver from dietary cholesterol and cholesterol derived from hepatic synthesis, and their main function is to aid absorption of dietary lipids and lipid soluble nutrients.However, bile acids are also important signalling molecules and are known to activate a number of nuclear receptors, including farnesoid X receptor (FXR), preganane X receptor, and vitamin D receptor, as well as the G-protein-coupled receptor TGR5, and cell signal pathways in the liver and GIT thus modulating their own biosynthesis, as well as glucose, lipid, and energy metabolism.In humans, 200-800 mg of bile acids escape enterohepatic circulation every day, pass into the colon and are metabolized by bacteria to secondary bile acids.In a mouse model, such secondary bile acids produced through the action of bacterial bile salt hydrolase (BSH) have been shown to regulate weight gain, lipid metabolism and cholesterol levels via regulation of key genes in the liver or small intestine.The gut microbiota has also been shown to inhibit bile acid synthesis in the liver by alleviation of FXR inhibition in the ileum.(https://www.daowen.com)
In recent years, there has been a growing appreciation for the ability of the gut microbiota to produce neurochemicals that can influence the peripheral enteric and central nervous systems.For example, gamma amino butyric acid (GABA) is a major inhibitory neurotransmitter in the brain and neuropsychiatric disorders, including anxiety and depression have been linked to GABA system dysfunction.Strains of culturable lactobacilli and bifidobacteria from the human intestine were shown to produce GABA, namely Lactobacillus brevis, Bifidobacterium dentium, adolescentis and infantis.Furthermore, GABA was found to serve as a growth factor for a previously uncultured gut bacterium, Flavonifractor sp., which was shown to ferment GABA.In the same study, the authors identified several gut bacteria capable of producing GABA, which included Bacteroides,Dorea, Parabacteroides, Alistipes and Ruminococcus species.A co-culture experiment revealed that GABA produced by Bacteroides fragilis was essential for the growth of a gut isolate termed KLE1738 which was believed to be an unreported bacterial genus.This led to the isolation of a variety of GABA-producing bacteria and the Bacteriodes species in particular were found to produce large quantities of GABA.Furthermore, in the same study relative abundance levels of faecal Bacteriodes negatively correlated with brain signatures associated with depression in patients with major depressive disorder.Bacterially-produced GABA was generated by the enzyme glutamate decarboxylase (GAD) which catalyzed the irreversible α-decarboxylation of glutamate to GABA and was believed to protect the microorganism against stomach acidity.GAD was also found in higher plants and animals.Interestingly, daily consumption of a GABA-producing Bif.dentium gut isolate was found to modulate sensory neuron activity in a rat faecal retention model of visceral hypersensitivity revealing that bacterially-produced GABA could modulate abdominal pain.GABA-enriched black soybean milk fermented with a GABA-producing fish gut isolate, L.brevis,generated similar antidepressant activity in rats as the common antidepressant drug, fluoxetine, but without the side effects normally associated with the drug, such as appetite loss and reduced body weight [98].
Serotonin (5-hydroxytryptamine, 5-HT) is a brain neurotransmitter and performs regulatory functions in the gut and other organ systems.It is derived from the amino acid tryptophan and plays an important role in the regulation of mood such that several antidepressants act on serotonin transporters in the brain.Human- and mouse-derived gut bacteria promote serotonin biosynthesis in colonic enterochromaffin cells which supply serotonin to the lumen, mucosa and circulating platelets.Spore forming bacteria, dominated by clostridial species, were found to elicit this effect.Furthermore, conventional mice were found to have 2.8 times more plasma serotonin levels than their germ-free counterparts.This peripherally produced molecule does not pass the blood brain barrier under physiological conditions but is an important signal molecule in the gut involved in peristalsis, secretion, vasodilation, pain perception and nausea, as well as promoting inflammation and being involved in neuron development and maintenance in the enteric nervous system, while platelet serotonin derived from the gut influences bone development amongst other functions.The mechanisms involved in gut microbiota-mediated serotonin biosynthesis have not yet been fully elucidated but are thought to be linked to microbial metabolite-stimulation of the enzyme tryptophan hydroxylase 1 in enterochromaffin cells, which produces a serotonin precursor that is subsequently metabolized to serotonin.In particular, rectal injection with the microbial metabolites deoxycholate, paminobenzoate, α-tocopherol and tyramine, were shown to increase colonic and serum concentrations of serotonin in mice [99].
Metabolism of tryptophan in the gut results in tryptophan catabolites which have profound effects on the host.Direct transformation of tryptophan by intestinal microbes results in the formation of several molecules, including ligands for the ligand-activated aryl hydrocarbon receptors (AhRs).AhRs, which are transcription factors, are expressed by several cells of both the adaptive and innate immune systems.Upon binding of AhR to its ligand molecule, the activated transcription factor translocates into the nucleus of the cell where it mediates cell-specific transcriptome changes.Thus,AhR signalling plays a key role in immune functioning in health and disease.Intestinal inflammation in mice harbouring a microbiota incapable of metabolizing tryptophan was attenuated following treatment with Lactobacillus strains capable of activating AhRs through the production of tryptophan metabolites.In the same study, faecal samples from healthy subjects induced significantly greater AhR activation compared to faecal samples from IBD subjects, the latter of which harbored significantly less tryptophan and tryptophan metabolites.The tryptophanmetabolizing strain Peptostreptococcus russellii provided a protective effect against colitis in mice,which the authors suggested was linked to its ability to produce the tryptophan metabolite and AhR ligand, indoleacrylic acid which mitigated inflammatory responses and promotes intestinal barrier function.The authors also noted a diminishment of tryptophan- metabolizing capacity in stool samples of IBD patients.The metabolite indole has been shown to enhance epithelial barrier function and attenuate indicators of inflammation [100].
The intestinal microbiota protects its host against colonization by exogenous pathogens and prevents the overgrowth of potentially pathogenic endogenous members, referred to as colonization resistance.This phenomenon is elicited through competition for nutrients and colonization sites,direct inhibition of pathogens through the production of antimicrobial substances, and indirectly through modulation of the luminal environment and via host-commensal interactions involving the epithelial barrier function, modulation of the host cell surface and the host immune system [101].
Members of the established microbiota are controlled by “substrate competition”, defined as the superior ability of a species/strain to utilize one or a few substrates over other species and the control of that population by the limited concentration of these substrates.Furthermore, one microorganism’s by-product can serve as substrate for another.In this regard, nutrient resources in the gut are in huge demand and simultaneously limited, making it challenging to become established or outcompete with resident microbiota.Indeed, nutrient utilization by the colonic microbiota of mice was shown to restrain the growth of Clostridium difficile, since it was incapable of competing with the mouse microbiota for the available carbon sources.Unfavourable environmental conditions created as a result of commensal fermentations can also inhibit the growth of undesirable microorganisms.The utilization of human milk oligosaccharides, particularly the dominant secretor associated oligosaccharide 2′-fucosyllactose, by infant strains of bifidobacteria led to an increase in their proportions, an increase in lactate concentration and a subsequent reduction in pH which was shown to decrease the proportions of Escherichia coli and Clostridium perfringens during in vitro anaerobic fermentations.Furthermore, consumption of butyrate by epithelial cells as an energy source has been implicated as important in maintaining a hypoxic environment in the gut lumen [102].
Decreased intestinal butyrate levels due to depletion of a commensal butyrate producer in a mouse model resulted in increased epithelial oxygenation and aerobic expansion of Salmonella enterica serovar Typhimurium.Butyrate has also been shown to down-regulate virulence gene expression in Salmonella [103].
The gut microbiota is a rich reservoir of bacteriocin producers.Bacteriocins are ribosomally synthesized peptides with antimicrobial activity against either a broad range of species or a narrow range of closely-related species.Their mode of action varies depending on the bacteriocin class, but they generally exert their antimicrobial activity by forming pores in the target cell (Classes I and II),by degrading cell wall peptidoglycan (Class III bacteriolysins) or interfering with cellular processes(Class III non-lytic bacteriocins).The genetic machinery for bacteriocin synthesis is encoded in gene clusters or operons where many of the genes are conserved.Based on this knowledge, 74 bacteriocin gene clusters within the genomes of the GIT subset of the Human Microbiome Project’s reference genome database using an in-silico approach of which the commonly identified were bacteriolysins, then lantibiotics and sactibiotics.Thuricin CD is an example of a sactibiotic bacteriocin produced by a human gut isolate, Bacillus thuringiensis.While it has a narrow spectrum of inhibition, thuricin CD is capable of killing a wide range of C.difficile isolates, its antimicrobial activity being as potent as the antibiotics vancomycin and metronidazole, but without the concomitant damage to other members of the microbiota.Bacteriocin production can also aid niche occupation of the producing strain.Indeed, bacteriocin production in Enterococcus faecalis harboring the bacteriocin-encoding conjugative plasmid pPD1 was shown to replace indigenous enterococci and out-competed E.faecalis strains lacking the plasmid in a mouse model.Supplementing mice with bacteriocin-producing strains resulted in transient advantageous changes,such as inhibition of Staphylococcus by enterocins and Enterococcus by garvicin and promotion of LAB by sakacin, plantaricins and garvicin [104].
Other antimicrobials generated by the gut microbiota can also aid colonization resistance.For example, a single bacterial species, namely Clostridium scindens, was shown to confer colonization resistance against C.difficile infection in vivo.In this instance, secondary bile acids generated by C.scindens from host-derived bile cells were found to inhibit the pathogen [105].
Commensal interactions with the host promote immune system maturation and immune homeostasis through complex microbiota-host networks, although much of our understanding of these mechanisms today have been extrapolated from animal or in vitro studies.We have already seen how several microbial metabolites play essential roles as signalling molecules for the immune system, such as the SCFAs and tryptophan metabolites.More specifically, a polysaccharide (PSA)produced by Bac.fragilis was shown to promote cellular and physical maturation of the developing immune system in mice.Interestingly, this species is an early colonizer of the infant gut and plays an important role in the development of the infant immune system.A 15 kDa protein produced by F.prausnitzii, a commensal microbe deficient in Crohn’s disease patients, was shown to have anti-inflammatory properties, decreasing activation of the NF-κB pathway and also prevented colitis in an animal model.M-cells are specific phagocytic epithelial cells which sample particulate antigens.Efficient induction of IgA is brought about by M-cell sampling of commensal microbes.Paneth cells (a specialized intestinal epithelial lineage) sense enteric bacteria through activation of the MyD88-dependent toll-like receptor which results in the induction of several antimicrobial factors that are essential for controlling bacterial translocation across the intestinal barrier.Colonisation by segmented filamentous bacteria was shown to induce the maturation of T-cell responses in a gnotobiotic mouse model, suggesting these microbes could play a role in the postnatal maturation of the gut immune system.These studies provide a snapshot of how commensals modulate host immunity.The importance of the microbiota for host immunity can also be appreciated from the consequences of its absence in germ-free animals.Indeed, germ free animals exhibit reduced expression of IgA and antimicrobial peptides and are deficient in Peyers’patches [106].
Goblet cells are specialized epithelial cells that secrete mucus, composed primarily of O-glycosylated proteins called mucins, resulting in the formation of a mucus layer whose composition and density is influenced by the commensal microbiota.The mucus layer creates a protective barrier for the epithelial cells, making it difficult for pathogens to gain access to epithelial cell receptors.Germ-free mice have been shown to have an extremely thin colonic mucus layer which can be restored to levels observed in conventional mice following exposure to bacterial products, including peptidoglycan and lipopolysaccharide.Mice treated with the antibiotic metronidazole were shown to have a thinning of the mucus layer which correlated with an increased attachment of the mouse pathogen Citrobacter rodentium.Certain commensals have been shown to modulate mucin gene expression and glycosylation patterns.This may be achieved through the activity of SCFAs which have been shown to increase expression of mucin-associated genes.Furthermore, the SCFA butyrate provides energy for the epithelial cells and has also been implicated in enhancing the intestinal barrier by up-regulating the tight junction protein,Claudin-1[107].