Emerging science of the human microbiome

Abstract

The human gastrointestinal tract hosts a large number of microbial cells which exceed their mammalian counterparts by approximately 3-fold. The genes expressed by these microorganisms constitute the gut microbiome and may participate in diverse functions that are essential to the host, including digestion, regulation of energy metabolism, and modulation of inflammation and immunity. The gut microbiome can be modulated by dietary changes, antibiotic use, or disease. Different ailments have distinct associated microbiomes in which certain species or genes are present in different relative quantities. Thus, identifying specific disease-associated signatures in the microbiome as well as the factors that alter microbial populations and gene expression will lead to the development of new products such as prebiotics, probiotics, antimicrobials, live biotherapeutic products, or more traditional drugs to treat these disorders. Gained knowledge on the microbiome may result in molecular lab tests that may serve as personalized tools to guide the use of the aforementioned products and monitor interventional progress.

Keywords: :

• microbiome
• metabolic disease
• dysbiosis
• immune system
• diet
• antibiotics
• probiotics
• prebiotics

The Human Body is a Community of Microbes

Only one of 4 cells of the human body is actually human in origin. The remaining 75% of cells are composed of the microorganisms that colonize the internal and external surfaces of our bodies. Although it is often stated that the number of microbial cells is approximately 10 times the number of human cells, recent studies have estimated we harbor 37 trillion human cells¹as well as at least 100 trillion microbial cells and a quadrillion viruses in and on our bodies.² Given these calculations, the ratio microbial-to-human cell is around 3-fold. The microbiota is defined as the full complement of microbes, including bacteria, viruses, fungi, and protozoa that reside in and on our bodies.^3-5 The microbiome has been defined as “the ecological community of commensal, symbiotic, and pathogenic microorganisms that literally share our body space.”⁶ The two may be distinguished as the microbiome includes the collective genomes.^3-5 In 1985 researchers demonstrated that bacteria which can be cultivated in the laboratory represent only a small fraction of the microbes present in the environment, and dubbed this phenomenon the “great plate count anomaly.”⁷ Recently, culture-independent high-throughput sequencing has greatly expanded our repertoire of known microbes.⁸ This has allowed for measurement of the structure and dynamics of microbial communities, the relationships between their members, what substances are produced and consumed, the interaction with the host, and differences between healthy hosts and those with disease. It is hypothesized that the composition of the microbiome may influence the host’s health by contributing to its metabolic and immune functions. In this review, we summarize the emerging work on the relationship of the gut microbiome with human health, its development and establishment, and the factors including diet, antibiotic use, and disease, that shape it.

The Microbiome and Human Health

An increasing level of evidence reveals that the human microbiome plays a major role in health. For this reason it is often referred to as the “forgotten organ.”⁹ All surfaces of the human body that are exposed to the environment including skin, respiratory system, urogenital tract and gastrointestinal (GI) tract, are colonized with microorganisms. The majority of microbes composing the human microbiome are in the GI tract³ and thus this review will focus on the human gut microbiome. The gut microbiome provides metabolic functions such as energy harvest and storage, fermentation and absorption of indigestible carbohydrates,^10,11 synthetic functions such as the production of vitamins K^12,13 and B,¹⁴ and immune roles such as the modulation of innate immunity, the promotion, maturation and development of cell-mediated immunity, and the maintenance of appropriate immune response to pathogens. In addition, the microbiome plays an important part in the preservation of the intestinal barrier function.^15-17

The gut microbiome expresses the enzymatic machinery to process otherwise non-digestible carbohydrates such as fructo-oligosaccharides (FOS), galacto-oligosaccharides (GOS), and inulin, and thus release monosaccharides that could be utilized by the host for metabolic purposes.^18-20 Some bacteria express colonization factors, which consist of polysaccharide utilization loci containing the machinery to degrade mucosal polysaccharides, and survive in epithelial crypts.²¹ The breakdown of mucosal polysaccharides results in the release of smaller sugars that can be consumed by other GI microorganisms. Additionally, fermentation of FOS and other dietary fibers by some of the bacterial symbionts results in the production of short chain fatty acids (SCFAs), specifically in the colon, which promote functions that are beneficial to the human body and provide an energy source for colonocytes. Microorganisms with such capabilities include bacteria from the Bacteroides and Bifidobacterium genera. For example, increases in SCFAs and glucagon-like peptide-1 (GLP-1) levels, were seen in humans after fiber intake.²² Conversely, GLP-1 levels inversely correlated with SCFA levels and the presence of a microbiome in a mouse study. This result was attributed to a GLP-1-dependent delay of gastric transit to improve energy harvest in germ free mice.²³ SCFA signaling has also been implicated in a number of immune and anti-inflammatory functions such as activation of regulatory T cells, downregulation of proinflammatory cytokines by peripheral blood mononuclear cells, promotion of chemotaxis and phagocytosis by neutrophils, and the maintenance of intestinal epithelial barrier integrity.^24-28

The gut microbiome also influences the host health status through enzymatic transformation of bile acids (BAs), natural detergents with novel signaling functions including regulation of cholesterol synthesis and absorption, modulation of inflammatory responses, and energy homeostasis.²⁹ Some microorganisms in the gut express enzymes that hydrolyse taurine- or glycine- conjugated BAs to change their physical and chemical properties. Other bacterial enzymes dehydroxylate or sulfate the resulting deconjugated primary BAs.³⁰ The composition of the gut microbiome determines the diversity and size of the BA pool in different host compartments.^31,32 As a result, transformed BAs can be re-absorbed into mesenteric circulation and exert signaling functions in different tissues through nuclear farnesoid X receptor (FXR) or G-protein coupled receptor (TGR-5) among others.³¹ It has been shown that BA signaling through FXR results in the reduction of triglycerides, cholesterol, and fatty acids,^33,34 improvement of glucose metabolism,³⁵ and hepatoprotection.³⁶ In contrast, BA signaling through TGR-5 plays a role in immune system modulation,³⁷ energy metabolism,^38,39 glucose metabolism,^39-41 intestinal motility, and gall bladder filling and fluid secretion.^42-44 These examples illustrate how the gut microbiome can trigger diverse mechanisms to maintain homeostasis, either by fermentation of food that is otherwise non-digestible by the host, or by transformation of metabolites in the gut.

Further, the effects of the microbiome in human health have been attributed to numerous mechanistic pathways. Specific functions, as outlined in Figure 1, include (1) degradation of dietary and mucosal polysaccharides with subsequent modulation of the microbial ecology and release of metabolites, and preservation of mucosal barrier integrity (2) regulation of host metabolic, inflammatory and immune responses through the release of SCFAs, (3) regulation of the host metabolism and inflammatory responses through modification of BAs, (4) enhancement of the host enzymatic functions by release of bacteria-derived co-factors such as vitamins, (5) development and modulation of the immune system by expression of extracellular molecular patterns, and (6) release of antimicrobial peptides with further shaping of the microbiome. Although evidence from in vitro studies as well as gene deficient, germ-free or gnotobiotic mouse models sustains these hypotheses, proof in animals with a natural microbiota is more difficult to obtain. Simplified models present an excellent opportunity to explain punctual mechanisms but fail to account for changes in bacterial populations caused by the release of metabolites as observed in wild type animals. Further, these studies are restricted to rodents, which differ from humans in both host genome and microbiome, and involve only a small set of genes within the gut microbiome, leaving most of the metagenome unexplored.

Figure 1. The microbiome is involved in immune, inflammatory, and metabolic functions through numerous pathways.

Changes to the Microbiome over Time

Understanding the stability of the microbiota within an individual over time is an important step in facilitating prediction of disease and the development of therapies to correct imbalance. It is known that several factors such as host genetics, diet, environment, antibiotic use, and age influence the development and composition of the human gut microbiome considerably.^45-47 While significant interindividual variability exists, the microbiota of most individuals can be categorized into one of three distinct variants of bacterial communities dominated by Bacteroides, Prevotella, or Ruminococcus, regardless of the geographic origin of the sample.⁴⁸ Interestingly, the abundance of each of these three dominant genera correlate strongly, either positively or negatively, with those of other genera, meaning different microorganisms co-occur or avoid each other. The enterotypes are thus determined by favored community compositions.⁴⁸ Overall, the microbiome is considered robust at both the phylogenetic and functional level. A 5-year-long study indicated that strains recovered from fecal samples may have been present in the hosts for decades.⁴⁹ Despite transient environmental perturbations caused by dietary changes, antibiotic use, or disease may result in significant variations in microbial populations, several studies report that the microbiome could be restored and is stable over time.^50-52

Development of the microbiome

The human microbiota starts developing shortly after birth, evolves and establishes itself through infancy and adapts to metabolic changes of the body even during old age.⁴⁵ Traditionally, babies have been considered sterile in utero and thus open to colonization by microbiota at each generation. However, the baby’s first intestinal discharge, the meconium, is not sterile and may contain a low diversity microbiome.^53,54 It is hypothesized that mothers may transfer their microorganisms in utero and no global differences were observed when comparing the meconia from babies delivered either via caesarean section or vaginally.⁵⁵ In contrast, other studies show newborn babies are exposed to microbes from different environments immediately upon birth and are rapidly colonized through vertical acquisition of the microbes depending on delivery mode.⁵⁶ Infants born vaginally have communities resembling those found in the vaginal microbiota of their mothers⁵⁶ while infants delivered by caesarean section harbor a microbiota characteristic of skin, which are dominated by Staphylococcus and Propionibacterium.⁵⁶ Delivery mode has also been hypothesized to influence immunological functions during the first year of life via gut microbiota development, with babies delivered by caesarean section having lower bacterial cell counts in fecal samples and a higher number of antibody secreting cells.⁵⁷ Diversity of bacteria in the infant gut is initially low with the early colonizers being aero-tolerant as the gut contains oxygen but are replaced by anaerobes typical of the adult gut microbiota.⁵⁸ Detailed time series show that the phylogenetic diversity increases gradually over time and is punctuated by major shifts in taxonomic compositions of the microbiota associated with events such as introduction of solid foods and antibiotic use.⁵² Interestingly, metagenomic analysis shows that the infant microbiome is enriched in genes to facilitate lactate utilization in milk and that the functional capacity to utilize plant derived glycans is present before introduction of solid foods.⁵² Once the microbiota reaches maturity it remains relatively constant until old age.^52,59 Finally, the elderly microbiota is generally less diverse and presents increased levels of the Bacteroidetes phylum as well as the Clostridia genus.⁵⁹

Establishment of the microbiome

The formation of a stable microbiome during the first years of life, followed by maintenance of a mature microbiome in adulthood, raises questions such as how the adult microbiome is established, what factors influence colonization and which organisms are involved. The most numerous organisms of the microbiome are members of the Firmicutes and Bacteriodetes phyla, which are appropriate candidates for studying the factors affecting colonization. Germ-free mice mono-associated with single Bacteroides species have been found resistant to colonization by the same, but not different, species.⁶⁰ In vivo genetic screening of a B. fragilis strain representative of Bacteroides identified a unique polysaccharide utilization loci, commensal colonization factor (CCF), which when deleted results in colonization deficit and reduced horizontal transmission.⁶⁰ This strain of B. fragilis penetrates the colonic mucus and resides within crypt channels. The CCF system of genes enables this microorganism to use crypt mucus as a nutrient source and was found to be required for colonization following microbiome disruption by infection or oral antibiotics.⁶⁰ Specifically, the crypts represent a preferred environmental niche and reservoir for B. fragilis allowing for durable and long-term colonization.⁶⁰ This is an example of how commensal bacteria have evolved molecular mechanisms to promote their own health in a symbiotic relationship with their human hosts. In agreement with these observations, it was shown that glycosylation patterns in the colonic mucus, determined by host genetics can modulate the composition of the microbiome, favoring species that utilize specific mucus terminal sugars as a source of energy. For example, in the absence of terminal fucose in the mucus as a result of the host’s deficiency in fucosyl transferase enzymatic activity, there is a significant increase in the populations of Bacteroides and a decrease in Clostridiales, as shown in a mouse study.⁴⁷ Additionally, fucosyl transferase mutations decrease the susceptibility to infections of Campylobacter jejuni, Helicobacter pylori, and Norwalk virus in humans.^61-63

Effects of host genetics, gender and environment on the microbiome have been analyzed in mice with different genetic backgrounds that have been housed with mice of the same strain, or co-housed with mice of different strains or gender. The study showed a highly significant contribution of genetic background and co-housing to the microbiome composition. Interestingly, strain genetic distances correlated positively with the microbiome distances.⁶⁴ The effects of host genetics on the microbiome were also described in co-twins, who share gut metagenomes with a core of redundant functionalities. Nonetheless, these metagenomic studies show monozygotic twins share less than 50% of species-level bacterial taxa, and only 17% of the gene clusters in the microbiome.⁶⁵ Despite equal genomes, many co-twin pairs have discordant health conditions which are associated with differences in the microbiome. Transplantation of cultured or uncultured microbiota from obese co-twins into germ free mice resulted in greater increases in the recipient’s weight and body fat as compared with the changes seen in mice receiving microbiota components from lean co-twins.⁶⁶ These series of elegant and well controlled studies show that the establishment of the microbiome is not only influenced by genes of the microorganisms that populate the gut and their mutual interactions, but also by host genetics. In another respect, the use of knockout mice to study the effects of host genetics on the microbiome addresses only the effect of one particular gene at a time in an inbred genetic background. Considering the more diverse genetic background of humans, it is difficult to confirm whether mutations in one gene are causing changes in the human microbiome; however, it does provide evidence of the potential for cross-talk. As mentioned above, emerging studies show other environmental effects, such as diet and antibiotic use, affect the microbiome as well, and are discussed in the following sections.

Diet affects the microbiome

Throughout life, diet plays a significant role in determining the gut microbial community. Dietary patterns associate with predominant variants, including Bacteroides, Prevotella, and Ruminococcus.⁴⁸ Bacteroides-dominated enterotypes are associated with a diet high in animal fat and derive energy primarily from carbohydrates and proteins through saccharolytic fermentation and proteolysis.⁴⁸ The Prevotella-dominated enterotype correlates with a diet rich in carbohydrates⁶⁷ and contains many species capable of desulphating and degrading mucin glycoproteins present in the mucosal layer of the gut.⁴⁸ The Ruminococcus enterotype is dominated by species which efficiently bind and hydrolyze mucins, as well as uptake the resulting simple sugars.⁴⁸ While the basis of enterotype clustering is unknown, it has been shown to be independent of nationality, sex, age, or body mass index (BMI). Adults who shifted from a high-fat/low fiber diet, generally a Bacteroides-dominated enterotype, to a low-fat and/or high-fiber diet display notable changes in the gut microbiota within 24 h.⁶⁷ Research combining cross-sectional and interventional studies show that long-term diet is correlated with enterotype clustering. However, while changes seen during dietary intervention are significant and rapid, the magnitude was shown to be insufficient to switch individuals between enterotypes clusters within 10 d.⁶⁷ Additionally, this study found that several factors such as BMI, red wine, and aspartame intake were significantly correlated with microbiome composition but not necessarily with enterotype, indicating that enterotype may not be predicative of BMI or dietary intake of individual foods.

The effect of diet on the microbiota composition was studied in gnotobiotic mice with well-defined diets composed of only four ingredients. Dietary perturbations evoked changes in microbial abundances and a statistical model developed from the experimental data could predict up to 60% of the experimental results.⁶⁸ Diets rich in plant polysaccharides stimulate the growth of bacteria within the Bacteroidetes phylum. The latter have richer genomes including glycan degrading capabilities to release monosaccharides from these carbohydrates. In the presence of polysaccharide-degrading Bacteroidetes, bacteria of the Firmicute phylum upregulate their amino acid and sugar transporter genes to perform glycolysis, and utilize acetate released by the former bacteria for energy harvesting. As a result, Firmicutes release butyrate, which is beneficial for human metabolism.⁶⁹ Moreover, in the presence of fructan rich diets, B. tetraiotaomicron produces acetate and formate, wherein the latter is consumed by the archaebacteria Methanobrevibacter smithii to release methane.⁷⁰

Recent studies have shown that diet-induced changes in the microbiome can occur very rapidly, notably after ingestion of an animal-based diet in comparison to a plant-based diet.⁷¹ Switching to a diet richer in fat and protein derived from animal products may increase populations of Bilophila, Alistipes, and Bacteroides genera in the gut microbiota. As a consequence, these microorganisms release SCFAs such as isobutyrate, isovalerate and valerate. In contrast, when the diet is changed to plant-based, the gut microbiota presents increases in the Roseburia, Eubacterium, Faecalibacterium, and Bifidobacterium genera, and their derived SCFAs such as acetate, butyrate and propionate.⁷¹ Whereas an animal-based diet was shown to trigger microbial expression of genes associated with vitamin synthesis, polycyclic aromatic hydrocarbons, or b-lactamase genes, a plant-based diet induced microbial expression of saccharolytic genes. It is believed that changes in gene expression in the microbiome are the combined result of regulatory and selective pressure.⁷¹

Consumption of inulin or FOS was shown to increase populations of Bifidobacteria by approximately 10-fold,⁷² which warranted studies on the effect of prebiotic intake on the microbiome of healthy human subjects.^73-77 However, in individuals suffering from gastrointestinal disorders, evidence of prebiotic intervention affecting the microbiome and/or clinical outcomes is mixed. In such cases, dose and type of prebiotic is critical to the clinical outcome where high doses may result in worsening of symptoms.⁷⁸ A review of dietary interventions, in the form of prebiotics, and their effect on the microbiome is shown in Table 1. Taken together, it is clear that long-term dietary habits and host genetic background⁴⁷ determine the types of bacterial communities that makeup the gut microbiome and that short-term dietary changes can have a significant and immediate impact, but may not be enough to change the composition and function of the microbiome in the short-term.

Table 1. Prebiotics, their effects on the microbiome and associated observed clinical benefit

Prebiotic	Effects on the microbiome	Clinical benefit	References
Dextrin	Increases Bacteroides Decreases Clostridium perfringes		76
Inulin	Bifidogenic	Reduced IBD scores in pouchitis Improved blood lipid profile in combination with fructo-oligosaccharides and soy product	74, 77 112 113
Galactooligosaccharides Trans-galacto-oligosaccharides	Bifidogenic	Infectious diarrhea IBS	114 115
Acacia gum Guar gum	Increases bifidobacteria and lactobacilli	Improved abdominal pain and bowel habits	73 116
Psyllium	Possibly bifidogenic		75
Fructooligosaccharides		Improved blood lipid profile in combination with inulin and soy product Weight loss, satiety	113

Antibiotics affect the microbiome

Observational and interventional studies suggest there is relative stability of the microbiome throughout adult life⁵⁹ and that long-term stability is maintained by genetic⁴⁷ and environmental forces⁷⁹ and governed by the laws of microbial ecology.⁶⁰ Oral antibiotic use results in immediate and significant alterations in the gut microbiota with the affected taxa varying between individuals.^46,80 There is at least partial recovery of taxonomic composition over time; however, some taxa do not recover even months after treatment and generally there is a long-term decrease in bacterial diversity.⁴⁶ After oral antibiotic use there is an immediate reduction in the stability of the microbial community, a collapse in diversity, oscillatory population dynamics, and colonization by opportunistic organisms followed by repopulation by the commensal flora.^46,80 As the microbiota recovers, there is a reduced resistance to colonization allowing foreign microbes to outgrow commensal organisms; however, most patients report normal GI function suggesting functional redundancy of the intestinal mirobiota.⁴⁶ It has been suggested that repeated use of oral antibiotics increases the reservoir of antibiotic resistant genes and overuse may be associated with an increase in antibiotic-resistant pathogens.⁸¹ Certainly, dysbiosis of the microbiome has been reported with both clinical and subclinical antibiotic use and is the cause of alterations in immune and metabolic functions. A healthy and rich microbiome protects the GI tract from bacterial or viral infections and antibiotic use often triggers the emergence of certain pathogens such as Salmonella enterica serovar Typhimurium or Clostridium difficile. These infections are facilitated by the fluctuations in microbial populations that result in transient increases of certain bacteria, such as B. tetaiotaomicron,⁸² which releases sialic acid from the intestinal mucus via sialidase activity. Free sialic acid is then utilized by the pathogen for propagation. Gnotobiotic mice colonized with a sialidase-deficient B. tetaiotaomicron demonstrated impaired expansion of the pathogen. This effect was reverted by oral administration of free sialic acid.⁸² Overall, antibiotic use disrupts the integrity and richness of the microbiome, the latter of which is not only beneficial for the host metabolism but also protects it from pathogenic infections.

Microbiome and the Immune System

Humans and their constituent microbiota have coevolved over millions of years. Approximately 70% of the human immune system is found in the GI tract.⁸³ The gut associated lymph tissue (GALT) is comprised of several types of lymphoid tissue that store immune cells, such as T and B lymphocytes, that carry out attacks and defend against pathogens.¹⁷ Innate and adaptive immune systems have evolved to require microbial interactions during their development. It has been shown that germ-free mice have reduced gut secretory IgA, defects in gut-associated lymphoid tissue development, smaller Peyer’s patches, and mesenteric lymph nodes.¹⁷ As well, it is believed that the progressive loss of microbial diversity, due to reduced transmission, may result in an adaptive immune system which is not adequately educated and in turn an increase in the incidence of autoimmune disease.⁸⁴ The innate immune system recognizes microbe-associated molecular patterns (MAMPs) that are present across diverse lineages of bacteria such as lipopolysaccharides (LPS), peptidoglycan, and flagellin.¹⁵ Toll-like receptors (TLRs) are one of several proteins that recognize MAMP antigens, and when not expressed the gut, immune systems do not form normally.⁸⁵ TLRs play an important function in host defense by either promoting appropriate inflammatory signals to pathogens via inflammatory cytokines such as interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α), or promoting immune tolerance through the production of anti-inflammatory cytokines such as IL-10.⁸⁶ The adaptive immune system is also programmed by the commensal microbiota and microbes have been shown to impact the differentiation of T cell populations.¹⁶ Pathogenic bacteria are capable of inducing the proliferation of effector T cells, whereas commensal bacteria induce the proliferation of T regulatory cell population, which can be educated by the commensal microbiota.¹⁶ Finally, microbes participate in a symbiotic relationship with their hosts, modulating the host immune system to promote their own fitness.¹⁶ Thus, it is clear that a primary function of the gut microbiome is to educate and maintain host immune function at multiple levels including maintenance of anatomical barrier function, development and maintenance of innate immunity as well as to temper the adaptive cell mediated immune response. The roles and relationships of the microbiome within the immune system have been reviewed extensively elsewhere.^24,87-90

Microbiome in Health and Disease

In the past, research on microbial interactions and disease has been related to single pathogenic microbes. While microbial communities benefit the host by providing functions such as digestion of nutrients or protection against infection,⁹¹ studies on the communities of non-pathogenic microbes were less prevalent as their influence on human health was deemed to pale in comparison with that of pathogenic microbes.⁹¹ Recent analysis has revealed that complex phenotypes are associated more appropriately with a dysbiotic gut microflora rather than the presence of a single pathogenic microbe.⁴⁵ Dysbiosis is a pathological imbalance in a microbial community and is becoming increasingly appreciated as a “central environmental factor” that is affected by host genetics, diet and antibiotic use. Furthermore, dysbiosis likely results from or is a cause of numerous disease states including atopy, autoimmune disease, inflammatory bowel disease (IBD), obesity, diabetes, non-alcoholic fatty liver disease (NAFLD), and hyperlipidemia.⁹² Emerging clinical studies comparing the gut microbiomes of healthy and disease populations using 16s DNA or metagenomic sequencing analyses have identified associations between certain bacterial families and disease, as presented in Table 2. Several studies show Firmicutes from diverse families, namely Clostridiales, Erysipelotrichaceae, Ruminococcaceae, Eubacteriaceae, and Lachnospiraceae have been associated with healthy populations.^66,93-96 In particular, bacteria from the Faecalibacterium and Roseburia genera, among the most abundant organisms in the colonic microbiome, are present in significantly lower numbers in diabetic, obese, and atherosclerotic populations. These genera express active bile salt hydrolases with significant activity in the hydrolysis of both glycine and taurine conjugated BAs⁹⁷ and may exert metabolic benefits to the host by modifying BA pool size and composition. In addition, Roseburia and Faecalibacterium are known SCFA producers, and deficiencies in these genera were observed in patients with ulcerative colitis.⁹⁸ These studies establish associations between disease and microbiota compositions, with microbial genes identified in some cases. However, there is little evidence of whether the composition of microbial ecology is causative of disease or a result of the host’s metabolism.

Table 2. Association of microbial species or families in health and disease

Disease state	Microorganisms associated with disease state	Microorganisms associated with healthy state	References
Diabetes	Betaproteobacteria Bacteroides Bacilli Akkermansia muciniphila Bacteroides intestinalis Clostridium boltae Clostridium hathewayi Clostridium ramosum Desulfovibrio sp. 3_1	Clostridia, Erysipelotrichi Clostridiales sp. SS3/4 Eubacterium genus Faecalibacterium genus Roseburia genus Erysipelotrichaceae family	95 94
Obesity	Clostridium hathewayi Parabacteroides distasonis Bacteroides ovatus Ruminococcus torques Clostridium cluster XIV	Bacteroides uniformis Parabacteroides merdae Alistipes putredinis Eubacterium desmolans Eubacterium limosum Clostridium symbiosum Clostridium cluster IV Bacteroides pectinophilus Faecalibacterium	66 96
Atherosclerosis	Collinsella	Roseburia, Eubacterium	93
Inflammatory bowel disease	E.coli	Bifidobacterium C. leptum	110
Autism spectrum disorder	Dysbiosis; altered populations of classes Clostridia and Bacteroidea: Porphyromonadaceae Prevotellaceae unclassified Bacteroidales and Lachnospiraceae	Erysipelotrichaceae Ruminococcaceae Proteobacteria	117
Rheumatoid arthritis (New Onset)	Prevotella copri	Bacteroidaceae	118
NAFLD	C. coccoides	Bacteroidetes	119
Obese NAFLD	Lactobacillus Dorea Robinsoniella Roseburia Kiloniellaceae family Pasteurellaceae family	Oscillibacter	120

NAFLD: Non-alchoholic fatty liver disease

Researchers believe that reduced microbial diversity, selective disappearance of the microbiota and coincident increases in metabolic and autoimmune disease may be the result of a loss of horizontal and vertically transmitted microorganisms.⁸⁴ One hypothesis helping to describe the finding of hereditary dysbiosis, is that there is a loss of maternal transmission of microbes to their offspring (i.e., vertical transmission) as well as transmission of microbes through fecally contaminated drinking and bathing water (i.e., horizontal transmission).⁸⁴ Part of this modern “hygiene hypothesis” is that a lack of early childhood exposure to infectious agents, symbiotic microorganisms, and parasites increases susceptibility to allergic diseases by suppressing natural development of the immune system.^84,99 Other recent changes in human ecology include cleaner water, an increase in caesarean sections, increased used of pre-term antibiotics, reduced breastfeeding, smaller family size, and widespread antibiotic use.⁸⁴ Diminished horizontal transmission resulting from changes in human ecology makes it more difficult to overcome losses in vertical transmission, and this then manifests as a birth cohort phenomenon. This is supported by the finding that over the past 50 years there has been a progressive decline in infectious disease coinciding with increased incidence of immune disorders. Furthermore, over this period there has also been a significant increase in the incidence of metabolic disease due to the widespread adoption of a more sedentary lifestyle and Western diet.^84,99 These changes are increasingly thought to be associated with the development of a dysbiotic microflora, and conversely a dysbiotic microflora has been shown to lead to changes in metabolism.^48,67,100

Dysbiosis in the form of reduced microbial diversity at the gene and species level does define a subset of individuals who are at increased risk of obesity related metabolic disorders.⁹⁶ Comparison of gene number across obese and non-obese individuals shows a bimodal distribution of bacterial genes in Danish⁹⁶ and French¹⁰⁰ individuals. It has been suggested that the obesity-associated signal in the human gut microbiome may be much more pronounced than that presently known in the human genome.⁹⁶ Based on microbiome gene richness there are two groups of individuals, including a group of high gene count (HGC) individuals and a group with 40% fewer genes called low gene count (LGC) individuals.⁹⁶ While dietary intervention with a high protein energy restricted diet has been shown to correct low gene richness as well as improve metabolic endpoints, it appears less effective in changing markers of inflammation.⁹⁶ Interestingly, low diversity of gut microbiota has been reported in a wide range of cohorts, including patients with IBD,^101-103 elderly patients with inflammation,⁵⁹ obese individuals⁹⁶ and individuals after oral antibiotic treatment.¹⁰⁴

Individuals with low microbiome gene richness are characterized by more marked adiposity, insulin resistance and dyslipidemia as well as a more pronounced inflammatory phenotype when compared with individuals with high bacterial richness.⁹⁶ An increase in the Firmicutes phylum and a decrease in Bacteroidetes has been shown to be associated with obese individuals in several studies, with others reporting no association. As well, increased Actinobacteria has been shown in obese individuals. Further, LGC individuals appear to have a phylogenetic shift in favor of Proteobacteria and Bacteroidetes as compared with HGC indivudals, who show increased Verrucomicrobia, Actinobacteria, and Euryarchaeota. Beyond metabolic dysfunctions, low-grade inflammation is associated with a plethora of chronic diseases. Whether a low gut bacterial richness is common in all cases could be revealed by exploring gut microbiota at a deep metagenomic level in a broad variety of these afflictions. Going forward, it may be possible to develop stratified approaches, based on gene richness, for treatment and prevention of chronic disorders.⁹⁶ In addition, knowledge of the microbiome in determined disease states supports the use of probiotics, as summarized in Table 3, either alone or in combination with prebiotics, to potentially affect phenotypes to which the microbiota is linked. The use of probiotics in the management of disease has been reviewed extensively elsewhere.^105-107

Table 3. Probiotics, their effects on the microbiome and associated clinical benefit

Probiotic Species	Effects on the microbiome	Clinical condition/benefit	References
Bifidobacerium infantis 35624		Irritable Bowel Syndrome	121
Lactobacillus reuteri protectis SD2112 (ATCC 55730)	Reduced E.coli and increased Lactobacillus-Enterococcus in in vitro colonic model	Infectious diarrhea treatment	122 123
Lactobacillus reuteri 30242		Hypercholesterolemia, Lipid profile, inflammatory markers Diarrhea, GI health Vitamin D status	124,125 126,127 128
Lactobacillus rhamnosus GG		Antibiotic associated diarrhea (prevention) Infectious diarrhea (treatment) Infectous diarrhea (prevention)	129 130 131
Lactobacillus casei DN-114001		Antibiotic associated diarrhea (prevention)	132
Saccharomyces Boulardii	Modified Bifidobacterium and Eubacterium cylindroides in patients with diarrhea	Antibiotic associated diarrhea (prevention) Infectious diarrhea (treatment) Infectious diarrhea (prevention)	129 133 134 135
Escherichiae coli Nissle 1917		Ulcerative colitis (maintenance of remission)	136
Combination of Streptococcus thermophylus, Bifidobacterium breve, Bifidobacterium longum, Bifidobacterium infantis, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus paracasei and Lactobacillus delbrueckii/bulgaricus	Decreased Clostridium cluster and changed composition of microbiota	Ulcerative colitis (induction of remission) Ulcerative colitis (maintenance of remission) Pouchitis (prevention and maintenance of remission)	137 138 139 140

Conclusions and Future Prospects

Understanding the interactions between host genetics, diet, and the microorganisms that live in the gut provides insight on environmental perturbations and how the microbiome changes over time. The use of advanced mathematical models of ecology and machine learning in combination with metatranscriptomics, metabolomics, and proteomics data will allow scientists to identify defined signatures or patterns associated with disease states. As described, a great body of evidence originates from animal models, sometimes using human fecal transplantation. Although these studies are well controlled, the use of murine models introduces concerns when results are extended to humans due to a number of reasons. For example, rats do not have gall bladders. Also, the BA composition of mice and rats differ from humans which is problematic when many of the beneficial mechanisms of the microbiome in health rely on BA metabolism and signaling through its receptors. Further, animal diets and metabolisms differ from those of the human, which may account for differences in microbial ecologies and in turn make results difficult to interpret. Nevertheless, these investigations provide compelling evidence that may warrant further human studies. It is also plausible that given the availability of knockout mice encompassing families of genes involved in metabolic, inflammatory, immune, structural, and developmental activities, future studies will include these resources to study the microbiome.

Another research question that should be better addressed is whether the dysbiotic microbiome is causative or a consequence of disease. The available proof of causation is based on a few animal studies where germ-free mice were transplanted with microbiomes from lean or obese individuals, as described above.^66,108 In order to answer this question, there is a need for more transplantation studies in animals as well as longitudinal studies in humans where the microbiome and host health are followed from the early onset of disease.

A better understanding of the microbiome should also result in the discovery of tools to modulate the microbiome composition toward a “healthy state” for the treatment of a determined set of metabolic or inflammatory ailments. In particular, knowledge on the effect of prebiotics on the gut microbiome can lead to the development of therapeutics (e.g., non-digestible fibers) that will favor the increase of certain commensal species with beneficial effects on the host that may be otherwise depleted in a disease state. In the same line of thought, specific microorganisms or a cocktail of select microorganisms including bacteria and yeast could be delivered to either enrich a dysbiotic microbiota, or supply metabolites or enzymatic activities that may be present at low levels in disease states. On the other hand, compositions of bacterial populations are regulated by antimicrobial agents as well as quorum sensing molecules released by the same native microorganisms. Thus, identifying genes that participate in the release of these molecules in healthy and disease populations may serve to introduce new therapies to control the growth of pathogenic species while favoring beneficial ones. This approach could, in certain cases, act as a useful alternative to broad range antibiotics that have detrimental effects on both microbiome and host health, as described.

In addition, identification of genetic markers within the microbiome that are associated with disease will lead to the development of cost-effective lab tests to evaluate the suitability of therapeutics in a personalized manner. Currently, evaluating the microbiome is performed through metagenomic sequencing, which is time-consuming and expensive, despite a significant cost reduction in recent years. For instance, the development of digital PCR-based assays for the quantification of specific microbial gene copy number or gene expression will reduce test times and make the analysis more accessible for patients and health care practitioners.

As an example, dysbiosis of the gut microbiome and impaired microbiota enzymatic activity has been observed in patients with IBD¹⁰⁹ and IBS.¹¹⁰ BA dysmetabolism, often observed in patients with inflammatory disorders, is commonly associated with a deficiency in bile salt hydrolase (BSH) gene derived from the Firmicute phylum,¹¹¹ Therefore, supplementing diet with a probiotic delivering high BSH activity may be a suitable solution for patients suffering from these maladies. As well, lab tests that are able to accurately detect GI tract BSH activity, gene expression or gene copy number may prove useful to determine who may benefit most from such a treatment. Further research in the field is well supported based on the available evidence.

Disclosure of Potential Conflicts of Interest

No potential conflict of interest was disclosed.

10.4161/gmic.29810