Role of the Gut Microbiota in Health and Disease

The gastrointestinal (GI) tract is colonized by a dense community of commensal microorganisms referred to as the gut microbiota [1]. Microbiota refers to the entire population of microorganisms that colonizes a particular location; and includes not just bacteria, but also other microbes such as fungi, archaea, viruses, and protozoans [2]. The gastrointestinal tract (GIT) is the most densely colonized organ in the humans, with the colon containing approximately 70% of all the microbes in the body [2]. Microbial colonization begins rapidly at birth, and during the first year of life shows large interindividual variations [3]. This initial gut colonization, influenced by numerous internal and external host-related factors, is instrumental in shaping the composition of the adult’s gut microbiota. The balance between the gut microbiota and host mechanisms has an important impact and influence on the attainment, development and stabilisation of the gut ecosystem [3]. There have been over 50 bacterial phyla described to date [4], and the human gut microbiota is dominated by only 2 of them: the Bacteroidetes and the Firmicutes, whereas Proteobacteria, Verrucomicrobia, Actinobacteria, Fusobacteria, and Cyanobacteria are present in minor proportions [5]. Both microbial density and diversity increase from the proximal to the distal gut, with individuals harbouring more than 1000 microbial, species-level phylotypes [6].

There has been significant research on the role of the gut microbiota in health and disease, demonstrating its involvement in human metabolism, immunomodulation and host protection [6]. The gut microbiota is sometimes referred to as the ‘hidden’ metabolic organ due to its abundance of functions and symbiotic relationship with the human host. The gut bacteria are involved in the production of a number of vitamins and amino acid synthesis, as well as the biotransformation of bile [7]. Biotransformation of bile by microbial enzymes is important for the metabolism of glucose and cholesterol [8]. In addition, the microbiome provides the much needed biochemical pathways for the fermentation of non-digestible substrates like fibers and endogenous mucus, promoting the growth of these microbes and the production of short chain fatty acids (SCFA) and gases. Bacterial fermentation takes place in the cecum and colon, where the SCFA are absorbed, stimulating the absorption of salts and water [7]. Acetate, propionate, and butyrate are the major SCFA produced, and are an important energy source for intestinal mucosa and critical for modulating immune responses and tumorigenesis in the gut [9].

The intestinal structure and function of the gut is ensured by the microbiota present within. The mucus layer present in the intestine plays an important role in the inhibition of pro-inflammatory mediators and preventing the uptake of antigens [10]. Additional protective features include the production of antimicrobial compounds by the intestinal bacteria, or what is otherwise known as the barrier effect. This important function results in competition for nutrients and sites of attachment in the gut lining preventing colonization by pathogens and enteroinvasive bacteria. Studies on germ-free animals [2] have revealed the critical importance of gut microbiota in the development of intestinal mucosa and the systemic immune systems. The intestinal epithelium is the main interface between the immune system and the external environment, and has a number of key functions in tailoring immune development of the host. Gut microorganisms induce the development of isolated lymphoid follicles (ILFs) which can function as sites for induction of mucosal immune response. Microorganisms also modulate immune cell differentiation, and regulate the production of cytokines and chemokines which influence the range of T cell activation processes both in the intestine and surrounding tissue [11].

Under normal physiologic conditions the host and its commensal microbes have a symbiotic relationship. The microbiota receives nutritional upkeep from the host while providing support in the form of intestinal homeostatic maintenance. However, in some cases the bacteria present in the GIT can acquire virulence and contribute to the development of a number of diseases. ‘Dysbiosis’ is the term used to describe a microbial ecosystem where the bacteria do not live in mutual accord, reflecting microbial imbalance or maladaptation. It is understood that this disturbance of intestinal microbial communities is caused by both intrinsic factors such as stress and genetics, as well as extrinsic factors such as diet and antibiotic use. It is clear that there is a multitude of potential factors in the pathophysiology of GI diseases, with certain individuals and populations at an increased risk of predisposed development. For example lifestyle has a strong influence, with the typical Western diet, associated with the consumption of high fat and sugar foods, implicated in dysbiotic events including endotoxaemia [12]. Interestingly, a recent study revealed that a Mediterranean diet, typically incorporating plant based foods and whole grains, had a beneficial effect in Crohn’s disease patients [13]. It may be the case that the consumption of high amounts of fat and sugar in the diet is a disturbance factor causing diseases in genetically susceptible hosts. The use of antibiotics is another disturbance factor, and may decrease natural defence mechanisms or cause an overabundance of pathogenic species leading to infectious diseases [14].

Chronic, relapsing inflammation of the GI tract is known as inflammatory bowel disease (IBD) and comprises of two separate but interrelated conditions known as Crohn’s disease (CD) and ulcerative colitis (UC). IBD is a worldwide healthcare problem, with an increase in the incidence of childhood-onset along with the diseases’ longevity posing future concerns [15]. The causes of IBD appear to be multifactorial, integrating the gut microbiota, host genetics, and the immune system as factors determining predisposition to disease. It has been shown that the diversity and richness of the gut microbiota are significantly reduced in patients with IBD, and that the accumulation of pathobionts has been shown in both human and animal IBD instances [16] [17]. A pathobiont is essentially a ‘pathogenic symbiont’ of the microbiota, and under normal conditions functions in a mutually beneficial relationship with the host. Pathobionts exert specific effects on the host’s mucosal immune system and are associated with the development of clinical disease. A recent review investigating pathobionts in the GI microbiota revealed that the combination of an immunocompromised state with colonization by pathobionts may be a risk factor in IBD, colon cancer and perhaps for diseases outside of the intestinal compartment [18]. This shift in homeostatic healthy flora to detrimental proinflammatory microbial species is referred to as the aforementioned dysbiosis, and is one of the key factors in the pathogenesis of inflammatory intestinal diseases. Improvements in DNA sequencing and applications have enabled investigations into the biodiversity of the IBD gut microbiome, with patterns revealing a reduction in the abundance and complexity of the anaerobic bacteroides (bacteroidetes phylum), eubacterium, and lactobacillus species (firmicutes phylum) associated with increased intestinal inflammation [19]. In the firmicutes population a decrease in the clostridium leptum groups, especially the beneficial commensal bacteria Faecalibacterium prausnitzii (FP) has been observed [20]. FP is is one of the most abundant anaerobic bacteria in the human gut and has strong anti-inflammatory effects, with results from one study revealing its potential in combating bacterial dysbiosis in CD patients [21]. Clostridium and bacteroiodes species are the main producers of SCFAs in the human colon. Among these SCFAs that are produced during carbohydrate fermentation, butyrate has key anti-inflammatory roles and serves as a major source of energy for colonic epithelial cells [22]. Therefore a reduction in butyrate production could be involved in the increased inflammatory characteristics of IBD, with butyrate at present considered at present to be of therapeutic value to IBD patients. A study by Frank et al. using 16S rRNA sequencing revealed an increase in the less dominant Proteobacteria and Actinobacteria phyla in mucosal biopsies taken from CD and UC patients compared to non-IBD controls. Additionally no differences were observed in the fecal and microbial bacterial population numbers between UC and CD patients [23]. Considering the diversity of IBD mucosal legions and disease course, no single pathogen can be isolated from the disease tissue and therefore insufficient evidence abounds that a single pathogen is the root cause of the disease. At the Enterobacteriaceae genus level, Escherichia coli was shown to be found in ileal mucosal lesions of CD patients, with this adherent-invasive bacterium less invasive in UC patients [24]. A second group of adherent and invasive bacteria is the Fusobacteria. The genus Fusobacterium spp. is principally found in the oral cavity but can also inhabit the gut, and has been found to be higher in abundance in the colonic mucosa of patients with UC relative to control individuals [25]. A study by Strauss et al. revealed that the invasive ability of Fusobacterium has been shown to correlate positively with the IBD status of the host [26]. These results illustrate the potential of this bacterial species in the pathogenesis of IBD. Indeed, Fusobacterium species were recently shown to be enriched in tumor versus non-involved adjacent tissue in colorectal cancer [27]. One of the highest risk factors for the development of colorectal cancer is IBD, therefore fusobacteria may potentiate a connection between these common and debilitating diseases.

Colorectal cancer (CRC) is the third most commonly diagnosed cancer in both men and women, with 1 in 22 men and 1 in 24 women estimated to be diagnosed with this type of cancer during the course of their lifetime. Although the incidence of gastric carcinogenesis has been directly and strongly linked to certain pathogens such as Helicobacter pylori (H. pylori) [28], possible infectious causes associated with CRC remain controversial. Similar to the incidence of IBD, CRC is characterised by a state of dysbiosis in the intestinal epithelium. Recent advances in metagenomics technologies have suggested evidence linking the imbalance in the diversity of intestinal microbiota promoting inflammatory conditions and the production of carcinogenic metabolites, leading to the abnormal state of neoplasia [29]. Studies in mice with altered immune and inflammatory responses suggest that dysbiosis could be sufficient to promote cancer [30], making it thus likely that the immune system is a key factor in the interactions between the gut microbiota and CRC. It is essentially unknown whether dysbiosis is a cause or occurs as a consequence of CRC. It may be the case that interactions between different bacterial strains in the host-compromised microbial state leads to pro-carcinogenic affects and the eventual development of adenocarcinomas. The microenvironment that characterizes CRC may on the other hand favour the growth of certain bacterial species that have potential carcinogenic effects and amplify the concurrent state of dysbiosis. Regardless of whether intestinal dysbiosis is as a cause or exacerbated effect of CRC, 16S ribosomal RNA (16S rRNA) sequencing of bacteria from stool or digestive tissues have shown colonic dysbiosis in patients presenting with CRC [31]. The first bacterium indirectly associated with CRC is Streptococcus bovis (S.bovis), with recent work by Abdulamir et al [32] showing increased levels of this pathogen in colorectal adenomas and CRC tissue, suggesting its potential involvement in colorectal carcinogenesis. Although as previously mentioned H. pylori has been strongly linked to gastric carcinogenesis, recent work has suggested the role of this group 1 human carcinogen in colon cancer progression. Using a specific 16S rDNA polymerase chain reaction (PCR) assay and pyrosequencing revealed the presence of high H. pylori levels in CRC tissue [33]. The facultative anaerobe Enterococcus faecalis (E. faecalis) has recently been classified as a human pathogen [34]. A study by Balamurugan et al. performing 16S rRNA real-time PCR reported higher levels of fecal E. faecalis levels in CRC patients compared to healthy controls [35]. There is strong evidence linking Fusarium nucleatum (F. Nucleatum) colonization in colorectal adenomas [36], and this may suggest that this anaerobic gram-negative bacterium is not only associated with CRC but likely plays a key role in the early steps of colorectal carcinogenesis promotion. Various studies have suggested a clear link between E.coli, a commensal bacteria of the human microbiota, and CRC [37]. This suggests that E. Coli may be a potential causal link in CRC progression. Other bacterial species have been researched and implicated for their roles in colorectal carcinogenesis. These include Bacteroides fragilis (B. fragilis) [38] and Clostridium septicum (C. septicum) [39]. In summary various alterations to the gut microbiota can result in reduced diversity and an increase in potential pathogenic bacterial species, eventually leading to the development of CRC. Due to recent advances in metagenomics technologies our understanding of the effects of these invasive species has drastically improved, helping to characterise the state of dysbiosis commonly associated with carcinogenic progression.

As previously discussed GI diseases are associated with a state of bacterial dysbiosis, reflecting microbial imbalance or maladaptation due to a number of intrinsic and extrinsic factors related to the host. Manipulation of the intestinal microbiota is an option in treating gut dysbiosis by reducing and potentially removing many of the symptoms associated with the disease states, thus restoring health. The concept of probiotics has been defined by the World Health Organization as ‘live microorganisms which when administered in adequate amounts confer a health benefit on the host’ [40]. For a probiotic to be beneficial in the gut microbiota of the host it must be capable of surviving passage through the intestinal environment, which involves the exposure to various gastric juices as well as hepatic bile [41]. The lactic acid bacteria – specifically the Lactobacillus and Bifidobacterium strains are the most commonly used in commercial products and have been the most widely researched, with mixtures common as researchers unearth the potential of improved efficacy by synergistic mechanisms. Specifically, in vitro studies have suggested probiotic bacteria to have a role in supressing the growth of microbial pathogens by stimulating the production of antimicrobial factors [42]. In terms of inflammation which characterises states of dysbiosis, probiotics have been shown to stimulate the production of anti-inflammatory cytokines [43] as well as suppress the production of pro-inflammatory mediators [44]. It is the Bifidobacterium genus that has been shown to have the greatest benefits in IBD disease states, influencing the host immune-system function and reducing inflammation in animal models [45]. A limitation is that the majority of the studies showing positive research outcomes have been performed in in vitro and animal models and have not been translated into clinically relevant outcomes in humans.

Prebiotics are another approach used to modulate the gut microbiota. The term ‘prebiotic’ was introduced by Roberfroid [46] and defined as ‘a selectively fermented ingredient that allows specific changes, both in the composition of and/or activity in the gastrointestinal microflora that confers benefits upon a host’s well-being and health’. While probiotics introduce beneficial bacteria in the intestinal environment, prebiotics act as a growth stimulant for these beneficial bacteria which include the aforementioned lactic acid bacterial species. Essentially prebiotics are a specialized plant fibres that are degraded by microbial fermentation to a mixture of gasses and the previously described SCFAs, including butyrate which has key anti-inflammatory roles and serves as a major source of energy for colonic epithelial cells [22]. Inulin, consisting of a long chain of fructose polymers, has been shown in in vitro and in vivo studies to have growth promoting effects on both bifidobacteria and lactobacillus [47]. Galactooligosaccharides (GOSs) are composed of between 2 and 8 galactose residues, and are commercially produced from lactose. Human in vivo studies have shown that supplementation with GOS resulted in increases in both beneficial bifidobacteria [48] and lactobacilli, as well as decreases in potentially harmful Candida [49]. There is strong evidence to indicate that both probiotics and prebiotics have important functions in modulating the intestinal microbiota. When they are are administered simultaneously it is referred to a synbiotic. The reasoning is that the prebiotic component improves the survival of the probiotic bacteria and stimulates the activity of beneficial bacteria inherent to the host, as has been shown with bifidobacterial numbers [50].

It is clear that the gut microbiota has a number of essential functions in host nutrient metabolism, immunomodulation and protection against pathogens. Imbalance of the normal gut microbiota is associated with a number of host-specific and environmental factors and has been linked to gastrointestinal disorders such as inflammatory bowel disease as well as colorectal cancer. There are a number of approaches to potentially modulate the microbiome present in the gut including the use of prebiotics, probiotics and synbiotics. While these approaches have shown limited effectiveness in reducing the incidence of the more debilitating gastrointestinal diseases outlined, the benefits shown to date from the larger randomized controlled trials are still useful adjuncts in therapy.