State-of-the-Art Lecture: Beyond the Bugs: What Makes the Gut (and Host) Healthy
Melbourne Veterinary School, University of Melbourne, Werribee, Melbourne, VIC, Australia
There has been a lot of focus on the interaction between mammalian hosts and their intestinal bacteria in recent years. It has been elucidated that the relationship between host and bacteria is a complex one, and that bacteria are essential for maintaining host health. However, focusing on just this interaction, this neglects the other components of the microbiome and the fecal components themselves.
Because the GI tract is a primary site of exposure of host organism to microbes, there is development of extensive crosstalk between the host and the luminal environment of the intestine. The other components of the intestinal microbiome (viruses, archaea, fungi) and the intestinal milieu (e.g. bile acids), also interact with the immune system. There is evidence that they can act in a beneficial manner to the host (by counteracting intestinal inflammation), maintain normal homeostasis or at times be deleterious.
Viruses outnumber bacteria 10:1 in most environments, but viral DNA usually only represents 2–5% of the total DNA in a microbial community. Until recently, it has been difficult to determine the full viral fingerprint in a community as most viruses are unable to be cultured, and they lack a consistent genetic fingerprint like the 16S ribosome that is common between bacteria. Advances in viral enrichment protocols, sequencing techniques and bioinformatic pathways mean that recently the GI virome is beginning to be documented in many communities and species, including the intestinal tract of cats and dogs.
The intestinal virome consists of eukaryotic viruses that can infect host cells, endogenous retroviruses and prokaryotic viruses (phages) that infect bacteria, archae and fungi. Most viral sequence data isolated from human faeces is unmatched in databases, with most unreadable sequences being phages. The predicted hosts of the phage populations coincided with the bacterial groups detected by 16S ribosomal RNA gene analysis in the same samples.
The viral genome appears to be unique to individuals, with little to no similarity between individuals, minimal clustering in households and with long-term stability. In a study of twins, the viral gut population remained stable and specific to the individual, with no clustering observed between twins or their mother, which contrasts with bacterial populations that tend to cluster within families. This is potentially one explanation for why there is variable disease expression of Crohn’s disease in monozygotic twins despite identical genetic and environmental factors being present.
Eukaryotic viruses have been identified in the feces of healthy dogs and cats, as well as cats in shelters. None of these eukaryotic viruses were associated with clinical disease. A further study of dogs with chronic enteropathy (inflammatory bowel disease) also identified eukaryotic viruses in affected dogs (Kobuvirus, astrovirus), but a wider prevalence study didn’t show any association with disease. The same set of studies also showed a higher number of bacteriophages in dogs with chronic enteropathy than healthy dogs, or dogs in shelters with acute diarrhea.
A landmark study showed that a eukaryotic virus, norovirus, may exert beneficial effects on the intestine. Germ-free mice were infected with norovirus, and vertical transmission was then allowed to occur. The presence of norovirus was associated with a reduction in intestinal inflammation that normally spontaneously occurs in germ-free mice, as well as following induction of colitis by dextran sodium sulphate or antibiotic administration. Although there were quantitative differences in the inflammation between strains, the overall effect was similar.
Phages are a very interesting area of future study, as they are the most abundant viral organisms in the gut, with very little known about their physiological function. There has recently been proposed a theory that the presence of phage within the mucus may also confer a protective effect. One in vitro study showed that phage abundance is dependent on mucus, and increased phage numbers protected the underlying epithelium from bacterial infection, independent of other factors. The enrichment of phage in mucus is via interaction between mucus glycoproteins and Ig-like protein domains that are exposed on phage capsids. Phages may have a beneficial effect on the development of strains of bacteria and be protective against pathogenic strains of bacteria. As a result, there may be the potential for phage therapy in the future of inflammatory bowel disease (IBD). The absence of phage in probiotics, as well as genetic variation, may be a possible reason for different responses in different studies to probiotics in CD. Similarly, the success of fecal transplantation for treatment of chronic Clostridium difficile infection may be in part due to the presence of phage.
Archaea and Fungi
Compared to bacteria and even viruses, the role of archaea and fungi in the normal intestinal homeostasis is poorly understood.
Of the two groups, fungi have probably been the most studied and are thought to contribute 0.1% of the GI microbiome. The most common fungi in the human gut is the Candida genus, and the mycobiome is greatly influenced by the environment and diet. Rodent models have shown that fungal dysbiosis can influence local and systemic inflammation and immune modulation. There is a small amount of evidence that fungi play a role in the development of chronic intestinal inflammation directly, although a competitive relationship with the resident bacteria is likely. More interestingly, it has been shown that when fungal overgrowth occurs in the gut secondary to antibiotic administration, airway sensitivity to mold spores occurs. Thus, the mycobiome may prime distant mucosal sites to disease. Archaea are unicellular organisms that unique ribosomal sub-units to be classified as a separate life domain. Methanogens are probably the best-known example of this domain, and are strict anaerobes found in many environmental samples as well as the intestine. Generally, archaea are resistant to most antibiotics, but their ubiquitous nature in the gut is only recently being explored, again due to the development of non-culture techniques such as fluorescent in situ hybridization (FISH) or metagenomic studies. Despite little being known about this group, they account for 10% of the anaerobic bacteria in the human gut and are vital to facilitate digestion, particularly of carbohydrates. There is also evidence that diversity of this group decreases with age, but it is unclear whether this is associated with a functional change. An emerging field of investigation is their use as probiotics to improve cardiovascular health in people; this is via reducing plasma triethylamine-N-oxide levels. However, there is also some evidence that methanogens are associated with constipation and inflammatory bowel disease in people. Whether these changes are opportunistic, or causative is unknown at this stage.
Bile Acids and Other Fecal Metabolites
To add to the overall study of the normal gut, the term ‘sterolbiome’ has been coined to describe the production of bile acids (BA) in the gut by the microbiome. Bile acids and the microbiome have bidirectional impact on each other. Primary bile acids are produced by the liver, and then modified by bacterial enzymes (bile salt hydrolases [BSH]) within the gut. Metagenomic studies have shown that BSH are produced in people predominantly by Firmicutes, Bacteroides and Actinobacteria, as well as methanogenic archaea. Secondary BA (namely deoxycholic and lithocholic acid) are produced in the gut by some specific clostridial species. The role of bile acids is increasingly being studied. In people with chronic Clostridia difficile associated diarrhea, secondary BA are down-regulated, whilst primary BA are up-regulated. Bench-top studies have shown that primary BA promote germination of C. difficile spores, whilst secondary BA suppress production of spores. The reduction in secondary BA with antimicrobial treatment, and lack of secondary BA in probiotic treatments is another compelling explanation for the success of fecal microbial transplantation in the treatment of C. difficile associated diarrhea. However, not all actions of secondary BA are as positive. A western diet is associated with an increase in both deoxycholic acid (DCA) and lithocholic acid (LCA), and higher serum concentrations of DCA and LCA have been identified in people with colon cancer. The mechanism by which this may occur is uncertain, but it appears that pro-inflammatory pathways (particularly COX-2) are stimulated. Bile acids can directly modulate the gut microbiota directly or indirectly. Therefore, it appears that host metabolism can be affected by microbiome manipulation of bile acids, particularly through modulation of FXR and TGR5 receptors. However, studies to date have shown massive differences between species, meaning that it will be difficult to directly extrapolate other studies. One study of dogs with chronic enteropathy identified low secondary BA in dogs with active CE and an increase following treatment. Whether this is a direct result of disease or associated with diet is uncertain, as diet has been shown to affect fecal BA concentrations. Other fecal metabolites that may have an impact on gut (and host) health and disease include catecholamines, short-chain fatty acids, amino acids and steroid hormones.
The synergy and complex interaction between the complex fecal environment and the host is a rich area of potential research for intestinal and systemic immune disease. The gut is now considered to be an endocrine and nervous system organ—one that has the potential to influence host health and immunity.
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