DE LA ACCIÓN DE PETICIÓN DE HERENCIA

Development of high throughput sequencing techniques have allowed probing of the incredibly complex HGM to explore the species composition, where previously it was only possible to identify species that could be cultured (Metzker, 2010). These advances gave way to the Human Microbiome Project (HMP) in 2008 with the aim of characterising the human microbiome, including the gut microbiome (genomes or metagenomes of the HGM). Originally those working on the project believed a core microbiome could be established for healthy individuals, but healthy individuals were found to possess incredibly divergent microbiomes. Three broad enterotypes were established during the MetaHIT study that were defined by variations in the prominence of the three dominant genera, Bacteroides, Prevotella and Ruminococcus (Arumugam et al., 2011), although this can be argued to be an oversimplification of an incredibly complex microbial community (Knights et al., 2014). The MetaHit and HMP studies generally use stool samples to probe the HGM, as this technique is much less invasive than endoscopy. Stool samples, however, may only give an idea of which species are present at the distal end of the colon. Animal studies using humanised microbiota have shown phylogenic diversity between the early and late gastro-intestinal (GI) tract compared with mid GI tract, where diversity was found to drop (Gu et al., 2013). Differences are believed to be due to variations in oxygen along the GI tract, where obligate anaerobes were found in locations

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more likely to provide anaerobic conditions (Gu et al., 2013). Similar data have shown microbe diversity at different locations in the GI tract of pigs, where the early GI tract was found to be Firmicute rich while the late GI tract showed dominance of Bacteroidetes (Kim and Isaacson, 2015). Sequencing of 16S ribosomal DNA was deployed to probe this vast population, results of which showed incredible diversity at the species level with 1000s of species present, while at the phylum level Bacteroidetes and Firmicutes represent 90% of the HGM (Figure 1.12) (Qin et al., 2010). Despite greater diversity at the species level 25% of gut microbial species belong to the Bacteroides genera, a significant proportion of this microbial community (Martens et al., 2009). The genus Bacteroides belongs to the phyla Bacteroidetes and includes over 20 distinct species. Although most species of Bacteroides are considered commensal under some conditions Bacteroides fragilis can become pathogenic. Bacteroides spp are anaerobic rod-shaped, bile-resistant, non-sporulating, gram-negative bacteria typically found in the gut (Wexler, 2007). Sequenced in 2003 (Xu et al., 2003) and 2005 (Cerdeno-Tarraga et al., 2005) respectively, B. thetaiotaomicron and B. fragilis both have a relatively low gene content for size of the genome, indicting a large number of high molecular weight proteins are expressed (Wexler, 2007). Bacteroides spp begin to populate the human gut approximately 10 days after birth, although Bacteroides spp are more prominent post-weaning or in infants which were not breast fed (Simon and Gorbach, 1986; Mackie et al., 1999). Products of Bacteroides metabolism, primarily SCFAs, provide a significant contribution to the daily energy requirements of the host (Hooper et al., 2002).

Another genus associated with positive health is Bifidobacterium. Belonging to the diverse phyla Actinobacteria, Bifidobacterium spp are gram-positive, non-motile, non-filamentous, Y-shaped bacteria without the ability to form a capsule (Barka et al., 2016). Interestingly, the Y-shaped cells are only maintained in clinical isolates from the gut, when cultured in vitro however, the cell revert to a rod shape (Barka et al., 2016). Bifidobacterium spp have been shown to exert antimicrobial activity by competitive exclusion while also adhering to the intestinal wall or mucus layer of the gut (Ouwehand et al., 2002).

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Although each individual host has a unique bacterial population, trends have been identified that are associated with particular host diets or phenotypes (Qin et al., 2010; Tremaroli and Bäckhed, 2012). Experiments investigating differences in gut microbe composition between genetically obese mice (ob/ob) and their lean littermates have revealed an association with increased Firmicute to Bacteroidete ratio in the obese phenotype (Ley et al., 2005), an observation which is mirrored in human studies (Ley et al., 2006). Further, in murine investigations have revealed genetically obese mice possess a greater intestinal SCFA concentration and reduced energy content of faecal matter than lean mice on the same diet, implying more efficient gut microbe composition in the obese phenotype. Metagenomic studies of the gut microbiota of obese mice demonstrated a greater capacity for glycan degradation and utilisation, which again is mirrored in studies on human gut microbes. Transplantation of the obese gut microbes into lean mice showed a two-fold increase in weight gain than lean mice given microbes from lean donors (Turnbaugh et al., 2006). Interestingly, germ-free mice, without any gut microbes, require up to 30% more nutrients than littermates with normal gut microbiota to grow at the same rate (Gilmore and Ferretti, 2003).

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Figure 1.12 Diversity of phyla and genera within the HGM of healthy individuals. The box plot shows

the thirty most abundant genera, while the inset shows abundance at the phylum level. Genus and phylum level abundances were calculated using reference genome based mapping using 85% and 65% cutoffs, respectively. Taken from Arumugan et al., (2011).

Host diet plays a major role in shaping the gut microbiota composition (Tremaroli and Bäckhed, 2012). A recent study contrasted the microbial composition from faecal samples of children from Africa and Italy. The African diet included high amounts of plant polysaccharides correlating with an increased Bacteroidetes to Firmicutes ratio in faecal samples, with Prevotella species being

particularly enriched. The Italian children gut microbiota showed higher levels of

Enterobacteriaceae. The African gut microbiota had adapted to maximise energy yield from the polysaccharide rich diet, selecting for species with greater glycan utilisation capacity, in this case Prevotella and other Bacteroidetes (Turnbaugh et al., 2009).

Studies in which participant diets are supplemented with resistant or non-fermentable starch have shown significant microbiota change in which Eubacterium rectale, Oscillobacter spp and

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Ruminococcus bromii were enriched (Walker et al., 2011). Each of these have been shown to utilise resistant starch. Bi. adolescentis also dramatically increased (Martinez et al., 2010), consistent with its ability utilise starch (Duranti et al., 2014). Drastic changes in HGM composition only seems possible with long-term dietary changes, as a study that monitored microbial composition as a result of dietary intervention showed no significant change during the 10 day experiment (Wu et al., 2011). The investigators did observe a selective increase for Bifidobacterium spp when daily doses of inulin, a prebiotic, were administered (Wu et al., 2011). In genetically obese mice enrichment for

Bifidobacterium spp correlated with reduced adiposity and a reduction in lipopolysaccharide, a known microbial-derived inflammatory molecule, when compared to ob/ob mice on the same diet without prebiotics (Cani et al., 2007). Interestingly, only a small number of species in the HGM appear to be effected by inulin dietary supplementation, and these organisms are in the genera Bifidobacterium and Atopobium (Costabile et al., 2010). A wider range of species were enriched when participants were supplemented with FOS rather than inulin. In these studies, Bifidobacterium and Bacteroides spp are particularly abundant post intervention, while Faecalibacterium prausnizii and Roseburia intestinalis were reduced in abundance (Benus et al., 2010).

Unlike dietary fibre, fat appears to have an indirect effect on HGM by modulating bile acid secretion and composition. Interestingly, the Bacteroides enterotype (defined in the MetaHIT study) positively correlates with intake of saturated fat (Wu et al., 2011). Polyunsaturated fats were found to affect adherence of gut microbes to the intestinal wall (Kankaanpaa et al., 2001), leading to a reduced presence of Bacteroides, E. rectale/Clostridium coccoides group and Bifidobacterium (Cani et al., 2007). Dietary proteins which reach the intestine are metabolised by microbial proteolysis or gut fermentation, generating gasses and SCFAs (Russell et al., 2011). Over a six week dietary

intervention, participants with high-protein low-carbohydrate diet were found to have reduced Bifidobacterium spp specifically and total bacterial abundance in the gut (Duncan et al., 2007; Brinkworth et al., 2009), potentially increasing the risk of infection with pathogenic bacteria, due to reduced competition from the HGM.

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