document

© CAB International 2014. The Human Microbiota and Microbiome (ed. J.R. Marchesi) 1

1 The Stomach and Small and Large Intestinal Microbiomes

Christian U. Riedel ,1 Andreas Schwiertz2 and Markus Egert3*1University of Ulm, Ulm, Germany; 2Institute of Microecology, Herborn, Germany; 3Hochschule Furtwangen University, Campus Villingen-Schwenningen, Germany

1.1 Introduction

This introductory chapter provides an updated overview on the composition of the micro-biome in the human gastrointestinal tract (GIT); that is, the microbiota of the GIT together with its entire genetic information and the microbe–microbe and host–microbe interactions taking place in this habitat. More specifi cally, recent scientifi c advances on the microbiome of the upper (stomach and duodenum) and lower GIT (jejunum, ileum, caecum, colon, rectum), particularly of healthy adults, will be discussed. However, where necessary, some studies performed with diseased patients or animal models will also be presented and integrated into the state-of-the-art-knowledge about the human GIT microbiome. In addition, an update on factors shaping the composition of the GIT micro-biome will be given. For a more functional or physiological discussion of the human intestinal microbiome, the reader is referred to Chapter 6, this volume. The structure and function of the microbiome of the uppermost part of the human digestive system, i.e. the oral cavity, are presented and discussed in Chapter 2 of this volume.

From a microbiological point of view, the human GIT can be regarded as the best

investigated ecological niche of the human body, although some diffi culties exist in obtaining representative samples from various parts of the GIT. Moreover, the human GIT probably represents one of the best investigated microbial ecosystems on earth. This fact can be explained due to the great importance of the GIT microbiota in maintaining and driving human health, disease and well-being: on a quantitative basis, humans can be regarded as a super-organism, consisting of 90% microbial cells and even 99% microbial genes, and the vast majority of the microbial diversity is located in the human GIT (Wilson, 2008). Con-sequently, the general importance of the GIT microbiome for human health and disease regarding digestion and general metabolism, gut development or immune status is un-doubted. Hence, a wealth of literature on the human GIT micobiome is already available, including several current and comprehensive review articles and reviewing book chapters (Wilson, 2008; Doré and Corthier, 2010; Marchesi, 2010; Gerritsen et al., 2011; Walter and Ley, 2011; Willing and Jansson, 2011). For a complementary overview including some of the more classical literature about the human GIT microbiome, the reader is referred to these articles.

*[email protected]

2 C.U. Riedel et al.

1.2 The Microbiota of the Human Stomach

1.2.1 Environmental conditions

The human stomach (Fig. 1.1) is a J-shaped structure with a volume of approximately 1.5 l. It can be diff erentiated into an upper part (fundus), the main body (corpus) and a lower part (antrum), which is connected to the duodenum part of the small intestine via the pyloric sphincter. The folded stomach epithelium is covered by a protective mucus layer of up to 600 μm thickness. The main functions of the human stomach are temporary food storage, mixture of food and gastric juice to chyme, pre-digestion of proteins by acidic pH and pepsin, and disinfection of the ingested food. The environmental conditions in the stomach are eutrophic – due to ingested food, mucus, desquamated epithelial cells and dead microbes – aerobic and acidic, with a more or less constant temperature of 37°C, i.e. the body temperature of the host. Pronounced daily fl uctuations in temperature, pH (from pH 1 to pH 5) and available nutrients are common and linked to ingestions of food and beverages. Bacterial viable counts are strongly

dependent on the actual gastric pH and range from 103 to 106/ml (Wilson, 2008; Walter and Ley, 2011).

1.2.2 Composition of the stomach microbiota

Data on the human stomach microbiome are usually collected by investigating biopsies, taken endoscopically after several of hours of fasting. Despite the harsh and antimicrobial environment, recent molecular diversity studies – in particular the widely cited study by Bik and co-workers – have shown, sur-prisingly, that the human stomach contains a diverse, unevenly distributed microbial community dominated by Proteobacteria, Firmicutes, Bacteroidetes and Actinobacteria (Bik et al., 2006). In endoscopic biopsies taken from 23 North American patients with symptomatic upper gastrointestinal disease, they identifi ed 128 phylotypes from 8 phyla by a 16S rRNA gene clone library approach. Several more recent studies corroborated that a remarkable diversity of bacterial genes could be amplifi ed and identifi ed from the human stomach (Andersson et al., 2008; Dicksved et al., 2009; Li et al., 2009;

Current knowledge

• remarkable undisputed bacterial diversity: 7–13 phyla, >> 100 phylotypes

• presence of Helicobacter sp. dramatically reduces bacterial diversity

• key genera: Helicobacter, Streptococcus, Prevotella

Key questions

• differentiation of truly resident from transient, i.e. food-, mouth- or oesophagus-derived species

• functional relevance of the resident microbiota (other than Helicobacter sp.)

• effect of presence/absence of Helicobacter sp., diet, ethnicity and gastric diseases (cancer) on community composition

Oesophagus

Small intestine

Fundus

Corpus

Antrum

Pyloric sphincter

Lower oesophagussphincter

Fig. 1.1. Current knowledge and key questions regarding the microbial ecology of the human stomach.

Stomach and Intestinal Microbiomes 3

Maldonado-Contreras et al., 2011). While investigating ten Helicobacter pylori-free patients with a Chinese background, Li and co-workers quite clearly corroborated several key fi ndings of the American-based study of Bik and colleagues (Li et al., 2009). With respect to the total number of detected phylotypes (133 versus 127), the number of phyla (8 versus 7) and the most abundant two genera (Strepto coccus and Prevotella), both studies yielded strikingly similar results. Anderson and co-workers even detected 262 phylotypes representing 13 phyla in biopsies of the stomach of three H. pylori negative patients with peptic ulcers (Andersson et al., 2008). As a consequence, the human stomach can no longer be considered a mono-associated environment.

In the thick mucous layer overlying the gastric epithelium, non-acidophilic bacteria can also be found, in particular H. pylori. When present, H. pylori usually dominates the stomach bacterial community (Andersson et al., 2008). So far, H. pylori is the only bacterium of the human stomach that can be considered unambiguously as a true resident and is considered to contribute to the development of gastritis, peptic ulcers and even gastric cancer (Dorer et al., 2009).

Several recent studies have tried to unravel correlations between the com-position of the microbial community in the stomach and the H. pylori status of patients. In a study by Maldonado-Contreras and colleagues, which was focused on patients from developing countries, a positive H. pylori status was correlated with increased relative abundances of (non-Helicobacter) Proteo bacteria, Spirochetes and Acidobacteria, while Actinobacteria, Bacteroidetes and Firmicutes were less abundant (Maldonado-Contreras et al., 2011). However, the study also showed that ethnicity had a stronger impact on the stomach community com-position than the H. plyori status. Focusing on H. pylori negative patients, Li et al. detected signifi cantly higher abundances of Firmicutes, in particular Streptococcus spp., in the stomach mucosa of patients with antral gastritis (Li et al., 2009). Interestingly, Strepto coccus spp., together with bacteria of the genera Lactobacillus, Veillonella and Prevotella, were

also abundant members of the stomach community in a study on patients with gastric cancer and a low H. pylori abundance (Dicksved et al., 2009). However, no stat-istically signifi cant diff erences were found between the stomach community of cancer and non-cancer patients.

1.2.3 Resident or transient microbiota?

Approximately 1010 microorganisms enter the human stomach every day. As a consequence, a clear diff erentiation of truly resident from just transient (swallowed) microbial species is diffi cult. Indeed, the majority of the 33 phylotypes identifi ed in the stomach of all three patients investigated by Andersson et al. were affi liated with the genera Streptococcus, Actinomyces, Prevotella and Gemella, which were also abundant in the throat community (Andersson et al., 2008). However, streptococci were shown to survive in the stomach and to adhere tightly to the mucosa, suggesting they might truly rep-resent resident stomach species (Li et al., 2009). Acid tolerance is clearly a prerequisite for (even just transient) microbial survival in the stomach lumen, and this is why particularly acid-tolerant streptococci, lacto-bacilli, staphylococci and Neisseria spp. have frequently been found in the stomach lumen. It was suggested that some of these bacteria be investigated in more detail for potentially benefi cial (probiotic) properties (Ryan et al., 2008). A similar suggestion was recently also put forward for propionibacteria: Delgado and co-workers cultured propionibacteria – mostly affi liated with P. acnes, but devoid of any clear pathogenic properties – from gastric mucosa samples of 8 out of 12 healthy patients and proposed them as true residents of the human stomach (Delgado et al., 2011).

So far, the functional relevance of the surprisingly high microbial diversity in the human stomach is still largely obscure (Lawson and Coyle, 2010). Its elucidation will require more long-term, dynamics-orientated and comparative analyses of mouth, throat and stomach communities and linking of particular physiological conditions, for example those associated with certain gastric


diseases and/or the presence/absence of H. pylori, with the composition of the micro-biota of the stomach. Eventually, such studies might prepare a basis for the defi nition of novel therapeutic targets (Lawson and Coyle, 2010).

1.3 The Microbiota of the Small Intestine


In the small intestine, the vast majority of food components are digested by mostly host-derived hydrolytic enzymes and sub sequently absorbed by the intestinal mucosa. The small intestine can be divided into three major parts (Fig. 1.2), with a more or less constant diameter (~3 cm) but considerable diff erences in length: i.e. duodenum (~25 cm), jejunum (~1.0 m) and ileum (~2.0 m). The entire epithelium of the small intestine is covered with a thick (up to 250 μm) protective mucus layer, secreted by goblet cells. In order to facilitate digestion and absorption, the surface area of the small intestine is greatly increased to almost 300 m2 by the formation of villi and

microvilli (‘brush border’). On transfer through the pyloric sphincter, chyme from the stomach is mixed with intestinal juice (combined excretion of epithelial cells), pancreatic juice and bile by peristaltic movements. Compared to the large intestine, microbial growth is hampered in the small intestine by relatively short food retention times, antimicrobial peptides secreted by paneth cells and bile salts. However, growth conditions for microorganisms improve towards the end of the small intestine. Consequently, the numbers of luminal micro-organisms increase from approximately 102 ml–1 in the jejunum up to 108 ml–1 in the terminal ileum (Wilson, 2008; Walter and Ley, 2011).

1.3.2 Composition of the small intestinal microbiota

Due to its restricted accessibility, the micro-biota of the human stomach, and particularly of the small intestine, has been investigated much less intensively than that of the mouth and large intestine or faeces. In particular, data on the small intestinal microbiota of

Current knowledge

• dominance of facultative and obligate anaerobes (Streptococcus sp., enterobacteria, Clostridium sp., Bacteroidetes)

• increasing cell numbers, microbial diversity and share of anaerobes from duodenum towards ileum

• significantly lower diversity but higher temporal variability of microbial community compared to colon

• competition for carbohydrates with host

Key questions

• functional relevance of the resident microbiota for the host

• suitability of stoma patients as models due to potential influx of oxygen

• development of appropriate sampling techniques

Stomach

Largeintestine

Duodenum

Jejunum

Ileum

Fig. 1.2. Current knowledge and key questions regarding the microbial ecology of the human small intestine.


healthy individuals are scarce. Until a few years ago, it was common knowledge that the lumen and mucosa of duodenum and jejunum were colonized at low density by only a few microorganisms, including acid-tolerant streptococci and lactobacilli. Towards the end of the ileum, the lumen was described as being dominated by streptococci, entero cocci and coliforms, while in the mucosa, obligate anaerobes (Bacteroides spp., Clostridium spp., Bifi dobacterium spp.) could also be found (Wilson, 2008, and studies cited therein). This knowledge has been broadened during the past few years.

In order to characterize the small intestinal microbiota in more detail by molecular means, Booijink and co-workers investigated the ileal effl uent of patients with so-called Brooke ileostomies, i.e. patients with an ileum ending in an opening of the abdominal wall, mostly because the colon had to be removed due to colon cancer (Booijink et al., 2010). They showed that the small intestine was characterized by a less diverse and temporarily more fl uctuating microbial community than the large intestine (Booijink et al., 2010). Based on community profi les obtained with a phylogenetic micro-array, the average community similarity of four patients over 9 days was just 44%. Notably, no Archaea were detected in the effl uent samples. Although the community of each patient was highly individual, a hypothetical common ‘core microbiota’ was defi ned based on these four patients. It comprised bacteria belonging to the genera Clostridium, Enterococcus, Oxalobacter, Strepto-coccus and Veillonella.

By comparing small intestinal lumen samples obtained from healthy subjects by means of an extended oral catheter with ileal effl uent samples, Zoetendal and co-workers very recently showed that the microbial composition of ileal effl uent might rather resemble the community in the jejunum (Zoetendal et al., 2012). They identifi ed bacteria belonging to the Bacteroidetes, Clostridium cluster XIVa and Proteobacteria as typical for the ileum. In line with previous studies (Booijink et al., 2010), they cor-roborated a lower species diversity and signifi cant tem poral fl uctuations in com-

munity composition, in comparison to the colon or faecal com munity. Additionally, using metagenomic, metatranscriptomic and metabolite profi ling in addition to community profi ling, Zoetendal and colleagues (2012) developed an ecological model of the small intestinal microbiota. They found genes coding for carbohydrate phosphotransferase system (PTS) transport mechanisms, central metabolism and biotin biosynthesis being over-represented in the small intestine. Interestingly, these genes were not only abundantly present in the metagenomic libraries, but also showed high-level in situ expression, as indicated by metatran-scriptomic analysis. Apparently, the small intestine is a habitat where the microbiota has to compete vigorously with the human host for carbohydrates, and consequently micro-organisms that possess rapid uptake and conversion mechanisms of simple carbo-hydrates become enriched.

In a quantitative PCR (qPCR)-based study on the ileal lumen of 17 patients that had to undergo small bowel transplantation, Hartman et al. could show that the ileal community before and after surgical closure of an ileostomy diff ered considerably (Hartman et al., 2009). Before the closure, it was dominated by facultative anaerobes (Lactobacillus spp., enterobacteria), while following the closure it was dominated by obligate anaerobes. They concluded that oxygen penetration into the terminal ileum was responsible for the community shift, thereby questioning the relevance, for healthy individuals, of community data obtained with ileostomy patients. Interestingly, the function of the small intestine itself was apparently not aff ected by this dramatic shift in microbial community composition.

Recent progress on disease-related changes in the small intestinal microbiota has been reviewed expertly by Co er (2011). For instance, elevated levels of Bacteroides spp., Clostridium leptum, Escherichia coli and Staphylococcus spp. and decreased levels of Bifi dobacterium spp., two other clostridial species and Faecalibacterium prausni ii were detected in duodenal biopsy samples of patients suff ering from paediatric coeliac disease (Sokol et al., 2008; Collado et al., 2009;


De Palma et al., 2010; Schippa et al., 2010). Lower duodenal levels of Bifi dobacterium catenulatum were found in patients with irritable bowel syndrome (Kerckhoff s et al., 2009). Finally, lower levels of F. prausni ii and Ruminococcus gnavus and elevated levels of E. coli and Roseburia spp. were found in patients with ileal Crohn’s disease (Willing et al., 2009, 2010).

Clearly, more research is needed to diff erentiate which community changes are causes and which are eff ects of certain disease states. Moreover, the inventory of the small intestinal species and their longitudinal and transversal spatial distribution is still far from being fully understood. This is, however, a prerequisite to defi ne a ‘normal’ or ‘healthy’ microbial community of the small intestine.

1.4 The Microbiota of the Large Intestine


The large intestine consists of the caecum, colon (ascending, transverse, descending and sigmoid), rectum and anal canal

(Fig. 1.3). In total, it is about 1.5 m long, 6.5 cm in diameter and has a surface area of approximately 1200 cm2. As in the small intestine, the surface of the colon is covered entirely by mucus under normal conditions. Early studies suggested that the thickness of the colonic mucus layer increased from about 30 μm in the caecum to 90 μm and more in the rectum (Matsuo et al., 1997). However, a very recent analysis has indicated that these values were underestimated and that the mucus layer of the colon might even be up to 450 μm thick (Gustafsson et al., 2012). The morphology of the colonic mucosa diff ers strongly from that of the small intestine. Permanent folds or villi, as present in the small intestine, are absent. By contrast, the colonic crypts, consisting of absorptive epithelial cells, are lined by a large number of mucus-secreting goblet cells and harbour defensin-producing paneth cells (Me -Boutigue et al., 2010). The main function of the colonic epithelium is the reabsorption of ions and water. As a result of water absorption, the chyme becomes solid approximately 3–10 h after having entered the large intestine and is then referred to as faeces. No digestive enzymes are secreted by the cells of the large intestine. Breakdown of

Current knowledge

• maximum microbial diversity (collective human GIT microbiota: up to 1800 genera and 15,000 species)

• suggestion of a core metagenome based on gene functions

• definition of three human enterotypes: Bacteroides, Prevotella, Ruminococcus

Key questions

Stomach

Smallintestine

Colontransversum

Colonascendens

Colondescendens

Sigmoid

Rectum

• microheterogeneity of the microbiota along the colon (longitudinal and transversal)

• effect of methodical biases on community composition results

• changes in microbial community composition in intestinal or metabolic diseases: cause or consequence?

• interactions of bacterial, archaeal, viral (phage) and eukaryotic (fungal) microbiomes with each other and with the host

Fig. 1.3. Current knowledge and key questions regarding the microbial ecology of the human large intestine.


dietary constituents, as well as mucus, shed epithelial cells and digestive enzymes is carried out by the resident microbiota.

Within the colon, the human microbiota reaches its numeral climax. The density of the microbial community in the colon is approximately 1012 cells per gram of intestinal content. The total microbial weight is estimated to be about 1.5 kg and totals 30% of the volume of the intestinal contents.

1.4.2 Composition of the large intestinal microbiota

Of all microorganisms inhabiting the large intestine, bacteria are by far the dominating ones, although an archaeal, viral and eukaryotic community is also present (Wilson, 2008; Dridi et al., 2009; Marchesi, 2010; Minot et al., 2011). Results obtained from culture-dependent and -independent approaches show that the microbiota of the colon is very complex. Culture-based approaches show that many species are present in very small numbers and it has been estimated that only some 40 species, belonging to a handful of genera, make up almost 90% of the colonic microbiota. However, the use of molecular-based and high-throughput techniques revealed an astonishingly high diversity, with more than 800 species or phylotypes from almost 200 genera (Eckburg et al., 2005). Recent results from 16S rRNA gene-sequencing studies suggested even higher numbers, with up to 1800 genera and 15,000 species-level phylo-types, for the collective human GIT microbiota (Peterson et al., 2008).

Regarding the proportions of the diff erent organisms present, the microbiota of the colon is dominated by obligate anaerobes. Over the last years, it became evident that two taxa, representing more than 80% of all phylotypes, dominated the colonic microbiota. These taxa are the Firmicutes and the Bacteroidetes. The majority of colonic bacteria affi liated with the Bacteroidetes belong to the genus Bacteroides, while the majority of the Firmicutes detectable in the human GIT fall mainly into two groups, the Clostridium coccoides group, also referred to as

Clostridium cluster XIVa, and the C. leptum group, also referred to as Clostridium cluster IV. However, members of the Proteobacteria, Actinobacteria, Verrucomicrobia and Fuso-bacteria are also present, albeit in lower numbers (Nam et al., 2011).

Even though the composition of the colonic microbiota varies considerably between healthy individuals in terms of absolute numbers and proportions of the diff erent taxa, in a single person it remains relatively stable over time (Flint et al., 2007). Table 1.1 shows the major genera usually found in the human large intestine by means of culture-independent methods. However, it has to be taken into account that our perspective of the colonic microbiota may be blurred. Most of the hitherto obtained data still come from the analysis of faecal samples (Wilson, 2008). But evidence suggests that the composition of the microbiota associated with the mucosa surfaces diff ers from that of the faecal microbiota (Zoetendal et al., 2002). Unfortunately, due to the fi ngerprinting technique used by Zoetendal and co-workers (2002), no conclusions on spatial diff erences on the genus or species level could be obtained. Thus, results obtained from the analysis of faecal samples cannot be interpreted as being representative of the total colon microbiota. In addition, one should keep in mind that colonic biopsies are obtained mainly from humans undergoing colonoscopy, which in general is preceded by a laxative preparation in order to clean the lumen. Such a treatment has a great infl uence on the composition of the gut microbiota (Mai et al., 2006). However, more studies addressing the spatial diff erences of microbial communities in the colon of healthy individuals are needed.

Assuming that the average genome size of a prokaryotic microorganism is 3.4 Mb and that approximately 92% of the genes of such a genome are coding for proteins, it has been estimated that the gastrointestinal micro biome is 47,000 Mb (Liolios et al., 2010), which is more than two orders of magnitude greater than the human genome. Thus, the micro biome is considered as an essential organ providing the host with enhanced metabolic capabilities, protection against pathogens, education of


the immune system and modulation of gastrointestinal (GI) development.

1.4.3 Core microbiota of the large intestine

Several groups have recently proposed the concept of a ‘core microbiome’, which is supposed to be present in all humans (Tap et al., 2009; Turnbaugh and Gordon, 2009). This ‘core microbiome’ consists of the most abundant phylotypes and is hypothesized to maintain the functional stability and homeostasis necessary for a healthy eco-system. Recent sequencing studies suggest that such a ‘core microbiome’ might exist at

the level of genes and that it shares genes of diff erent metabolic functions. These studies show that the human intestinal microbiome is enriched in genes coding for particular metabolic functions. More precisely, meta-bolic pathways responsible for energy conservation and biosynthesis of amino acids, nucleotides, carbohydrates, vitamins and secondary metabolites are highly represented (Verberkmoes et al., 2009; Turnbaugh et al., 2010; Gosalbes et al., 2011). In such a ‘core microbiome’, natural selection is refl ected: during the initial colonization phase of the colon, proliferation speed and energy utilization is essential, while later on, functionally more specialized microorganisms become established.

Table 1.1. Major groups of microorganisms detected in human faecal samples by molecular methods. Data compiled from Shen et al., 2010, and Arumugam et al., 2011.

Domain Phylum Order Family/genus Per cent of total

Eukarya Ascomycota Saccharomycetales Candida <1Archaea Euryarchaeota Methanobacteriales Methanobrevibacter <1

Bacteria Firmicutes Clostridiales Anaerostipes <1

Clostridium 1–2

Eubacterium 1–5

Ruminococcus 1–8

Roseburia 1–8

Dorea 0–2

Blautia 0–2

Faecalibacterium 5–15

Lachnospiraceae 2–8

Lactobacillales Streptococcus <1

Lactococcus <1

Lactobacillus 1–8

Total up to 50

Bacteroidetes Bacteroidales Bacteroides 5–35

Parabacteroides 1–3

Prevotella 1–5

Porphyromonas 1–2

Alistipes 2–8

Total up to 40

Proteobacteria Enterobacteriales Escherichia <1

Fusobacteria Fusobacteriales Fusobacterium <2

Verrucomicrobia Verrucomicrobiales Akkermansia 1–3

Actinobacteria Bifi dobacteriales Bifi dobacterium 1–10Coriobacteriales Collinsella 1–8


To identify the common genomic features of the human gut microbiomes of diff erent individuals, as well as the variations between them, Kurokawa and co-workers performed a large-scale comparative meta genomic analysis of faecal samples from healthy humans of diff erent ages (Kurokawa et al., 2007). The gut microbiota of unweaned infants displayed a lower degree of diversity, while adults and weaned children harboured a more complex com-munity. Moreover, the inter-individual dif-ferences were higher in unweaned children. In adults and infants, 237 and 136 gene families, respectively, were enriched, and only a small fraction of these families showed an overlapping enrichment in both groups. Prediction of the function of over-represented gene families in the two subgroups indicated an adaptation of the core functions of the adult and infant-type gut microbiota to its respective habitat. In adults, a prominent enrichment of genes involved in carbohydrate metabolism was observed: 24% of the commonly enriched genes had this function. For example, 14 families of glyco sylhydrolases for plant-derived dietary poly saccharides and host tissue-derived proteoglycans or glyco-conjugates and a large number of enzymes of the mono- or disaccharide metabol ism were over-represented. This fi nding sup-ports the commonly accepted hypothesis that the colonic microbiota breaks down and utilizes dietary polymers that are in-accessible to the digestive system of the host. In infants, about 35% of the 136 enriched gene families had a role in transport processes, with a high proportion of carbo-hydrate transporters and glycosyl hydrolases possibly involved in the degradation and uptake of the oligosaccharides delivered with breast milk.

Recently, Arumugam et al. proposed the concept of three general human enterotypes based on the gut microbiota present. According to this concept, the microbiota of an individual person can be assigned to one of these three enterotypes based on the identity of the most abundant bacterial taxa present (Arumugam et al., 2011). An enterotype is defi ned by a cluster of bacterial species

present in the human gut microbiome, independent of ethnical or geographical origin. The three enterotypes were assigned based on the relative abundances of the following three genera: Bacteroides (enterotype 1), Prevotella (enterotype 2) and Ruminococcus (enterotype 3). In terms of function, each of the enterotype-defi ning genera has been linked to nutrient-processing preferences: Bacteroides to carbohydrates, Prevotella to mucins and Ruminococcus to mucins and sugars.

The species and gene inventory of the human large intestine is probably still far from being complete. In addition, a deeper, more functionally orientated characterization of the microbiome by metatranscriptomic, metaproteomic and metabolomic/meta-bonomic approaches is now needed for a deeper understanding of the role of the human microbiota for maturation, in-fl ammation and nutritional processes within the large intestine.

1.4.4 Non-bacterial members of the colonic microbiota

Using an improved DNA-extraction protocol, Dridi and co-workers could recently show that the two long and well-known methano-genic members of the human colonic microbiota, Methanobrevibacter smithii and Methanosphaera stadtmanae, are obviously much more prevalent in the human GIT than previously thought (Dridi et al., 2009). In particular, M. smithii was detected in 95.7% of 700 investigated stool samples. Moreover, several culture-independent studies sug-gested that additional, yet uncultured eury- and crenarchaeal species might colonize the human GIT, as recently summarized by Dridi et al. (2011). However, their role in human health and disease remains to be elucidated.

In addition to prokaryotes, the human colon is also inhabited by members of the fungi kingdom. Pioneering molecular studies have shown that the human fungal gut microbiota is moderately diverse and that most of the detected phylotypes are affi liated with the phyla, Ascomycota and Basidiomycota (O et al., 2008; Scanlan and Marchesi, 2008).


As known for bacteria, pronounced dif-ferences between the mucosal and luminal (faecal) site of the colon appear to exist (O et al., 2008). Likewise, correlations between the fungal community composition and human disease states are more and more recognized. For instance, very recently Chen and co-workers reported that the diversity of enteric fungi determined by molecular methods was correlated positively with the disease pro-gression of patients with diff erent degrees of chronic hepatitis B virus infection (Chen et al., 2011). Undoubtedly, the eukaryotic per-spective has to be integrated into the human microbiome to gain a deeper understanding of its role in human health and disease (Parfrey et al., 2011).

Finally, it has to be mentioned briefl y that, besides Bacteria, Archaea and fungi, viruses (predominantly bacteriophages) rep re sent abundant and diverse entities in the human GIT. Reyes and co-workers have analysed the virome of stool samples from adult female monozygotic twins and their mothers at three points over a 1-year period (Reyes et al., 2010). They could show that the viromes were highly individual (irrespective of the degree of genetic relatedness) and quite stable over the period of 1 year. Minot and co-workers corroborated this pro-nounced individuality of the gut virome and showed co-variation with the composition of the bacterial microbiome (Minot et al., 2011). Moreover, they could show that similar diets lead to converging (but not identical) viral populations. Interestingly, no predator–prey-like oscillations in the densities of bac-teriophages and bacteria were observed. Clearly, the factors driving the composition and dynamics of the human virome are still largely unknown (Haynes and Rohwer, 2010).

1.5 Factors Shaping the Microbiome of the Human GIT

The composition of the intestinal microbiota is relatively stable in healthy adults over time. Nevertheless, there are a number of factors that may have an infl uence on the relative abundance of diff erent groups of intestinal

bacteria at certain points in the development and life of a human (Table 1.2). These factors include, among others, the mode of delivery and feeding of a newborn, diet, antibiotic treatment, genetic factors and certain diseases or surgical interventions.

1.5.1 The microbiota of neonates and children

It is assumed and widely accepted that the human fetus is sterile before birth (unless a prenatal infection has occurred) and the fi rst contact with microorganisms takes place during delivery. Thus, it is not surprising that the mode of delivery has an impact on the microbial colonization of the newborn. While vaginally born children are exposed to the vaginal microbiota and faecal ma er of the mother, the primary source of microbes for children born by Caesarean section is the skin microbiota of the mother and the nurses that handle the newborn. Using molecular and culture-based tech-niques targeting distinct bacteria groups, it has been shown that, compared to vaginally born infants, babies delivered by Caesarean section showed a delayed, reduced or absent colonization with important groups of bacteria of the normal intestinal microbiota including members of the genera Bifi do-bacterium and Bacteroides. Instead, they were colonized at higher numbers with bacteria of the Clostridium difficile group (Penders et al., 2006; Biasucci et al., 2010). A recent study employing high-throughput se-quenc ing of 16S rRNA gene libraries revealed that, across all investigated habitats, including the skin, oral cavity and GIT, the microbiota of vaginally delivered babies resembled that of the vagina of the mother, whereas the microbiota of babies born by Caesarean section was similar to the skin microbiota of the mother (Dominguez-Bello et al., 2010).

From classical culture-based studies, it was generally accepted that shortly after weaning, the colonic and faecal microbiota of children resembled that of adults, was subjected to no further major changes and that no or only li le day-to-day variations


occurred. However, a recent study char-acterizing the distal gut microbiota by a microarray-based technique surprisingly reported diff erence in the number of clostridia and bifi dobacteria between adults and adolescents (Agans et al., 2011).

Another factor infl uencing the development of the GIT microbiota is the diet fed to newborns during their fi rst months of life. Breast-fed infants are constantly exposed to bacteria present on the skin and in the breast milk of their mother. By contrast, formulas given to bo le-fed infants are

prepared mostly under sterile conditions and harbour no or only a few selected species with potential benefi cial characteristics. As a consequence, exclusively bo le-fed infants are more frequently colonized with E. coli, C. diffi cile, Bacteroides spp. and lactobacilli than breast-fed children (Penders et al., 2006). By contrast, in breast-fed children, the predominant group of faecal bacteria are bifi dobacteria (Kurokawa et al., 2007; Bezir oglou et al., 2011). Additionally, exclusive breast feeding increases the diversity of the bifi dobacterial community

Table 1.2. Summary of various factors shaping the composition of the human gut microbiota.

Factor Effects/observations References

Mode of delivery

Babies born by Caesarean section with delayed, reduced or absent colonization with Bifi dobacterium, Lactobacillus and Bacteroides, and higher numbers of the Clostridium diffi cile group l, compared to vaginally born babies

Grönlund et al., 1999; Penders et al., 2006; Biasucci et al., 2010

Infant feeding Exclusively formula-fed infants are colonized more frequently with Escherichia coli, C. diffi cile, Bacteroides spp. and lactobacilli than breast-fed children

Penders et al., 2006

Breast-fed infants with higher cell counts and diversity in the Bifi dobacterium microbiota

Roger et al., 2010; Bezirtzoglou et al., 2011

Ageing Increase in Enterobacteriaceae and Bacteroidetes, reduced levels of Bifi dobacterium spp.

Hopkins et al., 2001; Claesson et al., 2011

Antibiotics Antibiotic treatment results in rapid loss of diversity and a pronounced community shift, recovery after treatment is incomplete even after months

Dethlefsen and Relman, 2011

Diet High-fat diet is associated with an increase in faecal Enterobacteriaceae and LPS levels in serum, resulting in insulin and glucose resistance and obesity

Cani et al., 2007; Mehta et al., 2010

Type 2 diabetes

Reduced Firmicutes and clostridia compared to healthy controls

Larsen et al., 2010

Obesity Changes in the relative proportions of Bacteroidetes and Firmicutes

Ley et al., 2006; Schwiertz et al., 2010b; Million et al., 2012

Infl ammation Increase in the abundance of bacteria belonging to the Enterobacteriaceae and reduction in Faecalibacterium prausnitzii in human patients and animal models of chronic and infectious intestinal infl ammation

Kotlowski et al., 2007; Sokol et al., 2008

Host genotype TLR5-defi cient mice display changes in the levels of Bacteroidetes and Firmicutes and show hyperlipidaemia, hypertension, insulin resistance and increased adiposity

Vijay-Kumar et al., 2010

MyD88 knockout mice show increased levels of Lactobacillaceae, Rikenellaceae and Porphyromonadaceae and are protected from type 1 diabetes

Wen et al., 2008

Reduced diversity of the microbiota in patients suffering from familial Mediterranean fever

Khachatryan et al., 2008

LPS = lipopolysaccharide, an important part of the outer membrane of Gram-negative bacteria.


(Roger et al., 2010). Interestingly, the above-mentioned diff erences seem to disappear after weaning and with the introduction of solid food, and fi nally all individuals acquire an adult-type microbiota. However, it is tempting to speculate that diff erences in the composition of the GIT microbiota at the early stages of development may have an imprinting eff ect during the maturation of the immune system and gut physiology in general.

1.5.2 The microbiota of adults

The adult intestinal microbiota seems to be specifi c for an individual and is relatively stable over time (Eckburg et al., 2005; Costello et al., 2009; Jalanka-Tuovinen et al., 2011). Nevertheless, signifi cant changes in its composition are observed in response to diet, disease, travelling and, of course, antibiotic treatment (Dethlefsen and Relman, 2011; Jalanka-Tuovinen et al., 2011; Walker et al., 2011; Zimmer et al., 2011). It is generally accepted that during adulthood, bacteria acquired from the environment or taken up with the diet colonize the GIT only transiently. By contrast, changes in the microbiota on administration of antibiotics seem to be more profound and stable. After a recovery period, the overall composition is broadly similar to the composition prior to treatment. However, even after several months, clear diff erences can be observed (Dethlefsen and Relman, 2011).

The composition of the GIT microbiota also changes towards the later stages of life. In elderly persons, Enterobacteriaceae and Bacteroidetes tend to increase, and bifi do-bacteria are present at lower numbers (Hopkins et al., 2001; Claesson et al., 2011), pre sumably due to altered nutrition, decreased gut motility, increased use of medication and less mobility.

Obesity, insulin resistance and type 2 diabetes were linked to changes in the composition of the intestinal microbiota. Reduced proportions of Firmicutes, and in particular Clostridia, were observed in patients with type 2 diabetes compared to controls (Larsen et al., 2010). Some authors

described a relative increase in the abundance of Firmicutes and a decrease in the number of Bacteroidetes in (genetically rendered) obese mice (Ley et al., 2005) and overweight human subjects (Ley et al., 2006). Others have observed the opposite (Schwier et al., 2010b) or reported changes in the proportions of lactobacilli, bifi dobacteria and methano-bacteria (Million et al., 2012). The diff erences observed may be explained at least partially by the methods used, as the former two studies used pyro-sequencing and the la er two employed quantitative real-time PCR (see also concluding remarks).

Regardless of the nature of the above-mentioned changes, this raises the chicken-and-egg question whether the altered micro biota is the cause or the consequence of obesity. Interestingly, mice fed a high-fat diet showed an increased abundance of Enterobacteriaceae in their faeces and higher levels of lipopolysaccharides (LPS), a component of the outer membrane of Enterobacteriaceae and other Gram-negative organisms, in their blood serum. The authors refer to this state as ‘metabolic endotoxaemia’, which is associated with insulin resistance and obesity (Cani et al., 2007). All features of the metabolic endotoxaemia could be induced by subcutaneous, sublethal infusion of LPS, and mice defi cient for the LPS co-receptor CD14 were found to be resistant to metabolic endotoxaemia (Cani et al., 2007). Moreover, antibiotic treatment could reduce intestinal LPS levels and the signs of metabolic endotoxaemia in mice fed a high-fat diet (Cani et al., 2008). Similarly, acute endotoxaemia induced insulin resistance in humans (Mehta et al., 2010). All these fi ndings argue in favour of an altered microbiota as the consequence rather than the cause of obesity and diabetes. An additional link between diet and diabetes/obesity comes from studies using TLR5 knockout mice (Vijay-Kumar et al., 2010). These mice lack the gene for toll-like receptor 5 (TLR5), an important receptor of the innate immune system, they consume about 10% more food than wild-type mice and display a strong phenotype with typical signs of metabolic syndrome, i.e. hyperlipidaemia, hypertension, insulin resistance and increased adiposity. This phenotype is accompanied by


marked changes in the intestinal microbiota, especially in the composition of the Bacteroidetes and Firmicutes on the species level. These fi ndings also provide a link between host genotype and the intestinal microbiota resulting in obesity and diabetes. In principle, the observed changes in the microbiota in obesity are suspected to result in a more effi cient energy harvest from the diet. Thus, there seems to be some potential of a therapeutic alteration of the microbiota. Nevertheless, the primary reason for obesity in humans (and mice) is a positive energy balance (uptake of more calories than consumed per day) and it has been shown that by reducing the calorie intake, changes in the composition of the microbiota can be reversed (Ley et al., 2006).

Besides the observed changes in the microbiota in TLR5 knockout mice (Vijay-Kumar et al., 2010), the impact of the host genotype on the microbiota is increasingly recognized and has recently been shown in a few studies. This chapter is intended to describe the human microbiota. However, research on the infl uence of the host genotype is a relatively new fi eld and, as a consequence, most of the studies have been performed in mice. In non-obese diabetic mice, knock out of MyD88, an adaptor molecule involved in TLR signalling, resulted in major changes of the microbiota and protection from type 1 diabetes (Wen et al., 2008). In addition, the diversity of the microbiota of patients suff ering from familial Mediterranean fever (MEFV), a disease associated with a single nucleotide polymorphism in the MEFV locus, is reduced (Khachatryan et al., 2008).

Changes in the intestinal microbiota have also been observed during episodes of intestinal infl ammation, both in animal models and in human patients (Swidsinski et al, 2005; Heimesaat et al., 2007; Kotlowski et al., 2007; Wohlgemuth et al., 2009; Garre et al., 2010; Greenblum et al., 2011). In most cases, an increase in the number of Gram-negative bacteria of the Enterobacteriaceae has been observed, and in some cases could be traced down to a single microbial species or strain. Recently, a reduction in the F. prausni ii population was reported both in

adults and in infants suff ering from in-fl ammatory bowel disease (Sokol et al., 2008; Schwier et al., 2010a).

In patients with severe courses of infl ammatory bowel disease or colon cancer, parts of the colon, and sometimes ileum, are removed surgically. In cases where both small and large intestine have to be removed entirely, the last option is the transplantation of the small bowel. After removal of the diseased parts of the GIT, an ileostomy is constructed by bringing the end of the remaining small intestine to the surface of the skin, creating an artifi cial abdominal opening from which intestinal effl uent is collected into an external plastic bag. Alternatively, loops of the remaining small intestine are folded back on to each other, with the separating walls opened and stitched back together. This pouch is then connected to the anus, creating an ileo-anal pouch, which is thought to act as an artifi cial colon. In all cases, parts of the ileum become the terminal part of the GIT. This is refl ected by changes in the microfl ora. After surgery, bacterial numbers in the neoterminal ileum increase and the microfl ora in the ileo-anal pouch acquires a ‘colon-like’ composition (Neut et al., 2002; Kohyama et al., 2009). In this context, it has to be noted that the composition of the microfl ora in ileostomy effl uents is characterized by large numbers of facultative anaerobes (Neut et al., 2002; Hartman et al., 2009). Since this increase is at least partially reversed after closure of the ileostomy, it is hypothesized that a leakage of oxygen through the ileostomy into the neoterminal ileum might be responsible for this eff ect (Hartman et al., 2009).

Disease-related changes in the microbiota raise the question whether these changes may be reversed (‘rebalanced’) by the administration of ‘benefi cial’ bacteria, also termed probiotics, thereby restoring a normal microbiota and leading to an improvement of the disease. For a comprehensive review on the fascinating but also quite controversially discussed fi eld, which is beyond the scope of this chapter, the reader is referred to recent reviews (Gareau et al., 2010; Gerritsen et al., 2011).


1.6 Concluding Remarks

The advent of the new sequencing technologies has boosted the characterization of the human microbiota in any habitat (Proctor, 2011), but in particular in the human GIT. Parallel sequencing of hundreds of thousands of 16S rRNA genes allows the characterization of the complex microbiota of an individual within virtually hours and at very low cost.

1.6.1 New techniques – old biases

The technological improvements of the last decades have not resolved the long-known discrepancy between results obtained by ‘traditional’ culture-based techniques and ‘new’ PCR- or probe-based molecular tech-niques. Interestingly, a recent study indicated that the vast majority of the human faecal microbiota detected by high-throughput sequencing was comprised of microorganisms that could be readily cultured (Goodman et al., 2011). The authors compared microbial 16S rRNA gene sequences directly amplifi ed from human faecal samples with 16S rRNA gene sequences of about 30,000 colonies obtained by culturing the same samples. Surprisingly, approximately 90, 70 or 56% of the 16S rRNA gene sequences obtained from the colonies were also represented in the 16S rRNA gene collection from fresh samples on the family, genus or species level, respectively. On the one hand, this fi nding indicates that the portion of yet uncultured bacteria in faecal samples might be lower than previously suspected. On the other hand, the question arises whether the primers normally used in high-throughput sequencing studies capture all bacterial groups present in the samples with the same effi ciencies. In fact, this is not always the case, as recently shown by Martínez and co-workers (Martínez et al., 2009). They provided a plausible explanation why bifi dobacteria might be under-represented in some of the recent high-throughput sequencing studies on human faecal samples (Eckburg et al., 2005; Gill et al., 2006). Primer 8F (in some studies also referred to as 27F), which is typically used for high-

throughput sequencing of 16S rRNA genes, displays mismatches in three base pairs with the 16S rRNA gene sequence of bifi dobacteria (Martínez et al., 2009). However, bifi dobacteria represent important members of the human intestinal microbiota and are frequently detected at high numbers by classical plating techniques or by using FISH (fl uorescence in situ hybridization) technology, as summarized by Tannock (Tannock, 2010). Presumably, in studies using the 8F primer, bifi dobacterial 16S rRNA genes are amplifi ed with a signifi cantly lower effi ciency, leading to an under-representation of this group of bacteria. When looking at metagenomic libraries that take into account the entire genetic information rather than just 16S rRNA gene sequences, bifi dobacteria again become the third most abundant group of colonic microorganisms, dominated only by the Bacteroidetes and Firmicutes (Kurokawa et al., 2007), confi rming earlier culture-based studies (Tannock, 2010). This example illus trates that while studies employing high-throughput sequencing produce a tre -mendous amount of data and certainly are informative, they might be biased for some of the bacterial groups present in a given habitat.

1.6.2 Outlook

Undoubtedly, high-throughput sequencing technologies are a valuable tool to describe in great detail the complexity of the microbiota in a given habitat. However, the results obtained do not allow conclusions with respect to the relative abundance of certain bacterial groups, unless they have been validated by other methods that target the bacteria of interest directly either by selective cultivation or more specifi c primers or probes.

In addition, facing the strong in-dividuality of the human (GIT) microbiome, it seems reasonable to postulate that the highly improved sequencing capacity should rather be used for studying microbial diversity (e.g. based on 16S rRNA genes) in a greater amount of samples (human in-dividuals) than for investigating very high


numbers of sequences per single sample (Kuczynski et al., 2010). In this way, information about the large-scale pa erns or temporal dynamics of human–microbe associations and their statistical signifi cance could be gathered, which fi nally might turn out to be of greater interest for a deeper understanding of the nature of human–microbe interactions than the description of the actual depth of the microbial diversity in a single and highly individual human sample.

Finally, describing the structure or com-position of the complex microbiome in the human GIT is challenging, but it can only be the fi rst step on the way to gaining a deeper insight into the functional relevance of the human GIT microbiome for human health and disease. For this, more studies applying technologies summarized as ‘functional microbiomics’ (Egert et al., 2006) are needed, addressing not only ‘who is there’ but also ‘what are they doing?’, as recently reported (Kurokawa et al., 2007; Gosalbes et al., 2011; Greenblum et al., 2011; Zoetendal et al., 2012). For more information on the fascinating fi eld of the physiology and functionality of the human GIT microbiota, the reader is referred to Chapter 6, this volume.

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