Diversity of the Human Gastrointestinal Microbiota Novel Perspectives from High Throughput Analyses
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Diversity of the Human Gastrointestinal Microbiota Novel Perspectives from High Throughput Analyses Mirjana Rajilić-Stojanović Promotor Prof. Dr W. M. de Vos Hoogleraar Microbiologie Wageningen Universiteit Samenstelling Promotiecommissie Prof. Dr T. Abee Wageningen Universiteit Dr J. Doré INRA, France Prof. Dr G. R. Gibson University of Reading, UK Prof. Dr S. Salminen University of Turku, Finland Dr K. Venema TNO Quality for life, Zeist Dit onderzoek is uitgevoerd binnen de onderzoekschool VLAG. Diversity of the Human Gastrointestinal Microbiota Novel Perspectives from High Throughput Analyses Mirjana Rajilić-Stojanović Proefschrift Ter verkrijging van de graad van doctor op gezag van de rector magnificus van Wageningen Universiteit, Prof. dr. M. J. Kropff, in het openbaar te verdedigen op maandag 11 juni 2007 des namiddags te vier uur in de Aula Mirjana Rajilić-Stojanović, Diversity of the Human Gastrointestinal Microbiota – Novel Perspectives from High Throughput Analyses (2007) PhD Thesis, Wageningen University and Research Centre, Wageningen, The Netherlands – with Summary in Dutch – 214 p. ISBN: 978-90-8504-663-9 Abstract The human gastrointestinal tract is densely populated by hundreds of microbial (primarily bacterial, but also archaeal and fungal) species that are collectively named the microbiota. This microbiota performs various functions and contributes significantly to the digestion and the health of the host. It has previously been noted that the diversity of the gastrointestinal microbiota is disturbed in relation to several intestinal and not intestine related diseases. However, accurate and detailed defining of such disturbances is hampered by the fact that the diversity of this ecosystem is still not fully described, primarily because of its extreme complexity, and high inter-individual variability. The work presented in this thesis summarises the hitherto gathered knowledge on the diversity of the human gastrointestinal microbiota based on an extensive literature search that aimed to gather all reports of microbial life within the gastrointestinal tract starting from the first isolation in 1885. This resulted in the construction of the ecosystem-specific database of small subunit ribosomal RNA sequences of over 1,000 human gastrointestinal inhabitants based on which a phylogenetic microarray was designed. This newly developed HITChip (Human Intestinal Tract Chip) enabled comprehensive and high throughput analyses, and provided some novel perspectives on the diversity of this ecosystem, which include the following: (a) the first molecular follow up of the long-term microbiota dynamics showed that genetic, environmental and stochastic factors are shaping the microbial community within the gastrointestinal tract, and revealed that some members of Actinobacteria and Bacteroidetes are highly influenced by the genotype of the host, while determening the host-specific microbiota signature; (b) the analysis of the microbial diversity of intestinal contents of patients suffering from irritable bowel syndrome (IBS) and ulcerative colitis (UC) showed that both diseases impacted the microbiota, as its composition was distinctive from that of healthy adults, although the disturbance of the microbiota in relation to UC appeared to be more severe when compared to IBS, and was characterised, in addition to the different relative abundance of numerous members of the microbiota, by dramatically reduced richness of the microbial community; (c) finally, studying the development of the microbial community in a model of the human colon (TIM- 2) provided novel guidelines for improving the handling procedures that will ensure more accurate representation of the microbial diversity of healthy adults. In conclusion, the results indicate that a high throughput and comprehensive studying of the human gastrointestinal microbiota, as facilitated by the application of phylogenetic microarrays is a promising approach for further describing and understanding of this exceptionally relevant microbial ecosystem. Keywords: gastrointestinal microbiota, diversity, phylogenetic microarray, SSU rRNA Contents Outline....................…………………………………………………………......8 Chapter 1 General introduction…………………………………………………………....12 Chapter 2 Characterised microbes of the human gastrointestinal tract……………....24 Chapter 3 Diversity of the human gastrointestinal tract microbiota revisited..…….....50 Chapter 4 A generic design strategy for phylogenetic microarrays based on SSU rRNA gene sequence diversity – the human gastrointestinal microbiota as a case ................................................................................................66 Chapter 5 Evaluating the microbial diversity of an in vitro model of the human large intestine by phylogenetic microarray analysis...................................86 Chapter 6 Dynamics of the adult human gastrointestinal microbiota........................ 100 Chapter 7 Phylogenetic microarray analysis reveals specific faecal microbiota composition in patients suffering from irritable bowel syndrome..............116 Chapter 8 Diversity of the human gastrointestinal microbiota in relation to ulcerative colitis.........................................................................................134 Chapter 9 General discussion...................................................................................154 Bibliography..............................................................................................168 Supplements.............................................................................................192 Outline Evidence for the presence of specific microbes in the human gastrointestinal tract was provided at the end of the XIX century when Teodor Escherich isolated Escherichia coli from children's diarrhoeal faeces. Although this bacterial presence was considered as an infection, it soon became clear that a complex microbial community permanently resides in the gastrointestinal tract, and forms an essential element of the human body. A plethora of functions that contribute to the host's nutrition and health has been assigned to these microbes that collectively are termed microbiota. It has even been suggested that - because of its functions, dimensions and appearance - the microbiota can be considered as a virtual organ within the gastrointestinal tract. Furthermore, it seems that the disturbance of the delicate balance between numerous members of this ecosystem is associated with a number of intestinal and non-intestinal disorders. The appreciation of the importance of the gastrointestinal microbiota has generated significant interest in a further understanding of its dynamics, structure-function relationship and the possibility to modulate those. Although it is prerequisite for understanding and exploiting this ecosystem, the description of its enormous diversity is still not completed. 10 x Outline The work presented in this thesis aims to systematise the generated knowledge on the gastrointestinal microbiota diversity, and provide an improved insight into its relation to health and disease using a newly developed, high-throughput technology. Specific questions that were addressed by each of the chapters are listed below. Chapter 1 summarises the present perception of functions and dimensions of the gastrointestinal microbiota, and provides an overview of the currently available techniques for studying the diversity of this ecosystem. The following two chapters deal with the diversity of the human gastrointestinal microbiota. The results of cultivation-based studies, which are an important source of information because of their extensive use for the analysis of a large amount of samples are summarised in Chapter 2. This chapter presents a thorough analysis of gastrointestinal isolates, embedded in a curated phylogenetic framework, given through a historical perspective with the aim to clarify their importance. An alternative to time-consuming, laborious and expensive cultivation-based analysis of the microbiota’s diversity are molecular approaches, such as those based on small subunit ribosomal RNA (SSU rRNA) gene sequences. The application of molecular techniques has revolutionised our view of many microbial ecosystems, and their impact on the knowledge of human gastrointestinal ecology is portrayed in Chapter 3. This chapter also provides an up-to- date, systematic overview of the gastrointestinal microbiota’s diversity, built by integrating results that were generated by the application of culturing and molecular approaches, and that accumulated during more than a century long studying of this ecosystem. Chapter 4 describes a generic oligonucleotide probe design strategy based on two hypervariable regions of the SSU rRNA gene sequence. This approach is high throughput, can be readily applied to any complex ecosystem, and allows easy addition of probes that target its newly reported inhabitants, which is essential for not yet fully described ecosystems, such as the one that resides in the human intestine. The application of the described strategy on the human gastrointestinal microbiota resulted in the development of the HITChip (Human Intestinal Tract Chip), a phylogenetic microarray that was applied for the analysis of the human intestinal microbiota’s diversity described in the further chapters of this thesis. In vitro fermentation of intestinal contents is a widely used approach to study the metabolic activity of the human gastrointestinal microbiota. To clarify whether such