Community and genomic analysis of the human small intestine microbiota Bartholomeus van den Bogert Thesis committee Promotor Prof. Dr M. Kleerebezem Personal chair in the Host Microbe Interactomics Group Wageningen University Co-promotor Dr E.G. Zoetendal Assistant professor, Laboratory of Microbiology Wageningen University Other members Prof. Dr T. Abee, Wageningen University Dr J. Doré, National Institute for Agricultural Research, INRA, Jouy en Josas, France Prof. Dr P. Hols, Catholic University of Leuven, Belgium Dr A. Nauta, FrieslandCampina Research, Deventer, The Netherlands This research was conducted under the auspices of the Graduate School VLAG (Advanced studies in Food Technology, Agrobiotechnology, Nutrition and Health Sciences). Community and genomic analysis of the human small intestine microbiota Bartholomeus van den Bogert Thesis submitted in fulfilment of the requirements for the degree of doctor at Wageningen University by the authority of the Rector Magnificus Prof. Dr M.J. Kropff, in the presence of the Thesis Committee appointed by the Academic Board to be defended in public on Wednesday 9 October 2013 at 4 p.m. in the Aula. Bartholomeus van den Bogert Community and genomic analysis of the human small intestine microbiota, 224 pages PhD thesis, Wageningen University, Wageningen, NL (2013) With references, with summaries in Dutch and English ISBN 978-94-6173-662-8 Summary Our intestinal tract is densely populated by different microbes, collectively called microbiota, of which the majority are bacteria. Research focusing on the intestinal microbiota often use fecal samples as a representative of the bacteria that inhabit the end of the large intestine. These studies revealed that the intestinal bacteria contribute to our health, which has stimulated the interest in understanding their dynamics and activities. However, bacterial communities in fecal samples are different compared to microbial communities at other locations in the intestinal tract, such as the small intestine. Despite that the small intestine is the first region where our food and intestinal microbiota meet, we know little about the bacteria in the small intestine and how they influence our overall well-being. This is mainly attributable to difficulties in obtaining samples with the small intestine being located between the stomach and the large intestine. Therefore, the work in this thesis aimed at providing a better understanding of the composition and dynamics of the human small intestinal microbiota and to provide insight in the metabolic potential as well as immunomodulatory properties of some of its typical commensal inhabitants. Small intestinal samples used in the work described in this thesis were collected from ileostomy subjects, individuals that had their large intestine surgically removed and the end of the small intestine connected to an abdominal stoma, providing access to luminal content of the small intestine. Considering the importance of molecular techniques in contemporary ecological surveys of microbial communities, first of all, two technologies, barcoded pyrosequencing and phylogenetic microarray analysis were compared in terms of their capacity to determine the bacterial composition in fecal and small intestinal samples from human individuals. As PCR remains a crucial step in sample preparation for both techniques, the use of different primer pairs in the amplification step was assessed in terms of its impact on the outcome of microbial profiling. The analyses revealed that the different primer pairs and the two profiling technologies provide overall similar results for samples of fecal and terminal ileum origin. In contrast, the microbial profiles obtained for small intestinal samples by barcoded pyrosequencing and phylogenetic microarray analyses differed considerably. This is most likely attributable to the constraints that are intrinsic to the use of the microarray to enable the detection of predefined microbiota members only, which is due to the probe design that is largely based on large intestinal microbiota communities. However, the pyrosequencing technology also allows for identification of bacteria that were not in advance known to inhabit our intestinal tract. The pyrosequencing technology was used as the method of choice to study the total and active small intestinal communities in ileostoma effluent samples from four different subjects through sequencing the 16S ribosomal RNA gene (rDNA) and ribosomal RNA (rRNA) content combined with metatranscriptome analysis by Illumina sequencing of cDNA derived from enriched mRNA of the same sample set to investigate the activities of the small intestinal bacteria. The composition of the small intestinal bacterial communities as assessed from rDNA, rRNA, and mRNA patterns appeared to be similar, indicating that the dominant bacteria in the small intestine are also highly active in this ecosystem. Streptococcus spp. were among the bacterial species that were detected in each ileostoma effluent sample, albeit that their abundance varied greatly between samples from the same subject as well as samples from different subjects. Veillonella spp. frequently co-occurred with Streptococcus spp., indicating that the Streptococcus and Veillonella populations play a prominent role in the human small intestine ecosystem and their co- occurrence suggests a metabolic relation between these genera. Therefore, cultivation and molecular typing methodologies were employed to zoom-in on these groups, which revealed that the richness of the small intestinal streptococci strongly exceeded the diversity that could be estimated on basis of 16S rRNA analyses, and could be extended to the genomic lineage level (anticipated to resemble strain-level). From ileostoma samples 3 different Streptococcus species were recovered belonging to the S. mitis group, S. bovis group, and S. salivarius group, which could be further divided in 7 genomic lineages. Notably, the Streptococcus lineages that were isolated displayed distinct carbohydrate utilization capacities, which may imply that their growth and relative community composition may respond quite strongly to differences in the dietary intake of simple carbohydrates over time. This notion is in good agreement with the observation that the Streptococcus lineage populations fluctuated in time with only one Streptococcus lineage being cultivated from both ileostoma samples collected in a one-year time frame. Conversely, the cultivated Veillonella isolates from samples during that same time-interval consistently encompassed a single lineage. Furthermore, this Veillonella lineage could be isolated from both the oral cavity as well as the ileostoma effluent. Analogously, three Streptococcus lineages that belong to a single phylotype also appeared to be present in bacterial communities from the oral cavity as well as the small intestine. These observations suggest the representatives of the Veillonella and Streptococcus genera that are encountered in the oral and small intestinal microbial ecosystems are closely related and indicate that the oral microbiota may serve as an inoculum for the upper GI tract. The metabolic capacity of 6 small intestinal Streptococcus lineages, that were obtained from a single ileostoma effluent sample, was further investigated through the determination of genomic sequences of these lineages. The small-intestinal Streptococcus genomes were found to encode different carbohydrate transporters and the necessary enzymes to metabolize different sugars, which was in excellent agreement with what carbohydrates could be used by representative strains of the Streptococcus lineages. To further our understanding how the different streptococci as representatives of the dominant small intestinal bacterial populations may influence our immune system, human dendritic cells were stimulated with strains of the different Streptococcus lineages to study their immunomodulatory properties. The Streptococcus lineages differed significantly in their capacity to modulate cytokine responses of blood- monocyte derived immature dendritic cells. As Streptococcus and Veillonella frequently co-occur in the small intestinal ecosystem, pair-wise combinations of strains of these two species were also tested for their combined immunomodulatory properties. This resulted in considerably different cytokine responses as those that could be predicted from the stimulations with either Streptococcus or Veillonella, indicating that it is not trivial to predict gut mucosal associated immune responses and that the composition of the intestinal microbiota as a whole may have a distinct influence on an individual’s immune status. In conclusion, the work described this thesis provides an expansion to the accumulating knowledge on the human intestine microbiota. Whereas most studies focus on the microbiota present in the distal regions of the intestinal tract, this study targeted the microbiota of the poorly proximal regions of the intestine and also addressed its capacity to interact with the local mucosal tissue. The data presented here can be exploited to guide the design of future studies that aim to elucidate the interplay between diet, microbiota and the mucosal tissues in the human small intestinal tract. Table of contents Chapter 1 General introduction and thesis outline 1 Chapter 2 Microarray analysis and barcoded pyrosequencing 21 provide consistent microbial profiles depending on the source of human intestinal samples Chapter 3 Congruency of phylogenetic composition