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要約) Doctoral Dissertation Antibiotics Shapes Population-Level Diversity In ) Doctoral Dissertation Antibiotics shapes population-level diversity in the human gut microbiome ( ) Nishijima Suguru Acknowledgments I would like to express my sincere gratitude to my supervisor, Prof. Masahira Hattori, whose expertise, knowledge and continuous encouragement throughout my research. My sincere thanks also go to Assoc. Prof. Kenshiro Oshima (The University of Tokyo), Dr. Wataru Suda and Dr. Seok-Won Kim for their motivation, immense support and encouragement throughout my work. I am also grateful to all my collaborators, Prof. Hidetoshi Morita (Okayama University) for his fecal sample collection, DNA isolation and sincere encouragement, Prof. Kenya Honda and Dr. Koji Atarashi (Keio University) for mice experiments, Assoc. Prof. Masahiro Umezaki (the University of Tokyo) for support for dietary data analysis, Dr. Todd D. Taylor (RIKEN) for support for writing manuscript and Dr. Yuu Hirose (Toyohashi University of technology) for DNA sequencing. I also would like to thank all past and present members of our laboratory, Erica Iioka, Misa Takagi, Emi Omori, Hiromi Kuroyamagi, Naoko Yamashita, Keiko Komiya, Rina Kurokawa, Chie Shindo, Yukiko Takayama and Yasue Hattori for their great technical support and kind assistance. i Antibiotics shapes population-level diversity in the human gut microbiome () Abstract The human gut microbiome has profound influences on the host’s physiology through its interference with various intestinal functions. The development of next-generation sequencing (NGS) technologies enabled us to comprehensively explore ecological and functional features of the gut microbiomes. Recent studies using the NGS-based metagenomic approaches have suggested high ecological diversity of the microbiome across countries. However, little is known about the structure and feature of the Japanese gut microbiome, and the factor that shapes the population-level diversity in the human gut microbiome. In this thesis, to address the above questions regarding the human gut microbiome, I analyzed metagenomic data of fecal DNA samples from healthy Japanese individuals and compared the data with that from individuals in other countries. I obtained approximately 350 Gb of metagenomic sequences of the gut microbiome of 106 Japanese individuals in this study. By comparing the metagenomic data with that of 757 individuals from other 11 countries, I found that the Japanese gut microbiome showed more abundant in the phylum Actinobacteria, in particular in the genus Bifidobacterium, than that of the other 11 nations. Regarding the microbial functions, those of carbohydrate metabolism were overrepresented with a concurrent decrease in those for replication and repair and cell motility in the Japanese gut microbiome. The remarkable low prevalence of genes for methanogenesis with a significant depletion of the archaeon Methanobrevibacter smithii and significant enrichment of acetogenesis genes in the Japanese gut microbiome as compared to others ii suggested a difference in the hydrogen metabolism pathway in the gut between them. These data suggested considerable uniqueness in the taxonomy and function of the Japanese gut microbiome (Nishijima S. et al., DNA res., in press). To explore the factors that contribute to differences in the human gut microbiomes across the 12 countries, I further conducted an association study of the epidemiological data on dietary intake and antibiotic usage with metagenomic data of the 861 human gut microbiomes from the 12 countries. I found that the gut microbiome structure is significantly diverse across the 12 countries, which was strongly correlated with antibiotics as well as diet. Notably, the abundance of the major species Bacteroides showed a significant correlation with both antibiotic usage in humans and farm animals but not with diet; whereas, the abundance of another major species Prevotella showed a significant correlation with diet but not with antibiotic usage. Thus, the trade-off relation between these two major species appears to be a consequence of respective independent effects from dietary and antibiotic factors. The proliferation of antibiotic resistant genes, including the efflux pump, may underlie the positive correlation between Bacteroides and antibiotic usage. Collectively, these results suggest that antibiotics may have had a striking impact on the shaping of the gut microbiome structure of modern human populations (Nishijima S. et al., in preparation). iii Table of Contents Acknowledgement .............................................................................................................. i Abstract ............................................................................................................................. ii 1. Introduction .............................................................................................................. 1 1.1. Human gut microbiomes and study background .................................................. 1 1.2. Development of culture-independent method ........................................................ 2 1.3. International trends in the metagenomic study of the human microbiome ............ 4 1.4. Structure of the human microbiome ....................................................................... 6 1.5. Association with health and diseases ..................................................................... 9 2. The gut microbiome of healthy Japanese and its microbial and functional uniqueness ...................................................................................................................... 12 2.1. Introduction .......................................................................................................... 12 2.2. Methods ................................................................................................................ 14 2.2.1. Subjects and fecal sample collection ......................................................................... 14 2.2.2. Recovery of bacteria and archaea from fecal samples .............................................. 14 2.2.3. DNA isolation and purification ................................................................................. 15 2.2.4. Metagenomic sequencing of fecal DNA ................................................................... 15 2.2.5. Assembly and gene prediction for metagenomic sequences ..................................... 16 2.2.6. Publically available metagenomic data ..................................................................... 17 2.2.7. Country-specific metagenomic datasets of healthy individuals ................................ 18 iv 2.2.8. Construction of microbial reference genomes ........................................................... 18 2.2.9. Microbial composition by mapping of metagenomic reads to the reference genomes ............................................................................................................................................. 20 2.2.10. Comparison of microbial compositions among the countries ................................. 21 2.2.11. Assessment and comparison of different methodologies ........................................ 22 2.2.12. PCR detection of Methanobrevibacter smithii in the Japanese individuals ............ 22 2.2.13. Generation of a merged reference gene set of Japanese and integrated gene catalog ............................................................................................................................................. 23 2.2.14. Functional assignment of non-redundant genes ...................................................... 23 2.2.15. Quantification of the annotated genes in human gut microbiomes ......................... 24 2.3. Results .................................................................................................................. 25 2.3.1. Metagenomic sequencing of the Japanese gut microbiome ...................................... 25 2.3.2. Analysis of non-redundant genes of the Japanese gut microbiome .......................... 26 2.3.3. Structure of the Japanese gut microbiomes ............................................................... 30 2.3.4. Population-level diversity in the human gut microbiome ......................................... 33 2.3.5. Characterization of the Japanese gut microbiome ..................................................... 41 2.3.6. Functional comparison of the Japanese gut microbiome with the others .................. 43 2.3.7. Gene families enriched in the Japanese population ................................................... 49 2.1. Discussion ............................................................................................................ 53 3. Antibiotics shapes population level diversity in the human gut microbiome ... 56 3.1. Introduction .......................................................................................................... 56 3.2. Methods ................................................................................................................ 57 3.2.1. Collection of dietary intake data ............................................................................... 57 v 3.2.2. Collection of antibiotic usage data ............................................................................ 57 3.2.3. Statistical analysis ..................................................................................................... 57 3.2.4. Analysis of gut microbiomes of Asian children .......................................................
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