THE HUMAN MICROBIOTA ffffiirrss0011..iinndddd ii 22//77//22001133 1122::0033::1111 PPMM ffffiirrss0011..iinndddd iiii 22//77//22001133 1122::0033::1111 PPMM THE HUMAN MICROBIOTA How Microbial Communities Affect Health and Disease Edited by David N. Fredricks ffffiirrss0022..iinndddd iiiiii 22//77//22001133 1122::0033::1122 PPMM Cover Design: Michael Rutkowski Cover Illustrations: top fi ve panels © courtesy of David N. Fredricks; right side art © Mads Abildgaard/iStockphoto Copyright © 2013 by John Wiley & Sons, Inc. All rights reserved. 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QR46.H86 2013 579–dc23 2012015251 Printed in the United States of America 10 9 8 7 6 5 4 3 2 1 ffffiirrss0033..iinndddd iivv 22//77//22001133 1122::0033::1144 PPMM CONTENTS PREFACE vii CONTRIBUTORS xi 1 THE NIH HUMAN MICROBIOME PROJECT 1 Lita M. Proctor, Shaila Chhibba, Jean McEwen, Jane Peterson, Chris Wellington, Carl Baker, Maria Giovanni, Pamela McInnes, and R. Dwayne Lunsford 2 METHODS FOR CHARACTERIZING MICROBIAL COMMUNITIES ASSOCIATED WITH THE HUMAN BODY 51 Christine Bassis, Vincent Young, and Thomas Schmidt 3 PHYLOARRAYS 75 Eoin L. Brodie and Susan V. Lynch 4 MATHEMATICAL APPROACHES FOR DESCRIBING MICROBIAL POPULATIONS: PRACTICE AND THEORY FOR EXTRAPOLATION OF RICH ENVIRONMENTS 85 Manuel E. Lladser and Rob Knight 5 TENSION AT THE BORDER: HOW HOST GENETICS AND THE ENTERIC MICROBIOTA CONSPIRE TO PROMOTE CROHN’S DISEASE 105 Daniel N. Frank and Ellen Li 6 THE HUMAN AIRWAY MICROBIOME 119 Edith T. Zemanick and J. Kirk Harris 7 MICROBIOTA OF THE MOUTH: A BLESSING OR A CURSE? 135 Angela H. Nobbs, David Dymock, and Howard F. Jenkinson 8 MICROBIOTA OF THE GENITOURINARY TRACT 167 Laura K. Sycuro and David N. Fredricks 9 FUNCTIONAL STRUCTURE OF INTESTINAL MICROBIOTA IN HEALTH AND DISEASE 211 Alexander Swidsinski and Vera Loening-Baucke v ffttoocc..iinndddd vv 22//77//22001133 1122::0033::2244 PPMM vi CONTENTS 10 FROM FLY TO HUMAN: UNDERSTANDING HOW COMMENSAL MICROORGANISMS INFLUENCE HOST IMMUNITY AND HEALTH 255 June L. Round 11 INSIGHTS INTO THE HUMAN MICROBIOME FROM ANIMAL MODELS 273 Bethany A. Rader and Karen Guillemin 12 TO GROW OR NOT TO GROW: ISOLATION AND CULTIVATION PROCEDURES IN THE GENOMIC AGE 289 Karsten Zengler 13 NEW APPROACHES TO CULTIVATION OF HUMAN MICROBIOTA 303 Slava S. Epstein, Maria Sizova, and Amanda Hazen 14 MANIPULATING THE INDIGENOUS MICROBIOTA IN HUMANS: PREBIOTICS, PROBIOTICS, AND SYNBIOTICS 315 George T. Macfarlane and Sandra Macfarlane INDEX 339 ffttoocc..iinndddd vvii 22//77//22001133 1122::0033::2244 PPMM PREFACE The human body is a marvelously intricate machine, and the parts list includes tril- lions of microbial cells that colonize epithelial surfaces such as those found in the mouth and gut. There is increasing evidence that these microbes do more than just reside on tissues—they play key roles in human physiology and organ function. Indeed, there are 100 times more genes in our microbiome compared to our human genome, and these microbial genes code for proteins that impact diverse processes such as digestion, immunity, and development. The goal of this book is to provide an overview of the microbial diversity found in humans and to describe efforts linking microbial communities to human health. Attempts to understand human- associated microbial communities were given a boost by Human Microbiome Project (HMP) initiatives in the United States, Canada, Europe, and Asia. The National Institutes of Health in the United States has devoted more than $150 million to support these studies that are now maturing with release of data and a fl urry of publications. This is a time of unprecedented discovery, and although still young, the fi eld is suffi ciently advanced to warrant a book summarizing progress. Answers to many questions are now emerging. How do microbial communities differ across body sites? What is the variability in microbial composition across healthy and diseased humans at the same body site? How do certain microbial com- munities foster healthy tissues? What are the microbial community profi les associ- ated with disease states, and are these communities markers of disease or causes of disease? How can microbial communities be manipulated to optimize health and minimize disease risk? How do microbial communities change over the course of human development? What are the internal factors (genetic, anatomic, hormonal, physiologic) and external environmental factors (diet, sexual activity, hygiene) that shape human-associated microbial communities? With partial answers to these questions come many additional questions about the intimate relationships between human and microbial cells in our bodies. Our excursion into the human microbiome begins with an introduction to the Human Microbiome Project by Lita Proctor and colleagues from the NIH (Chapter 1 ). This chapter provides an excellent description of the HMP with its many research initiatives and early progress. It also provides some historical context and a vision for future research. We then shift to chapters focused on tools for studying the human microbiome, including a methodological overview chapter (Chapter 2 ) by Christine Bassis, Vincent Young, and Tom Schmidt that lays the groundwork for later chapters. Bassis and colleagues compare different genomic cultivation-inde- pendent methods for characterizing microbial communities, highlighting the advan- tages and limitations of commonly used techniques. They also consider the role of cultivation methods in the genomic era, and provide advice about designing micro- biome studies. In their chapter, Susan Lynch and Eoin Brodie delve more deeply vii ffpprreeff..iinndddd vviiii 22//77//22001133 1122::0033::1177 PPMM viii PREFACE into the use of phyloarrays for microbial community analysis (Chapter 3 ), a tech- nique that has some distinct advantages for microbial community profi ling. Manuel Lladser and Rob Knight round out our methods section by providing a mathemati- cal perspective on interpreting microbial community structure and diversity. This chapter (Chapter 4 ) is highly relevant in the era of high-throughput sequencing of phylogenetically informative microbial gene sequences [generated by polymerase chain reaction (PCR) or from metagenomic methods] for describing microbial populations. We then begin a tour of various microbial niches of the human body, such as the gut, respiratory tract, mouth, and genital tract. These chapters highlight the dif- ferent microbial populations found in different human tissues, and describe how microbial communities change with conditions such as gingivitis, infl ammatory bowel disease, and bacterial vaginosis. Dan Frank and Ellen Li start this tour with a description of how host genetics (immune response) and the gut microbiota may interact to facilitate Crohn ’ s disease (Chapter 5 ). They also introduce the concept of dysbiosis that will be used in other chapters. Edith Zemanick and J. Kirk Harris then describe the microbiota of the human respiratory tract (Chapter 6 ), focusing on the normal microbiota and alterations in conditions such as cystic fi brosis and ventilator-associated pneumonia. The oral microbiota is described in a chapter by Angela Nobbs, David Dymock, and Howard Jenkinson (Chapter 7 ). Here they note some of the physical and metabolic interactions among the 600 different bacteria species that live in the mouth. They also describe the connections between some oral microbial communities and local conditions such as caries, and systemic dis- eases such as endocarditis. Laura Sycuro and myself review the genital tract micro- biota of women and men, with a particular focus on the condition bacterial vaginosis that is associated with numerous adverse health outcomes in women and neonates (Chapter 8 ). Alexander Swidsinski and Vera Loening-Baucke end this section with a chapter on use of in situ hybridization methods combined with fl uorescence microscopy for describing the spatial relationships of microbes and human cells in the gut. This chapter (Chapter 9 ) is notable for moving beyond the description of “who ’ s there” to a description of the structural and functional features of the gut microbiota. Note that not every human body niche is covered in these chapters. Two chapters focus on the use of animal models to manipulate the microbiota and understand how changes impact health. June Round tackles the use of a variety of animal models to study host immunity, including the fruit fl y, zebrafi sh, and mouse (Chapter 10 ). She highlights key lessons that can be learned from these models regarding human immune responses to our indigenous microbiota. The team of Bethany Rader and Karen Guillemin describe new insights that have been produced by animal models, including fi sh (Chapter 11 ). Several important ques- tions are more easily answered with animal models, including how microbial com- munities assemble in space and time, and identifying the relative contributions of host genetics, environmental factors (such as diet), and stochastic sampling. Microbes and animals can be studied in the laboratory in ways that are not possible in human studies. The use of cultivation-independent methods suggests that many human-associ- ated microbes still resist cultivation in the laboratory. Karsten Zengler tackles this issue in his chapter on cultivation procedures in the genomic age (Chapter 12 ), reinforcing the challenges and rewards of cultivating microbes from the human ffpprreeff..iinndddd vviiiiii 22//77//22001133 1122::0033::1177 PPMM PREFACE ix body. So, how can we cultivate fastidious members of the human microbiota? The chapter by Slava Epstein, Maria Sizova, and Amanda Hazen (Chapter 13 ) provides many novel approaches for cultivating human microbes using cutting-edge tech- niques in order to resolve the “great plate count anomaly.” Finally, George and Sandra Macfarlane (Chapter 14 ) address a key issue in the microbiome fi eld: how one can manipulate the human microbiota. These investiga- tors provide a thoughtful and balanced review on the use of prebiotics, probiotics, and synbiotics to alter the human indigenous microbiota for the purpose of enhanc- ing health. What does it mean to be human? The authors of these chapters provide a com- pelling argument that we are far from alone, and that our microbiota helps mold the human form. Please enjoy the insights from this outstanding collection of inves- tigators who are unraveling the mysteries of the human microbiome. Special thanks to Sue Bartlett for helping to bring these chapters together and assisting the editors with organization of the project. D avid N. F redricks , MD Fred Hutchinson Cancer Research Center Seattle, Washington ffpprreeff..iinndddd iixx 22//77//22001133 1122::0033::1177 PPMM ffpprreeff..iinndddd xx 22//77//22001133 1122::0033::1177 PPMM