 Thank you for a more than generous introduction. I also want to thank all of you for being here and for being part of an enterprise which I think has yielded some really exciting science, insights, ideas, and a lot of momentum for a different way of thinking about the microbial world and about ourselves as well. What I hope to do in the next bit of time is to talk a little bit about some of the history and some of the ideas that have led to our current perspective on humaneness and our relationship to the microbial world and tell you a little bit about the themes that are shown here, diversity, stability, and resilience, but mindful of the fact that many more will follow me, many of whom are more than qualified to talk on any of these topics and so I will hope to simply introduce some of these items for you. I want to just start by reminding all of us that our perspective on the microbial world has been very much colored by history and by overt events that draw attention to themselves such as pestilence and pandemics. This is actually a painting of Bethesda in the 1940s, not really, but might have been actually. But it is a reminder of the way in which we have become aware of microbes around us and what that means for even the language about how we talk about our relationships with the microbial world. The fact is, though, that microbes almost to the single microbe on the planet live in communities and engage in beneficial activities. This is the rule. The last is very much the exception and despite all of that even today we still are gripped with fear and anxiety about the microbial world and I don't mean we in this room but our colleagues and family members out across the planet and certainly in this culture. You may not be able to read the caption, but I see it, but it scampers away from the light and there's still this mindset that the only good microbe is the dead one and that we mostly have much to fear and not much to gain from an encounter. This has been brought to light in a number of fora and I just wanted to highlight a few because this does have some history and it's sometimes well worth reading. This is a work by Theodore Rosebury published this book in 1969. It's a wonderful book by in fact the man for whom the wonderful genus of bacteria Roseburia was named the great butyrate producers in the human colon. But Rosebury was in many ways a pioneer and pointed out what I think we now recognize at least those of us in this room that in this nation and others especially in the developed world we are now a population of tub scrubbed and deodorized neurotics and he talks a lot about the vastly more beneficial kinds of relationships we have with the microbial world than we do with the harmful kind. In fact really takes us a bit further and talks about psychopathology and maybe its role in the origins of our thinking about fear and loathing of the microbial world talking a lot about anal and attentiveness and anal neuroses and sites Freud for a number of chapters. Well worth reading. They're a bit of history and I don't mean to dwell on this but of course the first observations of the human microbiome go back to 1683 and then have an interesting and checkered history through the subsequent centuries including I think the important reference to an observation that was made in the 1930s so called the great plate anomaly where there was an obvious disconnect between the number of cells that could be seen under a microscope and the number that could be grown and recovered as colonies on a plate and this then I think in my mind anyway led to an application to habitats of greater familiarity to us such as human. Renee Dubot in fact in the 1960 talked about the communities of organisms in the human body and their co-evolution over time with us and then shortly thereafter again the application of these older microbiological concepts to the human body with Moore's work right here outside in Virginia at VPI and then Dwayne Savage and his work and others in showing us that there were in fact so many more microbial cells associated with human than one might have thought in fact many more than we have cells of our own human kind. In fact the 10 to 1 ratio comes from the 1970s so why the interest today in the human microbiome. I would argue there are probably several factors one of which is this growing strong compelling sense that these microbial communities that make the human their home are a critical component of human biology. There is of course the growing evidence that these communities play roles in health but also in disease and we're now understanding that better. There's also the clear evidence that these communities help to determine an aspect of human individuality and that's of course a fundamental interest to many of us not only as clinicians but simply as living being sentient entities and then as you have already heard from Dr. Collins the whole notion that we are a collective and a meaningful collective not a random collective as I and many others will point out. Another factor is the opportunity to engage in approaches that would be provide novel therapeutics or preventive measures and the opportunity to apply what are now some very exciting kinds of science and ways of thinking to a topic that as I have just pointed out is really an ancient topic but it's a convergence of disciplines of technologies and of concepts. So the benefits that we now ascribe to our microbiota are numerous. These are some, they are familiar to many of you. Some of these may be less familiar and all of these will be addressed in subsequent talks especially later today and tomorrow. So I certainly won't dwell on these but simply remind you of some of the somewhat surprising aspects of what it is to have human physiologic properties that in fact can be written or attributed to the microbial world that lives within us. The other part of true commensalism or mutualism rather is the two-way benefit aspect and this is a part that we haven't talked about so much that is the benefits that our communities derive from living within us. They can be encapsulated in these three areas, nutrition, habitat, dispersal but there probably is much more that we could learn and should be learning in fact about the benefits that they derive because of course if you believe in at least half of the last slide and those attributes then it would behoove us to understand what it might take to nurture and sustain the communities that are providing these benefits to us. I mentioned the idea of disease association with the microbiome. This is an area that as you all well know is very much in the forefront today and certainly has led to a lot of associations where the idea might be that instead of a single organism as the pathogen it's the net effect of a community which has now become disturbed in some fashion. I think we have some evidence for such a concept in certain settings unless in others of course. Here are some that are associated but I will just hasten to remind all of us as we strive to understand what might be the other members of a list like this that in fact the issue here is how do we clarify the nature of these associations? Can we in fact impute a causative role to these communities, these disturbed communities or in fact are they the result of pathology that was produced or created by some other means? Or in fact is there no meaningful relationship, direct relationship between pathology and an altered microbiota? I think all of those three possibilities in that top line are certainly possible for some of those disease states as is the idea that if there were a causal relationship it might be of one of several different kinds including the role in initiating pathology as well as a role in simply propagating or contributing to ongoing pathology which I think is much more clearly a possibility in some of those disease states such as inflammatory bowel disease or any situation in which for whatever the reason one has an inflamed environment and now you have selection for certain clades of organisms that do very well, that compete very well in those conditions like the gamma proteobacteria and they then now begin to propagate their own form of inflammation for their own benefit but also as a very contributor to the pathology. And then remember if there is a causal role either in initiating or propagating the community may not have a necessary role, it may not even have a sufficient role, it may be neither and still be contributory. So this is clearly a complex area I think it's one that certainly deserves a lot more thought right now about how to address these important questions. In thinking about getting up here at the beginning of the first day worried about terminology I have this funny feeling in gazing out and seeing a lot of familiar faces that some of these terms may be very well known to most of you so perhaps those for whom these are not so familiar can read these faster than I can read them but I just want to point out that much of what we know today is based upon one way of looking at a complex community that is using a method that infers something about the phylogenetic or taxonomic composition of a community rather than some of the other features. And this is of course because approaches like 16S or other marker gene based survey capabilities are among the capabilities most available and easy to apply to many samples. But remember that there are other approaches many of you of course leaders in these other approaches but they include the means of assessing genetic potential or functional potential by doing metagenomic analyses as well as other kinds of so called downstream genomic approaches all of which get us a little bit closer to what's actually happening in the way of function but don't quite get us all the way there and that's something that I certainly will remind all of us about towards the end as I get to my goals and needs and challenges. So the questions that I want to address very briefly are the nature of the relationship between diversity in states of health of course disease matters here as well I'm going to spend more time on health how and to what degree is the microbiota stable what do we mean by stability really and especially during adulthood I am looking at the program and making certain assumptions thought perhaps that Ruth lay who follows me would talk about childhood and that may or may not be the case but anyway and then thirdly is the microbiota and the microbiome resilient meaning how well is it able to absorb a disturbance and recover or sustain its original functions. So first a little bit about diversity again mindful that you're going to hear much more about this I start with it with I think what is a pretty simple question but it has a complex answer because when one thinks about diversity and biology you have to ask well what is the aspect of diversity that might matter and how might you go about measuring that. So do we care about individual organisms meaning say strains or single cells do we care about clades of organisms do we care about their more fundamental features like their genes or their pathways or their products or activities and are we talking in some cases really about the community as a net some entity and really interested more in the comparisons between communities or between populations of hosts that bear multiple kinds of communities and then there's the issue of abundance and I think the answer is yes it really depends upon what it is that we're trying to understand that then determines what aspect of all of this diversity we might be most interested in measuring. Now what we see most are assessments of diversity that again take the taxonomic or phylogenetic compositional approach and classify at high levels of the taxonomic scheme meaning at the phylum level and that's of course because it's the easiest and the most straightforward and the thing for which we have great reference databases and so you end up with pie charts and stack bar graphs and other assessments of compositional state based upon the phylogenetic composition and in this case it's phylum and it's color coded by phylum and it makes the simple point that despite the fact that on the planet around us there might be something on the order of a hundred phyla level categories of organisms there are many many fewer of those categories the phylum categories within the human body. You see some of the similar colors across body sites but it's still a low number in total and it simply you know begs the question well why what is it about being a human that that leads to a very select subset of bacterial in this case bacterial diversity that that is found within the human body and I'll mention here something I glossed over which is that when we talk about diversity we're certainly interested in more than just the bacteria we're interested in viruses as you will hear about later today we're interested in archaea which about which we know something and we're interested in microscopic eukaryas as well but but again everything is not everywhere and even when one talks about body sites or body habitats this is a study from 2009 from Rob Knight's group Liz Costello and was in fact one of the first or the first to use modern techniques for assessing phylogenetic composition and then sampled on the same day multiple body sites within the same healthy individuals these are adults the clustering is is color coded based on habitat body site and what this tells us is that there is a very distinct compositional state that each of the major anatomic regions of the human body and you've already seen this many times and and heard it just this morning already even within the so-called body regions or body sites there are categories of habitats that might not have been apparent by just simply gazing at anatomy textbook this is work from Julie Segre as you well know showed that the skin can be divided into three kinds of habitats perhaps as distinct as would be desert grassland and forest to a plant ecologist but in this case it's sebaceous dry and moist now of course clinicians know something about these clinical categories as well but they also know that there are disease associations that track with these kinds of skin habitats and this sort of work in in understanding biogeography may have some important connections to our understanding of disease propensity disease location and disease causation with the possibility of interventions I mentioned that that really there are many lenses with which to look at diversity and this is a point well made in one of the HMP consortium papers from last year this figure and all you need to see is color on the top set of panels looks at body site each of those major panels going left to right and multiple individuals at each of these body sites and those of the small little columns within each of the panels the top set of panels shows a great deal of variation in the compositional state when looking at phylogenetic composition the bottom set of panels are the same habitats in the same individuals in the same samples but they were sequenced using a metagenomic approach and now they're assessing in a sort of high level fashion the genetic potential or the functional categories that are that are inferred from metagenomic data again for the same samples and body sites and what you see in the bottom is a much more even swath of color based upon these categories of genes and that tells us that even though there appears to be a great deal of diversity at the level of tax on strain species genus as shown here there is perhaps more even distribution of diversity at the level of genetic potential now Curtis Huttenhower who will speak later today will tell us a little bit I think about the insights that are revealed from work of this sort that now tell us a little bit about what is it about each of these body sites that either selects for or allows certain clades of organisms to do their things to demonstrate these different features of genetic potential because there is sort of segregation by body site with respect to the kinds of pathways and gene types that are that are displayed by the organism found there this color coding is based on body site and it reflects modules of genetic potential that tattoo is actually a tattoo that that dr. Collins proudly displays not really so just to summarize what are the sources of of variation diversity well it's body site habitat it's individual data haven't shown it's the health status of that individual are they healthy are they sick in what way are they sick it's genetics which is of course part of individual but so is environmental exposures and environmental history and life history and that encompasses these other features and that's all a part of what it is to be an individual having been born with a certain genetic compliment and then experiencing life in whatever ways and whatever places one does so what about stability how and what degree is the microbiota stable what's the relationship of stability to age age of the host and to immuno competence what are the determinants of stability and what do we really mean by stability to come back to an earlier question at the risk of maybe over playing a certain kind of imagery but because I think it's it's useful nonetheless I'm showing you a fitness or a stability landscape something that ecologists like to think about and use as a way of visualizing the state of an ecosystem the ecosystem is in some ways displayed as a circle here with a community and its position on this landscape is a reflection of how stable it is or is believed to be at that given time the deeper one of these valleys might be and the steeper the walls and the more narrow the more stable is the community but the landscape is not just one valley it's many and the and the terrain that separates valleys is varied and variable and so there may be multiple stable states called alternative stable states and they may be also associated with health or with disease but when we begin to think about stability and resilience it's this kind of notional idea that I think helps to help us to understand what it is we need to know even in the clinic as I will end with now despite the the significant important contributions of the first phase of HMP it provided relatively limited views of of temporal stability there were only a few time points selected at least in the the initial set of 300 subjects there are in fact some important but very limited examples of dense time series of sampling of humans and I just want to show you a few this is a study some of you know well it's again work of Rob Nights Greg Caparoso in his lab published two years ago two adults sampled daily as you can read six and fifteen months respectively and they were sampled at each of these three kinds of body sites the blue is guts fecal samples or poop samples as we like to say the green tongue swab samples and then the bottom yellow and reddish and orange is skin palm palm swabs the the x-axis is time and the y-axis is principal component one the value of any given community along that particular projection of the variability in the data so for those of you familiar with principal coordinates analysis this is old hat for those of you not the position here at least on the y-axis in this display and I've already shown you a few of these other displays in two dimensions indicates the relative relationship of one community to another with respect to in this case it's phylogenetic composition overall and the and the and the branch length within each of the communities and it's and the differences so this figure from the paper suggests that body habitat is coherent over time that's nice to know resuring it also shows some variability on a shorter time scale and it shows relative degrees of variability between each of these sites skin showing greater variability for example now if one looks at the same data in a slightly different way in this case by plotting on the x-axis time interval so this is the interval between any two samples pairs of samples and the y is again an ecologic distance measurement in this case it's the break Curtis or a derivative of the break Curtis distance now one sees instead of a relatively flat line a gradual upslope and the upslope indicates the apparent increasing dissimilarity between any two communities the further apart in time they are now these are adults and these are the data from the stool samples but the same is true for the tongue and for the skin the slopes are slightly different this slope is the steepest by the way it suggests the possibility in any case it's just two people and one way of looking at the data but it suggests the possibility that there's drift in the compositional state of the microbiota during adulthood now you may ask well maybe it doesn't really matter and it might not but this is another way of looking at the data now color coded by 45 day period during the course of collection and the first 45 days is the red towards the top and you can see this arrow that time is causing a shift in the position of the fecal communities downward and a little bit towards the right but interestingly it's also showing a much more transient much more dramatic excursions of the compositional state out towards the left within any 45 day period there are few days here and there when the community is very different meaning a much different PC one coordinate score than it typically has and I'll just tell you that when you look at the organisms that drive the separation of samples on this display it's along PC one it's a bacteroides predominance that takes you to the right and a prevatella and related organism dominance it takes you to the left so this would suggest that in a one given healthy adult during the course of a week there might be a day or two when instead of having a bacteroides dominant microbiota there's a prevatella dominant microbiota and for those aficionados this has some significance since these are the dominant types that help define some of the so-called intro types so we may have a a tendency towards one dominant type but we may spend days a few days here and there with a different type for reasons that at least here are not so clear this issue of stability is of course been addressed by others and most recently by Jeremiah Faith and Jeff Gordon's lab in this paper that's already been highlighted this morning and here one sees a decay in the degree to which one sample shares strains with another sample within the same individual across increasing time intervals but you can see this down slope tends to flatten off and in fact it follows a power law distribution such that one can make a prediction about how many strains might be shared within an individual if one were to look out over time intervals longer than the maximum here which is about five years and suggest that in this particular case through this particular lens of identifying strains that an individual probably retains a fair number of strains over long periods of time which is a again an important concept and one that of course certainly bears corroboration and further study strains probably are an important way of looking at diversity as I've already suggested perhaps in my previous comments and I just want to put one slide up that takes us back to childhood well doesn't take it takes us to the topic of childhood not me to my childhood these are daily samples from a particular time period in the life of one premature babies it turns out between days five and twenty four and the colors are taxonomic composition based on genus so just from the back of the room there are three relatively stable states is defined at the genus compositional level you can see several days with one a number of days in the middle and then number of days at the end and I won't talk to you about the details here except to remind you and perhaps lead to Ruth's comments about the model that appears to be at least relevant to childhood early childhood which is a model of punctuated temporary equilibria it's a model that's been certainly well studied and understood by ecologists and other systems this is a model that might apply to early childhood but I'm showing you this because in that last that third state one out here we in collaboration with Jill Banfield had opportunity to do deeper sequencing shotgun sequencing of several time points during this period when it appears this relative stability at least at the genus level and now what we see are very significant shifts in the relative abundance of two strains of the same species of Citrobacter in divergent manner the blue one is going down on day eighteen and back up by day twenty one the yellowish one is increasing in abundance and these are strains that that differ by less than the say the four percent overall sequence similarities that was used by Jeremiah faith and last paper I showed you and yet reflect differences that may be functionally important like deletions in intergenic regions that encode putative small RNAs or differences in the abundance or or composition of genes that encode for regulatory elements and adherence factors so we don't know if this matters but we wouldn't have known that it even happened where we not to look at diversity in this way and this gives us again another aspect of another feature of of stability so I'm going to turn finally to resilience and disturbance and just again give you a few highlights we have tended to think about disturbance as a negative thing something that does harm but in fact from an ecological point of view it can do good because it provides opportunity for replacement or for or for various kinds of turnover that may be favorable or at least potential for favor for a favorable outcome and yet we know that disturbance is is much more common we think in today's world for our microbiota than it used to be although again there may be aspects of lifestyle that we don't fully understand in days gone by and and there may have been plenty of disturbance of other sorts when we talk about resilience we're talking about again about the capacity of the ecosystem to absorb a disturbance and retain function or structure or some other feature that we think is important so on this notional landscape scheme the the the feature resilience is proportionate to the the width of the valley it's also proportionate to the the time for a community to recover from a disturbance which again might be the steepness of the valley so again just part of the the imagery here and and the ideas and concepts I often turned to to buzz haulings for for some of the most interesting early on discussion of what this really means for an ecosystem to very quick just snippets of of data here this is from a study that Katie Shaleff has been has undertook in my labs now writing up this was a study of of time series data at four different subgenival pockets in the human mouth for the mouths are healthy for were disease with chronic periodontitis and you can see the time series here begins two weeks prior to a major disturbance and then three months after and the major disturbance was this sort of medieval torture event that we all undergo when we go to our dentist and have cleaning done and scaling of our teeth so these are the data for two mouths the four sites and each the red sites are the four sites from a periodontitis mouth the four blue community samples are from a healthy mouth and these are their positions in again two-dimensional space on a principal coordinates like plot and this is their starting position two weeks before so what then happens is an interesting sort of aspect of dynamics which is this this is what's happening over the course of the two weeks before the disturbance the the disease sites from this one mouth have sort of deviated and and and moved off to to the right and upwards and the blue ones downwards and to the right so the overall effect of the two-week average data is to define what is a slightly different disease compositional state than than a healthy one which may not have been apparent at any one point in time so now comes the the cleaning and here's what's happening to these sites they clearly show a disturbance but a relatively rapid recovery this is seven days now and even by three days there was near complete recovery so what do we learn from data like these well there is not only site specificity to the patterns of diversity but there's site specificity to the response to the same disturbance there in fact and Katie's now looked at all of the mouths and and a lot more data there is a larger response or displacement or disturbance at sites with greater clinical disease scores at the start there is relatively rapid recovery in all cases much the dismay of dentists and but the possibility that might be able to predict the like