 Hi, this is Julie Segre. So today I'm going to talk about my own research project, specifically examining the microflora, which are the bacteria, the fungi, the viruses so on that live on your skin, and also give an overview of the larger project. So what's really important to remember about this and on slide two, I don't seem to have control over the slide, Sarah. On the next slide of this presentation I wanted to, okay, I wanted to introduce some of the goals of the larger NIH Roadmap for Medical Research Human Microbiome Project. And what's really important to realize here is that we tend to think of ourselves as humans that we're these human cells, but in fact we're really made up of human cells that are living together with bacteria, fungi, and other small microscopic organisms. And it's really remarkable because in and on our bodies these microbial cells actually outnumber the human cells by about ten fold. So for every human cell that you have in your body you actually have ten more microbial cells. But it actually, it's not that they outnumber us, but they don't actually outweigh us because each human cell has about a thousand times more DNA and is about a thousand times bigger or at least a hundred times bigger than a microbial cell. So we actually outweigh them even though they outnumber us. Now as an example the bacteria that live in the gut and the intestine, we think that because of this microbial diversity and this large number of bacteria that the bacteria and the other fungi and the other small organisms, they probably have a hundred times more genes than our own human genome. So there's a tremendous amount of diversity and this microbiome is really the, microbiome means the total microbial DNA is really an important part of our genetic landscape and also of our bodies. So for example when you take a drug you probably think that it's your human cells that are metabolizing the drug, but in fact the bacteria that live in your gut are part of what metabolizes this drug. And so it's really important for us to understand the complexity of our human DNA but also to understand the complexity of our microbial DNA. So the overall goals of this project are really the human microbiome project are really to understand how the microbiota, small organisms interact with our human cells and how that maintains health and disease. So I just talked about drug metabolism by a bacteria, but of course these bacteria also break down food and aid and digestion, and on our skin they can break down the proteins that are made by the human cells of the skin and create sort of a, you know, linkage to creating natural moisturizing factor that keeps your skin supple and smooth. It's true on the skin, they also have waste products that create part of what we think of as body odor. So, you know, we have to think about the full range of how the bacteria contribute to our health and to disease states. And that really is the goal of the larger human microbiome project written here. We are bringing in 250 normal individuals and sampling them at five different body sites, the gut, the nose, the oral cavity, the vagina for women, and the skin. And this is giving us a baseline so that other investigators, including myself, are then doing clinical trials where examining the skin microbiota in people with common and rare skin disorders. But in order to know what the differences are in a disease state, we have to first know what is normal, how much variation there is between people, and how much variation there is between different sites of our body. In order to do this, we are assessing the microbial diversity of 250 individuals. And then skipping ahead to the third point here, our ultimate goal is metagenomics. Let's say that I could just scrape the, you know, the dead layers of my skin off that like sort of white, flaky stuff that makes those dust bunnies in your house. If I could scrape off those dead cells or could take, you know, a swab inside your nose, I'd like to just directly sequence that. But the bacterial diversity is so complex that we need to take some baby steps to get there. And that's why point number two is that we're sequencing bacterial reference genomes. So that means that we're determining the entire bacterial sequence for different isolates and using that to then form a springboard for future microbial studies. Because with metagenomics, where we analyze the combined coding potential of a complex mixed environment, that's what we'd ultimately like to achieve. And we'd like to achieve that as stated in point number four, to correlate the changes in the microbial community with disease states. And another arm of this study is really to explore the ethical, legal, and social implications of this new field of research. Because every human genome project that we embark on, we devote time and energy to understanding how this information is going to be communicated to the public and how this is going to be absorbed by people and understanding that there may be a contribution of their microbial communities to their health or to their disease. And as such, I'll get to, at the end, what are really the questions that we'd like people to think about in terms of how this research is impacting their own lives. So let me now take you into my laboratory's specific research project, which is to really understand that the skin is a barrier to infection of pathogenic organisms, but the skin is also an intricate home for microbes. And those are what I'll call the commensal microbes, or the healthy microbes, the good microbes, the ones that just normally live on your skin and serve the role of breaking down human proteins and keeping your skin moist and also serve the role of keeping pathogenic organisms from being able to adhere to or attach to your skin. So when people think about the skin, they, you know, it's actually, I show you here a cross-section of it. And underneath your skin are these blood vessels, which are the red and the blue, and you know that because if you scratch yourself, you can bleed from your skin. Overlying that are these orange cells, which are really the stratified epidermis, the top layer of your skin. And these go through a process where they lose their nuclei and they become dead cells on the top of your skin that provide the skin barrier. That's what you see on the upper layer of your skin. Well, this is really, we think that there is a relationship between the skin cells, the immune cells that can sense if there has been like a wound or an abrasion, because as you think about it from an evolutionary perspective, of course it's very dangerous to have an open wound. You could have a bacteria get in and your body could, you know, develop sepsis. Well, now of course we have antibiotics to treat that. So it's less of a risk, but antibiotics have only been around since the 1940s. So the body still responds as if this is a tremendous risk. And so the third type of cells that we'll be talking about today are really the microbes, these small microscopic organisms. Well, we ask the question about how do you know what microbes live on your skin. And the way we traditionally do this is by looking on petri dishes and seeing what we can culture. Well, there's been a new method that's been developed, but this is new and old and just being altered so that it has greater specificity. What we do is that every bacteria has a 16s rRNA gene. Now the 16s r stands for ribosomal. It's a ribosomal RNA gene. It doesn't get made into a protein. What it does is it stays as an RNA and it helps guide other RNAs through the ribosome. The ribosome is where proteins are made, so mRNAs are translated into protein. This stays as just an RNA and is a guide. But if you examine the sequence of the 16s rRNA, the ribosomal RNA, which is shown on the left here, what you see is that it actually has the stem regions, which form a lot of double-stranded base pairing. And from those, that actually puts a fair amount of conservation constraint on those base pairs. And then there are the loop regions, which are more variable. Now, when we look at the sequence of the stem regions, those are conserved and that serves as what we consider an evolutionary clock. So if we looked at the conserved regions, we can say, well, there is some change in them, but that allows us to say this is a staphylococcus, this is a streptococcus, and assign what type of bacteria it is. The sequences in the loop region will change even faster than that. But these sequences of the 16s gene allow us to identify what type of bacteria it is. So the orange highlighted sequences are how we amplify this gene out of a bacterial genome, and also we use the purple sequences. Those are highly conserved, and then we examine the intervening sequences, and that allows us to identify what type of bacteria this is. Now, here I show you an example of how we compare the data that we obtained with our DNA survey sequence identification with our culture methods. So what we did here was we had healthy volunteers come in, and we looked at two different sites of them, and actually we did 20 different sites of them, as you'll see, but I'm just showing you the examples from two different sites here, from the face and from the belly button. Now, on the right side of each bar graph, you see what we found when we brought these swabs down to the microbiology lab and tried to culture everything that we could. Well, the dark blue is called propionobacterium from the face, and that's an oily, loving bacteria, and so the skin is a little bit oily, and that would make sense that those are the types of bacteria that live there. As well, the orange is the staphylococcus. That's like staphylococcus epidermis, which is one of the most common healthy bacteria that we can grow. And if you compare what we found on the face from culture versus survey, you can see that we did a pretty good job, but we completely lack the cornflower blue and the lighter blue. Those are actinobacteria, and you can see the chart on the right-hand side. We failed to culture them. These bacteria are actually the carinobacterium and other actinobacteria. They're very hard to culture because they're very slow-growing. Sometimes we grow them after about six days, but by six days, the staff and the propionobacterium grow so well that they've almost overgrown the culture plates. And you see this even more when you look at the samples that we collected from the belly button of this person. Well, culturing, it seems like the orange and the red are the firmacutes. So those are the staff and the strep and other bacteria that fall under the greater taxonomic name of firmacute. And so that's what we can culture from this person, but when we look at the survey, what we see is that really 50% of the bacteria are the carinobacterium. And as I said, we just had a very hard time culturing them. So the take-home message from this is that we can get greater information and different information if we use these DNA-based surveys that have less of an ambiguity to them and there is less of a bottleneck that the strains go through. So the information is more precise. Now, let's talk about how we would use this type of information. Well, I'm just going to show you one example of an animal model that we made, and then I'll get into our studies with human subjects. So as you can see from this mouse, this mouse has scaly skin, and you can best see it on their ears. In the bottom corner of the picture of the mouse, they show you what a wild type or what a normal litter mate would look like, and you can see their smooth skin on the ear. When you look at the histology of this mouse, you can see that the dark purple and pink layer on the top is thicker in the mutant mice. And then it has where the arrow is pointing is that sort of thick basket weave, and that's the scale. So the question is, is this related to, you know, how does this then contribute? How does the bacterial community contribute to this? Because we were using this as an animal model of the very common eczema, which has that sort of scaly rash-like skin. So now here I show you what is the bacterial survey that we find by sequencing. Well, if we consider this an animal model for eczema, one of the things that we know is that in eczema patients, there's a large amount of colonization by the staphylococcus. And now the staph is the firmacute, so that's the third over. And what you can see is that the mutants have about 11% firmacute. So they do have an increase in firmacutes over what one bar over you see is the wild type litter mate. So that's what we pick up when we culture. These mice have an increased amount of these staff and strep and other types of firmacutes. But in fact, even though that's what we pick up on the culturing, that's not the whole story because if you move over to the actinobacteria, what you see with these mice is that they have a huge increase in the crinobacterium, which are shown as the green bacteria. Well, I just told you those are really hard for us to culture. So if I didn't know to look for them, which my survey data tells me that there's an increase in crinobacterium, I would think that there's an increase in the firmacute. And I would mystify that there was an increase in the actinobacterium. And perhaps when you give antimicrobial treatment, you know, antibiotics, you decrease the amount of staff and you think you've cured, you know, the person's microbial disorder. But in fact, maybe it's really acting on the crinobacterium. And so that's an equally plausible hypothesis. From this data, I can't tell which is more likely to be causative to increase in firmacutes or the increase in actinobacterium, but I now have two hypotheses to test. And in fact, I have a third hypothesis to test, because if you look at the first set of bars, the proteobacterium, what you see is that we've had an increase in firmacutes and an increase in actinobacterium, but they have come in and they have selectively pushed out the pink bacteria, the pseudomonas. If you look at that bright blue bacteria, that genfinobacterium, that's about 35% in the normal littermates and about 32% or 31% in the mutants. So those levels have stayed the same as have the other components of that bar, the dark blue, the orange, and the yellow. What has changed is that the pink, the pseudomonas have been pushed out. Well, what about if those pseudomonas provide some beneficial effect to this skin? Then that's a third hypothesis to test. So it's really about generating more ideas to test. So now let's look in human skin. And as I've shown you before, there are different levels to the human skin as there are to mouse skin, and this is the histology. So to assess things from the human skin, we took a swab, which has a very superficial bacteria. Then we scraped off without drying blood, we just scraped off the dead cells from the top of the skin, and those tell us what lives inside that scrape. And then we took a punch biopsy from a few sites to look at the full thickness because there are bacteria that live all the way down into the hair follicle that goes all the way down into your thick, into the dermis. And what we found was that if we used a swab, we could recover 10,000 bacteria per square centimeter. If we used a scrape, we could recover 50,000 bacteria per square centimeter. And if we used a biopsy, that yields 1 million bacteria per square centimeter. So that means that when you're washing your hands, you know, like a swab would be, you're really only removing 1 in 100 bacteria. When you're scraping, which I promise you is, you know, less... I mean, it's less harsh than even washing your hands. You can remove about 1 in 20 bacteria. But it's these bacteria that live down in the hair follicles that can then come back and repopulate, and those are the healthy bacteria. So we looked at the 20 different sites on the human skin to say, and now this is all done with scrapes because we decided that scrapes were as effective as punched biopsy and certainly less invasive. So when we look at the scrapes, what we see is that there's a great variety of bacteria. But in fact, the bacteria are determined by where you are on the skin. So the blue sites are what we consider oily sites. And those are very similar to each other. They share a lot of the dark blue and light blue bacteria called the propionobacterium that can live on the lipids. But then what you can see is in the middle of the body, there's a lot of moist sites that are more sweaty. And in those sites, those have a lot of the green bacteria, the proteobacteria. And then you can see the cornflower blue, which is the kind of bacterium, more at the base. There also are sites that have a lot of the orange bacteria, the staphylococcus. And what we found was that the human body is really an ecosystem. So imagine it like a dry desert, but then there are sites that are streamed. Those are the moist sites, the creases, and then there are the oases. Those are places like inside your nose or inside your umbilicus that really just harbor a huge amount of diversity. And what we found was that the sites like the left arm and the right arm are most similar to each other on the same person. But then my left arm is most similar to your, or my arm is most similar to your arm. And my arm is more similar to your arm than my arm is to my chest region. And I've given all the anatomical correct terms, so I, instead of saying chest, we call it manubrium. But you can see here that the manubrium, which is five sites down on the left, is more similar to the side of my nose, because those are both oily sites than either one is to the axillary vault, the underarm, which is considered a moist site and not an oily site. So this is another example of this, of the data is just shown here on the next slide where what you see is that the back of every person is very similar. Rhetoricular crease is behind the ear. It's very similar to each person, although you can convert it. So you can see that healthy volunteer number four, that site has been converted to being really staff colonized. And that's a term that you sometimes will hear from your physician if you have a sort of what we call really below threshold infection, where this is, you know, it's not causing you any problem, but that site has become overgrown with death. You can see the anti-cubital crease, which is the bend of the elbow is really a lot of the proteobacteria. And then actually what you can begin to see here with the nares, which is inside the nose and the umbilicus, which is the belly button, these are really diverse complex sites. And that's shown actually in the next chart, where you can see that there is a range. Now here where I show you these different anatomical sites and then they match up with a two-letter code, there are sites that are really very complex, like the volar forearm, which is the forearm has about 44 species. Each person has about 44 species living there. The site right before that is the umbilicus, the belly button that has about 40 sites on every, 40 species for every individual. Whereas some of the sites, like all the way over on the left behind the ear, the retroridular, is actually kind of simple in the back of the next site. Those have about 15 species and so there's a variation of the complexity of these different sites. So I'd like to just give us a summary how we think about the microbiome project and how we hope that this can serve as an educational tool. First of all, we'd like this to really put forth the idea that as I showed you on slide number six, if you compare the survey and the culture data, they're both accurate. When we culture, we culture multilaystaphylococcus and that is true, but that is based on culture. When we survey, we can find that there is an increase in the amount of crinobacteria. So what we understand about scientific facts is really relative to the methodology that we employed or how we determined that information. And as science evolves and we find better tools, we can also find out new things. This is a real process of exploration for us. So we can learn new things about our microbial contribution when we have new tools to examine that. And that's really the process of science, understanding what is known and what is unknown. We hope to use this project as a way to educate consumers about what is health. And in that regard, I think we need to lose the language of warfare with pathogenic microbes. We should not just think about all bacteria as bad, but remember that bacteria also do contribute to our health and that our goal should be to promote the growth of the healthy bacteria while maintaining low levels of exposure to any pathogenic microbes. And of course, our goal is also to educate physicians about how to make better diagnosis and treatment decisions. And so I'll ask you, you know, really, why is, I mean, this is sort of at the crux of it, is we have to have this relationship between health and disease. And I see in the culture right now that everyone wants to sterilize their exterior with using these hand sanitizers, but then eat probiotic yogurt or take probiotic pills. And I think we need to really balance this and to understand that our goal is to balance the healthy bacteria and the pathogenic bacteria. But it's not really just to sterilize our exterior because the bacteria will come back and it's really about promoting the growth of healthy bacteria while maintaining the pathogenic bacteria. And so finally, in conclusion, I want to emphasize that this study is a very thorough relationship and transdisciplinary investigation that is being led by the Human Microbiome Project, so HMP, but it is a scientific endeavor pursued by physicians in clinical medicine and infectious disease combined with colleagues in microbiology and colleagues who have an expertise in sequencing DNA and analyzing that information. So these are all the groups who are involved in the study, but it's really a three-legged stool between DNA sequencing, microbiology and clinical medicine. And it is the strength of that three-legged stool and the scientific disciplines that really is powering this project. So thank you very much. All right. Well, thank you, Julie. That was excellent. And I definitely learned a lot about things I never knew. So I want to open the call to any questions that we might have from the group, and I need to just let our hosts know that that's what we're doing. So if you would just hold one minute. Can everybody hear me? Well, I could. Oh, you could? Yes. Well, potentially they can hear me, but we can't hear them yet. We're just waiting to open the line to be able to ask questions. And so what we'll do is we'll have an open session. People can ask questions to Dr. Seigre. And we'll just ask that you obviously go one at a time. One question that was emailed to me, Julie, that we can probably talk about before we get everybody else on the line, is just whether there's, I think you did this to a certain extent, but whether there's more, can you translate, the question is, could you translate some of the bacteria speak into more of the diseases that we typically hear of? Right. Sort of everyday public health, kind of the laundry list that was up. Yeah. So there are several projects that are being investigated. Here we're looking at common eczema and what is the contribution of bacteria to eczema. We're also looking at patients who have recurrent infections of MRSA, the methicillin resistant staph aureus. We're looking at whether there are some people who are exposed to MRSA but never develop an infection and other people who would develop an infection. And is that related to the MRSA that they're exposed to or is that related to something else in their microbiome? Maybe if you get exposed to MRSA but you have a corinobacterium that the corinobacterium can actually keep the MRSA in check because actually bacteria have a lot of ways that they have figured out to control the growth of other bacteria. So that's an interesting question for us. Now other projects in skin are exploring acne, psoriasis, and then in the gut there's projects about inflammatory bowel disease, Crohn's disease. A lot of these diseases that are being treated with antibiotics but we don't really see a clear infectious agent so we don't really understand but we know that if we treat with antibiotics the disorder can get better. So that's what we're really trying to understand a lot of is what are the infections really, what are the antibacterials really treating. So we're looking at projects that have to do really with all of the systems, the gut, the esophagus, all of the digestive system, the oral cavity, and trying to understand really a wide variety of diseases. Excellent. Now I was told that the line should have opened so if somebody has a question and they want to try asking it, hopefully we can all hear you. Okay, well we actually, I have gotten a number of questions over email and if that's something that anybody would rather do, that's fine so I'll just ask a couple more questions and so one of them has to do with the slide, one of the last slides that you put up and that is that there has definitely been a, it has to do with the picture of the Activia yogurt that you have and why that of all kind of yogurts of all, what has made that special in terms of having a pretty big campaign and you kind of see it, everybody knows the jingle and why has that necessarily been any more special than any of the yogurt we've had before. So I think here we really need to get into what is science-based and that is what we're trying to create the foundation for, is to understand really who would respond to these probiotic yogurts and who is this really benefiting. And in the same way that we do double blind studies, placebo studies and try to test most drugs, we would like to have that same kind of science-based, evidence-based approach to other things that we consider that we're taking to increase our health. And I think that there is a great potential for understanding probiotics for many systems of our body and to really understand what on an individual level is making each of us healthy. But I just think that this is something which we really want to understand not as a marketing strategy but as a scientific endeavor. And in that same way, what we have, I mean Activa is just adding probiotic to a yogurt. But the Purell, the hand sanitizer, has really substituted now for soap and water and washing your hands. And so it's great in that we often have access to having clean hands when we might not otherwise, you know, if you're traveling or something in the airport or something like that. But we just need to, I mean, we just need to understand what these products are really doing and how they're affecting our health and how they're preventing us from getting sick. Great, thank you. Are there any other questions from participants? Okay, then I have one other question that has come up. Hear me? Oh, yes, hi. Oh, okay. This is JJ at CDC. Just a simple question. Have you all gotten to the point where you can see if natural microbiota, the composition is preventing certain infections, such as MRSA, I mean the ideal being the bacteria, the natural ones would occupy niche, preventing colonization. Have you gotten that far yet? So we have not gotten to the point where we know which bacteria are really having an effect of keeping others out. We recruit those patients to the NIH clinical center, and patients we recruit are at an increased risk for developing staph aureus infections, in some cases because they have eczema, and we recruit the entire family, you know, in to try to see if anyone else who has been exposed to MRSA is also a carrier for MRSA. And so that's really the goal of our study is to do an in-depth analysis of exactly that type of question. How do we control the transmission of MRSA and can that be controlled by other microbes and promoting the growth of other microbes? Because you can imagine that it could be very effective to try to overgrow the MRSA rather than trying to wipe out the entire bacterial colony. And so those are the goals, and even one step harder than that is going to be to figure out what is causing these diseases, but if we could even just figure out what's correlated with a protective effect, we could start to target that. So no, this project, I should have said that, this project was launched just one year ago with the goal of trying to develop the basic understanding that would enable us to treat disease and also to control infection. Great. Any other questions from our audience? So I did just receive one question from Tim Berg at Access Genetics, and he had a question about, he mentioned you had three hypotheses about skin flora and their cause for skin disorders, which is assuming that eczema is caused by a lack of some bacteria or an overload of others. And so he poses the question of what if it's the other way around, if the skin disorder is causing a certain variety of bacteria? And so I completely agree with that, and that actually takes us back to the first slide that I used to launch into the question about what are the types of cells that inhabit the skin, and there's the human cells and, you know, the skin cells, the immune cells and the microbes, and that it may very well be that the skin disease, which is caused by a defect in the human cells, are then causing the scaly skin, and that causes the microbes to be different. And if it's only the skin cells then affecting the microbial flora wouldn't change that. But we know that in the case of eczema, what are the most common treatments for eczema are steroids, corticosteroids, antibiotics, sometimes even giving the kids bleach baths so that we can just reduce the microbial load. And that seems to make the kids look and feel a lot better. So it is true that the root cause may be something in the human cells that maybe makes them make a less good barrier. But we already know that the treatments that we use are more likely affecting the microbial community than they are the human cells. So it may be that the primary cause is something in the change in the human DNA. But what I'm looking for here is how to really affect the greatest health for people with skin disorders or other disorders. And so if I can intervene and give them, you know, a topical therapy that increases their health, even if it's not getting at the root cause, even if it's just getting at some other part of the pathway, I still think that that is our goal, is to really be promoting health. And so it will be very difficult to untangle what is causing the disease, but really the outcome that we're looking for is to improve the health. Excellent. So I appreciate this. There are a number of you asking questions over the web portion of this webinar, which I think is excellent. And I'll just bring it to your attention to those of you who maybe haven't seen it. But we did get a question from Ke Chan at Boston University. The question is, are you considering the impact of environmental factors, such as the level of sun exposure or living in an area of more damp or dry area, to the relationship of human cells and microbial cells in your study population? If so, how and what type of impact would these environmental factors have? So that's another, and these have all been just great questions. And that is another thing that's just made for so complex. When we're trying to capture what is normal, of course, we are finding individuals who live in a dry community, people who, I mean, even a dry environment like, you know, living in Arizona, and people who live in a moisture community. And even that can change between if it's the wintertime and you're spending your days inside, you know, a heated environment which dries out your, you know, is a dry environment. So there are many variables here. It may be important, you know, the sun exposure. All those things may really be important. And we're actually trying to capture as many of those variables as we can. And we're asking people, you know, who they live with. We're asking them, do they have a pet? It turns out people have a lot of contact with their pets. And so we're asking them all those questions. I am not sure that we will have a great enough population that we can determine if there is statistical significance to any one of those factors. And it may be that what we find out is that the variance is just so great that you really can't tell a difference. But we are capturing all of that information in our questionnaires to see if there is a correlation between where someone lives and who they live with and, you know, what they eat and what allergies they have and those types of questions to try to see if those are affecting their microbiota. Great. So are there any other questions from the audience? All right. Well, I want to thank Dr. Segre for speaking today. I think this was very interesting and certainly added some insight into one of the many programs that are going on here at the NHGRI. This is a series of talks that we will be holding pretty much every other month for the next, for the significant feature. And so we will be announcing more of these webinars as time goes on. So we hope to see you again. There is a website that I believe went out with some of the emails that you would have received from me that would have information on our other webinars that happened last year. So, again, we look forward to seeing you in future webinars. And if, in fact, you would have other ideas of other topics that you would like to hear from, I would be very excited to hear about those as well. So you will receive more information from me. Again, thank you very much for participating. And I hope you all have an excellent afternoon. Thanks very much. Bye-bye.