 Okay. Hello, everyone. I'll also thank Leader Proctor for inviting me and also for extending the break by 15 minutes, especially before a topic like this that food can move on in its way. I'm going to start with a case that got me into this field. I have some disclosures. This was a patient I saw in clinics. She was a 61-year-old woman referred for evaluation of chronic diarrhea for eight months when I first saw her. She already was eight months into the course. Her symptoms originally started following treatment with cephalosporine and quinolone antibiotics for back surgery and inflammatory infection. During these eight months, she had several hospitalizations for IV hydration. In the documentation, the diagnosis was not entirely clear from the history. With her colonoscopy showed ischemic colitis on biopsies, which can actually be seen with C. difficile infection, which undoubtedly this is what it was. Intermittently, she was treated with variable success with metronidazole and vancomycin. She had bowel movements when I saw her in clinic every 15 minutes. With urgency and tenezimus, she lost 27 kilograms of weight by that time and was simply living in the wheelchair. I've been doing this fecal transplant work for a couple of years now, and my sense of humor sensitivity may have gotten blunted. Hopefully, the next slide will work. This is the pathophysiology of recurrent C. difficile infection syndrome. This really is a syndrome as the incidence of C. difficile infection has increased and new toxigenic and more virulent strains have emerged. The morbidity and mortality with this infection has increased about four-fold. The recurrent syndrome is one of the most difficult challenges that we face in clinic. The overview of pathophysiology is fairly simple or understanding. Antibiotics will...this is what normal is. There's obviously microbes there. Antibiotics will decrease the density and alter the composition of the microbiota. That provides a window of opportunity for spores to come in, which is usually the way it's acquired. And then the vegetative forms of bacteria grow up, produce toxin. That results in pseudomembranous colitis in at least some patients, or variable symptoms and syndromes that present may not be necessarily as severe. You treat that with antibiotics and you hope to come back here, but in these recurrent C. difficile infection patients, the cycle just keeps repeating because you're never quite able to break the loop. And so the solution, which has its own history that goes fairly far in the past, is to administer the normal microbes back and reconstitute. So as I said, my sense of humor may have changed, so hopefully this will go okay. This is the mechanistic version of what we do at least a couple years back. So we need a donor. This man has put bad poop. He used to be good poop and fast. And we need to take their good poop and put it into the other patient, in place of the bad poop. This will save his life for some reason. I'm a doctor. Actually, the doctor that is often credited, at least in Western medicine with doing this, was No Slouch. Dr. Ben Eismann was chief of surgery at the time of the publication of the first paper that's usually cited in 1958 at the Denver VA hospital at age of 36 or so, which is younger than most people get their R01 these days. He was a founding chairman of surgery at a couple of institutions, had a very active academic life, wrote more than 450 scientific articles, published seven books, including one book that came out very close to his death on the care of the aged. He was participated for wars and active duty. He was a rear admiral in the Navy. He retired in 1974. He was an active academic through 2012 when he passed away. And we actually did have a communication from him, and that's what he said in an email. In the early days of oral antibiotics, we were plagued by frequent diarrhea in our patients, due presumably to killing off intestinal bacteria. I was chief of surgery at the VA and simplistically considered merely in reintroducing normal organisms to counter such absence. Those were days when if one had an idea, we simply tried it. Seemed to work and I wrote it up, made a small splash, best wishes. This was before the FDA started regulating the drugs, before the agency appeared. So the technique goes back in Western medicine into the 50s, in Chinese medicine probably 4th century or so. So this was back to the case. I thought if we're going to do something like that, we better study it since that's one thing that hasn't been done through all those years. And so we collected fecal material before, as well as the donor. The donor in this case was her husband of 40 some years and after. And this material headed off to Sweden where Janet Janssen helped us to do TRFLP analysis, which is what this was. And it was published as a case report back in 2010. So this is the patient on day minus seven and you can just look at it as a barcode. This was at the time of the procedure. The material was introduced by colonoscopy. Looks a little different, but I think it's probably because the colonoscopy itself changes things and stuff coming out from small bowel into the colon. This was just an aspirin. That's the donor material on day zero and this is the patient two weeks after and then one month after. So there's a fairly striking resemblance between the donor and the recipient, which quite quickly led to the acceptance of the notion that this procedure results in complete engraftment that is stable and donor-like. So part of the notion here is these patients that get these multiple infections and I think it's an important point. They get carpet bomb with antibiotics and the patient that I presented, she was ill for eight months and it was another seven months before I did that procedure, so it was over a year. I talked to IRB and all that and in the meantime I thought I could get her out with antibiotics but our average patient gets a year of antibiotics which is an incredible pressure on the gut microbiota and this is kind of reflected here. So we're looking at a handful of controls. This is back from 2008. People with initial C. diff infection as well as people with recurrence C. diff infection and you look at the diversity of microbiota or bacterial species and it doesn't take much work to reach a plateau for those with recurrent disease because there isn't much left. Compared to controls and the ones that have the initial infection are somewhat intermediate. This is more recent data and we have a poster here that's presenting this. So this is a collection of 14 patients. We have donors over here. These are recipients. These samples taken usually a couple days before the procedure and then about a week afterwards. These are actually two donors but they're sampled on different days. We're looking at 16S ribosomal RNA gene profiling. So as you can see, as you would expect they're dominated by the two major phyla, firmacutes and bacterial adides. And the proteobacteria is just this tiny red band here which is what you would expect. Our patients, however, proteobacteria expand and become dominant. And I don't actually think this is completely benign as when we look at the species here and it's all the kinds of things that infectious disease doctors know which is probably not a good sign. They're proteas, glapsiella, E. coli, intrabacter, things like that. And sometimes these patients, after having this antibiotic pressure for a long time, start running into other infections which were caused by these organisms. Bladder infection, for example, is a common one and it assumes its own recurrence cycle and then the two infections start playing tag team with each other. Anyway, then shortly after the procedure, this is about one week after, it looks essentially donor-like. If we look at the family, I'll just call attention to these proteobacteria here that fall into an enterobacteria family which is more of those pathobionts are found. If we do look at diversity, these are our donors, that's our recipients, markedly reduced diversity as you would expect and then that quickly reconstitutes after the procedure by PCA plots. It does support that original case report. The patients before the procedure fall into their own cloud here. The donors and recipients afterwards are not distinguishable. And you saw a version of this when Rob Knight presented a movie that was sampling patients daily and it was kind of similar. They shifted toward the donor and then each patient, each recipient, settled in their own little cloud close to the donor but not quite the donor. There's still some individual fingerprint left. So what about the yuck factor? That's what journalists like to talk about and it piques certain attention. I actually don't want to talk about it because that's what it looks like for me at this point and this is frozen material in a cryobag. At this time we just use rigorously tested volunteer donors. We're aware of the scientific literature that ties microbiota to all kinds of problems. So we do screen and test for not just infectious disease but for metabolic syndrome, neurologic disorders, autoimmune problems. We're basically looking for Greek gods. The material can be cryopreserved. Some of these tests trickle in in days, weeks, sometimes a month. So all the tests have to be to satisfy all those constitute release criteria for when this material is ready for use. This material actually virtually has no donor. It passes through a bunch of filters. It's washed. The microbes are there but they are not actively producing hydrogen sulfide or whatever makes the smell. We've standardized it so there's the same number of bacteria at least per dose. We look at viability by membranes, permeability and such. And this material is now manufactured under GMP conditions at an FDA registered site at the university. So I can be courageous about this. All right. So how does it actually work? It sounds simple but you have to study it a little bit and it's not necessarily that. This picture is from a wonderful little children's book written by Dr. Arthur Kornberg, Nobel Prize winner and illustrated by Adam Allen. So that's my view of microbiota. So they could work directly inhibit C. difficile or they could work somehow through a host loop to do the same. And C. difficile has a number of steps to go through to cause its problems. Sporulate, spores have to germinate. It has to be vegetative growth, adhesion to epithelial cells, toxin production are some of them. And on the host side there could be a number of factors that affect C. difficile cycle including immune system, short chain fatty acids, bile acids as we've heard about already. Competitive niche exclusion. This comes from the same book. I wish we actually knew what the niche was and how narrowly it's defined for C. difficile but I don't think we do. So I, for looking for some figure I reached into my immunology past and I used to think about things like that in lymphocyte homeostasis where normally you have a lot of T-cell clones and they exist in a particular space of lymphoid tissue and they either have unique resources which is T-cell receptor or common resources for example cytokines like IL-7 and IL-15. There could be a lymphopenia inducing insult, viral infection or radiation or whatever and that results in death of many clones and if you're young enough to have a stymus you can recover back to normal diversity. If you don't the best you can do is at least re-expand and re-occupy the space with what you have left. But life is not necessarily that simple so here's an autoimmune looking clone that gets extra TCR stimulation and under these conditions it can expand out and cause autoimmune disease so you have a link between lymphopenia which is kind of paradoxic and autoimmunity in this case. One can think through the same kind of logic if this is a microbe and antibiotics etc. except we don't know what the mediators are. There could be more direct interaction not just competition but direct killing of C. difficile so there's a paper on bacteriocin that's found from manufactured by a normal constituent of gut microbiota bacillus through inges and it has very narrow activity against C. difficile so if you compare that with metronidazole metronidazole of course causes in a bioreactor bloom of proteobacteria just like we saw in patients so major disruption of microbiota and on the other hand this other antibiotics derived from microbiota is much more narrow spectrum it doesn't cause nearly as much disruption another idea is illustrated in this paper this was just an in vitro work but this particular bacterium and strain is able to inhibit cytotoxicity as well as adhesion of C. difficile to epithelial cells this is the cytotoxicity assay it happens to be a soluble factor and other strains could not do it and then it also inhibits this particular strain once again this factor inhibits the adhesion to epithelial cells but it does not affect vegetative growth so you can grow them together and they seem to grow fine but cytotoxicity is altered on the host there could be immune mediated colonization resistance and this has been explored just a little bit and of course we know many mechanisms that exist antimicrobial peptides for example alpha defensants neutralize C. difficile toxin beam perhaps that's why the small bowel is relatively protected against C. difficile disease of the colon because you have panacells in the small intestine but not really in the colon there could be other mechanisms a lot of this is mouse work which has joined the field with all the tools that it has so we know there's a role for not one in myD88 and some of these like IL-1, CX-Cl1 are dependent on microbiota being present to provide this colonization resistance there's a role for adaptive immunity as well we know that patients with recurrence C. difficile infection do not develop as high titers of antibody against C. difficile toxin although most of them don't really have a major immune defect like we know the common variable immunodeficiency patients are prone to this condition but majority of patients don't have anything that's that dramatic and our own focus has been on bile acids so this has been introduced before primary bile acids are made in the liver and then they are modified by microbiota in the colon so you have this hydroxyl group at position 12 this one at 7 and you have a conjugation site that can get conjugated to amino acids like glycine and torene and here it is again and I'll just point out that for example toricolic acid one of the main primary bile acids produced is a pro-germanent of C. difficile it's like fertilizer and I became aware of it once we started growing it in the lab a light bulb went on and olythicolic acid which is one of the secondary ones is a known inhibitor of C. difficile germination if you look at germ-free mice versus conventionally raised mice in germ-free mice you just see as you would expect only the primary bile acids in their fecal material as all the secondary ones show up with conventionalization interestingly I should know kids are born they're nearly germ-free and they primarily have the primary bile acids at first and those newborns are highly colonized with C. diff about 30-50% you'll find C. diff in them for some reason they don't develop the clinical syndrome so there's actually some work done on role of bile salts in mice the sport germination C. difficile quickly run through that so in the lab this is germination assay you need toacolic acid to germinate C. difficile if you take in small intestinal contents there is some germination activity there which is augmented by antibiotic lynda mycin on the other hand if you take material washings from the colon they don't have that germinant activity anymore unless the animal has been exposed to antibiotics and that's fecal pellets even with clindamycin don't work very well this germination factor is small it's heat stable, water soluble sensitive to coast tyrimine coast tyrimine was used in the past as one approach to C. difficile infection was thought to bind up the toxin probably another role that should be considered is binds up bile acids and salts again as you might expect there is primary bile acids are the only ones seen in clindamycin treated animals and when you look at these assays you basically find that fecal bacteria block this pro germinant activity of toacolic acid so this is our own data again to the poster we looked at fecal bile acid composition before and after the fecal transplant and before the procedure again these are patients that are bombarded with antibiotics for a long time all we find is primary bile acids in their fecal material after the procedure they're gone the pattern is fully reversed with secondary bile acids deoxicolic acid, lithicolic acid so before the procedure we don't find any secondary bile acids but afterwards they come back and they're indistinguishable from the donor so these are the same patients that I showed with the 16S data on and then we actually also did untargeted metabolomics looking for in the fecal material and loamy hold the major things that drive the difference before and after were once again bile acids toacolic acid came up which is of course the one of interest so in this summary we see the procedure is associated with increase in lithicolic acid which inhibits germination and decrease in toacolic acid which promotes it so our little simple model this would be a normal situation where primary bile acids are illustrated as green they go through the small intestine and here the microbiota convert them to secondary interns color in this illustration on the other hand and you see a couple of cdiv spores that just can't do very much after antibiotics this is what happens when you lose your secondary bile acids instead your colon is sprayed with this fertilizer for cdiv and you have a bloom of cdiv and its spores so our current directions are further development of standardized full-spectrum microbiota for therapeutic transplantation we're expanding our GMP manufacturing no trivial task and of course we're interested in mechanism-based development of these targeted microbiota therapeutics and for my own gaps and challenges I thought I would bring up this issue which is not new and it's the challenge between basic science and translation and I grew up scientifically in this world and I love this world it's so intellectually stimulating and linear and thinking about these tools to work with and it takes a lot of courage to cross this bridge and this is the risk of what can happen and it wasn't easy to turn around my own career and then you come to this place and there's a difference in infrastructure one can sometimes see it on the campus we have these beautiful buildings that are called translational research but there's not a single patient there it's all most work happening in the clinics and NIH cannot fix this but it has to be sensitive to these realities the academic health centers are competing with private clinical entities they're just trying to survive and the research priority is falling behind and there is not as much investment made into that so while we have all this science being built in the basic sciences building the infrastructure on the clinical side is still somewhat prehistoric and hasn't changed very much so we come to a place where science instead of leading is following whatever is happening within the field in the fecal transplant example you have physicians performing these procedures without any particular protocol or with some and it's just whatever happens happens sometimes patients just do it themselves all this knowledge and resources toss the side one would think that this donor pool that we have that is going into patients should be considered a great resource to study we are saving their samples and such but there could be a whole bunch of biomarkers that could be collected on them and this material could go into a variety of studies and there could be some biomarkers that will identify a particular composition that could be interesting in autism or diabetes or heart disease or whatever else that people are talking about here this is kind of happening already I receive emails daily from patients who are doing this procedure on themselves for a variety of indications because they've heard about the science it's usually not C. diff they do it for IBS, they do it for rheumatoid arthritis autism it just sounds a little crazy and I hope that somehow we can be in front of this pack and lead them with scientific insight so for acknowledgments I particularly would like to highlight Alexa who's done all the work I've presented here and her poster this of course is a multi-disciplinary science and that's been the most gratifying part of it that I'm a clinician, immunologist but now I work with microbial ecologists who studied all kinds of species in the woods and lakes and rivers and has not studied humans before but now we're at the same team Chi Chen from Food Science and Nutrition did our metabolomics Matt Hamilton is our current head of production in the GMP facility and there are other collaborators including Janet Janssen that helped us with the first case I think that's all I have for you Thanks a lot but because of the time constraint we will take question in the open session our next speaker is Elaine Petrov from Queens University, Canada and the title of the talk is use of microbial ecosystems to treat recurrent clostridium difficile infection