 Thank you very much. I'd like to thank the organizers for inviting me and actually I was a bit surprised and honored To be invited because I am a newbie in this area I was introduced to the microbiome world a couple of years ago When I became involved in a research consortium led by Jeff Gordon at Washington University in St. Louis called the breast milk gut microbiome and immunity project and And and my part in that consortium is really because of work that I've been involved in in Africa with child malnutrition and So I've had to kind of do a crash course in the microbiome field in order to prepare for this presentation So I hope that my colleagues in the audience who know more about some of this than I do will chip in during the discussion now I Myself work at the far end of the translational spectrum when it comes to my research So I'm involved in human population research in communities And I'm I hope that the discussion today and tomorrow can lead us even further in the direction of linking the very exciting work that we've seen presented today at the basic level to its translation into application in the field and for many different types of populations We've just heard about the elderly and I'm going to talk about the other end of the age spectrum Just one last thing to say about my background I was originally trained trained as an ecologist and so for me it's really delightful to see the human body now viewed as an ecosystem and I only wish that more MDs would see it that way So when thinking about what to review for this presentation I think that the key questions in my mind were how does diet in early life influence the microbiome How does the microbiome influence child nutritional status and how do differences in microbiome structure and function effect nutritional outcomes during the first two to three years of life as well as long-term health and developmental outcomes and What I was able to find was really very scant and really almost nothing on the third question listed there So I'm going to start first with something you all know We've heard a couple times previously that the be the beginning stage for the infant is Theoretically at least an a sterile environment in the infant intestine and that various factors then influence the Individuals gut microbiotic composition thereafter including gestational age delivery mode maternity ward or neonatal clinic feeding mode other foods and fluids antibiotic use paternal skin and the skin of other caregivers and the environment and This study shown in this slide has also been referred to before but I want to mention it again for one specific reason This was the cross-cultural study done by Yatsunenko and colleagues published last year in which they characterized bacterial species present in fecal samples from three very different populations around the world and they were able to show that the Differences between the younger Profiles and the adults Narrowed with age so you can see that in all three of the populations the children were quite quite different from the adults and then Achieved more or less the adult type by about three years of age So the first three years of life are a period of very rapid transformation of the microbiota and An important period in life for us to focus on in that same study They were able to show that this Fundamental sort of change that occurs in the first couple of lives a couple of years of life is in the diversity in the microbiota and If you look at that blown up over here in all three of the populations That was the trend between zero and three years of age Now of course the very first diet that we hope the infant will receive is breast milk And this plays a fundamental role in influencing the infant's microbiome And I'm going to talk about three aspects to that first of all there are microbes in breast milk That have been hypothesized to seed the infant's GI tract There are many many constituents in human milk that are considered prebiotic that are very important at promoting the growth of certain types of bacteria and There are quite dramatic differences in the microbiome between breastfed and formula fed infants So starting with the first one. I'm just going to show you this one study by Cabrera rubio at all In which they characterize the milk of my probe microbial community for 18 lactating women within two days of childbirth And then again at one and six months postpartum now they found several hundred species of bacteria and Or that they suspect there are several hundred species and that these are compositionally distinct From other human niches and they conclude that they are not simply contaminants from the skin They found that colostrum has a higher microbial diversity than transitional or mature milk and in the colostrum sample is the dominant types of bacteria shown here and that by one and six months the typical Inhabitants of the oral cavity became more prevalent in the milk and they Speculated that this was due to bacteria from the infant's mouth colonizing the milk ducks and the areola Interestingly, they found that milk from obese mothers tended to contain a different and less diverse Bacterial community compared to milk from normal weight mothers and they also found that milk samples from mothers who went through elective but not Non-elective c-section Contained a different bacterial community than the milk samples from mothers with a vaginal delivery Because it was only seen in the mothers with elective c-section They concluded that it was the process of at least going through some labor the physiological stress of that That linked the mothers of vaginal delivery and the non-elective c-sections in having some commonality in their micro Microflora in the milk I'm not going to spend a lot of time on the prebiotics in human milk because you're going to hear a wonderful lecture tomorrow by my colleague David Mills And I am showing just one slide from one of their papers recently published Characterizing the some of the prebiotics in human milk and particularly the human milk oligosaccharides and glycoconginates That are quite diverse many many many different types of these that are very abundant in human milk and pretty unique to human milk And are known to be utilized especially by bifidobacteria So this is a structural Example of a human milk oligosaccharide. These are some of the other glycans of different types and over here a glycol lipid and David will explain to you how all of those are Operating in terms of their impact on the micro flora So as a result of some of these differences in the experience between breastfed and non breastfed infants There are differences in the microbiome by feeding mode, but the results from different studies are actually somewhat mixed Just showing two examples here. This is a study of a fairly large sample of more than 600 from European countries Which found that bifidobacteria dominated the microbiota of the breastfed infants Whereas the formula fit infants had higher proportions of bacteroides and members of the Clostridium and Lactobacillus groups In this study, which is much smaller They found that formula fed infants had increased richness of species with overrepresentation of Clostridium difficile, but no difference in bifidobacteria compared to breastfed infants So there has been some mixture in findings and there was a review conducted a few years back that Contrasted the studies done before 1980 and after 1980 With the purpose of making the point that methodological differences as well as differences perhaps in the definition of breastfeeding And how Subjects were categorized may have led to differences in some of the findings So for example here in the bifidobacteria line, you can see before 1980 Five out of six studies found those were increased in the breastfed infants But after 1980 that was not as commonly demonstrated On the other hand, there are other types over here that are lower in the breastfed infants And that's fairly consistently found including bacteroides and Clostridia So I think that we don't fully understand all these differences, but certainly there are differences in microbial Composition in the gut by feeding mode Now to straighten out some of these Some of this confusion. I thought it might be helpful to look at one Study from an animal model in this case a primate Which is very close to humans. This was just recently published And in this case, they looked at rhesus monkey infants who were randomized to receive breast milk from their mothers or formula Exclusively from birth to three months of age. It's a small sample when you're working with monkeys. That's the way it is But the value of this particular model is that nutritional needs of Rhesus monkeys are actually quite similar to those of human infants And they found that the formula fed monkeys had more rapid growth and higher serum insulin That's also true in human infants who are formula fed And they had higher levels of bacteria from the ruminococcus genus and lower levels from lactobacillus genus And along with these differences, they found elevated levels of many cytokines, chemokines and growth factors at four weeks of age So in addition to characterizing the microbiome in this study They also did metabolomics work and you can see here. I'm not going to go into the details But that there's quite dramatic separation in both serum and urine in The profiles that they were able to find so this difference between receiving breast milk or receiving formula has fundamental effects on the child's metabolism For the next phase in terms of what the infant would experience to be the introduction of solid foods And this also plays a role in the changes that occur in the microbiome This was a study I mentioned before from Europe in which they assess the fecal microbiota composition of infants from five different countries They sampled them at six weeks of age Before any solid foods were introduced and again four weeks after the introduction of solid foods And in this particular set of infants 59% were fully breastfed 27% fully formula fed and 14% were mixed fed at baseline You can see in this slide that after solid foods are introduced. There was a reduction in bifidobacteria As well as in some of the clostridia species And in the enterobacteria here and there was an increase in couple of other species of clostridia So that shift makes a difference what we don't know is what happens And when you give different types or different composition of solid foods and that's been Uninvestigated to the best of my knowledge the other study I wanted to mention along these lines is one from Defilipo et al and this was mentioned in an earlier presentation today And in this case they contrasted the gut microbiota of children in Burkina Faso in West Africa and children in Europe and they found that the Composition of the microflora diverged after weaning in both populations breastfeeding was fairly common in very early life and When they started receiving other foods they received very different types of diet So in the Birkenabbi diet, this was low in fat and animal protein rich in starch fiber and plant polysaccharides and predominantly Vegetarian the European diet being higher in fat animal protein sugar and starch and lower in fiber Now I wanted to mention that this is an agricultural population here So I would not consider this to be any kind of model diet Certainly not an ancestral diet if you go back to more of the evolutionary past for humans It's a cereal-based diet and nutritionally Inadequate in many different ways, so I don't want to give you the impression that this is a better diet than than the European one in any way As I said the differences in the microbiota became evident after the period of predominant breastfeeding and at that point They found higher microbial richness and biodiversity in the Burkina Faso samples they found a greater representation of actinobacteria and bacteroid deities and In contrast in the European children greater abundance of fermicutes and proteobacteria and this shows in color The very dramatic difference in the microbiota in these two populations over here very much dominated by prevetella and other bacteroid deities and over here you can see a lot more representation of The ones in this area here Now there were some other correlates to these differences in the microbiota in the Burkinaabe children. They had greater Total short-chain fatty acids in fecal samples. We just heard about the importance of short-chain fatty acids that are produced or During fermentation by intestinal microbiota Based on plant polysaccharides these substances are very important precursors for many things that are Potentially have health outcomes and have been theorized to prevent the establishment of potentially pathogenic intestinal microbes and have a role against gut inflammation in this particular situation I'm not sure we could argue that the Burkinaabe children were protected against Some of these pathogenic microbes because diarrhea rates and other illnesses are very very common there I want to move on to think about specific nutrients because a lot of times when people think about nutrition and the microbiota They're thinking in very general terms about total protein total fat carbohydrates, maybe fiber, but they're really not thinking very often about micronutrients and In nutrition we think about the huge number of different nutrients that we think about every day There's probably 40 or more nutrients that we might consider There's very very little in the literature on the specific nutrients and how they might interact with the microbiota I'm actually just going to mention two that I could find studies related to and one is iron and the other is fatty acids In the case of irons is an important one to consider because it's an essential nutrient for many gut microbes But some of the beneficial barrier bacteria in particular lactobacilli do not require iron So we introduced iron into the diet in large amounts. You can shift the profile of the bacteria in the gut And in particular for some enteric gram negative bacteria and some of these are pathogens Iron acquisition plays in a central role in the virulence and colonization of the pathogenic strains With regard to fatty acids, there's just a very little information But suggesting that the omega-3 long chain polyanthesaturated fatty acids may modulate growth and adhesion of lactobacilli and May impair the growth of certain species of bacteroides So I wanted to show you one important study was published just recently by Nancy Krebs and her group in Colorado looking at effects of different complementary feeding regimens on iron status and enteric microbiota in breastfed infants in this case It was a randomized trial with four to five forty five exclusively breastfed five-month-old infants randomized one of three feeding groups either commercially available pureed meats Iron and zinc fortified infant cereals or iron only fortified infant cereals and they followed them until nine to ten months of age and In the last 14 infants in the study. They were able to get samples to characterize the enteric microbiome And they got fecal samples monthly between five and nine months One of the important aspects of the study is that the cereal groups had much higher iron intake than the meat group And that's shown here in this slide Right here is the iron and zinc fortified cereal. This is the iron only fortified cereal and this is the meat group This is dietary iron between six and nine months of age You see a huge jump With both of the fortified cereals and a much smaller increase in dietary iron in the meat group Concomitantly with that they found differences in the microflora in this relatively small sample of infants But only in the iron only fortified group. So in that case, they had a reduction here in the actinobacteria and they had increase in the bactiroid it is a decrease over here in the lactobacilli and The only difference that the meat group showed was an increase in Clostridia group a clade in this particular case So these are I would say probably fairly preliminary data showing that the Type of complementary food in a particular the iron content of that food could actually make quite a large difference in the microflora to Illustrate that point further. I have one slide from an older age group. This was conducted in school age children in Cote d'Ivoire in Africa by Michael Zimmerman and his group This was a double-blind randomized controlled trial in which they received either an iron fortified or a non fortified biscuit for six months And those biscuits contain 20 milligrams of iron per day given four times per week as electrolytic iron when they looked at the Gut microflora, they did not find a significant difference in the bifidobacteria This is the control group at baseline at six months iron at baseline and at six months But they found an increase in the iron group in entero bacteria a decrease in lactobacilli and Along with those changes in the floor They found quite a large increase in a marker of gut inflammation, which is fecal cal protectin So there are some concerns that providing iron May shift the flora in such a way that it may be disadvantageous to the child with that said now should back up for just a second and say that We worry about iron a lot because iron deficiency is probably the most common nutritional disorder in the world and we estimate probably about 50 percent of young children are iron deficient and similarly very large percentage of pregnant women globally in low-income countries and so that's why fortified products are one of the strategies being used to Reduce iron deficiency and iron deficiency anemia, but we need to be concerned about whether there are potential side effects from those kinds of strategies Now as I mentioned the other nutrient I could find some information about were fatty acids And this is just one study I found on the effect of fish oil supplements on fecal microbiota from nine to 18 months This was conducted in Denmark with a hundred and thirty-two healthy infants And they were looking at molecular fingerprints of bacterial DNA And so they were characterizing the outcomes here in terms of sort of general types I don't have genus, but these were both of these particular Categories were presumed to be Bacteroidetes, and they were modified by the breastfeeding status of the child. So what they found is that In those who were not breastfed at baseline at nine months There was a greater increase in one of these types in the fish oil group and a greater increase in the other type in The sunflower oil group. It's a little bit hard to interpret what that means in terms of potential health consequences But what it does mean is that the fatty acid composition of the diet can Cause shifts in the micro flora in the infant's gut So switching to the second way of looking at this. How does the microbiome influence the child's nutritional status? Going to Mention a study that has also been mentioned by previous speakers Just published in science this year in which the gut microbiomes of Malawi and twin pairs discordant for a form of severe malnutrition called kwashyorkor were Compared and in the study they compared Or they examined 317 twin pairs that were followed through the first three years of life And in this case 50% of the twin pairs did not develop acute malnutrition 43% became discordant which means one twin pair of the twin of the pair did and the other did not and seven percent Both developed acute malnutrition Whenever one or both of the members of the twin pair developed malnutrition. They were both treated with Therapeutic food a peanut-based ready to use therapeutic food And so in this study they assess the microbiomes of nine same gender twin pairs without malnutrition and 13 pairs discordant for kwashyorkor and And in this slide They were able to show that the microbiomes of the twins with kwashyorkor were less mature compared to their Non-malnourished co-twins. So these are the co-twins who remained Non-malnourished. I don't personally call them healthy because in that environment That's the exception rather than the rule But they didn't meet the definition of severe malnutrition and you can see that there's an increase over time in the diversity of the microbiome And in the healthy co-twin of the pair where one of them developed kwashyorkor that same sort of trend was exhibited But in the twin with kwashyorkor There was a transient increase in maturity when they received the RUTF, but that was not sustained So they ended up not really having that same temporal maturation as their co-twin and And as you know in this study they were able to take the fecal samples from these pairs of twins and implant them Into neurobiotic mice and I won't go over again how that method Works, I think most of you here are familiar with it, but I do want to show a slide that's been shown before just to reiterate that in fact the results they found in the mice who are recipients of these microbial transplants Exhibited the the phenotype of losing weight only when they were on the Malawian diet And this slide doesn't show that when they were on a control mouse chow They did not exhibit a phenotypic difference. So there is a diet microbiome interaction and I think it's very important for all of us here to remember When thinking about Consequences so here's the group that got the Transplant from the twin with quash your core And then this is from the co-twin that did not develop quash your car and you can see that these mice lost weight during that period of time They recovered that during the period of feeding with our utf, but then did not really Sustain that weight gain over time and similar profile was seen in the fecal microbiota very dramatic Reconfiguration during the period of refeeding with our utf But then became more similar to the the co-twin thereafter now in terms of the types of Species and taxa that changed When the mouse received the microbiota from the quash your core twin There were increases in certain types of bacteria and decreases in others And some of the taxa also changed in the mice that got the healthy Co-twin transplant And I'm not going to go into the details here, but you can just see visually that this is the period during the Malawi diet This is on our utf now. You see a lot of changes occurring and this is back on the Malawi diet over here So where do we go from here? this is Really what what drew me into the field is the project that is attempting to bring together various disciplines to look at all of the relationships between the breast milk gut microbiome and immunity using populations in populations with a lot of malnutrition and The goals of this project are to identify and validate new pre and probiotic interventions to improve the health and development of infants and children in the developing world and then demonstrate a process by which new such interventions can be identified and validated in the future and The the point here is that as several people have mentioned before it really requires lots of different disciplinary expertise to Put this all together to take the information From the trials that have been done in humans to then look at what that means in animal models to identify Identify leads for pro pre-pro and symbiotics then go back and test those in animals and eventually Test those in human populations. So that's where we are aiming. I Was asked to identify gaps. So this is my last slide I just wanted to say that apart from the studies comparing the breastfed and formula fed infants There's very little information on how dietary composition or nutrient intake affects the microbiome of children So I would very strongly urge greater funding and research in that area and Then in terms of the emerging link between malnutrition in the microbiome This is something that we need to understand much better in terms of what the causality and the direction of that causality is As well as the effect of different types of interventions and lastly we really desperately need Prospective studies that include long-term follow-up of intervention trials to look at consequences later in life of these changes in early early childhood And just like to finish by thanking the BMMI team that introduced me to this world and to thank all of you for your attention Thank you. Thank you Katherine. That was excellent. I'm going to take a moderator's Privilege and ask one quick thing relative to the Comparing or doing research in formula fed babies versus breastfed Babies formulas that are now being enriched with probiotics at an increasing rate in in the United States and Prebiotics as well and this has been a trend within the last five years or so So there needs to be that caution to control for that and it's going to be difficult to compare to Historic studies because of that Yeah, thank you for bringing that up I think part of the reason there's so much diversity in the findings of studies doing this is that formulas have changed dramatically Over the last 30 years not even just the last five or 10 years with a lot of different Compositional changes and so it's very difficult to compare Across time as you say Hi, you may have said this it went by kind of fast But for the case of bacteria in mother's milk and appearance of those lineage is in babies What's the what's the overview of studies on that reason? I ask we did a study with Grace Aldrovandi Where we tracked otus and mother's milk and otus in baby poop and we really didn't see any Linkage between the two babies poop was knows more similar to their own mother's milk than somebody else's mother's milk and vice versa And I was wondering what the what the line of studies is on that Well, I think you probably know better than I do. I mean, I don't believe the study that I was mentioning Tried to look at how the microbes in in the mother's milk were related to the infant's micro flora And I think that's a good point if it is seeding the gut is that really contributing to the growth of Bacteria in the infant over time. So I think it's an open question unless somebody else Wants to answer that with regard to the microbes in mother's milk I mean, how is there a sort of special organ that cultivates the microbes or is it just sort of distributed throughout the gland? I mean, it seems to me like this is some sort of a evolutionary Devet, I mean Some some need to have these microbes in there And yet nobody actually talks about what's maintaining them and how they're actually kept in the breast and how they accumulate Where do they come from originally? Is it is this sort of spores that they have all life and suddenly wake up later I mean any ideas on them. Well, those are great questions I mean, I can only speculate that that there is a portal to the outside world Even before the mammary gland starts to lactate That right, but once they get inside at least the ducks then they have this wonderful food To start flourishing on which is not present on the skin or in other parts of the body So I'm just speculating here if anybody wants to to jump in Please go ahead. But those are great questions. Thank you Let's let's give it another round Thank you And it's my pleasure to introduce the last speaker for the session Dr. Johanna Lampy from the Fred Hutchinson Cancer Research Center And she's going to talk about gut microbial metabolism of food constituents modulating human dietary exposure