Scientist Stories: Trevor Lawley, Early Life Microbiomes

Hi, my name is Trevor Lawley. Today I'm going to talk to you about some research going on in my lab at the Sanger Institute. The lab's called the Host-Microbiota Interactions Lab. We use a variety of tools and approaches to understand how the human microbiome impacts health and disease. We have a big focus now on early life microbiome, that is referring to babies, neonates, the earliest stages of acquisition of bugs by children when they're born, and the impacts on longer-term health outcomes. And this is a big question. We're thinking, how can the early life microbiome impact things like obesity, asthma, and other growth-stunting conditions and syndromes? So it's a big question, big question we're addressing here. What today I'll do, I'll break it down and give you a rationale or approach. And there's something specific I want to talk to you around today too, this idea of spreading health through maternal transmission of adaptive Often we think of spreading of bacteria, we think of pathogens. I'm going to talk to you about a slightly different concept and how these highly evolved adaptive conventions can be beneficial to us and how understanding maternal transmission will be really key to understand the acquisition assembly of the early life microbiome. So human microbiome sciences is a maturing field of research and What's so exciting around this is it's the combination of biomedical research and ecology coming together. And so the title of this slide is "The Human Body is an Ecosystem." So we now know that approximately, we have approximately 1 bacterial, 1 microbial cell for every eukaryotic cell, human cell, in our body. We are half microbial. It's important to remember these aren't random bugs. These are bugs that have co-evolved with humans to provide beneficial functions. This is what our interest is understanding symbiosis. Now, if you look at this little cartoon here, I want to illustrate some numbers and how we look at this. If you look on the left, we have an illustration of our visible organs, if you will, and that includes human genome. And we, we know that there's about 23,000 genes in the genome. Now, we see that we have this invisible microbiome. These are much smaller than human cells, maybe a tenth of the size, and we have approximately the same amount, as I mentioned, 10 to the 14. But this actually equates to about 9 million genes. So we have under 100-fold more unique genes in our human microbiome than our human genome. This is immense, and we should start to understand how this integrates together to get the complete human genome and microbiome together for understanding the normal function of the human body. I include a couple facts here on the human microbiome, more general stuff. For example, Each person has 1 to 2 kilograms of microbiome, mainly in the digestive tract. Bacteria dominate the biomass, and I'll focus a lot on bacteria today, but also archaea, fungi, viruses are also present. The bacteria are really the metabolic engine. That's why we're focusing on this at this point. The microbiome contains commensals, mutualists, pathogens. It is an ecosystem, and they all live there, sometimes in harmony and sometimes not. We have distinct microbiomes in our gut, our our lungs, the urogenital tract, the oral cavity. And what's interesting, I'll show you this in the next couple slides, is out of over 100 phyla of bacteria, it's really 4 phyla that dominate the human microbiome. I think this is quite interesting. We'll talk about this in a couple slides. And this idea of dysbiosis, uh, it's a term used to describe a pathological imbalance in our microbiome, in our gut, and it's linked to a growing list of poorly understood syndromes IBS, for example, and also a lot of diseases, IBD, and also checkpoint inhibitor therapy in melanoma patients. Very exciting stuff happening. I'll talk about this a bit too. Now, the gut microbiota is our big focus now, and, and when I'm asked what is the key— what are the key functions of our gut microbiota— I often start with digestion. We have a lot of, uh, a lot of the bugs in our guts actually break down a lot of food that we ingest. We actually— our human genome doesn't have— they don't have the genes for it. Our carbohydrate metabolism, for example. Metabolism releases vitamins— vitamin K, vitamin B12, for example. All these are produced by the human microbiome in our gut. One of the major functions— I— and it's one of the topics of really of interest in my lab, and we'll talk about this later on— how pathogens and commensals interact. And what we— one reason we want to understand this is the idea of pathogen colonization resistance, the ability of a healthy microbiome to exclude or block pathogen colonization. And we'll end the talk, the seminar today, talking about some of our data and what this means. Immune development and a response, and also cognitive development, the neurodevelopment in the brain, and how it's linked to the gut. These are emerging ideas. So that's the general background I want to share with you today, but I want to leave with the message that I think studying human microbiome co-evolution and symbiosis will provide insight into the function of the human body. I think this is important. Now, I described the human body as an ecosystem, and in ecology, you often think of open and closed ecosystems. So I wanted to make this point of an open ecosystem, the idea that material, matter, energy, other things can enter and leave the ecosystem. And why I'm bringing this up is because we also have to understand how these commensals transmit faithfully from humans. This is a big, big problem that we have. We're trying to address because most of these bugs are strictly anaerobic, meaning they die in the presence of oxygen. So if you have this donor here, ecosystem 1, if you will, there's often excretion of vegetative cells or spores. These are— this is the big conundrum we're trying to understand better, how they establish themselves in the environment, spread. And we know some bugs make spores, very, very resistant structures that can allow the bacterium to survive this harsh environment. The ingestion of spores and the transit to the intestine is very important. And bugs, we now know that, for example, spores respond to bile acids to germinate, to grow, start to colonize the gut. So we have to understand this whole process. And here we have a little cartoon how a pathogen or any commensal could integrate into a microbial community. And there's a lot of scenarios to expect. So the point being, the bugs colonize and they transmit, and these are highly evolved processes that we're trying to understand. Now, human gut membership is not random. There are very specific bugs. Sometimes these bugs are only found in humans. So the title of this slide is that they are— their human microbiome membership is highly adapted and diverse bacterial species. So if you pay attention to this graph on the left, this figure on the left here, this is— I want to talk about the, the human microbiome. These are metagenomes, and so what we do is we can extract DNA from feces. We have a lot of processes to do this and get high-quality DNA sequences. But when we sequence it, we break it into small pieces, shotgun sequencing. And what that gives us is small little pieces, 1 to 200 base pairs, that represent the entire ecosystem. Now the challenge is in how you interpret that. So one thing we do is we actually map them to reference genomes. We do mass culture and map them to reference genomes. And this is an example of that. We have other methods, very clever methods, about building MAGs, metagenomic assembled genomes. Which serve another purpose. But here is reference genome-based mapping. Here's a paper from the lab here that, uh, where this figure is from. It's a really nice paper by Sam Forrester. Now what you have here down the left are a number of species. Now these species are found in the human gut microbiome in over 13,000 individuals. These are common abundant species. That's one thing I wanted to mention. And you probably— most people probably recognize E. coli, Bacteroides vulgatus. This will come up later. We have very cool bugs in here. We have Bacterium prasnitii is in here, very common bug that's possibly a spore former. Point is, most of these bugs are in us. All of us are playing a role in our biology, our health, and we know almost nothing about them. I'm really excited about the future of human microbiome sciences and trying to understand the biology, the beneficial properties the bugs provide humans to help us develop properly and also resist disease. Now, these are the highly specific and highly adaptive bugs I'm talking about. Now, I'll put this slide on here too. What I wanted to do was map these lineages of bacteria, these, these 4 phyla. And what I've done over here is the taxonomic hierarchy here, and I mentioned species down here, here, these, these names here, and up phyla. These are high, high levels, and we find 4 phyla out of 100+ phyla in nature. So this is not an accident. I bring this up, I want to show you the diversity here. So these are eukaryotes, and humans would be down here. There's actually not even a spot for humans. I think in the big, big picture of evolution and diversity of life, humans— there's not much notable of humans in this particular— I'm glad I'm a human and we're quite remarkable, but the big picture, there's a lot of diversity in the tree of life. The archaea are here, and we, we expect that maybe the root of the tree, maybe the origin of life, is somewhere around here. That splits the archaea and eukaryotes from the bacteria. You notice the bacteria group is massive. We have groups here that haven't been cultured but been sequenced, metagenomic sequenced from rare environments, the really cool and extreme environments. So there's a discovery process going on here, bringing whole new clades under the tree of life. The bugs, the groups I want to talk about are Firmicutes, Actinobacteria. Now these are gram-positives. Firmicutes include the spore-formers, things like Enterococcus, Lactobacillus, a very common bug in babies. Actinobacteria is a very different group. It includes the, the heroes of the early life microbiome, the bifidobacteria. We'll talk a bit about those later. They're not spore-forming bacteria though. You come down here and you follow the backbone of this tree and you get the gram-negative Bacteroidetes. These are amazing bugs that have co-speciated with primates. That means if you took the genomes of all kinds of Bacteroidetes species and build a phylogeny and compared to humans, they actually co-evolved together. Very interesting. This is how deep this evolution is. And so this is a tree of life, and there are going to be references to phylogeny throughout here, so I just want to take a quick second to mention the key things are the branching points and the length of the tree, of each branch. And closely related organisms will sit close to each other, and distantly related organisms, for example, proteobacteria down to humans, very far apart. So we'll see some of these later on, and we'll talk about how we can track transmission of pathogens through these tools. Now I want to make a couple points on this slide. The human adult microbiome— I want to come back to the adult aspect of this— but the link to diseases, novel clinical treatments, and also biomarkers, it's phenomenal what's happened in the last 5 or 6 years in the human microbiome space, human microbiome sciences. I think we can agree now it influences every organ in the body. We have a lot of chemicals and metabolites in our blood that are strictly from bacteria in our gut. It impacts every aspect of our body and our development. I believe recognized drivers of many diseases. And here are some around this cartoon here. The brain, for example, the liver, the GI tract. IBS is a very interesting condition. And I think it's interesting for a number of reasons. One is it's linked. Gut and the mind. Often depression, anxiety are linked to these gut conditions. It's very fascinating. We still don't quite understand the mechanisms, but there are many diseases and poorly understood syndromes linked to this dysbiosis, pathological imbalance in human adult gut microbiota. A couple other points here. FMT stands for fecal microbiotic transplantation, this idea of taking donor stool and putting into a patient who is ill, has issues in their GI tract. C. difficile is probably the most a common example, but it's very, very strong clinical responses for C. difficile, ulcerative colitis, a number of diseases show that if you can transplant and replace the microbiome and graft healthy bugs, beneficial bugs, you can cure disease. It's a fascinating area, fascinating times. Defined LBTs, and these are defined consortia of 7, 8, 10 bacteria that we're developing as a medicine, and there's a number of disease, IBD and infections. Very exciting times. We have a defined consortium bacteria that's going to be a medicine. The idea is these bugs can engraft into our microbiome and give the beneficial effects and help promote this healthy state in humans. Key role in drug response. One of the things I'm referring to, these are melanoma patients undergoing checkpoint therapies. We now know the microbiome is a key determinant of how these patients respond. And also that I know there's a lot of predictive biomarkers coming from stool samples. So tremendous amount of potential. Inner microbiome is a target for treating disease now, and so tons of information around the status of the human body and the health status, I believe. Now I mentioned the adults, and I'm going to bring that up on this slide. The point is we have a developmental— throughout our life, a developmental program. We know, we know this very well from the human body, but we know nothing about how the microbiome develops and assembles over our lifetime course. And so most of the work has been on adults. A lot of the basic science and the commercial has been— and I think we have to think beyond in different, different parts of life. The elderly, for example, there might be— there's some interesting ideas out here that our microbiome, it could be impacted in aging. We're going to focus, say, on babies. Babies come out sterile, they're born sterile, and they quickly require a microbiome from the mom and environment. We're going to talk through some of this today. And this is a very interesting problem, and I put down bottom here— and this is a nice illustration from Sprocket Review from a few years ago— and the idea, if you accept that the human body is an ecosystem, we're thinking now about the formation of the human ecosystem. You're born sterile, babies are born sterile, and you have this environment. Of course, the mom— we're going to explore in this talk today how the mom donates bugs or transmits bugs that are highly adapted to do that. And how they ultimately lead to the formation of the gut microbiome. But we're also going to look at other variables, other sources of bugs as well. Why we're doing that is we think that there is a program, there's a deep evolution in health and ecology, how microbiome is transferred from mother to child. And there's a lot of modern-day derailers of microbiome assembly, and these are in the last 70, 80 years really. Remember, humans have evolved for millennia with the microbiome, and just in the last several decades that we start doing things to, to damage the microbiome itself and assembly the microbiome. Antibiotics, the emergence of AMR pathogens, C-section, diet, or family size, hygiene. A lot of modern, modern lifestyle changes are probably impacting microbiome assembly. The result, there are links here to malnutrition, for example, growth stunting, wasting, obesity, and being overweight. Here's some illustrations here of these child. This is a child with asthma getting an inhaler. This is a child here that's experienced growth stunting. And this is a child here with severe wasting. How does the microbiome in the early stages, I'm talking the first month, lay the groundwork for the development of the child? These are the sort of things we wanna talk about. Now this slide talks about the global trends in elective cesarean sections. Now what you look at, you see this map, You can see using the scale at the bottom here, it's colored based on the percentage of elective cesarean sections that are happening in each of these countries. And what you see here is, for example, in Latin America, there's very high rates of elective cesarean sections. In Africa, for example, it's underused. And these are the patterns. And we think this is a very important process that's impacting the assembly of the child's microbiome. Here are some facts. It's a life-saving intervention. It's important to remember. Maternal antibiotics are given around, right around the time of birth as a prophylactic. It's on the rise. Elective cesarean sections are on the rise around the world and are performed without medical indication. WHO guidelines suggest about 10-15% would be at the population level the ideal rates of C-sections. Now, this is the last point here I want to discuss a bit further, is there epidemiological association with the longer-term outcomes of these children? And the data is astonishing. There's so many papers out. So this is a slide I put together. I was writing a grant and doing a lot of reading and writing several months ago, and I had on my screen all these papers. I just took snapshots and put them down just to give examples of some of the literature out there looking at the risk of a C-section, for example, in the onset of childhood type 1 diabetes. Uh, this one's an interesting because it gets into cognitive development. Tons of information. And I added also a book here. I think this is very important. Anyone interested in the topic, this is about the massive antibiotics given to children. More than that, but there's a couple of chapters in there that are really important. It's a book by Martin Blaser. Martin Blaser. It's really good. If you're interested in this area, I'd recommend this book. It's very nice. So the data is astonishing, epidemiological links. So this is a slide that talks around maternal-infant immunological development dynamics around the time of birth. Now this illustration across the top shows a timeline. So this is when the baby's in the mom, there's the trimesters, the lactation period when breastfeeding, for example, and then weaning. And these are the very important inputs into the child. We talked about ecosystem, and this— these are the nutrients going in. I'll talk about that in a second. In parallel, there's a lot of things happening in the child, the mucus and the gut barriers being strengthened. There's more mucus on the surface, which is very important for the digestion in the child. Intestinal permeability— a lot of stuff happened around the mucosal surface. Neutrophils, adaptive immunity kicks in. Now what happens around the time of birth when the child comes out? The cells are there, but they need the signal to develop properly, right? And as we get a microbial colonization, and we think that this is going to be critical stage, particularly in the first month or two. And we're going to look at this, and this is where the ecology comes in again. We have ecosystem assembly here, and this just shows a generic cartoon just to illustrate the point that a primary ecological succession is when one organism can come in and impact either the acquisition or the blocking of another organism as the ecosystem builds up. We think there's parallels happening in the child's gut during the early stages. And also, we talked about nutrients and energy. Breast milk is chock-full of carbohydrates, different antibodies, cytokines, lipids, very high in lipids. And these are— this is what Mother Nature wants our gut bugs to have. And this is the interaction we're going to start looking at in more detail. Now, this is several years ago we started this consortium. It's been about 9 years now. This is with Nigel, Alison, and Peter. It's called the UK Baby Biome Study. It's a multi-center study. And these are, these are the hospitals, the 3 hospital folks. And I'm here, I'm the messenger here, but the amount of work in the background— this is an operation. And huge credit to the nurses and midwives at these hospitals in the UK. All-star crew. And this is the workflow here. I want to look at the timeline across the top here. And what, what, what the team has done is actually gone and recruited women. These are, these are full-term child deliveries. They got recruitment, the birth samples, fecal, a lot of other samples came into a centralized biobank. We collect a lot of metadata around the child, we'll talk around that later on, and that's been stored centrally. After about a year, we get more information, more questionnaires, and more biosamples, and then this is accumulated into a large cohort dataset that we study. We'll talk around this in the next few slides, how we analyze it. We're also in the background, this is amazing stuff, Getting access to electronic data. The idea we can use e-healthcare data sets to incorporate, to look at the health of the child long-term. Amazing stuff. Amazing stuff. Now, this is a large scale. This is the one I want to show you, the type of sampling that's involved. Now, every one of these dots here, red or blue, represents one fecal sample, stool sample, collected either from the mom or the child. We have a timeline here. This is mom right around birth, and we have a child at day 4, 7, 21. And went to 6 months. And this is a critical window that hasn't been studied yet, and we need large data sets to really get the type of questions we want to ask. And so this is a beautiful study. We have a little bit, uh, information here— healthy full-term newborns. The neonatal period is really a deep focus here, although we have longer sampling. And I call— what I'm going to talk about today is the UK Baby Biome 1 Phase 1, where we analyze the microbiomes almost 600 children. And we've expanded that. We have a BioBaby, BabyBiome 2. We've doubled the number of children, infants, but I'm not going to talk about this today. But later on in the year, I think we'll start sharing some of this, and it's very interesting what we're finding in these large data sets. And using discovery science to go into these can be very powerful approaches from the microbiome sciences. And here just shows the distribution of the vaginal versus varying children, almost 50/50. Very, very important. Very nice for the statistics. And these are the recruitment rates around the 3 hospitals. Now this is Jan. Jan was a PhD student in my lab, now he's a staff scientist within the group, and he led the analysis on this. He's leading the analysis on BabyBiome II as well. And the first thing I want to talk about is the assembly dynamics of the neonatal gut microbiome. As the microbiome comes together, we see very cool patterns. And these are the vaginal-delivered children and these are the C-section-delivered children here. First, look at the purple. This is the mom. This is an adult-like microbiome. If you come over here, you see that there's this greeny color, blue, and we get this red color and green. This is the assembly of the microbiome as the child ages, coming up and going more towards adult-like microbiome. This sometimes takes 3 or 4 or 5 years depending on the environment of the child and the diet, a number of variables. We see a very similar pattern here with the C-section babies. The adults over here in the same pattern as assembled.. Now, you could argue that there's no difference at this level, but this is a very high-level analysis, very global view. So what we've done next is we zoomed in, and this is where it's pretty clear what happens with the C-section babies compared to the vaginal delivery babies. There's a delayed commensal microbiotic colonization after C-section birth. So these are, this is relative gene abundance, and these are all the children from day 4, 7, 21, and you can see around 6 to 9 months in general, The vaginal-delivered children, you see a lot of reds. Bifidobacterium, I mentioned this earlier, highly adapted to humans. And then we have other bugs like Bacteroides, I mentioned them earlier as well. And then we get up to the top here, these are— the blue is a little concerning to me. You see things like Enterococcus, Apococcus, Klebsiella, Enterobacter. I worked in healthcare infections before, and these are some common names you'd find in a hospital setting. And that's where the— when you look at the C-section, the T-section babies, it's quite striking the amount of blue here in the day 4, straight away, like early, early days. This child, they're highly colonized and they have very few of these beneficial bugs. They do seem to— they do recover, but there's a stunt in this acquisition of some of the microbiome that we're going to focus on more. And these are types of bugs here, these at the genus level, mind you. So we could drill down even further. We talked about the taxonomy, the genus level. We'll go down to strains, even species, and strains now with this analysis. So what Jan did is he actually looked at the number of strain-sharing mother-infant pairs. And what he did is he looked at the vaginal-delivered children, and we'll start there, and he asked, well, which strains are in the mom and in the infant? We can use genomics to really get down to high resolution and make that call. What we found is actually things like Bacteroides vulgatus, I referred to this earlier, is very commonly transmitted between mother and child. Bifidobacterium, I mentioned, these are very common. Now, in the C-section babies, there's almost nothing. This is being blocked and stunted. We're going to try and tease it apart in a few slides, but there's something about the C-section process that's inhibiting this transmission, we believe. You can look at the list of bugs here and what you'll notice is most of them, there's 18 here, 14 are either from Bacteroides or Bifidobacterium, highly adapted species. And instead we get other bugs down here that spore formers that come in the C-section babies. Now this slide talks about what happens in the C-section babies. What we think is the C-section promotes colonization by healthcare-associated pathogens. Now the two, two figures on here, the left shows the relative abundance within individual children. This is the frequency. So you see that in blue are the, the pathogens inside the C-section babies, the vaginas of the babies. We also have the mother on here. We took samples from the mom. The first thing is we don't see a lot of these pathogens in the mom. We think it's probably coming from the environment. We'll talk about that next couple slides. What you see is Enterococcus faecalis, Escherichia pyropharyngens, Enterococcus cloacae, Klebsiella oxytoca, Klebsiella pneumoniae, and then combined. Now these are, these are not the type of bugs you'd want in anyone to be to be clear, but they're always higher in the C-section babies, and combined, it's almost a third of their microbiome are these opportunistic pathogens. This is very high. Mind you, we are seeing in the vaginal delivery children too, to a much lesser extent, but it is happening. Frequency is another concern as well. You find that almost 80% of the C-section babies have these bugs in them, and maybe about 50% of the vaginal delivery babies. So we think that the C-section itself is shifting it away from the beneficial bugs and allowing these opportunistic pathogens to colonize. Now, what we've done here is this is a different type of sequencing. What we did here is we have these abilities to culture bugs from feces, and so what Jan did is said, well, let's go and target these bugs, pathogens, and let's culture a lot of them and sequence them. Because what we can do with whole genomes, we can look at various antibiotic resistance genes at a high level you can't really do as well with metagenomics data. So Yann did mass culture on all these pathogens I just mentioned, but I'm going to show just one example here, Enterococcus faecalis. Cultured 356 of these, whole genome sequence. And what we can do now is we can take all these public datasets available around Enterococcus faecalis and combine them with our UK baby dataset. So we also have Enterococcus faecalis from adults in UK hospitals, and then we have a global collection. We can build this tree, and we talked around trees here. Here's the root. If two nodes are close to each other, high related. If they're far apart, they're distantly related. And what I want you to take a look at is around the outer circle, these colors. Remember, all the UK babies in our study, the Baby Biome Study, are in red. The blue is from a global collection. This yellow is from UK hospitals from adults, blood from infection. The first thing you notice is there's no discrete clustering of the UK baby microbiome, or Enterococcus from UK babies. It's all around the phylology. We think it's probably coming from the environment. It's different sources. There's not one distinct lineage. Of Enterococcus faecalis. A couple other things we can look at too is the prevalence of virulence genes. These strains do have a lot of virulence genes, cytolysis, for example, in half the strains, a number of other virulence factors, and we have a lot of antibiotic resistance. However, we don't have any vancomycin, which is a good sign. These are the VRE, vancomycin-resistant Enterococcus, is not found, not common in these children. However, some of the other pathogens did have extended beta-lactamase. Extended-spectrum beta-lactamases. Those type of antibiotics are very concerning. So that's what we're finding in the children, and this is showing high resolution. Now, something I didn't talk around yet is we have a lot of metadata. I mentioned it earlier. I alluded to it. And what we can do with this data now is start to combine it with the microbiome data and a number of different clinical variables. And so we have a long list, and what Jan did is analysis to actually highlight the different covariates that are significant, have a significant impact on the variation of microbiome. What he's done here, this is a perinatal analysis where he's actually stratified by age here. Before it was based on C-section, vaginal delivery, now it's age. So different. And a couple things, for example, the delivery mode had the biggest impact on the variation microbiome, effect size of 8%, very large, and dissipates with time. Goes down. Maybe the bugs are acquired from the mom, maybe, speculating a bit. The second category, there's antibiotic given to the mom, so the C-section, the mom gets antibiotics, and they also get the antibiotics around birth for other reasons, and we have all that data. Again, a big impact early on, it dissipates, it levels off a bit. And the second thing I'm going to talk around is partial and exclusive breastfeeding. We have this data as well, and we can link it to the microbiome composition and variation. So there is a significant impact, and I think not one of these— delivery mode has a massive— but we got to think of all these, how these things all contribute to the overall picture. And that's what I get from this type of analysis. But we can drill down a couple of these points and tell you where we are with our thinking. First off, the effect of maternal intrapartum antibiotics on neonate microbiome is quite interesting. We do see two groups. So these are microbiome from children that were born vaginal delivery. These children here, their moms had antibiotics, and we can actually see patterns in the disruption of microbiome in these children through the mom. These are— we've seen these, went through this in detail earlier, but we do see a specific disruption of transmission of bacteroides from mother to child. That's very distinct here. You maybe can't see at this level, but that's very important. We also see higher loads of opportunistic pathogens in the children whose mom had got antibiotics. So what we can do, if you remember back to earlier, the slides of C-section babies, the blue was actually maybe 80%. So it seems to be an intermediate between a C-section baby and a vaginal delivery baby where there's no antibiotics involved. So they're all contributing in different ways. And I wanted to just go into a little more detail around this. Neonate pathogen colonization is linked to the amount of antibiotics and show you some data underpinning those claims. These are, again, the combined. These are the pathogens. And you can see the differences. For example, combined, there's a statistically significant difference. It's a higher level of pathogens in these children whose mom received the antibiotics. And also, the frequency is much higher. Very interesting. Now, with the BabyBUCA/BabyBiome II analysis, we're going to double the number of children. We hope to get more of a signal here, a stronger signal. This is a long-term goal. Breastfeeding influences microbiome. Later in infancy. So this is a— I'll take a second to walk through a lot of information here. We have the C-section baby, let's cover that first. So the C-section babies, these are that were breastfed and these that were not breastfed. Very interesting, we actually see not that much of effect on these children because the key ones aren't there that expand, maybe the bits of those are missing. But we do see an effect on vaginal-delivered children who are breastfed compared to non-breastfed, but it's the, the difference actually happens later on in infancy, you know, 6 to 9 months. So it's a long-term impact, we think, probably after the neonatal period, maybe the antibiotics given or not, and it settles in and starts to assemble. This is the idea at the moment. So I'm going to summarize the key messages here from Phase 1 in UK Baby Biome. And so we have our newborn child, this new ecosystem if you will, and as the baby comes in, it's to the world, uh, you have— this is what Mother Nature wants. This is deeply evolved, the transmission of bugs, commensals. And there's other, other body sites as well, I'm sure, combined to the acquisition assembly of the microbiome in the child. And the breast milk is probably the food that these bugs— and these are co-evolved for millions of years. Now what happens with modern lifestyles, things like C-section, antibiotics, and non-breastfeeding We see that there's a shift from these commensals towards opportunistic pathogens that are likely in the local environment. So this is more or less what we think so far. I want to say that this was a UK study that we talked on, the UK Baby Biome. I put this slide in to raise a point. I don't think what we're seeing is specific to the UK. I think it's probably broader. Now, this is a nice paper from Christine published a few years ago. And this looks at the DALYs, the Disability Adjusted Life Years. Stratified by different ages, and what they're looking at is the burden of infections across the EU. I want to point out here, children under the age of 1, they take the large— the highest burden across Europe. There's something, something going on in an antibiotic infections in early life that we should start to understand better. So I think it's, it's not just UK, it's a broader— now if we take a step further back and we say, well, What about children in low and middle-income countries? This is what the scene looks like if you compare the UK up here down to Africa, for example. And babies from low and middle-income countries are the most vulnerable across the globe. So we're thinking global health and baby microbiomes here now. I want to talk you through a few slides here and tell you our longer-term plans in this area. So we have a deep focus on malnutrition and growth stunting. So you may notice the two, two, two maps overlapping in these sections here around Africa. Now these green, green countries are highlighted— 34 countries that harbor 90% of child malnutrition spending. About a third of these children, 19 million, with severe malnutrition, they fail to respond to therapy. These are diet— these are nutrient supplements that are designed to give exactly what the child needs. They don't respond. Maybe the microbiome is not there to convert it to useful energy, nutrients, perhaps, and subject to risks of about 1.5 million deaths per year with considerable mortality and morbidity. So this is the problem. This is the situation. I want to tell you about— it's a newer project here— the CHANGE study. I'm not going to talk over any of these. I just want to highlight— and this is a very exciting area and a bigger picture of what we're trying to accomplish by generating large datasets for global health. Now, this is Jay Berkley. He is the co-lead with Judd Walson. And this is the team here. This is Caroline, Bonface, and James, and they've all been to my lab or will spend time in my lab helping analyze this dataset. And what the study is looking at is 9 different sites, 7 different countries through Africa and Asia. It's a beautiful study, and what we're looking at is how hospital admission is a significant risk factor for acutely ill babies with wasting. So the situation is the children with severe wasting, and we saw a picture of a child with severe wasting earlier, when they go in the hospital, there's a significantly high mortality. And this shows here the time from admission, and this is the cumulative mortality hazards here. 20%. With moderate wasting, it's still significantly high but quite a bit less. And these are children with no wasting, and this is what you expect from these settings, these 9 sites we talked about. So we're looking at the microbiome and trying to understand, is there something with the microbiome that's explaining this? One, the severe wasting, but also how, how they're vulnerable to something in the hospital. Could be an infection picked up, for example. So this is the type of question we're asking. It's also addressing another question, another issue, I should say. The low- and middle-income countries are neglecting, you know, microbiome studies. And this is a snapshot of the publicly available data set. There are almost 70,000 metagenomes in the public domain, probably even more now. And this just looks at percentage of metagenomes based on continents. And you can see South America is very low. I mentioned one way we're trying to address this. And Africa is very low. And so what we're trying to do is correct this and make sure that these public metagenomes are made public so people can access them, use them, and get on with research. So something very exciting is part of what's one of the the main aims, or a major aim of my academic lab at the Sanger. So I'm winding down now. I want to end on this point and the idea of building human microbiome genomic basis for global health. So I talked about the lack, the dearth of metagenomes in some countries. And these are the LatinBiota sites. So LatinBiota is led by Gregorio, who used to be at Sanger. He's based in Uruguay. And he set up these study sites all through South and Central America. And these are the sites that we're sequencing metagenomes now, and eventually will be in the public domain for everyone to use. These are the sites in Africa. And we think this is going to be really important, and we think long term it will impact and help the maturation of human microbiome science. So really excited around this. And here are our key partnerships. Everything we do is underpinned by key partnerships, long-term partnerships. Nigel, Alison, Peter, Gregorio, we have Jay, James, Boniface, and Caroline. So huge credit to them as well. So I'm going to wrap up here, and this is a longer-term vision. How can we use gut microbiomes to nurture childhood development? Is this an option? We're starting to put the science, the data together. I believe there is. There's a long way to go, but I want to summarize what we think is happening in when a child is born, there's an acquisition of microbiome, assembly of microbiome, and maybe it goes along this trajectory. We know about the maturation follows almost maybe an age or timeline, and a lot— there's a rapid amount of development going on in the child, uh, immunologically, metabolically, neurologically, and this is actually part linked to the microbiome and how it evolved. However, we think there's things going on with pathogen colonization. These aren't uh, Enterococcus mucosae, these pathogens I mentioned, they come in. I think what they do is they interfere with the ability of these beneficial commensals, the Bifidobacteroides, to colonize. There's a competition. I know these pathogens we spoke about are highly evolved to transmit and move around hospitals, so this is really something we think we're going to focus on now, how these pathogens and commensals interact. And we can use the commensals ultimately to shift a newborn or a young child's microbiome back on the right trajectory towards health. And these are some things that we think about when we develop live bacterial therapeutics to purposely inoculate a child at birth. Maybe a child had to have a C-section for whatever reason. But can we rationally design diet improvements? I know the Gates Foundation and a number of others are very interested in this. Very interesting. The idea that can we develop predictive biomarkers and diagnostics around the microbiome? Could we actually profile a child's microbiome birth and predict their developmental trajectory and their growth trajectory. These are interesting ideas we're pursuing. And also, I think, you know, at the end of the day, I think the data, what I see coming now, I think we will be able to offer guidance to inform clinical practice and policy, especially around antibiotic use in children. We need the data sets to really make these decisions. And here's my final slide, the acknowledgments. I am the messenger. I'm part of a large team and a number of partnerships. And here's a A nice picture of my lab. Amazing group of talented scientists, ambitious, and they get huge credit for doing this. I talk a lot about Jan's work here and a number of others involved with the other partnerships we have. Good partnership with EBI, Rob Finn. We've had a lot of successful publications. The Gates Foundation, Chain Study, and the Baby Biome Group, the whole operation, if you will. Huge thank you to them. They get big credit. Thank you for your time, and I think we'll have some, uh, some time for questions now. Thank you very much.