 Thank you very much, Rob, for your kind introduction. And I want to thank also Lita Proctor for inviting me to be here today. So site-specific communities have co-evolved with hosts. And their genes conform unity with the, in this case, the human genome. And we know that as the hollow genome. How do we know that they have co-evolved? There are different evidence of work. One was given by the group of Phil Hugenholz, who studied the evolutionary relationships in wild hominids. On this phylogenetic tree we see the humans, these are the host, two species of chimpanzees, and two species of gorilla. Then he sequenced the fecal communities. And we see in this tree the humans, the two species of chimpanzees, and the two species of gorillas. So the fecal microbiota recapitulates the topography of the phylogenetic tree. What this means is that in this case, genes have to be transmitted within species. And for that, from the microbiota point of view, vertical transmission has to be very important. We know or we believe that the mammalian fetus develops in a bacteria-free environment. But the mom prepares and the mother microbiota changes in the vagina and in the intestine, as Ruth Lee told us yesterday. So we asked the question, how does the microbiota assemble early in life? And Ida Pantoja, my former student at UPR, at the University of Puerto Rico, studied six mothers and six individuals of the offspring during days one to 21 of development. This is the winning time in mice. So in this cluster analysis, we see two branches. In one branch, we have the maternal vaginas and the feces of the offspring at day one to day nine. So they are clustering with the communities of the vagina. In the other cluster, we have the feces of the offspring at day 21 and the mothers. So what this tells us is that the first microbiota in the babies is vaginal. We can see in the rarifaction curves what happens with the richness. In the pink box of the first two plates, we have the maternal vagina and feces. And then individuals are in different lines with different colors. The babies at day one, at day three, at day nine, and at day 21. We can see a decrease in richness in the number of OTUs or species or phylo-types. And all babies converge to decrease the diversity. And this period is streak lactation, which in these mice lasts for 14 days. After 14 days, they start leaking solids. By 21 days, it's the winning and they recover the diversity. When we look at the taxa, we have the dominant taxa in the top panel and the rare taxa. We have the mothers in the pink box. And what we see is that the babies are born high in streptococci, which decreases to be, and coincides with the bloom in lactobacilli. We don't, so we can see that mothers provide the first vaginal inoculum. That streak lactation markedly reduces the microbiota diversity. And that fecal-like microbiota develops after the introduction of solid food. In humans, we don't know really what is the importance of the vaginal inoculum or the importance of the early microbiota during streak lactation. And we can see here the diverse cultural manifestations that lactation has in humans. But we do impact, in our culture, we do impact both severely. By doing lots of C-sections and by breastfeeding. This graph evidences the increase in C-section rates in the US from 1996 to 2007 in mothers of different ages, age groups, or different ethnicity. Everywhere, the increase has been about 50% in this period of time. This is in the US, but in the world, we can see very alarming figures too. The countries that have the lowest C-section rates, the lowest is the Netherlands. And then we have Scandinavian countries, usually countries highly technological, highly developed. We see the rest of the world having above 25% of the babies being born by C-section. And they show different levels of development. The top countries in the world are Brazil, with 46% of the kids being born by C-section, Dominican Republic, and Iran. The US is in the group of 30 to 40%. So this is generalized tendency to which some European countries have resisted. And basically, doctors and mothers think there is no cost associated to C-section. C-section is okay. There is no biological cost or no collateral effects. That's their belief. So we ask the question, what is the difference in the newborn microbiota by delivery mode? And we did this study in collaboration with Rob. And this slide was shown by Rob and Ruth. So we have the mothers delivering vaginally or by C-section. There isn't much difference in their mouth, skin, or vagina microbiota. But the babies born vaginally have a pattern similar to the mother's vagina. The babies born by C-section acquire human skin bacteria. We can see this in this principal coordinate analysis. Again, the mother oral communities in green, vaginal in red, and skin in purple. Babies, any side of the body of the newborns born vaginally, clustered with vagina. Babies born by C-section, clustered by skin. We are now doing the follow-up study. We are following babies for one year to see when there is a recovery. We don't know the implications for health directly because there are no longitudinal studies. But we have epidemiological evidence that C-section is associated with diseases that involve malfunction of the immune system or high inflammation, including type 1 diabetes, celiac disease, asthma, and obesity. We also have learned, as Dan Liedman greatly explained yesterday, that the human microbiota educates the immune system. And recently there is a very nice work in which they show how the bacteria and TLRs sensing bacteria modulate circadian gene expression. And we also know that the microbiota is important in metabolism and even in brain development and function. So our multiple impacts, we do compounded perturbations on right at birth and later. Not only we do C-sections, we give antibiotics in C-sections and all mothers that are strep B positive. Then typically we bottle fed, especially the C-section borns baby, tend to be more bottle fed. Then we will give antibiotics because the baby has otitis. And when the baby is able to eat food, we give the baby processed food. And we have learned from basic principles in ecology that the way you impact the ecosystem depends on the ecosystem status. So if you impact an ecosystem that is mature, the resilience will allow recovery. But if you perturb, and before it recovers, you perturb again, that can lead to extinctions. The same if you start with a not normal ecosystem, that can lead to extinctions. So how different, how impacted is the human microbiota that we know best that the HMP has studied? In a collaboration with Jeff Gordon, we studied the gut microbiota across geography. And we compare the Amerindian guajibos from Venezuela with Malawi people and people in the U.S. And we, in this principle coordinate analysis, we can see how the U.S. clusters really apart with the other people from the two continents clustering together. Also, we can see that although babies had the lower richness as shown in the rarefaction, with no differences between geography, geographic locations. In adults, U.S. had the significantly lower richness in relation to the other two groups, and the Amerindian had the highest richness of fecal diversity. This is in the gut, but in the skin, we also collaborated with Marty Blazer, and we studied Amerindians and U.S. people. We can see the cluster of the skin microbiota here. U.S. people cluster separately from the Amerindians. And basically, our skin in this country is dominated by actinobacteria, whereas the Amerindians were not dominated by actinobacteria. They could either be dominated by staphylococcus or by proteobacteria. In the case of the proteobacteria group, which we call group B, we could see that they had significantly more diversity than the rest. So this shows that there are differences by lifestyle. It's not necessarily by geography or ethnicity because of the clustering in the two different continents. But we ask, what happens? Why are these differences? And what happens with transculturation? So in an attempt to do a more controlled study, the Sloan Foundation is funding us to do a study at the same latitude, and the Amazon is a region of the world where at one latitude, we can find the whole range of transculturation gradient. From people living very deep in the jungle to rural communities, to towns, to modern cities. And we compare Achuar communities that live in the border between Peru and Ecuador on the Peruvian side. We compare them with rural towns also in the Amazon basin. Iquitos, which is a big town, very isolated but big. And Manaus, which is a very modern city in Brazil. We went there and we studied not only the people, but also their homes. Across this gradient, we studied 10 houses per location. We went with a team including architects, environmental engineers. So with the architect Umberto Cavallin from University of Puerto Rico, we are interested in studying architectural parameters that are relevant to microbes. So we can see, for example, from the jungle town Checherta to Manaus, and across the gradient, we tend to decrease the density in the houses and increase privacy. We also study other parameters that are relevant to microbes, such as air exchange rate. In Checherta, in the jungle, the air exchange rate is as high as 100 per hour. This is changes, exchanges of the air of the house per hour. To Manaus, where it's as low as 0.8. In North America, the normal figure is 0.5 per hour. So we lose ventilation of the houses. We also change the materials from 100 percent biodegradable to a very high proportion of synthetic. This is all relevant to microbes. We sample the houses and the equivalent spaces, functional spaces in hats. We sample the humans and the animals to study the microbes. We still don't have those results, but we have results from the first expedition we did to check that that was a good place to study. In 2011, we included the same community, Checherta, which is outside the scope of the Peruvian Health Ministry. We included Secunza, which is a Chuar community, that is inside the scope of the Health Ministry, but still in the jungle. The Little Rural Town, Puerto Almendras, and we are comparing with Puerto Rico as a reference. We see the richness curves show that there is a decrease in diversity in the gut microbes, fecal bacteria cluster apart by location, especially Checherta in one end, Puerto Rico really segregated. We can see in the taxa that the structure of the communities is really different, and as we saw in Africa, their microbiota in the feces is dominated by Prevotella, in Puerto Rico it's dominated by Bacteroides. There are also other taxa that are high in the Checherta end, which is shown in red, including, for example, succinibibrio, which decreases in Peru as trash cultivation occurs, and disappears in Puerto Rico. So this is in the gut, we observe remarkable differences. We did also the skin, same tendency, Checherta highest diversity, Puerto Rico the lowest, they cluster apart, and we can see the taxa in the skin that explain those differences, and it's remarkable that Prevotelas, just like in the feces, the skin of these people are higher in Prevotelas than the skin in Puerto Ricans, and in Puerto Ricans we have more Bacteroides, and there are other species that vary, marking red for the Checherta end and in yellow for the Puerto Rican end. So again, the gut and the skin are different in the gradient of transculturation. We were very lucky to be able to have access to samples from un-contacted people. Venezuela is a rich country, and because they live on oil in the 60s, early 60s, they declare the Amazon a national park without access, with restricted access to a certain point, and complete isolation is a national park with military, you require military permits. These days you are not allowed to go if you're not Venezuelan. So we know Yanomami communities, they live in the border between Venezuela and Brazil. They were traditionally mountain people, now they live in the rivers, and those are the communities we know, but there are still many communities in the mountains that haven't been contacted, and in 2008 the Ministry of Health decided to fly over the very extensive jungle and find and contact communities in the mountains. They went to 11 remote communities of which eight were first time contacted. We were, we sent our team in Venezuela, a team with which I have worked for 20 years in that expedition, and they were able, from a community of 57 people, they were able to swab 28 people and get 12 people, get visas from 12 people before the doctors gave vaccines to the kids or antibiotics to the patients that needed. This is a great natural experiment, we would love to go back and see what happened after the antibiotic introduction. They haven't, nobody has ever gone back yet, and we made a big team of people to study the microbiota of these un-contacted Amerindians. We will probably never have another opportunity. We are doing community studies, composition, resistome and antibiotic resistance, metagenome, and making a reference strain collection to study whole genome and characterize each cultivable bacteria. These Amerindians cross, their ancestors crossed the Bering Strait 14,000 years ago, and there is no record of previous contact of these people before this expedition. Interestingly, they knew about us, they knew they were non-Yanomami who flew those things, they had a Yanomami name for the helicopters and planes, but they knew the word medicina in Spanish, and they learned that from Yanomamis who were told by other Yanomamis who were told by other Yanomamis. Medicina is extremely popular, and in all of our Indian villages have been, it's very welcome and wanted. So we started the microbiota in three body sites, the skin, the feces, the mouth. We are comparing them with U.S. people from Colorado who were extracted the DNA the same way, sequenced with the same platform. We can see that by body site they cluster in the right site in relation to the U.S., and we can see the lower diversity in the oral microbiota of these Amerindians in relation to skin and feces, and we see the typical patterns for each body site. When we compare to the U.S., their feces, their fecal microbes, we can see here the beta diversity in four panels. The first panel, we do the assignment against reference, which is green genes database, but we get a lot of unknown sequences which we recover assigning with RDP. We still keep the clustering apart. Then the unassigned sequences are these, and this is the global sequence data. So in all cases, we see remarkable clustering apart. The Amerindian feces have much higher diversity than the U.S. They differ, this is the phylum level structure or genus level, and they differ in a very different way that we saw in Peru or with the Malawian. They have a dominance of pre-Votela, U.S. have dominance of bacteroides, and succinibibrio is high in these peoples and absent in the U.S. Then we said what are the 100 most important bacteria that explain the differences, and we can see them in this heat map. In the top, we have the clustering with the U.S. people, 157 people versus our 12 people who donated feces in that community. What we can see is that all these Americans have, of the 100 autos, they have a minority of them, about a third, of which Amerindians have some, as we can see here, but the Amerindians have a lot of OTUs that are completely absent in the U.S. Not only U.S. people lack some bacteria that they have, but in this curve, which we call a prevalence curve, what we show is the abundance, the logarithm of the abundance of OTUs that are above .005% in the gut, and how many people have them, prevalence. As we increase the abundance, less people have that abundance as expected, but the Amerindian curves are shifted to the high abundance side. What this means is that they have more of the OTUs they have in relation to Americans. Using pie crust, we predicted the metagenome of the feces and we compare the Amerindians in blue here with the Americans. Again, a lot of more genes are present in the Amerindians than in the Americans. Remarkably, Americans have a lot of genes related to lipid metabolism, much more than the Amerindians than the Amerindians, and Amerindians have a diversity of genes in more abundance that are related to carbohydrate metabolism. The abundance curves are also shifted to the right, indicating more expected genes based on the 16S data. As I said, we culture in different media and different aerobic and anaerobic conditions, and we want to characterize this valuable collection of strains and compare with the strains that are circulating in the rest of the world. We started with E. coli and as a proxy to whole genome, which we still haven't done. In collaboration with James Walter, my students, Elena Rodriguez, went last summer and did multi-local sequence typing of the Amerindian E. coli strains. They are shown in bold and the Amerindians were assigned to 19 sequence types, 12 of which were novel. Six novel alleles were discovered. More than 50% of the estates detected in the Amerindians were novel as well. Interestingly, no strain fell into the B2 group, which is very common in western societies. So they seem to have unique E. coli strains. We did antibiotic resistance test on these strains against 29 antibiotics, and we find they were all sensitive. 56 strains that we tested where there were no antibiotic resistance. Gautam Dantas from the University of Washington in St. Louis and Erica Ferson, his student, studied four people, fecal and oral samples, and they studied the resistance, the presence of resistance genes in these people, and they did find resistance genes. 29 antibiotic resistance genes, the majority having high identity with HMP. So they have functional strains, but are silenced, seem to be silenced. So probably they might be activated upon exposure to antibiotics. So we think based on this body of work that we have lost, we have impacted our microbiome, and that generation to comes will obtain impacted microbiomes from impacted mothers. And for this, we should be thinking in ways to restore. So we are doing restoration in some instances. For example, fecal transplant is a way of restoring an impacted ecosystem. But for C-section, more babies, we are doing a study in which we are restoring the vaginal microbiota of the mother into the newborn, born by C-section. The study has been done by Cassandra de Jesús Lavoy in Puerto Rico. We are sequencing now the first months of the study comparing vaginal delivery babies, C-section born, or C-section with vaginal inoculation. Puerto Rico has 50% of C-sections. Half of the babies are born by C-section. That implies about 30% of elective C-sections with no medical indication. So the moms enrolled in this group have to be mothers with elective C-section, no medical indication, strep B-negative, HIV-negative, healthy pregnancies, and vaginas dominated by lactobacillus with a low nugget index and high acid pH. So what we do is we, before the procedure, which has a fixed time and everything is very easy, this is the easiest group, the C-sections, we sample the mom, we introduce a gauze in the vagina for one hour previous to the delivery, then the doctor, a surgeon extracts the gauze, when the baby comes out, we expose the baby to that gauze, and then we sample the babies as we do in the other groups. This is a Smithsonian documentary recently aired called Aliens Inside Us by Chad Cohen, and I'm showing the surgeon and the time, the first baby of the study of this group comes out, they are brought to the side where I was there waiting with the gauze, we pass it first through the mouth, then the face, then the rest of the body. We hope to demonstrate that these babies get a normal microbiome at birth with the first results of the first month of the data. So we know very little about the unimpacted microbiome and microbiota of the people in the world and we need that knowledge because if we lost species we have to know what to restore, what species or strains to restore, we have to find out ways of restoration either as the true human probiotics that have co-evolved with us or as microbiome vaccines to expose the babies to the right antigens at the right time. There are lots of gaps and challenges in this field. We don't know really what is the developmental role of the early microbiota during those six months in humans when the babies really develop their sensorial motor, their brain until the baby is able to grab for solid food and put it in the mouth. What are the microbial changes associated with trans-culturation? What functional changes we caused? What are the plastic versus the permanent changes? What is the role of eukaryotes that are very common in all these ancestral people, people living ancestral lifestyles? Is there lost diversity? What tax and functions to restore? Because the HMP and most current research is still done on impacted people. So the human microbiome project should be called the Western People Microbiome Project. We have the technology to increase that knowledge, globalize the scale. We need to act fast because these ancestral communities are disappearing very fast and we think that knowledge is really fundamental to public health and understanding and reverting the current tendencies of diseases in modern society. With that I would like to thank my collaborators of the Sloan Project, my students at University of Puerto Rico and my colleagues which are joining efforts with me to discover the microbiome of ancestral people. Thank you very much for your attention. Okay, thank you for a fabulous talk as always and we have time for a few questions. Please use the microphone. Yeah. So wonderful talk. There seems to be an assumption over here. There seems to be an assumption underlying your work that an ancestral microbiome would actually be good for us in Western society to have and isn't it possible that the microbiome in the Amazon communities has evolved to the lifestyle of those people as ours has as well. Is it really a foregone conclusion that their microbiome would be better for us as well? Yeah, that's a very good point. That wasn't my conclusion. I was trying to be clear that we don't know but the point is we have evolved during 100,000 years as human homo sapiens species. 14,000 years ago they moved there and they've been isolated and it's only really the last few hundred years that we have changed dramatically our lifestyles. So evolution doesn't work that fast. We are impacted in ways we don't think we are co-evolving where microbes are acquiring the fitness and the mature ecosystems because we all these impacts have happened very recently. We need to understand in healthy people of those communities. We study only healthy people. What are the differences and what we have we may have eliminated with practices that are antimicrobial and then determine that we need to study a lot to conclude that we need restoration. In the case of the C-section we know that those babies shouldn't have the wrong intervention is the C-section and that's why the IRB allowed us to do the intervention but evolutionary speaking is our impacts are too recent. Robby. That was a fascinating talk on the geographic distribution. What is the average lifespan of the Yamamoto? They live very shortly mostly because young boys die in accidents. Women die a lot there is a lot of cervical cancer related to viruses and they die of infections because they don't have antibiotics obviously. So we don't know what would be the lifespan if they weren't subject to those risks. They were some old people so they don't count they don't keep track of time and of age but other Janomamis estimated the ages and there were people up to 70 years old estimated but we really don't know the lifespan. Right so I think that's a consideration and as Western society has changed and there is disease if you look at the average lifespan it continues to increase so I think it's I agree with you it's extremely complex issue I think we have to take more variables into consideration you know as we start to sort out these complex issues. Yeah absolutely their focus though should be that we are increasing diseases that before were uncommon related to abnormal immunological response and that's where we think that focus should be. We have new epidemics associated to modern lifestyle. Yeah take a question for my player. Thank you Maria for a really outstanding talk and congratulations on doing some very very difficult work in a culturally appropriate and sensitive manner. I do think I want to just echo the cautions of some of the interpretations of the Caesarean data. If I remember correctly your study on Caesarean versus vaginal birth was done in Venezuela in a limited cohort of individuals and extrapolating and generalizing that to larger Western cultures is may have some some dangers with it. The term elective Caesarean as a many years practicing obstetrician gynecologist not just in the U.S. but Malawi is a dangerous term because we code for the term elective Caesarean to indicate women who've had a prior Caesarean and are choosing a repeat Caesarean either because their local hospital does not offer vaginal birth after Caesarean or because it's contraindicated for other reasons so elective Caesarean does not mean that we are choosing to do a Caesarean and in fact the American College of Obstetrician Gynecologists have tracked now for almost 10 years the number of obstetricians who are willing to do Caesarean on demand meaning a woman who has not labored and is electing to do a Caesarean with no indication whatsoever and it's fewer than 3 percent that is not true throughout certain regions of Central America and South America but that is true in the U.S. Yeah I agree with your comments absolutely and I also think it's really important to remember we do Caesareans for indications in life-saving reasons having delivered tens of thousands of women I can't think of a single occasion where it hasn't been a very difficult decision for me as her provider and her as an individual and if we go to these regions of the world if we take that same latitude of the Amazon basin and move it to Malawi one in 29 women die during labor here our maternal mortality rate is right now about 1 in 500,000 so comparing and saying that the introduction of Caesarean has really harmed our society I think is a dangerous statement to make yeah absolutely I want to make clear that I'm not saying that doing C-section harms our society so please don't take me wrong yeah what I'm saying is that in Puerto Rico half of the babies are born by C-section and there are elective C-sections by mothers decision and doctors without previous C-section those are not the cases in our study because we we these mothers have a previous C-section and so 50 percent that means in comparison to the Netherlands that there is at least 35 percent of the women who in the Netherlands would have had the babies vaginally yeah there is a lot in Puerto Rico of moms that say I don't want to go through birth Puerto Rico is part of the US it's not and they in New York 50 percent of the babies are born by C-section so I really look forward to seeing how this data plays out over time in larger population cohorts and many of us are engaged in to see how this works at a population based level and then also what the impact is when we look at six months of life one year of life three years of life and on into the future so thank you very much for your work you're welcome so we're a little over time if any if anyone has a really quick question we could maybe fit in one more Claire thank you Rob thank you for a great talk just want to make one comment it was clear from your data that in a lot of the rural populations that you looked at the Yanamamo that you saw a lot of evidence for Prevetella dominated communities and I know when we look at urban populations Prevetella dominated communities are unusual just one comment we did see a significant amount of Prevetella dominated gut communities when we looked at the old order Amish what was interesting was that those communities were almost exclusively found in farmers and farmers wives so it might be worth considering I'm thinking of the slide that you showed where you're talking about some of the impacts of modern life exposure to antibiotics availability of processed foods we might want to also think about including isolation from animals that may be something that is driving the communities and maybe worth considering yeah thank you very much good points okay I'm sorry but we're running a bit behind schedule this lot of interest in this and perhaps you could talk to Maria Gloria after the break so let's let's thank Maria Gloria again for fabulous and now I'll turn things over to Michael Gray from NIDDK and and it's Annette Reithamal from NIAID who will be chairing the next session