AMR and the Microbiome PACCARB Day 2 Part 3

Welcome back to day two of the 28th PACCARB public meeting. I hope you had an opportunity to enjoy lunch and take a break. We will now reconvene and continue with our afternoon program. I'll turn it over to Dr. Paul Plummer to introduce our final panel. Thank you, Sarah. Our final panel will examine how natural restoration approaches can strengthen microbiome resilience and support better health outcomes across humans, animals, food animals, and the agricultural system. To help close the day, we will hear from Dr. Saurabh Mehta from Cornell University. Following that, we'll hear from Dr. Marcia Costa from the University of Montreal. And then we'll conclude the panel with a presentation by Dr. Phil Myers from the University of Tennessee. Dr. Mehta, we welcome you to begin when you're ready. Good afternoon, everyone. How's the volume? I'll try to hold this. Thank you again for the council to include me in this panel. I'm going to talk about one of our recent studies, recent publications on food-based strategies to strengthen microbiome resilience in children. Next slide, please. Here are my disclosures. Just to point out, nothing relevant to this, but this research was largely funded or supported by the Harvest Plus of the International Food Policy Research Institute and the United States Department of Agriculture. I'll also start with the caveats. Since the morning session became a lot more about nutrition, I'm not going to tell you what I eat. I'm also not, I'm also not going to give you a personalized prescription or anything like that or endorse anything, even though we recently published an op-ed on that space for Neon. And I'm also, most importantly, not going to judge what you eat. With that said, sorry, next slide. I think we have already covered this over the last few days, that the links between what are the mechanisms that tie the gut microbiome to antimicrobial resistance and how by strengthening colonization resistance mechanisms, we can potentially protect against antimicrobial resistance. So I won't necessarily go into that here. Next slide, please. In this study, we focused on the first two years of life. And why focus on this space? Because early ecological disruption may reduce colonization resistance. It might enable opportunistic or end-stage pathogens to get a foothold and then increase exposure to mobile antimicrobial resistance. We also think diet is the primary modulator of the gut microbiome. It's a primary non-pharmaceutical way of being able to alter the gut microbiome. That was kind of our interest in looking at this session. Next slide, please. This is a study. Actually, this was my first big grant when I was a junior investigator. And it was funded about 12, 13 years ago. And it was a randomized control trial in the context of urban slums in Mumbai, which, of course, if you have been to Mumbai urban or any most international settings, I think now 60 percent of children live in urban slums around the world and or what would be categorized as urban slums. So a lot of livestock exposure, a lot of overcrowding and everything else that goes with being in an urban slum. The context for this was that we were trying to set the groundwork to diversify the procurement for the government programs that have integrated child development schemes which provide a supplemental ration, but not very dissimilar to what happens in the U.S. or what needs to happen in the U.S. And so setting the stage for that this can be done, this can be done at scale. And there is a whole pipeline that can be established from farm to kind of port in a way. So we worked with a lot of ag folks. We worked with crop developers and farmers to get a whole several tons of crops planted that we tested out for how much the iron and zinc levels were in those crops. And then we procured about nine tons of crops to prepare a whole slew of recipes, a cyclical menu for about two weeks to be able to deliver that in this context. Again, where it was delivered was three times a day to these children, about five minutes, not more than five minutes walking the street from their homes. So that's why the urban slum setting is also more attractive. And so we were so that continued for nine months. At the same time, there were studies that came out both in the U.S. and internationally which showed that giving iron supplementation and again iron is very critical during the first few years of life for child development and brain development and so on. And in that period, these studies came out which showed that giving iron as supplements was shifting the microbiome composition towards a more pathogenic kind of portfolio. And again, suggestive stuff. And so we hypothesized, I think this was back in 2016, that food-based, the food-bio delivery of iron will not have that adverse effect. So we proposed that we can set up the secondary analysis within this randomized controlled trial and we were able to get funding, secure funding for that. And that's what the results today are. So what are the big picture findings? We looked at two different things. What in the overall cohort, what's happening in the second year of life, mostly to establish biological plausibility that are the normal things that we would expect for microbiome maturation, are those happening here? And we see that as the diet became more diverse and so on, as kids get older, you can see that microbiome adapting to diverse types of inputs, what the graphic really is showing the tech pathways or the pathways that predict what function the microbiome components might have. They are not actually representative of whether it's functioning that way or not, because then you need metabolomic studies and others to do that. But at least it's talking about whether there is functional capacity. And so you can see that there is increased capacity to amino acid metabolism over the second year of life, increased carbohydrate processing, lipid metabolism, biosynthesis, folate and B vitamins were mentioned this morning as well, and increased xenobiotic degradation. The second piece, the second piece that we wanted to address was that did the intervention have any differential impact on these things? We were happy to see that the iron delivered through a food-bound approach didn't have the adverse impact that we had seen with iron supplements. And and what we saw actually was some of this is exploratory again. So with those caveats that we saw that with the intervention, there was increased antibiotic biosynthesis, increased enhanced invite to this panel, I guess. And increased antioxidant metabolism and also increased benzodiade degradation and so on. What the implications of all this. All this work is that there is enhanced colonization resistance potentially, and it aligns with lower pathogens and next. Again, a lot of caveats. The trial was not designed for this as a primary outcome. So there is antibiotic use and illness reporting was not the specific target. We collected morbidity data, but it was not, again, a primary purpose of this trial. Results remain speculative without in vitro validation. The absorption measures were not directly assessed. And is it generalizable to other populations? Having said that, 60 percent of kids are living in urban slums. We think it is, but it still needs to be tested. And since the first study on looking at microbiome with biofortified crops, we did do shotgun metagenomics, so those are strengths. We were able to do functional inference and also read time points across over a nine month period. So those are strengths of this study. Next slide, please. In summary, the metabolic profile associated with the intervention was consistent with increased secondary metabolite and antimicrobial biosynthetic capacity, greater antioxidant capacity and abuse abundance of potential pathogens, potentially stronger colonization resistance mechanisms. We also want to point out that food based nutritional interventions, they can support food systems already challenged, they can provide a culturally appropriate approach to nutritional interventions. There are no adverse effects on the microbiome compared to the conventional varieties that were already there. And also have simultaneous end goals or simultaneous things that can be achieved, like addressing our indeficiency while promoting optimal child gut health and microbiome development. Our findings are exploratory and hypothesis generating, and they definitely support the rationale for testing whether food based biofortification can influence pathogen carriage and gut resistance. Next slide, please. antibiotic stewardship system, but they are not a replacement for typical antibiotic stewardship strategies. With that, I will switch to thanking my team, and this work was really led by Dr. Samantha Hui and Daniel Cole, both on my team. Sam will have given this presentation, but she's out in maternity leave, and also crediting some of the functional analyses by Dr. This is a partnership with Rob Knight's team at UC San Diego as well. And I will thank the funders as well as some others and children. Thank you, Dr. This time, Dr. Costa is virtual. Get your slides loaded up and you're free to have the stage. Thank you very much. Thank you for this invitation to share a little bit of my knowledge with you about this topic today. Next, please. So when we talk about supplements in animals, we really need to distinguish animals between two categories, companion and farm animals. We do want to use antibiotics in pets when they are sick or they have a disease. But for farm animals, we use antibiotics mainly to promote growth and to enhance performance. So that's where the majority of the antibiotics are used worldwide. And we know that if we, the antibiotics selecting some profiles of microbiome, if you feed them to mice, for example, they will gain more weight. So it's very clear that the microbiome is capable of enhancing performance. But despite the measurements we've been taking to decrease the use of antimicrobials, we have not seen a decrease in the rates of infection and also neither in the use of antimicrobials in farm animals that now are more likely to be used therapeutically. So it's clear that we need to find alternatives to replace antibiotics. And we're going to be discussing a little bit of those options we have in the next slides. Next, please. So probiotics are for sure the most used. However, the evidence that they work is weak. And I state that because for every study that shows a beneficial impact of the probiotic, we have another one that shows no difference with control groups. And we also need to consider that there is a big publication bias, as most of the studies that didn't work don't end up at the publication process. And we need to distinguish also between bacterial and yeast-based probiotics, because if you use yeast-based probiotics in high doses, we have been showing that you do actually can change or cause microbiome changes that's believed to be caused by the cell wall molecules that those yeast have, and that can be fermented by bacteria, and that can modify the environment. And you can actually have some impact on the ecosystem. Now, it was pointed out yesterday that probiotics for humans are poorly regulated, and the same is true for veterinary products, where a lot of them do not contain viable bacteria or organisms that are claimed to be present in the label, and also have been shown that some of them even have some antimicrobial-resistant genes. So we need to be careful with those types of products. Now, when we talk about prebiotics, there are actually fibers, or as I like to say, food for bacteria. They do have a high capacity to modulate the microbiome, but however, it lacks specificity. That means it's going to enhance the good bacteria, but it can also enhance or increase the amount of potentially harmful species. And I have seen, as a reviewer, some publications where the authors tend to highlight the good changes that those products can cause, but they completely forget to mention or to give attention also to the potential harmful impact of those products. And when we talk about the use of prebiotics in pets, we have been using with success, like molecules such as celluloses, rhabdicillium, and most or almost all of the companies have now invested a lot of research to include those type of fibers into their diets. Next, please. Other supplements such as macro and micronutrients can be used to modulate the microbiome. For example, in this study, we were comparing a probiotic with manzoic acid and zinc oxide to control diarrhea and piglets. And what we can see in this plot, as it has been explained before, it's a big separation of the microbiome that is driven by the use of zinc oxide. And not like in the other, in the previous presentation, here we are talking about high doses of zinc. And we just have to pay attention because it is believed that the selection of tolerant bacteria can actually induce co-selection of antimicrobial genes. So this is widely used now in the industry, but in the future, you might find that as a problem. Next, please. In this other study, what we did was to induce experimentally infection with Clostridium perfringens in chickens, which is the causative agent of necrotic enteritis, a huge problem, economic problem in poultry production. And what I'm showing here is that the groups that receive the phytogenic additive and a postbiotic, which are byproducts produced by the bacteria, for example, butyrate, they tended to have statistically less lesions in the intestine compared to a group receiving probiotic and a control group. Next, please. Now, the question everybody wants to answer is what are the profiles that are more resistant to disease? But this is very difficult to answer because the profiles I'm finding here might not be the same as other researchers are finding, mainly because of the limitations of the technologies we use to microbiome characterization. That means most or the vast majority of these studies, they use short-read sequencing, which is limited to classify bacteria to the genus level. If I find one group has more Clostridium, I cannot tell which one of the 250 species of Clostridium are actually enhanced in that group. Some of them might be good, some might be bad. And also, for every difference in the methodologies, I might find one result that is not true compared to other pipelines used by other researchers. And even when we try to use long-read sequencing, what we found is that the databanks are completely based on human bacteria, and what is in the gut of animals remains completely unknown, at least at the species level. Okay, but let's suppose we are able to identify patterns of resistance to diseases. Now, to implement that, that would be really difficult, especially when we are talking about a healthy microbiome, healthy animals that we want to modify or modulate the microbiome. So, some people have tried to do that, but it becomes very different. And it has been mentioned over those two days that there are products available, for example, for humans to restore the microbiome. But what hasn't been mentioned is that the price for that treatment is extremely high, around $20,000 per treatment, where some people would pay that to treat their dogs or their pets. But when we talk about farm animals modulating that microbiome, it becomes a little bit prohibitive. Next, please. So, having that in mind, we also are trying to find solutions, which is the use of some bacteria that are less conventional probiotics. Let's put this way. In this study, we used bacteria isolated from organic chickens to colonize day-old chicks. And we could see that, on the contrary of the majority of the probiotics, these bacteria could actually establish colonization. So, we measured them with qPCR, and they were present in their guts 10 days later. And what was also interesting is that they were able to completely modify the overall microbiome composition, which at this point, we aren't sure if it's good or if it's bad. But next slide, please. When we establish which bacteria can actually colonize them in the right window, what we can do in the future is to use genetic engineering to, for example, knock out some resistance or virulence genes, and also to insert some beneficial pathways. For example, we know that a lot of bacteria, they produce bactericins that are able to kill other species of bacteria, or even to produce some molecules that have immune competence to stimulate the local immunity. However, it's very difficult to obtain regulation for commercialization of those products. When we present ideas like that to the industry or to probiotic companies, they tend to not see that with good eyes because it's a very long process, a lot of work, and especially strains originated from animals, they do not tend to invest on the development of those products. Next, please. So, to conclude, we do can use supplements to modulate the intestinal microbiome. Animals, they are not as potent as antimicrobial drugs, but we can see that it is possible, especially if you use the right products in the right window of intervention. Probiotics, despite the lack of evidence, continues to be the most used supplement for pets and farm animals. What we need now is to focus maybe more research into how prebiotics can be selected or modulated to actually cause more beneficial impact on the microbiome. We do need more discussions about policies to facilitate the commercialization of non-conventional and genetically modified microorganisms. I believe I finish here. Yes. Thank you for your attention. Thank you, Dr. Kast. And for our final speaker for this session, we're going to have Dr. Meyer. All right. Thank you very much for having me today. I'm a rumen microbiologist, and so today we're going to talk about fiber. Fiber is right up my alley, right up their alley. And so I'm glad for today for you all to be able to allow me to talk about a lot about the livestock perspective today with the microbiome, and there's been a lot to ruminate on today, so thank you for allowing me with that. I had to do that. Next slide, please. So as we begin to talk about really what strengthens the microbiome, the rumen microbiome itself, I feel like lends itself to be a very good model system to study resilience, but resilience and strengthening seem to be somewhat of a nebulous trend, right? With that, when I like to step back and think about it, I like to think about it in a number of ways. These aren't sequential. These are individual and obviously overlapping, but I like to look at it from the standpoint of adaptation and acclimation. These microbial communities are obviously part of the G by E interaction with genome and environment. The host, heritable microbiomes and diet are a large part, especially part of a rumen ecosystem. The other side we like to think about is the stability. So whether that ecological organization, that network, which I'll talk about a little bit in a couple of slides, and how that persists and develops under prevailing conditions. The beef production system lends itself to several different production cycles and dietary inclusion. So again, good model for what qualifies that as being a strong microbiome in context of those different production systems. And then finally, function. When we think about the microbiome supporting fermentation, we want that then to also make that has a host outcome for that animal health and productivity. So in general, this resilient microbiome doesn't mean an unchanged microbiome. A lot of the times we think that it's going to have to be some sort of stable interaction, but we'll see that in livestock communities, there's a lot of different types of interactions that we can categorize. Next slide. And so with this diet lends itself, like I said, there's obviously the G by E interaction, but commonly and historically throughout research on the ruminant and livestock side, diet, because of the nature of the ruminant, ecologically, environmentally, and evolutionarily perfect ruminant fermentation that lends itself to be very deterministic for substrate availability. I always like to tell my students and producers that we're basically, we're feeding the microbes that then feed the animal. These, I think was mentioned earlier today, obviously the fiber is not naturally degraded in the live species, right? And so with a lot of our microbes, that allows us to degrade things such as, you know, oranges and grains, proteins, cellulose, hemicellulose, xylose. Prove those microbial communities to ferment different byproducts, give the animal volatile fatty acids, microbial proteins, and other nutrients to be absorbed, whether that's food in the ruminant or in the small and large intestine. And the result of that helps us get a strong supportive animal that hosts a microbial metabolism that's helpful for feed and to be able to give those nutrients to the animal. So microbial adaptation and in the context here, not only that, but resilience and stability are normal part of this ruminant biology and critically important when we're talking about microbial strengthening. So next slide. So interestingly, the fiber part of my talk, if you want to get towards this, you know, I like to give a couple of anecdotes of some of the work that we've done to try to look at dietary transition models, you know, with the production system of cattle, you know, a lot of our southeast producers and whether they are grown and fed on grass or grass-finished or grain-finished, there are a lot of different dietary inclusions that occur across the production cycle. And so this high-grain or forage-based model, whether that is through a more fibrous to carbohydrate-based diet, really lends itself as a great model to study the stability adaptation function of the ruminant microbiome. Even when we do some of our work historically, a lot of nutritionists have always looked at, you know, introducing a new diet. Two weeks later, the animal's responding and that's good and that's all that we really lend ourselves to. But as we'll see, going from a forage-based diet with baseline nutritional environment, controlling that change into a new profile and a new adapted microbial state, we can look at how those changes may not correspond as rapidly as we thought. Next slide. So some of our work and some of the summarized slides here was looking at a lot of our ruminant microbial temporal stability over about 10 to 12 weeks of time. And through a lot of that work, responding from a forage-based diet to introducing at the beginning of this trial to a more substrate or a more carbohydrate-based diet. The interesting part is, as you can see through the top line there of a representative of we have microbial adaptation and responses really soon. We also see physiological changes in the animal. May not be weight gain yet, but we'll see, you know, blood vascular changes. We'll see a number of gut changes, but we'll also see rapid microbial adaptation. But what we don't see initially is that we will have a shift toward a final bacterial community around week four. So it takes 10 to 12 weeks on this extreme dietary change to even have a relatively stable microbial community. So that resilience must be evaluated over time. And I think that's a key point when determining if we would have taken some of these samples early, you're not getting an accurate community. Next slide. But what it does point out to us is that microbial stability also is not necessarily an indicator of change or untreatment. You know, we went from a community A under a certain diet to a community B. And we went through a lot of different adaptations or acclimations and then also dysfunctional if you want to call that, and stability and timing. And so this resilience we're talking about strengthening in the microbiome, especially in the ruminant system, doesn't necessarily, different doesn't mean dysfunctional. Stable doesn't mean unchanged. Timing is important. But resilience has to be defined as a successful adaptation and function and making sure that we can measure that community at the right time and taking those things into account. Next slide. And so when we start to finally look then what we can do to actually intentionally influence this ecology of the adaptive ecology, the stability that I mentioned prior, some of the other work we've been doing is, as we mentioned prior with some probiotic or direct end microbial work, is trying to use targeted native microbes that are known to isolate and colonize the ruminant ecosystem to strengthen the microbiome through interaction. Earlier it was talked about what is a normal microbiome, what is not. We have an interesting way of looking at it, not necessarily based on abundance, but actually through increased microbial organization and strengthening. So through our dosing of six microbes, I can get back to those later on what those microbes were, but through those native microbes to a fish-related, ruminant fish-related sphere, we were able to dose those through animals and conduct this research trial to where we saw in the control versus the native microbiome supplementation, we were able to see increased relationships, node sizes, and even some microbial taxon, again, with the summary table right there. But the biggest part was that we had increased connectivity and centrality, meaning that basically we had increased correlations among microbes, and the centrality meaning that we had increased relationships of impact on the community. So even the five out of the six microbes, for example, had reduced taxon abundance absolutely by the end of the trial. But had increased between networks centrality, meaning they were highly impactful by the end. So again, we were able to get a greater microbial connectivity and a stable microbiome to give us very good animals. So taking this together, we look at this as, yes, we have the geobody interaction, dietary interactions, huge part of this ruminant microbiome. But thinking about stability, adaptability, and function in context of this is the key part. I like to tell a lot of our students that there's not going to be a key ruminant microbiome when we know that. I think of it as a tree where the trunk is, if you want to call it, the core. And a lot of the things up here between diet, environment, stability, adaptability, and function are our branches and our leaves and our twigs. So if we can manipulate those to help strengthen the microbiome, I think that's where we start to stand in the production process. Next slide. And so finally, in context of today for potential pathways, I think the biggest part in animal agriculture is that we support normal means of maintaining healthy, normal functioning animals as possible. Meaning that through our curve that the greater proportion of what we have healthy animals in the herd, flock, whatever you want to call it, that they can respond appropriately through supplementation or dietary inclusion, the greater likelihood we can have a positive outcome. I think this correlates to a normal digestive and bacterial function, helping organize and maintain more healthy and resilient animals, and reduce circumstances requiring some antimicrobial intervention. Obviously, those, though, do have to be tested. But the hypothesis that strengthening these early on will help with upstream AMR. So overall, I think the take home with everything today is that with the rheumatic microbiome as a good model of the system, fiber, and a number of other things, being able to strengthen that rheumatic microbiome with adaptation, function, resilience, G by E, and diet. If we support normal, it's a great prevention strategy to start testing what we can do for AMR. All right. Thank you, Dr. Meyer. At this time, we'll open it up for council questions. As in the previous sessions, if you have a question, please, if you're in the room, place your pink card on the end, or if you're online, raise your hand. Please give your name at the beginning of the question, Dr. Pantaleon. Thanks very much for interesting talks. And you know, Dr. Meyer, you talked about strengthening the rheumatic microbiome. I thought that was a pretty interesting concept. And like Dr. Costa talked about, you know, things that we can put into the room and try to strengthen, it's not going to work. So maybe you're talking more about dietary strategies rather than probiotics, prebiotics, that sort of thing. Is that correct? And have you seen any, you know, with these strengthening strategies, have you seen any improved in weight, daily weight gain, or decreased disease prevalence? Have you looked at some of those key indicators to, you know, translate these to improve productivity? Yes. And thanks for that question. It's, I think a lot of it comes functionality to maintain. I think our approach with some of our collaborators was making sure that things are adapted, well-adapted, native, or acclimated to the rheumatic. Common times, even supplementing things like certain enzymes, historically, a number of things, they're not optimized for rheumatic pH. They're not optimized to even get through that four-chambered summit to be able to be impactful maybe downstream in the lower gut. So our approach here was to look at the idea of going through and isolating previous trials, some impactful microorganisms. Actually, the trial itself was supposed to be used for an acidosis trial for a lower pH incidence, and what we ended up finding that they were a lot better for average daily gain. We had a very significant outcome from that, and have been able to reproduce that as well through these microbes. So the key part about that is the native part as well, is why we monitored not only relative abundance of the community using a lot of amplicon work and metagenomics or reduced representation metagenomics, is that we also wanted to make sure that we did some absolute counting, at least of the microbes themselves that we administered, to be able to follow them through, be able to say that, hey, that they're still prevalent, because the theory again was that maybe we don't have to keep dosing them. And at some degree, it's probably best to keep dosing them. The ones that aren't native will more likely wash out at some point if they do competition. But regardless, we did see persistence of those microbes. Like I said, five out of the six actually had a reduced absolute abundance by the time the trial ended, but they impacted the community more. So that's sort of our little network model. Thanks to the speakers. Dr. Mehta, congrats on that very large, important trial you did. When I saw your presentation, it took me back to one of my former colleagues at Wash U, Dr. Maneri, who had done nutritional studies in a low-income country where antibiotics were used. I'm just curious your thought in regarding to your nutritional work, because obviously they had a nutritional component, too, with theirs. And how do we, you know, that trial actually had, you know, protective benefit by being on antibiotics. And there's always the debate in the antimicrobial resistance world, like, oh, how could you do that? But you're saving children's lives. That's why you do it. I'm curious your thinking in regards and on just this concept of what you're doing with this nutritional model. Where does antibiotics fit in these certain areas? And can we get away without using antibiotics? I realize it's a big question. That's what I think. Thank you. I'm not sure I'll have a satisfactory answer either, because I think the context matters. And the background, you know, I think the trial that you're referring to was done in a very different kind of a context. And this is relatively a free-living, apparently healthy kind of a context. So it's slightly different. But I think it's an open question. All right. I have a question, and Dr. Meyer, you brought it up, but it hasn't come up in any other places. So you brought up the genetic component. So I'm interested, you know, we've talked a lot about microbiome, but we haven't talked a lot about the microbiome post-genetic interface. So I'm interested from any of the speakers on their thoughts around that. And maybe I'll start with you, Dr. Meyer, just knowing from a ruminant standpoint what you see there. Yeah, that's some of our actually recent work that we've been doing probably for the past five to seven years. Pretty much tackling the idea of a heritable microbiome, what members there are, whether that is an actual microbial taxon or that's going to be a microbial feature. And what we're getting through several years of studies is that we do have heritable components of at least the, in this system, we'll say Angus sphere system of heritable taxa or heritable microbes and microbial features. And importantly, that we're taking into account not only this heritability, but newly coined terms in the past couple of years of microbial ability, for example, and the heritability of those microbial features across not only just ruminants, but how that impacts the system. And we include things like microbial ability with heritability, for example. We can get better predictive answers to phenotype. And we increase our ability to be able to predict a more feed-efficient animal. That's what we're starting to see throughout. Now we're actually starting to tie that to individuals host cell types, trying to figure out in the ruminant current research of how those individual cell types in the ruminant through single cell work, how they interact with the ruminant microbial ecosystem. I may add something also, if you allow me. That's very interesting because we also did a study where we tried to transformate ruminal fluid from cows into sheep, small ruminants. And what we see is that after the perturbation of the ecosystem, a few months later, the microbiome returns to the baseline. So that's the species driving the composition of the microbiome. And in another study, now talking about fecal microbiome, we follow calves during their development and colonization of the microbiome. And we compared purebred with crossbred living in the same pasture, and we could still see a significant difference in their microbiome. So there is no doubt that this brings us to the personalized medicine where the genetics play a role, and it's very important too. All right. Dr. Payal. Yes, thank you. I have a question for both Dr. Meyer and Dr. Mehta, similar question. This came to mind yesterday when we were looking at someone who was presenting on FMT, and they had a vegetarian and non-vegetarian kind of approved stool sample. And as I was looking at my fellow vegetarians, the cows, on your slides, I was wondering if you've seen in vet, in some of the work that you've done, Dr. Meyer, and then same with you, Dr. Mehta, some differences in vegetarian kids or vegetarian animals versus non-vegetarian. Yeah, I'll be happy to speak in a second. But yeah, it's a very good point. I can't necessarily speak on the aspect of children, but the diet sampling in the model that we talked about I think is very important when speaking about dietary changes. And I think it does translate though to other species to where the rumen system is so impacted by diet that a lot of those drastic examples that I gave in my talk that not only do they start to impact pH, which was mentioned earlier, which we mentioned we can measure activating as mentioned yesterday about making sure that we take a board today about a number of different measurements to make sure that we have predictive microbiome components. But also tied with that, it does obviously start to select for different domains of life, different kingdoms. And so we get drastic changes across the board on a lot of these various areas of impactful microbes. And so with that, they become very tailored to those specifics. But in that extreme example, on the side of vegetarian diets or Mediterranean diet versus a number of others, again, those are going to be impactful, obviously, for the community themselves. I still kind of stay true to my little tree aspect that we do have. Those are always there. Those abundances may change. They may disappear. But we're trying to influence the rest of that tree to our outcome. Thank you. This has certainly been a big interest area for us. I wish I could answer it based on this study, because theoretically, because the problem is not just whether somebody is vegetarian or not, then are they functionally vegetarian or versus by choice? And then what's the quality of meat and what's the quality of animal source food they're getting? So even though we have three forms of data, we have dietary recalls, we have the infant and young child feeding questionnaires all through the nine months. And then we have weight food records for what we fed them. What we fed them is all vegetarian. But what they self-reported, they are 70% are non-vegetarian in this community. And parsing that out and trying to figure out whether there is, because when they say non-vegetarian, is it once a month, which is true for a lot of them? What kind of meat? What kind of animal source food? It's a little bit trickier to just bend it into two category kind of a response there. I wish I had a better way to investigate. So in a newer study, we have dug into this a little bit more, where we have moved the age group to six-month-old infants and also included their mothers. So breastfeeding mothers and child diets to investigate this a little bit more. So hopefully, in another six months to a year, I might have something to report. But no promises. All right. Dr. Jam. Thank you so much. Dr. Mehta, I had a question, and I briefly chatted with you about this yesterday. But so as we talk about the types of foods that folks eat, I start to think about food insecurity. So like what we're not eating, does that also have an impact on what we would consider for the microbiome? I asked that question because I did a study, and we happened to see like a small but significant increase in urinary tract infections in individuals that were more likely to be food insecure versus those that were food secure. So I wanted to know what your thoughts were on that in any work that you're doing in that space. Thank you. And again, for food insecurity, I think the whole, it's not just the food insecurity and whether it's tied to poor nutritional intake or quality of nutritional intake, or is it representing a lot of other systemic and other factors that, you know, whether access to health care and everything else. It's harder to parse that out without, but that said, food insecurity is a major factor both in the US and internationally for a lot of different health outcomes, sometimes directly, sometimes indirectly. And in this case, food insecurity, again, this study was done across 20 urban slums. And so Mumbai has a collection of slums, and there are slum slums, and then there are, you know, just slums because they're categorized that way because of space and crowding, considering it's one of the most expensive cities. So. I have one more question. Dr. Mehta didn't talk today about some of the work that he's done, but he's shown that food has been important in TB, decreasing TB rates. So I was wondering, with this work that you presented, what are the next infections that you might be considering can show some link between what you study and other sorts of infections that you're thinking about studying in the future? Thank you. Yeah, that's where I started. My PhD work was largely HIV and TB. And so looking at, so I think there is a lot of literature out there. I don't think I'm spearheading a lot of that from that. I think the food and TB work is the editorial based on the ration study that was done in India, where they're providing a food ration, about 750 calories, reduced the household transmission of TB within the household. So we are building up on that. We are looking at, actually, one of my postdocs, who is on one of the NIH AID 32s, she looked at the metabolomic profile based on response to diet at TB treatment initiation. And we do see different levels of metabolic scarring in there. But again, very nascent, very early stage from a mechanistic point. From a social protection point, I think it's pretty established that giving that food is a huge thing from a social medicine perspective. Dr. Pantaleon. So this is maybe for Dr. Costa, but maybe Dr. Mayer could add to that. And you did mention, Dr. Costa, that the use of antibiotics in animal agriculture as growth promoters, even though they have been banned in the US and Europe and some of the developed countries, they do work. They're effective in increasing weight gain and production. Could you expand a little bit more about the mechanisms by which antibiotics do that? And is there anything we could learn from that? And we know that there is not one thing in production animal, you know, health, breast, vaccination. All those things will compound and have a good production system. But could you explain a little bit more about the antibiotic use? Yes. Growth motor and what we can learn from that, and maybe translate it to others there. Absolutely. What farmers learned is that if they give low-dose antibiotics, the animals will grow faster. At the beginning, there was a lot of hypotheses about is that the actual antibiotic molecules promoting growth is this control of infection. Now we believe that it's control of infection and microbiome modulation. If you take the microbiome from animals treated with antibiotics and you colonize mice, they gain more weight. They accumulate more fat. They grow faster. There is no doubt that the microbiome that has been selected is the one actually with an active role in digesting food better, let's put it this way. What we do see is a decrease in diversity. That means we kill a lot of species of bacteria and we allow some species to dominate the environment. We believe that we are actually killing the competition. These bacteria that have been selected, they are better to digest food. They have no competition and they can extract energy better from the food. Those are all hypotheses that we work with to explain how this microbiome can enhance performance. There is no doubt that they also, to a certain level, they will control disease, especially in poultry and pigs because, like I said, we moved from giving those drugs as growth promoters to give them as a therapeutic treatment of disease. Now we are not preventing disease, but we are treating the problem. That's my point of view about how they work. We still have some time, so if anybody in the room or virtual online at the table has another question, feel free. In the meantime, I'll ask another question. Again, it's kind of broader than just these talks, but we've talked a lot about bacterial microbiomes. We haven't talked as much about other potential organisms, protozoa and other things in there, so I'm interested in any of y'all that have comments on that. Yes, we have to talk about, yes, because we've been talking specifically bacterial, right, and microbiome is an abomination of that. Needless to say, a lot of because of the ease and number, especially in the ruminant ecosystem, obviously we focus on bacteria. We do, in our group and a lot of others, a lot of work on at least a little bit of the fungal side, yeast and molds, depending on how that can work out. Yeast usually are exogenous to the ruminant, but more of a black box in some of the activity. That activity is more on breaking down some of the cellulose and more physical, whereas a lot of protozoa work out there recently, especially with the methane mitigation research and tied to new organelles that have been discovered, for example, not only the hydrogen but just other hydrogen-producing organelles, which are tied to nitrogen work and making methane as well, where that system is pulling back one kind of box by the end of the hybrid. So, that in itself, we're talking about nutritionally stable microbiomes, and we've seen a lot of research in the past where, you know, they've defaunated the rumen to get rid of protozoa and interesting results. Nitrogen's important, which is part of a lot of the protozoa work. And you'll see a lot of meta-analyses, a lot of pluses and minuses, just as you would assume if you had a whole area of life out of this. So, with that, a lot of the targeting technologies talked about today, I think are really impactful. Obviously, not, you know, bacteriophages are going to be part of the bacterial system, but still, thinking about how they interact, that whole network model I discussed, I think it still applies across the board, and I can't tell you one without one. If I may just add a comment about archaea that also are not bacterial. When we do bioinformatics analysis, we actually exclude those reads from our analysis, and they are actually the main producers of methane. So, I don't know. Dr. Meyer is a specialist on that. It's not my field, but I know that in the rumen, archaea are kind of understudied. Did you have another comment, Dr. Meyer? Yeah, I can follow up. I mean, yes, understudied, the percentage, obviously, even just low, and yeah, we usually try to as well. The hot topic right now, right, and I think there's a lot of ways to analyze and to work with that. They're obviously very difficult, but very good point. Yeah, I guess. You didn't have anything, Dr. Meyer? I thought you were getting ready to say. No, I was just going to say that I think some of those methods have not been as well standardized, so we have the data. For example, like any of us who does that collection and sequencing, so we have the data, but if anyone wants to pop in and look at that data, we're open. Great. We'll go to Dr. Jenkins online. Thank you very much. Dr. Meyer raised a semantic question when he asked, what does it mean to strengthen the rumen microbiome? And I think that's a really interesting question, and we just heard about from Dr. Costa about growth promotion, fattening the animal. And, you know, when we're talking about humans, we would consider obesity as a bad thing rather than a good thing. So I just wonder how much consensus there is in the language and terminology we're using when we're talking about microbiota and the consequences of trying to perturb microbiota. Are we all talking about the same thing, using the same language and using the same definitions? Thank you. And that's a very good point. I always kind of blast me what my job is. I say it's the opposite of what we want to do in humans, right? I would love to eat less and gain more weight, or wait a second, eat more and gain less weight. But either way, you know, those communities, I think we're still talking about the same question and the same answers. It's just when I phrase the resilience as an idea of adaptability, stability, and function, I think that's still all relative to the system you're examining. In the cattle realm, in the livestock, beef side, that, you know, I mentioned earlier that that ruminal ecosystem is evolutionarily perfect. I mean, it has evolved to take in, like you said, it also biomass and be able to convert it into for itself, weight gain, a number of other things, and obviously production outputs. And so when we look across other species, systems, humans, again, I think those terms for strengthening still allow themselves to be taken with the same definition, but as the context of what it is, I think that's where we can have some leniency. If I may add, it really depends on the species we are talking about. For example, poultry and swine, the slaughter age is really early in their lives. So poultry, chicken, we're going to be slaughtering them at 30 something days. So there is not enough time for the fat to deposit on those animals. Rather than cattle, because they live longer, yes, then the treatment with those drugs can actually be associated with a higher fat covering, which independent of the breed, it's actually desired because the industry wants the fat layer can protect the cold chambers in the industry. So it is, depending on the intention of the species and the breed, yes, it is a good thing to have the position. If we still have time and you want to talk about the modulation we do by the antibiotics, like I said, it's actually by decreasing the diversity. And in humans, this is counterintuitive because we believe this is a good thing, right? The more diverse, like we see in First Nations and tribes, they all have higher diversity. And what we try to do with animals is actually to decrease the diversity so that they can gain weight faster. But we don't know the consequences because they don't live long enough to have chronic disease or anything that lack of diversity may cause. Right. There's no other questions. We'll give this group another round of applause. Thank you for some great presentation. And I'm going to send it back. Thank you, Dr. Plummer. Thank you, presenters and council members. Again, great discussion and great presentations. We're now going to move to a short break. If I can ask those in the room to reconvene in about a minute at 2.22. And then we'll reconvene online about 2.27, around 2.30. Begin again. Thank you all. Produced by the U.S. Department of Health and Human Services.