Combating Antibiotic Resistant Superbugs | Gautam Dantas || Radcliffe Institute

Combating Antibiotic Resistant Superbugs | Gautam Dantas || Radcliffe Institute

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Hello. Everyone and, welcome we. Want to thank those intrepid souls who actually crossed, over to. Radcliffe today, despite. The weather winter. Is fully upon us thank you very much I. Want. To welcome you to our science lecture series my name is Sean O'Donnell, I'm the, associate director of academic, ventures and I help, to oversee, the lecture series I have the opportunity and privilege of working with, Immaculata. DeVivo and, Alyssa. Goodman our science faculty directors, whose, vision, has really, helped launch. A year-long exploration. Of this, idea, of what. Is the undiscovered, in science, there's. Clearly so much more for us to know but, in practice of course for scientists, that off it means that they are setting off to find one thing and what, happens when they find something completely. Different this. Notion of serendipity, is not just romantic but, it's also a very practical experience. And we. Are really honored today to have professor. Gowtham. Dantas with us today whose story about his own surprising. And unexpected, discovery. Really. Redirected. His research and led, to a whole. Decade, worth of. New. Research program, I will, that Professor Dantas. Tell that story himself, of course but, I do want to briefly. Introduce him. And. I'd like to do so if you don't mind by reading his Twitter handle. He. Is a bowtie, enthusiast. He, is a husband, a father. Microbial. Ecologist, general genomicist. Computational. Biologist, he's also a homebrewer, gardener. Foodie, and pretentious. Cocktail, mixologist. I. Think. Well I'm hoping serving up some wonderful cocktails. For us today in. His stories, and should. Also point out perhaps that he is at, the same time the, professor of pathology and, immunology, and biomedical, engineering and molecular microbiology, at. Washington, University School of Medicine in st. Louis and he. Is no stranger to Harvard he was here. In George church's lab and. He, met with our students today he clearly has a desire for mentoring students. And leading them and, directing. Them in this process of becoming scientists. And inquisitive minds, and with, that I hope that you will join. Me in welcoming professor. Dantas. To the stage today thank you. Well. Thank you Shawn for that very kind introduction, and thank you to the Radcliffe Institute for this opportunity to come, back to an old home before, I get started I want, to say it's something that I personally believe and I think reflects. The opinion of you know most of my good colleagues and that is all. Good sciences team science, and if, there's anything of importance, that I say today it's because of these fine folks that, I have the privilege of working with, I'm. Really here as their spokesperson. Before. I get to the science aspects, that I do want to mention this. Role of serendipity, and everything, that you hear today. So. I was a postdoc in George church's lab from 2006. To 2009, and I came here to ostensibly study. Microbial. Engineering. For biofuels. Right this idea that we could convert plant sustainable. Manner into. Replacements. For petroleum and, approached. That myself and this grad student Morgan summer took but, I tried to enrich microbes, from the soil that, could survive, on plant-based toxins, as their sole source of carbon with. The hope of understanding that, genetic machinery and we. Thought would be good scientists, and set up an appropriate negative, control, and, luckily. Our naivete, led us to this idea that okay the, type of compounds, that no microbes, should be able to consume as a sole carbon source would be antibiotics. So, we went to off-the-shelf, drugs, that we could find in George's lab set. Up the experiments, and lo and behold a week later we got very poor results and the plant-based toxins, but, we had gangbuster. Growth on all of the anti microbials. We. Were surprised. Most. Reasonable, advisors would have said that's great why don't you focus on what you're doing George.

Is Wonderfully, not that type. He, got as excited, as we did him and said stop, the bath fields work for a while and figure, this out and the. Rest is history basically, everything. I'll tell you about today, every part, of our interest in antimicrobials, even, the ones that will seem like they were, designed, from scratch with, this you, know oppressions. A priori really, was because we decided to follow up on that slightly. Weird experiment. So. Let's. Step back for a second and think about what, we're actually going. To discuss and that is, anti. Microbials, or anti biotics so the definition, of an antibiotic just, to get so, the language straight, is any chemical, that it either inhibits, the growth of or kills microbes. And. Today I'm going to exclusively, be talking about the, group of anti. Microbials, there are antibacterials. These compounds. That can be used to kill bacteria. One. Thing that you should note, is, what. They're not are. Is. They're, not anti virals right and so this is a CDC warning, that, very clearly tells you and you really should heed this that. Anytime you have a viral infection you, certainly would be doing no good if you take antibiotics for, those viral infections, and I'll show, you pretty soon that, you probably do something bad. So, one thing to recognize is though why these particular drugs work is because, they target, the most key and conserved, processes, of bacterial life, they. Destroy. The bacteria bacteria's. Ability to, have, a cell wall so their guts bleed out if they are impacted. By things like penicillins. Or. They gunk up the ability for them to replicate, and. The reason I bring this up rather than for me to discuss molecular, mechanism, is to. Mention that Paul Ehrlich called antibiotics. Magic, bullets but, we really should be thinking of them more as magic shotguns, on magic, nuclear bombs because every. Bacterium, has this target, so, they ingest, an antibiotic, thinking, that you're going to be targeting a specific pathogen you're, actually impacting, every, single microbe, in that ecosystem for instance in your gut and so we, realized that even though it might be warranted use of this really, important chemical, it going to have potential, collateral damage, and it's through that lens that really I'm going to be describing work, that people in my lab have done to.

Understand, What that collateral damage might be you, do both warranted, an unwanted unwarranted, use of antimicrobials. One, thing that we have observed in, that particular vein, especially. By looking at retrospective. Studies is anytime, that antibiotics, are used in for instance the human population, that, those antibiotics do have impacts and all of the good bugs as well as the bad bugs so, down here you look at this particular, plot, and. You could observe that, you. Know virtually, at any point in life, at. Any time point you'll notice, that, there. Are some kind of malady. Of some sort of problem. That occurs on the human side even, if the antimicrobial was warranted. Now. Perhaps the greatest response, that a microbe might have to. Be trying to be killed by an antimicrobial is, to, respond to become resistant, to it and, this idea of antibiotic, resistance, is something you see is a, common. Feature that I'll discuss through the rest of selection it, turns out the history of antimicrobial use, in the, human population is intricately, linked with antibiotic. Resistance, so we're here to talk about serendipity. That. The whole field of antimicrobial is, really thanks, to a serendipitous, accident, at the hands, of Fleming. Who, happened, to have one of the greatest opportunities to win a Nobel Prize because he got a little bit sloppy when he went on vacation right so. You left off these plates that we all have heard about found. The Penicillium mode wiping. Out this potential, pathogen, but, the interesting part for this is that this natural product antimicrobial. Penicillin. First. Discovered by, Fleming. In the 1920s. The first actual treatment because, of, the amount of developer took was about in the 1940s. But, the first reports, of resistance, to this compound, predated. That by over, a decade, and. This is something I keep coming back to is that antimicrobial, resistance. Even, though we heard about any scary stories in the media as being associated with the clinic is actually. A natural feature, of all. Microbes. You. Also take the, example of the. Sulfonamides, discovered. By this german, chemist guerra domak the. Sulfonamides were a synthetic class of compounds, their first treatment was in the mid, 30s and. This is a little bit more intuitive that the first reports of resistance, what, four or five years later though we discovered, that that, was again an underestimate, resistance, to those particular compounds, was well before humans came on the scene and. So this particular plot, shows you that. Sort of zoom not view for every class of antimicrobials. So what you see on the x-axis is time you're. Seeing along the particular plot every, single important class of antimicrobials, views in the clinic and when. Blue shades the red is the, very first reports, of clinical resistance another.

Way Of interpreting what you're seeing on this particular plot is, antibiotic. Resistance and pathogens, is not, a question of if it's just, a matter of when death, taxes, antibiotic, resistance right all of them are predictably. Going. To come up very very fast. And. This encodes, an incredible. Burden on the human population, now, some of these numbers have been challenged, so, I'm not going to focus too much in the specifics, but we do know that you know a large, number of people almost a million people are on the planet right now died, due to drug-resistant. Infections and. This. Might scale up if things don't change by, about 2050, based. On this UK prime ministers report from a few years ago to, maybe one, person died. From a drug-resistant, infection, every, three seconds, on the planet now. Of course the human life is the most important. In this, equation, but it also costs a lot of money so, the US economy is estimated to lose about 55 to 60 billion dollars per, year because of the treatment of drug-resistant, infections and, that 10 million number. Is. To be believed and also scales to about a cumulative, hit to the global economy by, 2050, of, a hundred trillion dollars, so. Hundred trillion dollars for those of you like me who don't have access to that many zeros is, about. 6 years of the u.s. GDP wiped, out, and. I will warn you I'm a little bit sorry that this is going to be a slightly bad news bear stuff. That. Exactly. At this time when, we've got this massive increase in, drug-resistant. Infections, exactly. When we need new drugs to be coming to market we, don't there's. Lots of reasons for this so, what, we've decided to do as a group is. Focused. Not only on trying, to understand how we might come up with new antimicrobial. But take the the parallel, complimentary, view of saying if we would have discover, key, features about antimicrobial resistance. That we might be able to counteract that could be part of this particular pile. Now. This gets a little bit molecular in these details and I'll try to keep this at the level that is still relevant to this discussion and, there is to ask now. That we know that resistance, is a problem how is it that bacteria, evolved, resistance right when they see these anti microbials, how do they make changes in their genomes to become resistant, to these compounds, and they do it in the way that bacteria evolved any property, and it's, in one of two ways one, way is the way that we evolved that is we have we. Pass on our genes over, to our progeny bacteria. Do this by dividing and then mistakes. Occur during those replication, processes, and if, a particular mistake, a genetic, mutation happens. To give your progeny a selective, advantage it's. Going to now take over the population and, say, that selection happens to be antibiotic, resistant, now, that, particular resistant. Mutant that resistant, a, kid, if you will in the bacterial population will. Take over that's called vertical. Transfer. But. Bacteria, and archaea. The other two domains of life can, do something, that's much more spectacular, something. That unfortunately. So far we can't do as humans and that is in one fell swoop exchange. Large chunks, of DNA the, genetic material, with their neighbors so they don't have to wait until they have kids they can pass on traits to other. Members, in their population, and as it turns out if those particular pieces of DNA and code antibiotic, resistance, in very, short timescales, you can take an entirely, susceptible. Population, of microbes and convert, them to be multi drug-resistant and. Analyses. That many groups have done now suggest that show that. Because, of the huge selection, pressure of surviving, under antibiotic, stress microbes. Then become you. Know use this particular method this idea of horizontal. Gene transfer suppose, the vertical, transfer, to, become antimicrobial resistant. And. So what I'm going to talk, about are a few stories that we've, used modern genomic, methods and computational, methods to understand, the burden, of horizontal, gene transfer in, terms, of who's the source of these resistance, genes and how do they eventually end up in the to cause the type of problems I just talked about. Now. One thing that we as biomedical, scientists, have learnt over the last say 10 or 15 years is a better scientist worth your salt you need to ohm at the end of what you study the. Genomes, the epigenome. Not. To be left behind, we, are the group, that study antibiotic, resistance, were, fortunately, given the resistive, and actually, a very elegant concept, put forward by Jerry, Wright from McMaster University, about 12 years or so ago to describe, all of the genetic material, and any arbitrary, microbe, that are allow you to be resistant, to a particular, antibiotic so.

How Do you study the resistant, how do you study all of these resistant genes well, you could certainly turn, to the. Foundation, of microbiology, this idea of domesticating. The microbes were bringing them into the lab and we should certainly happy that this occurs because of a clinical microbiologist. Wanted, to diagnose, your infectious, agents this is what they would do in, the modern eight of era, of genomics, you could then sequence, the genome of that organism in further, resistance properties, the. Problem, though is we've really. Realised over maybe the last two decades that most. Microbes, live in microbial ecosystems. All over. In the soil in our bodies, virtually. Anywhere we look and most of those have not been cultured so, how do you study things that have not been cultured, rather that we've not been able to domesticate. Well we can turn to sequencing. This, idea of now looking just the DNA the genetic material. In the organisms, and infer, what type of functions they might have in this, case the function of antibiotic, resistance, so, at the practical level this is literally just smashing. Up all of the microbial cells harvesting. Their DNA putting them through a sequencer, and then inferring, what the resistance might be and this might seem like you've got the whole part of the pie. Unfortunately. You're, only iterating, on unknown so, what does that mean it, means that all of our databases, of known antibiotic. Resistance, genes have. Been inferred, from that small tiny minority of bacteria, that have been cultured, and so, they're gonna use sequencing, methods you're just relearning, the few tiny ones that you've known before so. What we've done is, try. To complement. That part of the cryptic, resistant the, part of the pie that is the undiscovered. To try to improve our ability to not only harvest. Out and bio prospects, that. Might be a problem in the clinic but also to try to be sort. Of prescient, about what, might eventually, occur in the clinic so. What is this complicated. Term called functional metagenomics. There is the the. Method that we use so. The method itself was. Co-developed, by Jo. Handelsman who's, at Wisconsin, and John cloudy who's here at Harvard Medical School. Back in the late 90s. Where. They discovered, that, much. Like the natural process, of horizontal, gene transfer if. You could come up with a method that forces, that to occur in large, scale then. You might be able to use, model. Organisms like. E.coli, and test. Their ability to express random. Chunks of DNA from any organism, to, encode resistance, and the steps are the following you, extract, the DNA you, clone it in to. A library, in e.coli this is an, easy-to-use micro. Bacterium. You get millions of these random chunks. Of DNA in e.coli, and then you turn back to culture so. In this case much, like a clinical microbiologist. Would do you use petri dishes that have antibiotics in there the, back here that grown now have a new chunk of DNA there is a resistance, gene and then you figure out what's going on right. What are those genes by sequencing, well, our lab is basically done is taken that basic engine married. It with sequencing, technology, and, effectively. Reduce. The cost of doing this about a thousandfold, now. Let's say for the sake of argument that we completely, zoned that for those last two slides and don't, care about any of those technical, details the only thing that you need to take away from, what I just said is that with this method you can start out with any arbitrary, group of microbes, whether, they're culture or not and after, pumping them through this particular pipeline which is now a lot cheaper to use you'll, get a catalog of all, of the genetic material that, is compatible, with horizontal gene transfer to. Make a, particular. Bug like, e.coli resistant, to antibiotics, so we apply a method of this type to then, interrogate, all sorts, of different microbial communities, for, their potential, to contribute antibiotic. Resistance, to, Clinic. So. This is one way in which we view the world, this, cartoon, schematic, of.

Interacting. Habitats, and, the reason I put this up and, you'll see this a couple times is to, recognize, partly, that microbes, don't care about our definitions, right, we might define something as a habitat boundary, microbes. Can traverse those, and so what we really like to understand, from an ecological perspective, is, that, even though resistance, really is most acutely, problematic. In the clinic because. Microbes, and different habitats, could, themselves transfer. Over eventually into the clinic or their genes could transfer, over we, would like to understand all of the interactions, that are shown here so, I'll walk through a couple of habitats, that we've particularly, focused on to, discover, resistance, genes to, assess, their risk of getting into the clinic and hopefully, also to mitigate, those particular risks right, and, I'll start out by not going into too much detail and just giving you a couple of highlights that, we've published, on from, our investigations. Of the, so-called commensal. Microbiome, so, these are the good bugs for the most part that live in and on our bodies right, the, one that students study the best is the gut microbiome, so. Again this is the trillions of microbes that live in us they're basically another, organ, and, because they're, constantly, subjected to, antibiotic, pressure it makes, sense that they might be a source through which pathogens. Could pick up resistance, genes, so. One of the things that we were surprised, to find in collaboration. With Rob, Knight's group as well as Maria. Gloria Dominguez, was, that through, them we got access to fecal. Samples, and oral samples, from an Amazonian, tribe at first contact I can't. Tell you how do you convince a group of people who have never seen the outside world to give you a fecal, sample I think that's probably how office, alien contact will be you'll be taking microbiome, samples from us but. Erika Pearson a. Genetics. Graduate student my lab access. Those samples, used, the methods I just described and we were shocked to find at least people who had no history of any antibiotic. Use resistance. Genes against modern, antibiotics, and. You see when I get to the soil why this makes sense but we thought this is an important thing to recognize that. Resistance. Against, compounds. Is not, something that we invent, through I use it, already exists, as a natural, feature of these ecosystems we, just get selection, pressure by using the antibodies for them to amplify, okay.

Now, I'll, give you another example of something that's at the other extreme, of the spectrum, where. We know and vulnerable populations, they get a ton of antibiotics. And that, is for, instance in this case preterm. Human infants so, we know that rates. Of prematurity, are going up and especially, when we look at very low birth weight infants so these are infants that are born about ten weeks too early these, kids are highly immature, in terms of their immune systems and because. Of their risk of infections, we give them a ton of antibiotics. Well not me I'm not a physician but the field and. So for instance in this Court which is describing, kids. Born in st. Louis at our Children's Hospital, virtually. A hundred, percent of these kids born ten weeks too early get, antibiotics as soon as they're born for the first couple days of life and if, you see in the plot next to the histogram the top. Two drugs that they get while they're in the neonatal ICU are antibiotics, four. Of the top eight drugs and they get our antibiotics, so we step back again from an ecological perspective when, you think about the fact that these, kids that require, this, commensal, microbiome, to set up what you're doing with each of these insults, is your carpet-bombing, a, an. Ecosystem. That should be setting up normally, so, we wanted to assess what the collateral damage might be and. So one, thing we found and this was work led by Molly Gibson, a graduate. From a computational biology, program in. Collaboration, with a couple of pediatricians at, Wash U Barb Warner and filter we. Had access to a large cohort of fecal samples, they actually have about 75, thousands, had fecal samples banked which. Is probably, the largest collection of poop in one particular location, and. We, access, only 400, of those and two, groups of kids ones that I had lots of antibiotic, exposure and, ones, that didn't have very much and, the thing that I just want to emphasize you. Know for the non clinical. Microbiology aficionados. That, the main thing that Molly discovered, that was surprising, to us was the identity. Of the bacteria, that was surviving, all of these inserts in these kids and so, again don't worry about the specific name, but you never take my word for the fact that the, bugs dominating, the guts of these very. Vulnerable kids, are the, bad guy bugs on the CDC list right, these are not back here that we associate, with good gut health they're, the bugs that usually cause no sat-comm in the hospital acquired infections, and perhaps. Now that you think about the selection pressure it's not all that surprising, who else can survive those insults except they're really hardcore drug, resistant, bugs right, but, the silver lining there is, that through a series of computational. Approaches. You. Know the again. The aficionados, call it machine learning that's. Partly you know because it sounds fancier, it's, really pattern recognition, using computer, algorithms so, what Molly did was she took all of this data that she had gathered in terms of who was there in the microbiome, and their resistance genes and trained, the model and was fortunately, able to predict how, the microbiome, would change based on just a couple of these features with, about 85%, accuracy why, is that something to consider, as being important because it suggests then, a potential, future, of personalized, medicine where.

Each Of us anytime I get sick we might be able to get a microbiome, sequenced, as these set of methods, improve your. Physicians, might be able to take the information about your specific microbiome. State and tailor. Your antimicrobial, therapy, in a way that not just kills the infection but, protects, your microbiome right, so we're hoping that this is where this will go, now. A picture, tells you know a, thousand. Words and I. Can't think of anything besides this particular questionable. Parenting, event and, really telling you that resistance cross habitats, right. And. And so we decided to. Take this concept and this is the sort of slightly more boring version of that slide where. Fortunately. Through a number of international collaborations. Led. By Erica who already mentioned, and Pablo Tsukiyama another graduate student my lab we. Access, samples, from a village, in El Salvador, and a, Slama outside Lima, Peru to, look at potential. Interactions. With microbes and resistance genes between, people so there are fecal samples and as many environmental, samples, we could get at the same time with GPS, coordinates right so so soil samples, and sewage. Samples, and subsistence animals, and pets. And again I want the Labour all of the incredible. Data, analysis, that these. Folks did and this jumped to the conclusion that, we think has impacts, and Public Health and that, was they were able to identify using, this analysis, key, hotspots, for resistance, gene exchange, right, so, what this means is they identified, particular features, particular habitats, between, these people in the environment then, clear, evidence, for having the same resistance, genes in those habitats, in humans. As well as the environment and we found two such key habitats, again which could be really, impactful for Public Health in El Salvador and in Peru, so across the top panel you'll, see pictures. Of chicken coops in El Salvador in the village and that appeared to be one of these hotspots so this is the droppings. From the chickens were subsistence, animals. At. Exactly the same resistances and the humans as well as the soil implicating. Those chickens, as a route, between moving, between humans in the environment, the, reason this is important to consider especially here. In the u.s. in terms of its translational, impact is the. Folks in our salvador in this village don't use any anti microbials, in growing, their chickens but almost any chicken that you or I would eat has, probably been grown under, the pressure of lots.

Of Antibiotics if you think of what's happening here consider. What might be happening when we're eating you know chicken, under antibiotic, pressure the, story was a little bit different in Lima in terms of what we found there there, we were able to implicate the sewage treatment system as a hotspot for such exchange, right, so this is a massively, crowded. Ecosystem. All of the effluent sewage goes through a single treatment, plant it. Gets cleaned up based on pretty sound civil engineering principles, and then, once that water is cleaned it, has to be dumped somewhere now, in Boston that's dumped in your harbor far, out but it's there in your harbor right in. Lima being, a desert, that, water is used to irrigate every, single field and. Every single. Place. That requires, water and so, you might now consider, that even though the, civil engineering system was working to destroy certain types of microbes because. It was allowing other microbes, to thrive that, I picked up resistance, genes from the human microbiome they, are now disseminating. Those genes all over, Lima, so this, is where again these these kind of, bioprospecting. Methods. To hunt for resistance, genes comes. Into play to have potential. Public health impacts, okay. So, I'm going to leave the human microbiome and spend a little bit more time and a habitat, that maybe a lot of people may, not consider as a really key part. Of antibiotic, resistant exchange and that is the soil or the soil microbiome, so. Why look at the soil was, it turns out there's a long history decade's, worth of really. Important, work implicating. The microbes, in the soil perhaps. The most diverse ecosystem, of microbes on the planet in, not. Just the production of antibiotics, but also on antibiotic, resistance, and I'm only gonna give you three examples of work by other people that really inspire our work one. Was this beautiful. Story by Jerry Wright and colleagues when they went into the Canadian Beringia permafrost. Cored, out samples, and they could carbon date to be thirty thousand years old sequence. The DNA there, and then, interrogated, that DNA, from whether they could find antibiotic, resistance, and lo, and behold they found resistance, to modern antibiotics, providing. Clear genetic evidence, that resistance, in environmental. Microbes, vastly. Predates, any human, use of antibiotics so, kind of putting the nail in the coffin of saying resistance. Existed, before we came up with antibiotics, or discover them, another. Piece of evidence that's important, is put forward by kind of the grandfather of the antibiotic, resistance, field Julian, Davies who now more than 40 years ago, recognized. That almost, all of these compounds, that we call antibiotics. Are natural. Products of soil bacteria, so. All of those Nobel Prizes that were given for the discovery of antibiotics came, from people hunting, him in the environment, this is the case with amino. Glycosides is, an example. Where. You know you found these extracts, through the soil and realize, whoa they're able to kill these particular pathogenic. Bacteria, it's because, of these small molecules and so what Julian. Posited. And and it's, been supported since is that these. Producers, must be the original, evolutionary. Progenitors. Of antibiotic. Resistance, and why is that because, these anti microbials, target, as I said key, processes, of better lie so if they didn't have resistance at the same time as production they, would commit suicide so it would not be a terribly interesting evolutionary. Process, to figure this out without having resistance just in time since. All of this happens, you know millions of billions of years ago they, have also given all of their neighbors the, pressure to evolve. Resistance hence soil, is the original source of resistance and. Then finally the show that we as humans can actually have an impact in a bad way this. Is evidence from European, archival, soils were about 70, years where.

This Group led by David Graham was able to measure the abundance, of resistance, genes and find. That over the years of human, use their, their, abundance, has gone up so. Based on all of this evidence the one, thing that you might speculate, is that. For this reason every resistance. Gene every resistance, element that you find in a pathogen, a disease-causing. Microbes, must, be the same as the one they must be identical, to things that we find in the clinic sorry. In the soil and bizarrely. That's, not what we found right so when we started working, on this and looking through databases. Virtually. All of the resistance, genes that people have work hard to discover in soil microbes, was vastly, different, from the type of microbes, that, we see causing infections, so this seemed like a bit of a conundrum and. Our, hypothesis. Was maybe we've. Just been looking at the wrong slice of the pie now. One. Thing that Jerry. Writes group had done to really emphasize, that. The bugs in the soil are highly resistant, was they, went and they isolated. In, the seminal paper from 2006. A, whole. Group of microbes that they know are the producers, of antibiotics, so, all. They did was culture, them up know that they produce of antibiotics, and then tested, to see how, resistant. That is non-pathogenic. Bacteria to, anti microbials, and, shockingly, on average. These bacteria were resisted, as seven or eight different classes, of antibiotics, even though they're not pathogens, so that's actually more resistant, than most pathogens, so, now this should be used as evidence probably, that these guys the guys who are producing the resistance, genes right but. Again you stumble across this problem where when, people analyze, the genes that these producers, have they, look completely different, for the genes of the pathogens. So how do you reconcile, that particular, disparity, between the phenotype, of high resistance, and the genotype of being very different so one way in which you could reconcile, that is to say okay I buy, the argument, that these guys are the original, donators, of resistance, but it happened so long ago millions.

To Billions of years ago that, it's not really relevant in terms of us treating, clinical, problems so that's one hypothesis, the, other hypothesis, that maybe there's still another missing, link maybe there are microbes, in the soil that are contributing. We just still haven't found them out yet and so, this now jumps over to this crazy experiment, that we did when we were in George's lab where. We said okay, what, about these weird bugs that are able to eat antibiotics. Right so you've got the producers, you block the resistors, and now you've got the eaters so, in this slightly. Again ridiculous, experiment, we try to enrich microbes, in the soil that, could again not just resistant antibiotics. They were using them as their sole source of energy at. This you know there's a it wasn't a terribly elegant, experience experiment. It was just a lot of culturing, and. When. We were, able to successfully identify, all of these microbes I could eat antibiotics, we then tested their resistance, so now what I'm showing you in the bottom and blue. Is the same data from Jerry right where his producer, organisms, on average. Resistant, about seven or eight antibiotics. Now, you look at the eaters. On the right hand side they're. On average. Resistant. To 17. Or 18 antibiotics, out of, 17, or eight antibiotics, to which we could test basically, they don't care about the antibiotics, actually do care about them because they're eating them right, so, we think now we had finally stumbled, across that missing link the, the potential, that these eater organisms. Might, be contributing, to resistance. In the clinical now. Before, I get to showing how we actually did that I'm going to jump ahead ten years from that original discovery, which is how long it took us to figure, out how it was these bizarre bugs, who eating antibiotics, how are they surviving on the antibiotics the, way that most bugs survived on glucose, and so this was work that was led by many. People, but. Primarily by Terrence crafts a recent postdoc. In my lab where, he went to about five of these soil bacteria, and used. A battery of techniques that really pin down. How the heck they were using those compounds, which normally kill bacteria as their, source of food right, and and, it's visit mr. it. Almost. Brushed past it if I don't point. It out one. Of these bacteria can. Eat penicillin.

As Its sole source of carbon but it can't use glucose, doesn't. Like sugar likes, antibiotics. Right. And. So I'm, not gonna belabor again the molecular biology all I'll say is that through a lot of different people's contributions, we were able to figure out there are three key, steps of how these bacteria eat antibiotics, and, the important, part is that the committing, step is an, antibiotic, resistance, gene so the very very first thing that these bedbugs, need to do is still, detoxify. This toxic, compound so they break it open exactly, the way the pathogens, do and then, they digest, it using a couple of different methods and just. To show that we really truly understood, it one way in which to show, that you know the. Genes. That underlie this particular phenomenon, are sufficient. For this particular trait. As you could transfer, that trait to another bactrim that doesn't have this right, so that's something else that we did led. By Terrence was we were able to take all of the influence he made from these soil microbes transfer. Those genes into e.coli one. Of these benign microbes, in the lab at least and he, was able to convert a coli into, an eater of penicillin, now, we're, not doing this because we're crazy you know if someone were to ask why the hell you turning a coli which came you know some versions make. People sick why you making it eat antibiotics. When, this case our motivation, was partly to prove that we really understood how it worked but, also because it might be used in the future for cleanup of antibiotics, right, not with this basic, work but, perhaps in the future where if you can have microbes, that, have these abilities to destroy, these antibiotics, which can contaminate, systems, like we had seen in the sewage systems maybe, this is a next-generation approach, to, clean up much like we clean up our sewage using. Microbes to destroy these antibiotics before they go out into the the, wild, okay. So, now let's step back to say right, so that showed that we, know how to now we can figure out how to eat the antibiotics but, what about this fundamental, question are they, the missing link between the, soil and the, clinic and to do that we. Step back and this, was work done by Kevin forsberg a graduate in our genetics program and Alejandro. Reyes who. Was a graduate student in Jeff, Gordon's lab so they applied that method many, many slides ago where we, go and discover, new antibiotic, resistance, genes and they applied it to these antibiotic, eating bugs and said okay using, this method that doesn't require a priori. Knowledge of what resistant genes are looking. For cryptic resistant genes can. We discover, the. Missing link and sure, enough they did in a single experiment they're now in, increased. Evidence by, an order of magnitude of, the. Number of genes in the soil there were exactly, the same as resistant. Genes and pathogens that cause disease, and. Again I won't talk about the specific details of the resistance, genes in terms of what their names are but, the key features that certainly, made us a little bit scared was the fact that between, those ten genes they knock out four or five classes of antibiotics we, find them not only in those bugs also in pathogens, and in that map that you see any country, that shaded dark is a is a country, that has deposited, or someone in that country has deposited a pathogen, with a resistance, gene that was identical, to those genes we discovered, in u.s. soils, and, to, emphasize that this was particularly. Problematic what, is seeing here is a comparison, of all, of those genes compared, to genes that have been found in pathogens, and you'll, see that there are many genes there it's across the bottom are the genes that come from the soil across, the top of the genes that come from four or five important, pathogens, and initially, anyway you see great shading that's, identical, a hundred percent DNA, identity, and, what we observe here is that not, only do we find that they're the same single genes across these bugs but, actually they cluster together so. Anything in red that you see is an antibiotic resistance, gene anything. In yellow is a gene involved in horizontal, gene transfer so. They're the vehicles, that loop genes around so, we're seeing here is evidence that. Bugs in the soil and bugs, that cause disease can. Move large chunks, of DNA between themselves that. Can knock out three four classes of antibiotic, resistance, in horizontal. Gene transfer events, that, could happen on the order of minutes right. So, again now taking, all of this together, reconcile. With the day there was Ford suggests that we have discovered the missing link and these.

Are The bugs that we might want to focus on as we, consider challenges, to the clinic now I, will. Again preface. All of this I guess but. We're saying that we stumbled, across this, right this was not something we originally set out to do because you, know only someone insane would think about looking for antibiotic, eating bugs, but. Since we had this and now we had this data in hand we, wanted to ask how, much of this is this the exception versus, the rule that. Is to say because we had gone through this bizarre experiment, of culturing. Our bugs that could eat antibiotics. Which. We discovered, were these multi drug-resistant, Proteobacteria. Now, Kevin, who led that study teamed up with some Keith Patel in the lab to ask again, when, we stumbled across those weird bugs are they representative. Of all bugs in the soil or are they sort these weirdos right, and basically, the short, answer that story is they are weirdos. And. Really the only important, part of this sort of complicated, slide was we, now collaborated with a couple of ecologists, in Colorado, Rob Knight and Nora fear got, access to a much larger number of soils, about 20 soils apply. The same methods, discover, thousands, of new resistance genes in the soil but, luckily most, of those resistance, genes are not, ones that we found in pathogens, right, so. It appears really it's only that tiny weirdo minority, there of Proteobacteria, that are the ones that are the missing link the other, thing we discovered is all of, those genes we found from these these bugs that are not the same as in pathogens, they, don't have those same linkages, with mobilization, elements, that cause those genes to hop around right, so we know that in pathogens, for those genes to move between different bugs they require those accessory, elements to actually move the DNA back and forth these guys in the storm didn't have them so how do we put all of this together towards, something that could be impactful, for work in the future we. Think what we've done is we've, discovered, indeed, that the soil is rife with antibiotic. Resistance, fields probably. Reflecting, this evolutionary, pressure of antibiotics, being produced in the soil but, fortunately.

Most, Of those genes are not at risk for being acquired, by pathogens. But. There are these very specific bugs, but now we know the identity, that, we can focus on so that we can prevent them from traversing, this particular boundaries. Okay. But. As it turns out there's, still something interesting, to learn from these particular bacteria. That, cause that, that has and, from the soil that had these cryptic, resistance, genes so, one hypothesis, that we came up with at the time is to say could, these be bellwether, events, could, these genes that we have seen that haven't popped into, pathogenic. Bacteria, yet could we stat craft them could we evaluate. Their risk across. Those thousands, for the few that, we think might emerge them to the clinic in a few years so why would you want to do that right why would you want to know what is gonna happen in the future is because, if you know which ones are at great risk those. Are the ones you can focus on for mitigation the ones that you might try to destroy, the one that you might try to diagnose, this. Is the story I'm going to tell you about one such class of enzymes and that's, against, the class of antibiotics, called the tetracyclines. The, tetracyclines. Are a very very important class of antibiotics they. Were discovered in little certain late 40s, and. They, continued to be one of the major classes of antibiotics used in the clinic and agriculture. And aquaculture and, the way they work is they, gunk. Up a better ability, to make proteins, right, by targeting this thing called the ribosome so. Big surprise because they've been used against pathogens pathogens. To figure out how to be resistant, to them and the two main ways that they do that is they either pump the drug out or they, protect their ribosomes, what. Hadn't been observed, very much though was a third mechanism of resistance which, a lot of bacteria use and that is destroying the chemical itself right, and that seems to be a pretty good idea if you're a bacterium, in terms of wanting to be resistant, if you destroy the chemical it can't hurt you in a contour to your population that's. How for instance those beta lactams those penicillins, that's how they're destroyed by most pathogens, there are enzymes that cleave them and break them apart so. We. Had we found evidence of only one such instance there was a, non-pathogenic. Bacteria from, from, human guts that have been shown to have this particular enzymatic, activity, so, we decided to say we've got this treasure trove of newly discovered resistance. Genes in the soil could, we find evidence. That these guys might, actually be lurking somewhere beneath the detection threshold.

Motivating. This was again as I mentioned the fact that the tetracyclines. Are a unique class but not only they old but, they've kind of gone through this Renaissance of, being. Modified. And getting newer and newer versions of them including. And this is something that's really important to keep in mind the, last three drugs have been approved by the FDA in the US as antibiotics are all, tetracyclines. One, in August. Of 2018. And two, in October, of 2018. There are really important, emerging class of antibiotics. One, other thing to note is that those drugs that are coming to market have, not been considering, obviously. A resistance. Mechanism, that have not been seen in the clinic yet so, we want to make sure we can enable the protection, of these particular compounds. By. Seeing whether there again is this lurking, danger, against. Them so. To do that Kevin, who had done the soil work basically, discovered, this new class of enzymes across his soils again I won't walk you through all of the data all I'll, say is that he was able to show that, about ten such enzymes existed. That, can destroy, the tetracyclines. And a whole bunch of soil bacteria, they. Were completely, novel they were not related for instance to the ones that will sit in this soil in the human, gut before and, the only gene or enzyme that we could find in any database that, they were similar to was, a gene in Legionella, so Legionella, causes, Legionnaires, disease, it's a soil derived pathogen, and sure, enough this was something lurking in the genome of Legionella, that no one knew was a resistance gene that we were able to now show can, cause Legionella, to no longer respond to tetracycline, therapy, so. That's. All well and good what is the clinical relevance, so. It turns out drew Gasparini you recently graduated from my lab went, through all of those selections that had been performed in my lab across many, many many different habitats, from human fecal samples, and soils, and suet, samples, from El Salvador and Peru and started, looking to see whether maybe. More of these are there in fact through just a quick cursory survey. He found that there was 70, more such enzymes that existed, just in our freezers, right, and in, this case good. For our funding and really, bad for humanity. Just. In the time that we started working on this those. Resistance. Genes started. Popping in to pathogens right. Exactly. What we expected. That would occur really, hope that I wouldn't but it did right, so this is why we've now decided to take a very concerted, effort to understand. Those enzymes, understand. Their vulnerabilities. To hopefully, destroy them and so that's exactly what we're able to do turning. Back to something that we've learned from the penicillins, so, one way in which the, chemists. Have helped rescue, the penicillins, a little bit is to recognize, that yep the penicillins of the middle atoms are degraded. By these particular professional, professional, enzymes called beta lactam aces so if you can come up with chemicals. That break the resistance, you can reactivate, the antibiotic, right so this is the idea of a beta lactam. Which is the antibiotic, and a beta-lactamase. Inhibitor combinations. Of breaking the resistance so, we said hey why not try that let's try to find compounds, that, are you know kind of looks similar to the tetracyclines.

That Might be able to drunk up those tetracycline. Destruct aces and again. This is where serendipity. Comes into play you, may not believe me but the very first compound we tried had, that property, and. It, was happens to be a compound called an hydro tetracycline, the, names are all that important, but again what we're able to show is that if we add an hydro tetracycline, into, the mix when these enzymes are working to degrade the tetracyclines, they, don't work anymore okay. And what, you're seeing here is something that you know that's a little bit more relevant to the clinic. There's, a way in which to measure the resistance that ink is encoded, in our organism, by. Looking at how well it will grow in the presence of the antibiotic, so, on your, left hand side you've seen what's called an e test strip this is basically a piece of paper that has an antibiotic, across the gradient that we put on a lawn, of growing bacteria if the, bacteria can't. Grow close to it they're, susceptible they'll, be killed by the nante barrack they go really close to it they're resistant, so you can see in the first panel the bacteria growing really really close to it right so, this is e coli, with one of these enzymes growing. Close to tetracycline, they don't care a tetracyclines. There we, add this newly discovered inhibitor, and lo, and behold tetracycline. Can work again right, and so we are now, really. Heartened, by this discovery, and the collaboration by, a couple of other researchers, at Wash U and organic. Chemist Tim. Ben Savage who, with Chris Walsh here and nearest. Olia structural, biologists, we had now been able to take this, initial. Sort, of pre discovery, almost make, new, synthetic analogs, to that originally, discovered enzyme, so, I originally, discovered inhibitor, and now, we've been able to make, even more potent, inhibitors, of these particular enzymes, and again. Now. That we were spending, more time looking at you know more and more of these genes or they're seeing here about you know twenty or so of the genes that we've been working, on so far the ones in red are the, ones, in the soil the, ones in blue are ones that now we've discarded live. In the, guts of healthy humans one. Of the things that's again, motivating. Us to sort, of work fast on this is already told you some, one of these enzymes of the soil bit fan of Legionella, just. Last year we discovered that one of these enzymes, that, destroys, every, single, tetracycline.

We've Thrown at these particular enzymes, including. The the the, the compounds, that were just approved by the FDA they. Destroy, those two that. Gene, that enzymes, we found in a cystic, fibrosis patient. Sample, in. Pseudomonas. Right, so, that's really scary but, the good news is our hypothesis. Came true by, focusing, four or five years earlier and trying to find those inhibitors we, now have inhibitors, that can, reset, eyes that Pseudomonas. Isolate through those latest generation, tetracyclines. Okay. So. I'm. Gonna end my talk now in the next couple slides with. A simple, question why, are we not dead yet, right, with. Everything, I've just shown you why, are we not dropping like flies every time some highly, drug-resistant, victim comes around and certainly. The, cdc has this almost Pokemon. You know card, deck of bad guys that tells you you should really be scared you know in terms of their hit, points you. Know if you're a sonido back and carbapenems. Resistant, tantrum x-ray and Pseudomonas these, beautiful, pictures of things that if they get any they're gonna kill you right, so. What can we do about this what you know I've showed you one strategy do. We have anything, in our arsenal to be able to fight, back against, these multi drug-resistant, organisms so. It turns out there, are some strategies outside, of just coming up with new antibiotics, that we might be able to use. So. And. This is what we turn to some evolutionary, biologists, and systems, biologists, to think sort of out of the box a little bit that, to, figure out what our general, strategies, we might have. To. Kind. Of flip that the, table, on the drug-resistant, organisms and, so, what idea is called. A, selection. Inversion. So, what does that term actually mean it. Means trying, to come up with some kind of magic sauce, that, transiently. Allows the, drug susceptible. Version of the organism, the one that you can kill to, out-compete, the drug-resistant version right. Why would you do that because as you're shown here normally. You've got the susceptible, and the resistant, guys mixed, together in this case the yellow bugs are susceptible the. Blue bugs are resistant, normally. What happens is you bring in the antibiotic.

Sure, Enough the susceptible, ones by definition the yellow ones get wiped out the, blue ones survived and now you've got a drug-resistant, population, selection. Inversion this magic sauce is somehow to. Suppress. The blue resistant, guys to, let the yellow ones take over and then you kill them with the antibody so how do you do that there, are a few strategies they've been proposed one. Is. Drug. Cycling, so. If you, were able to find drugs that work in very different ways what. You might do is use drug a until. You get a resistant, population, to drug a and then, much like crop rotation, you switch to drug B because. Those resistant mechanisms, don't necessary talk to each other you can go back and forth back and forth and kill, off these these differently, resistant, populations, this. Is only unfortunately, going to work for a short period of time because, as I showed you in the soil case sometimes. Multiple, resistance. Genes can transfer at the same time and then you've lost this particular ability, right, so, another strategy is to recognize, that drugs, of. Different, classes sometimes. Can combine, together to be better than the trivial some of their parts through. A property called synergy, right, so what you're seeing here in this particular panel is normally. You take a population, then, you beheaded with drug a it doesn't care individual, beheaded with drug B it doesn't care but when you combine drug, a and B together there's, something magical, about that combination where. They potentiate. Each other and they can synergistically. Combination. Give those better so that's strategy 2 and then, strategy, 3 which, i think is actually the most elegant, is this concept called collateral. Sensitivity. Right, so, we try, to explain what that term is imagine. For a second that as a bacterium. Becomes. Resistant. To one class of drugs Class A the. Mechanism. That it uses to become, resistant, opens. Up an Achilles heel something. About what it's doing to become resistant, to drug a opens. Up a vulnerability, against, drug B that's, the collateral, sensitivity, because, of resistance 1 you've come susceptible, rosa resist drug. 2 if you are a, st.. Louis summer analogy, for, collateral sensitivity. Is imagine. For a second you want, to go out into the st. Louis summer and, you don't want to be hit by mosquitoes, or bitten by mosquitoes and. You, also you. Know don't want to drop, dead because. Of the heat now imagine that what you decided to do is to make, sure that you don't want. To get bitten by those mosquitoes, you put on a big moon suit and you walk out of the st. Louis. Summer you've certainly protected, yourself against, that mosquito, but, you're gonna drop dead because that moon suit is going to suffocate you in terms of the heat that's, the kind of weak analogy for collateral. Sensitivity. So. I'm just going to end by saying we were able to implement, this against one of these skirts pathogens. Methicillin-resistant. Staph aureus on Mercer so Mercer is a is a you know deadly, bug it. Killed something like eleven thousand Americans, when. It gets into the bloodstream it's resistant, to the. Important class of beta lactams, is penicillins, and so, we thought could be used some of those features that i told you about before in combination, to try to knock out myself and so this in this story that, gonzales a graduate. Student genomics, in my lab, was. Able to find a combination, of three beta lactams, so that same penicillin. Category, completely. Fda-approved, generic. Drugs that. On their own are completely, useless against, Mercer but somehow in combination. They, work together to overwhelm. Ourselves defenses. And are able to wipe it out right. To. This process of synergy, but. Perhaps, more, importantly, they, had this process or this this property, of collateral sensitivity. That is to say if Mirza, becomes resistant or any one of those drugs it now becomes more susceptible to the other two and why, is that important, because if you imagine you're a bacterial population, you've got these three drugs around you, think you're clever and you become resistant to one now, you've got two other drugs still around and I'm much better at killing you but locks, them against resistance and we tried very hard to make these guys resistant, we couldn't and. That was fortunate, and then finally in collaboration. With Malin. Chang and Sharma, bas three researchers, in University. Of Notre Dame we, tested, to see whether this really, interesting test, tube result could actually translate, into an animal model so, they happen to have an animal model of a, really, severe, bloodstream infections, with MRSA and we're, able to fortunately show by working with them that this completely, cleared that infection, this triple antibiotic, combination, within, 24 hours of treatment and so we're pretty, jazzed.

About This because again this is composed of three, generic, drugs and. So we have a lot of information about how they work fortunately. They're all Co delivered in the same way currently, and, so we're hopeful that this might be one particular. Quiver, in you know the arsenal against. This deadly infection, but also sets up a paradigm, of how we might be able to repurpose other existing. Drugs to, come up with at least stopgap, measures against infectious, diseases, so. With that I'll end by not. Addressing this particularly elephant, in the room and, that is to say everything, I've said is focused kind of on human use of antibiotics but, in this country still it's, estimated that something like eighty percent of antimicrobials. Wai-wait are still used in the rearing of food animals, and I. Would argue without. Getting into that debate just right now that, that's perhaps not a great use of this natural resource, right, and there are different ways in which to mitigate that but I just want to say that this is something I'm not discussed, earlier but I think is very germane, to this particular discussion with. That I also say that sometimes I look like this but sometimes also, look very Missouri, with my lab, and. I'd. Be remiss not to again say that the really anything of importance, that I might have said today is because, of the wonderful people that I have the, working, with both, the people in my lab alumni. Who have gone on to bigger and better things people, at Wash U and all around the world and obviously the. Funders that are generous. To, support this work and. If there's one sort of PSA, thing that you remember, when you leave. Today it's this please don't use anti microbials, for viral infections I'm happy to take any questions. Thank. You very much that was great, I have, a question about teaching I'm so part of the reason that we started this undiscovered, series was to talk about how we have to teach science and how we have to acknowledge these.

Kind Of unexpected. Findings so I'm just curious in, your teaching now genetics, is a constantly. Evolving you know relatively, new subject. How do you incorporate this, kind of thinking when you teach especially undergraduates. Yeah. That's a very good question and I think there. Are already great measures, in place and, doing, this at Wash U where. Part. Of our undergraduate curriculum has, a discovery, element. Right off the bat so this is amazing thing called phage. Hunters which a lot of universities around the country, have. Committed, to and anyone interested in even if they not declared biology, majors they, have this sort of experience-based class where. You'll still learn the fundamentals, of microbiology. And bioinformatics and, sequencing, but they do it by going out into the field for instance harvesting. Soil getting, their own Phaedrus these you know the viruses, that infect bacteria. You. Know going to the isolation, process, going. Through the sequencing, process and the annotation, so I think I. Think, sometimes the best way to teach that. The sort of wonders of science is to actually perform the science yourself not everyone, has the opportunity to, immediately join a research lab so I think engaging students. Very very early on in discovery, based science, you. Know it's of course exceptionally, important to teach them the value of hypotheses, but, also to have, that that kind of exciting moment to say no matter what you do in a phage hunter's class you're, going to find something new and then, once you have something you can test a particular biological question, so that's, one aspect that you know we find pretty useful the other is, my. Lab always, hosts a pretty large cadre, of high, school students, and undergraduates. All. Through the year you. Know sometimes, the people in my lab complain about this because we have a dozen students that come in additionally, in the summer but I try to convince people that you're, doing this for multiple reasons there's, no other way to maintain, the steady pipeline of students interested in science without, allowing them to do the science themselves, and also. The only way to learn to be a Eastham mentors and, so, that's why I strongly. Encourage every. Single person in my lab independent. Of what courier, stage they're at to, take on an undergraduate or a high school student to be able to see what it is like to teach so I think those are two ways in which good work.

Thank. You again that was fantastic so the. General, mechanism of, drug resistance, or the way, the vector bacteria, has become, the superbug. Is. Pretty, well understood. Thanks, to you and others so what, is the the practical. Solution. Other. Than you know to tell people not to take antibiotics to, sort of sit out the, cold is. Is, something that most. People are not going to in. Practice follow so. Well, you know in general what would you suggest yeah. There are a few things actually. I would say that education. Campaigns, in. Countries, that have tried them to pretty. Openly, discuss, when it is appropriate to take an antibiotic Canada. As an example have, been successful, it's just that the, timelines, of turning, people's opinions around are not on the order of weeks or months they could be years right, and so I think, reframing. Antibiotics. As a natural, resource as something. That's finite, and that's, something that really requires a sort of conservation. Mentality, and. Then tie that to the fact that there is collateral risk, that you're taking when you sort of willy-nilly take an antibiotic I think a large, part of, being, able to turn that table, to to, sort of preserve those antibiotics when read them is education, but, when it when I say education I just don't mean to the consumers. Of the antibiotics, but it's also to empower, physicians. Pediatricians. For. Instance to, say here is why you, know ultimately you're, still going to have to respond, to the really aggressive parent, who says I don't really care I want this drug for my kid fine, but, if you can spend maybe a few minutes to explain, to the, parent here's why here's a brochure here's something that you should consider here's, the risk that your kid five years down the line because of this unwarranted, antibiotic, has a greater, chance of something bad happening I think that is a large, part of of change and practice, I think for, turning the tide there I think the other also, is sort. Of better reporting, structures, right

2019-02-28 13:36

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