This Podcast Will Kill You - Ep 50 Antibiotics: We owe it all to chemistry!

Episode Date: May 12, 2020

Fifty episodes. That’s fifty (sometimes) deadly viruses, bacteria, protozoa, parasites, and poisons. And don’t forget the fifty quarantinis to accompany each! What better way to celebrate this momentous occasion than talking about something that may actually save you: antibiotics. In this, our golden anniversary episode, our ambition tempts us to tackle the massive world of these bacteria-fighting drugs. We explore the various ways that antibiotics duel with their bacterial enemies to deliver us from infection, and we trace their history, from the early years of Fleming and Florey to the drama-laden labs of some soil microbiologists. Finally, we end, as we always do, with discussing where we stand with antibiotics today. Dr. Jonathan Stokes (@ItsJonStokes), postdoctoral fellow in Dr. Jim Collins’ lab at MIT, joins us to talk about some of his lab’s amazing research on using machine learning to discover new antibiotics, which prompts us to repeat “that is SO COOL” and “we are truly living in the future.” We think you’ll agree. To read more about using machine learning to uncover antibiotic compounds, head to the Collins’ lab website, the Audacious Project site, or check out Dr. Stokes’ paper: Stokes, Jonathan M., et al. "A deep learning approach to antibiotic discovery." Cell 180.4 (2020): 688-702. See omnystudio.com/listener for privacy information.

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But what if we didn't get the whole story? I would just be made to fit. The moment you look at the whole picture, the case collapsed. What if the truth was disguised by a story we chose to believe? Oh my God, I think she might be innocent. Listen to Doubt, the case of Lucy Letby, on the IHeart Radio, app, Apple Podcasts, or wherever you get your podcasts. I remember the astonishment when the first cases of pneumococcal and streptococcal

septicemia were treated in Boston in 1937. The phenomenon was almost beyond belief. Here were moribund patients, who would surely have died without treatment, improving in their appearance within a matter of hours of being given the medicine and feeling entirely well within the next day or so. The professionals most deeply affected by these extraordinary events were, I think, the interns. The older physicians were equally surprised, but took the news in stride. For an intern, it was the opening of a whole new world. We had been raised to be ready for one kind of profession, and we sensed that the profession itself had changed at the moment of our entry. We knew that other molecular variations of sulfanelamide were on their way from industry,

and we heard about the possibility of penicillin and other antibiotics. We became convinced overnight that nothing lay beyond reach for the future. Medicine was off and running. Oh, I loved that. Right? I got chills reading it. Oh, that's very cool. I really, I searched high and low to try to find, maybe my search terms were off,

but I wanted to read like a whole collection of doctors who were using penicillin and sulfanillamide and other antibiotics for the very first time, because that, was such a revolution. Right? Oh, my gosh. Hi, I'm Erin Welsh. And I'm Aaron Olman Updye. And this is, this podcast will kill you.

We are thrilled about today's episode. Absolutely thrilled. Today we're talking about antibiotics. Yes. Like, it's a huge topic. Oh, it's too massive. It's too massive. And so just like we did with the vaccines episodes,

we're splitting this into two separate episodes. And so this week we are talking about antibiotics themselves, how they work, how they were developed, and sort of what the current status is of antibiotics today. And we are very excited to bring on a very super cool researcher who does super cool work on antibiotics. Yeah, that's very true. Super, super cool. So stay tuned for that.

And the next episode, we're going to talk about antibiotics. resistance. Right. And how resistance works, the history of resistance, et cetera, and then hopefully another super cool guest. Yeah, it's such a huge topic. And I think to, I mean, everyone wants to know what's going on with antibiotic resistance because that's like in the headlines. It's a big deal. But in order to understand antibiotic resistance, you have to understand antibiotics. And so that's what this episode is about. Plus, antibiotics are cool as heck. They are so cool. Like, I am legit, very thrilled for this episode. Because I also, like, I know the history, obviously, from reading about it.

But there was only little glimmers of insight into how they worked in the history book. So. It's really cool. I can't wait to go over it. Well, first of all, before we can fully express our excitement about antibiotics, we have some business to take care of. Of course. first of all, it's quarantini time. Of course it's quarantini time. We are drinking this week, very appropriately, the drink called penicillin. So we didn't come up with this recipe.

We're not reinventing the wheel here. Uh-uh. And because we don't need to, because this is such an excellent cocktail on its own. So what is in the penicillin? It is scotch, which we haven't done a scotch quarantine. for a very long time. Season one, I think, actually. I think so.

And I like scotch. Like, I don't know why we've avoided it so much. It's not intentional. And then honey ginger syrup and lemon juice. Super simple. And we will post the recipe for that quarantini and the non-alcoholic placebo librieta on our website. This podcast will kill you.com as well as posting it on social media channels.

Mm-hmm. And then we have one more little bit of business. And that is related to our last normal season episode on Eastern equine encephalitis. And one of the things that we sort of discussed was why horses experience such a higher mortality rate. And first of all, Aaron, we really need to get a vet on here. That was our bad. Yeah, 100%. In the future, we promise.

So a veterinarian reached out to us to kind of shed some light on our question about how horses have higher mortality rates. And so they reached out to other equine specialists in the vet community and got a general consensus for that answer. Ooh. Are you ready to hear it? I'm so ready. It's pretty interesting. Okay. So this is a quote from the email.

It's not well known whether there is a difference in the immune response between species, but most likely the increased mortality in horses is related to the difficulty in providing nursing care to a 1,000 to 1,500 pound recumbent animal and the issues that are. from the horse being down. So like pneumonia, pressure sores, self-trauma. Horses are also dangerous to work around when they are neurologic and having seizures. And even if the horse recovers, most people cannot afford to support an animal that has continued neurological deficits and will never be ridden again. Right. Yeah. So because Tripoli causes these neurological deficits. And so it seems to be that euthanasia is often sort of what must be done for some of these cases. So there you go. So yeah, thank you so much for reaching out and sending us that information. Yeah.

It's very interesting. All right. Now that that's out of the way. Let's talk antibiotics, please. I can't wait. We'll take one quick break first. Dinner shows up every night, whether you're prepared for it or not. And with Blue Apron, you won't need to panic order takeout again.

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And for anyone with an adjustable base, this is a game changer. Their stay fit corners are the best I've ever tried. No slipping, no popping off, even when the bed moves. When your days are demanding, Sheeks helps you wake up restored, not run down. Try them for yourself with their 60-night, risk-free guarantee. Upgrade your sleep at Sheeks.com. Obviously, we've already said this is a massive topic. So here's how we're going to break it down when we talk about the biology of antibiotics. First, we have to talk about bacteria, right? Yeah, of course. We have to understand what bacteria are, how we classify and identify them, and then we can kind of understand, we can start to understand how we target those bacteria using antibiotics, right? I'm very excited. I love bacteria. Me too. And what's really fun about this is that all of our other episodes where we've covered a bacterial pathogen, I kind of gloss over a lot of this part of it. So this is kind of like you'll understand more about,

many of our past episodes now. If you... Yeah. Anyways. Okay, let's get into it. All right. What is a bacterium? It's a single-celled, prokaryotic, something.

Okay, yeah. Yep. Yes. So what's a prokaryote then? That's the first question, I guess we have to answer. The term prokaryote, it's not a great term, but we still use it because it's still useful. It's basically how we separate.

separate bacteria and archaea from eukaryotes, which is plants and animals and fungi. All right? Mm-hmm. So let's talk about some of the differences between the cells of prokaryotes and eukaryotes. So our cells, for example, are basically bags of water, right? And these little bags of water are surrounded by a membrane. This membrane is made of lipids, which are fats, and they have some proteins. interspersed in there, okay?

Mm-hmm. Inside of our cells, so human cells, animal cells, plant cells, fungus cells, we have things called organelles, which are also bound by membranes, so they have little lipid membranes around them, and those carry out all of the functions of our cell, okay? And that includes in our cells a nucleus, which is where all of our DNA is. That's our genetic material. Bacterial cells are a little bit more. simple. So there's still a bag of water, okay? They're still surrounded by a lipid membrane,

but here's one of the first differences. Inside of that bag of water, there's no other organelles, okay? There's just a little round piece of DNA and then a bunch of ribosomes, which are essentially RNA and protein mixed together. Okay? Yep. And then the other big difference between bacterial cells and animal cells is that on the outside of their plasma membrane, that lipid membrane, they have a cell wall. That's a big huge difference. And it's really important because anywhere that there's a difference between bacterial cells and human cells, for example, that means we can use that as a target to kill those bacteria. So now let's talk about the ways that we classify these bacteria. We're not going to get into like evolutionary relationships because that's

way beyond my capacity. What we do want to talk about though is the way that we can classify bacteria in order to identify them so that we can choose the right antibiotics to use to treat them. Okay. Right. All right. So one of the very first ways, and this, if you've ever heard one of our podcast episodes, you've heard me say these words, we can first class. classify a bacteria by is it gram positive or gram negative? Mm-hmm. Right? And normally, I just say that means it's pink or purple under the microscope.

It actually means a whole lot more than that. Yes, it does. So let's actually define what that means, okay? So gram staining, when we say the term gram stain, this is a tool that we use to visualize bacteria under the microscope. And basically what you do is you mix two different dyes. You put a purple dye over a bunch of bacteria, and then you wash that purple dye off, and then you put a pink dye on, okay?

And what happens is that purple dye gets stuck on the cell walls of these bacteria. Remember I said bacteria have cell walls around their membranes. So if bacteria take up that purple stain in their cell wall, then they're what we call gram positive. Okay? Yep. If they don't take up that purple stain, then they'll look pink under the microscope because they'll take up that pink stain. And those are what we call gram negative, all right, because they don't pick up that gram stain. So what is the difference between these gram positive and these gram negative bacteria?

They're cell walls. They're cell walls. So they both have cell walls that are made of the same basic stuff, and that is generally a substance called peptidoglycan. This is something that our cells don't have. Right. Okay. So it's very different than eukaryotic cells. But gram positive bacteria have a really thick layer of this peptidoglycan, and it sits right outside their plasma membrane.

and then that's the end. There's nothing else. Okay? Gram-negative bacteria have a thinner layer of this peptidoglycan, and then on the outside of that cell wall, they have another membrane. Mm-hmm. And this membrane is made usually of something called lipopolysaccharide LPS. That's not as important. But it's basically another barrier between the inside of that bacterial cell and the outside world.

Okay. Right. So that's a really huge and important difference because these cell walls, they act as kind of like an exoskeleton almost, right? They give the bacteria its shape and structure. But they also, in the case of gram positive bacteria, they're fairly permeable to small molecules. Okay. So a lot can still get in and out of gram positive bacteria that just have this peptidoglycan. cell wall. Yeah. Graham-negative bacteria, on the other hand, have an extra membrane on the outside,

so it's harder for things to get in and out. The way things get in and out of those cells is by little proteins that are along the surface called porins. Want to guess what they do? They're pores, their channels. They make a pore. Okay. We're really creative here in naming. Okay. So those are gram-positive versus gram-negative. bacteria. Importantly, some bacteria have entirely different cell walls. So they might not take up either of those pink or purple stains, right? So that's something like mycobacterium, like tuberculosis, that we've talked about a lot, right? Right. All right, Aaron, other ways that we classify bacteria that we've talked about a lot. We can look at their shape.

Are they round? Are they rod shaped? Are they little? spirals, okay? This is helpful when we are trying to identify specific bacteria. Again, because the antibiotics that we're going to use, we want to make sure target the right bacteria so that they're actually effective. And then the other thing that we can look at is how and where do these bacteria live? Are they aerobic, meaning they need oxygen in order to survive? Or are they anaerobic, meaning they can't live in the presence of oxygen, like claustridium botulinum that we talked about recently. So those features of bacteria are common across pretty much all bacteria. So one thing that's important to keep in mind with antibiotics is that depending on the antibiotics you use,

they might be killing a lot more than just the pathogen that you're targeting, right? Right. And so I think when we say bacteria or think of bacteria, I think we're, a lot of us are used to thinking of pathogenic bacteria. Right. And so instead of saying, oh, well, the pathogenic bacteria, we say bacteria and assume it's going to, you know, lead to an infection or death. But as we have increasingly become more aware in the past 30, 40 years or so, there's, you know, humans, animals, plants have a microbiome. And so these are bacteria that may themselves not be pathogenic or they may be opportunistically pathogenic, but they are also an unintended target of antibiotics. And that can lead to issues. Yeah. The vast, vast, vast, vast majority of bacteria are not pathogenic. It's a very small

subset of bacteria that are actually able to colonize, infect, and cause illness in animals, plants, et cetera. So, and it's kind of a whole separate topic, like what those specific mechanisms are that allow for bacteria to make us sick. And we kind of touch on those when we talk about specific bacteria, right? Right. All right. So those are bacteria. So hopefully you understand now listeners how they are different from ourselves because those differences are what we're going to exploit to be able to use antibiotics to kill those pathogenic bacteria. All right. So what is an antibiotic? We don't have to do the etymology of this, right? like anti-bio, anti-life.

Pretty easy, pretty easy. Pretty straightforward. So antibiotics are generally small, low molecular weight, so really small molecules that have the action of either killing or halting the growth of bacteria. Originally, the term was specifically for compounds that are produced by other living organisms was the intention. But then it's sort of now it's been it's been more widely expanded as synthetic antibiotics have been developed. Isn't that interesting now? Interesting. All right. So I've said this a couple times already, but when we're thinking about antibiotics, we have to make

sure that they're specifically targeting bacterial cells and hopefully not causing too much damage to animal cells, our cells. And so we tend to target things that are specific to these bacteria. And then the other thing is that understanding the mechanisms, the specific mechanisms of action of these antibiotics can tell us not only what groups of bacteria they're likely to work against, but also how antibiotic resistance can eventually evolve. All right. So in general, there are four broad picture ways.

that we target bacteria in order to kill them with antibiotics. These four are. We can target their cell wall synthesis, right? Right. Since we don't have cell wall. You can't build a wall. You can't exist. Yeah.

It's not true for humans, but true for bacteria. We can target their DNA replication. And you might say, but Aaron, don't human cells also? replicate their DNA? And you'd be right if that's what you said. But we'll talk about some differences. We can target their protein synthesis, which again, our cells, of course, make protein, but there are some differences in the way that bacteria make protein and the way that we make protein. And then we can also target some specific elements of bacterial metabolism that are very different from animal and plant metabolism. Right. So those are the four major targets. Let's get into the nitty-gritty,

shall we? We shall. So, cell wall synthesis. I said this. Most bacterial cell walls are made of peptidoglycan, and this is a substance that animal cells don't produce. The way that bacteria make these cell walls is they have to make chains of peptidoglycan. And, this is, and And then they have to crosslink those chains together in a specific way in order to build a strong wall. It kind of is similar to the way that we make collagen in our bodies, if you remember the scurvy episode, right? Yep. So if you can mess up the way that that cell wall crosslinks, then you basically make an ineffective cell wall. Like you're peeling apart the pull and peel twizzler?

Yeah, exactly, just like that. So it turns out that the beta-lactam antibiotics, which include penicillins, the most famous, I can't wait for you to tell us the story of penicillin, Aaron. It's a good one. It is. Penicillins, cephalosporins, which you've probably heard of, carbapenoms, and monobactams. All of these are beta-lactam antibiotics. They target a very specific part of this.

cell wall synthesis. They block the enzyme that catalyzes that cross-linking reaction. Okay. So it's really similar to the scurvy situation where if you don't have vitamin C, you can't cross-link collagen to make your good collagen. If you block this enzyme, you can't cross-link peptidoglycan. You can't make a cell wall. Okay. Right. So it turns out that we named this enzyme a penicillin binding protein. There's a bunch of different penicillin binding proteins, a lot of different versions of these enzymes, and they all do the different steps of pepto-glycan synthesis and cross-linking in slightly different ways. So we have a variety of different beta-lactam antibiotics that can target the different processes. Does that make sense? Mm-hmm. So knowing that

beta lactams block cell wall synthesis, what types of bacteria are they most likely going to be able to be really, really effective against? Grandpas. Right, because grand positives have that cell wall right there and it's really, really important to them. So beta lactams historically are really, really good at treating grand positive infections like strep, like staff, etc. Okay? Mm-hmm. Graham negative bacteria, on the other hand, still do have a cell wall. So you still can target them with beta-lactam antibiotics, but because that cell wall is surrounded by another membrane, it's a little bit harder to get in and to get those beta-lactams through. Right. Right. So that's why we develop better and better, like they are called second and third and fourth and fifth generation beta-lactams

that have slightly different structures that can do a better job of getting in to treat those gram-negative infections. Okay. Cool. Cool. Now, there are other bacteria that don't have peptidoglycan cell walls, like mycobacterium tuberculosis. Their cell wall is made of a different substance called mycolic acid. So obviously, beta-lactam antibiotics are going to be entirely useless against those types of bacteria. But we do have other antibiotics that are specific to mycobacterial cell walls. So they do the same thing just on a different substance. Cool.

Cool. So that's cell law synthesis inhibitors. Bam. I think that's so fun. And it's such a beautiful target because we don't have peptidoglycan, right? Right. Oh, it's very cool. So the next thing that we could target, how about proteins?

We've talked a lot on this podcast about how cells' basic function is to make proteins. Proteins are how we do all the things that cells do. So if we could target bacterial protein synthesis, then we could stop bacteria from making proteins that will eventually make the cells die because they won't be able to do their job. The only problem is that our cells also make proteins. But the good news is that it turns out that the specific ribosomes that bacteria use to make protein are different in their shape and structure and function than human ones are. So we can target those.

This is really good news. So there's two steps that we can target in terms of protein synthesis. First, we can target the process of transcription. This is where we take DNA and turn it into RNA, which serves as the template for making proteins. This is what the rithomycines do. This targets bacterial RNA preliminaries, so it basically blocks the ability of bacterial cells to make RNA.

So it totally is going to kill them. That's great. But that's just sort of one class. The vast majority of antibiotics that we have that target protein synthesis block the ribosomes, which are integral in making protein. How exactly do they block the ribosomes? So there's a bunch of different ways. Ribosomes have two parts.

They have a small subunit and a large subunit. So depending on the antibiotic class, they're either going to bind to the small subunit, or the large subunit and basically inactivate them. So they bind to those ribosomes and they block those ribosomes from like acting on the RNA to make it into protein if that makes sense. Right. That makes sense. Yeah. And there's a lot, a lot of different antibiotics that do this, whether they target the large or the small subunit. So those are the amino glycosides like streptomycin, gentimicin,

topromycin, the tetracycline, like doxycycline, also the macrolides, which are like erythromycin, azithromycin, okay? So, in the case of these, whether it's refampin that's blocking RNA synthesis or any of these classes that are blocking the ribosomes and blocking protein synthesis, what types of bacteria do you think that we could target with these antibiotics? Lots of them. Lots of them. Pretty much any of them. We use these antibiotics, immunoglycosides, tetracycline, macrolides for like so many different infections, gram positives and gram negatives. Which also means that they like do a very good job of wiping out microbiome stuff. We should do an episode on the microbiome. It's a whole, it's such a separate topic. Yeah. It's just hard not to

think about it every time I like think about an antibiotic used. Oh yeah. use. Absolutely. So anyway. You should think about it every time you think about an antibiotic, though, because it's absolutely, it's absolutely a consideration. Yeah. All right.

Interesting. Okay. So that's blocking protein synthesis. If we take a step back from protein synthesis, we have DNA, right? So we could block DNA replication itself. Mm-hmm. So long as bacterial DNA replication is different from eukaryotic DNA replication. It is mostly the same, but there are a couple of enzymes that are different.

So we can target those specific enzymes, all right? And it turns out that we have antibiotics that do exactly that. Fluoroquinolones, which are a synthetic group of antibiotics, they target a bacterial enzyme called DNA gyrase that eukaryotic cells don't have. So that's great. And so they can block that enzyme and thereby inhibit all of DNA replication. If you can't replicate DNA, you can't make a new cell. Boom.

Boom. Boom. Over. That one is short. We don't have a lot of those. It's mostly fluoroquinellates. All right. The last big way that we could target. And this one's really fun, even though there's very few antibiotics that we have that do this. is we could block some of the metabolism of bacteria, specifically folate synthesis. Okay. So most people have probably heard of folate, right?

Because you've heard of folic acid that you need to, if you're pregnant, you have to make sure you're getting enough folic acid. That's the context that most people have heard of it. Right. Okay. So folic acid is vitamin B9. this in both bacteria and eukaryotes is very heavily involved in the actual synthesis of DNA building blocks. Okay, so you don't use folic acid itself, but it's necessary to make the building blocks of DNA. Right.

So if you don't have enough folic acid, you can't make DNA. We have to get folic acid from our diet. We have to eat it. We can't make it ourselves. bacteria, as it turns out, make their own folic acid. That's very cool. It's very cool. And so if we can block bacteria's ability to make folic acid, they can't uptake it from

their environment, so then they're going to die because they can't make DNA so they can't replicate. All right. So, turns out we have antibiotics that inactivate enzymes in the folic acid. synthesis pathway in bacteria. So those are the sulfonamides and a antibiotic called trimethyprim. And so the sulfonamides and trimetoprim target two different steps in the synthesis of folic acid. So we actually often use them together in combination. You've probably heard trimethypren sulfomyphalmithoxicide. I don't think I've heard of that. Oh, you haven't?

Sure. Have you heard of Bactrim? Yes. There you go. Then you have heard of it. Which group is chloramphenicol? Chloramphenicol is a protein synthesis inhibitor. Okay, thank you.

Just curious. Yeah, it's not in one of those big groups that I talked about, but it is its own protein synthesis inhibitor. Okay. So, yeah, that's kind of the big broad strokes of the different classifications of antibiotics and how they work. antibiotics can be either bacteriostatic or bacteriocidal. Mm-hmm.

So bacteriocidal, like pesticide, means that they kill the bacteria. Okay. Whereas bacteriostatic means they just stop the growth of bacteria. And then we rely on our immune system to come in and finish the job. Right. Okay. It's a little bit more nuanced than that because some antibiotics are bacteriostatic, against some organisms and bacteriocidal against others.

Interesting. Yeah, it's a little bit complicated, but it's partially because of how we define bactericidal, which is basically you have to kill 99.9% of bacteria within 24 hours. So you might be bacteriocidal, but it actually takes longer than 24 hours, so you're not technically bactericidal. Gotcha. Okay. So that's basically antibiotics in a nutshell. Well, those are like your pharmacology course and your microbiology course.

But overall, what I think is kind of the most important takeaway is that there aren't any good antibiotics or bad antibiotics. You might hear people say like, these are big gun antibiotics. That's a terrible term, actually, even though people use it all the time. There are the right antibiotics and there are the wrong antibiotics for any given infection. There are antibiotics that are going to work, and there are ones that aren't going to work. And so we can see, based on these mechanisms of action, that some of these antibiotics will work against a large number of pathogens, and we call those broad spectrum antibiotics. Whereas others work against a more narrow spectrum of pathogens, right? Mm-hmm.

And then on top of that, some antibiotics are more potent, so they might need less of a concentration, in your system in order to kill the bacteria. But some of those that are more potent might also be more indiscriminate in killing maybe our own cells, right? Like they might have adverse side effects on our own cells. Right. And of course, pretty much without a doubt, antibiotics end up killing our own microbiome as well as the pathogenic bacteria that they're supposed to be targeting.

So when we're thinking about what antibiotics we use in certain situations, it depends both on the severity of the infection, what that infectious organism is or is likely to be, and what the overall antibiotic resistance looks like in that area. Yeah. And that's how we have to decide what antibiotics we use for specific pathogens. Well, and it can be, I'm sure we're going to talk about this much, much more. Like there are so many questions that I wanted to ask about antibiotic resistance. I know.

This is really hard to not talk about resistance. And so I'm sure we're going to talk about this more in that resistance episode. But I think it really also comes down to like if somebody is very ill from what seems to be a bacterial infection, then oftentimes it's not possible to sort of like choose this antibiotic is going to be the best one. Exactly. So then what we do is we, it's called treating them empirically, right? So you look at, okay, what type of infection is this? Where do we think this infection likely came from? Because then we can start to narrow it down. Do we think that this is a gram positive or a gram negative infection? Or are we not sure? Do we think we're trying to treat aerobic bacteria or anaerobic bacteria, right? What was the source of these bacteria? Where are they growing?

And then we also, yeah, we have to look at how sick the person is because if someone is really, really sick, then we might accept. using an antibiotic that has greater range of side effects if it's going to be more effective at killing those bacteria. Right. Right. So, and then the other thing, too, is that in general, you want to use the most narrow spectrum antibiotic that you can in the situation. So sometimes you might start out when you're not sure what the infection is with one antibiotic. And then once you have a clearer picture, you can switch to another antibiotic. But so, for example, there are some antibiotics that we talked about already that are really, really effective, and they're effective against a really wide range of pathogens. For example, refampin, this targets RNA polymerase,

so it's effective against tons and tons of bacteria, and it also actually has relatively few side effects. It's a pretty good drug, but it's such a good drug that we don't want to use it against just anything. Right. So we save that for use in very severe infections. We generally use it just to treat tuberculosis and meningitis as well. If we had discovered that one first instead of penicillin, I mean, we wouldn't be using it at all because of resistance. Yeah, because resistance also develops really rapidly to refampin as well. And so it's also often used in combination with other antibiotics. Right, right.

So, yeah, it's a complicated but really fun topic, I think. It's really interesting. And I also, I wish I had read more about sort of the history of microbiological developments because I feel like, you know, and we're going to talk about this later on in the episode, but like how the development of new antibiotics has really slowed down. And I wonder if part of that is because our knowledge of the differences between, like, Like, we've already kind of taken out all the low-hanging and middle-hanging fruit when it comes to differences between bacterial cells and eukaryotic cells.

Totally. And so now it's like, do we even have, like, what targets do we even have left to identify? Absolutely. Yep. So, it's interesting. It's interesting. So, Erin. Oh.

How did we come up with these? Where do antibiotics come from? Tell me everything about them. Okay. I can't wait. We'll take a quick break first. Anyone who works long hours knows the routine. Wash, sanitize, repeat. By the end of the day, your hands feel like they've been through something. That's why O'Keefe's working hands hand cream is such a relief. It's a concentrated hand cream that is specifically designed to relieve

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15% off their first order of O'Keefs. Just visit O'Keef's company.com slash this podcast and code this podcast at checkout. It's cozy season and nothing compares to wrapping yourself in a Minky Couture blanket. Luxuriously soft, perfectly warm, thoughtfully made. From movie nights to chilly mornings, Minky Couture turns everyday moments into pure comfort. Once you feel it, you'll understand why it's called the original best blanket ever. Visit minky couture.com or a store near you and make this cozy season your softest one yet. China's Ministry of State Security is one of the most mysterious and powerful spy agencies in the world.

But in 2017, the FBI got inside. This is Special Agent Regal, Special Agent Bradley Hall. This MSS officer, has no idea the U.S. government is on to him. But the FBI has his chats, texts, emails, even his personal diary. Hear how they got it on the Sixth Bureau podcast. I now have several terabytes of an MSS officer,

no doubt, no question, of his life. And that's the unicorn. No one had ever seen anything like that. It was unbelievable. This is a story of the inner workings of the MSS and how one man's ambition and mistakes opened its fault of secrets. Listen to the Sixth Bureau on the IHeart Radio app, Apple Podcasts, or wherever you get your podcasts. From defense to offense, from art to science, this is the story of antibiotics.

I couldn't resist. It's hard to know exactly where to begin this story. Do we, for instance, start with Lister and a use of, carbolic acid to disinfect wounds? Or do we start with Fleming and penicillin? Or do we go back in time before germ theory to when people used moldy bread to treat infected cuts? Yeah, that happened. What? That happened? I did not know that. But like you talked about, the word antibiotic simply means against life. An antiseptic, like Lister's carbolic acid, may effectively kill bacteria, making it antibacterial, but it also kills human and animal cells. So it's not really an antibiotic.

And also, it shouldn't be injected into your body, which unfortunately does need to be said, considering some recent statements made. The world that we live in. By someone. Plus, I would also really love to tell the story of Lister and antiseptics and surgical practices one day. So let's put a pin in that and come back to it in another episode. Perfect. Instead, I would like to go back to the earliest known use of mold to treat infections. And it's surprisingly widespread that practice. That's so weird.

So there are descriptions of using moldy bread for various ailments found in ancient Egypt, China, Serbia, Greece, Rome, Central America. Like, it's amazing. And some of these are topical treatments, like rub moldy breadcrumbs on a postular scalp infection. Yep. And others actually instruct you to make a moldy bread mixture to eat to, quote, soothe the pipes if you have like bladder or urinary tract inflammation. Oh, I thought it meant the like pharyngitis. Oh, maybe that too. Any kind of pipe. But for whatever reason, these uses of mold to treat infections kind of fell out of style or were forgotten about for a few thousand years. And before going into the modern history of antibiotics, I want to paint a little picture of what the pre-antibiotic era was like.

Oh, gosh. That sounds depressing. It is. But it also shows how far we've come. So before antibiotics, three out of 10 pneumonia patients died, nine out of every 1,000 births led to the mother's death. Untreated ear infections and strep throat led to hearing impairments. and heart failure. And as you can imagine, war was an absolute feast for pathogenic bacteria.

In the American Civil War, more soldiers died from typhoid fever in dysentery than directly from combat. And similarly, in World War I, more people died of dysentery and typhus than the fighting itself. The modern history of antibiotics starts, or at least I'm going to start it, with a lecture given by Paul Errolick in 1907. and this name may sound familiar to you for any number of reasons. One of those reasons may be this now infamous lecture

because in that lecture he used the term magic bullet to describe this ideal drug that could be used to kill bacterial infections. And at this time, it was still a hypothetical concept. Up to this point, early 1900s, germ theory had been well established and discoveries were still pouring in. The understanding of how infections, diseases worked had really grown over the past 50 years, and technology for carrying out microbiological studies had also advanced a ton. And so that really helped. So microscopes and

lab equipment made it much easier to find and identify bacterial cells, as well as create vaccines for both bacteria and viruses. But even though many horrible diseases could not be prevented, there hadn't been much progress in terms of treatment for those diseases. And so this had led to a really interesting shift that I hadn't really thought about before in the philosophy or attitude of many physicians. So before germ theory, let's say like the late 1700s and the mid-1800s, the predominant strategy for medicine in much of Europe and the U.S. was called heroic medicine. And so what this consisted of was essentially trying to shock the body back into balance.

Huh. So we're talking excessive bloodletting, purging, sweating, you name it. Basically, extreme, extreme intervention was the mode. And as you can probably imagine, most of the time, the recipients of this heroic medicine were treated to death. But then in the mid-1800s, germ theory came around, and with it came this recognition that, for these infectious diseases, there was often no amount of intervention that could stop their progress. Could you prevent these through vaccines sometimes? That was great when that could happen. And you could also lay out a timeline of what an infected person would experience at each excruciating stage of an infectious

disease. But you couldn't do anything most of the time to stop fate. And so doctors went from this extreme interventionist heroism. medicine to what has been called therapeutic nihilism or fatalism. So it's like, well, we can't do anything. Like it just so hopeless. So on the one hand, that meant fewer people being bled to death, but on the other, if your patient developed a fatal infectious disease, there was nothing you could do but ease their passing. But fortunately, researchers and physicians weren't satisfied by that. Enter Paul Ehrlich. Erlich. Erlick had spent his dissertation, exploring the use of dyes for staining various tissues and cell types, and one of his

important discoveries was that you could selectively stain certain types of bacteria. Side note, Erlick's fascination with dyes made him a really recognizable figure in all the places that he worked because his fingers were always stained and his clothes were always stained. And if you watch the show Charate, which we both have watched and loved, they depict him as such. She has like stains all over his lab coat and stuff. It reminds me of my high school French teacher who always had like chalk dust on his fingers and the edges of his pockets. One of my favorite things in like middle school in high school is when a teacher would be have like chalk dust, like a chalk dust handprint that they would put in some very like dorky place.

And I loved it. Wow. Wild times high school. Chalk boards, you know. But his discovery that you could selectively die certain types of bacteria had huge implications for medicine. Because basically, if you could get a dye to recognize those specific bacteria, maybe you could get a magic bullet then to target and destroy them. So, Ehrlich went to work with Robert Koch in Berlin in 1891 to get started on this. And there he collaborated with Bering, if you remember Bering from our Diphtheria episode. to work on this diphtheria antiserum, among other things. But Erlich also had recognized the limitations in antiserums in terms of safety and scaling

at production, and also because not all bacteria produced toxins for antiserum development. And so it's kind of like not a rich field for treatment. Right. And so he partnered up with a chemical dye company and went in search of a chemical compound that could deliver the same targeted blow. as anti-serums. And he began looking at treatments for tropanosomyasis, African sleeping sickness, which is caused by a protozoan parasite. And he looked at this because these protozoan parasites were a bit easier to see and identify under the scope. And so he and his collaborator,

Sahacirohata, tested out many, many different compounds and versions of compounds. And eventually they found success with compound 606. And that is not, as it is often told, the 606th compound to be developed, but it was the sixth version of the sixth compound tested. But anyway, they found it worked to cure sleeping sickness, and this alone was fantastic. But then Erlich made a fortuitous mistake. He thought that syphilis was also caused by tropanosomes. It's not, as we know from our syphilis. episode it's caused by the bacterium trepanema pallidum. And I wonder, like, my pet theory is that he

just got the names confused. Trepanene. I don't, I can't remember when syphilis was called trepanema, but like, tropanism and trepanema, they sound similar. That was the case. Pretty cool. Anyway. But he tested it out. He tested out 606 on syphilis. And this compound 606, which would later be called Salvarsan, or nowadays Arsphenamine, was hailed as a wonder drug because it worked. This was the first real synthetic chemotherapy drug, and it was widely prescribed all over the world. And it wasn't great, as you may remember, from our syphilis episode, but it was adequate, and Neo-Salversan was a slight improvement when it was developed a few years later. And this would be followed by other synthetic drugs that targeted malaria, for instance.

and this incremental progress continued through the early 20th century. But these drugs were super specific and sometimes had really, really nasty side effects. And the hunt was still on for something that could be more widely used to kill bacteria, but success was really hard to come by. So for example, a physician named Iago Galdson wrote that by 1930, it was the universal opinion of physicians that nothing could be. discovered which would be effective against the ordinary diseases produced by bacteria? Nothing.

Nothing. Nothing. Nothing. We have no hope. It's maybe a bit of a pessimist. And so, a year later, in 1931, a team of researchers headed by Gerhard Domek at Bayer, check out our aspirin episode for more on that company. He was working on a super lethal strain of, of strep, and he found that if he combined an azzo compound, which is a type of synthetic dye, with sulfonylamide, an organic sulfur compound, they had a drug on their hands that could

wipe out the super strep in the mice that they had tested. And it also proved effective in humans to treat strep and non-strep infections like spinal meningitis and gonorrhea. But maybe most impressively, prontazel, which is the commercial name that this drug got, was found to be effective in treating strep pyogenes, which is the cause of puperal or childbed fever, which was horrible. And we'll do an episode on it at some point. With this drug, mortality from childbed fever fell from around 20 to 30 percent to just 4.7 percent. Whoa. Yeah.

Can you imagine? That's, wow. Yeah. We owe it all to chemistry. Our whole lives are chemistry. They are. They are. And for this, Domach got the Nobel Prize.

Okay. Anyway, it turns out that the azodi in prontazel has absolutely no antibacterial effect. Does it do anything? It's just the sulfanillamide. That's hilarious. Which is, you know, it's good news for the world, but it was bad news for Bay. because they couldn't, you know, copyright or patent this drug anymore because sulfonylamide had been in the public domain for a few decades because it had been identified and published in a doctoral

thesis back in the early 1900s. Yeah. So this meant that these sulfa drugs could be made pretty easily around the world. And that satisfied demand much more effectively than if Bayer alone had gotten their patent like they had wanted. Right. And these drugs were viewed as a miracle, rightly so. No one had ever seen such incredible and rapid improvement of patients who seemed on death's door, and dreaded strep infections were no longer this death sentence that they had previously been. But there's a dark side to the widespread production of sulfa drugs,

and that is the regulation, or more accurately, the lack of regulation. One company in the U.S. combined sulfa, raspberry flavoring, saccharine, and diethylene glycol to create a sweet little sulfa syrup. Oh, no. Yeah, your face was very telling. You don't want to eat ethylene glycol. No, no. So diethylene glycol is a compound found in break fluid, coolants, resins. Antifree.

And when it's ingested by animals, it produces dizziness. Intoxication, nausea, elevated heart rate, muscle spasms, and ultimately kidney failure. And death. So this, however, didn't stop the launch of the drug, the sulfa syrup, in October of 1937. And almost immediately deaths were reported. And the FDA launched into action and tracked down the almost the entirety of that initial shipment. Oh, thank gracious. So, I mean, this company was brought to trial over this, but they were not brought to trial because their drug had killed a bunch of people.

The only thing that they could be, like, fined or sued for is because they had mislabeled their drug as an elixir. And it wasn't an elixir because by law, elixirs were required to contain alcohol, and this did not. I'm I can't I know I don't even have any words right now quite honestly I know

so they were you know they were fined that's that's about it this was 1937 yeah okay cool cool cool cool cool cool cool cool cool mm-hmm and one good thing did come out of this

and that was the 1938 federal food drug and cosmetic act that introduced some much, much-needed regulation and oversight into the manufacture and sales of medicines. Yeah. It's about dang time. Sulfa drugs continued to be widely used and were a key component in fighting infections during World War II. And so it might not be that surprising that these were the first antibacterial drugs that we see antibiotic resistance towards. For example, in the late 1930s, 90% of soldiers treated for gonorrhea with this drug were cured, but by 1942, that had fallen to 75% and would continue to drop.

Hooie! Just a few years. But this concerning development was somewhat overshadowed by the introduction of an entire suite of antibiotics. Snaps. Here we go. I'm into it. I feel like this is where most people probably expected this story to. begin. Oh, I apologize. Yeah. I think I think the whole story of sulfa drugs is not as well known as

penicillin for sure. Oh, spoilers. Oh, yep. Whoopsie. Yeah, and there's a whole book about it that I have to confess I haven't read, but I wanted to, if I had had more time, called the Demon Under the Microscope. And that's about sulfa drugs. Cool. But anyway, okay, so yes. I think, yeah, Most people are at least somewhat familiar with the story of Fleming's accidental, quote-unquote, discovery of penicillin. But just in case, I'll take us through it because there's also some fun things that I learned. Of course, Aaron, there always are. There always are. All right.

So in 1928, Alexander Fleming was a researcher in a lab in St. Mary's Hospital in London. And by this time, his work during World War I on the role of anaerobic bacteria in, in battle wounds and the harm that antiseptics and wound treatment can cause, as well as his discovery of the digestive enzyme, lysosyme, these things had established him as an intelligent and insightful scientist. He also claimed to have discovered lysosine by when a drip of snot from his nose accidentally fell on a plate of bacteria he was culturing. And then they, what happened?

And then they died. And so he's like, oh, there must be something in. my snot. And then so he discovered lysisime. Okay. That's pretty cool. Yeah. So he was just admitted to the world that he had a snotty nose and he just let drip everywhere. Yep. Yeah. Yeah. I mean, he was known by his coworkers to be pretty messy. Oh, yeah. Well. Yeah. Not that having a snotty nose makes you messy, but like this is just, I think, in addition. So in August of 1928, as the story goes, left for a vacation in Scotland, leaving petri dishes of staff cultures just out on the bench and a window open.

When he came back a couple of weeks later, he found spots of fungal contamination on one of his plates, and the fungus probably, he assumed, blew in through the open window. And around the spots of fungus, technically mold, actually, was a clearing, so a ring where all the staff cultures on these plates had died. Fleming recognized that this mold represented some amazing possibilities in terms of killing bacteria, and so he's set to work trying to cultivate it. And he later discovered, actually was told by another colleague, that it was penicillium notatum, now penicillium chrysogenum.

I think that's how you pronounce it. I'm not sure. And he reasoned that if there was something, which he referred to as mold juice, produced by this mold that inhibited the growth of bacteria. And he called that penicillin. And he published this finding in March of 1929 in an article called On the Antibacterial Action of Cultures of a Penicillium with special reference to their use in the isolation of B influenza.

What an amazing story of accidental brilliance and insight. You know, what? Or was it? It's, I will say, what's impressive is like you pay that much attention to the plates that you supposedly just forgot and left lying around. I'll open and empty. Right. You come back and you're like, let me inspect this. Ooh, I see a fungal growth and a small clearing around it.

Like that's pretty good. Yeah, it's pretty, I mean, I am by no means saying that this is not impressive. I just think, and this is not a thought unique to me. This is definitely something I picked from these books that I read. This probably wasn't as accidental of a discovery as he claimed it to be. The insight was still brilliant and amazing. But let's just go through some of the points of the story that don't make a lot of sense. First of all, that window that just happened to be open,

according to other people in the lab, it was like never opened. So it would be kind of strange that he would leave it open for like a bunch of weeks at a time. Yeah. And then the timeline itself is a bit fuzzy. So first, Fleming said he was gone for at least five weeks. And then it was no more than two. And that, you know, could just be the not remembering well if you're recalling this, you know, years into the future. But the biggest plot hole lies in the biology.

of this penicillium species. So although Fleming wouldn't have known this, the staff on a plate would have killed the mold before it would produce penicillin. If the plates already had those cultures there, like they did before he left, and then the penicillium blew in through the window, that staff would have killed the mold

before it could produce the compound penicillin. Huh. And so those rings weren't possible. So what actually was going on? Well, Fleming was a rather inventive guy who liked to play games. He would paint pictures of the Union Jack or the logo of St. Mary's using different bacterial species. And this was in the 1920s when you would have had to have encyclopedic knowledge of bacteria to be able to pull that off. Yeah.

That's pretty cool. Yeah. And he was also painfully shy and hated discussing his methods or results with everything. anyone. So the best guess from the author of the book that I read about this is that he invented the story so as not to have to describe his process of discovery. And he may have actually been looking at more sources for lysosyme and thinking maybe it's found in mold or penicillium as well.

Because basically they're saying he would have had to basically plate the mold before he plated the staff orias to be able to actually kill staff with that mold. Right. Exactly. I mean, but regardless of how he arrived at this discovery, he still discovered it and connected those dots. Yeah. That's pretty cool. They're impressive dots to connect.

That's for sure. Super impressive. I just don't know why he would have chalked it up to like serendipity. I don't know. Maybe it's like more fun to have a light bulb moment than like. you know, the incremental progress and like years of hard work and insight? I don't know. I don't know. Yeah, I don't have a single idea. But if there's one name that we associate with penicillin, it's Fleming, right? And if there's

one era, it's in the first half of the 20th century, right? However. Oh, however. However. It turns out that the bacteria-killing quality of penicillium molds had been observed before, as early as the 1800s. When Sir John Scott Burden Sanderson, Joseph Lister, and John Tyndall all observed separately that bacteria would not grow in media that had been contaminated by mold. And Lister and Tyndall went as far as to describe the mold as a penicillium species. Huh. And there are other instances of people recognizing the power of mold. So what made Fleming's discovery a breakthrough while the others remained simply observations?

Right. So one reason is that Fleming saw the implications that this could have for treating infections, and also because he set out trying to isolate his mold juice compound, basically turning his lab into this penicillin farm. Okay. And when they, they meaning Fleming and his assistant Stuart Craddock finally had enough mold juice to test it out, they realized that it killed not just Aflacocchi bacteria, but also streptococchi and a bunch of other groups of bacteria, gram positive bacteria. They also realized that some bacteria were immune to penicillin, gram negative, among others. And the final really important realization was that it was not harmful to non-bacterial cells.

Yeah. That was the big one. Right. This was finally like, was this the magic bullet? Seemed to fit. But all of these observations were made through benchwork alone. Fleming never tested out penicillin on an animal. Maybe he never thought of it.

maybe it was just too difficult to isolate the compound. And finally, the lab where he worked at St. Mary's, it simply wasn't equipped to do the kind of work that he was doing. There were very few chemists working there. And overall, it was much, much less funded than the chemical dye companies who microbiologists were partnering with in Germany. So there was only so much Fleming could do. But where Fleming left off, Florey picked up. In 1935, Australian Howard Florey was professor of pathology and fellow of Lincoln College at Oxford and was leading a research group to investigate why bacteria could not penetrate the wall of the GI tract or did not seem to.

He suspected lysosimes, which you remember from Fleming's discovery, and to carry out his research, he realized he needed to form a strong collaboration with chemists. And honestly, Aaron, I think I had no idea that this episode was going to be propaganda for chemistry, but like I'm one over. I'm there. I'm here for it. Okay. And anyway, he ended up partnering with a couple of chemists, one named E.A.H. Richards, who would purify lysosyme, and another named Ernst Chain, who would identify its substrate. These efforts were part of a larger goal of the lab that had begun in 1937, which was to survey,

this is like a massive undertaking, to survey all of the antibacterial substances produced by microorganisms. Just all of them. Just all of them by all of the microorganisms. Yep, cool, cool, cool. How'd that work out for them? Actually, pretty great. because penicillium molds were on that long list. But in the eight years since Fleming had published his paper in 1929,

not much substantial work had been done on penicillin, and Fleming had all but abandoned it. At one lab group afternoon tea, Florey was talking about what a difficult time Fleming and a biochemist named Ray Strick had in trying to stabilize penicillin. And Shane was like, well, they must not have been very good chemists and took it as like a personal challenge to try to do it himself. I relate to that. And Shane, but Chain at the time had his hands full with a bunch of other

chemical experiments on penicillin. So he called on biochemist Norman Heatley, who by all accounts was the nicest, most humble, like, kind person in this group. And, So he wanted Heatley to work on growing enough of this penicillium mold to make research feasible. Okay. And where Fleming had lacked the equipment and chemical mines to make forward progress with penicillin, Flore's lab just lacked money, period. Wow. And eventually the Rockefeller Foundation awarded a nice grant to help them along,

and it was just in the nick of time because the penicillin project had been yielding some very promising results. So Heatley had been able to extract larger quantities of penicillin as well as stabilize it, which made experiments much easier to perform. And finally, on May 25, 1940, penicillin was injected into four of eight mice that had been infected with strep pyogenes. And the four that received treatment survived and seemed absolutely 100% healthy. And the four that did not died. Whoa. Yeah. And this was great news.

I mean, not for the mice that died, but for the world. But there was still this huge problem of production. Right. Because although Heatley had made amazing progress in the stabilization of penicillin, he simply could not make enough of it to keep up with demand. And it's not like the lab had the funds to supply the materials or equipment to ramp up production. So he resorted to stealing. He took bedpans from.

the hospital supply cabinet and baking trays from the kitchen to supply this huge amount of space that he needed to grow the mold. And he basically like, MacGivered his way through the purification process through a collection of just like random junk that he found in the lab. I love that. It is amazing. He just literally needed containers to grow mold in. He's like, I'll use this, I'll use that. Cool. Great. You're not using this? You haven't baked in a while. Yeah. Yeah, it's really impressive. And in the book that I read, there are some, like, figures that show his setup.

And it's, like, just amazing. It's amazing. There's, like, so much more to all of these stories. And this is already a very long story. But it's just fun. And it's fun to, like, know about the individuals themselves and the personalities and how much personality plays a role. Yeah. It's very cool.

Yeah. So in August of 1940, Flory felt that he had enough to go public with this penicillin news, and he published this research in an article in a Lancet titled Penicillin as a chemotherapeutic agent. But at this point, penicillin hadn't yet been tested in humans, just mice. First up was someone with terminal cancer, but no bacterial infection. After receiving a shot of penicillin, she developed a high fever and seizures. And so that led them to realize that the process of concentrating penicillin also had this unintended side effect of concentrating impurities.

So they fixed it. But instead of just testing to see whether it was safe for injection, they also needed to test whether it was effective. They needed someone with an infection. And they found one, and an Oxford policeman named Albert Alexander, who had scratched his face with a rose bush while gardening. Over the course of a few months, that small cut, that tiny cut, led to a massive infections all over his body. He was, quote, oozing pus everywhere. Gross. Like his eyes, like everywhere, just everywhere.

One day, after just one injection of 200 milligrams of less than 5% pure penicillin, he had made a miraculous improvement. The pus had stopped flowing, and the fever had gone. But the problem was still in keeping up the supply. So the researchers had known that penicillin is excreted by the kidneys, so they started to collect the policeman's urine and rush it back to the lab to reprieve. purify and then back to the hospital to re-inject. But they just couldn't keep up with demand.

They couldn't do it. And so the policeman died five days after once they ran out of penicillin, which is sad. But another person, a young kid that they treated survived later on. And so this was proof enough that it worked as long as you had enough of penicillin. But there was never enough. Never, never. Flory and Heatley needed more funds if they were ever going to make penicillin a feasible treatment for infections. And so they turned to a country that had the funds and the agricultural research infrastructure that they needed, the United States.

After meeting with some friends slash fellow researchers about the work that they wanted to do, they came to the conclusion that they needed to head to the foremost site of agriculture. cultural research in the country, the northern regional research lab in Peoria, Illinois. Peoria! Yeah. That's like this is, Peoria is the site for penicillin, like, from when it went from a novel potential thing to actual penicillin. Actual penicillin.

Yeah. Wow. Peoria. Man. Peoria. I wonder if they have that just, like, blazoned everywhere, like all their bridges. It's like, penicillin. Penicillin was found here.

It wasn't found there, but. To the outskirts of Peoria, I've never traversed the city streets. So how exactly did penicillin go from medical curiosity to world-changing substance? Yeah. There are three big developments that would make this transformation possible. Number one, finding the strains of penicillium that produced the most penicillin, Number two, developing the best protocol to rapidly grow the mold. And number three, improving the fermentation process that actually led to penicillin.

To find these turbostrains of penicillium, a bacteriologist at the lab named Mary Hunt went to Peoria's markets, like every day, every weekend, to find moldy fruits and vegetables. Like the veg market. She's just like walking around farmer's market, like, don't wear I'm here for work. Yeah. Yeah. Well, and she hit peter. She hit absolute peter in 1943 with a moldy cantaloupe. Canalope. The mold on that cantaloupe was so powerful that it became the source for basically all of the world's penicillin. And as for a better growth medium, the Midwest U.S. is known for what type of food, Aaron? Corn. Corn. Corn. Corn. Corn. Corn and corn. Corn. Corn. Corn and corn. Corn. Corn. Corn and corn.

And it turned out that growing penicillium in something called corn-steep liquor plus sugar produced 1,000 times more penicillin than the previous method. Wow. That's a lot. Way to go corn. Way to go corn. Corn for the win. But once again, it came time to ground truth penicillin.

On Valentine's Day in 1942, a woman named Anne Miller was in the hospital in New Haven Connecticut after experiencing a miscarriage. She had developed blood poisoning, aka hemolytic streptococcal septicemia. She had like fevers of 107. She was not coherent. It was really bad. And her doctor ended up through a series of pleading phone calls, he ended up securing a small glass vial containing 5.5 grams of penicillin, which had come from that research lab. 5.5 grams at the time, 1942, was half of the entire amount of penicillin in the U.S. Whoa. Uh-huh.

He guessed at a dosage because there were no guidelines, right? And he injected the drug into Anne. She survived the night and the next day and the next day and the next 57 years after that. Wow. Yep. Her chart, her like medical chart from this time is actually in the Smithsonian's National Museum of American History. So you can go and see it. I love that museum. Yeah. Oh, it's amazing. Yeah. And this marked a real turning point in penicillin's history. By 1942 and 1943,

many pharmaceutical companies in the U.S., including Merck and Pfizer, got into the penicillin biz and worked on their own production processes, meaning that mass manufacturing was just around the corner, and so were massive profits, just like heaps and tons and loads of money. Money, money, money, money. And whereas the successful treatment of Anne Miller with penicillin had led to the biomedical industry taking note of the drug, a horrific fire would wake up the public to it. On November 28, 1942, an artificial palm tree at a Boston nightclub called the Coconut Grove caught fire. And within minutes, the entire club was consumed.

Whoa. 492 of the over 1,000 partiers died. Isn't that? It's one of the deadliest fires in American history. That's terrifying. It's horrible. And hundreds more were horrifically burned. At Mass General Hospital, the doctors decided that rather than debreedment of the burns, they would try to administer penicillin and sulfa drugs. And it's hard to say whether the penicillin did perform the miracles on these burn victims,

as the newspapers later claimed, but regardless, it had now become firmly established in the public's eye as a wonder drug. Demand for penicillin reached new heights, but the mounds of this dried brown powder weren't just there for anyone to use, because there was a war going on, right? And so basically, until the war was over, penicillin was strictly reserved for troops, allied troops, with most of the drug being used for wounds received in battle and also gonorrhea. Most civilians, and there were a few exceptions, wouldn't enjoy the benefits of penicillin until after the war was over.

And Australia would actually be the first company to open up its use to the public. Even with all of these amazing advancements in penicillin production, there was still one big piece of the puzzle that had yet to be solved, that of its structure. So chain had made some progress towards this, but only in that he could produce crystallized degradation products, which he offered up to biochemist Dorothy Crowfoot-Hodhatchkin, who had already made amazing, amazing discoveries about the structure of many large complex organic molecules, such as cholesterol, testosterone, pepsin, insulin, and many others using x-ray crystallography. So with the crystals that chain had given her, Hodgkin used x-ray crystallography to get a clearer idea of the different components of penicillin. But a clearer idea is not the

same thing as a clear idea. And without that clear idea, penicillin would never be synthesized in a lab where it could be produced in larger quantities that were more stable, more pure, and more efficient. The breakthrough came when Hodgkin proposed a beta-lactam ring. Yeah. And that's amazing. And by all accounts, just like Heatley, she was not only an amazingly brilliant researcher, she was also really kind and well loved by everyone who knew her. Oh, I have a baby book that she is the letter D for Dorothy Hodgekin.

Nice. Oh, that makes me happy. Yay. So anyway, penicillin wouldn't be synthesized until 1957, but knowing its structure was integral to not just making it in a lab, but also in trying to look for other compounds that have similar structures and could be used to treat bacterial infections. So the reason that I spent so much time talking about penicillin in this history of antibiotics as a whole is because it provided this new framework for thinking about what these different antibiotics might look like and where you could look for them. Right. So namely in the existence of other living things. But as you talked about, Aaron, penicillin acts on only a subset of bacterial species, gram-positives.

It's not effective against gram-negatives like ursinia pestis or chlamydia trachomatous or vibrio cholera or acid-fast bacteria like mycobacterium tuberculosis. And up until the early 1940s, the second leading cause of death in the U.S. was bacterial pneumonia, which could be caused by a variety of gram-positive and gram-negative bacteria. and tuberculosis wasn't much farther down the list. It took sixth place. So the war on bacteria was far from over, and it's spoiler still not over today. And besides penicillin, the war years yielded more than just penicillin in terms of antibiotics. Two other drugs named Tyrothriacin and Gramicidin were developed from compounds produced by a soil bacterium. But there's a reason that those two might not sound that familiar.

even though they're still occasionally used today. So one of these works by stopping proteins from being made, as you had described, and the other makes cell membranes impermeable. Both of these things will definitely kill bacterial cells, but in these cases, they also killed animal cells. So their use was pretty limited. But one important thing they did was give people a reason to look in soil for other possible antibiotics. And whereas Fleming's discovery of penicillin had been a possibly fortuitous accident,

the hunt for antibacterial compounds and soil was grueling systematic trial and error search full of long days of hard work, which is, I mean, let's face it, that's how most scientific developments actually happen. Right. One of the people doing these long days of work, and often even sleeping in the lab, was a PhD candidate named Albert Shatz. His particular obsession was with actinomycides, which is a group of soil bacteria and trying to find a compound to kill mycobacterium tuberculosis. And if you listen to our tuberculosis episode from way back in season one, this story may sound a bit familiar to you.

As we know, tuberculosis is very deadly, and at the time there was no cure. So Shats was basically exiled to do his work in the basement by himself. Oh, gosh. Because it was dangerous. He was like culturing tuberculosis. Oh, culturing tuberculosis. Okay. Yeah. But this isolation, I guess, paid off. On October 19th, 1943, he discovered that a bacterium named Streptomyces, Grisias, produced a substance that killed mycobacterium tuberculosis. Oh, yeah. And that substance is what we know as streptomycin.

Stereptomycin. But just like with the early days of penicillin, there was a production issue. How do you make enough of the stuff to actually perform meaningful experiments? And that's where Shatz's advisor, Selman Waxman, leaned on the lab's connection with Merck to enlist their help in ramping up production. So eventually they were able to make enough streptomycin to test it on guinea pigs infected with tuberculosis. And guess what? It worked. It worked.

It also worked on humans, which was, again, viewed as a miracle. It's a common thread in this. But who would get credit for this miracle? Not the PhD student. Never. No. No. I mean, interestingly, Merck gave up the patent rights to Shats and Waxman, and they filed the patent on behalf of Rutgers University on February 9th in 1945.

Both swearing under oath that they were co-discoverers of the drug. But if you were living then and following the news about streptomycin during this time, you only heard one name, Waxman. Waxman had dozens of newspaper articles calling him a hero and man of the soil, while Shatz's name was nowhere to be found. Shats was not okay with this and told Wachsman this, but what he got in reply was, quote, you must therefore be fully aware of that fact that your own share

in the solution of the Structomycin problem was only a small one. You were one of many cogs and a great wheel in the study of antibiotics in this laboratory. there were a large number of graduate students and assistants who helped me in this work. They were my tools, my hands, if you please. Oh, gosh. That's why doing a PhD is so depressing. Your advisors like that. That's not a very great way to look at your students.

Nope. And this conflict spilled over into the financial side of things. So they both had supposedly signed away their patent rights for $1 each, but Waxman had made a side deal that had earned him 20% of the profits if he got Schatz to sign away his rights. What? Yeah. That's so gross. I know.

I know. I know. Ugh. And of course, there was the fact that Waxman was awarded the Nobel Prize, and Shats was not mentioned, either by the committee or in Waxman's acceptance speech as more than just one of 20 graduate students or lab texts. This is the problem with Nobel Prizes, guys. It is true that Waxman was a brooklyn.

brilliant scientist who made many other incredible discoveries, and he is actually the one who came up with the term antibiotic. But this is not great visuals. Not great visuals. In any case, by the late 1940s, there were now two super powerful antibiotics at work, completely reshaping the health of the world. And the dive into soil bacteria kind of opened the floodgates even more than penicillin. The first broad spectrum antibiotics that worked on both gram positive and gram negative

bacteria were discovered, so called tetracyclines. And then in 1949 came erythromycin, also from a soil bacterium, streptomyces erythreous. Chloramphenicol was another that came along that received immediate popularity. But its popularity wouldn't last too long because when people realized that this super powerful antibiotic resulted in some people developing a plastic anemia, it was kind of not used as much. Yeah. Aplastic anemia means that your body stops making blood cells, like all of them.

Yeah. So it's really bad news. Yeah. It's also very deadly for babies. Uh-huh. Yeah. Yeah. It was especially in kids, they were seeing this happen.

Mm-hmm. And then in 1948 came the discovery that there was a massive, so far untapped, market for antibiotics. Agriculture. Oh, gosh. Yeah. Oh, gosh. An early experiment showed that chickens receiving broad-spectrum antibiotics grew much bigger, much more quickly than those that didn't get the drug.

And almost immediately pharmaceutical firms jumped on this, producing tetracycline-derived nutritional supplements. Yeah. Up to 25% of all of the antibiotics ever manufactured have been for use in animals. Wow. That is a lot. It's a lot. And the doses that they were given were not therapeutic doses. Right.

Like the amount needed to cure an infection. These were super tiny doses given to promote growth. And those tiny amounts of antibiotics led to massive amounts of resistance, which I'm going to talk more about in our next episode. But this is a great trend towards antibiotic use throughout the 20th century. Do you have any sort of complaint or anything? infection, whether it be viral or bacterial. Sprinkle some antibiotics on it.

Sprinkle, sprinkle. And the philosophy was that even if it might not be, and the philosophy still kind of is, I think some people, that even if it's not a bacterial infection, the antibiotics couldn't hurt, right? But that's where we might be wrong. We are definitely wrong. Not only does this overuse of antibiotics and misuse lead to antibiotic resistance, but we're also finding fewer and fewer effective antibiotics.

And so we're literally running out of the ones that we have. And we're also, as I mentioned earlier, learning a lot more about our own microbiome and the huge role that it plays in our health. Great. So my, I mean, my story kind of stops there because it's just like antibiotics continued to be developed. And then now it's kind of fallen off. And I'll pick up in our next episode on the history of resistance, which is a fascinating one that definitely bleeds into current times. Oh, definitely. Big time. But for now, Erin, tell me what's going on in the world of antibiotics. Oh, I can't wait to. We'll take a quick break first.

A lot of the modern story of antibiotics is antibiotic resistance. And we're not going to talk about that today. So we'll just sort of focus on what is the status of sort of antibiotic. development today. So how do we use antibiotics today? You mentioned in agriculture. The use of antibiotics in agriculture is so intense and so massive. In 2010, there were something like 63,000 tons of antibiotics used in livestock. And it's on the rise. It's projected that by 20,000, 30, it'll be 105,000 tons. Yeah.

It's, it is eye-opening to be sure. Most definitely. So what about in humans? How do we use antibiotics? Between 2000 and 2015, overall antibiotic consumption has increased 65%. Really? Yeah.

We're just increasing our use of antibiotics. even though we know we shouldn't be. I mean, and this is now, so antibiotics, penicillin, when it was first introduced, you could just go to the store and buy it. And in a lot of places, that's still the case. That's true. A lot of parts of the world, you can still buy antibiotics just over the counter. So, yeah, overall global consumption is really increasing, and this is concerning for a lot of

reasons, resistance chiefly among them. But the other thing is, and you kind of mentioned this a little bit, Aaron, we haven't been good at coming up with new antibiotics for a really long time. So after the discovery of penicillin and then quickly after that, the discovery of so many other groups of antibiotics, right, streptomycin that acts in a totally different way, we know, on protein synthesis rather than the cell wall, once we came up with those four classes of antibiotic mechanism, there haven't really been. new classes of antibiotics approved. Between 1960 and 2000, no new classes of antibiotics were approved.

Ooh, right? That's, yeah. And that means while we came up with plenty of new antibiotics, they were just variations on a theme. Right. Which means that resistance might be less of a, it might be an easier leap than it would be for a whole new class of antibiotics. Exactly, right. So it's basically just trying to one up the resistance that we're seeing, right? Let's change the structure of that beta lactam just a little, so, et cetera. Yeah. The good news is that between 2000 and 2014, there have been a couple of new antibiotics approved, including a few new classes. The oxazolidinones that I mentioned earlier, linazolid, so that's a synthetic drug. It still inhibits protein synthesis, but its mechanism of action is different than other classes, like the amino glycosides and

macrolides. And another one called diaryl quinolone. I hadn't heard of that before. It's a drug that we use for TB, I guess. It's relatively new. And this disrupts energy metabolism in some new way that I don't know a lot about. Cool. So we're making progress. But there's still a lot of challenges, right? You mentioned how we used to screen and discover new antibiotics, right, by digging through soil samples and trying to isolate compounds, etc. It's a grueling process. It's a very long and drawn out. Labor intensive.

Labor intensive. Expensive. Yeah. Difficult process. So I guess one of the big questions is have we come up with any better ways to identify antibiotics? I think that we have. We have, which is absolutely thrilling.

And we don't want to tell you about it. We want to have someone tell you about it who knows more about it. So for that, we're very, very fortunate to interview Dr. Jonathan Stokes, who has worked on antibiotics extensively and who recently published a paper about his work on discovering new antibiotics using entirely different methods than what we have used in the past. It is so mind-blowing. I love it. Very, very cool.

It's like this is the future. It is the future. We are living in the future. We are the year 2000, the distant future. Okay. Yes. So we'll let him introduce himself. So I'm John Stokes.

I'm a post-doctoral fellow in Jim Collins Lab at MIT and the Broad Institute. I did my PhD at McMaster University in Hamilton, Ontario, under the supervision of Eric Brown. So by training, I consider myself more of a biochemist, like an antimicrobial biochemist. And I find myself now in Jim's Lab doing a lot more systems biology type work, which is cool because it kind of complements what I did during my PhD nicely, I feel. That sounds awesome.

So we talk in our episode about how antibiotic discovery has kind of, decreased over the last few decades. But of course, the need for antibiotics hasn't decreased, if anything, it's getting worse. So could you talk a little bit about some of the challenges in the traditional ways that we search for new antibiotics? Yeah. So historically, you know, we had our heyday of antibiotic discovery between the 1940s, 1940s and the 1960s, the golden era of antibiotics, where we were able to find a wide structural and functional collection of antibiotics through screening secondary metabolites from soil-dwelling microbes, right? However, come the mid-1960s, we ran into a problem in that we kept rediscovering the same

antibiotics over and over again. You know, and so in more recent decades, we shifted to high-throughput screening of synthetic chemical libraries in an attempt to find new antibiotics. However, you know, for I would guess perhaps 30 years maybe of high throughput screening, we haven't found a single new clinically used antibiotic through that approach. The other one, you know, beyond the scientific drawbacks are the economic drawbacks, right? So if you're a drug company, it costs just as much money to make an antibiotic as it does to make, you know, a blood pressure medicine or an anticoaguline or something, like something that patients are going to be

on for decades, as opposed to, you know, you're going to take a 10-day course of an antibiotic. And furthermore, if let's say, you know, let's say we start a company, you find the best antibiotic the world has ever seen, right? Physicians aren't going to want to use it. They're going to want to save it, right? So you've invested, you know, a billion dollars or two billion dollars or something to develop this drug that is never going to be able to generate the revenue to recoup your costs. So, you know, the economic model for antibiotic discovery and development is fundamentally broken

as well. So you mentioned high throughput screening for potential antimicrobial or antibiotic compounds. What are some of the other innovative new ways that people have looked for new antibiotics? Yeah, so for my old lab, one approach that we took in our lab was looking for molecules that have unconventional activities. Another one is adjuvant screens, right? So instead of looking for one molecule at a time that inhibits the growth of a bacterium of interest, what happens if we start combining two at a time? There's been some work looking into phage therapy for the treatment of bacterial infections, but not just using what we would call like natural phage or wild type phage,

but there have been instances where investigators have engineered phage to deliver toxic payloads. What else? Antivirulence molecules. In this case, you're not eradicating your population. You're just inhibiting a bacterial cell process such that they won't make you sick. So there's a whole lot of ways that people are trying to kind of address this problem. outside of conventional antibiotic discovery as we would define it. Awesome.

So we found you through your recent paper that was published in Cell that was on deep learning approaches to antibiotic discovery. I had literally never heard of the term deep learning before reading this paper. So could you tell us a little bit about this project specifically and kind of explained maybe for us who have no idea what machine learning is? Yeah. So at a high level, machine learning is the discipline in which computers are programmed to learn patterns and data sets. In our specific case, we can use that as a concrete example. We wanted to predict antibiotics. What we wanted was a case where if we showed a

computer the structure of a molecule, it would be able to predict whether it was antibacteri, or not, right? Like at the simplest form, in order to develop a machine learning model that can do that, what you first have to do is train that model. So the model that we trained is called ChemProp. It was developed a couple years ago maybe by Regina Barzali and her lab. So in our case, we had 2,500 molecules that we trained on, and each one of those molecules had a biohic. activity score. Did it inhibit the growth of E. coli? Yes or no. So during training, the model was walking around all 2,500 molecules and then learning the relationships between the structures of every molecule and whether it inhibited the growth of bacteria or not. And then we ran predictions in a library

that's housed at the Brode Institute, which is called the Brod Repurposing Hub. It's about 6,000 molecules, either in preclinical development, in phase one, two, three trials, or on the market. And all the model did, right, was look at every molecule in that library because we had the structures for all 6,000 molecules. And then for everyone, it gave us a number between zero and one. If it was close to zero, it was unlikely to be antibacterial. If it was close to one, it was likely to be antibacterial based on the model that we trained using the 2,500 compounds. So the way that we did it through the message passing approach, it was not user-defined. The model could find any molecular features within any given molecule that would be strongly

predictive or not predictive of antibacterial activity, right? Like it was not human constrained. It was free to explore the molecule and interpret the molecule in any way that it found fit to make right predictions. But yeah, like so they can make all of these interesting, you know, quote-unquote discoveries, even though we don't necessarily know what they're doing, they know what they're doing, to predict molecules that you would not, to be antibiotic that you would not necessarily think to be antibiotic just looking at the structure.

Wow. That is amazing. So you mentioned that this model did find a couple of potential antibiotic compounds. Can you talk a little bit more about those and how they work to either inhibit the growth or kill bacteria and what kind of bacteria they seem to. to be effective against? Yeah, yeah. So the first one that we found was Hallison, right? So that was actually from the drug repurposing hub, that library of 6,000 I just mentioned. So we had set out to find molecules, like we wanted to predict molecules that were antibacterial, but also we wanted to find molecules

that didn't look like conventional antibiotics, right? Because we wanted to find structurally and functionally new stuff that would be able to overcome a lot of the currently existing resistance mechanisms. When we ran our predictions on the drug repurposing hub, we actually put two constraints on it. The first thing was obviously it had to have a high prediction score. The molecules had to be strongly predicted to be antibacterial. But the second constraint was they couldn't look like conventional antibiotics. And then we trained another model to predict toxicity. So we wanted the molecules to be not toxic, predicted to be not toxic. The molecule that fit the bill for those three criteria was Hallison.

And Hallison was a really interesting molecule to study. So we trained our model in E. coli. We also found that it was bacteriocidal in E. coli rather than bacteri static. So it killed E. coli cells rather than simply inhibiting their growth. One of the coolest features to me about the molecule is that it had, this really interesting ability to eradicate antibiotic tolerant cells. So most antibiotics, they were discovered using growth inhibition. However, there are states in which bacteria can reside in which they aren't undergoing a lot of biological activity. So what happens is if you take those

cells in stationary phase or nutrient deprived or whatever in any other way, and you give them, expose them to a bactericidal antibiotic, most of the time, with a couple of exceptions, those bacteriocidal antibiotics don't work because the biological processes that they inhibit are not doing anything. Oh, man. That is amazing. It's like trying to crash a parked car like it doesn't really work. Oh, my God.

That's a great analogy. But we found, you know, we could take E. coli, put it in conditions that didn't have food in it. They weren't dividing. They weren't doing anything. and we were observing that Hallison was still eradicating these cultures. Right. So to me, that was like, okay, like there's not a whole lot of molecules that do this.

And that's what got me pretty excited about this compound. That is so cool. That is really, really cool. We're going to set a record for the number of times we say that's so cool, I think, Aaron. Oh, gosh. Right. So that was all an E. coli. So that's fine and good.

But it's lab strain E. coli. So the next question becomes, does it work? in clinically problematic species, right? So can we take isolates that are multi-drug-resistant, you know, Klebsiella, acinetobacter, staff, mycobacterium tuberculosis, like these types of bugs? So we asked, does Halison retain activity and multi-drug-resistant versions of all of these really nasty pathogens? And it did. We observed that it had retained activity and multi-drug resistant. So carbopanem resistant enterobacteriae, right? Whoa. That's major.

Multi-drug resistant acineobacter bomanii. MRSA, mycobacterium tuberculosis, C. difficil. That is amazing. I don't want to just say that's so cool again, but like, whoa. Oh, man. So when we saw this, it became very obvious, but the next thing that we have to do is figure out how it was working. And I kind of have a playbook for how I like to. to figure out how molecules work.

And the first thing that I like to do is evolve resistance to it, right? Because if you can evolve resistance to something, sometimes, not all the time, but sometimes it will point you in a direction to telling you what it's doing, right? What is the target? However, something that was super cool, and at the time, slightly annoying occurred. And it was in the laboratory, in liquid culture over the course of 30 days. I ran just like a typical evolution experiment. liquid media and I couldn't evolve resistance to Hallison over the course of 30 days.

And the control experiment for that was Cyprophloxasin, right? So I ran the same experiment in parallel with syprofloxacin and over the course of the same 30 day experiment, I think we evolved resistance to Cypro like 200-fold or something like that. Like it was outrageous. Oh my gosh. So then we also did an experiment with. where we try to evolve resistance on solid media. And again, we were unable to see colonies after seven days on Halison supplemented media, whereas we were able to get, you know, dozens or something on CIPRO. All right.

So we wouldn't be able to play, you know, the evolution trick to figure out how Hallison was working. So what we did next was of RNA sequencing, right? We just asked what is the cellular response when you take E. coline expose it to Hallison? And what we observed, it was a very obvious transcriptional response. What was happening is cells were immediately downregulating genes involved in motility and flagellular biosynthesis. And it turns out that that's a very common transcriptional response when you dissipate the proton gradient across the cytoplasic membrane. So it appeared, based on a whole bunch of experiments that we did, that Hallison, was dissipating that proton gradient that is essential for viability across the cytoplasmic membrane.

So cells did not have that proton gradient that is essential to do things like turn like flagella, flagellar motors, couldn't turn ATP synthase, so they couldn't make ATP. And, you know, that proton gradient is important for moving like solutes across the membrane. So it was like all these different functions that are dependent on this proton gradient weren't working. that resulted in a loss of viability. Oh, my God. That's amazing. So in terms of like having a very clear effect, like a clear bacteriocidal effect on those cells, would it have a similar effect or did you test for a similar effect on eukaryotic cells?

So we didn't test specifically, but we did put it in a mouse. So we tested Hallison in two mouse models, right? So we tested it in an osteobacter bomaniae skin infection model. And we also tested it in a C. difficile gut infection model. So the abomanii model, we set up an abrasion on like the dorsal surface of mice. So just on their back, we infected it with abomanii. And then we treated topically with halison. We had six mice in the control group and six mice in the treatment group.

And I think in 24 hours or 25 hours, five of the six mice that received halicin, the number of astinobacter bomania cells was below the limit of detection. And in the control group, they had like 10 to the 8 cells per gram of tissue or something like that. Like it was a lot. And then in the C-DIF model, we tested it against both a vehicle control group and metronidazole. And we observed that Hallison eradicated the C. difficile infection in mice over and within like, I think it was four days, something like that. That's huge.

Yeah. Yeah. So that was cool. But then after that, right, we have this model that appear to work fairly well, better than fairly well, I should say, quite well. So then we wanted to test it on like a very large chemical library, right? So we tested it against the zinc 15 database. So the zinc 15 database is a virtual repository of 1.5 billion molecules. Whoa.

Right? But instead of running predictions on the entire $1.5 billion, we curated 107 million. We predicted that they had antibiotic-like physical chemical properties. And then we ran predictions on that $107 million. It took us four days to run predictions on that. And I actually did a calculation not too long ago. So the model jogged through those molecules in four days. And I calculated that if I had access to 107 million molecules, which nobody does.

And I screened all day every day. It would take something, I think I calculated it was like 14 and a half years to screen that many molecules empirically. Oh my. gracious. Right? Wow. We are living in the future. This is the year 2000.

Wow. Right. So then it was when we ran predictions on the zinc, it was like a similar game plan as we did for the drug repurposing habit. It was run predictions, take the molecules that are the most likely to be antibacterial and find molecules that are structurally dissimilar from conventional antibiotics. Based on those criteria, we were able to curate 23 of such molecules for testing in the lab. Eight of those, 23, ended up working against one of panel of different species of bacteria. And two of them are actually really cool that we're continuing the study.

They're both bacteriocidal and they're both broad spectrum. And right now, those are at the stage of mechanism of action elucidation. So that's what we're focused on right now with those two. Oh, my goodness. So then what are the next sort of steps? And does the fact that was that a choice that you used this repository where you're maybe like further along in that drug development pathway? Was that intentional? And so what are the kind of next steps for Hallison?

Yeah. So we're trying to design analogs of Hallison that work better against TB because we saw like this, really rapidly bacterial biocativity against TB. We're seeing if we can both increase its potency, right? So decrease the concentration of drug required to do that, as well as perhaps explore chemical spaces around the core structure of Hallison such that it can eradicate those cells even faster.

But also, like, we have some, you know, late stage pre-clinical work that we're aiming to finish up with halosin. So these would be experiments that would allow for, an I and D filing prior to hopefully moving into phase one if everything looks good. And we've also recently started a large initiative leveraging this platform to find antibiotics against a wide phylogenetic spectrum of species. So basically we took the top seven bacteria pretty much on the WHO hit list and we're trying

to find new antibiotics against each of those using this machine learning approach and other similar ones as well. That's awesome. So machine learning, we are, I think Aaron and I are both completely one-over. Like we're all in for machine learning. And it seems like there are so many different amazing applications. Are there any drawbacks to machine learning that you could shed some light on? Yeah. So pros for drug discovery anyway, it's cheaper. You don't have to run giant screens. It's faster. You know, four days versus 14 years or whatever. And you can assay, quote unquote, virtually assay, you know, orders of magnitude more molecules than you could feasibly do in the lab, right?

Cons, at least for drug discovery, it's way better to run predictions for molecules that have some biological activity and chemical spaces that the model recognizes, right? So imagine like a giant circle, right? So that giant circle is the chemical space of all the molecules that you want to run predictions on, let's say. And let's say when you train your model, it's like a tiny, tiny little circle within that larger circle at like the bottom edge of it, right? If the model has only seen like a very narrow substructure of the possible chemistry within the prediction space, it's not going to be able to do something called generalize very well, right? So the other, another con, and I wouldn't even say it's a con. It's more just something to be aware of.

ML is like the epitome of like garbage in, garbage out. Like if the data that you're using for training isn't pristine, like, and you have complete control over it, you don't know what your model is going to be predicting. And that's something that I developed a very strong appreciation for throughout the course of this project. Wow. So I know your background is in kind of antibiotics more specifically, but I wonder if you could just briefly talk about some of the other ways. It seems like this type of machine learning could be used for obviously a lot more than just antibiotic discovery. Oh, yeah, for sure. So one thing that a team at MIT is working on now is using this approach to find molecules for COVID, right? So antivirals is another obvious. one. And something else that we're also working on is building algorithms not only to predict whether something is antibacterial or not, but being able to predict the mechanism of action of that

molecule. But I mean, like ML in general, I mean, it's permeated like every aspect of our life, right? So like we have self-driving cars and like what we want to watch next on Netflix. Like that's all thanks to ML algorithms, right? So it's just natural that ML is going to permeate, obviously, you know, biomedical research and health care too. If you pick your favorite biological problem, I'm sure you can envision a way that ML might be able to assist. If not now, then in some number of years when we get even better at this. Thank you so much, Dr. Stokes. That was great. We are. Is it okay to say we were, we were stoked to talk with you.

How many times have people made that joke? That's really good. Oh, really, though. That was phenomenal. How, it's one of those ingenious things, you know, you're like, how come people didn't think of it? Well, maybe they did, but they didn't have the technology for it. Right, right. It's amazing.

It's so cool. It's so cool. Awesome. Well, sources. Sources. is. So I mostly leaned on a couple of books and that were great. One was called Miracle Cure by William Rosen. And this is such an excellent book that talks about the history of antibiotics. I loved it. It was really well written. And then another one was called Big Chicken, as I mentioned

earlier, by Marin McKenna. And then I also read a book called Missing Microbes by Martin Blazer, which talks mostly about sort of the overuse of antibiotics and the microbiome disruption. And there are a few papers that I'll also post. I also used books this time, which is rare for me. But there are a couple of books I found actually very useful, and they have a lot more detail if you're interested on the different mechanisms of antibiotics. One was by Rosaline Anderson at all. That was the editors.

It's called Antibacterial Ate. agents, chemistry, motive action, mechanisms of resistance, and clinical applications. And the other one, he actually has two books, and I kind of used them both, but one is more recent. It's by Christopher Walsh and Timothy Wensowitz called Antibiotics, Challenges, Mechanisms, Opportunities. He also has one from 2003. That's antibiotics, actions, origins, resistance. They're both great. And then a number of papers.

If you'd like to read Jonathan Stokes' paper, it's called A Deep Learning Approach to Antibiotic Discovery, and we'll post that on our website as well. Mm-hmm. We will. Awesome. Well, thank you to Bloodmobile for providing the music for this episode and all of our episodes. Thank you again to Dr. Stokes for coming on the podcast to tell us everything about their work.

And thank you listeners for listening to this very long episode about antibiotics. If you made it this far, you are, we love you. We love you. Even if you didn't make it this far, we still love you. But they won't ever hear that, so. I know, but it still exists, Aaron. The feeling is still there. It's true.

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