The world is fighting two pandemics at the same time – COVID19 and the silent pandemic of antimicrobial resistance (AMR). AMR occurs when bacteria become resistant to antibiotics, making their infections harder to treat. In Europe, approximately 25.000 people die every year because of drug resistant infections. It is estimated that AMR will kill an extra 10 million people globally by 2050.
In this episode we will continue to talk about AMR, this time with three researchers, who all tackle the AMR problem from a different angle. We will talk to Dr. Sam Linsen (02:27), a Dutch researcher who started Squared Ant in China, a business focusing on antibiotic residue, testing and data gathering in order to map the extent of the problem worldwide. Then we will talk to Dr. Nathaniel Martin (21:16), Professor in biological chemistry at the Leiden University. With his research group, he tries to understand bacteria at a molecular level in order to be able to design new antibiotics and develop new molecules that will block resistant pathways bacteria have developed. Our last guest is Professor Arjan de Visser (40:16), evolutionary biologist at Wageningen University, who is working on models to predict evolutionary pathways. By understanding the determinants of evolution he tries to get insights in why bacteria evolve resistance.
[00:00:00.710] – Host
Welcome to the second episode of Innovation Matters, a podcast organised by the Netherlands Innovation Network. This episode we’ll continue to talk about antimicrobial resistance and is broadcasted from Shanghai, China. In our last episode, we spoke extensively about the problem of antimicrobial resistance, also called AMR antimicrobial resistance happens when microorganisms such as bacteria change, when they are exposed to antimicrobial drugs such as antibiotics. Together with Dr. Maarten van Dongen of the global information platform AMR Insights, we spoke about the different sectors involved in the problem and about the five strategies on how to curb AMR. We spoke with Dr. Yonghong Xiao, co-writer of the Chinese AMR Action Plan, about China’s goal of developing new innovative drugs like new antibiotics or alternative treatments like phage therapy or protein drugs by the end of this year. This episode we’ll go more in-depth and talk with three different researchers who all tackle the AMR problem from a different perspective. We will talk with Nathaniel Martin of Leiden University. He’s a professor in biological chemistry and with his research group, he tries to understand bacteria at a molecular level. This should help in designing new antibiotics and in developing new molecules that will block resistant pathways bacteria have developed. We will also talk with Professor Arjan de Visser, evolutionary biologist at Wageningen University, who is working on models to predict evolutionary pathways. He tries to understand the determinants of evolution and by doing this, get insights in why bacteria evolve resistance. But we’ll start with our first guest, Sam Linsen. We will talk about antibiotic pollution in our environment, in rivers, our drinking water and milk. Surprisingly, this is not yet measured adequately. While mapping the issue is a key part of finding a solution Sam Linsen is a Dutch researcher and started his business Squared Ant in China, which focuses on antibiotic residue, testing and data gathering in order to map the extent of the problem worldwide. By gathering enough data, his company should be able to track sources of antibiotic pollution in our environment. Welcome Sam Linsen. Could you please briefly introduce yourself?
[00:02:27.180] – Sam Linsen
So my name is Sam Linsen and I’m the founder of Squared Ant. It’s a Chinese company that measures the antibiotics in food, water and the environment. And in fact, we are a technology company that’s developing a platform to test for antibiotic residue in food and water in order to inform, inform the public about antibiotics and bacterial activity in their environment.
[00:02:53.100] – Host
So you’re constructing a platform that should give an overview of the antibiotic residues in the products we consume on a day to day basis, but also the residues in our environment.
[00:03:03.510] – Sam Linsen
Yeah, so in fact, it’s mostly about antibacterials, anti microbials that are, for instance, used in food production. And then we specifically think about animal animal protein production, which could be meat, which could be shrimp, which could be fish, which could be milk, which could be eggs, because the animals that produce these products, they often receive antibiotics during their life in order to treat, of course, infectious disease, but also to prevent infection disease.
[00:03:37.620] – Host
Is the use of antibiotics in livestock the main cause of the pollution of our environment?
[00:03:43.020] – Sam Linsen
There are many sources of antibiotics in the environment. And yeah, animal food production is one of them, but definitely not the only one. Antibiotics actually come from microbes because in the end, microbes produce antibiotics. They use antimicrobials. They have been doing that for billions of years.
[00:04:04.030] – Host
OK, hang on. Maybe we should go back to basics here first. So antibiotics are actually a natural compound?
[00:04:10.620] – Sam Linsen
Yeah, I think that’s a little bit of a misunderstanding. And I think it’s also not completely solved in what actually is meant with what definition. But I think in general, you could say that there is a sort of a family of antimicrobials and antimicrobials with target microbes, and these are microbes actually also not very well defined, the group of organisms. But in general, we we think that viruses and bacteria and algae could be a microbe family. Well, those if those microbes, they produce the anti antimicrobials themselves. But then we talk about antibiotics. So antibiotics are like just natural products that inhibits the growth of microbes or kill the microbes.
[00:05:01.340] – Host
So antibiotics are naturally present in nature, but the pollution is a result of human misuse. You mentioned livestock as an important source. What other sources are also contaminating the environment?
[00:05:16.270] – Sam Linsen
Yeah, so these antibiotics they in in the bigger countries that produce antibiotics and they are active pharmaceutical ingredients. It is likely that some of those traces would come from those factories. Those producers are then mostly located in India and in China. And these producers there, there are factories and up to 50 percent of the ingredients they produce and end up in the region because of the waste water production that they have and the fact that they do not feel very well. So that’s and of course, you know, looking at the human pipeline, that is the first point where antibiotics and the environment. But then, of course, we also have the food production. But also don’t forget the medical sector and you know, where a lot of antibiotics are used in many countries. We also look at pet animals. They use antibiotics. Yeah, so everywhere where there’s use or production, there will be pollution.
[00:06:21.090] – Host
Do we already have an idea of the extent of the pollution?
[00:06:25.830] – Sam Linsen
Yeah, well, I mean, if you if you have a decent idea on the scale of the problem, I would say yes and no, because. So if so, let’s let’s look at drainage systems, right. If you look at the lakes, rivers and coastal areas. You can actually get a good idea on how how pollutants in general spread, you can see some of the dynamics. For instance, if they are available to our system. So if, for instance, you have pollutants in one area, which is a source, of course, at that source, well, it may have a certain effect. But if the same pollutant that ends up in coastal areas and wetlands, it may have a different effect. So that’s why it’s not only important to check only measure at the source, for instance, because many antibiotics may actually just bind themselves to clay or to stones and just remain there for a long time. But like a certain part of those of those waste products will also find a way down the rivers, the lakes and the coastal. So if you look at those drainage systems, they actually see that basically in Asia and Europe. In the United States. Yeah, a couple of examples in South America. I mean, yeah, basically there’s not a single river like a main water body that does not have anti biotic rescue in it, because in those huge water bodies, the situations are very, very low.
[00:08:00.940] – Sam Linsen
We always try to measure in nanograms per mililiter or maybe even picograms per mililiter, which are extremely low concentrations, like one in a billion or less. But if you if you would actually calculate that into like an item that you would recognize, which is a pill like a pill to ampicillin that you would take 500 miligram, then for many of those main water like big rivers, it would be strange to find about one to 10 pills a second floating by. So this is just free floating antibiotics.
[00:08:40.480] – Host
That is very impressive, if you put it like that. So this is a measure of the antibiotic levels in rivers, free floating in our rivers. Can we also measure how much our, let’s say, unintended antibiotic intake is that we consume through our food and our water? Can we say, for instance, on a yearly basis we consume X amount of pills of antibiotics?
[00:09:05.770] – Sam Linsen
Yeah, that’s a that’s a very interesting question. And I think that also comes closer to actually what I’m doing, even though this, of course, also no simple answer to that. Like, the overarching answer would be, of course, to measure. And I think especially these days, every sane person on the planet would actually appreciate that measuring, measuring is a start like a, of making good decisions. But then, of course, the question is how to measure. Right, because we don’t want to be like this measurement to be intrusive. But having said that, there have been some groups, including one here close to where I live in Shanghai, that have been following, for instance, schoolchildren and and measured how much antibiotics they had in their urine. And they found that actually the exposure of antibiotics was relatively common for the school. So I think about seventy four percent of these kids had antibiotics in their urine, but not 74 percent of those kids actually was on an antibiotic regimen doing the measurement. So, yeah, where did the antibiotics come from? Question number two could be water that they drink or the meat that they eat. But this anyway, could be could be a way to measure the exposure to other ways. Of course, are more you know, it’s you could try to build on antibiotics in the environment around you. We try to generate an overview of, of course, the sources of pollution, the trace amounts you find in the water and also the, you know, the water that the animals drink and maybe the urine that animals produce that that in the end will reach the consumer market. And by such an overview, I think it would also be possible to to to fine tune some of the risks and also inform the public more to to push them into a direction than to actually start to be more demanding on antibiotic use in the products that they use and eat in their daily lives.
[00:11:19.240] – Host
And you are working towards that strategy you mentioned. So next to that that platform that you’re building, you’re also working on developing a test, a so-called chip to measure the levels of antibiotics in fluids. And how does that test compare to current tests that are already available?
[00:11:38.620] – Sam Linsen
So what is my solution then? So what I believe is is a platform to measure antibiotic residue, primarily in water. We also work on milk and later we will also go to other fluids. And if we are possible to also build able to chip, we also bring a solution that that’s actually consumers can use themselves to test for antibiotics in whatever they buy. So there is this is something that can be done in many, many different ways and has been done by different companies all over the world successfully to, for instance, help the industry to remove or to to measure antibiotics in the dairy industry before milk goes into all kind of production cycles for which you need bacteria to grow healthily. Yeah. So I’m also building such a tool. And the key question for me was with what is there on the market? Can we actually maybe make an improvement step? To add some value to the data itself that is produced by such a such a tool, because what we see now is that you would get, for instance, like you would get a tube from, you know, for instance, the delvotest. You would add some milk. You you incubated at a certain temperature for a couple of hours and then you read the color and then you really get a yes or no answer. So, yes, there are antibiotics and there are no antibiotics, but it won’t tell you what the antibiotic is. And it may also not tell you where it comes from, because for for every antibiotic from which the system that you test for is sensitive, you will just get one color. So I think anyway, for antibiotic residue testing, there’s a lot of there’s a lot of room for different types of testing because we look at antibiotics and antibiotics are defined based on the function, not so much based on the structure. And that means that we ought to measure the activity. Actually, you should measure the function. But if you are if you’re interested in just the presence or absence of a certain molecule, then you can actually test for structure. So that means that analytical techniques to test for them separate themselves into two. One is targeted, one specific molecule or a family of molecules. Let’s say it is there, yes or no, and maybe also quantitate and the other one is you just actually just less biological systems like cells that you have in the freezer tell you if there is an antibiotic and then buy, buy, buy, buy by telling that, you know, if there’s antibiotic activity or sample. But you actually don’t know what the antibiotic is. You don’t you cannot identify them. So I thought there is one thing missing here. Actually, you you can actually go into a sample, but you know nothing about you do not make any assumptions. You want to test for antibacterial activity, but you also want to be able to say something about the specifics of that particular activity. So you want ideally you want to also be able to identify those. But what if you can identify those? You can at least make the outcome of your essays unique in a way or so diverse that different mixes of antibiotics generate different outcomes. So maybe then that is something that I develop then we cannot necessarily identify each specific antibiotic, but we can measure antibacterial activity and we can make them response unique to a certain combination of materials. So we expand it. And actually, we we build a panel of all kinds of like different types of sensors and by that generate patterns and patterns can, of course, be used for clustering that can be used for source tracking and by that, and of course, it can also be standardized. So we can actually compare samples are taken on different places in different times, in the same or in different areas. Basically, I would say our platform would definitely be a data tool for which we built our own data collector. We can start to map all areas of the delta of the of the of the long river or lake district or maybe areas around farms, together with people who are interested in keeping the antibiotic footprint of their activities low, or people who are interested to actually control the inputs in, for instance, the ducks they breed or the cows they produce. And the same will be true for other food producers. All of this data then in the end is is meant to to to to inform the public about where can you expect to get safe and healthy produce. And so how you can actually base your purchasing decisions on that.
[00:16:46.200] – Host
So the chip test that you are developing is more extensive in the sense it could test a wide variety of antibiotics instead of just giving you a simple yes or no answer to the question whether or not the food you’re testing is polluted. And also the test data is used more effectively through data gathering, which in turn can be used to start tracing actual sources of pollution. I actually thought such platforms already existed.
[00:17:16.470] – Sam Linsen
So platform cities do exist. Yes. So they can be based on the microbial inhibition that can be based on immunoassays and elisa and they can be based on mass spectrometry, but there’s no platform that integrates all these different types of data. And it’s actually also no platform that allows the consumer to say, hey, let’s compare my own footprint or fingerprint, so to say, with with all the other fingerprints that are produced by other people. All the other techniques are much more vague and also won’t steer you in the direction of where the rest of you that you find actually comes from.
[00:18:00.180] – Host
So all current tools out there actually simplify the problem.
[00:18:05.310] – Sam Linsen
I know people people just think things very pragmatically. And I also talk to a milk farmer at some point just saying, like, you know, if the level of antibiotics in my milk is too high, I just mix it with milk, with less antibiotics. That is a practical solution for a working class solution for a middle class problem. But of course, for the for free for consumers. This one, I mean, the consumer will know. And if if the milk that they drink, which is not processed apart from being pasteurized, does contain some some antibiotics, you know, they they, I think, are much more concerned because they can mix it with something that doesn’t contain antibiotics.
[00:18:48.690] – Host
If I get this straight, we as consumers should be more aware of the consequences of pollution and urge our governments to take action. And then governments will be seeking tools that provide help and Squared Ant, your company could be one of them, is that right?
[00:19:05.260] – Sam Linsen
Yes. Yeah, absolutely. I could I could provide like a standardised platform to sample like everywhere, basically in as low as a starting point for source tracking and defining like high risk areas.
[00:19:21.480] – Host
What do you have to say to the people that are listening right now and want to take responsibility, too? I mean, I would also like to demand more transparency for this. Is there somewhere they can go to sign up? What if they would like to conduct the tests at home? What should they do?
[00:19:39.000] – Sam Linsen
Well, for me at the moment I’m in the R&D phase, and it will still take some time before, you know, the product will become available for all the consumers, but at the same time antibiotic residue pollution, it’s not something that will go away in the coming years. It is something that is here to stay. And therefore, I would like to invite you, the listener, to come to my website Squared Ant and register yourself so you will receive updates and insights on what we’re doing at the moment and whenever we ready. We start sampling in your area or do measurements and we’ll get in touch and then we will ask you to contribute because just sending some of the water into your environment to us, which then, of course, will be a huge impact for us or our developments and also probably, hopefully for the situation around where you live.
[00:20:31.200] – Host
Thank you Sam Linsen for taking the time to talk about the development of your test and data company. So if you would like to follow Sam and support his journey towards more transparency, then please go to his website SquaredAnt.com and subscribe. Like Sam said, the problem of antibiotic residue pollution will not go away and mapping the issue is a start of the solution. However, as we already live in a post antibiotic era, we do need other solutions as well. I’m going to talk about it with my next guest. My next guest is Professor Nathaniel Martin from the Leiden University, and he is running a research group which, on a very fundamental level, focuses on the resistance mechanisms that bacteria develop. Welcome professor Martin, could you please introduce yourself?
[00:21:16.860] – Nathaniel Martin
Sure. My name is Nathaniel Martin. I’m a professor of biological chemistry at Leiden University in the Netherlands. And my background is actually as an organic chemist. I did a PhD in organic chemistry at the University of Alberta in Canada. And from there I decided to branch out beyond pure chemistry and look at the way ways to apply it to infectious disease. And so that led me into the field where I now work. And that’s that’s trying to develop new antibiotics. And my background as a chemist is very influential there because, of course, as an organic chemist, one can understand things at the molecular level and try to develop solutions at the molecular level. So a big part of the research in my group, in Leiden, is designing new antibiotics to overcome antibiotic resistant bacteria. We also try to develop new molecules that will block resistance pathways and in drug resistant bacteria. And if that’s successful, then you can in principle re sensitize drug resistant bacteria to conventional antibiotics. So this is this is basically the two strategies we take in the lab trying to develop new antibiotics or trying to disarm resistance in bacteria to overcome overcome antibiotic resistance.
[00:22:33.120] – Host
Let’s assume not everyone who’s listening has a background in biology. Could you please tell me more about what kind of mechanisms can bacteria develop to become resistant?
[00:22:44.010] – Nathaniel Martin
Sure. So you have to envision bacteria as these small single celled organisms that that multiply very quickly. And they’re actually all around us and they’re they’re part of normal, healthy human life, too. In fact, one of the startling statistics is that we have more bacterial cells in our body than we actually do have human cells. So we’re actually more bacterial than human. And that speaks to how important bacteria are in our body, specifically in our digestive system. And this is the whole area of the microbiome you may have heard of. But what can happen is if you have a dis balance in the bacterial population in your body, or if you have an infection by a pathogenic bacteria that maybe gets in your bloodstream, then you have a problem and these bacteria will circulate and of course, multiply and will make you sick. Causing infection and resistance to antibiotics takes many forms. I mean, antibiotics. When they were first brought to the clinic in the in the mid nineteen forties, really revolutionized the way that we could treat infections because it was for the first time really recognized that one could take a drug that would specifically kill bacterial cells and not harm our own human cells. So that’s the so-called magic bullet. And in the 20 or 30 years that followed the discovery of penicillin or let’s say the clinical implementation of penicillin, many different antibiotics were discovered and many of them kill and target bacteria by different mechanisms. And in subsequent in the last 50, 60 years, bacteria have begun to adapt and in doing so, become resistant to these antibiotics. And you ask what type of resistance mechanisms are at play? Well, in some cases, bacteria can actively degrade or destroy antibiotics. They’ve developed ways of doing that. In other cases, they also have ways of spitting antibiotics out so the antibiotic may get into the bacterial cell. But before it can harm the bacteria, the bacteria is able to just spit it back out. And in other cases, bacteria are also able just to modify the target that antibiotics would normally hit so that the antibiotics no longer are able to bind to the bacterial cell. So those are those are three main types of resistance. I would say the destruction of the antibiotic, the spitting out of the antibiotic or the bacteria actually altering their own structures so that the antibiotics are no longer able to to bind them.
[00:24:59.100] – Host
And which resistance mechanism do you primarily focus on?
[00:25:03.540] – Nathaniel Martin
We focus primarily on two of those three. One one major area is trying to develop, what we call inhibiters, these are inhibitor molecules that can block the mechanisms that some bacteria use to destroy antibiotics. So if you have an inhibitor that that blocks such a resistance mechanism, it prevents bacteria from destroying conventional antibiotics. And those conventional antibiotics in turn, can then be useful again at treating these types of bacterial infection. So that’s one line of work that we’re actively engaged in. And then another one is the developing of new antibiotics or let’s say improved antibiotics that function again against bacteria that has altered their targets. And this is where a background as a chemist is very insightful, because if you understand at the molecular level how bacteria have structurally altered their targets, you can then in principle also look at the antibiotic. And in the lab we can alter the structure of the antibiotic to try and help it, then overcome the modification that the bacteria has introduced as target. So it’s a bit of an arms race. The bacteria does one thing, then we do another to keep pace and back and forth it goes.
[00:26:11.740] – Host
Is it like changing the structure of antibiotics so the bacteria will not recognize it as its enemy and therefore will not attack it?
[00:26:20.820] – Nathaniel Martin
Exactly, yes. Yes. This is this is a big part of it. So bacteria over over years or decades of exposure to an antibiotic will develop ways, as you say, of recognizing the antibiotic as being something that’s toxic to them, and they can then destroy it or block its action. So if we modify the structure of an antibiotic, as you say, we can also then in some sense trick the bacteria or the bacteria doesn’t sense the modified antibiotic the same way it does the original antibiotic.
[00:26:48.210] – Host
If we are able to reverse the resistance mechanism of bacteria. Does that mean that all bacteria will be susceptible again to all the antibiotics that became obsolete over time?
[00:27:01.890] – Nathaniel Martin
Not I wouldn’t say that they would become sensitive to all antibiotics, but generally speaking, you talk about specific resistances to specific classes of antibiotics. And the other thing to remember again is, of course, bacteria adapt to to pressures that we put on them. And antibiotics are a pressure. Now, if you have an antibiotic that’s become resistant or if you have a bacteria that’s become resistant to an antibiotic, and we then can develop a new inhibitor that blocks that resistance mechanism and then re sensitizes that bacteria to the antibiotic, there’s no there’s nothing saying that the bacteria in response will also evolve to become resistant to the inhibitor that we’ve developed to the first resistance mechanism. So you really do have to see this as a ever progressing or ever sort of changing almost arms race between us. But in principle, your assessment is correct. That is, if we develop inhibitors against known resistance mechanisms, we will be able to then once again use antibiotics that maybe haven’t been very effective for the past two years. And that that’s that’s also an important strategy and a viable strategy. There are in some cases, clinically used combinations of what we would call older antibiotics, plus a newer, more recently invented resistance blocking drug molecule. So these these are established methodologies for for certain types of resistance.
[00:28:20.640] – Host
So even though your research is quite impressive, this is definitely not a silver bullet.
[00:28:25.770] – Nathaniel Martin
Yeah, there’s there’s no such thing as a silver bullet in the sense that it’ll be forever a silver bullet. The best we can hope for is to have a bag of silver bullets or to have an ever adapting and growing line of silver bullets so that when one stops being effective, we’ve got a new one that can take its place. It’s really the only way that you can ever really maintain a proper arsenal of antibiotic drugs is to recognize that the minute we use an antibiotic is also the minute we start providing bacteria, the challenge to evolve towards it, that we’re against it. And this is a completely normal process. I mean, Alexander Fleming in his Nobel Prize speech acknowledged that it’s fairly easy to make bacteria resistant to penicillin. We can do this in the lab. We do do this. In fact, when we just when we work on new antibiotics, one of the standard tests we do is how easy or difficult is it for a bacterium to become resistant to this new antibiotic. And you do this simply by exposing bacterial cells to low levels of the antibiotics so less than what would normally kill the bacteria. If you do this over a period of days and weeks, you will often see that bacteria on day 30 now require much more antibiotic to be killed than they required on day one. So this is this is something that is is widely known in the field. It’s, as I say, a natural process. So this is why also people need to recognize that even if we’re very responsible with our use of antibiotics and limit how widely we prescribe antibiotics, it’s still more or less inevitable that resistance will will be developed. The best you can do is maybe hope to delay it as long as possible by prudent use of antibiotics. But ultimately, if we want to maintain our standard of medicine in the world, we also need to acknowledge that antibiotics and research and develop of new antibiotics, is quite foundational to being able to maintain our health care systems.
[00:30:14.290] – Host
When can we expect your findings to reach patients?
[00:30:18.510] – Nathaniel Martin
Well, the standard number that you always hear in the drug drug development field is 10 years from the moment a drug is first made to the first moments that it will be used in a human patient. And that’s, of course, because there’s such a long and expensive trajectory when you talk about the sort of clinical preclinical validation, demonstrating efficacy, but also safety. And then that first needs to be done in a number of animal animal studies before you can get to human trials. Now, when you talk about antibiotics that work against really dangerous pathogens, there is some opportunity for this process to be accelerated, especially if you’re talking about maybe a new antibiotic that works against bacteria that have become pan-resistant, so for which there are no proper treatments. And in those cases, there are now initiatives that are being taken and have been implemented by the FDA and the states to try and accelerate the approval process. But at the end of the day, you still need to first go through the clinical trial process to demonstrate safety and efficacy in humans. And that is an expensive and time consuming process. There are not that many companies chomping at the bit to invest in this type of development, work for antibiotics because of the uncertainty associated with their being able to turn a profit or recoup their costs at the end if they bring it to market. So there’s there’s the framework is there for how to do it. But the as long as the economics remain so dismal, it’s hard to really predict how quickly promising compounds that comes out of my lab today would actually ever reach patients. So this is a big question mark. And I think I think governments around the world actually need to step up and think about how they can address this financial barrier to antibiotic development, because the science is there. I mean, we are doing good stuff, but certainly the best people to discover new antibiotics would be the people working at drug companies, a professional drug hunters, if they are properly incentivized to do so, they will be able to develop new antibiotics. I believe that. I think as an academic scientist, what we can do is sort of keep things going, train young scientists so that if and when companies are properly incentivized to work in the field, they’ll be also qualified researchers to do those jobs. And of course, the other thing that academic science is very good at is understanding processes and mechanisms of disease and of resistance. So as I mentioned, it actually does start with understanding how a bacteria becomes resistant. And if you understand that, then you can start imagining ways to overcome that resistance. So I think there’s there’s, of course, always going to be a role for academic researchers like myself. But I do worry about the lack of incentives for companies to work in this space. And I think until that’s properly or effectively addressed, it’s very difficult to imagine timelines for new antibiotics coming to the market. And and certainly in the meantime, bacteria aren’t waiting there. They’re continuing to evolve their resistance mechanisms to our existing classified abiotic. So, as you say, there are some reasons to feel rather dismal about the prospects here unless unless we can sort of coordinate our efforts internationally and create some some global incentives for companies to work in the space.
[00:33:41.410] – Host
Do you have any funding issues or other problems to get new compounds or products validated?
[00:33:47.470] – Nathaniel Martin
Well, I mean, funding is difficult for everybody, ourselves included. And it’s something of a rat race, if we’re honest. I mean, everybody thinks their research is the most important thing. And I think a lot of people acknowledge that antibiotics research is important. But I’m competing with scientists who are also interested in trying to develop effective medicines against cancers for which there are no treatments or for other chronic diseases. And because there are not big companies available to collaborate with on antibiotics, it’s actually the case. I think that it’s rather difficult to find funding compared to other disease areas where there are huge profit motives. If you look at, for example, research in certain emerging cancer therapies, there’s all kinds of funding available there. Fact is, though, if you don’t have proper antibiotics backing up your health care system, a lot of innovative chemotherapies are very dangerous to use. A lot of people don’t recognize that. But when you are treated with chemotherapy, oftentimes you’re also treated with an antibiotic sort of as a protective effect because many chemotherapies can deplete one’s immune system. So you need to also then take a prophylactic antibiotic. And without effective antibiotics, these fancy and amazing new cancer medicines actually start to come with some increased risks. Same goes for for a lot of other hospital procedures, by the way. So I think this is this is eventually a scenario that is going to start to bite us from from multiple sides and so definitely there should be more funding available for people doing the innovative discovery and development work. And to be honest, that’s a relatively small amount of money compared to what it’s going to take to really properly incentivize companies to want to do the later stage development work. So I would love to see more funding coming in. For those of us that are doing the discovery at the early stage, that’s obviously essentially if you can’t develop an antibiotic candidate that hasn’t yet been discovered. So, yes, I would love to see more more funding come in that direction. There are initiatives, but I think we need to do more. And I believe also if companies are properly incentivized to get back into this space, then it stands to reason that they would also then look to work together with with research groups and other small companies interested in developing antibiotics. So there would be more resources from that side as well.
[00:35:59.430] – Host
I think it’s quite surprising. We’re talking about a lot of money, perhaps billions of dollars. But on the other hand, if we’re talking on a global scale, on a government level, it’s not that much, especially when you consider the impact. It’s such a pressing issue. And yet apparently it’s still lacking urgency.
[00:36:16.980] – Nathaniel Martin
I think covid really kind of shines a light on this, too, because we know we’re spending trillions globally on just measures to try and contain or repair the damage to the economy and the society that Covid has done. If we invest a few billion earlier on, you may be able to avoid that later on. So I think there might might my hope anyways is that coming out of the Covid situation, people might also acknowledge let’s appropriately invest and provide some incentives to develop new antibiotics now rather than 10 or 20 years from now when the problem is really out of hand and the costs are astronomical.
[00:36:51.130] – Host
Yes. So this will maybe make us more willing to invest in prevention.
[00:36:54.760] – Nathaniel Martin
We do this for other things. I mean, the classic example would be fire extinguishers or fire trucks. We don’t wait until a house is on fire before we go by a fire extinguisher or build a fire truck. We have those things we’ve invested in, those things that we hope we never need to use them. But when they’re necessary, we have them at our disposal. And you call the fire service and in two minutes their at your house to put your fire out before your house is burnt to the ground or the fire extinguisher can put the fire out in the kitchen before your whole house is on fire. Think think of having a proper antibiotic arsenal in our hospitals like that to prevent future outbreaks of really difficult to treat antibiotics, being able to treat them when there’s a small number of people who are infected rather than when they spread and get out of control.
[00:37:32.220] – Host
Yes. So this will maybe make us more willing to invest in prevention. So in order to wrap up, what stage of development are you currently in?
[00:37:39.210] – Nathaniel Martin
So as a as an academic research group are very fundamental, very early stage, we would we would say discovery stage and maybe preclinical. That would be the two areas that that we were able to get to. This is driven largely just by the costs that are involved in drug discovery and development. So the place we try to get to in our research is demonstrating efficacy in a relevant animal model. If we can do that and convince ourselves and others that there is promise, then the idea, the hope is that outside parties would be then interested in licensing these these discoveries. And then, of course, you get involved with the technology transfer offices of universities and they come into play, too. So, yeah, this this, as I say, the discovery to actually design and synthesis of the new antibiotics. And then we can do what are called in vitro tests in the lab. That’s simply where you see to these new antibiotics kill drug resistant bacteria in a petri dish or in a ninety six well plate. So we do that every day. There’s there’s people in lab right now those types of experiments on new antibiotics. And if they work well in an ninety six well plate, then we can escalate, as I say, up to an animal model of infection. And one of the benefits actually of working in an area like antibiotic research is the animal models are fairly predictive for how well you might expect a new antibiotic to work in higher animals or in humans. This is one of the things that that actually makes antibiotic research I won’t say easier, but but certainly the models are more predictive. And so we try as much as we can to validate our compounds up to the point where we see that they work well, for example, in a mouse model of infection.
[00:39:19.220] – Host
Professor Nathaniel Martin from Leiden University, thank you for taking the time to tell us more about your research and its importance in staying ahead of AMR in our search to understand and overcome the resistance mechanism that bacteria develop, we could, like Professor Martin is doing, focus on the most important components on a molecular level and see if we can manipulate them or overcome them. This is a more analytical approach of the issue. Another possibility is to go even more fundamental by focusing on the mechanism that causes evolution in the first place. So instead of merely looking at, let’s say, binding receptors and finding solutions to re sensitize them, we focus on the evolutionary process of natural selection. My last guest is Professor Arjan de Visser. He’s an evolutionary biologist at the Wageningen university. Welcome, Dr. de Visser, could you please briefly introduce yourself?
[00:40:16.270] – Arjan de Visser
My name is Arjan de Visser, I’m a biologist, an evolutionary biologist, and we work on models to try to predict the evolutionary pathways. So and for that, I’m not a modeler. So I work with people who are theoreticians that build the models. But we do the experiments. And the way we do this is we use bacteria and fungi in so-called laboratory evolution experiments to try to understand exactly what are the determinants of evolution. So what determines that bacteria evolve in a certain way?
[00:40:58.030] – Host
You try to predict the evolutionary pathways of bacteria. Could you tell me why this is relevant for the AMR topic?
[00:41:07.630] – Arjan de Visser
Well, this is relevant, we hope, because if we can predict how bacteria evolve resistance, we may be able to control this process better by designing therapies that then can counter, say, the evolution that we predict that will occur.
[00:41:27.400] – Host
We just spoke with Dr. Martin about the several mechanisms bacteria developed to defend themselves against antibiotics. One of them is creating enzymes that will attack the medication. And you try to understand at a very fundamental level the genetic evolution of this enzyme. So actually, it’s not the bacteria itself that mutates, but it’s his weapon, the enzyme, the beta lactamase.
[00:41:55.390] – Arjan de Visser
Exactly. Yeah. So this is but this is one approach we take. So there we focus on the evolution of the beta lactamase and becoming more resistant to this new antibiotic.
[00:42:06.040] – Host
OK, so this is one of the two focus areas. But first, about the antibiotic you use in your experiment, is that an antibiotic that is commonly used?
[00:42:15.760] – Arjan de Visser
So the Beta Lactam that we use typically is a cefotaxime. That’s a so-called third generation cephalosporin. So this is this is one member of this big group of Beta Lactam antibiotics that started with penicillin in the 1940s. And now it’s and then later synthetic versions of penicillin have been developed by by chemists. And so this class of antibiotics this Beta Lactam group of antibiotics is the most widely used group of antibiotics worldwide.
[00:42:48.730] – Host
So Beta Lactam is the antibiotic and the beta lactamase is the enzyme, which is produced by the bacteria that kills the antibiotic. So could you tell me how you try to understand the determinants of the evolution and therefore try to predict its evolution path?
[00:43:07.890] – Arjan de Visser
We do that in two different ways. So our focus is on the evolution of Beta Lactam resistance. Beta Lactam is the largest U.S. group of antibiotics worldwide and we study the evolution of Beta Lactam resistance in two different ways. The first way is a very simple and controlled way where we only focus on one mechanism that leads to resistance to the Beta Lactams that we use, where we only focus on the evolution of the enzyme involved in the breakdown of this antibiotic called beta lactamase. And this we do by making that, by introducing mutations in the gene for the enzyme in a PCR machine. So outside the bacterium.
[00:43:54.880] – Host
And what is a PCR machine?
[00:43:56.790] – Arjan de Visser
A PCR machine is a machine that copies DNA. And so we put the original gene, the DNA of the gene for the beta lactamase in the machine. We make copies. The copies contain mutant’s mutations. And so some of these mutations cause the enzyme to work better in breaking down the new antibiotic. So that is that is called in vitro evolution.
[00:44:19.840] – Host
OK.
[00:44:20.130] – Arjan de Visser
And then we put the the mutated versions of the of gene back into the bacteria. We challenge them with the new antibiotic and we select for resistance. And in this way, we we can see the pathways where the enzyme is involved in resistance to the Beta Lactam and how many there are and what the influence of environmental factors is on the choice of these different pathways. But we also have a broader approach where we where the enzyme is just one of the possible mechanisms of resistance to be tied to the antibiotics, and for that we use a more conventional methods by putting the gene for the enzyme on a plasmids in the strain of E. coli that we use. And we challenge both the the the plasmids and the chromosome with all the other possible mechanisms that may lead to resistance with the same antibiotic. And now we look at the whole broad range of the pathways involved involving different mechanisms of resistance. And we are interested in the same questions. What how many different pathways there are and what makes certain bacterial populations choose from one pathway over the other.
[00:45:39.050] – Host
Let me try to summarize that. So the first angle of research is the most fundamental and solely focuses on the pathways of the enzymes, which is the main mechanism bacteria use to evolve to resistance. Then we have the second research angle, which is broader, that research recognizes enzymes as being part of a broader biological mechanism, a part of the whole bacteria, which as a whole mutates as well. Then my next question is, what are the external factors that cause mutations of enzymes into certain directions?
[00:46:22.010] – Arjan de Visser
Mutations occur just spontaneously when bacteria copy DNA. It’s also true that in an environment where the antibiotic the Beta Lactam is present, mutation rates may slightly go up because the antibiotic itself is somewhat mutagenic. But in terms of which of these mutations then survive and determine the route towards resistance that the population takes, we are particularly interested in the effects of population size. How many mutations are present in a population from which the population can choose? That’s one very general factor we’re interested in, and the other is how the different resistance mutations that the bacterium may generate, affect the fitness of the bacteria themselves. So how how does it affect their growth rates, if you want, because it is by natural selection that these mutations get selected, which are not selected for the resistance effects directly there. They’re selected for their effects on the fitness, the survival and reproduction rate of the bacteria that causes them to become dominant or not.
[00:47:36.580] – Host
Do we already have enough understanding of the evolution process? I actually mean, can you already reproduce the same paths of mutations?
[00:47:47.980] – Arjan de Visser
You mean that we see results in our experiments back and say clinical isolates? That is indeed what we what we see with with the in vitro, with the simple version of our evolution experiments, the in vitro evolution of TEM-1 Beta Lactamase so then we repeatedly find in our experiments an allele with three mutations that are exactly the same mutations as in ten fifty two, an isolate that is found in many clinical or a version that’s found in many clinical isolates. So there apparently we are able to repeat the natural evolution of this beta lactamase. But in general, this is this is a long term perspective to, to, to, to try to predict exactly what’s going on in nature, if you want to in the clinic. So we are mainly focusing now on trying to find organizing principles that that’s with some general applicability, for instance, the role of population size in the choice of different resistance mechanisms having to do with what drives these different mechanisms, are these high rate mutations or high benefit mutations for the bacterium. And so with that, if we find this for certain antibiotic, this may apply to other mechanisms as well so also to other antibiotics, we hope. And so this may be relevant for understanding the natural evolution of resistance also towards these other antibiotics.
[00:49:25.930] – Host
Will these research insights contribute in optimising the use of existing drugs or will it help in the design of new ones?
[00:49:34.120] – Arjan de Visser
Well, that’s that’s that’s a long haul. But so one principle that that people are investigating and that we could contribute to is this principle of collateral sensitivity. So this is what occurs when the bacterium becomes resistance resistant to drug and thereby becomes more sensitive to drug B. So if you know this is happening, we can with drug A we can, of course, try to predict a an effective therapy with the combination of these two drugs where you first apply A and then B to kill off the resistant bacteria to A.
[00:50:15.250] – Host
So it’s some kind of manipulation of the direction of its evolution path.
[00:50:21.580] – Arjan de Visser
It’s yeah. So well it’s basically trying to understand how bacteria deal with these different resistance mechanisms, that that are available, so if resistance to to to one drug comes with higher sensitivity to another drug, we can, of course, exploit this. And with our evolution experiments and the protocols we develop, we can we have tools now to kind of analyze that at large scales so we can for instance, we use small droplets in which we put bacteria that are challenged with with antibiotics that we use by pipeting robots to to transfer many populations of bacteria in parallel. And so these these Large-Scale experiments are really very good at exploring a whole range of conditions and antibiotics for their effects that they may have also on the sensitivity to other drugs. So interactions between drugs.
[00:51:27.040] – Host
Of what I understand, most of the experiments are conducted manually. Trying to truly understand the evolution path means that one is able to repeat the exact same path of mutations over and over again. If this is done manually, then it sounds like a very repetitive procedure. Isn’t there a way to automate this process?
[00:51:48.990] – Arjan de Visser
Yes, that’s indeed a very big challenge now. So we indeed want to get an overview of the mutational pathways towards resistance and thereby we want to explore them as broadly as we can. So with with with very high replication of our experiments and we use two different tools for that. So in one tool we we have bacteria evolving in small droplets inside a machine. So these bacteria are in a small tube filled with oil. The bacteria are then the small water droplets in this in this oil. They’re pumping back and forth and they’re detected with by the fluorescent markers that they have. So we can see how many are these droplets. And they’re are challenged with antibiotics. And so we can repeat the growth process within these droplets by getting them out and putting small fractions back into new droplets again. And so this is one machine to look at the highly replicated evolution of bacteria challenged with antibiotics and another another tool we use in collaboration with the group in Cologne at University of Cologne in Germany is a pipetting robot where we where we evolve bacteria in so-called micro tidal plates in small volume, sitting in a small dense into a big plate, and they are transferred automatically by this robot. So this is another tool to study these highly replicated evolution towards antibiotic resistance.
[00:53:26.740] – Host
How common is this research topic focusing on the most fundamental aspect of a disease? Are there other research groups researching this as well, or is there any international collaboration on the topic?
[00:53:39.910] – Arjan de Visser
The topic of the predictability of evolution is is certainly one that is that is receiving growing attention. So we are part of a bigger consortium at the university that’s coordinated at the University of Cologne in Germany, which is a mix of biologists, physicists and medical people that have united to to work on this problem of the ability of evolution. And so we we focus on the predictability of antibiotic resistance, evolution, but others focus on the predictability of cancer evolution or the evolution of the immune system there. And so this is a mixture of experimentalists and theorists that then build models based on our experiments to try to predict the evolution of all kinds of different unwanted processes like antibiotic resistance, cancer, but also the evolution of new pathogens such as influenza or currently covid.
[00:54:45.820] – Host
Thank you, Dr. de Visser. Thank you for your time. And I also want to thank my other guests, Dr. Sam Linsen of Squared Ant and Dr. Nathaniel Martin from Leiden University. More information on the speakers and their research can be found on our website netherlandsinnovation.nl, here you can also leave a comment. If you would like to reach us, please send an email to China at NetherlandsInnovation dot nl. With this episode, we finished the topic of AMR for now and our next episode we’ll focus on other Sino-Dutch technologies and innovations. I hope you’ll be tuning in again and thank you for listening.