[Narration] Geoff Marsh
Hello, and welcome back to the Eppendorf Young Investigator Award podcast. And once again, this year’s celebrations are being held virtually. But luckily, as restrictions were lifted slightly in the UK, I was able to go and meet this year's winner, face to face, just a few hours from London, in a suitably prestigious location.
[In office] Geoff Marsh
I'm here now at the William Dunn School of Pathology at the University of Oxford to speak with Tanmay Bharat and we're in a very lovely office.
Tanmay Bharat
Yes, indeed we are. This was the Office of Henry Harris, the director when the first antibiotic known to man was developed at the Dunn School. So this was the department where the antibiotic era started.
Geoff Marsh
The last two years have obviously been largely dominated by the fight against COVID-19. But let me hear your sense of the importance of one of the other big challenges that we face as a society, and that is antibiotic resistance.
Tanmay Bharat
So I should say, even within the COVID-19 pandemic, many patients that end up in hospital die of bacterial infections. So it is a problem even within the current situation. But in general, antibiotic resistance is a very slow moving pandemic. It’s extremely serious and if it's not dealt with right now, it will lead to a return of the Dark Ages, when antibiotics don't work and there are no treatments against bacterial infections. Then when people get infected, the only treatment is physical removal of tissues, including amputation or people just dying. So it's extremely urgent to invest in fundamental and translational research at this moment in time to develop new antibiotics. In our laboratory, we study a process known as bacterial biofilm formation, which is a process in which single bacteria become part of a multicellular community with the secretion of some kind of an extracellular matrix. This extracellular matrix secretion is accompanied by bacteria becoming extremely tolerant to a wide variety of environmental stresses, including antibiotic treatment.
Geoff Marsh So am I right in saying that recently, we've started to understand these biofilms as almost the new battleground for the fight against antibiotic resistance?
Tanmay Bharat
Yes, indeed, because it has been estimated that over three quarters of human bacterial infections proceed with bacterial biofilm formation, which means that whenever we get a bacterial infection in our bodies, it's mainly by formation of these infectious aggregates known as biofilms. So indeed, understanding antibiotic resistance is important in this framework, because this is the framework in which the bacteria need to be treated within a medical setting.
[Narration] Geoff Marsh
Tanmay and his team are interested in the fundamental mechanisms of biofilm formation. But his relationship with these bacterial colonies hasn't always been so abstract...
[In office] Tanmay Bharat
I'm very familiar with this physical removal process, having been through it myself, quite recently. So when I moved here to Oxford, I got infected with a bacterial biofilm in the bone in my leg and the Oxford bone infection team had to basically drill and remove pretty much a third of my leg’s bone tissue to remove the infected tissue because the antibiotic treatment wouldn't work.
Geoff Marsh
It's probably not often that scientists come up so close and personal with their area of study. Did your personal circumstances have any influence on your research, do you think?
Tanmay Bharat
Yes, it did, actually, because I had the episode of this bacterial infection when I was 14, so many, many years ago so since then, it's been on my mind to work in this area. And it was just by chance that I moved here and had this surgery. I think my boss feared that I would never return back to the lab, but I think in the end everything worked out.
Geoff Marsh
I feel like I have an intuitive sense of what we mean by a biofilm, but just in case I've got it wrong - what does a biofilm actually look like, physically?
Tanmay Bharat
Pseudomonas biofilms typically look like greenish slime. So you might mistake them for some algae or some kind of moss but they are antibiotic-resistant, they are pathogenic bacteria, and they give off a very characteristic smell.
Geoff Marsh
Can you bring that to life for us?
Tanmay Bharat
It's a very sharp smell of rotten food, I would say. So it can infect lung tissue, bone tissue etc. And whenever a bacterium gets into one of these places, it can then grow, multiply and form this kind of slimy film, which is the biofilm. And once it's in this biofilm status, it’s extremely difficult to treat because it's resistant to high doses of antibiotics and in some cases, you have to physically go in and remove the infected tissue, that's the only way you can actually treat the infection.
Geoff Marsh
So that's really interesting because when I hear about antibiotic resistance, I've always really imagined what we're talking about is a sort of chemical way of evading antibiotics. But this sounds so physical.
Tanmay Bharat
Exactly. So our work is focused on the extracellular matrix that is secreted by the bacterial cells, which consists of molecules that either bind to antibiotics, preventing them from reaching the bacterial cell, or molecules that provide a physical barrier, also preventing the antibiotic from reaching the bacterial cell, thereby neutralising the threat and allowing the bacteria to survive under heavy antibiotic treatments.
Geoff Marsh
And there's another twist to this tale with Pseudomonas aeruginosa isn't there, because it's not just proteins and different chemicals being secreted, it's actually viruses, bacteriophages...
Tanmay Bharat
Sure. So many people think of bacteriophages as a way to treat bacterial infections because they usually kill bacteria. But in this case, a phage molecule is secreted by the bacteria themselves, and it has a symbiotic relationship with Pseudomonas aeruginosa. In fact, it forms these liquid crystalline droplets around Pseudomonas aeruginosa cells and makes this physical barrier, preventing the antibiotics from reaching the bacterial cell surface, allowing the bacteria to evade the antibiotic treatment as a result.
Geoff Marsh
Now, you're clearly being rewarded for your unravelling of this mechanism. But there's another reason I think that judges were impressed by your work and that's the techniques behind how you look at these things. Can you talk us through that because it sounds incredibly challenging to study tiny viruses inside bacterial cells. It all sounds very tiny. Talk us through your process.
Tanmay Bharat
Sure. So we employ cryo-electron microscopy, or electron-cryomicroscopy, which is a technique in which an electron microscope is used, operating at liquid nitrogen temperatures, which allows us to image these bacteria and these molecules at extremely high resolution. We combine these microscopy techniques with some high-end image processing methods, which allows us to resolve high resolution structures and understand the molecular mechanisms at play at a resolution where we can observe single molecules. So that is the technique we are using - cryo-electron microscopy and a particular flavour of cryo-electron microscopy called electron tomography.
[Narration] Geoff Marsh I wanted to hear more about how these viruses were helping to protect the Pseudomonas aeruginosa from antibiotics. But first, Tanmay was kind enough to show me the electron microscope he and his team used to image the microscopic samples. So we put on our masks, which you might be able to hear via our muffled voices, and made the short walk around the back of his office to the microscope. [Walking into microscope laboratory]
Geoff Marsh
So we are here at the centre for cellular and molecular imaging...
Tanmay Bharat
Yes, also known as ‘COSMIC’ - the Central Oxford Structural Microscopy and Imaging Centre. So that is the sound of the computers that you can hear, churning away, collecting the data from the microscope. And that’s the microscope itself.
Geoff Marsh
Oh, wow.
Tanmay Bharat
As you can see, it’s quite big
Geoff Marsh
It looks like a 12 foot tall, futuristic fridge.
Tanmay Bharat
Yes, it does. And as I said, the size of the specimen that you're looking at is inversely correlated with the size of the machine.
Geoff Marsh
So where do you put the samples?
Tanmay Bharat
Yeah, so they go into this cassette which carries the small microscopy grid, and that can then be moved into this column, which basically shoots electrons on your sample.
Geoff Marsh
Tanmay has just opened the front door, and we're looking into the belly of this microscope. It looks incredibly complicated, like the inside of a supercomputer or something.
Tanmay Bharat
They are very complicated machines. They cost a lot of money and they also cost a lot to maintain. As you can imagine, there are all sorts of electronics and apertures and lenses that can have a defect and need special engineers to come and repair.
Geoff Marsh
And then this is all operating at really, really low temperatures?
Tanmay Bharat
Precisely. So the sample is always at liquid nitrogen temperatures. So you press this green button. Now the sample is going up into the autoloader.
Geoff Marsh
Do we have to close this up at some point?
Tanmay Bharat
Yes we can shut that. And then over to the computer.
Geoff Marsh So this is Pseudomonas aeruginosa?
Tanmay Bharat It is indeed. This is Pseudomonas aeruginosa bacteria. And these are surrounded by these liquid crystalline droplets.
Geoff Marsh
But this is less spectacular looking than the images in your paper.
Tanmay Bharat
Yes, I think those images were on a very heavily optimised sample, obviously. And to get that image quality, a significant amount of image processing is needed, which we obviously are not doing right now.
[Narration] Geoff Marsh
Well, the images that have been processed are pretty spectacular, and you should go and find Tanmay’s paper to see them. But what do these amazing pictures tell us about antibiotic resistance in these bacteria?
[In office] Tanmay Bharat
So I should say that because everything is operating in liquid nitrogen temperatures, it’s frozen, so we only get a high resolution snapshot of what is going on. And in our images we can observe the bacterial cell surface, and how the molecules are arranged at the surface. And in the case of these liquid crystalline droplets made of these phage molecules, we observe that the phage molecules basically surround the bacterial cells, come in close proximity to the outer membrane of the bacteria, and form as heath around the bacterial cell, kind of like a forcefield in Star Wars, I suppose. So we actually don't know what these liquid crystals actually look like in an infection setting right now but we are developing models to image that. So perhaps in a few months, we can report how that looks like
Geoff Marsh
What is a liquid crystal? When I hear crystal, I think of a solid material?
Tanmay Bharat
Yes, so a liquid crystal is an arrangement of molecules where there is orientational ordering, but positional randomness, which means that although the constituent molecules, in this case the bacteriophage molecules, are roughly oriented in a lens-shaped droplet, they have positional randomness, meaning they can move quite a lot within this arrangement.
Geoff Marsh
I’m visualising what you're talking about here and it sounds really sci-fi. Is it these liquid crystals in this sheath that physically protects the bacterium from marauding antibiotics?
Tanmay Bharat
Yes, I think this is what we have shown, that these liquid crystals protect the bacteria from antibiotics. Because we have measured that bacteria that were not surrounded by these liquid crystalline droplets had a much higher probability of being killed on an antibiotic challenge. What the exact mechanism is still something we are actively researching - whether it's purely a biophysical phenomenon or whether there is a chemical component, and whether the liquid crystals actively bind to the antibiotics in a chemical way, because the antibiotics that we tested in our experiments were aminoglycoside antibiotics which are typically positively charged. One of the theories in the field previously was that these positively charged antibiotics bind to negatively charged DNA, which is within these bacteriophages. So we have to do more research to figure out exactly what the mechanism is, but the presence of the liquid crystals absolutely protects the bacteria from antibiotic attack.
Geoff Marsh
Do you have a sense of what's orchestrating this process? Is it the bacteria making the bacteriophage do it, or is the bacteriophage going to form this sheath around anything the right shape?
Tanmay Bharat
Yeah, I think it is the bacteria that are pulling the strings because these prophages are encoded in the bacterial genome, and they are overproduced during biofilm formation. So the phages wouldn't be there if the bacteria didn't harbour them within the genome and they wouldn't be there if the bacteria hadn't produced them and secreted them into the extracellular matrix. So I think, yeah, it would be fair to say that the bacteria are driving this antibiotic tolerance through production of the phage.
Geoff Marsh
And to be clear, what we mean by that is that they actively up-regulate the genes which spew out these phages. Is that right?
Tanmay Bharat
Yes, that's what we believe. So bacteria go into a biofilm lifestyle, and when they go into this biofilm lifestyle, it's accompanied by secretion of these molecules into the extracellular matrix, such as this bacteriophage, which protects the bacteria from antibiotic treatment
Geoff Marsh
Now that we have this new appreciation of the fact that it’s these tiny bacteriophages that are actually causing this really effective defence against antibiotics, could those bacteriophages themselves become a therapeutic target?
Tanmay Bharat
Yes, precisely. So if we target those bacteriophages, then their protective effect will be removed. And if that treatment is provided alongside an antibiotic treatment, then together, they could be very powerful in treating a bacterial biofilm disease that has occurred. There are many ways bacteria can avoid antibiotics and it depends on the type of antibiotic - there are antibiotic efflux pumps which pump antibiotics from bacteria to the outside so that they don't accumulate in the cytoplasm of the bacteria, there are these extracellular matrices involved in biofilms... A large part of research on biofilms concerns biofilm dispersal mechanisms, which are ways in which the cells within the biofilm signal to each other that now is the time to disperse and break up the biofilm, and there are many other types of mechanism, which just goes to show that antibiotic resistance is a multifactorial, complex issue that really needs a community effort with many people working on it from many different perspectives. So even if the bacteria are able to evade one particular antibiotic in a particular setting, you could treat the bacteria with a cocktail of antibiotics so that it just overloads the system and they get treated. So we use a kind of reductionist approach where we take one particular aspect and reduce it to one liquid crystal and one bacterium to try and explain it, but in reality, it's a multifactorial problem. I think we just have to keep looking and keep trying to outpace the bacteria. Because the bacteria are actively evolving and using new methods, which they can pick up from other bacteria or from other organisms so we have to be one step ahead.
Geoff Marsh
Now, I'm sure that this award means a lot to you and your team. Could you just paint a picture for us of how you got the news and how it was taken?
Tanmay Bharat
Yes, I mean, it was very surprising. Actually, I was unsure whether I should even apply for this award. But our head of the department, the person whose office we're sitting in right now, he encouraged me to apply. And then I got a phone call. I did think it was a prank, because it was completely out of the blue. But they did say that they would send me an email confirmation as well. So it seemed legitimate, and it was followed up by an email.
Geoff Marsh
That would have been a very specialised, incredibly cruel prank.
Tanmay Bharat
Yes, indeed. And I should also say, I share this award with the achievements of my laboratory and my colleagues in the group who are incredibly talented, and who work incredibly hard. This is reflecting on their work, really,
Geoff Marsh
What will this award practically mean for you and the group?
Tanmay Bharat
It gives us an opportunity to highlight our work in this podcast, for example, or the press release that the Eppendorf Award is accompanied by. It just gives more spotlight to our work and gives some validation to the importance of the research that we are involved in. Although we don't do the work for any kind of accolades, when they do come along it is extremely nice because it validates that your original goal was a worthy one, and a noble one that people find important and as important as you do, which is nice.
