Kryptonite or Bacteriophages?

**Intro Music** Tony: Hello and welcome back to another episode of Microbe Matters, presented by IDPITTStop, where we discuss, dissect, and demystify topics in Infectious Diseases with our experts here at UPMC and the University of Pittsburgh. I’m your host, Tony Morrison, media specialist here at Pitt ID and I’m just as curious as you may be about navigating through a world full of microscopic organisms. Please join us as we examine both the dangerous and beneficial microbial microcosms that surround us, promote public health, and showcase research and treatment of modern infectious diseases.  **Fade out Intro Music** **Fade in Holter Monitor and Respirator Sound Effects** Tony: Breathing- something that we all do to survive can often be taken for granted. For 25-year-old Mallory Smith, it’s something that she’s struggled to do her whole life. Diagnosed with Cystic Fibrosis at a young age, Mallory was all too familiar with frequent hospital stays and breathing through a respirator. The genetic disease, which affects about 40,000 Americans, is characterized by a mutation that makes the lungs, pancreas, and other organs fill up with a thick, sticky mucus. Though Mallory was able to successfully receive a lung transplant, her condition continued to worsen.  You see, Mallory was battling against Burkholderia- a gram-negative bacterium that had colonized her body at age 12. The organism had lingered in her upper airway and sinuses and crept back into her new lungs. This superbug was unresponsive to all antibiotic treatments. To overcome an infection of this caliber, a different approach was necessary. Phage therapy, a treatment that uses viruses that specifically target bacteria, was Mallory’s last hope.  Researchers were in a race against time to produce and deliver a phage cocktail to save Mallory’s life. Unfortunately, it was too late, and Mallory had passed away from the infection. ** Holter Monitor Flatlines and Respirator stops ** ** Sound effects Fade Out** **Uplifting Music Fades In** Tony But although phage therapy was unable to save Mallory’s life, it did, however, save Isabelle Carnell-Holdaway- a 17-year-old girl also with cystic fibrosis and a super bug infection in her lungs. It also helped to recover another teenage girl in London, a man in San Diego, and many others across the globe. **Uplifting Music Fades Out** Tony: Mallory’s story is one that has sparked interest in phage research and treatments. But what exactly are bacteriophages and where do they come from? Joining me today to talk about the growing research in phages is Daria Van Tyne, Assistant Professor of Medicine here at the University of Pittsburgh, who’s research investigates how bacteria evolve to become superbugs, using comparative genomics and functional analysis.  Welcome Daria, thank you for being here today. Daria: Thanks for having me! Tony: Ghady: Thanks for having me. Tony: Before we get into the topic, let’s clear up some questions our listeners may have about phages. Daria, what are bacteriophages? Where might one find these organisms? Daria: So, bacteriophages, simply put, are viruses that infect bacteria. So, viruses are all around us. We care a lot about viruses that infect US. But bacteriophages are viruses that are specific for bacteria. So, viruses want to use host cells as factories to replicate themselves. And bacteriophages are no different. They are looking for bacteria to infect and turn into factories to make more of themselves. Bacteriophages are highly abundant, and this means that they are everywhere. They’re in the air that we breathe, they’re in the water that we drink, the food that we ear. We are exposed to bacteriophages – ALL the TIME. They’re generally believed to be inert, and don’t interact with the cells in our own bodies. They’re basically looking for bacteria that they can infect to replicate more of themselves.  Tony: So, you mean to tell me that these organisms are everywhere, right under our noses? Fascinating! But if they’re so abundant, why are we just hearing about them now and not 10 years ago or even 100 years ago?! Daria: That’s a good question. In fact, bacteriophages have been known for the last hundred years or so. They were originally discovered in the early 20th century and were developed into antimicrobial therapies around the same time that Alexander Fleming was discovering penicillin. And there are actually active bacteriophage research programs in Eastern Europe. And in these countries people have been studying bacteriophages and using them for phage therapy throughout the last thundered years. In the western world, there was a very rapid shift of focus away from bacteriophages and their antibacterial properties and toward these small molecule antibiotics, like penicillin and others. But now we’re in a place where we are returning to the idea of bacteriophage therapy as a potential treatment for antibiotic resistant bacterial infections.  Tony: Ghady, can you weigh in here about phage therapy? How are phages different from antibiotics and why should we care about them? Ghady: Yeah, I think Daria covered very nicely how phages are different than antibiotics. I mean, they have technically been around for similar periods of time, but just because it was much easier to use and mass produce and study antibiotics, everything shifted towards that. In recent years, though, there’s been a problem with antimicrobial resistance, which has become a crisis not just in the us, but also worldwide. Hundreds of thousands of people are infected with multi-drug resistant organisms across the globe. These bacteria result in hospitalizations and in lots of deaths, and especially for people who have co-morbidities: people who are older, have underlying conditions, have immunocompromising conditions and things like that. And you’ve really gotten to a point where a lot of these infections are “untreatable.” And it’s difficult to keep up with the evolution of bacteria. Every time a new “last resort antibiotic” comes along, we see the bacteria become resistant to it very quickly. And so, you’re constantly laying this game of catch up. And so, you’ve ended up in a scenario where in some situations, existing and even future antibiotics are simply not going to be able to treat certain kinds of really resistant infections. And that’s why I think there’s been a resurgence in the interest in the use of phages. I would say that the case that you just talked about, as well as this other case of someone named Tom Patterson, who was infected with an extensivity drug-resistant strain of a bacterium called Acinetobacter, while on vacation in Egypt. And all of this info is online for people who want to fact-check this, but this infection was in his pancreas, and it really was extensively resistant and failing everything. And then the patient did ultimately end up getting phages for this infection and ultimately survived and is now alive and well. And so, I think these two cases have really brought phages back from the background to the forefront of our fight against antimicrobial resistance.  Daria: I often say that phage therapy right now is like a Back to the Future kind of scenario. I want to expand just a little bit on the biology of how bacteriophages differ from antibiotics. So, antibiotics are small molecules, they’re compounds generally used to inhibit the growth of bacteria or kill bacteria. Antibiotics are usually quite broad spectrum, so they kill lots of different types of bacteria. And the most antibiotic, like the highest antibiotic concentration that you’ll find in the body of a person that takes antibiotics, is right after they take their does of antibiotic. And then the amount of antibiotic will go down over time. Some antibiotics go down very quickly over time, and some go down more slowly. But there’s a peak and then there’s a decay of the amount of antibiotic in the person.  Okay, so phages on the other hand, are viruses looking for hosts that they can use to replicate more of themselves. So, phages are very specific. They are very picky in the types of bacteria that they can use as a host. So, antibiotics are more broad spectrum and phages are much more specific and narrow spectrum. An if a phage finds its bacterial host in your body, it will infect and make more of itself. So, phages are self-replicating. It also means that when the phages can no longer find bacterial hosts to replicate in, they won’t replicate anymore and their numbers will go down pretty quickly. So, we call phages se-replicating as well as self-dosing because they will vary their abundance based on how many of their hosts are around. And so these different properties between phages and antibiotics are important to understand when we think about how phages could be used for therapy. Tony: So, to clarify, bacteriophages are among the most common and diverse entities in the biosphere. They are ubiquitous and found everywhere bacteria exists. And though we’ve known about them for quite some time, we’ve redirected our focus onto antibiotics- which is partly the reason why we’re coming back full circle to bacteriophages as a therapy.  Ghady, can you weigh in here about phage therapy? How are phages different from antibiotics and why should we care about them? Ghady: Yeah, I think that there’s a few answers to this question. One is what we regard as an ideal candidate today, based on the cases that have received phage today. But two, what we would like phages to be used for and the potential candidates in the future. There really is no FDA-approved phage product. You can’t just go to a pharmacy and order a phage in this country and in most countries. So every single phage that a patient receives is unique, because they’re targeting the specific organism that is infecting that patient. Every single case is approved by the FDA individually through the process called Emergency Use Investigational New Drug Application, and some other processes as well. And because of that, we haven’t really been able to scale-up the use of phages to all potential patients who might qualify. As in various kinds of patients with resistant infections. Thus far, the candidates for phages have been patients who are infected or colonized with really resistant bacteria that have typically failed to respond to other rounds of antibiotics. What I’d like to see happen in the future is for the phages to be used a little earlier, as in before patients run out of all the other options, and to hopefully turn them from this treatment of last resort when all other things have failed to something that may be a bit more routine or given more up front, even paired with antibiotics and things like that. We’re not there yet, so currently in 2023 an ideal candidate would be someone who has failed to respond to other drugs or who’s isolate is now resistant to everything else. And theses have been the patients that we’ve treated and these have been the referrals that we’ve received from other hospitals. So actually, about the referrals from other hospitals, Daria’s lab has now been receiving lots of requests from physicians from all over the US to help find phages to treat their patients. And she’s also done the same for, I want to say 7-ish, of our patients here. So Daria, can you walk us through he process of how and where you identify phages for these patients. Daria: Sure! So to do phage therapy, you need phages. These phages have to come from somewhere. I said earlier that phages are abundant; they’re all around us. But phages are also very specific. And so in order to do phage therapy and treat patients with phages, we have to find phages that are specific for the bacteria that are making those patients sick. So the way that we do that is we build libraries of phages that are available for testing against isolates from infected patients. And so where do we find the phages for these libraries? When I started my lab here at the University of Pittsburgh just about five years ago, we took bacterial isolates that came from patients with bacterial infections and then sampled waste water from the Pittsburgh area and it turns out that if you take this waste water and you remove all of the solids and you filter sterilize it to get rid of any bacteria or fungi that are in it, you’re left with a filtrate that has a bunch of bacteriophages in it. So if you take this filtered waste water and incubate it with bacteria, then if there are phages in the water sample that can infect and replicate on the bacteria, we can see that in the laboratory. So we can test water samples and clinical bacterial isolates and find phages that are active on those bacteria so we can passage and isolate those phages in the laboratory and then study them further. So in my laboratory, we routinely sequence the genomes of the phages that we isolate to try to learn more about their biology. And then once we’ve characterized a new bacteriophage, we add it to a library of phages that can all target a particular species of bacteria. Some of the bacterial species that we have libraries for are pseudomonas aeruginosa, Burkholderia, vancomycin-resistant Enetrococcis, and some other bacteria as well. So we have found that waste water is a very rich source of bacteriophages for a lot of the bacteria that we wish to target with phage therapy.  Tony: Huh, so I guess bacteriophages are so abundant that you can actually just filter them out of waste water? Ghady: Yeah, and are there any other organisms that you haven’t been able to find phages for or that are just harder to find phages for? Daria: Yeah, for sure. We’ve received a lot of requests for phage screen for patients with a lot of different species of bacteria. And depending on the bacterial species, some of them are easier to find active phages for and some of them are harder. A prime example of this is Burkholderia, the type of bacteria that Mallory Smith was infected with. So, for reasons we don’t fully understand, Burkholderia bacteria are just very hard to find phage for. And this could be for a number of different reasons. It could be that the water samples that we are looking for phage in are not actually the right place to be looking for phages against Burkholderia. It’s possible that Burkholderia phages are abundant in some other environment that we aren’t sampling with our phage-hunting approach. Another possible reason for this is the Burkholderia bacteria themselves, because of their unique biology, are less amenable to becoming infected and killed by bacteriophages. If you look in the genomes of Burkholderia bacteria, you see a lot of genes that belong to these things called prophages. And prophages are actually bacteriophages that have integrated themselves into the genomes of bacteria. And when a phage gets into the bacterial genome, it’s basically hitching a ride on the bacterial genome, and it gets replicated along with the bacteria. Often, these prophages can refer protection to their host from additional infection with other phages. So one of the questions that my lab is interested in is whether the prophages that are inside these Burkholderia isolate genomes might protect them from infection with other phages, and that this might be one of the reasons it is difficult to treat Burkholderia infections with phage therapies at the moment.  Tony: So I guess the hardest part isn’t necessarily finding phages in the environment, but finding the right specific phage for the usage.  Cosnidering where these phages come from, I suppose I have to ask: Ghady, are phages safe to use on patients? Ghady: The short answer is yes. The long answer is yes, as long as we comply with some safety regulations that the FDA wants us to comply with. And it’s specifically with something called the endotoxin content. As Daria just described, you essentially isolate phages to give to patients by growing them on the bacteria that you infect. When the phages kill the bacteria and explode them, you end up with all these “toxins” or evil humors from inside the bacteria that you want to get rid of. Otherwise you are essentially injecting the patient with not just the phage, but with all these toxins from the bacteria. Endotoxin is one of the reasons why you get into things like fever, a low blood pressure, shock, and things like that. And so, the FDA has specific guidelines of how much endotoxin one can safely inject into a human being. It’s generally less than 5 endotoxin units per kilogram per hour. So we just make sure that we comply with that when we give phages. But aside from that, yes phages have been safe. That’s been our experience, that’s been our colleagues’ experiences across the globe, and that’s also been what’s been reported in the published literature. There’s been a case or two here and there, where someone appears to have an immediate reaction to phage. The thing is that I don’t think this is unexpected; people can also have immediate reactions to antibiotics, for example. Long story short, phages are generally safe to give, yes.  Daria: So Ghady, you invoked the idea of picking a dose of phage to give. So, I was just talking to my lab the other day about antibiotics and antibiotic dosing and how there’s a therapeutic window of concentration of antibiotic that you can five that will kill the bacteria and treat the infection. And then there’s also a window that’s much higher (hopeful higher) that will hurt the patient. So there’s this toxicity window and a therapeutic window. Ideally, like a good antibiotic will have a big distance between those two windows. Now if we turn to phage, and we think about- you just said that phages are largely say, so there’s not a large toxicity window, but what about the therapeutic window? Ghady: Yeah, unfortunately we don’t really know a lot. A lot of what we do when it comes to phage dosing is just based on, again, case reports and case series and what other people have done. The Antibiotic Resistance Leadership Group, or the ARLG- and Daria was actually a co-author on this guidance document- does outline preliminary recommendation for phage dosing. And it’s something along the lines of 10th to the power 8-ish plaque forming units per dose is generally thought to be required for effectiveness, as in these doses appear to kill bacteria. But again, there have never been these really well-done phases 1 and 2 and so on trials to really inform this. And when we first started phage therapy here about five or six years ago, we really relied on the experience of our colleagues from other venters in the US when it comes to guidance on how specifically to dose phages and how much phage to give. I think you just need more and more trials and you and I are trying to work on these trials to just try to find these really basic questions of how much phage to give, where does the phage go? The other unknown too is how to give it, as in do you give it as a pill, do you give it through the IV, if someone has a lung infection do you just give it inhaled, or do you give it through the IV and inhaled? If you give it IV how much is it going to hone into the lungs and is it going to replicate in the lings? All of these are unknown questions. It does make me happy to see that it’s not just us, but other colleagues across th4e country and across the world are trying to answer these questions. But there are a lot of unknowns and a lot of the time we are kind of going by our best guess, or common sense, by our own experience, and by what we can glean from our experience from our colleagues and from case reports and case series. Daria: So you mentioned some of our colleagues and I just want to take a minute to highlight some of our colleagues in our local community here at University of Pittsburgh and UPMC that are also involved in phage therapy. A couple of years ago, we organized under an umbrella called The Pittsburgh Phage Project, and this was a group of over 20 researchers, clinicians, and other investigators at Pitt and UPMC that all were interested in bacteriophage therapy but were each approaching the problem from a different angle. But I have to say that early on in my lab, a lot of dir3ection in building our own program came from our colleague, Graham Hatful, who is a professor in the department of Biological Sciences here at the University of Pittsburgh. His lab has really been a pioneering group in developing phage therapy, in particular for mycobacterial infections. One thing in particular that Graham’s lab has done is quite a bit of phage engineering. So they have done some genetic manipulation of some bacteriophages, which initially were not very good candidates for use in phage therapy. What grahams lab did was perform genetic engineering to remove specific genes in the bacteriophage genomes that would make them more highly active and more deadly toward the bacteria they wanted to target. I want to highlight this because the idea of engineering phages really expands on their potential for use in treating resistant infections.  Ghady: Well, I don’t want to put you on the spot, but when do you think your lab might be ready to engineer some phages? Daria: That’s a great question. We have not started actively working on it yet, but I have to say there’s actually another way that you could try to do this engineering through experimental evolution. And what we mean by experimental evolution is that we might take a particular phage and a particular bacterial host, and we will put them together in a test tube and let them grow together. Because these two partners will be put together and will both be trying to survive, we have this evolution that happens. And the bacteria can mutate to try and become resistant to the phage, and the phage can also mutate to try to infect the bacteria more quickly. We can take this to one extra level and sprinkle in some chemicals that cause more rapid mutagenesis and then do an evolution experiment to try and select out phage that have a particular property that we’re interested in. Long story short: we’re not deeply involved in phage engineering yet in the lab, but I think there are a couple of different ways to go about it and hope to start doing it soon. Tony: Well it sounds to me that phages are a very valuable approach to alternative treatments to antibiotic-resistant organisms. I do have to ask both of you, and this is probably something we shouldn’t need to worry about for quite some time, but if phages become a mainstream therapy is it possible for these organisms that are so good at opposing treatment to develop anti-phage resistance in the future? Daria: Yeah that’s a great question. I agree with you that it will probably be a while before phages become mainstream therapy. But to the question of whether bacteria can become resistant to phage, that happens very quickly. All it takes is one day. Even though phage resistance happens very quickly in the laboratory, of the patients that we’ve treated with phage therapy here at Pitt so far, we actually haven’t seen a lot of evidence of phage resistance developing during the course of treatment. One possible approach for dealing with phage resistance is to just have a very very large library of phages and assume that if resistance evolves, you might be able to find another phage that targets the resistant bacteria. Another thing that happens when bacteria become resistant to phage is that they often give something up in the process, and we call this a trade-off. So bacteria might become resistant to phage by changing something on their cell surface that the phage needs to use as a receptor for infection. But when they do that, there’s often some sort of trade-off. So we often see among phage-resistant mutants that we generate in the lab is that these resistant mutants are now newly susceptible to antibiotics that the original strain may have been resistant to. They also sometimes look like they are less virulent, so they’re less dangerous to a person that might be infected with them. And so, we’re very interested in trying to understand the push and pull between antibiotic resistance and to try to figure out if there’s a way that we could leverage all of this to our advantage. For example, could we combine phage and antibiotics together and achieve a better outcome for the patient?  Ghady: And I think what you just said highlights two things:  That life finds a way – as we have learned from Jurassic Park  **Dinosaur Roar Sound Effect** Ghady: And two: that there’s still so many unknowns. And not just when it comes to dosing and safety and efficacy, but even down to phage biology. To the point that you just said about the trade-off between phage-resistance and antibiotic—resistance, we just had a paper published of a patient who received Burkholderia phages for a severe infection and unfortunately died, but we also had access to the patient’s Burkholderia isolates over the span of approximately 20 years or so and it was really interesting to see this trade-off. It also leads us to question the design of phage trials. One strategy that’s been proposed when it comes to dosing phages is not just to give the phage alone or give a phage with antibiotics, but also might you: result in more effectiveness   or Maybe prevent some of the emergence of bacterial resistance to phage if you use phage cocktails, as in give the patient more than one phage that’s active against their organism.  And these things have been tried in case reports and case series, but we haven’t scaled that up yet to see. To answer the question of if you give more than one phage at once, are you more likely to prevent the emergence of anti-phage resistance among bacteria- people have looked into this with antibiotics and it hasn’t really panned out, but I genuinely don’t know what the answer would be when you do it with phages. And I will also say that another kind of unexpected reason that patients fail phage therapy is that they become immune to them. And that was unexpected because it just wasn’t on our radar and it’s not something that I ever thought would happen with antibiotics, for example. I mean, these are viruses ultimately, and you can definitely develop an antibody response to a virus. We really don’t know, but these are kinds of the translational questions that we’re trying to answer. Other groups have observed the same thing as well with the development of an immune response against phages.  Daria: So Ghady, you and I have been working together as a team researching phages and also doing phage therapy treating patients with phage. It’s been very rewarding to see how for some of the patients that have received phage therapy, there’s been a really clear improvement in the patient’s clinical course and in the quality of life. One question I want to ask you though, is what you think is the biggest barrier to progress in the field of phage therapy at the moment? Ghady: Well, all of the above. But so a few things- one it’s not mainstream yet. I think it’s still relatively unknown. And people who do know about it, and ‘m talking here from the clinical care perspective, you often don’t hear about them until it might be too late in many cases. There’s the regulatory hurdles too, meaning that since we cannot just prescribe phage from the inpatient pharmacy, we have to go through an entire process with paperwork and all this stuff. Which at this stage, we’ve gotten streamlined, but it’s still a lot of work to actually do. To get the phages from your lab into the patient is not a trivial task and requires a lot of regulatory work. And then there’s the issue of all of the unknowns. And these will simply require large clinical trials. And you’re going to have to design trials for various things. Also the other barrier too is how to really define what “phage susceptibility and resistance” is. We still have a long way to go. Maybe in the next decade all of these barriers will have been overcome and it really does become something mainstream.  Tony: With all this talk of antimicrobial resistance lately with things such as MRSA, P. aeruginosa, and quite infamously C. auris, it’s a huge relief to know that there are alternative counterstrikes in development for the never-ending war on infectious diseases.  Daria, thank you for your insight on phage research. **Theme music fades in** Daria: Thanks for having me! Tony: And Ghady, thank you as well for your clinical perspective and expertise. Ghady: Thank you as well for having me. There’s more talk of antimicrobial resistance to come on this season of Microbe Matters.  Thanks for listening and be sure to check us out on social media at IDPittStop. **Theme Music Fades Out**