Hi. I’m Paul Turner from the Department of Ecology and Evolutionary Biology at Yale University, and the Microbiology program at Yale School of Medicine. Today, I’d like to talk about phage therapy. It’s an old idea for how to treat bacterial infections in humans and other organisms, and it’s an old idea that’s gaining resurgence due to the rise of antibiotic resistance in bacterial populations. Now that antibiotics are failing widely, we’re turning to this old idea as a possibly good new one to combat the spread of bacterial pathogens and the rise of antibiotic resistance. The key question is whether evolution thinking can improve virus applications such as phage therapy. So, what I’ll present… our data that relate to this idea of, can you use phage therapy effectively to treat bacterial infections? But, as an evolutionary biologist, I’ll talk about this in the context of understanding evolutionary biology, and how we can more rationally choose candidates to use, which it… within phage therapy so that we choose the right virus to employ in these types of methods with an eye to evolution that is destined to happen in the future, and yet have the method prevail through time. As a reminder, the evolution of antibiotic resistance is a profound challenge to humans and biomedicine, domesticated agriculture, both plants and animals… this is a rising problem where we see that antibiotics… in the past, they were used very effectively to treat and kill bacterial infections, but, lately, this is not happening at all well, and that’s because, largely, of an uncontrolled mass experiment that we’ve done around this world. When antibiotics were discovered in the ’50s, we immediately started using them in a large degree to treat infections in humans and also, prophylactically, to introduce them into domesticated animal populations, to protect them from bacterial infections before they ever occurred. Well, what happened is this imposed selection on bacteria to be resistant to the antibiotics, and unfortunately, through time, what we’ve seen is that bacteria are able to not only become resistant to some antibiotic that is used presently on them, they’ll maintain the resistance to that antibiotic when we roll out some new antibiotic that they subsequently also become resistant to. So, through time, you can think of bacteria creating this problem because they are collecting antibiotic resistance genes at no fitness cost, and therefore we’re running out of options for antibiotics that we can use to treat them. A particularly important kind of a bacterium that is showing the spread of antibiotic resistance is Pseudomonas aeruginosa. We see in this system that multidrug resistant bacterial genotypes are on the rise. Now, this is a bacterium that all of us encounter in our homes and it’s even in soil and waterways, so it’s a very prevalent bacterium on the planet, and people who have normal immunity and strong health are able to simply fight off an infection from Pseudomonas aeruginosa if it enters the body. But there are very many people who cannot do this. Cystic fibrosis patients, those with severe burns, and anyone who’s immunocompromised is at risk of seeing this very prevalent Pseudomonas aeruginosa bacteria, and have it enter the body, and opportunistically cause an infection. There’s a main reason for this. This is because Pseudomonas aeruginosa has a lot of what are called efflux pumps. These are combinations or complexes of proteins that exist, that permeate the cell, spanning the inner and the outer portion, and their function is to bring things actively in and out of the cell, including antibiotics. So, if antibiotics get into a Pseudomonas aeruginosa cell, it’s actively pumped out through an efflux pump. And this works in a wide variety of antibiotics, but it’s important to remember that efflux pumps have a large number of functions for Pseudomonas aeruginosa. In terms of pathogenesis, this protein complex is also important for host colonization, the ability for bacteria to form a very tough layer called a biofilm that’s very difficult for antibiotics or anything else to get through, and they also function in allowing these bacteria to evade or avoid host immune responses. Now, these are chromosome encoded and they seem to be highly prevalent or, in other words, genetically conserved in Pseudomonas aeruginosa genotypes. And, a wide variety of antibiotics, as I said, get pumped out from efflux pumps, such as these antibiotic classes — macrolides, aminoglycosides, tetracyclines. And here’s a typical structure of an efflux pump, which is at least three proteins spanning the outer and the inner portion of the cell. Now, let’s get back to phage therapy and how the use of phage therapy might be able to help us overcome the problem of opportunistic pathogens such as Pseudomonas aeruginosa. So, the idea of phage therapy is that you can use a phage, or a virus that is specific to a bacterium, to kill the disease bacterium instead of some other type of a therapy. So, this picture is showing phages or these virus particles in relation to a typical bacterial cell in terms of their relative size. Phage therapy is an old idea. So, even before antibiotics were ever discovered by Alexander Fleming and used widely by the human population, long ago, people discovered that phages… in the early 1900’s, these are some of the earliest viruses ever discovered and described… that these could be placed in a human or an animal to in… to combat bacterial disease. At that time, the Russians were the main ones who were developing the technology of using phages to combat disease. They were so enamored of the idea that they even outfitted their soldiers in World War II with little vials of phage that they could use, in the field, to treat wounds and to prevent those wounds from being colonized by bacteria, because, at that time, it also was unlikely for soldiers to be able to even reach some place that they could get health care and, in the case of an opportunistic bacterial infection that they encountered in the field, this might invade the body and even kill them before they could reach any medical help. It is important to also know, even in the early discovery of phages, these experiments placed phages in animals such as chickens and humans, which are both types of organisms that can suffer from cholera bacteria infections, and, yes, we know that rehydration therapy is very key to get people through a cholera infection, but phages are able to actually get in the body and find these cholera bacteria, kill them, and destroy them, so that someone can recover from cholera much faster, even in the absence of rehydration therapy. So, now, I want to give you some of the details, or remind you of the details, of how a lytic phage, one that is lethal to a bacterium, undergoes its normal growth cycle. And I’ll further explain how understanding how evolution of resistance to phages by bacteria can help us, if we find the right phage, to overcome the problem of antibiotic resistance. So, a phage encounters a cell — a bacterial cell in this circumstance — and, if it can infect and get in… I’m showing in this diagram how the phage enters the cell, hijacks the metabolism of the cell, makes copies, the cell bursts open, and those phage particles can go on and infect new cells. So, in this diagram, you’ll note that, at the exterior of the bacterial cell, I’m showing these recognition proteins in black, and this is a protein binding that allows the phage to infect the bacterium. The normal circumstance, and one that’s very understandable from the… in light of evolutionary biology, is that this selection pressure of phage infection is going to favor mutants of the bacteria that are able to escape. In other words, they’ll have phage resistance. So, now, I’m showing this escape mutant as surrounded by blue proteins, and the phage is unable to infect. So, here’s a key question: Suppose that if a phage interacts with bacteria and selects for the bacteria to become resistant to the phage, suppose that, following along with that, it made the bacteria somehow sensitive to antibiotics? That would be a great thing for phage therapy, because, if the phage kills the bacteria, that’s what you intended to happen, but if evolution kicks in and the bacteria escape the phage infection, if they are now suddenly vulnerable to antibiotics, especially antibiotics that are normally useless in killing the bacteria, you have a double-edged sword. You have not only the ability of the phage to infect and kill under normal circumstances, but, even when the inevitable evolution happens where resistance to the phage occurs, you still have sensitivity following along for these bacteria to become sensitive to antibiotics and to die in that fashion instead. So, what would this really indicate? It would indicate a trade-off between phage resistance and drug sensitivity in bacteria. Obviously, that would improve antimicrobial therapy options and, importantly, it would extend the lifetime of current antibiotics in our arsenal. So, these would be chemical antibiotics that are approved by, say, the US Food and Drug Administration as safe and effective in people, but what we have as a biomedical problem is that they are failing widely. If we could intervene somehow and find a phage that prompts this kind of a genetic trade-off, then this would lead us to use those currently useless antibiotics over again, and we wouldn’t have to continue searching for new antibiotics — we can simply trot out the ones that are failing currently. So, we found just such a phage. It’s abbreviated as OMKO1 and it actually came from a lake in Connecticut. We took the old school idea of go out into a natural environment and try and find a phage that kills your target bacterium. Well, we used this method with an eye to understanding evolutionary biology. We found phages that are able to infect and kill multidrug-resistant Pseudomonas aeruginosa, and when they fail to kill, because the bacteria have become resistant, we find that this sensitivity to antibiotics that results from the trade-off is a great way to further kind of a rational design behind phage therapy. So, the diagram shows, when the phage is countering the efflux pump, the key thing is that we found phages that bind to and enter cells because they are binding initially to the outer-most exposed proteins in the efflux pump. In this one example, that protein is abbreviated as OprM or outer membrane protein M, and OMKO1 is abbreviated as outer membrane knockout 1, meaning that when we challenged the virus to infect knockout strains of the bacteria, the only one that had failed to infect is when OprM is not made on the cell surface. That indicates that it must be involved in the binding property of the phage. So, now, this is wonderful, because we have this genetic trade-off. The phage-sensitive bacteria efflux antibiotics but they’re killed by the virus. The phage-resistant mutants have an impaired drug efflux ability — this makes them vulnerable to antibiotics that are normally useless against this bacterium. So, looking a little bit at, how do you measure this in the laboratory? How would you even know what’s going on? This is a typical agar plate, where the surface of the plate is coated with a high density of bacteria — that’s called a lawn — and what you can do is you can take some commercially available thing like a disc of paper that’s been soaked in antibiotic, and you place that on the agar surface, so if these bacteria are multidrug-resistant, including able to grow in the presence of this drug that’s leaching out from that paper, then they’ll grow up right next to the edge of the paper. They completely shrug off the effect of the antibiotic — it’s… has no effect in controlling their growth. What we observed is, after we exposed those bacteria to this phage, they gain a mutation that makes them resistant to the phage, and instead, now, they have a very different growth ability in this assay. Now, a much lower concentration of that drug is adversely affecting the growth of the bacteria, and technically that’s called a minimum inhibitory concentration. That means that there’s a lower level of this drug that is now impacting their growth in this way. So, now, I’ll walk you through a table that shows and summarizes the key data from our study. Let’s begin at the first line of this table. In the column beginning here is an antibiotic, for example, and the class of antibiotic that it’s drawn from. Next column is the minimum inhibitory concentration of these multidrug-resistant bacteria in the presence of that drug. And then, the column that follows is, how does that MIC change when the bacteria see the phage and they become resistant to the phage, and how has it affected their MIC? In these first two examples, you’ll see that there was a huge impact — the fold-increased drug sensitivity is a very large number. Through simply exposing the bacteria to the phage, they become resistant to the phage and it makes them much more sensitive to the antibiotic. Now, these first two examples are not by accident, because we know through prior scientific data that tetracycline and erythromycin are examples of two popular antibiotics drawn from two different antibiotic classes, for which efflux pumps provide the main mechanism of resistance for Pseudomonas aeruginosa to remove those antibiotics from the cell. So, we’re not quite certain, in the terms of other antibiotics drawn from other antibiotic classes, whether explicitly efflux pumps are always doing this, or only sometimes doing it. Well, when we looked at four more examples, drawn from different antibiotic classes, you see mostly the same result, where there was an increased fold drug sensitivity. Now, you may not be as impressed by these lower numbers, but I’ve shown you in asterisks that the clinical resistance has reversed to sensitivity. This means that a doctor, a physician, could actually administer these antibiotics successfully against these phage-resistant bacteria, and that’s because the bacteria are now sensitive to these drugs at a clinically relevant level. So, the last line on the table is more or less a control. Here, we have ampicillin, which is known to be not efflux pumped out of the cell for resistance. So, one would not expect these values to change when the bacteria see or do not see the phage. And, indeed, there is no change in their fold-increased drug sensitivity. So, these data, taken together, suggest that there are a wide variety of antibiotics drawn from different antibiotic classes for which this phage exposure makes the bacteria sensitive to antibiotics that are currently useless in controlling multidrug-resistant Pseudomonas aeruginosa. So, what we would like to do next is, at least… we know for now that the trade-off is… is observed in a wide variety of Pseudomonas aeruginosa genotypes, and that gives us a lot of… an indication, a strong indication, that this should be broadly useful in targeting a wide variety of genotypes. So, this worked not only in laboratory model strains PA01 and PA14, it worked in a wide variety of clinical samples, including individuals who had suffered otitis, or ear infections, diabetic foot ulcers, osteomyelitis, etc. We took clinical samples from these individuals, put them into our assay, and we got data that suggests that those clinically relevant bacteria are experiencing the same trade-off as in the model strains. And, even more interestingly, if you just simply take Pseudomonas aeruginosa from the environment, and you can find multidrug-resistant forms of it in the environment, and estuaries, and even in human homes, these also experience the exact same trade-off as the other strains. So, ideally, then, we can now expand this out even further, look at a very, very large repository of clinical isolates, and I would expect that these data would also be supportive. But that’s a goal for our near-future work. Already, we’ve obtained the ability from the US FDA to use phage cocktails in chronically infected human volunteers, where these could actually only be the single phage that we found, or in combination with other phages. And, by that, I mean these are human individuals who have suffered chronic infections with MDR P. aeruginosa, and they have essentially run out of options for treatment. There are no antibiotics that will help them. In some cases, it’s actually remarkable that these individuals are still alive, and they are very willing to undergo experimental treatment if this is going to help them. Well, we are identifying individuals at Yale New Haven Hospital and other locations, and working with physicians and surgeons, in order to use this experimental treatment where these volunteer patients are bravely looking to this option to see if it will improve their health. Already, we’ve had one successful event, where a man had an aortic arch replacement, which is a pretty routine thing that is a surgery that leads to the replacement of a very key portion of the heart, that kept this man alive, but, in any individual, and this is increasingly seen in medical science… is that we have these routine surgeries that bring into the human body artificial substrates, and these make great places for bacteria to form a biofilm, a very tough infection that’s hard to treat. And, in many cases, these are multidrug-resistant bacteria. Well, this man did not have… he did not have any other options and he volunteered for treatment, and we placed the phage in his chest near the site of the infection, and one administered treatment was enough to remove his bacterial biofilm, and he is now completely off antibiotics and in much better health. We talked about his one case as well as our general approaches to taking an evolutionary eye to phage therapy in several radio shows, which you might want to listen to for further information. But our big goal is to test… test the safety and efficacy of this whole approach in a mouse model. In some ways, what we were able to achieve was a bit backwards. We found some human patients that were very excited about this potential therapy, and it did work safely, obviously, in the one case that we’ve tried, but now we’d like to take a step back and see how broadly useful might this be for say lung pneumonia infections in immunocompromised patients, or in cystic fibrosis patients, and it’s very easy to create a mouse model dataset in a laboratory to test the safety and efficacy of whether this works. So, that’s underway right now. If that works, then we’re at the stage where we can actually help humans on a grand scale, ideally. So, this would be a clinical trial that would happen in humans. Perhaps we could use humans who are suffering hospital-acquired pneumonia, or cystic fibrosis-associated pulmonary infections, and see if we can improve their quality of life by administering the phage and the antibiotics simultaneously, and that might reduce, even just… for individuals who are on chronic antibiotic therapy, which has a lot of bad consequences and side-effects, even using the phage to help prevent the bacteria from infecting or removing them entirely, it should expose those individuals to less of these chemicals that can adversely affect their health, but are helping keep them alive. I’d like to acknowledge the people who helped on the study. Ben Chan was the first author on the paper that we produced in 2016, that looked at not only the initial discovery of this phage but also a subsequent paper where we described the case study of the individual who we helped with experimental therapy. And two other key individuals who worked with me on this were Deepak Narayan, the surgeon at Yale School of Medicine and Yale New Haven Hospital, who was able to find this patient who bravely volunteered for the treatment and is also helping us administer the treatment to other individuals who have run out of options. Also, my long-term collaborator, John Wertz, who’s at the Coli Genetic Stock Center at Yale, was another key architect in the study. And, without them and the help of my lab group, none of this work could have happened. We’re also thankful to those who funded our work externally, especially federal agencies and private foundations.