Virus Ecology and Evolution: from Virus Adaptation to Phage Therapy
Transcript of Part 3: Phage Therapy
00:00:14.24 Hi. 00:00:15.24 I'm Paul Turner from the Department of Ecology and Evolutionary Biology at Yale University, 00:00:20.00 and the Microbiology program at Yale School of Medicine. 00:00:22.14 Today, I'd like to talk about phage therapy. 00:00:25.18 It's an old idea for how to treat bacterial infections in humans and other organisms, 00:00:31.07 and it's an old idea that's gaining resurgence due to the rise of antibiotic resistance in 00:00:36.02 bacterial populations. 00:00:38.02 Now that antibiotics are failing widely, we're turning to this old idea as a possibly good 00:00:43.21 new one to combat the spread of bacterial pathogens and the rise of antibiotic resistance. 00:00:51.05 The key question is whether evolution thinking can improve virus applications such as phage 00:00:56.07 therapy. 00:00:57.07 So, what I'll present... our data that relate to this idea of, can you use phage therapy 00:01:03.10 effectively to treat bacterial infections? 00:01:06.02 But, as an evolutionary biologist, I'll talk about this in the context of understanding 00:01:11.02 evolutionary biology, and how we can more rationally choose candidates to use, which 00:01:17.11 it... within phage therapy so that we choose the right virus to employ in these types of 00:01:23.10 methods with an eye to evolution that is destined to happen in the future, and yet have the 00:01:28.18 method prevail through time. 00:01:32.05 As a reminder, the evolution of antibiotic resistance is a profound challenge to humans 00:01:38.04 and biomedicine, domesticated agriculture, both plants and animals... this is a rising 00:01:44.04 problem where we see that antibiotics... in the past, they were used very effectively 00:01:49.23 to treat and kill bacterial infections, but, lately, this is not happening at all well, 00:01:55.17 and that's because, largely, of an uncontrolled mass experiment that we've done around this 00:02:01.13 world. 00:02:02.13 When antibiotics were discovered in the '50s, we immediately started using them in a large 00:02:08.08 degree to treat infections in humans and also, prophylactically, to introduce them into domesticated 00:02:13.24 animal populations, to protect them from bacterial infections before they ever occurred. 00:02:18.23 Well, what happened is this imposed selection on bacteria to be resistant to the antibiotics, 00:02:25.13 and unfortunately, through time, what we've seen is that bacteria are able to not only 00:02:30.10 become resistant to some antibiotic that is used presently on them, they'll maintain the 00:02:35.07 resistance to that antibiotic when we roll out some new antibiotic that they subsequently 00:02:40.15 also become resistant to. 00:02:42.05 So, through time, you can think of bacteria creating this problem because they are collecting 00:02:47.07 antibiotic resistance genes at no fitness cost, and therefore we're running out of options 00:02:52.14 for antibiotics that we can use to treat them. 00:02:55.12 A particularly important kind of a bacterium that is showing the spread of antibiotic resistance 00:03:02.15 is Pseudomonas aeruginosa. 00:03:04.22 We see in this system that multidrug resistant bacterial genotypes are on the rise. 00:03:09.13 Now, this is a bacterium that all of us encounter in our homes and it's even in soil and waterways, 00:03:18.00 so it's a very prevalent bacterium on the planet, and people who have normal immunity 00:03:23.01 and strong health are able to simply fight off an infection from Pseudomonas aeruginosa 00:03:28.21 if it enters the body. 00:03:30.11 But there are very many people who cannot do this. 00:03:33.09 Cystic fibrosis patients, those with severe burns, and anyone who's immunocompromised 00:03:39.00 is at risk of seeing this very prevalent Pseudomonas aeruginosa bacteria, and have it enter the 00:03:44.17 body, and opportunistically cause an infection. 00:03:49.18 There's a main reason for this. 00:03:52.17 This is because Pseudomonas aeruginosa has a lot of what are called efflux pumps. 00:03:59.06 These are combinations or complexes of proteins that exist, that permeate the cell, spanning 00:04:06.23 the inner and the outer portion, and their function is to bring things actively in and 00:04:11.05 out of the cell, including antibiotics. 00:04:14.09 So, if antibiotics get into a Pseudomonas aeruginosa cell, it's actively pumped out 00:04:20.06 through an efflux pump. 00:04:22.04 And this works in a wide variety of antibiotics, but it's important to remember that efflux 00:04:27.22 pumps have a large number of functions for Pseudomonas aeruginosa. 00:04:31.16 In terms of pathogenesis, this protein complex is also important for host colonization, the 00:04:38.19 ability for bacteria to form a very tough layer called a biofilm that's very difficult 00:04:43.22 for antibiotics or anything else to get through, and they also function in allowing these bacteria 00:04:49.12 to evade or avoid host immune responses. 00:04:53.07 Now, these are chromosome encoded and they seem to be highly prevalent or, in other words, 00:04:59.03 genetically conserved in Pseudomonas aeruginosa genotypes. 00:05:02.21 And, a wide variety of antibiotics, as I said, get pumped out from efflux pumps, such as 00:05:08.24 these antibiotic classes -- macrolides, aminoglycosides, tetracyclines. 00:05:14.14 And here's a typical structure of an efflux pump, which is at least three proteins spanning 00:05:20.00 the outer and the inner portion of the cell. 00:05:23.07 Now, let's get back to phage therapy and how the use of phage therapy might be able to 00:05:28.21 help us overcome the problem of opportunistic pathogens such as Pseudomonas aeruginosa. 00:05:34.04 So, the idea of phage therapy is that you can use a phage, or a virus that is specific 00:05:39.18 to a bacterium, to kill the disease bacterium instead of some other type of a therapy. 00:05:45.13 So, this picture is showing phages or these virus particles in relation to a typical bacterial 00:05:50.20 cell in terms of their relative size. 00:05:54.17 Phage therapy is an old idea. 00:05:56.15 So, even before antibiotics were ever discovered by Alexander Fleming and used widely by the 00:06:02.04 human population, long ago, people discovered that phages... in the early 1900's, these 00:06:09.01 are some of the earliest viruses ever discovered and described... that these could be placed 00:06:14.06 in a human or an animal to in... to combat bacterial disease. 00:06:19.06 At that time, the Russians were the main ones who were developing the technology of using 00:06:23.20 phages to combat disease. 00:06:26.03 They were so enamored of the idea that they even outfitted their soldiers in World War 00:06:30.06 II with little vials of phage that they could use, in the field, to treat wounds and to 00:06:36.24 prevent those wounds from being colonized by bacteria, because, at that time, it also 00:06:42.16 was unlikely for soldiers to be able to even reach some place that they could get health 00:06:47.01 care and, in the case of an opportunistic bacterial infection that they encountered 00:06:51.09 in the field, this might invade the body and even kill them before they could reach any 00:06:55.02 medical help. 00:06:57.06 It is important to also know, even in the early discovery of phages, these experiments 00:07:02.05 placed phages in animals such as chickens and humans, which are both types of organisms 00:07:08.01 that can suffer from cholera bacteria infections, and, yes, we know that rehydration therapy 00:07:13.16 is very key to get people through a cholera infection, but phages are able to actually 00:07:18.11 get in the body and find these cholera bacteria, kill them, and destroy them, so that someone 00:07:24.20 can recover from cholera much faster, even in the absence of rehydration therapy. 00:07:30.17 So, now, I want to give you some of the details, or remind you of the details, of how a lytic 00:07:36.24 phage, one that is lethal to a bacterium, undergoes its normal growth cycle. 00:07:41.05 And I'll further explain how understanding how evolution of resistance to phages by bacteria 00:07:47.12 can help us, if we find the right phage, to overcome the problem of antibiotic resistance. 00:07:51.16 So, a phage encounters a cell -- a bacterial cell in this circumstance -- and, if it can 00:07:58.10 infect and get in... 00:07:59.10 I'm showing in this diagram how the phage enters the cell, hijacks the metabolism of 00:08:04.18 the cell, makes copies, the cell bursts open, and those phage particles can go on and infect 00:08:11.07 new cells. 00:08:12.07 So, in this diagram, you'll note that, at the exterior of the bacterial cell, I'm showing 00:08:17.23 these recognition proteins in black, and this is a protein binding that allows the phage 00:08:23.06 to infect the bacterium. 00:08:25.20 The normal circumstance, and one that's very understandable from the... in light of evolutionary 00:08:30.17 biology, is that this selection pressure of phage infection is going to favor mutants 00:08:37.16 of the bacteria that are able to escape. 00:08:40.02 In other words, they'll have phage resistance. 00:08:42.11 So, now, I'm showing this escape mutant as surrounded by blue proteins, and the phage 00:08:47.24 is unable to infect. 00:08:50.08 So, here's a key question: Suppose that if a phage interacts with bacteria and selects 00:08:58.00 for the bacteria to become resistant to the phage, suppose that, following along with 00:09:04.04 that, it made the bacteria somehow sensitive to antibiotics? 00:09:08.24 That would be a great thing for phage therapy, because, if the phage kills the bacteria, 00:09:15.02 that's what you intended to happen, but if evolution kicks in and the bacteria escape 00:09:20.04 the phage infection, if they are now suddenly vulnerable to antibiotics, especially antibiotics 00:09:26.03 that are normally useless in killing the bacteria, you have a double-edged sword. 00:09:31.07 You have not only the ability of the phage to infect and kill under normal circumstances, 00:09:35.20 but, even when the inevitable evolution happens where resistance to the phage occurs, you 00:09:42.00 still have sensitivity following along for these bacteria to become sensitive to antibiotics 00:09:48.12 and to die in that fashion instead. 00:09:50.22 So, what would this really indicate? 00:09:53.22 It would indicate a trade-off between phage resistance and drug sensitivity in bacteria. 00:09:59.03 Obviously, that would improve antimicrobial therapy options and, importantly, it would 00:10:04.09 extend the lifetime of current antibiotics in our arsenal. 00:10:08.00 So, these would be chemical antibiotics that are approved by, say, the US Food and Drug 00:10:12.18 Administration as safe and effective in people, but what we have as a biomedical problem is 00:10:18.06 that they are failing widely. 00:10:20.04 If we could intervene somehow and find a phage that prompts this kind of a genetic trade-off, 00:10:25.17 then this would lead us to use those currently useless antibiotics over again, and we wouldn't 00:10:30.23 have to continue searching for new antibiotics -- we can simply trot out the ones that are 00:10:36.06 failing currently. 00:10:37.19 So, we found just such a phage. 00:10:41.06 It's abbreviated as OMKO1 and it actually came from a lake in Connecticut. 00:10:47.06 We took the old school idea of go out into a natural environment and try and find a phage 00:10:52.23 that kills your target bacterium. 00:10:54.24 Well, we used this method with an eye to understanding evolutionary biology. 00:11:01.00 We found phages that are able to infect and kill multidrug-resistant Pseudomonas aeruginosa, 00:11:08.04 and when they fail to kill, because the bacteria have become resistant, we find that this sensitivity 00:11:14.00 to antibiotics that results from the trade-off is a great way to further kind of a rational 00:11:20.04 design behind phage therapy. 00:11:22.04 So, the diagram shows, when the phage is countering the efflux pump, the key thing is that we 00:11:28.18 found phages that bind to and enter cells because they are binding initially to the 00:11:34.08 outer-most exposed proteins in the efflux pump. 00:11:38.14 In this one example, that protein is abbreviated as OprM or outer membrane protein M, and OMKO1 00:11:47.13 is abbreviated as outer membrane knockout 1, meaning that when we challenged the virus 00:11:52.21 to infect knockout strains of the bacteria, the only one that had failed to infect is 00:11:59.01 when OprM is not made on the cell surface. 00:12:02.07 That indicates that it must be involved in the binding property of the phage. 00:12:06.05 So, now, this is wonderful, because we have this genetic trade-off. 00:12:11.10 The phage-sensitive bacteria efflux antibiotics but they're killed by the virus. 00:12:17.24 The phage-resistant mutants have an impaired drug efflux ability -- this makes them vulnerable 00:12:23.14 to antibiotics that are normally useless against this bacterium. 00:12:27.23 So, looking a little bit at, how do you measure this in the laboratory? 00:12:32.13 How would you even know what's going on? 00:12:34.16 This is a typical agar plate, where the surface of the plate is coated with a high density 00:12:39.13 of bacteria -- that's called a lawn -- and what you can do is you can take some commercially 00:12:44.12 available thing like a disc of paper that's been soaked in antibiotic, and you place that 00:12:50.19 on the agar surface, so if these bacteria are multidrug-resistant, including able to 00:12:56.03 grow in the presence of this drug that's leaching out from that paper, then they'll grow up 00:13:01.05 right next to the edge of the paper. 00:13:03.18 They completely shrug off the effect of the antibiotic -- it's... has no effect in controlling 00:13:08.24 their growth. 00:13:10.09 What we observed is, after we exposed those bacteria to this phage, they gain a mutation 00:13:16.12 that makes them resistant to the phage, and instead, now, they have a very different growth 00:13:21.06 ability in this assay. 00:13:22.23 Now, a much lower concentration of that drug is adversely affecting the growth of the bacteria, 00:13:31.10 and technically that's called a minimum inhibitory concentration. 00:13:35.07 That means that there's a lower level of this drug that is now impacting their growth in 00:13:40.14 this way. 00:13:41.14 So, now, I'll walk you through a table that shows and summarizes the key data from our 00:13:47.13 study. 00:13:48.16 Let's begin at the first line of this table. 00:13:50.15 In the column beginning here is an antibiotic, for example, and the class of antibiotic that 00:13:56.23 it's drawn from. 00:13:59.00 Next column is the minimum inhibitory concentration of these multidrug-resistant bacteria in the 00:14:05.02 presence of that drug. 00:14:06.23 And then, the column that follows is, how does that MIC change when the bacteria see 00:14:12.10 the phage and they become resistant to the phage, and how has it affected their MIC? 00:14:18.09 In these first two examples, you'll see that there was a huge impact -- the fold-increased 00:14:23.07 drug sensitivity is a very large number. 00:14:27.02 Through simply exposing the bacteria to the phage, they become resistant to the phage 00:14:32.20 and it makes them much more sensitive to the antibiotic. 00:14:35.18 Now, these first two examples are not by accident, because we know through prior scientific data 00:14:41.07 that tetracycline and erythromycin are examples of two popular antibiotics drawn from two 00:14:47.06 different antibiotic classes, for which efflux pumps provide the main mechanism of resistance 00:14:52.23 for Pseudomonas aeruginosa to remove those antibiotics from the cell. 00:14:56.23 So, we're not quite certain, in the terms of other antibiotics drawn from other antibiotic 00:15:01.20 classes, whether explicitly efflux pumps are always doing this, or only sometimes doing 00:15:07.04 it. 00:15:08.04 Well, when we looked at four more examples, drawn from different antibiotic classes, you 00:15:12.08 see mostly the same result, where there was an increased fold drug sensitivity. 00:15:18.10 Now, you may not be as impressed by these lower numbers, but I've shown you in asterisks 00:15:24.20 that the clinical resistance has reversed to sensitivity. 00:15:28.13 This means that a doctor, a physician, could actually administer these antibiotics successfully 00:15:34.14 against these phage-resistant bacteria, and that's because the bacteria are now sensitive 00:15:42.11 to these drugs at a clinically relevant level. 00:15:45.19 So, the last line on the table is more or less a control. 00:15:49.20 Here, we have ampicillin, which is known to be not efflux pumped out of the cell for resistance. 00:15:56.23 So, one would not expect these values to change when the bacteria see or do not see the phage. 00:16:03.09 And, indeed, there is no change in their fold-increased drug sensitivity. 00:16:07.16 So, these data, taken together, suggest that there are a wide variety of antibiotics drawn 00:16:14.04 from different antibiotic classes for which this phage exposure makes the bacteria sensitive 00:16:21.10 to antibiotics that are currently useless in controlling multidrug-resistant Pseudomonas 00:16:27.14 aeruginosa. 00:16:28.14 So, what we would like to do next is, at least... we know for now that the trade-off is... is 00:16:35.09 observed in a wide variety of Pseudomonas aeruginosa genotypes, and that gives us a 00:16:41.04 lot of... an indication, a strong indication, that this should be broadly useful in targeting 00:16:47.04 a wide variety of genotypes. 00:16:48.19 So, this worked not only in laboratory model strains PA01 and PA14, it worked in a wide 00:16:54.20 variety of clinical samples, including individuals who had suffered otitis, or ear infections, 00:17:02.02 diabetic foot ulcers, osteomyelitis, etc. 00:17:05.12 We took clinical samples from these individuals, put them into our assay, and we got data that 00:17:11.04 suggests that those clinically relevant bacteria are experiencing the same trade-off as in 00:17:16.24 the model strains. 00:17:17.24 And, even more interestingly, if you just simply take Pseudomonas aeruginosa from the 00:17:22.16 environment, and you can find multidrug-resistant forms of it in the environment, and estuaries, 00:17:27.16 and even in human homes, these also experience the exact same trade-off as the other strains. 00:17:35.01 So, ideally, then, we can now expand this out even further, look at a very, very large 00:17:41.04 repository of clinical isolates, and I would expect that these data would also be supportive. 00:17:47.06 But that's a goal for our near-future work. 00:17:50.13 Already, we've obtained the ability from the US FDA to use phage cocktails in chronically 00:17:58.09 infected human volunteers, where these could actually only be the single phage that we 00:18:04.00 found, or in combination with other phages. 00:18:06.15 And, by that, I mean these are human individuals who have suffered chronic infections with 00:18:12.03 MDR P. aeruginosa, and they have essentially run out of options for treatment. 00:18:17.05 There are no antibiotics that will help them. 00:18:20.07 In some cases, it's actually remarkable that these individuals are still alive, and they 00:18:24.07 are very willing to undergo experimental treatment if this is going to help them. 00:18:28.17 Well, we are identifying individuals at Yale New Haven Hospital and other locations, and 00:18:34.02 working with physicians and surgeons, in order to use this experimental treatment where these 00:18:39.24 volunteer patients are bravely looking to this option to see if it will improve their 00:18:44.16 health. 00:18:45.16 Already, we've had one successful event, where a man had an aortic arch replacement, which 00:18:51.11 is a pretty routine thing that is a surgery that leads to the replacement of a very key 00:18:56.21 portion of the heart, that kept this man alive, but, in any individual, and this is increasingly 00:19:02.13 seen in medical science... is that we have these routine surgeries that bring into the 00:19:06.24 human body artificial substrates, and these make great places for bacteria to form a biofilm, 00:19:13.12 a very tough infection that's hard to treat. 00:19:16.02 And, in many cases, these are multidrug-resistant bacteria. 00:19:19.15 Well, this man did not have... he did not have any other options and he volunteered 00:19:23.14 for treatment, and we placed the phage in his chest near the site of the infection, 00:19:29.06 and one administered treatment was enough to remove his bacterial biofilm, and he is 00:19:33.17 now completely off antibiotics and in much better health. 00:19:38.08 We talked about his one case as well as our general approaches to taking an evolutionary 00:19:44.11 eye to phage therapy in several radio shows, which you might want to listen to for further 00:19:49.05 information. 00:19:51.11 But our big goal is to test... test the safety and efficacy of this whole approach in a mouse 00:19:58.05 model. 00:19:59.05 In some ways, what we were able to achieve was a bit backwards. 00:20:02.12 We found some human patients that were very excited about this potential therapy, and 00:20:07.03 it did work safely, obviously, in the one case that we've tried, but now we'd like to 00:20:11.02 take a step back and see how broadly useful might this be for say lung pneumonia infections 00:20:17.04 in immunocompromised patients, or in cystic fibrosis patients, and it's very easy to create 00:20:22.00 a mouse model dataset in a laboratory to test the safety and efficacy of whether this works. 00:20:28.12 So, that's underway right now. 00:20:31.01 If that works, then we're at the stage where we can actually help humans on a grand scale, 00:20:38.15 ideally. 00:20:39.15 So, this would be a clinical trial that would happen in humans. 00:20:42.14 Perhaps we could use humans who are suffering hospital-acquired pneumonia, or cystic fibrosis-associated 00:20:48.18 pulmonary infections, and see if we can improve their quality of life by administering the 00:20:54.05 phage and the antibiotics simultaneously, and that might reduce, even just... for individuals 00:21:00.00 who are on chronic antibiotic therapy, which has a lot of bad consequences and side-effects, 00:21:05.22 even using the phage to help prevent the bacteria from infecting or removing them entirely, 00:21:12.09 it should expose those individuals to less of these chemicals that can adversely affect 00:21:17.07 their health, but are helping keep them alive. 00:21:19.07 I'd like to acknowledge the people who helped on the study. 00:21:23.00 Ben Chan was the first author on the paper that we produced in 2016, that looked at not 00:21:28.04 only the initial discovery of this phage but also a subsequent paper where we described 00:21:33.08 the case study of the individual who we helped with experimental therapy. 00:21:38.10 And two other key individuals who worked with me on this were Deepak Narayan, the surgeon 00:21:43.09 at Yale School of Medicine and Yale New Haven Hospital, who was able to find this patient 00:21:49.22 who bravely volunteered for the treatment and is also helping us administer the treatment 00:21:54.06 to other individuals who have run out of options. 00:21:56.23 Also, my long-term collaborator, John Wertz, who's at the Coli Genetic Stock Center at 00:22:02.06 Yale, was another key architect in the study. 00:22:05.11 And, without them and the help of my lab group, none of this work could have happened. 00:22:10.07 We're also thankful to those who funded our work externally, especially federal agencies 00:22:16.03 and private foundations.