NEB Podcast #84 -
Transcript
Interviewers: Lydia Morrison, Marketing Communications Manager & Podcast Host, New England Biolabs, Inc.
Interviewee: Dr. Graham Hatfull, Ph.D., Eberly Family Professor of Biotechnology, University of Pittsburgh; HHMI Professor; National Academy of Sciences
Lydia Morrison:
Welcome to the Lessons from Lab and Life podcast, brought to you by New England Biolabs. I'm your host, Lydia Morrison, and I hope this episode brings you some new perspective. Today, I'm joined by molecular biologist Graham Hatfull, who is the Eberly Family Professor of Biotechnology at the University of Pittsburgh. He's also been an HHMI professor since 2002, and he was elected to the National Academy of Sciences in 2024. Dr. Hatfull has spent decades studying the molecular genetics of mycobacteria and their bacteriophages. He joins us today to share some of his findings and to talk to us about the use of therapeutic phage to treat bacterial infections. Thanks so much for being here today.
Graham Hatfull:
Oh, thank you. Happy to be here. Delighted.
Lydia Morrison:
Excellent. Well, we're delighted as well. And I heard your seminar that you presented earlier, your lab studies mycobacteria and their bacteriophages. I'm curious what pulled you toward phages, and in particular, why the phages of mycobacteria?
Graham Hatfull:
Oh, that's an interesting question. It's a common question that students often have is how the heck did you get into studying the things that you do? And principally by luck, serendipity, not necessarily by design. I got interested in the phages because I was looking for a particular type of DNA recombination system that I might want to study mechanistically. And I learned about one particular phage that likely had a system that might be of interest. I started working with that phage and studying it in some detail, ended up sequencing its genome, which was a fairly substantial task back in 1988 when we started. And so that took us into figuring out how much information and insights and things that we could do with just one phage and one phage genome. And that quickly led us to want to study more and to understand issues of their diversity and evolutionary mechanisms, et cetera.
So principally, it was just serendipity. And once we really started working with the phages, they were easy to love and easy to work with, and we just moved on right from that starting point.
Lydia Morrison:
Well, I find that that's often the way with science, right? There's a lot of serendipity involved in where scientists end up and where study falls. So I think that part of it is the innate curiosity of scientists, and part of it is the timing of the findings. For many of our listeners who might not work with mycobacteria, and I have to be honest, I feel like prior to this, a lot of what I knew about mycobacteria was from my time in tissue culture and being like, "Oh no, we have a mycobacteria infection. Everything has to be scrapped." So for listeners who might not know everything that you know about mycobacteria, which I can only assume is most of them, I'm curious, this is the genus that contains the tuberculosis bacterium. So why are mycobacteria so hard to study?
Graham Hatfull:
The mycobacteria are a group, a genus of bacteria. And there's lots of different species, probably a hundred or more, I don't remember the number, some of which we know more about than others. Perhaps the best most recognized is Mycobacterium tuberculosis, the causative agent of human TB, which is a very substantial health-wide issue. But there are others. Lesser known is Mycobacterium leprae, which is the causative agent of leprosy, a relatively rare disease now, but still an important one. And then there are other bacteria and bacterial species of mycobacteria that are opportunistic pathogens. Most healthy people wouldn't get infected with them, but if you have other underlying particular situations and conditions, then you can become susceptible and those are important infections and they're relatively difficult to control with antibiotics. They are a little bit funny to work with in the lab from a couple of perspectives. If you want to work with Mycobacterium tuberculosis itself, well, first of all, it's a dangerous pathogen, so you have to use it in a BSL-3 containment facility and it grows extremely slowly.
So if you want to grow a colony of bacteria on a plate, it takes like three or four weeks to incubate to see that. But with all of the mycobacteria, they tend to be very clumpy. If you try to grow a culture of bacteria, the cells all clump together. So instead of getting a nice dispersed culture of single cells, they all clump together, which makes doing microbiology and genetics a pain in the neck. And so we have to do various machinations in order to deal with those. Sometimes it involves adding some detergent. Sometimes we have to sonicate the cultures or pass them through a syringe with a needle, for example, to try to disperse them. And so we get used to applying these various tricks, but there are things that make the mycobacteria often a little bit trickier than many other bacteria that people work with in the lab.
Lydia Morrison:
That's interesting. Certainly some particular challenges to mycobacteria. I know we started talking about phage and then we started talking about mycobacteria. And so could you give our listeners a quick definition of what a phage is and what a mycobacteriophage is and why it's important to study these phages?
Graham Hatfull:
So a bacteriophage is a virus and bacteriophages have all of the common properties of viruses that many of us I think are familiar with. For example, a virus cannot grow by itself and a phage cannot grow and replicate by itself. It is completely dependent upon the host cell that it can infect. Bacteria have their viruses that we call bacteriophages and bacteriophages just infect bacteria. There's lots of different types of bacteria and lots of different types of bacteriophages, but there is specificity. So the phages that infect, for example, E. coli, a common gut bacterium, do not infect Bacillus subtilis, a soil organism. And so the specificity is a characteristic feature of all viruses and definitely of bacteriophages. We use the term mycobacteriophages just to refer to that subset of phages that infect mycobacterial hosts. And so we use it generically. In fact, there's lots of different types of mycobacteria and many, many different types of mycobacteriophages.
And we study them for all sorts of reasons. They're a useful entry point to try to understand viral diversity. How many different types of viruses are there? How many different types of phages? How do they evolve? So we use them to address those questions. We also use them because they're just really effective toolboxes. If you study the phages, you can find lots of components, parts of them, genes, particular aspects that are useful for studying the bacteria themselves. And so we use them as toolboxes that we can dip into them and we can pull out parts, develop them and use them, and make them as tools that other people can use to study the bacteria.
And then finally, there's the idea of using the phages as actual therapeutic agents to try to treat and to control infections that people would have with different types of bacteria. In our case, our interest is mostly with mycobacterial infections and the question as to whether the phages may be useful therapeutically to control those infections.
Lydia Morrison:
Yeah, a really interesting question, and I think one that has a lot of import to individuals who are immunocompromised and tend to be infected with these mycobacterium infections that can be really hard to treat and resistant to antibiotics, right?
Graham Hatfull:
Yes. That's one of the central issues with dealing with mycobacterium infections is that they are very challenging to treat with antibiotics. Mycobacterium tuberculosis infections, if you have TB, typically those infections can be controlled with antibiotics, but you have to take at least three antibiotics for six months, right? And that's considered short-course therapy for TB. So seriously challenging infections to control. And of course, when trying to control those infections in that way, it's very common for the bacteria to become resistant to the antibiotics. And so for TB, that is a particularly specific concern.
For other types of mycobacteria, and we have a particular interest in a type of mycobacteria called Mycobacterium abscessus, often found as infections of persons with cystic fibrosis and some other kinds of underlying conditions that can leave people susceptible to them. They can be controlled in some instances with antibiotics, but in at least half of the cases, the physicians try really hard for many years often with different antibiotics and different routes of giving the antibiotics with all sorts of intolerance and toxicities, which make them extremely challenging to treat from a clinical perspective.
Lydia Morrison:
Yeah, absolutely. And so I think exploring different treatment methods is so important for those. When you are in your lab and your students are starting to sequence phage genomes, I know they sequence some from M. smegmatis, what discoveries surprised you the most?
Graham Hatfull:
Well, I think the most amazing thing is that back in 1988, we sequenced the first genome of a mycobacteriophage. It was called L5, not a very glorious name, but it'd been isolated in Japan quite a few years ago. And I thought at the time that it was a lot of hard work to sequence this particular genome at that particular time with the existing technologies. And I thought, boy, this is the only mycobacteriophage genome sequence that we'll ever do and we'll be studying this for the rest of our career, I guess. But what's happened is we started sequencing more phage genomes. And as time has gone on with the programs and the education programs we've been involved in, we now have something like 2,800 genome sequences, phage genome sequences of the phages that were isolated just on this one strain of Mycobacterium smegmatis. And the amazing thing is that basically they're all different.
And so the variation, the diversity is truly amazing genetically. I think there are only two instances where we can say we know that exactly the same phage was isolated twice. Okay?
Lydia Morrison:
That seems shocking.
Graham Hatfull:
It is amazing. Everything else is different. Now, the way they differ and the ways they differ is itself very varied. They may differ by a few nucleotide differences, a few base changes, or they may have whole blocks of genes that are shared with the rest being different and every possible imagination, imaginable type of variation you can see. So that's what really is mind-boggling is that there are so many different types, so many different genes that make up these types of phages. And to see that diversity in the phages of one particular strain, of one particular bacterial species, is really stunning. Think of what that would look like if you amplified it to all of the other gazillion strains of different types of bacteria, all of what we know about in the biosphere. It just gives you a sense that the phage population is truly amazing in terms of its diversity and how many different types and how many different genes, the vast majority of which we would think of as the dark matter of the biosphere, the stuff that we really have yet to discover what it's all about.
Lydia Morrison:
Yeah. It's a really mind-boggling amount of diversity of these tiny viruses that exist in the world. Could you explain a little bit more about phage therapy, what it is and how you got involved with treating the first cystic fibrosis patient that you treated with phage therapy?
Graham Hatfull:
Yeah, I think it's a relatively simple concept that the bacteriophages, phages we call them for short, infect bacteria and can replicate and typically can kill the bacteria. So phages are inherently and can be inherently antibacterial in their nature. So it makes sense that you would be able to use phages similarly to the way that you use antibiotics to treat a patient with a particular infection and have the phages find their way to where the infected bacteria are, and then for the bacteriophage to essentially have at them, to replicate on them, kill them, and have a benefit to the patient by clearing out the infection. And so it's actually a very old idea. It's phages were discovered a little over a hundred years ago and the idea of using them in this way was tested shortly after that. And it's been an idea that's remained of some interest over the years, although interest in it has waxed and waned.
The fact that in the past hundred years, people haven't really figured out how to get phages into routine therapies is complicated. Well, it's an easy question with a complicated answer, I think. But there's been a lot of renewed interest in it, especially in the Western or developed systems, societies that most commonly use antibiotics to control infections. As the pathogens have become resistant to the antibiotics, there's clearly a need for new types of therapies, new strategies, new ideas as how to control these because it's helpful to think about, to cast our minds back to what the world was like before antibiotics. The lifespan was about half of what it is now. And so antibiotics have had a huge impact on health of societies in general by being able to control these infections. And so if you lose the use of antibiotics, the health consequences at a global level are really a big concern.
And so people are starting to appreciate the magnitude of that concern, that problem, and to ramp up the idea of looking at alternative solutions. And I think that bacteriophages have shown some promise as therapeutic options, which to me is not a surprise. The question is how can you use them most effectively? What types of infections and what types of patients, what types of bacterial pathogens are best suited for addressing or being able to use therapeutic options with phages? I suspect that phages will never replace antibiotics. I think that's very unlikely. I think it's likely that phages may only fairly rarely be used by themselves, but in combination with antibiotics, I think they really have the potential to add helpful clinical solutions to some very challenging infections.
And I think that it's helpful to have a sense of being modest of the expectations. I think that as we gradually move towards perhaps in certain circumstances, introducing the use of phages in combination with antibiotics, that in and of itself may help to reduce or to minimize the appearance of new resistance to the antibiotics. Phages may simply help to extend the lifetime by which we can have good use of the antibiotics before we lose use of them altogether.
Lydia Morrison:
Which could be a very powerful application of it. Absolutely.
Graham Hatfull:
Absolutely. Yeah.
Lydia Morrison:
And so tell me a little bit about how you first got involved in the treatment of the first cystic fibrosis patient who received this phage therapy.
Graham Hatfull:
It was towards the end of 2017, and I had a colleague that I'd known for a time who worked at a London pediatric hospital and he sent me a message that they had two patients that had rather similar clinical conditions with cystic fibrosis and had both had a lung transplant and then had acquired these infections with this bacterium called Mycobacterium abscessus. And these had disseminated throughout their bodies. Antibiotic therapies were tried in many different combinations, but essentially did not succeed in clearing the infections. And the patients were sent home on palliative care essentially with all treatment options essentially exhausted. And so one of the parents had contacted the physicians there and said they'd been looking around for alternative therapies online and had stumbled upon this thing called phage therapy and they wondered whether that might be an option for their daughter who was one of these patients.
And so when James Soothill contacted me, he said, "Would you be interested in looking to see if you had phages for these particular strains?" And I said, "Well, why not? Send them to us, and we like looking at phage infections and different bacteria. And I doubt that this would ever be a therapy, but we'll take a look." And so we received those strains and we began a search to see if we could find phages that might work. For one of those strains, we didn't find any at all. And unfortunately that patient passed away. For the other patient, we after a fairly extensive search found three phages, which seemed to work quite well. We had to do further work. We had to engineer the phages and do some genetics. But nonetheless, in a few short months, we were able to come up with three phages that we thought would be therapeutic candidates.
And after some complicated discussions to think about how we might move forward with a therapy and some advice from people who understand the clinical aspects certainly better than we do, and with the team in London that worked exhaustively to try to get regulatory permission to use the phages for the, we'd call it a compassionate use therapy and experimental therapy, that all succeeded and moved forward. And about seven months or so after we'd first received the strains, those phages were administered to the patient and it was very well tolerated. And although clinical improvement takes some time, even after about six weeks, there was all sorts of good indications. The scans that showed where the infection was were looking much improved and the other clinical aspects really showed a good response to the phage. And she was able to basically return to as close to a normal life as you can for someone who still has cystic fibrosis and has to manage the other challenges and other types of infections.
And so that was probably about the best outcome that we could have expected for an experimental therapy, which as far as we know, had never been tried before in that kind of way.
Lydia Morrison:
Yeah, that's amazing. And I'm so happy to hear that it was effective. You mentioned that there were three phages that you had identified and a couple of them needed to be changed or altered slightly. Why did you need to edit those phages before you tried to use them?
Graham Hatfull:
It's a good question and it's not easy to answer without going a little bit into the weeds. So just hang in there, okay? Many phages, including the ones that we study, can be grouped into what's referred to as being temperate phages. And temperate phages have a particular type of behavior where when they infect a cell, a bacterial cell, they can replicate and kill that cell, but they don't always do that. Sometimes when the bacteria are infected by the phage, the phage essentially just hides away inside the bacteria. So it doesn't kill the bacteria. The bacteria survive, the bacteria are perfectly happy, and in fact, they carry a copy of the phage DNA inside its own bacterial genome. So the problem with temperate phages therapeutically is we're trying to kill all of the cells. We don't want these surviving cells coming through and not being killed by the phages.
And so fortunately, we've spent a lot of time over the years studying the biology and the genetics of how these systems work. And so we knew when we saw these behaviors that there are particular genes that we could delete from the genome in order to prevent them from doing that second pathway. In other words, to convert them from being a temperate phage to one that we would call being lytic, that only results in what's called lysis or death of the bacteria.
We had also, over the years, developed specific engineering tools, genetic engineering tools that enabled us to actually alter the genomes in the desirable way. So that's what we did. We used the tools that we developed. We deleted the genes that we needed to remove in a very precise and controlled way, and then could demonstrate that we had indeed, in the lab, now made phages which have the desirable property of efficiently killing the bacteria when we simply add the phage to the bacteria. We wanted to be able to show that the phages could kill the bacteria efficiently in the lab so that we would have essentially a baseline to think about how that phage might infect and kill the bacteria in the patient.
Lydia Morrison:
And since that first patient, you and your collaborators have been approached by many other patients. Can you tell us about that experience? How have those treatments gone? And I guess why do you think some infections respond well to the phage treatment while others perhaps are less responsive?
Graham Hatfull:
Yeah. So first, I mean, these types of infections, the patients and people that have these kinds of infections with Mycobacterium abscessus in particular, there's a lot of them. There's not just the two patients in London. They're not uncommon, especially with patients that have cystic fibrosis. That's a common underlying cause, but not the only one. And so when we wrote a report of the first case and what we had learned from it, we started getting regular requests at a tune of about a hundred a year from physicians who have patients that they're looking after who are in similar circumstances, essentially asking if there's a possibility that we might have phages for that particular bacterium that is infecting that particular patient. And in each case we've said, "Well, if you send us the strain, we will screen it against a panel of phages and to see if we have any that infect and kill efficiently. And if we do, then we can have a follow-up conversation about how that might be implemented therapeutically, the paperwork, the regulatory permissions that have to be asked for, et cetera."
And so that's the process that we've been engaged in. We've screened about 650 bacterial isolates. We haven't found phages for all of those, but we found phages potentially for about a hundred of them, a little over a hundred, and then had follow-up conversations. There's a lot of parameters that go into deciding whether you do this experimental medicine or not. So it is definitely not trivial, but we've now have identified and provided phages for treating about 54 patients, which feels like it's a fairly large number. This is all since about 2019.
And in terms of the outcomes, we're still collating the data from those 54, but we have assembled the data and published what we saw from the first 20. And I think the data are fairly clear. For five of those, we couldn't make an adjudication for one reason or another. Many things go on with these patients. They're complicated clinically and patient may have had to stop taking phage for some reason other than the phage, but just other things that was going on. So five of them, we didn't have an insight into good or bad outcomes. For four of them, it appeared like it didn't make any difference. We don't know why, but for 11, we saw favorable outcomes, improvements in clinical conditions, reductions of the infection, et cetera. In this type of study, every patient is different. This is not a clinical trial. Every observation is essentially anecdotal, and we should be very cautious about how we interpret that as to even if a patient gets better, what the cause was. But we know that these are really difficult cases to control with antibiotics.
And by definition, we're only giving the phages to those patients because they are out of options and the antibiotics haven't worked. So there's, I think, a fairly at least strong indication that as those patients experience those clinical or microbiological improvements, that it's likely that the phages were having some kind of beneficial effect. And so we have a lot more to learn.
We think as we prepare the collation of the larger number of patients, of 54 or so patients, the profiles are going to look quite similar to what we saw in the first 20. So I don't think those first 20 was biased in any particular way. So we don't know why it doesn't always work for every patient, but the fact that out of those 20, those 11 patients who saw improvements, they were 11 patients that had no other options. So we think that there's at least a signal there that there's potential clinical benefit from the use of the phages and now figuring out how to broaden that and to try to understand why and how it may have a benefit. Those are the kinds of the questions that we really need to try to understand.
Lydia Morrison:
Yeah. It sounds initially very promising and I can't wait to hear more about the results of the larger cohort when those are published. I know we talked a little bit about your work with students and gathering the genetic information around these phages to have this catalog to be able to look at. You're one of the most prolific mentors that I've ever met in terms of the number of students that you have working on these projects and getting some hands-on experience. I know you started the PHIRE program at Pitt in 2002, and then it became what's now called the SEA-PHAGES program in 2008. What was the original idea behind that, and what did you expect to be the outcome?
Graham Hatfull:
So again, luck and serendipity came to play a role here, but I was very fortunate to be able to submit an application, a proposal to what is essentially a new grant program that the Howard Hughes Medical Institute was starting. They called it their Professors Program, looking for people to develop initiatives to improve science education. And that was all at about the same time that we had started sequencing a few phages, we're beginning to get a glimpse into how interesting and diverse and potentially useful they are. We needed more of them. And so we had a scientific motivation. We needed somebody to go and isolate new bacteriophages and sequence the genomes to expand that view. And we just realized that this was just a very good type of program that would be suitable for, we'd call them novice scientists, students, or in fact, anybody who wants to get an experience and an insight into actually doing scientific research.
Isolating phages and characterizing them doesn't require you to have any prior scientific knowledge, doesn't require you to have any real prior technical knowledge. You just have to go and do it. And so we developed that program in this, what we call the PHIRE program, which is an acronym for Phage Hunters Integrating Research and Education. So you can get a sense of what we're trying to do here. We're trying to align our goals in science education with our scientific goals so that by students being engaged and doing these particular research programs, it advances a science and it advances their involvement and their interest and excitement in doing science. And so the PHIRE program really ran locally in Pittsburgh and we developed it there and learned about how that might work. And that was then the basis and the rationale for then packaging it in a way that it could be used at other schools and universities in what is now part of the SEA-PHAGES program.
The SEA-PHAGES program works in a way that much of the programmatic issues, the training of the faculty, the protocols to be used, advice about which kinds of materials, the databases that store and share all the information about the phages and the program, that all is centralized. So if you're a school, a community college, tribal college, an R1 university, it doesn't matter. You can essentially apply to the SEA-PHAGES program and tap into it and take advantage of the common infrastructure that supports the program. And then you can have students out there isolating their phages and characterizing them, doing it not in their own little isolated environment, but as part of a community. We call this overall structure, we call it, so we love our acronyms, we call it an iREC, which stands for an inclusive Research Education Community. So four really important concepts there.
There's the research, there's the education, the sense of community of all these people doing it together as a community. And perhaps most importantly, it's inclusive. And it's inclusive because anybody can do this. Any faculty member can help to teach it, any student can participate, and any institution, whatever their infrastructure is, can participate. And so that's what's enabled the program to have a much larger impact. I wish I could say I had personally mentored some 70,000 students. I haven't, but I'm very pleased. It's a point of pleasure that the program has developed and has been able to develop in such a direction that a very large number of students have been impacted and they've isolated collectively the largest phage collection in the world.
Lydia Morrison:
Yeah. They've essentially helped fill this huge knowledge gap that existed, which is incredible. I know that the collection is now thousands of sequenced phages. I'm curious who else uses this catalog of information and what are they using it for?
Graham Hatfull:
So actually quite a lot of people use it and for all sorts of different reasons. And so we frequently get requests for access to some of the phages. And what people do with it is, as I said, enormously varied. For example, there are new techniques in microscopy, especially cryo-electron microscopy that lets you look at things like phage particles and actually see them and view them at very high resolutions. So you can essentially model in where the atoms are in there. And because we have so many and they're so diverse, we have essentially an endless repertoire of phages which are proving to be incredibly interesting and useful to look at using the cryo-electron microscopy. So we have a number of colleagues who write to us because they want to use phages for that. Or alternatively, phages often, they have to infect bacteria and the bacteria are trying to become resistant to them in the natural world.
And phages have developed ways to counteract that. And so people contact us because they want to have access to some of the phages because they're in the process of trying to discover what kinds of counter-defense systems phages have. And so that's just really two examples and there are others. They are I think a very valuable resource and there's enough there to keep a lot of people going for a very long time trying to understand these questions.
Lydia Morrison:
Absolutely. A very valuable resource that you definitely helped create and put into the world. I know you've been recognized by the Royal Society, the National Academy of Sciences, and the European Society of Mycobacteriology. What are you most proud of and what's the question you're still chasing?
Graham Hatfull:
Those accolades are fine and they're obviously humbling and pleasing. I don't see them as accolades for me personally as much as the collective group of, I mean, it's all of the students who have isolated the phages and the many people who have worked in my lab who have done the work that have made the advances. And so I'm very pleased that that collection of contributions has been recognized. I think I'm mostly just proud of all of the students. And when we have a chance to get together as we have over the years with students, often other institutions, if you just see, I think just the joy and the engagement that they've had from being able to step in as a first-year undergraduate student and make a contribution to the scientific endeavor, just seeing that is totally precious. And I feel like as an individual that's working and doing science, there's only certain set of contributions that you can do personally, but you can amplify it for all the people that you train and hopefully influence and impact in a way.
That's where the real impact, it's from what they do in the future to advancing the science, which is going to far outweigh anything that I've personally contributed to the endeavor. So that's what I'm most proud of is the thousands of students, many of which I obviously don't know personally, but it's a rare opportunity. Not many of us find ourselves by a stroke of good luck being able to positively impact a large number of really bright young students.
Lydia Morrison:
Yeah. I think that you are a great example of how that amplification can really affect the greater scientific community and patient therapies. And I just want to thank you so much for being here with us today to share your work and to share where it's headed in the future. And we can't wait to continue following you on social media and finding out what great things the future holds and how phage therapies are brought to more patients and help improve their lives.
Graham Hatfull:
Well, thank you very much. It was a total delight, and thanks.
Lydia Morrison:
Thank you for joining us for this episode of the Lessons from Lab and Life Podcast. We invite you to check out the episode's transcripts on neb.com for lots of helpful links from today's discussion, and we hope you'll be with us next episode.
