Authors: Colin Hill (APC Microbiome Ireland and School of Microbiology, University College Cork, Ireland)
What are bacteriophage?
The first thing you need to know about bacteriophage (phage) is that they are gorgeous! They are beautifully constructed, single minded, evolutionarily perfect nanomachines. When a phage encounters a sensitive bacterial cell it binds to specific receptors on the surface, inserts its nucleic acid genome and subverts the cellular machinery to begin to create new copies of the phage. It takes one bacterium about 4 hours to multiply to become 256 bacteria, but in that same time one phage can give rise to 10,000,000,000,000,000 new phage copies!
As a result of the speed of replication, and the extraordinary numbers generated, the phage can afford to be a little careless in terms of replication fidelity, mutating individual bases at a high frequency and constantly selecting for even ‘fitter’ progeny. This explosive replication rate permits us to witness evolution in real time. Over billions of years this has led to an extraordinary diversity of phage – in terms of their morphology (shapes and sizes), their nucleic acid content (ssDNA, dsDNA, ssRNA, dsRNA) and their genome size (from a few kb to more than 500 kb). They can also adopt different lifestyles (lytic, lysogenic, carrier state, pseudolysogenic). It is no wonder that they are the most abundant biological entities on the earth (estimated at 1031 individual phage representing billions of individual phage species). Phage are the undisputed winners of the survival of the fittest contest that has been run over the lifetime of the planet, but they are also the least well characterized in terms of taxonomy, genetic content, and most scientists can’t even agree on whether they should properly be characterized as being ‘alive’ or not.
Given all of this, one might think that they would also be among the most exploited biological entities, harnessed for multiple applications in agriculture, food, medicine and nanotechnology? But this is not so. Phage were used in some former Soviet states for treating infection (phage therapy) , a few phage have been approved for use in foods in the USA and other countries to prevent foodborne illness, but in general they have been almost criminally underused. In this short piece I will focus on the advantages and disadvantages of using phage in medicine, specifically in treating or preventing infectious disease.
What are the advantages and disadvantages of using phage to treat infection?
One aspect of phage biology can be classed as both an advantage and a disadvantage, and that is the relatively narrow spectrum of most phages, especially in comparison with broad-spectrum antibiotics. This has the disadvantage that you either have to know which pathogen you are trying to kill before treatment is initiated, or you have to use a broad cocktail of phages to cover the most likely pathogens. On the other hand, given our better understanding of the importance of the microbiome to human health, and the emergence of antibiotic resistance in non-target species, a narrow spectrum could also be classed as a significant advantage of phage over antibiotics. Our ability to identify pathogens quickly, using more sophisticated bacterial diagnostic tools, has made this less of a problem than it was in the past. Equally, in the case of chronic infections or an infectious outbreak, rapid strain identification may not be a significant problem.
A second advantage is the self-replicating nature of phage. Theoretically, you only have to deliver one phage particle to the site of infection where it will then multiply until all the pathogens have been destroyed (without collateral damage to the surrounding microbiome). Once the pathogen has been eradicated the phage will have no host and will itself be rapidly eliminated – the perfect solution to an infectious event. Therefore, dosing should not really be a problem, and there are no off-target effects once the primary pathogen has been killed. Of course, real biological systems do not always follow theoretical models. In a complex large organ like the human gut for example, it may be difficult for phage to come into contact with the infectious agent in the first place, and so a high number of phage may have to be delivered to ensure an initial contact. Phage will also bind to mucous, be inactivated by the low pH in the stomach or bind to insensitive bacterial hosts, among other fruitless outcomes.
A significant disadvantage is bacterial resistance to phage [2,3]. Phage and bacteria have not co-evolved for billions of years without the bacterial cells acquiring strategies to resist phage attack. There are many resistance mechanisms, which include preventing phage binding to the surface of the cell, chopping up the phage DNA as it enters (restriction enzymes and CRISPR), and even committing suicide to block phage replication and save your ‘sister’ bacteria. Resistance can be expected, but there are ways to limit this. For example, a cocktail of phages can be used, since the possibility of becoming simultaneously resistant to two or more phages is significantly less than becoming resistant to one. This is the approach that has been used in most successful examples of phage therapy.
Another advantage of using phage is that they do not kill eukaryotic cells and have no record of toxicity in mammalian systems. As large protein structures they can be immunogenic but I am not aware of any instance where they have been shown to have an inflammatory effect, or have caused any adverse events. However, this may be the result of a lack of properly controlled clinical trials that would have to be conducted to identify any such adverse effects.
Phage and the microbiome
Most microbiome studies involve next generation sequencing of DNA isolated from a particular environment (biome), followed by mapping the millions of resultant short ‘reads’ to well curated databases [4–6]. In this way you can identify the microbes present in a microbiome and calculate their relative abundance. Phage are not normally included in microbiome studies, largely because they are so diverse they are not well represented in databases. Even if you physically separate the phage away from the rest of the microbes before sequencing, it is not unusual to have to describe more than 90% of the resultant reads as ‘viral dark matter’ . As an example, in a recent longitudinal study of ten individuals over 1 year we identified more than 60,838 phage/viral genomes or partial genomes, of which only 307 resembled known phage/viruses (0.5%) . This illustrates the diversity and complexity of the phage component of the microbiome. While this presents a significant challenge to microbiome scientists, the bigger question is what role phage might play in overall community structure and function of the microbiome . Do phage offer the possibility of precise microbiome editing? Can we alter the composition and function of the microbiome using phage or phage cocktails? This is an area which I believe will be the subject of a lot research in the next few years.
There are a number of additional challenges to be overcome if phage are to become evidence based alternatives in medicine. Firstly of course, we need the gold-standard evidence of well-conducted randomized controlled trials. There are decades of observational evidence from those countries that have been using phage to treat infections, but these will not convince the US FDA or EMA that phage are ready for primetime. There have also been some recent high-profile cases where phage have been successfully used in single individuals on compassionate grounds [9,10], but we have no idea how many times they have been tried but failed under these types of circumstances. Regulators will be rightly wary of administering a therapy where the biological entity is evolving even as it is being prepared (every preparation of phage will contain mutants), and where even basic taxonomy for phages is still under debate [11,12].
Equally, once administered the phage can continue to mutate as it multiplies within the human or animal host. In an ideal scenario, the clinician or pharmacist would have a flexible panel of phages and would choose the appropriate combination rationally once the bacterial target has been identified. But if every phage has to be prepared and maintained under GMP conditions, this would be prohibitively expensive. One useful suggestion is the to use the concept of magistral preparations. Under EU law, a magistral preparation is defined as “any medicinal product prepared in a pharmacy in accordance with a medical prescription for an individual patient”. This provides a practical way for a pharmacist to personalize a treatment from a bank of phages prepared to a particular standard. I refer the reader to the article ‘The magistral phage’ by Pirnay et al. for further information .
The future for phage
I am very optimistic for the future of phage in medicine. There are barriers to be overcome, but I cannot believe that we can continue to ignore the most successful biological entity on Earth as we seek new solutions for human health. Phage can be used to deliver therapeutic molecules, to display specific antigens , to sculpt microbiomes and to selectively eliminate pathogens. I hope that these next few decades will come to be known as the ‘Age of Phage’.
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