Phage therapy

Definition

Phage therapy involves the use of viruses that attack bacteria to treat pathogenic bacterial infections. The advantage of such viruses, known as bacteriophages or phages, is that they selectively target and destroy certain bacteria without harming the host organism or other beneficial bacteria, such as gut flora, thereby minimising the possibility of complications. Most therapies use lytic phages, which take over the machinery of the bacterial cell and then destroy the cell.

The diagram shows how a phage infects a bacterial cell. Once a phage infects a bacterium, it takes over its cellular machinery to shut down its defence mechanism and synthesise new phage particles. The number of phage particles synthesised eventually reaches a point where they rupture the bacterial cell which allows them to be released into the environment to infect a new host.

Importance

Phage therapy is now seen as a major alternative treatment to the use of antibiotics. Spurred on by the rising incidence of antimicrobial resistance (AMR) in recent years, phage therapy is now considered an important means to treat various bacterial infections not amenable to antibiotics. With AMR predicted to cause up to 10 million deaths by 2050, far exceeding any other infections or even cancer (O’Neill), phage therapy is one of several approaches scientists are intensively exploring to treat bacterial infections. Indeed, as Table 1 shows phage therapy offers some advantages over the use of antibiotics.

Phage therapy has already been shown to be effective in animals for treating various multidrug resistant pathogens, such as Acinetobacter baumannii, Pseudomona aeruginosas, Escherichia coli and Klebsiella spp. All three pathogens are serious threats to patients in the hospital setting and to those with weakened immune systems. A. baumanii is associated with life-threatening diseases such as pneumonia (inflammation in the lungs) and meningitis (brain and spinal cord infection). P. aeruginosa is no less deadly, a frequent cause of chronic lung infections.

Just how much interest phage therapy is now generating can be seen by the amount of money currently being invested in such treatment. Armata Pharmaceuticals alone (a merger of AmpliPhi Biosciences and C3J Therapeutics) recently received over $130 million in funding towards the development of engineered phages and other antimicrobial approaches (Schmidt). In addition to corporate funding, between 2013 and 2017 the European Union invested 5 million euros in research to investigate the use of phages to prevent skin infections in burn victims (Phagoburn). Similarly, in 2018 the University of California San Diego School of Medicine awarded a three-year $1.2 million grant towards the advancement of phage therapy through the Center for Innovative Phage Applications and Therapeutics (Dall).

For more information on what phages are and how they work watch 'The Deadliest Being on Planet Earth - The Bacteriophage' by Kurzgesagt'

Discovery

The discovery of bacteriophages dates back to 1915, when Frederick William Twort, a British bacteriologist based at Brown Institution in London, experienced difficulties growing the vaccinia virus, a key component of the smallpox vaccine, on agar plates. The only thing that he could find on the plates were contaminating bacteria which soon after died. Twort noted that the death of the bacteria appeared to be linked to the presence of some sort of viral agent that, unlike bacteria, could pass through a porcelain filter. He published his findings in 1915 but was unable to take his work any further because of the First World War and financial constraints. By 1917 Félix d’Herelle, a French-Canadian microbiologist, had independently discovered what appeared to be a similar substance while working at the Pasteur Institute in Paris. He recovered it by filtering stools taken from recovering dysentery patients. Promisingly, it appeared to be capable of killing shigella bacteria. Concluding the substance to be a virus particle, d’Herelle called it a ‘bacteriophage'. Both d'Herelle and Twort are now credited with the discovery of bacteriophages.

The first medical application of phages in humans was undertaken by d’Herelle. In 1919 he administered phages to four patients at the Hospital des Enfants-Malades in Paris. They were suffering from dysentery, an intestinal infection often caused by Shigella bacteria, which can cause bloody diarrhoea, stomach cramps and a fever. D’Herelle had already demonstrated the safety of phages by administering them to himself, his coworkers and his family members. Each patient was given an oral dosage of the anti-shigella phages and quickly showed signs of recovery within a day. This was just the start of a number of experiments that d’Herelle undertook (d’Herelle).

While d’Herelle took time to publish his results, his work proved sufficiently inspiring to Richard Bruynoghe, a physician at Katholieke University Leuven, Belgium, and Joseph Maisin, his student, to try out the use of a phage to treat staphylococcal skin disease in patients. Reporting on their experiment in 1921, they found clear evidence of recovery within 48 hours after injecting an anti-staphylococcal phage into the patients’ infected areas (Wittebole, De Roock and Opal).

In 1925 d'Herelle’s treated four individuals in Egypt diagnosed with bubonic plague caused by Yersinia pestis bacteria. All four patients made a full recovery within a month after having been injected with d’Herelle’s anti-pestis phage mixture. The tale of this success caught the attention of Dr. A. Morrison, the British representative of the Quarantine Board in Egypt, who engaged d’Herelle to work with the Quarantine Board on phage therapy. By December 1926, d’Herelle had been requested to help with the Bacteriophage Inquiry set up by the Indian Office in collaboration with the Indian Research Fund Association (IRFA), several hospitals and research institutes. Begun in 1927 and completed in 1936, the Inquiry was designed to study the effect of phage therapy as a treatment for cholera, an intestinal infection that causes severe watery diarrhoea. Three studies were carried out in Kolkata and Kasauli within the first year. With all three showing promising results, phage therapy gained attention as an item of ‘particular importance’ at the 7th Congress of the Far Eastern Association of Tropical Medicine in Kolkata in 1927 (Summer, 1993).

Despite the Inquiry’s initial success, d'Herelle left it in 1927 to become a professor at Yale University. Igor Asheshov, a Yugoslavian bacteriologist, continued in his place. The Inquiry thereafter suffered a series of setbacks, hastened on by Asheshov’s shift in focus from clinical studies to basic research. To make matters worse, the IRFA cut its funds from 64,260 rupees to 8,500 rupees between 1932 and 1934. The Inquiry’s last study was completed in 1936. By this time Asheshov had managed to provide extensive classifications of different cholera phage strains and their various biological properties as well as to establish how they could be used for therapy. However, he had failed to fulfill the main object of the Inquiry which was to determine their usefulness for the treatment or prevention of cholera. His efforts in this area had been hampered by poor cooperation from both staff and patients and poor design of the trials, all of which served to undermine the end results. Increased political unrest in India in the mid-1930s further undermined the project (Summer, 1993).

The difficulties experienced during the Bacteriophage Inquiry took place against a wider controversy about the nature of phages and the immune system. Throughout the 1920s a number of hypotheses were put forward about the nature of phages. On the one side was d’Herelle who saw phages as a living parasite or virus, which after infecting bacteria went on to destroy them as part of their life-cycle. And on the other side there was Bordet, who viewed the phage as a self-perpetuating chemical enzyme produced by bacteria. Having been awarded a Nobel Prize in 1919 for his work on antibodies and immunity, Bordet’s phage theory carried far more weight in the scientific community than that of d'Herelle who then lacked any real institutional base.

Initially, scientists had very little understanding of how phages destroyed bacteria. Most observations in the early 1920s were confined to lytic phages, a strain that triggers the disintegration of the bacteria’s cell membrane, a process known as lysis. The lytic phage does this following its replication within the bacteria’s cells. This process is known as the lytic cycle. Another pattern, called the lysogenic cycle, was discovered in 1925 by Bordet and colleagues in another type of phages known as the temperate phage. In this situation the phage injects its DNA into a bacterial cell where it integrates into the host’s genome and becomes what is known as the prophage. This prophage will continue to replicate alongside the host genome without causing any harm unless the host conditions become unfavourable, in which case the prophage may initiate the lytic cycle.

Despite the advances in understanding how phages worked, arguments raged over their efficacy as treatment. This surfaced strongly in 1934 and 1940 when the American Medical Association (AMA), the largest association of physicians in the United States, issued two reports on phage therapy. What troubled the AMA in particular was the experimental design used by d’Herelle and others to test out the therapy because they had failed to include any reliable control groups in clinical trials.

While criticism of the efficacy of phage therapy continued to rumble on, it soon became clear that d’Herelle’s vision of phages as viruses held true. This was prompted by the availability of the first electron microscope image of a phage in 1940. Acceptance of phages as viruses, however, took time to materialise. In part this was because World War II prevented the widespread distribution of the new electron microscope image. Strikingly, the AMA only acknowledged d’Herelle’s concept of a phage as a virus in its third report, issued in 1945 (Morton, Engley). By the time the first electron microscope images of a phage had been produced, a number of steps had been taken to commercialise phage therapy. Amongst those leading this effort was d’Herelle, who in 1928 gained a position at Yale University. By the early 1930s d’Herelle had founded his own commercial laboratory, Laboratoire du Bacteriophage, in Paris, where he produced the first commercial phage preparations against various bacteria that cause diarrheal and upper respiratory infections. The preparations were marketed by Robert et Carriere, a French company later acquired by L’Oreal (Pirnay et al. 2012). Major companies such as Parke Davis, E.R. Squibb and Sons, Swan-Myers and Eli Lilly in the US and Western Europe soon followed suit by developing their own anti-staphylococcus phage therapies. Such products were directed toward treating various infections, including abscesses, festering wounds, upper respiratory tract infections and mastoid infections (Sherman).

Despite the initial commercial enthusiasm for phage therapy, the approach was largely abandoned in North America and Western Europe by the 1940s. In part this was due to the arrival of antibiotics. The spread of phage therapy was also hindered by a lack of convincing clinical data. Poor understanding of the biology of phages and phage-host specificity all contributed to poor results collected in many trials run up to this time. Added to this was the difficulty of securing standardised therapies. Characterisation of the preparations was also poor. Most were characterised as either strong or weak, depending on how completely they could clear an infection. Many were in fact subsequently found to be inactive. Varying instructions were also given as to how the treatment was to be given. All these factors made it difficult to accurately assess the efficacy of products in clinical trials (Summers 2001).

The one place where the idea of phage therapy was kept alive was in the Soviet Union, helped in part by d’Herelle who in 1933 took up a visiting post at Eliava Institute of Bacteriophages, Microbiology and Virology. D’Herelle moved to the Institute, in Georgia, following a dispute with Yale University over his funding and research plans. Founded in 1923 by George Eliava, one of d’Herelle’s protegees, the Institute had a strong partnership with the Kharkov Mechnikov Institute, a Ukranian institution founded in 1886 to combat infectious diseases among soldiers. The two institutes joined together in 1934 to advance the development of phage therapy, and in 1936 received support from the Communist Party to begin construction of a new research institute and accommodation for both d’Herelle and Eliava. The Georgian Institute, however, suffered a major blow during the Great Terror in Soviet Union between 1936 and 1938, which saw d’Herelle return to Paris and Eliava being executed on the drummed up charges of espionage for France, sabotaging vaccines and poisoning drinking wells with bacteriophages.

Despite the setback, the new research institute was built and merged with the Research Institute of Microbiology, Epidemiology and Bacteriophage in 1939, known as the Tbilisi Institute of Microbiology, Epidemiology, and Bacteriophage (IMEB). Several phage therapy research programmes were launched through the IMEB, financed by the Soviet state and led by Alexander Tsulukidze, who had worked closely with Eliava and d’Herelle, and Elena Makashvili, a long-standing assistant of Eliava. The scientists soon developed methods for the mass production of phage therapies, including for the treatment of cholera, dysentery and typhoid.

Astoundingly, in 1942, the Soviet microbiologist, Zinaida Ermol’eva, managed to set up her own production of cholera and dysentery phages in the midst of the prolonged siege of Leningrad which saw many people struck down with such enteric diseases. Despite some diversion of funding into antibiotics after the Second World War, Georgia remained an important centre for phage therapy into the 1950s (Myelnikov). Phages continued to be routinely used for the treatment of several bacterial pathogens in Soviet Union and other parts of Eastern Europe as a cheaper alternative to antibiotics throughout the latter half of the 20th century. Just how widespread it became can be seen by the fact to this day Russians and Georgians can buy over-the-counter phage remedies to treat upset stomachs, urinary tract infections and many other disorders. In some clinical trials phage therapy have proven effective in treating patients previously not responsive to antibiotics (Sulakvelidze, Alavidze; Sachs; Myelnikov).

Attitudes towards phage therapy began to change in the West by the 1980s as a result of the rising threat of antibiotic resistance. One of the first to pursue phage therapy in the West was Herbert Williams Smith, a British veterinarian and experimental scientist at the Institute for Animal Disease Research in Houghton, Cambridgeshire. From the 1950s he began to investigate the rise of AMR in livestock which he concluded was caused by the overuse of antibiotics both as food additives to promote growth and as a preventative against infection. Familiar with d’Herelle’s work, Smith was convinced that the failure to demonstrate the therapy’s success was because the wrong methods had been used in the past (Datta). In 1981, together with Mike B. Huggins, Smith launched a series of experiments in different animals to explore the possibility of phage therapy. The animals were infected with diarrhoea-causing E.coli and successfully treated via intramuscular injection or oral administration of a phage mixture. Their work not only dispelled the stigma of the inconsistency of phage therapy, but also demonstrated phages to be potentially more effective than antibiotics. On publishing the results, Smith pointed out that one advantage that phages had over antibiotics was that once introduced they multiplied so continuous dosage was not necessary (Smith, Huggins 1982, 1983, 1987; Datta).

The success of Smith and his colleagues with phage therapy was soon repeated by other researchers in other animals. This work helped to rekindle interest in testing out phage therapy in the West to treat bacterial infections in humans. One of the key figures behind its promotion was Alexander Sulakvelidze, a Georgian molecular biologist trained at Tbilisi State Medical University, who took up a postdoctoral research fellowship with Glenn Morris, an American epidemiologist, at the University of Maryland Medical Center in 1993. Arriving just at the moment when Morris was struggling to contain an outbreak of vancomycin-resistant enterococcus which was causing the death of many patients, Sulakvelidze suggested, based on the routine use of phage therapy in Georgia, that a solution might lie in phage therapy. Within three years the two scientists had managed to isolate ideal phages for such purposes, sourced from the Baltimore Inner Harbour, and had formed an ongoing research partnership with the Eliava Institute. They also set up a new company, Intralytic, in 1998, which began developing phage-based products for use in livestock. The company won FDA approval for its first phage product, ListShield, in 2006, a food additive that targets listeria in meat and later in 2011, for its second product EcoShield, which targets E. coli O157:H7 (Sachs; Martin; Lang).

Application

For every strain of bacteria, there exists several phages that can kill it. One of the biggest challenges is to find the right phage strain for treatment. Luckily, phages can be found wherever bacteria exist, including in the soil, rivers, plants and animals. When isolated from the environment, the phages are tested against common antibiotic-resistant pathogens. This typically involves infecting a bacteria culture with the isolated phages. Phages that are capable of lysing the bacteria culture are recovered and purified. Several strains of phages are often administered together in the form of a cocktail. This method provides a means to compensate for the high specificity of each strain. Most cocktails tested in clinical trials consist of at least three phages with overlapping host strains.

Efforts are also now underway to genetically engineer phages to enhance the bacterial killing capacity of antibiotics. In 2009 Lu and Collins demonstrated that it was possible to genetically modify phages to suppress the SOS DNA repair system bacteria use to ward off the effects of antibiotics. Such a method could pave the way to the use of phages in combination with antibiotics. The phage engineered by Lu and Collins, for example, helped increase the bactericidal effect of the antibiotic ofloxacin.

Numerous clinical trials are now being conducted to test out phage therapy in humans. In 2009 the FDA approved the first phase I clinical trial to assess a cocktail of eight phages to treat venous leg ulcers in humans. The phage mixtures were demonstrated to have no adverse effects in 42 patients (Potera; Rhoads et al.). In the same year, the UK also held its first phase II phage clinical trial to assess the efficacy of phages against chronic otitis, an ear infection caused by an antibiotic-resistant P. aeruginosa. The phage treated group of participants received a combination of up to six phages who had a significantly lower bacterial count after 42 days (Wright et al.).

Phage therapy is also being explored for the treatment of milder diseases such as acne. While seemingly harmless, up to 60 per cent of Propionibacterium acnes are antibiotic-resistant and can be a persistent cause of discomfort. In 2012 a team of dermatologists at the University of California identified a variety of phages effective against a broad range of antibiotic-resistant P. acnes. This study, however, discovered that the bacteria causing the acne soon developed resistance to the phage therapy. Such resistance was due to the presence of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), an immune mechanism that bacteria use to cleave any foreign DNA. Coincidentally, the phages used in this study contained a DNA sequence recognised by the P. acnes’ CRISPR system, resulting in phage DNA being cleaved and inactivated upon entry into the cell. This resistance, however, was circumvented by genetically engineering phages to remove the recognised DNA sequence (Marinelli et al. 2012).

Although phage therapy has yet to receive regulatory approval to treat humans in the European Union and the United States, it can be used in these regions for compassionate treatment where all other conventional interventions have been exhausted. There have been 29 reports of compassionate use of phage therapy since 2000, half of which were published between 2017 and 2019. The therapy has been most frequently used to treat patients with S. aureus infections, followed by P. aeruginosa and E. coli. One of the best known cases is that of the 68-year-old diabetic, Tom Patterson, who in 2017 was suffering from necrotising pancreatitis, an extreme case of inflammation of the pancreas. His condition was caused by a multidrug resistant A. baumannii infection with no effective antibiotic treatment available. Patterson was successfully treated with a combination of phages contributed by various research institutes across the US and Patterson (Schooley et al.; McCallin et al.).

Beyond treating bacterial infections, the high specificity of phages allows for them to be harnessed as tools for cancer therapy. Although phages only normally target bacteria, Przystal et al. for example, have demonstrated that the M13 phage can be modified to specifically bind to human tumour cell receptors. They also incorporated an adeno-associated virus genome into the phage, a therapeutic gene which triggers cell death upon activation within tumour cells. A combination of modified M13 phages and the conventional chemotherapy drug temozolomide have shown significant improvement in suppressing growth of glioblastoma, the deadliest form of brain tumours with only 2 per cent 5-year survival rate.

In addition to their therapeutic applications, a phage’s ability to home in on specific bacteria is a useful characteristic for diagnosis. One example is that of fluorophages. These are phages that have been genetically engineered to carry a fluorescent protein that glows when they infect their target bacteria. In 2012 a team led by Jain used such phages to target Mycobacterium tuberculosis, the cause of tuberculosis (TB). The fluorophage allows for the detection of TB in less than a day, in contrast to the conventional cell culture tests that can take up to two months (Jain et al.).

Issues

Although phage therapy has been around for nearly a century, phage therapy has yet to obtain regulatory approval in the US and Europe. Part of the problem stems from the fact that phage therapy does not mimic conventional industrially-made pharmaceutical products. The very fact that phages can self-replicate in the body makes the therapy very different from other drugs like antibiotics where its concentration in the body diminishes over time. Moreover, phages are highly specific and often administered in the form of a cocktail to cover a wide range of target strains. The use of several phages at once makes it difficult to monitor their replication in the body, which is the indication that the phages are working. Just how difficult it can be to get an approval was emphasised by James Soothill, a consultant microbiologist at Great Ormond Street Hospital who in May 2019 reported the successful use of phage therapy to treat a 15-year old cystic fibrosis patient who acquired a Mycobacterium infection after having a double-lung transplant (LeMieux). He suggests that individual patients should be initially treated with low doses to allow phages to be counted. This may provide useful and perhaps the only evidence of phage’s efficacy in individual monitored treatments, where scientists cannot make the comparison between the treated and placebo groups like in clinical trials. The establishment of phage therapy organisations in possession of well-characterised phages that could oversee the entire process would be valuable. This would facilitate monitored treatments where low doses of specific phages could be administered and evidence of multiplication sought (Interview with Soothill).

Preparation of a phage cocktail is no easy feat either. In some cases the phages need to be shipped, which poses biosafety questions. Regulation of their production is also challenging, as they are usually prepared outside of a pharmaceutical company setting (Fauconnier). Belgium is currently pioneering a pragmatic framework for phage therapy that allows phages to be used in magistral preparations as long as they are prepared by a certified Belgian Approved Laboratory. 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 means for doctors to prescribe personalised treatment using unapproved ingredients. As of 2018 such magistral preparations could only be used to treat patients in Belgium, but other countries might adopt a similar scheme in the near future. A possible pitfall in Belgium’s strategy is that phage therapies are not currently eligible for reimbursement and therein lies another major challenge in developing phage therapy (Pirnay et al. 2018).

Development of phage therapy is expensive due to the cost of identifying suitable phages and purifying phage preparations. Purification is essential to its safety. The process removes any endotoxins released by bacteria upon lysis. Such endotoxins can trigger inflammatory responses in patients. Moreover, as is the case in the development of other drugs, phage therapies need to be produced in certified laboratories to ensure appropriate quality control and that they are safe for human use. All of this adds to the cost of phage therapy. Most treatments are also highly individualised for each patient who require a lot of monitoring during treatment. The total cost of such care can run up to tens of thousands of US dollars. In some places like the US, France and Australia, patients can get treatment free of charge based on compassionate grounds (McCallin et al.).

Difficulties in securing intellectual property (IP) protection further discourages entrepreneurs from investing in phage therapy. Patent law varies between different countries. For example, patenting a phage product in the UK mainly requires the invention to have a specific novel inventive step. This was achieved by Martha Clokie (2019), a researcher at University of Leicester who patented therapeutic bacteriophages to treat Clostridium difficile in August 2013 based on the fact it was counterintuitive to isolate a particular combination of phages in the environment that would target clinically relevant strains (JUSTIA Patent). In the US however, it is harder to patent natural products (Pirnay et al. 2012), however her patent has also been granted there.

As in the case of antibiotics, bacteria can develop resistance to phage therapy. Good stewardship in the development and handling of phage therapy is therefore important. Such resistance needs to be taken into account to design the best possible phage combinations to reduce the extent of this problem (Clokie). Yet bacterial resistance to phages is less of a problem than with antibiotics. Firstly, phages outnumber bacteria ten-fold and have enormous diversity. Secondly, the development of resistance to phages can reduce the virulence of the bacteria towards their host as well as lower their growth rate, shorten their life-span, and reduce their ability to attach to or invade the mammalian cell. Lastly, it is possible to genetically modify phages to make the evolution of such resistance harder. For example, phages can be engineered to carry plasmids, a small circular strand of DNA, designed to inhibit the action of a bacterial defense mechanism, such as the CRISPR system which aims to cleave phage DNA (Roach, Debarbieux).

While genetically engineered phages have gained traction in recent years, they have their own problems. Firstly, as with most genetically modified products, the release of engineered phages into the environment could have unforeseen negative consequences on the dynamics and ecosystem of the bacterial community (Nair, Khairnar). Some of the technical challenges were also highlighted by Antonia Sagona, a researcher at Warwick University involved in the engineering of K1F fluorescent phages. Among the difficulties she encountered was that most engineered phages are unstable and tend to ‘to mutate back to the wild type’. Such phages also have short shelf lives (Interview with Sagona). This will pose problems for manufacturers should phage products become commercially available in the future. Another concern was raised by Clokie regarding the regulation of such products. Regulating naturally-occurring phages is complex to begin with and this becomes more so for genetically modified products (Clokie). One approach to making mutants in a way that is more compatible with the current regulatory systems is through the use of techniques such as Bacteriophage Recombineering of Electroporated DNA (BRED) system (Marinelli et al. 2008). The BRED system makes it possible to delete a region of the phage DNA without introducing any marker genes. Interestingly, such mutants are not always classified as genetically modified. It was this method that Hatfull’s team used to develop a lytic derivative of a temperate phage to treat their patient with cystic fibrosis (Dedrick et al.).

This piece was written in November 2019 by Lara Marks and Jakrin Bamrungthai with generous input from Martha Clokie, Antonia Sagona and James Soothill.

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K.E. Thorpe, P. Joski, K.J. Johnst, ‘Antibiotic-Resistant Infection Treatment Costs Have Doubled Since 2002, Now Exceeding $2 Billion Annually’, Health Affairs, 37(4) (2018).

F.W. Twort, 'An investigation on the nature of ultra-microscopic viruses by Twort FW, L.R.C.P. Lond., M.R.C.S. (from the Laboratories of the Brown Institution, London)', Bacteriophage, 1/3 (2011), 127-129.

X. Wittebole, S. De Roock, S.M. Opal, 'A historical overview of bacteriophage therapy as an alternative to antibiotics for the treatment of bacterial pathogens', Virulence, 5/1 (2014), 226-235.

A. Wright, C.H. Hawkins, E.E. Anggard, D.R. Harper, 'A controlled clinical trial of a therapeutic bacteriophage preparation in chronic otitis due to antibiotic-resistant pseudomonas aeruginosa; a preliminary report of efficacy', Clinical Otolaryngology, 34/4 (2009), 349-57.

Phage therapy: timeline of key events

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