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“Tiny and deadly bacillus has enemies still smaller”

Full article by Joshua D. Jones

  • Summary
  • Introduction
  • References

One hundred years ago British virologist Frederick Twort made a remarkable observation, he noticed that small clear areas appeared on some of his bacterial plates. Upon closer investigation he deduced that a filterable agent was responsible for killing the bacteria at these sites. However, soon after his discovery Twort joined the army to serve as a medic during World War One, and did not pursue his finding further after the war. Several years later, a remarkably similar observation was made by the Frenchman Felix d’Herelle.

Although neither knew exactly what they had found, both Twort and d’Herelle had independently discovered viruses of bacteria – termed bacteriophages by d’Herelle.It has been estimated that there are 1030 bacteriophages – also known as phages – on the planet, some 10 billion times the number of stars in the universe. Phages have been, and continue to be, extensively studied in molecular biology, and were used in the famous 1952 Hershey-Chase experiment to demonstrate that information was inherited in DNA instead of protein. Crucially, phages are extremely specific viruses, only able to bind to the surface molecules of the particular strain of bacteria to which that virus has adapted. Replication occurs via either a lytic or lysogenic cycle; lysogeny being the integration of the phage genome into the bacterial DNA, with the potential for later excision.

Although not glamourous, the phages to treat many antibiotic resistant infections lurk amongst our waste, with their isolation being relatively straightforward. However, there remain key challenges if phages are to ever become a routine therapeutic option. Current regulatory frameworks have been designed to assess traditional molecular therapeutics, not dynamic biological agents. Public opinion too may need convincing of the use of bacteriophages to treat recalcitrant infections. Crucially the development of phage therapy is reliant on attracting sufficient funding, and present investment from major pharmaceutical companies is scant. Alongside continuing research into phage therapy, phage-based alternatives are also being examined, these include the potential of using isolated phage proteins as antibiotics, such as holins and lysins which degrade the bacterial cell membrane and wall respectively.

This article is a summary of the winning talk given at the Liverpool Medical Institute History of Medicine Prize evening on the 10th of November 2015.
One hundred years ago British virologist Frederick Twort made a remarkable observation, he noticed that small clear areas appeared on some of his bacterial plates. Upon closer investigation he deduced that a filterable agent was responsible for killing the bacteria at these sites. However, soon after his discovery Twort joined the army to serve as a medic during World War One, and did not pursue his finding further after the war. Several years later, a remarkably similar observation was made by the Frenchman Felix d’Herelle. An autodidactic scientist of insatiable curiosity and drive, d’Herelle had travelled extensively in pursuit of microbiology. When a locust invasion struck the Mexican agave plantation he was working on in 1910, d’Herelle noticed some locusts suffered a diarrhoeal infection and he hoped to employ this as a form of biological control. During this research, he examined bacterial cultures from locust faeces and noted the presence of clear areas on his plates. D’Herelle continued his research at the Pasteur Institute in Paris, and in 1917 he demonstrated that a filterable agent present in the stools of convalescent shigellosis patients was capable of lysing Shigella cultures. Although neither knew exactly what they had found, both Twort and d’Herelle had independently discovered viruses of bacteria – termed bacteriophages by d’Herelle.
It has been estimated that there are 1030 bacteriophages – also known as phages – on the planet, some 10 billion times the number of stars in the universe. Phages have been, and continue to be, extensively studied in molecular biology, and were used in the famous 1952 Hershey-Chase experiment to demonstrate that information was inherited in DNA instead of protein. Crucially, phages are extremely specific viruses, only able to bind to the surface molecules of the particular strain of bacteria to which that virus has adapted. Replication occurs via either a lytic or lysogenic cycle; lysogeny being the integration of the phage genome into the bacterial DNA, with the potential for later excision.
Following his discovery of phages in 1917, d’Herelle continued his research, and began to explore the application of phages as a therapeutic. By 1919, after conducting successful studies in fowl, d’Herelle was ready to apply his novel therapy to a human case. The first patient of phage therapy was a young boy, Robert K, who was admitted to the children’s hospital in Paris with severe shigellosis. D’Herelle administered phages to the boy, and ingested a dose 100 times greater himself to prove the safety of the treatment. Robert made a full and rapid recovery, heralding the beginning of the era of phage therapy. News of the case spread across Europe, and throughout the 1920s and into the 1930s many laboratories and hospitals experimented with phage therapy. In the early 1930s large field trials were conducted in the region of Assam, India, in which two districts annually afflicted by cholera epidemics were studied. Phage distribution replaced vaccinations and well disinfection in the district of Nowgong, while measures to tackle cholera remained unchanged in the control district of Habiganj. Come the annual cholera epidemic Nowgong was relatively spared, while Habiganj was ravaged. Although poorly controlled, these studies broadly demonstrated the efficacy and wider potential of phage therapy. With continued studies into phage therapy, awareness of this novel treatment grew in the public mind, such that the title of this article appeared on the front page of the New York Times on the 27th of September 1925.
Nevertheless, phage therapy began to run into seemingly insurmountable challenges. Given the highly specific nature of phage infection, clinicians were required to conduct phage typing to deduce which phages could infect that particular strain of bacteria. However, some clinicians began to omit phage typing, thereby rendering their administered therapy ineffective and contributing to the growing number of conflicting reports surrounding the efficacy of phage therapy. Industry too ran into challenges, with pharmaceutical companies often finding that their preservatives inactivated the phages. There was also substantial inconsistency in the number of phages used, with volumes of an undefined phage stock often being quoted in the literature, further hindering reproducibility and efficacy. Moreover, many of the studies into phage therapy used small sample sizes, leading some to question their significance. Studies were often poorly controlled too, and some researchers were cautious that the phages were responsible for clearing the bacterial infection, instead suggesting that the inevitable coadministration of bacterial components primed the immune system to fight the infection. Today we know this was a prescient question, with toll-like receptor stimulation being the basis for many of the adjuvants used in modern vaccines. The uncertainty that remained over the nature of phages further contributed to the growing scepticism surrounding phages. Phage particles were first observed using electron microscopy by Helmut Ruska in 1939, and while many in the preceding years,
including d’Herelle, had believed phages to be viruses some contemporaries had promoted the idea that phages could be an enzyme or chemical entity which was capable of dissolving bacterial cultures. Much of the criticism surrounding phage therapy was summarised in an influential 1934 paper in the Journal of the American Medical Association that reviewed the available research; the authors suggested phage therapy was of limited use beyond the treatment of localised staphylococcal infections or cystitis – however later reports in the literature were somewhat more positive about the prospects of phage therapy.
Ultimately during World War Two it was imperative to treat injured or ailing soldiers for return to the battlefield as quickly as possible, and scientists on both sides turned to bacteriophages. Germany quickly embarked on production, but ran into challenges. The Americans meanwhile took a more meticulous approach to phage research. However, serendipitously discovered by Fleming in 1928, penicillin offered an alternative to phage therapy, free of laborious phage typing, comparatively easier to produce and consistent. Developed further by Florey and Chain in Oxford, who later together shared the 1945 Nobel Prize with Fleming, the allied mass production of penicillin began in 1943. Consequently Western research into phage therapy declined in favour of the pursuit of antibiotics. Further east, the USSR invested in sustained production of phages, helped in part by the friendship between Felix d’Herelle and the Georgian scientist George Eliava, who founded the Eliava Institute in Tiblisi in 1923. At its peak during WW2 the Eliava Institute produced around two tonnes of phage product per week, the majority of which went to the Red Army. The Institute still exists today and holds an extensive library of phages; phage products are still used in Georgia, with phage therapy being issued to Georgian soldiers as recently as the 2008 dispute with Russia.
The use of phage therapy remained largely forgotten in the West until Smith and Huggins, a pair of veterinarians working near Cambridge, published two elegant studies in 1982 and 1983. The first of these studies involved infecting 30 mice with 100 times the lethal dose of Escherichia coli K1, after eight hours the mice were treated with eight doses of an antibiotic or one dose of K1 phage. Eight doses of tetracycline, ampicillin, chloramphenicol or co-trimoxazole were only sufficient to rescue up to four mice; streptomycin was more efficacious, with eight doses rescuing 27/30 mice. However, of the mice given one dose of K1 phage 28/30 survived, demonstrating the superior efficacy of phages to many antibiotics. Crucially, Smith and Huggins also performed a key control experiment, administering lysed E. coli bacteria to mice infected with 100 times the lethal dose of E. coli K1. Only 2/30 of these control mice survived, demonstrating that the phage, not the adjuvant effect of the coadministered lysed bacteria were responsible for clearance of the infection. These experiments reignited interest in phage therapy in the West, which has been further stoked by the spread of antibiotic resistance. Unlike antibiotics, phage therapy is biologically dynamic and although bacteria can evolve resistance to phages, phages have spent millions of years evading and adapting to bacterial resistance strategies. Nevertheless, phage therapy has remained largely in the realm of veterinary science. Today phage products, such as those manufactured by the American company Intralytix, are available for the control of foodborne bacterial pathogens including Listeria monocytogenes and Salmonella. Human applications of phage therapy include the development of a biodegradable bandage impregnated with a phage cocktail to treat wound infection, known as ‘Phagebioderm’, the result of a collaborative effort between American groups and the Eliava Institute.
Faced with the growing spectre of recalcitrant antibiotic resistant it is unsurprising that interest in phage therapy continues to grow. Although not glamourous, the phages to treat many antibiotic resistant infections lurk amongst our waste, with their isolation being relatively straightforward. However, there remain key challenges if phages are to ever become a routine therapeutic option. Current regulatory frameworks have been designed to assess traditional molecular therapeutics, not dynamic biological agents. Public opinion too may need convincing of the use of bacteriophages to treat recalcitrant infections. Crucially the development of phage therapy is reliant on attracting sufficient funding, and present investment from major pharmaceutical companies is scant. Alongside continuing research into phage therapy, phage-based alternatives are also being examined, these include the potential of using isolated phage proteins as antibiotics, such as holins and lysins which degrade the bacterial cell membrane and wall respectively. The development of the CRISPR/Cas9 gene editing technology has also raised the possibility that phages could be used to engineer antibiotic susceptibility in bacteria during infection – essentially ‘treating’ the pathogen, not the patient. Ultimately, antibiotic resistance has the potential to dramatically turn the clock back on modern medicine, and it therefore remains imperative that all potential alternatives be explored.
Dr Joshua D. Jones is a molecular virologist with a broad interest in infectious disease. He completed his undergraduate and postgraduate training at Christ’s College, University of Cambridge, and is currently a second year medical student at the University of Liverpool.

Selected references & further reading

  •  Hausler, T. Viruses vs. superbugs: a solution to the antibiotic crisis. Macmillan, 2008.

Selected academic papers

  •  Abedon, S. T. Phage therapy of pulmonary infections. Bacteriophage 5, e1020260, doi:10.1080/21597081.2015.1020260 (2015).
  •  Abedon, S. T., Kuhl, S. J., Blasdel, B. G. & Kutter, E. M. Phage treatment of human infections. Bacteriophage 1, 66-85, doi:10.4161/bact.1.2.15845 (2011).
  •  Loc-Carrillo, C. & Abedon, S. T. Pros and cons of phage therapy. Bacteriophage 1, 111-114, doi:10.4161/bact.1.2.14590 (2011).
  • Nobrega, F. L., Costa, A. R., Kluskens, L. D. & Azeredo, J. Revisiting phage therapy: new applications for old resources. Trends Microbiol 23, 185-191, doi:10.1016/j.tim.2015.01.006 (2015).
  • Wittebole, X., De Roock, S. & Opal, S. M. A historical overview of bacteriophage therapy as an alternative to antibiotics for the treatment of bacterial pathogens. Virulence 5, 226-235, doi:10.4161/viru.25991 (2014).

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