The Legacy of Bacteriophage

Early Discoveries

Bacteriophage have been used as therapeutic agents since the early 1900’s. Ernest Hanbury Hankin reported the first observation of phage activity in India in 1896. He noticed a marked anti-bacterial action in the waters of Indian rivers Ganga (Ganges) and Yamuna (Jumna) against Vibrio cholerae. This activity destroyed cholera bacteria in culture. He demonstrated that it could pass through fine porcelain filters and was destroyed by boiling. He suggested that this activity might be responsible for restricting the cholera outbreak among the people that consumed the river water. He, however, did not probe the phenomenon any further.

Twenty years later Frederick Twort in England, and Felix d’Herelle from Canada, working at the Pasteur Institute in Paris, reported isolating similar filterable entities capable of destroying bacterial cultures. It was d'Herelle who named these entities, "Bacteriophages" (bacteria eaters) and pioneered the use of phages for treating Shigella dysentery in rural France.

Felix d`Herelle

D'Herelle was asked to investigate the outbreak of dysentery, which was afflicting soldiers engaged in fighting World War I. He quickly found that the dysentery was caused by bacteria (Shigella). He cultured the bacteria to study their growth and noticed that sometimes, clear areas (plaques) could be seen on plates of bacteria. He realized that something was killing the bacteria and he wondered if he could use whatever it was as a treatment to cure the dysentery.

So, d'Herelle started monitoring an individual patient carefully. Each day, he took samples of the man's feces and filtered them through a porcelain filter to remove any bacteria. He mixed samples of filtrate with bacterial cells and spread them on agar plates. Initially he saw nothing but on the fourth day he started to see plaques. He now performed a direct test - he recovered the material from a plaque and mixed it with a flask containing a growing culture of bacteria

In d'Herelle's own words- "The next morning, on opening the incubator, I experienced one of those moments of intense emotion which reward the research worker for all his pains: at the first glance I saw that the culture which the night before had been very turbid, was perfectly clear: all the bacteria had vanished, they had dissolved away like sugar in water. As for the agar spread, it was devoid of all growth and what caused my emotion was that in a flash I had understood: what caused my clear spots was in fact an invisible microbe, a filterable virus, but a virus which is parasitic on bacteria." (The Bacteriophage by Dr. Felix d'Herelle, Science News 14: 44-59 (1949). (Translation by J. L. Crammer)

His success stimulated the commercial production of phages for treating a variety of bacterial infections both in Europe and the United States in the 1920's and 1930's.

Felix d'Herelle came to India in 1927 and demonstrated the efficacy of anti-cholera phage in reducing the mortality rate in the Indian state of Punjab from 63% in the untreated group to 8% for the phage-treated group. Encouraged by these results, J.Morison, Director of the King Edward VII Pasteur Institute in Assam, tried phage therapy with impressive success. Whereas 300 to 500 people died during the cholera epidemics in 1925 - 1928: View THE CHOLERA CHART & FIGHTING CHOLERA & PLAGUE IN INDIA, following the use of phage in 1929, the death rates fell to less than ten per year in the period 1930 - 1935, the duration of the study (Transactions of the Royal Society of Tropical Medicine & Hygiene, Volume 28, pp 563-570, 1935 cited in Felix d'Herelle and The origins of Molecular Biology by William C. Summers, Yale University press, New Haven 1999; pp.125-144).

In the 1930s and subsequent decades, pioneering virologists such as Luria, Delbruck and many others utilized these viruses as model systems to investigate the nature of genetic material. These relatively simple agents have since been very important in the development of our understanding of all types of viruses, including those of man which are much more difficult to propagate and study. They are still a paradigm for many areas of biology.

The Beginning Years of Phage Therapy

Whereas Felix d'Herelle was highly successful in treating dysentery in rural France, cholera in India and later cured diseases like typhoid fever, bubonic plague, wound infections, avian typhosis and hemorrhagic septicemia of the buffalo using phage therapy, other early attempts to treat infections with phage gave mixed results. A review of phage therapy commissioned by the Council on Pharmacy and Chemistry of the American Medical Association concluded in 1934 " the evidence for the therapeutic value of lytic filtrates is for the most part contradictory " (Eaton, M.D. and Stanhope, B-J, Journal of the American Medical Association, Volume 103, pp. 1769 - 1776; 1847 - 1853; 1934 - 1939). The use of the phrase " lytic filtrates " in place of phage reflects the Commission's bias that the nature of phage was not yet known. This assessment had adverse effects on further exploration of phage therapy in the United States and the advent of antibiotics later in that decade (1940) effectively stopped the development of phage therapy except in the former Soviet Union and Eastern Europe. In USSR, thousands of bacterial strains and bacteriophages that killed them were collected, isolated and characterized. The most promising ones were selected for therapeutic studies and tested. Phage mixtures such as "intestiphage" and "piophage" were routinely available in the pharmacies throughout USSR. However, little was known of this work in the western world. Clinical studies done in Poland (1981-2000) documented in detail the efficacy of bacteriophage therapy. These studies showed that the therapy had 75-100% success rate (92% overall) and 84% of patients demonstrated full elimination of the suppurative process and healing of local wounds. (B. Weber-Dabrowska, M. Mulczyk and A. Gorski, Arch Immunol. Therap.Exp.48, 547-558, 2000).

Significant discoveries in genetics are associated with phage research. For example: that DNA is the genetic material, the involvement of messenger RNA in protein synthesis, DNA and protein are co-linear in prokaryotes, the use of a triplet genetic code, discontinuous synthesis of DNA, chemical reaction as the underlying basis of life, the existence of restriction enzymes and so on.

Therapeutic use of phages in animals

H. W. Smith and R. B. Huggins in Great Britain reported successful treatment of experimental E.coli infections in mice and diarrhea in calves using phages. They highlighted the general superiority of phages over antibiotics on counts of significantly smaller dosage, complete stoppage of the associated fluid loss and 100% success rate.

Smith, H. W. and R. B. Huggins (1982) Successful treatment of experimental E.coli Infections in mice using Phage: its general superiority over Antibiotics. J. Gen. Microbiology 128:307-318.

Smith, H. W. and R. B. Huggins (1983) Effectiveness of Phages in treating Experimental E.coli Diarrhoea in calves, piglets and lambs. J. Gen. Microbiology 129:2659-2675.

Smith, H. W. and R. B. Huggins (1987) The Control of Experimental E.coli Diarrhea in calves by means of Bacteriophage. J. Gen. Microbiology 133:1111-1126)

Soothill, J. S., ( Burns, 20, 209 - 211, 1994) performed several experiments demonstrating the utility of phages in preventing/treating experimental infections with P.aeruginosa and Acinetobacter in mice and guinea pigs.

Vijayashree Ramesh, Joe A. Fralick and Rial D. Rolfe(1999) Prevention of Clostridium difficile-induced ileocecitis with Bacteriophage. Anaerobe 5: 69-78

Biswas, B. et al(2002) Bacteriophage Therapy Rescues Mice Bacteremic from a Clinical Isolate of Vancomycin-Resistant Enterococcus faecium. Infection & Immunity, 70: 204-210

Safety profile

Phages have been used successfully as therapeutic agents in Eastern Europe and the former Soviet Union, as well as in the United States before the antibiotic era. Phages have been administered to humans by a variety of routes, including intravenously. There are no reports of serious adverse effects associated with their use (Sulakvelidze et al Antimicrobial Agents & Chemotherapy, 45, 649 - 659, 2001).

In the United States, the phage phi X174 has been used as a marker for evaluating the immune response in patients with persistent generalized lymphoadenopathy (Ohs et al, J. Clin. Immunol. 8: 57 - 63, 1988), in adenosine deaminase deficient patients (Blood, 5: 1163 - 1171, 1992), for analyzing the importance of cell-surface associated molecules in modulating the immune response(intravenous administration; Ohs et al, Clin. Immunol. Immunopathol. 67: S33 - S40, 1993) and in a recently completed study evaluating the immune competence of HIV patients sponsored by the National Institute of Arthritis and Infectious Diseases (NLM identifier NCT00001540 ).

Owing to their extraordinary specificity for bacteria, they appear to have no effect on mammalian cells. Phages are ubiquitous in the environment, abundant in aquatic environments (2.5 x 10^8/ml, Bergh et al, Nature, 340, 429, 1989) and are regularly consumed in foods. Lytic phages pose no known risk to anything other than their specific bacterial host.