The Forgotten Belgian Bacteriophage Pioneers
Richard Bruynoghe
Alveringem, 4 November 1881 – Leuven, 26 March 1957.
Richard Bruynoghe received his doctorate in medicine in 1907 and became a full professor in 1918. He was founder and head of the Leuven Bacteriological Institute, a clinical research laboratory.
Reports of the English bacteriologist Frederick Twort (1915) and the French-Canadian microbiologist Félix d’Hérelle (1917), describing the capacity of certain bacterial filtrates to inhibit growth of specific bacteria, triggered his scientific interest.
In contrast to the Belgian Nobel Prize-winning Immunologist Jules Bordet, who attributed the observed bacterial inhibition to autolytic ferments (endogenous bacterial enzymes), Bruynoghe had always considered bacteriophages as ultramicroscopic microorganism. In contrast to d’Hérelle, who believed that only one type of bacteriophage existed that could adapt to various bacteria, Bruynoghe’s research team demonstrated that many different bacteriophages existed. They came to this conclusion because they observed that bacteria that had become resistant against a certain bacteriophage could still be inhibited by another bacteriophage. Since this observation was also made in the opposite direction, and was also observed for many bacteriophage – bacteria relationships within one bacterial species, they could conclude nothing but the existence of a multiplicity (or plurality) of distinct bacteriophages.
They also observed that animals injected with bacteriophages produced an immune serum, which neutralized these bacteriophages in such a way that growth of the bacteria was no longer inhibited. This neutralizing activity was specific for the applied bacteriophages, which was again an indication for the presence of many different bacteriophages.
In 1921, Richard Bruynoghe and his student Joséph Maisin authored the first ever scientific publication on the use of bacteriophages in a human therapeutic context. They reported on the successful application of bacteriophages to treat patients with Staphylococcus aureus wound infections.
In other words, already in 1921, Richard Bruynoghe’s team had shown that (i) “Le Bactériophage of d’Hérelle” in fact consisted of a plethora of diverse bacterial viruses; (ii) the application of bacteriophages readily resulted in the selection of bacteriophage-resistant strains, and (iii) bacteriophages could elicit a neutralizing immune reaction in mammals.
About two decades later, in 1940, bacteriophages were first visualized using electron microscopy, confirming that they are indeed diverse bacterial viruses. Unfortunately, Bruynoghe’s research had only been published in short notes in society proceedings and was quickly forgotten.
René Appelmans
Sint-Kwintens-Lennik, 29 April 1896 – Leuven, 5 November 1953.
René Appelmans graduated as doctor of medicine in 1922. During his studies, which were interrupted when he volunteered for military service in the First World War, he frequented the Richard Bruynoghe’s Bacteriological Institute. Immediately after obtaining his diploma, he left Bruynoghe’s laboratory to embark on a new path, the practice of surgery.
However, the few years he spent in the research laboratory in Leuven were enough for him to make a big impact on the bacteriophage field. For one, he and had a large part in demonstrating the plurality of bacteriophages. In addition, in 1921 he communicated two important phage-related studies in the “Société de Biologie”. First, he mapped the distribution of bacteriophages in the organism after oral administration and after injection. As such, he demonstrated already in 1921 that phages injected intravenously disappear from the bloodstream within a few hours.
Second, he developed a bacteriophage dilution method that is still used today to dose bacteriophages and to pre-adapt or evolve bacteriophages to exhibit increased virulence (titers), delayed bacterial resistance evolution, and/or a broadened host-range. This method is known today as “The Appelmans Protocol”. Using his dilution method, Appelmans demonstrated that bacteriophages were “living”, replicating organisms, and he was able to select bacteriophage variants able to resist to some chemicals otherwise toxic for most individual clones in the bacteriophage population. He also used his dilution technique to evolve bacteriophages capable of infecting other bacterial species, while maintaining their specific antigenicity in immune neutralization experiments, an additional clue in the direction of the plurality or multiplicity of bacteriophages.
André Gratia
Saint-Gilles (Brussels), 8 July 1893 – Nyon (Switzerland), 6 October 1950.
In 1910, André Gratia started to study medicine at the Free University of Brussels (Université Libre de Bruxelles), but his studies were interrupted in 1914 by the First World War. He joined the Belgian army and distinguished himself as an auxiliary doctor. After the war, in 1919, he graduated as a medical doctor and started a research career at the Rockefeller Institute for Medical Research in New York. During his research, he isolated the first bacteriophage of staphylococci and demonstrated that the phenomenon of Twort (1915) and of d’Hérelle (1917) were two manifestations of the same agent. Gratia published what was the first work on bacteriophages carried out in the United States in the American scientific literature as early as 1921. Unfortunately, because the article was published in a medical journal rather than microbiology journal, it escaped the attention of microbiology historians.
In 1921, he joined the Department of Bacteriology of the University of Brussels, which was headed by Jules Bordet. In 1922 he published results on the treatment of about fifty cases of bacteriophage therapy of staphylococcal infections in Brussels hospitals in which he had observed undeniable clinical improvements.
In 1924, André Gratia and Sara Dath published their observations on the lysis of Bacillus anthracis (Anthrax) by a mycolysate, a secretion of a mould (actinomycete). In 1925, they observed the lysis of an athrax culture by another lytic agent produced by a variety of Penicillium glaucum. Unfortunately, after what was the likely first discovery of an antibiotic (Alexander Fleming discovered penicillin in 1928), Gratia became seriously ill and had to abandon his research. The active substance might have been penicillin, but the molds were not preserved, so we shall never know. Fleming said, when interviewed for the Belgian radio, “I cannot refrain from mentioning one other Belgian bacteriologist my good friend Andre Gratia, and I mention him for the special reason that, but for circumstance, he might well have been the discoverer of Penicillin”. Gratia resumed his work in 1929 and went on to discover colicin, the first known bacteriocin.
Gratia developed new techniques in virology, such as ultracentrifugation and fractional centrifugation, and introduced the first electron microscope at the University of Liège, where he was offered the chair of Parasitology and Bacteriology (1932) and became full professor (1934).
In 1936, he developed what is known today as the double agar layer method. This method improved plaque assays by incorporating bacteriophages and target bacteria into a low-density top agar layered upon a high-density bottom agar layer.
André Gratia, passed away in 1950, likely due to exhaustion, when he arrived in Nyon (Switzerland) to enjoy some rest. André Gratia is not only a forgotten pioneer of bacteriophage, but also of antibiotic research.
In conclusion, the “forgotten Belgian bacteriophage pioneers” were (among) the first to:
- Demonstrate that bacteriophages are “living” organisms.
- Demonstrate bacteriophage plurality.
- Demonstrate the selection of bacteriophage-resistant bacteria.
- Demonstrate bacteriophages immune neutralization.
- Develop an improved bacteriophage enumeration method (double agar layer method).
- Develop a dilution method for bacteriophage enumeration and pre-adaptation (Appelmans protocol).
- Monitor the distribution and half-life of bacteriophages upon clinical application.
- Publish on bacteriophage therapy.