The text itself indeed offers a clear and well argumented case which defines the diversity of phage in nature and reflects the existing discussions between the renowned researchers in this budding research field. A clear advocate of the Delbrück school of thought on the existence of the bacterial virus (contrary to Bordet’s views), his results obviously stem from his experimental work on lytic bacteriophages. The views of Bruynoghe are excellently summarized in the final paragraph of his manuscript, which offers a nuanced and critical view of the role of bacteriophages in medicine and the environment in general. From that perspective, his conclusions can be considered as surprisingly accurate, even by today’s standards. See .
Richard Bruynoghe (A) was affiliated as a professor at the K.U.Leuven within the Bacteriological Institute (B). He provided an overview of his research for a lay-audience within the local University magazine, Onze Alma Mater (C).
In the bacteriological institute, investigations of a scientific nature are performed, besides the investigations which directly benefit our patients. It is difficult to determine the scale of these activities.
In recent times, analyses of a clinical nature have increased strongly. It no longer suffices to determine the nature of the infective bacteria, often their susceptibility towards antibiotics (penicillin, streptomycin, aureomycin, chloromycetin,…) has to be determined. Add to this the numerous serological tests, requested by the hospital and doctors. To give you an idea about the work being performed, I should point out that we weekly perform about two hundred Bordet-Wassermann reactions [antibody test for Syphilis], eighty Rhesus determinations [blood typing] and numerous Widal reactions [diagnosing Typhoid fever], Wright stain [blood cell type differentiation], etc.
Our scientific research aims at gathering new information on various puzzles or to confirm recent discoveries of interest to us. So many topics have been studied within the Bacteriological institute, that it’s impossible to fully report on all of them. This is why it’s my pleasure to summarize our bacteriophage research. This research led to my 1931 prize for Medicine, which is awarded every five years.
The bacteriophage was discovered by Twort (1915) and d’Herelle (1917). These scientists observed that filtrates of certain cultures had the capacity to inhibit growth of specific bacteria for a certain time. According to d’Herelle this inhibition was caused by a virus which penetrates into the bacteria to make them sick. Professor J. Bordet had a different take on this phenomenon: according to him these filtrates damaged the bacteria, producing an autolytic ferment.
We have always considered bacteriophages as viruses and have observed that:
1. Contrary to the assumption made by d’Herelle, who thought a single bacteriophage existed that could adapt to various bacteria, we observed that many bacteriophages exist, which differ from each other like microbes do
We have come to this view because microbes which have become resistant against a certain phage, can still be inhibited in their growth, just like normal microbes, by another phage. Since this observation was also made in opposite direction, one had to assume the multiplicity of phages. Since this observation also was done in the opposite direction, the multiplicity of phages had to be accepted. To make this easier for you to understand, I should note that in 1921 we had two phages which inhibited the growth of the same Typhoid culture. The Typhoid culture which had become resistant against bacteriophage L (hence in its presence grew as on normal media) was inhibited in its growth by bacteriophage St. Since we could observe the same phenomenon in the opposite direction (i.e. the Typhoid culture, resistant against St, was inhibited by bacteriophage L), this could only be explained by the multiplicity of phages.
If this had occurred only in one direction, one could have explained the observation by assuming that one of the phages was more active than the other. However, in view that the growth inhibition occurred in both directions, this could only be explained by assuming that both phages are different, like the Typhoid culture differs from the paratyphoid bacteria.
Persons, cured from typhoid infection [Ebert’s bacillus (tubercle bacilli)], are no longer susceptible to this microbe, but can still be infected by the paratyphoid bacillus. Similarly, persons cured from paratyphoid infections still can be infected by Typhoid, simply because we are dealing with two different microbes.
We have also proved the diversity of phages by neutralisation experiments. An animal, injected with phage L., produces an immune serum which neutralises this phage in such a way that growth of the microbes is no longer inhibited. This occurrence is specific, such that the immune serum only neutralizes the bacteriophage used for immunisation.
The resistance of phages against heating also supports their multiplicity. Scientists have obtained different results: one claimed that a heat treatment at 60°C for one hour destroyed the phages; others found larger resistance and noticed phages could withstand heating at 70°C. We have explained these results by demonstrating that these experiments were not carried out by one and the same phage.
2. The multiplicity of phages implied that phages isolated with normal techniques often have a complex composition
(To isolate phage one adds, e.g., rincing water to broth. After 24 hours of incubation, the obtained culture is filtered in a porcelain filter, which retains the bacteria but let through the phages (virus). This filtrate has the above-mentioned properties: it inhibits susceptible bacteria, at least temporarily, in their growth).
We have observed this by neutralisation experiments. A serum obtained from animals which were immunized against bacteriophage A, neutralized this as well as another phage (which we indicate as B). The latter, injected into rabbits, produced a neutralising serum against bacteriophage B, but not against phage A. One may explain this if one assumes that bacteriophage A has a more complex composition, and includes, among others, bacteriophage B.
This complex make-up of certain bacteriophages has also been shown by heating experiments; bacteriophages heated to 65 degrees for one hour often show a simpler composition compared to phages heated at lower temperatures. This can be explained by the destruction of some sensitive phages. For this reason, the immune serum obtained by injecting animals with phages heated at higher temperatures, was not capable to neutralize the original phages. We should add that we have pointed out a technique to count phages in cultures. Their number usually ranges between 10 and 100 million per cubic centimetre. If one exceeds the dilution where no phages are found per cubic centimetre, then one will not find any phages in one or another cubic cm. This is because phages are corpuscular elements, which, once their maximal dilution is reached, can no longer spread in a homogenous fashion.
3. In our investgations we could also demonstrate the independence of phages
For example, a phage, cultured in symbiosis with coli bacilli, remains the same when it is co-cultured with entirely different microbes, like typhoid, paratyphoid and paradysenteric bacilli, etc. This would not occur if phages, as claimed by Professor J. Bordet, were a secretion product of microbes; in that case, their properties would shift in relation to the origin of the microbes, which is not the case if the phage remains independent. Similarly, we have observed that microbes infecting animals maintain their autonomy even when they infect humans. All these observations have contributed significantly to acceptance of the “bacteriophage-virus” concept.
We have, moreover, done other observations to support this view. We’ve noted that phages possess a power to adapt against harmfull influences, like heating at high temperature, like the impact of concentrated glycerin. Adaptability definitely is a characteristic of something alive.
Let us add to this that, since the introduction of the electron microscope, one has been able to assess the multiplicity of bacteriophages.
Furthermore, this view fits well with general biology. It would a peculiar phenomenon if nature only contained pathogenic bacteria against humans, animals and plants. For some disease-causing microbes, we have numerous saprophytic germs which are of use to man, or even essential to his existence.
Bacteriophages are now saprophytic viruses which are also useful to us. However, one should not exaggerate their role, like d’Herelle did, and attribute the recovery of illnesses to phages only. According to d’Herelle, immune substances which form during the disease course, are not the cause of the cure, but its consequence. Although we consider bacteriophages as useful to us in certain cases, we’ve never taking this exaggerated point of view. Certainly, these phages do play an important role in the destruction of germs which are spread with the faeces on the earth, in the water, etc., in other words, in the self purification occurring in nature.