Abstract
Both of the well-established and curated bacteriophage co-discoveries by Twort 1 and by d'Herelle 2 took place as side results of research projects that were not aimed at searching for possible viruses of bacteria. The inquisitiveness of both Twort and d'Herelle is commendable as neither of them had lost heart due to the technical problems associated with their bacterial cultures not growing well. This compliment first goes to Twort whereby even the bacterial growth in his experiments could be considered as undesirable.
Twort's and d'Herelle's observations brought the scientific community close to what is arguably one of the most intriguing and fruitful biological phenomena that allowed us to test and understand key concepts in virology and greatly contributed to the emergence of molecular genetics (reviewed in Letarov). 3 However, both Twort and d'Herelle already knew of and were thinking of viruses (independently discovered in 1892 by Ivanowski 4 and in 1896 by Beijerinck 5 and Bos 6 though, probably, not of the viruses of bacteria.
The field of microbiology was rather well developed by the end of XIX century with intensive research going on in most developed countries and practical applications from routine medical bacteriological testing to relatively large-scale microbiological production of the bacterial biomass for vaccines and therapeutic sera making. So researchers and laboratory workers inevitably had to encounter manifestations of bacteriophages but presumably overlooked the phenomenon. In some cases, descriptions of bacterial lysis very similar to that mediated by bacteriophages are found in old literature.
Thirty putative phage publications appeared before 1917 as identified by Abedon et al. 7 The article by Nikolai Gamaleya 8 is listed the second oldest in the aforementioned collection of the phage prehistory references. This study included an essential component necessary to attribute the induced bacteriolysis to bacteriophage activity—the report of propagation of the lytic agent over multiple passages. The oldest “possible phage” observation is by Hankin in 1896 who described water in the Ganges as having “healing properties,” although this is plausible due to the amount of bacteria in the water is arguably associated with a large amount of phages, there is no component of phages being shown to be propagated.
Sadly, the description of the experimental procedures given by Gamaleya is not exact enough to make it possible to easily reproduce the study. This lack of precision was not a weakness but reflects the tradition of scientific writing at this time, which did not have the rigor of a clear methods section. However, from my point of view, even if Gamaleya had provided a more exact technical section, it would not help much since it appears highly likely that the presumptive bacteriophage activation in his cultures was not due to his manipulations but to the inadvertent contamination of the bacterial cultures by phages.
The interpretation of the data presented by Gamaleya is purely biochemical and by no means allows to consider him as a bacteriophage co-discoverer, but it is fascinating that he most likely observed this phenomenon nearly two decades before his more famous scientists did. By the time of this study Nikolai Gamaleya was already known for his work as the creator of the first Pasteurian rabies vaccination station in the Russian Empire, and during his later career he proved himself to be a very talented scientist, being one of the most recognized Russian microbiologists in the beginning of the 20th century. Therefore, revisiting episode when Gamaleya has overlooked the discovery of bacterial viruses that was apparently laying on his hand is not only interesting from historical perspective but may also be instructive for researchers nowadays.
Although the Gamaleya's article has been already introduced to the contemporary community by Bardell, 9 for unknown reasons the full English translation of the document was not made available for the public. Preparing our translation, we tried to keep as close as possible to specific style of Gamaleya's writing that may now sound somewhat archaic and difficult to read to let the reader to experience sensation of this romantic period of the history of microbiology.
The author is grateful to Dr. S. Abedon for provisioning a copy of hardly accessible Gamaleya's article (in Russian) and to Prof. M. Clokie for critical reading and linguistic editing of the article.
Footnotes
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N.F. Gamaleya (Odessa)
Bacteria—as I have mentioned by several occasions—represent a very convenient object for the research regarding general pathology. One problem, which I had occasionally to work on, is connected to the importance of inorganic salts for life and to phenomena of the mineral starvation. To feed bacteria with organic compounds without any cinder besides some other compounds, I used casein, which I purified by repeated precipitation by acetic acid subsequently redissolving it in ammonia. During this work I have noticed that the casein produces clearly seen morphological alterations in bacteria. Under the influence of this compound bacteria lose to larger or smaller extent the ability to get stained by nuclear aniline dyes; the pale nonstained patches can be found in them. This effect can be explained in the following way: the bacteria lose the compound featuring the affinity to the alkalic aniline dyes that is referred to as chromatin. Thus, the result of the casein action on bacteria described earlier can be called chromatolysis. A similar degeneration of bacteria was observed long time ago as a result of the action of bactericidal serum on bacteria. However, the degeneration could be observed also as the effect of distilled water on some bacteria. Finally, in the old cultures of diverse bacteria one can find a lot of exemplars that have lost the ability to get stained by the nuclear dyes. I could, however, easily confirm that the casein acts on bacteria in a much more vigorous manner compared with the distilled water. For more detailed study of the casein-induced chromatolysis, I have fixed myself on the most convenient object—the anthrax bacterium (bac. anthracis)—one of the largest bacteria, therefore clearly demonstrating the signs of the degeneration.
It has to be added that earlier I had already observed in the anthrax bacterium the chromatolysis caused by the caffeine, and at that time established the main conditions required for the study of the chromatolysis phenomena.
The conditions are the following:
First, the reaction of the milieu in which the chromatolysis is performed should be neutral. Sharp acidity and, even to a larger extent, alkalinity hinders the chromatolysis.
Second, to undergo the chromatolysis the bacteria should be intact. Not only boiling but also a heating above 50° yields them unchangeable in the caffeine. The other antiseptic compounds such as phenol, mercuric chloride, formaldehyde, naphthylamine, and volatile oils have a similar effect. All these compounds kill bacteria and, at the same time, obviously, fix them and fix the chromatin in their bodies.
Third, there is only a single poison that does not prevent the chromatolysis. It is chloroform.
Fourth, different neutral salts—sodium chloride, nitrogenous potassium, and magnesium sulfate prevent the chromatolysis in anthrax bacteria inducing in them a completely different alteration—the stromatolysis, which we will discuss later.
Finally, it should be added that the Loeffler's blue turned to be the best dye to revealing even feeble signs of the chromatolysis.
Based on these experimental data I have built the following method for the chromatolysis <observation>. On the one hand [such expression was used by Gamaleya—A.L.], the most dense suspension of the bacteria under the test should be prepared (in our case bac. anthracis), the bacteria should be collected from the agar and suspended in the distilled water. If few drops of chloroform are added to this suspension and it is placed into a tightly sealed vessel, the suspension can be stored for an unlimited time (however, I usually use freshly prepared bacteria). On the other hand, a neutral solution of the compound to be tested for the ability to cause chromatolysis should be prepared in distilled water. This solution should be placed in a test tube, the emulsion of the bacteria should be added along with few drops of chloroform, and the tube should be placed into the thermostat at 37° (I usually add to 5–8 cm cube of the solution under the test the amount of the anthrax bacteria grown on the surface of a single agar slant). After 6–12–24 h I examine the dried slides made of this suspension and stained with Loeffler's blue.
Using this method I have studied chromatolytic activity of a series of compounds over the anthrax bacteridia [Gamaleya calls bacterial cells or cell aggregates in such way—A.L.].
Here are the main results obtained.
The plant alkaloids do not produce any chromatolysis. It also could not be caused by the compounds similar to caffeine such as theobromine, xanthine, uric acid, and creatinine. A strong activity was observed for bacterial alkaloids, for example, ptomaines, such as methylamine, ethylamine, triethylamine, and ethylenediamine. They extract the chromatin from the anthrax bacteria. Nucleohistone (from gl. thymus) does not act on bacteria, nuclein also produce only a small effect, but the nucleic acid (i.e., its ammonia salt) produces a sharp chromatolysis.
Histone, for example, fixes the bacteridium. Casein, as it was mentioned, causes the chromatolysis. However, if concentrated casein solutions are used to enhance the effect, it causes the separation of the degrading bacteridia of some casein-coagulating compound. The coagulated casein surrounds the bacteridia filaments forming around them a capsule and thus preserves them from further chromatolysis. I have tested the action on bac. anthracis of the pellet forming in course of pepsin digestion of casein. This pellet being dissolved by the means of ammonia exerted a very strong chromatolytic action. Following this pathway, I decomposed casein by the boiling with strong hydrochloric acid.
Among the products of such decomposition I have found the substances that could be precipitated by the acetic acid and redissolvable in ammoniac. These compounds produce the chromatolysis much stronger than all the substances described to date. Under the influence of these substances the bacteridia lose all their chromatin within few hours and turn into stainless shadows. Chemical reactions of these substances indicated that I am dealing with amido-acids. And indeed, one of the amido-glutaric acids that can be produced out of casein, more precisely the amido-glutaric acid or glutamic acid (i.e., its ammoniac salt), has, as it was revealed, an amazing chromatolytic effect.
What chemical reactions underlie the chromatolysis, that by this point studied only morphologically? Is the chromatolytic substance consumed? Does it form any complexes?
The response to these questions was the obtaining of the enzyme, destroying bacteria. In the liquid in which the chromatolysis took place, the acetic acid produces a precipitate. This precipitate collected on the filter and redissolved with the help of the ammoniac, represents the destroying bacteria enzyme. In contrast to the aforementioned compounds, this substance not only induces the chromatolysis but also causes complete destruction and dissolution of the bacteria—the bacteriolysis. In other words, not only chromatin gets extracted from the bacteria, but also the connections between bacterial filaments, between individual rods in the filaments are broken. The rods then get split into pieces, undergoing further decomposition into small angular barely stainable fragments, and the bacteridia flocs are replaced by the formless debris. This allows the observer to follow the process macroscopically. Dense cloudy bacterial emulsion in the solution of this enzyme turns within a few hours (6–12) into a transparent liquid with barely seen opalescence.
From this liquid that has dissolved the bacteria it is possible to precipitate the enzyme by the acetic acid for the next time, and so on. Since the enzyme gets all the times regenerated from the used liquid, it is obvious that some elements of the dissolved bacteria are included in its composition. In other words, the enzyme, I think, is produced by the interaction of a chromatolytic substance with some bacterial product. What is this bacterial product?
If it is produced from the dissolved bacterial chromatin, this means that it should be found in any chromatolysis. And indeed, whatever the type of chromatolysis the anthrax bacteria were subjected to, be it distilled water, caffeine, or nucleic acid action, or even if one merely takes old culture in the broth, all the time the solution contained specific substance. This substance can be precipitated by the acetic acid and redissolved in ammoniac. This substance features a strong bacteriolytic activity. Let us call this substance chromatinin for brevity.
So, it is possible to think that anthracolytic ferment is a complex of the glutamic acid and chromatinin of the anthrax bacteria. The destruction of the bacteria by this ferment can be represented as a double decomposition reaction between the ferment, on the one hand, and the compounds forming the stroma and chromatinin of bacteria, on the other hand. This idea will be developed later.
The facts that I have just described raise a lot of questions, which I am going to discuss now.
Is this ferment specific? Does it represent a universal dissolving agent active on all the bacteria or, in contrast, does it work only on the anthrax bacteria from which it was produced? The facts support the second hypothesis.
First of all, the ferment easily undergoes the invasion of the saprophytic bacteria that can multiply in its solution.
Next, our ferment, although it can dissolve some other bacteria, does not act so vigorously on any of them as it does on bac. anthracis.
On many bacteria it does not work at all.
Finally, for different bacteria I could produce different ferments.
I think that for each bacterium a specific ferment should be found. The pathway for doing this is clear: it is necessary just to obtain chromatinin from the bacterium of interest and to conjugate it with the casein derivative or with glutamic acid or with a different amido-acid, and the ferment is ready.
Indeed, from many bacteria, for example, from cholera bacteria, as well as from diphtheria or tuberculosis bacteria, it is easy to obtain chromatinin, precipitable by the acetic acid. With the help of such chromatinins, I have prepared the corresponding bacteriolysins for the indicated three bacterial types (and I have in my view several other).
Choleralysin dissolves the cholera vibrions in the absolutely similar manner, but converts them before into cocci, as it was described by Pfeifer for their destruction in the guinea pigs' peritoneum.
I have still little data on the diphtheria enzymes because I used all my time to study the tuberculosis ferment. †
The problems of the ferments specificity and practical significance of the enzymes found are in tight connection. Do the bacteriolysins play any role in elimination of the bacteria in a living body? There are already a lot of data supporting the positive answer to this question.
First, morphologically the action of the ferments is very similar to the action of the bactericidal serum, but it is much more intensive.
Second, in contrast to other antiseptic compounds, these ferments act in the animal sera as well as they do in the distilled water.
Third, as I confirmed for the tuberculosis ferment, it can also destroy the bacteria in the organism of an animal.
Fourth, in the animal organism these ferments can be produced out of vaccines in the same way as it occurs in our test tubes from the animal products and bacterial chromatinin.
Getting back to the practical significance of the bacteriolysins, we have to point out three possible applications that were found in our experiments.
First, bacteriolysins dissolving the bacteria in the test tubes liberate the toxins contained in these later ‡ thus facilitating the studies on bacterial poisons.
Second, bacteriolysins been injected simultaneously with bacteria into an animal organism will liberate the toxins inside of the organism. This may yield a new vaccination method.
Third and finally, due to their destructive action on bacteria, infecting the organism, bacteriolysins represent a new method to treat infections.
I have to add that bacteriolytic ferments in such doses that are effective against the bacteria in the organism are harmless for the animal cells. § This was, however, expected since the ferments are specific.
To understand bacteriolytic process, it is of importance to know the means of defense against the chromatolysis that bacteria possess. Under the action of alkali, strong concentrations of neutral salts or some other reagents bacteria undergo slim or, better to say, hyaline metamorphosis. The substance forming the wall and stroma of the bacteria swells and spreads in a form of a gel-like mass. Some bacteria, such as b. Friedländera, b. pyoceaneus, and b. typhosus, develop this stromatolysis particularly intensively. Bac. pyoceaneus is turned during this process into a gel-like mass in which formless chromatin clots can be found under the microscope. It is obvious that such stromatolysis is a completely opposite process in respect to the chromatolysis. I could observe that the bacteria forced to live under the conditions causing the chromatolysis, they dissolve the substance forming the aforementioned gel in their growth medium. It is possible to think that the capsules and the colonies have similar origin.
Getting back to the action of the enzymes, destroying bacteria, we may hypothesize that the action of these enzymes may be due to both chromatolysis and stromatolysis processes.
Here I should stop, leaving to the next occasion detailed presentation of my experimental data as well as two general questions: of the physiological significance of bacteriolysins and of their relation to the ferments.