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Louis Pasteur (1822–1895)
Microbes and Infection 5 (2003) 553–560 www.elsevier.com/locate/micinf
Review: on the shoulders of giants
Louis Pasteur (1822–1895) Guy Bordenave 1 Unité d’immunophysiologie et Parasitisme Intracellulaire, Institut Pasteur, 25, rue du Dr.-Roux, 75724 Paris cedex 15, France
Abstract In Louis Pasteur’s scientific career it is striking to note the exponential character of the research he introduced in all the fields he opened up. He offered fabulous opportunities to stereochemistry. He is acknowledged as one of the founders of microbiology. He established the possibility of anaerobic life. He pointed the way to epidemiology, public health, and the bacteriologic fight. He struggled against the idea of spontaneous generation of life. He irrevocably substantiated the microbial theory of infectious diseases. He demonstrated that bacterial virulence could be attenuated, he evidenced immunity and generalised the vaccination principle. He also was an incomparable experimenter. © 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. Keywords: Molecular dissymmetry; Microbiology; Aerobic and anaerobic lives; Rebuttal of spontaneous generation of life; Immunity; Vaccines; Microbes and infectious diseases; History of science
1. Introduction What more can be said about this exceptional man who has already had so much written about him2 [1]? It is true that every generation has a duty to remember the immense contribution made by some of our predecessors. Pasteur’s patriotism, for example, has been widely discussed and evidence of the influence of his father—a former soldier of the First Empire who later became a tanner craftsman—is sought in vain within this patriotism. His mother appeared to have a heightened sense of the relativity of human concerns as, shortly before her death in 1848, she wrote the following words to him: “whatever happens, never let yourself get down. Little is of great importance in life” [2]. The human species is made in such a way that it is easily fascinated by everything that relates to the major work of so-called eminent people. We can therefore ask ourselves if, in this case, it was the conservatism origins of that characterised Pasteur and, under Napoleon III, inspired him to talk about “the exaltation created by a great reign”, while Victor Hugo was himself in exile. However, it is difficult to assess whether such reasoning is really worthwhile. The most sensible approach would E-mail address: [email protected] (G. Bordenave). 1 G. Bordenave is a retired Institute Pasteur scientist. For correspondence and reprints please contact G. Milon at the given address. 2
Pasteur’s correspondence and other documents: Annick Perrot, Conservateur, Musée Pasteur, 25-28, rue du docteur Roux, 75524 Paris cedex 15; E-mail: [email protected]. © 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. DOI: 1 0 . 1 0 1 6 / S 1 2 8 6 - 4 5 7 9 ( 0 3 ) 0 0 0 7 5 - 3
probably be to try and limit ourselves to an impersonal description of his scientific work. But is this really possible given that he was such an exceptional being? How can we possibly define a scientific career in relation to such work? In any case, this account could only ever approach such a subject with great humility. Dole, a small sub-prefecture in the Jura region, can be proud, and rightfully so, of having witnessed, in 1822, the birth of the one who, indisputably, belongs to that category “of children whose genius immortalises the memory of civilisations”.
2. Molecular dissymmetry It was in 1844, at the age of 22, while he was studying chemistry at the Ecole Normale Supérieure in Paris, that Pasteur made one of his first, key discoveries. It was already known that quartz crystals were able to deviate polarised light either to the right or to the left. This property was directly linked to their crystalline configuration: some facets of the crystals—the hemihedral facets—are inclined in relation to the edges which support them, sometimes in one direction and sometimes in the other. Biot, an expert in crystallography and optical phenomena, noticed that tartaric acid deviated polarised light both in its liquid state and crystalline form. Two crystalline forms of tartaric acid were identified: tartaric acid and paratartaric acid. Mitscherlich, another reputable chemist, had shown that these two forms as
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Today, we are more aware of the importance, in pharmacology for example, and even in perfumery, of the two enantiomer forms (the mirror image of each other) of a same substance—one can be active, whereas the other remains inactive [1,4]. 3. Fermentations and microbiology
Fig. 1. Facets of the double sodium ammonium paratartrate crystals: some are inclined to the right and others to the left. Photo courtesy of Institut Pasteur.
well as those of their respective salts—tartrates and paratartrates—were identical in all respects except in their ability to deviate polarised light. The tartrates deviated light to the right while the paratartrates remained inactive. Pasteur noticed small facets similar to those of quartz crystals on the tartaric acid and diverse tartrate crystals (Fig. 1). He also noticed that all the facets were orientated to the right in the tartrate crystals, while in the double sodium ammonium paratartrate crystals, as in those studied by Mitscherlich, some were inclined to the right and others to the left. He divided up the two categories of crystals with tweezers and used them to make separate solutions. Both solutions deviated polarised light—one to the right and the other to the left. Mixed in equal quantities, they formed an optically inactive solution. The concept of molecular dissymmetry was born from the interpretation of this observation. The age of stereochemistry was beginning. In comparison to Biot’s often repeated exclamation: “My dear child, I loved science so much during my life it makes my heart flutter” (Biot who imposed draconian measures on the experiment), Pasteur’s reaction is moving in other ways. So violent was the shock, that he rushed out of the laboratory and, when he came across a chemistry technician, he grabbed him and cried out: “I’ve just made an important discovery... I’m so happy I’m trembling all over, I can’t even look through the polarimeter”. What an extraordinary moment of elation! What researcher, even when his find is more modest, has not experienced such emotion? This is a joy dominating over all others, an inexpressible and absolute joy! Pasteur was truly won over and this impetuous whirlwind was going to take over his whole life. Pasteur’s choice of science was significant, as during his adolescence he also showed an undeniable talent for painting, proof of which is shown in the pastel pictures he left [3]. Pasteur was convinced that molecular dissymmetry was one of the most fundamental characteristics of living organisms.
After being a professor in Dijon and then Strasbourg, another challenge awaited Pasteur in Lille. Here, he was drawn to study the problems that industrialists in the region were having with their sugar factories or breweries. He was faced with the problem of fermentation and, from this confrontation a new discipline would be born: microbiology. The scientists who prevailed in this field at the time were Berzélius, Liebig, Helmholtz and Berthelot. Using all the influence that their wide fame gave them, they fought against what was known as the “vitalist theory”, i.e. the involvement of living organisms in fermentations. They attributed this involvement to chemical agents, which they referred to as ferments and which they considered independent of the vital processes. Pasteur studied ethanolic, lactic, butyric and acetic fermentations. He began by explaining lactic fermentation because, of all fermentations, it is chemically the most simple. On fission of the glucose molecule, lactic fermentation only consists of two lactic acid molecules. He isolated the lactic bacillus from the greyish deposit which accompanies the souring of milk and he cultivated it in a clear, nutrient broth. Once a sufficient quantity had been produced, the population of microscopic living organisms (which were all alike) ensured that lactic acid was formed from glucose. As for ethanolic fermentation, he began by showing that glucose was not only transformed into carbonic gas and ethanol—something that had been accepted since Lavoisier—but that other products were also found as a result of this fermentation such as glycerine, succinic acid and amyl alcohol. Above all, he showed that the yeast, which was very different from the bacillus involved in lactic fermentation, induced ethanolic fermentation in a liquid which only contained glucose and mineral salts. There was total parallelism between fermentation and yeast multiplication. The agent responsible for butyric fermentation appeared in the form of small rods, which were capable of undulatory vibrations and could therefore move. Whilst observing a drop of liquid undergoing butyric fermentation between a slide and a coverglass, he noticed that the bacilli at the edge of the coverglass were losing their mobility while those in the middle retained it. This situation was the reverse of what he was used to recording with other microorganisms, as they did the opposite and gathered on the edges where there was more oxygen. He was convinced that life without oxygen was possible as butyric fermentation could be stopped by blowing air into the liquid where this fermentation was occurring. He came up with the terms “aerobic” and “anaerobic” to refer, respectively, to life in the presence of oxygen and life without it. Pasteur still examined acetic fermentation and here he also
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introduced the vitalist theory even though it was known that vinegar was the result of a simple chemical reaction: the oxidation of ethanol in acetic acid. He demonstrated that the transformation of wine into vinegar depended on the activity of a bacterium whose population formed a thin layer, which floated on the surface of liquids. It is this bacterium which incorporates oxygen from the air into the ethanol [1,5]. In 1897, 2 years after Pasteur’s death, Büchner managed to extract a soluble fraction from yeast, which was able to trigger ethanolic fermentation in the absence of living cells. It seemed that the catalytic theory of Liebig and Berzélius, etc. was basically correct. The two great trends of scientific thought during this period—the physiological and chemical theories of fermentation and metabolism—were going to be reconciled. Microscopic living organisms produce ferments—enzymes, which act either inside or outside the microbial cell. Enzymes can carry out chemical transformations in the absence of the living organisms which produced them but, under ordinary conditions, these transformations give the living organisms energy and basic elements which are essential to their metabolism. Pasteur had never ruled out this possibility as, for example, in 1878 he wrote “I should not be surprised to see the yeast cells produce a soluble, alcoholic ferment... soluble ferments have only yet been produced by a vital function”. In any case, the products of ethanolic, lactic, butyric and acetic fermentations were linked to bacterial life. 4. Rebuttal of the idea of the spontaneous generation of life Having studied microbial life, Pasteur found himself drawn into the controversy surrounding the idea of the spontaneous generation of life. He joined this debate on the origin of life in 1859, the year Darwin’s “The Origin of Species” was published. Here, he fully demonstrated his talent as an exceptional bench scientist. From the concept of specificity, born out of the study of fermentations, was derived that of hereditary characters which, in turn, depended on normal generations. He naturally admitted that it was possible that, somewhere in the universe, life could be reproduced but it was necessary to check the assertions of those who claimed to have witnessed the birth of life. In 1859, Pouchet, Director of the Natural History Museum in Rouen, claimed that he could bring about spontaneous generation whenever he wished. His demonstration consisted of taking a flask of boiling water, sealing it tightly and then plunging it upside down into a bath of mercury. Once the water had cooled down, the flask was opened in the mercury and half a litre of pre-sterilised oxygen as well as a little hay infusion were introduced. A microbial population regularly developed after a few days. Pasteur demonstrated that by eliminating the mercury from the experiment, he avoided contaminating the liquids and the air. He then conducted those famous experiments in his swan-neck flasks (Fig. 2). Having introduced a culture broth into a flask, he heated the neck in a flame and
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Fig. 2. Louis Pasteur observing a culture broth sterilised in straight and swan-neck flasks. Painting Robert Thom. Photo courtesy of Institut Pasteur.
then bent it into the shape of an “S” (hence the name “swanneck”). Vapour from the boiling broth expelled the air via the opening in the s-shaped tube. The flask cooled down and the outside air entered slowly, without having been heated or filtered, but having been sterilised by the humidity of the tube which retained the germs which were unable to get past the bend in the swan-neck. As the s-shaped tube remained open, the air on the inside and outside of the flask communicated freely but the broth remained sterile indefinitely. The lack of air was therefore not responsible for the fact that life did not appear. Moreover, the broth retained the elements that were necessary for the development of life as, when the neck of the flask was broken, microbial life appeared quickly. It was with straight-neck flasks that Pasteur discovered the uneven distribution of microorganisms in the atmosphere. Flasks containing a nutrient broth underwent sterilisation by boiling, which also helped to expel the air due to the stream of water vapour. The flasks, which were closed using a flame while the water vapour was still coming out, were almost empty of air, and the nutrient broth remained sterile as long as the flasks were sealed. Pasteur opened these flasks in many different places and took extreme care not to contaminate them. By breaking the neck of the flasks, air from the outside was allowed in. The flasks were then re-sealed and put into an incubator. Some of these flasks remained sterile while in others, microbial life appeared. The evidence of the uneven distribution of microorganisms in the atmosphere was proven when it was noticed that the number of flasks with a contaminated content was higher at low altitudes, near inhabited areas and cultivated lands than in the high mountains or places where the air remained still for a long time. This also proved, once again, that non-heated air could only trigger microbial growth if it contained living germs likely to culture the nutrient broth [1,5]. These demonstrations led to aseptic manipulation, sterilisation and autoclaving techniques, and the Englishman, Tyndall, played a key role in founding them. It was at this time that scientists also became aware that if air contained microorganisms, then water and solids must also contain them and maybe even greater numbers of them.
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After the death of Claude Bernard, a renowned physiologist, notes were published relating to some experiments that he conducted on the fermentation of grapes. In particular, he stated that, contrary to Pasteur’s opinion, fermentation was possible independently of living processes and he seemed to implicitly suggest that yeast could be the consequence of fermentation rather than its cause. Pasteur detected there a disguised resurgence of the idea of the spontaneous generation of life and he devised, by way of response, a fabulous experiment. He owned a small vineyard in the district of Arbois. Using glass, he isolated a section of the vines at a time when the yeast germs were not yet deposited on the grapes. It was impossible to obtain ethanolic fermentation with ripe raisins from the protected bunches, but the opposite was true of the ripe raisins from the exposed bunches. If the protected bunches were placed on vine-stocks left in the open air, fermentation was easily obtained with their ripe grapes. Nothing had been discovered about the conditions in which life came about. All that was shown was that microbial life did not occur in a properly sterilised nutrient medium, which was protected from outside contamination. Furthermore here is what Pasteur wrote on this subject: “I’ve searched for spontaneous generation in vain for twenty years. No, I don’t think it’s impossible but what leads you to believe that it is the origin of life?You put matter before life so matter has therefore existed since the beginning. How do you know that the incessant progress of science will not force scientists in a century, a thousand or two thousand years time... to assert that life, and not matter, has existed since the beginning. You go from matter to life because your current knowledge... does not allow you to understand things differently. Who can assure me that in ten thousand years time we will believe it impossible not to go from life to matter...?”[6].
5. Wine, beer and silk worm diseases It is Pasteur who rightly received the credit for having discovered that ethanolic fermentation diseases were caused by microorganisms which entered into competition with yeast. He went to great lengths to prevent microorganisms from developing in finished products, i.e. after the involvement of yeast. He tried several antiseptics and then used heat as a sterilisation agent. Very wisely, he knew how to take advantage of the vulnerability of microorganisms by combining the slight acidity of wine with a moderate rise in temperature (55 °C), over a short period of time (and better still without oxygen), to obtain partial, but generally sufficient, sterilisation. This principle would later be applied to a very large number of perishable liquid foodstuffs (wine, beer, vinegar, milk, etc.) or solids, and would lead to what is now known throughout the world as pasteurisation. His work on the role of microorganisms in fermentations and then in wine and beer diseases, would gradually lead him to the microbial theory of infectious diseases and give this remarkable unity and outstanding direction to his scientific career [1,7].
Fig. 3. Silk worm on mulberry-tree leaves. Drawing by Lackerbauer, published in “Maladie des vers à soie”, Paris, 1870. Photo courtesy of Institut Pasteur.
It was then that he returned to Paris, to the École Normale Supérieure, and was called upon by Dumas, one of his former masters/“maîtres” to study the silkworm disease which was causing devastation in the sericulture industry in the south of France (Fig. 3). When suffering from the disease, the worms weaving the cocoons from which the silk was extracted stopped growing at very different stages. They showed small marks, which resembled specks of pepper on their skin—hence the name “pebrine”, which was given to the disease. Pasteur noticed that only butterflies from sick worms showed the characteristic corpuscles, which were easily detected under the microscope. He set up a method of selection based on this observation to sort eggs from healthy butterflies, which could be kept for reproduction, and eggs produced by affected butterflies that had to be destroyed. The healthy eggs made healthy worms, which wove cocoons with healthy chrysalises. Today, it is known that pebrine is caused by a protozoan parasite. At the time it appeared that the problem was more complicated than first thought because at least two simultaneous diseases were present: pebrine and flacherie. Flacherie was only really identified when it persisted on its own—pebrine having been eliminated from the breeding of healthy worms. The general view today is that flacherie is above all caused by opportunist infections which accompany a benign viral attack. These varied infections resulted in diverse and disturbing forms of the disease. Pasteur acquired a certain understanding of flacherie and devel-
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oped appropriate techniques for keeping it under control. One of his recommendations was not to retain eggs from worms which appeared languid during the stage preceding the weaving of the cocoon or from those which died in large numbers at this time. It was a contagious disease problem and it was necessary to keep healthy worms in non-infected premises using non-infected material. From this approach emerged the principle—elementary in infectious diseases but not yet implemented—of separating the healthy from the affected [1,8]. (This principle was applied to humans by Emile Roux and his colleagues for the first time ever at the Pasteur Hospital). Pasteur learnt much from this work and was now in a better position to tackle the problem of infectious diseases in “superior” vertebrates. The period when the laboratory moved to the Cevennes has been described as a probably happy time in Pasteur’s life, although it is easy to imagine that the word “happiness”, in the most general sense of the term, did not feature frequently in the vocabulary of a man who was so intellectually and morally demanding. He called on his whole family to help with his silk worm research. To people who clung to traditional practices and who protested at the difficulty of new methods suggested, Pasteur would reply that he knew of a child (his daughter Marie-Louise, then aged ten) who excelled in the art of telling healthy butterflies from sick ones. This period spent in the countryside must have made a wonderful holiday for this young town girl! His wife, Marie, was also mentioned. She took notes and wrote up the reports of his experiments, which he dictated. She inquired into problems, followed the progress attained and also probably made suggestions, because in this field and in such an exciting atmosphere, it is difficult to remain passive or indifferent. The image of the two of them in the calm surroundings of this provincial environment (rather than in a relationship that revolved around domination of one by the other as has been too often suggested) paints a peaceful picture. It is satisfying to imagine the hours of deep communion and the oases of serenity and intimacy known to this household where science seemed to take centre stage. 6. Chicken cholera, sheep anthrax, swine erysipelas and the birth of immunity In 1879, Pasteur succeeded in cultivating the bacillus involved in chicken cholera and showed how the disease could be triggered by injecting a pure suspension of this bacillus. With this material, Pasteur demonstrated that, unlike chickens and rabbits, adult guinea pigs resisted the infection, developing only a local abscess, which was nevertheless the local outbreak site of the same bacilli, whose full virulence subsisted in chickens and rabbits. This concept of a healthy host opened the way for investigation into the wider problem of epidemiology. It is said that the fundamental observation was discovered by chance—if this was not so it must be said that Pasteur’s foresight was all the more astonishing—but, whatever the case, the main point was that
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the issue could no longer be avoided. Pasteur’s observations highlighted immunity and led to a general understanding of the vaccination principles. His research was interrupted for a while and the suspensions containing the chicken cholera bacillus aged in the laboratory. When he resumed his research, it became apparent that the chickens injected with the bacillus taken from these old suspensions not only survived, but went on to resist a second injection of a fresh suspension of the virulent bacilli which resulted in the rapid death of untreated chickens. Pasteur understood that the chickens had been immunised and that the bacilli responsible for causing the disease could be attenuated. Attenuation was achieved by exposing the culture to the air for some time periods (and at a suitable temperature), as the same culture, which was attenuated when exposed to the air, retained all its virulence when enclosed in an airtight container. Virulence was therefore not a permanent characteristic but rather an unstable property that could be lost and even regained without affecting any of the other bacillus characteristics. Pasteur established a connection between this observation and the one made at the end of the 18th century by Jenner, an English country doctor, who succeeded in protecting humans against smallpox by inoculating them with the substance contained in “cowpox” pustules. Cowpox or vaccinia was a similar disease found in cows but which was harmless in humans. At that point, it was the only application known to man. In order to combine the two discoveries, the name “vaccination” was given to the phenomenon brought up to date by the chicken cholera experiments. Pasteur then became aware of the existence of a general rule, which he had already suspected: the possibility to make an animal less susceptible to the virulent form of a microorganism by putting it in contact with the attenuated form. This research would now have an impact on the wider issue of attenuating virulence in germs. While Pasteur was studying the problem of sheep anthrax, the disease was causing devastation on sheep, goat and cow farms. Koch, a young German doctor, had already won recognition through his work on the sheep anthrax bacillus. He proved, among other things, that the anthrax bacillus actually caused the disease and he also ascertained the complete life cycle of this bacillus: immobile rods which develop into long filaments and then into round granules—the spores (or the resistant form) which, in the right conditions, changed back into rods. It was at the beginning of his research into the anthrax bacillus that Pasteur discovered large amounts of another bacillus in the tissues of some of the animals infected by the disease. He named this second bacillus (which is rare in blood) “septic vibrio”. He showed that it was a strict anaerobic bacteria, which was responsible for causing both abnormal death and erratic results in animals which had purposely been contaminated with anthrax, using the blood from animals that had died from the disease. The vibrio septic bacteria killed the animals before the anthrax bacillus had a chance to grow. Pasteur also used the anthrax bacillus to prove that the presence of a pathogen in an organism did not necessarily mean that the disease was manifested itself,
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and that the environment could be crucial in influencing its development. Although chickens appeared to resist anthrax, when their usual temperature of 42 °C was lowered to 38 °C, they could be made almost as susceptible to the disease as rabbits or guinea pigs. The anthrax bacillus turned out to be extremely sensitive to temperatures of around 45 °C, which explains why it did not multiply or did not multiply well in chickens. If a chicken, which had been purposely infected with anthrax by artificially lowering its temperature, then had its usual temperature raised back to 42 °C, it was restored back to health. Pasteur also concluded that anthrax bacillus spores could survive over long periods of time in the earth around where the animal that had died from the disease was buried, and that worms in this earth were involved in bringing the spores back up to the surface. He injected earth taken from the worm’s intestines into guinea pigs, infecting them with anthrax. Pasteur pointed out that stubble left behind after harvest could injure the animal and thus provide the infection with a means of entry. This research into the etiology of anthrax has been widely acknowledged as exemplary work. The main incentive behind it was, of course, to develop a vaccine. However, the task turned out to be more difficult than for chicken cholera. At the most usual temperatures, the bacillus is present in its rod form (i.e. sensitive to virulence attenuation) but the spore form of the bacillus (i.e. resistant to attenuation process) is also detected. The problem was how to keep the bacillus alive while at the same time preventing the spores from developing. After much trial and error, the objective was achieved by storing the cultures at 42-43 °C inside shallow containers with oxygen present. Eight days in these conditions resulted in producing bacilli that were harmless to guinea-pigs, rabbits and sheep. Before reaching this final state, the bacilli went through various stages of attenuation in which they could be maintained. Pasteur discovered that it was best to vaccinate against anthrax in two steps: an initial inoculation using bacilli from a culture with very weak virulence followed by a second inoculation, 12 d later, using bacilli from a more virulent culture. This process protected the guinea-pigs, rabbits and sheep against the most virulent form of the bacillus. This led to a public experiment—a memorable event that took place at Pouilly-le-Fort in the Spring of 1881, in the presence of a crowd of journalists and inquisitive on-lookers. The challenge was to vaccinate 24 sheep, one goat and six cows and then to inject them with the virulent strain of the anthrax bacillus. At the same time, a similar group of unvaccinated, control animals was also injected with the same anthrax bacillus strain. The experiment was a complete success—only the vaccinated animals survived. Evidently, critics protested at the time against the principle of vaccination. Some of these criticisms came from Koch, the famous German microbiologist. He criticised Pasteur, who did not use his solid culture medium method, for working with impure bacillus cultures. Koch remained unfamiliar with this emerging new discipline—immunity— perhaps because he had apparently failed in his own attempt
to protect against the tuberculosis bacillus. He remained relatively sceptical to the possibility of attenuating pathogen virulence, even though his own work on the anthrax bacillus and his important discoveries (the tuberculosis bacillus and the human cholera vibrio) contributed a great deal to establishing the microbial theory of infectious diseases. Most of the ill feeling between Koch and Pasteur stemmed from the extreme sensitivity of scientists to anything related to their own personal work, to say nothing of the severe FrancoGerman bitterness that existed at the time. It would appear that a scientist’s sensitivity could be as acute as his contribution to progress was crucial. The German school of medical bacteriology attracted considerable international interest. It discovered various bacteriological agents of infectious diseases. It had an advantage over the French school because—so it is claimed—it was larger and better organised. However, through its research and particularly through Pasteur’s contribution, the French school can be credited with the discovery of streptococcus (1879) (which was causing damage in maternity wards under the name of puerperal fever), staphylococcus (1878), pneumococcus (1881) and septic vibrio (1877). This is what Roux said on the subject: “A small, round organism, made up of clusters, that cultivates easily in nutrient broth, can be found in the pus of warm abscesses and furuncles. It can also be found in infectious osteomyelitis in children. Pasteur affirmed that osteomyelitis and furuncle are two forms of the same disease and that osteomyelitis was really a furuncle of the bone. This assertion caused much amusement among surgeons in 1878. Pasteur never gave botanical names to the microbes he discovered, but named them according to their shape or culture. Thus, for him, the furuncle microbe was the “grain cluster microbe” and the puerperal fever microbe was the “grain chaplet microbe”. These same discoveries are better known in the world of bacteriology as Staphylococcus and Streptococcus pyogenes” [9]. And they also enabled certain people to claim them as their own discoveries. The discovery of the plague bacillus by the pasteurien, Yersin, could also be added to this never-ending list. As far as research into erysipelas or swine fever was concerned, the problem was of a different nature. If the swine fever bacillus is injected from rabbit to rabbit, the bacillus acclimatises to the rabbit. All the rabbits end up dying after a few days, whereas initially the rabbits always got sick but did not die with the same frequency. If pigs are then injected with the blood from this group of rabbits, the virulence for pigs gradually decreases over several successive rabbit inoculations and this injection no longer causes the pigs to die but just makes them sick. Once they recover from the disease, the pigs are immunised. This must have been enormously encouraging for everyone who worked alongside Pasteur in this amazing development, i.e. the application of essential principles: the use of attenuated, living bacilli in relation to a recognition system that was gradually being discovered and which ensured that
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the “superior” organism being studied was protected in some way. Two fundamental approaches, centred around a concern for public health, immediately set the Pasteur school apart from other bacteriology schools—firstly, research into microbial agents that cause infectious diseases and secondly, using this research (with the aim of attenuating virulence as soon as the disease agent is exposed) to prepare a vaccine for prophylactic use and afterwards, a serum for use as a cure. As far as virulence attenuation procedures for microorganisms were concerned, initial attempts dramatically showed how diverse they could be, as no less than three different attenuation methods were designed under Pasteur’s supervision for the first three bacterial vaccines. One of the main reservations at the time was based on the fact that people remained reluctant to the idea that these tiny and apparently insignificant beings—bacilli—could have such a devastating effect on “superior” organisms [1,10]. 7. Human vaccination against rabies Without a doubt, these experiments required an enormous amount of intellectual courage, of courage in every sense of the word. There has been much debate about the decision to carry out research into rabies. Clearly it was Pasteur’s choice. We can assume that the determining factor was the fact that rabies was a disease common to humans and animals and that it was possible to experiment on animals. The least that can be said is that the experiments involved several rather startling activities. Firstly, they involved a virus that could not be detected under an optical microscope. Secondly, the disease had a long incubation period—over a month from the moment of contamination to the appearance of the first symptoms. This period was used to try and obtain resistance by vaccination, even after the infectious bite. The first positive result was the discovery that the virulence was based in the nervous system and that the best inoculation route was via dura mater after trephination. By regularly transferring contaminated spinal cord via this route, from rabbit to rabbit (a process requiring around fifty passages), a fixed virus was obtained which had a regular incubation period of 7 d. The second positive result was that viral virulence could be attenuated and extinguished by exposing the spinal cord from contaminated rabbits to dry, sterile air (Fig. 4). It was not denied that this could have been due to a reduced amount of rabies virus rather than a reduction in virulence as, unlike bacteria, it was impossible to quantify the virus. It is easy to imagine the moral dilemma that Pasteur must have faced when 9-year-old Meister came to him on 6th July 1885 from an Alsatian village where he had been bitten on the 4th July. Imagine what a request for help from Alsace must have felt like in France in 1885. The child was suffering from 14 wounds and his death appeared unavoidable. The decision was made to apply the treatment that had been continually successful in dogs. Roux was working on the same problem and we can credit him for his idea of gradually drying out rabbit spinal cord in air inside sterile flasks.
Fig. 4. A painting by Edelfelt showing Louis Pasteur inspecting a rabbit spinal cord drying out in a sterile flask. Photo courtesy of Institut Pasteur.
Pasteur adapted this idea by adding potassium particles to speed up the dehydration process. Roux believed that the animal experiments had not been perfected enough for use on humans. He left the laboratory to mark his disapproval and pointedly got back to his seat when the diatribe against Pasteur reached its paroxysm. The method involved injecting subcutaneously suspensions of spinal cord via the peritoneal region. First non-virulent spinal cord was used, then more and more recent spinal cord until finally an extremely virulent spinal cord was administered. The treatment began on 6th July 1885 and continued until the 16th. This is Pasteur’s brief account: “For each of the different spinal cords used, we also inoculated two new rabbits by trephination in order to monitor the virulence of the spinal cords.... Spinal cords dated from the 6th, 7th, 8th, 9th and 10th July were not virulent as they did not cause rabies in the rabbits. All the spinal cords from 11th, 12th, 13th, 14th, 15th and 16th July were virulent and the virulent matter became progressively stronger and rabies broke out in the rabbits.... Joseph Meister therefore avoided not only the rabies that his bite wounds may have otherwise caused but also the rabies that I inoculated him with..., a more virulent form of rabies than the kind found in stray dogs...” [1,10]. This description, which remained true for all subsequent individuals treated for the disease, meant that there was no basis for the allegations accusing the treatment of being ineffective. Strangely enough, however, the same allegations still exist today.
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8. By way of conclusion What more can be said about this scientific career when all attempts at describing it are feeble in comparison. The most striking element is the exponential nature of every area of Pasteur’s research and the fact that—due to a lack of time owing to the choices made—he was not able to investigate further, or perfect every area of his work. He opened up fantastic opportunities for stereochemistry. He is recognised as one of the founders of microbiology. He discovered that life without oxygen is possible. His observation that chicken cholera bacillus, deadly for this species of animal yet harmless in guinea-pigs (which could carry the bacillus without becoming sick), provided a foundation for epidemiology. Lister, the English surgeon, revered as the founder of antiseptic techniques, always remained grateful to Pasteur for his intellectual contribution to medicine. Pasteur did not lay down any specific rules concerning hygiene but we can consider that he left it up to others to enforce such rules after he had brought the problem to light. He noticed that microorganisms in soil could affect the anthrax bacillus to such an extent that it no longer spread the disease. He realised that this discovery could lead to therapeutic applications. He played his part in the bacteriological struggle, offering, albeit unsuccessfully, to provide the Australian Government, whose territory was infested with rabbits, with the chicken cholera bacillus as he knew that rabbits were sensitive to the bacillus and that it could destroy them. He identified the principle of immunity and appreciated that all animals were indelibly marked when they came into contact with a bacillus. He also discovered that it was possible to attenuate the virulence of such bacilli. Even if we only consider that he showed medicine the microbial approach to infectious diseases—an approach marked by rigorous experiments—he would have been one of the very few who, during their lifetime, must have noticed the enormous repercussions their scientific work had on the improvement of the human condition. As well as being enormously generous in his contributions towards public health, Pasteur also gave rise to a long tradition of scientists who were uninterested in money. We have heard that he invested all the money from registered patents for the pasteurisation of beer, vinegar and wine back into the public domain, and he did not benefit personally from any of the industrial tools developed either. After the Pouilly-le-Fort experiment, he allocated to his laboratory the earnings from sales of sheep anthrax vaccinations in France, reserving only the proceeds from foreign markets for himself and his closest partners. He must have felt intensely nostalgic at not being able to travel to distant lands and study infectious diseases peculiar to different regions and contribute to their eradication. One of his major concerns was to send some of the most eminent students from his school instead. In this way Instituts Pasteur developed and prospered, and they still form a unique network in the world today. Some of them operate in the same way as the Parisian model, which Pasteur founded and devised policies for.
His life is a constant example of relentless demands of originality and quality, a permanent invitation to anxiety and cross-examination, a glorification of willpower. Pasteur’s fascinating self-assurance encourages us to ask ourselves what state of perfect equilibrium he must have reached and whether or not he belonged to those Plato enthusiasts for whom “to discover means to remember”. Better than anyone else, he demonstrated the presence of that strange energy that exhorts humans to surpass themselves and prevents them from sinking into indifference. Finally, he wished to see Bossuet’s philosophical principle engraved on the front wall of all laboratories: “the most unsettling thing for the spirit is to believe in things because we want to believe in them ...”. On his death at Villeneuve-l’Etang, in September 1895, he left his successors vast areas of research to investigate—all the different future prospects that he had laboured so hard at creating. It seems appropriate to apply to Pasteur those verses that Malraux borrowed from Hugo for the epigraph of his book on de Gaulle [11]. Oh! What a terrible sound from the dusk Oak trees felled for Hercules’ stake!
Acknowledgements This article was translated from the French by Victoria Skewes. My warmest thanks go to Geneviève Milon and David Ojcius for their help and encouragement during the writing of this article. I also wish to thank Chantal Brulé, Sylvie Delassus and the Publications Service of the Institut Pasteur for their assistance.
References [1] [2]
E. Duclaux, Pasteur, Histoire d’un esprit, Charaire, Sceaux, 1896. R. Dubos, Louis Pasteur, Franc-tireur de la science, La Découverte, Paris, 1995. [3] R. Vallery-Radot, Pasteur, Dessinateur et pastelliste (1836-1842), Emile Paul, Paris, 1912. [4] L. Pasteur Vallery-Radot, Œuvre de Pasteur. Volume 1: Dissymétrie moléculaire, Masson & Cie, Paris, 1933-1939. [5] L. Pasteur Vallery Radot, Œuvres de Pasteur. Volume 2: Fermentations et générations dites spontanées, Masson & Cie, Paris, 1933-1939. [6] L. Pasteur Vallery Radot, Œuvres de Pasteur. Volume 7: Mélanges scientifiques et littéraires, Masson & Cie, Paris, 1933-1939. [7] L. Pasteur Vallery Radot, Œuvres de Pasteur. Volume 3: Études sur le vinaigre et sur le vin. Volume 5: Etudes sur la bière, Masson & Cie, Paris, 1933-1939. [8] L. Pasteur Vallery Radot, Œuvres de Pasteur. Volume 4: Études sur la maladie des vers à soie, Masson & Cie, Paris, 1933-1939. [9] E. Roux, L’œuvre médicale de Pasteur, In Centième anniversaire de la naissance de Pasteur. Institut Pasteur, Hachette, Paris, 1922. [10] L. Pasteur Vallery Radot, Œuvres de Pasteur. Volume 6: Maladies virulentes, virus-vacccins et prophylaxie de la rage, Masson & Cie, Paris, 1933-1939. [11] A. Malraux, les chênes qu’on abat, Gallimard, NRF, Paris, 1971.
Review: on the shoulders of giants
Louis Pasteur (1822–1895) Guy Bordenave 1 Unité d’immunophysiologie et Parasitisme Intracellulaire, Institut Pasteur, 25, rue du Dr.-Roux, 75724 Paris cedex 15, France
Abstract In Louis Pasteur’s scientific career it is striking to note the exponential character of the research he introduced in all the fields he opened up. He offered fabulous opportunities to stereochemistry. He is acknowledged as one of the founders of microbiology. He established the possibility of anaerobic life. He pointed the way to epidemiology, public health, and the bacteriologic fight. He struggled against the idea of spontaneous generation of life. He irrevocably substantiated the microbial theory of infectious diseases. He demonstrated that bacterial virulence could be attenuated, he evidenced immunity and generalised the vaccination principle. He also was an incomparable experimenter. © 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. Keywords: Molecular dissymmetry; Microbiology; Aerobic and anaerobic lives; Rebuttal of spontaneous generation of life; Immunity; Vaccines; Microbes and infectious diseases; History of science
1. Introduction What more can be said about this exceptional man who has already had so much written about him2 [1]? It is true that every generation has a duty to remember the immense contribution made by some of our predecessors. Pasteur’s patriotism, for example, has been widely discussed and evidence of the influence of his father—a former soldier of the First Empire who later became a tanner craftsman—is sought in vain within this patriotism. His mother appeared to have a heightened sense of the relativity of human concerns as, shortly before her death in 1848, she wrote the following words to him: “whatever happens, never let yourself get down. Little is of great importance in life” [2]. The human species is made in such a way that it is easily fascinated by everything that relates to the major work of so-called eminent people. We can therefore ask ourselves if, in this case, it was the conservatism origins of that characterised Pasteur and, under Napoleon III, inspired him to talk about “the exaltation created by a great reign”, while Victor Hugo was himself in exile. However, it is difficult to assess whether such reasoning is really worthwhile. The most sensible approach would E-mail address: [email protected] (G. Bordenave). 1 G. Bordenave is a retired Institute Pasteur scientist. For correspondence and reprints please contact G. Milon at the given address. 2
Pasteur’s correspondence and other documents: Annick Perrot, Conservateur, Musée Pasteur, 25-28, rue du docteur Roux, 75524 Paris cedex 15; E-mail: [email protected]. © 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. DOI: 1 0 . 1 0 1 6 / S 1 2 8 6 - 4 5 7 9 ( 0 3 ) 0 0 0 7 5 - 3
probably be to try and limit ourselves to an impersonal description of his scientific work. But is this really possible given that he was such an exceptional being? How can we possibly define a scientific career in relation to such work? In any case, this account could only ever approach such a subject with great humility. Dole, a small sub-prefecture in the Jura region, can be proud, and rightfully so, of having witnessed, in 1822, the birth of the one who, indisputably, belongs to that category “of children whose genius immortalises the memory of civilisations”.
2. Molecular dissymmetry It was in 1844, at the age of 22, while he was studying chemistry at the Ecole Normale Supérieure in Paris, that Pasteur made one of his first, key discoveries. It was already known that quartz crystals were able to deviate polarised light either to the right or to the left. This property was directly linked to their crystalline configuration: some facets of the crystals—the hemihedral facets—are inclined in relation to the edges which support them, sometimes in one direction and sometimes in the other. Biot, an expert in crystallography and optical phenomena, noticed that tartaric acid deviated polarised light both in its liquid state and crystalline form. Two crystalline forms of tartaric acid were identified: tartaric acid and paratartaric acid. Mitscherlich, another reputable chemist, had shown that these two forms as
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Today, we are more aware of the importance, in pharmacology for example, and even in perfumery, of the two enantiomer forms (the mirror image of each other) of a same substance—one can be active, whereas the other remains inactive [1,4]. 3. Fermentations and microbiology
Fig. 1. Facets of the double sodium ammonium paratartrate crystals: some are inclined to the right and others to the left. Photo courtesy of Institut Pasteur.
well as those of their respective salts—tartrates and paratartrates—were identical in all respects except in their ability to deviate polarised light. The tartrates deviated light to the right while the paratartrates remained inactive. Pasteur noticed small facets similar to those of quartz crystals on the tartaric acid and diverse tartrate crystals (Fig. 1). He also noticed that all the facets were orientated to the right in the tartrate crystals, while in the double sodium ammonium paratartrate crystals, as in those studied by Mitscherlich, some were inclined to the right and others to the left. He divided up the two categories of crystals with tweezers and used them to make separate solutions. Both solutions deviated polarised light—one to the right and the other to the left. Mixed in equal quantities, they formed an optically inactive solution. The concept of molecular dissymmetry was born from the interpretation of this observation. The age of stereochemistry was beginning. In comparison to Biot’s often repeated exclamation: “My dear child, I loved science so much during my life it makes my heart flutter” (Biot who imposed draconian measures on the experiment), Pasteur’s reaction is moving in other ways. So violent was the shock, that he rushed out of the laboratory and, when he came across a chemistry technician, he grabbed him and cried out: “I’ve just made an important discovery... I’m so happy I’m trembling all over, I can’t even look through the polarimeter”. What an extraordinary moment of elation! What researcher, even when his find is more modest, has not experienced such emotion? This is a joy dominating over all others, an inexpressible and absolute joy! Pasteur was truly won over and this impetuous whirlwind was going to take over his whole life. Pasteur’s choice of science was significant, as during his adolescence he also showed an undeniable talent for painting, proof of which is shown in the pastel pictures he left [3]. Pasteur was convinced that molecular dissymmetry was one of the most fundamental characteristics of living organisms.
After being a professor in Dijon and then Strasbourg, another challenge awaited Pasteur in Lille. Here, he was drawn to study the problems that industrialists in the region were having with their sugar factories or breweries. He was faced with the problem of fermentation and, from this confrontation a new discipline would be born: microbiology. The scientists who prevailed in this field at the time were Berzélius, Liebig, Helmholtz and Berthelot. Using all the influence that their wide fame gave them, they fought against what was known as the “vitalist theory”, i.e. the involvement of living organisms in fermentations. They attributed this involvement to chemical agents, which they referred to as ferments and which they considered independent of the vital processes. Pasteur studied ethanolic, lactic, butyric and acetic fermentations. He began by explaining lactic fermentation because, of all fermentations, it is chemically the most simple. On fission of the glucose molecule, lactic fermentation only consists of two lactic acid molecules. He isolated the lactic bacillus from the greyish deposit which accompanies the souring of milk and he cultivated it in a clear, nutrient broth. Once a sufficient quantity had been produced, the population of microscopic living organisms (which were all alike) ensured that lactic acid was formed from glucose. As for ethanolic fermentation, he began by showing that glucose was not only transformed into carbonic gas and ethanol—something that had been accepted since Lavoisier—but that other products were also found as a result of this fermentation such as glycerine, succinic acid and amyl alcohol. Above all, he showed that the yeast, which was very different from the bacillus involved in lactic fermentation, induced ethanolic fermentation in a liquid which only contained glucose and mineral salts. There was total parallelism between fermentation and yeast multiplication. The agent responsible for butyric fermentation appeared in the form of small rods, which were capable of undulatory vibrations and could therefore move. Whilst observing a drop of liquid undergoing butyric fermentation between a slide and a coverglass, he noticed that the bacilli at the edge of the coverglass were losing their mobility while those in the middle retained it. This situation was the reverse of what he was used to recording with other microorganisms, as they did the opposite and gathered on the edges where there was more oxygen. He was convinced that life without oxygen was possible as butyric fermentation could be stopped by blowing air into the liquid where this fermentation was occurring. He came up with the terms “aerobic” and “anaerobic” to refer, respectively, to life in the presence of oxygen and life without it. Pasteur still examined acetic fermentation and here he also
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introduced the vitalist theory even though it was known that vinegar was the result of a simple chemical reaction: the oxidation of ethanol in acetic acid. He demonstrated that the transformation of wine into vinegar depended on the activity of a bacterium whose population formed a thin layer, which floated on the surface of liquids. It is this bacterium which incorporates oxygen from the air into the ethanol [1,5]. In 1897, 2 years after Pasteur’s death, Büchner managed to extract a soluble fraction from yeast, which was able to trigger ethanolic fermentation in the absence of living cells. It seemed that the catalytic theory of Liebig and Berzélius, etc. was basically correct. The two great trends of scientific thought during this period—the physiological and chemical theories of fermentation and metabolism—were going to be reconciled. Microscopic living organisms produce ferments—enzymes, which act either inside or outside the microbial cell. Enzymes can carry out chemical transformations in the absence of the living organisms which produced them but, under ordinary conditions, these transformations give the living organisms energy and basic elements which are essential to their metabolism. Pasteur had never ruled out this possibility as, for example, in 1878 he wrote “I should not be surprised to see the yeast cells produce a soluble, alcoholic ferment... soluble ferments have only yet been produced by a vital function”. In any case, the products of ethanolic, lactic, butyric and acetic fermentations were linked to bacterial life. 4. Rebuttal of the idea of the spontaneous generation of life Having studied microbial life, Pasteur found himself drawn into the controversy surrounding the idea of the spontaneous generation of life. He joined this debate on the origin of life in 1859, the year Darwin’s “The Origin of Species” was published. Here, he fully demonstrated his talent as an exceptional bench scientist. From the concept of specificity, born out of the study of fermentations, was derived that of hereditary characters which, in turn, depended on normal generations. He naturally admitted that it was possible that, somewhere in the universe, life could be reproduced but it was necessary to check the assertions of those who claimed to have witnessed the birth of life. In 1859, Pouchet, Director of the Natural History Museum in Rouen, claimed that he could bring about spontaneous generation whenever he wished. His demonstration consisted of taking a flask of boiling water, sealing it tightly and then plunging it upside down into a bath of mercury. Once the water had cooled down, the flask was opened in the mercury and half a litre of pre-sterilised oxygen as well as a little hay infusion were introduced. A microbial population regularly developed after a few days. Pasteur demonstrated that by eliminating the mercury from the experiment, he avoided contaminating the liquids and the air. He then conducted those famous experiments in his swan-neck flasks (Fig. 2). Having introduced a culture broth into a flask, he heated the neck in a flame and
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Fig. 2. Louis Pasteur observing a culture broth sterilised in straight and swan-neck flasks. Painting Robert Thom. Photo courtesy of Institut Pasteur.
then bent it into the shape of an “S” (hence the name “swanneck”). Vapour from the boiling broth expelled the air via the opening in the s-shaped tube. The flask cooled down and the outside air entered slowly, without having been heated or filtered, but having been sterilised by the humidity of the tube which retained the germs which were unable to get past the bend in the swan-neck. As the s-shaped tube remained open, the air on the inside and outside of the flask communicated freely but the broth remained sterile indefinitely. The lack of air was therefore not responsible for the fact that life did not appear. Moreover, the broth retained the elements that were necessary for the development of life as, when the neck of the flask was broken, microbial life appeared quickly. It was with straight-neck flasks that Pasteur discovered the uneven distribution of microorganisms in the atmosphere. Flasks containing a nutrient broth underwent sterilisation by boiling, which also helped to expel the air due to the stream of water vapour. The flasks, which were closed using a flame while the water vapour was still coming out, were almost empty of air, and the nutrient broth remained sterile as long as the flasks were sealed. Pasteur opened these flasks in many different places and took extreme care not to contaminate them. By breaking the neck of the flasks, air from the outside was allowed in. The flasks were then re-sealed and put into an incubator. Some of these flasks remained sterile while in others, microbial life appeared. The evidence of the uneven distribution of microorganisms in the atmosphere was proven when it was noticed that the number of flasks with a contaminated content was higher at low altitudes, near inhabited areas and cultivated lands than in the high mountains or places where the air remained still for a long time. This also proved, once again, that non-heated air could only trigger microbial growth if it contained living germs likely to culture the nutrient broth [1,5]. These demonstrations led to aseptic manipulation, sterilisation and autoclaving techniques, and the Englishman, Tyndall, played a key role in founding them. It was at this time that scientists also became aware that if air contained microorganisms, then water and solids must also contain them and maybe even greater numbers of them.
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After the death of Claude Bernard, a renowned physiologist, notes were published relating to some experiments that he conducted on the fermentation of grapes. In particular, he stated that, contrary to Pasteur’s opinion, fermentation was possible independently of living processes and he seemed to implicitly suggest that yeast could be the consequence of fermentation rather than its cause. Pasteur detected there a disguised resurgence of the idea of the spontaneous generation of life and he devised, by way of response, a fabulous experiment. He owned a small vineyard in the district of Arbois. Using glass, he isolated a section of the vines at a time when the yeast germs were not yet deposited on the grapes. It was impossible to obtain ethanolic fermentation with ripe raisins from the protected bunches, but the opposite was true of the ripe raisins from the exposed bunches. If the protected bunches were placed on vine-stocks left in the open air, fermentation was easily obtained with their ripe grapes. Nothing had been discovered about the conditions in which life came about. All that was shown was that microbial life did not occur in a properly sterilised nutrient medium, which was protected from outside contamination. Furthermore here is what Pasteur wrote on this subject: “I’ve searched for spontaneous generation in vain for twenty years. No, I don’t think it’s impossible but what leads you to believe that it is the origin of life?You put matter before life so matter has therefore existed since the beginning. How do you know that the incessant progress of science will not force scientists in a century, a thousand or two thousand years time... to assert that life, and not matter, has existed since the beginning. You go from matter to life because your current knowledge... does not allow you to understand things differently. Who can assure me that in ten thousand years time we will believe it impossible not to go from life to matter...?”[6].
5. Wine, beer and silk worm diseases It is Pasteur who rightly received the credit for having discovered that ethanolic fermentation diseases were caused by microorganisms which entered into competition with yeast. He went to great lengths to prevent microorganisms from developing in finished products, i.e. after the involvement of yeast. He tried several antiseptics and then used heat as a sterilisation agent. Very wisely, he knew how to take advantage of the vulnerability of microorganisms by combining the slight acidity of wine with a moderate rise in temperature (55 °C), over a short period of time (and better still without oxygen), to obtain partial, but generally sufficient, sterilisation. This principle would later be applied to a very large number of perishable liquid foodstuffs (wine, beer, vinegar, milk, etc.) or solids, and would lead to what is now known throughout the world as pasteurisation. His work on the role of microorganisms in fermentations and then in wine and beer diseases, would gradually lead him to the microbial theory of infectious diseases and give this remarkable unity and outstanding direction to his scientific career [1,7].
Fig. 3. Silk worm on mulberry-tree leaves. Drawing by Lackerbauer, published in “Maladie des vers à soie”, Paris, 1870. Photo courtesy of Institut Pasteur.
It was then that he returned to Paris, to the École Normale Supérieure, and was called upon by Dumas, one of his former masters/“maîtres” to study the silkworm disease which was causing devastation in the sericulture industry in the south of France (Fig. 3). When suffering from the disease, the worms weaving the cocoons from which the silk was extracted stopped growing at very different stages. They showed small marks, which resembled specks of pepper on their skin—hence the name “pebrine”, which was given to the disease. Pasteur noticed that only butterflies from sick worms showed the characteristic corpuscles, which were easily detected under the microscope. He set up a method of selection based on this observation to sort eggs from healthy butterflies, which could be kept for reproduction, and eggs produced by affected butterflies that had to be destroyed. The healthy eggs made healthy worms, which wove cocoons with healthy chrysalises. Today, it is known that pebrine is caused by a protozoan parasite. At the time it appeared that the problem was more complicated than first thought because at least two simultaneous diseases were present: pebrine and flacherie. Flacherie was only really identified when it persisted on its own—pebrine having been eliminated from the breeding of healthy worms. The general view today is that flacherie is above all caused by opportunist infections which accompany a benign viral attack. These varied infections resulted in diverse and disturbing forms of the disease. Pasteur acquired a certain understanding of flacherie and devel-
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oped appropriate techniques for keeping it under control. One of his recommendations was not to retain eggs from worms which appeared languid during the stage preceding the weaving of the cocoon or from those which died in large numbers at this time. It was a contagious disease problem and it was necessary to keep healthy worms in non-infected premises using non-infected material. From this approach emerged the principle—elementary in infectious diseases but not yet implemented—of separating the healthy from the affected [1,8]. (This principle was applied to humans by Emile Roux and his colleagues for the first time ever at the Pasteur Hospital). Pasteur learnt much from this work and was now in a better position to tackle the problem of infectious diseases in “superior” vertebrates. The period when the laboratory moved to the Cevennes has been described as a probably happy time in Pasteur’s life, although it is easy to imagine that the word “happiness”, in the most general sense of the term, did not feature frequently in the vocabulary of a man who was so intellectually and morally demanding. He called on his whole family to help with his silk worm research. To people who clung to traditional practices and who protested at the difficulty of new methods suggested, Pasteur would reply that he knew of a child (his daughter Marie-Louise, then aged ten) who excelled in the art of telling healthy butterflies from sick ones. This period spent in the countryside must have made a wonderful holiday for this young town girl! His wife, Marie, was also mentioned. She took notes and wrote up the reports of his experiments, which he dictated. She inquired into problems, followed the progress attained and also probably made suggestions, because in this field and in such an exciting atmosphere, it is difficult to remain passive or indifferent. The image of the two of them in the calm surroundings of this provincial environment (rather than in a relationship that revolved around domination of one by the other as has been too often suggested) paints a peaceful picture. It is satisfying to imagine the hours of deep communion and the oases of serenity and intimacy known to this household where science seemed to take centre stage. 6. Chicken cholera, sheep anthrax, swine erysipelas and the birth of immunity In 1879, Pasteur succeeded in cultivating the bacillus involved in chicken cholera and showed how the disease could be triggered by injecting a pure suspension of this bacillus. With this material, Pasteur demonstrated that, unlike chickens and rabbits, adult guinea pigs resisted the infection, developing only a local abscess, which was nevertheless the local outbreak site of the same bacilli, whose full virulence subsisted in chickens and rabbits. This concept of a healthy host opened the way for investigation into the wider problem of epidemiology. It is said that the fundamental observation was discovered by chance—if this was not so it must be said that Pasteur’s foresight was all the more astonishing—but, whatever the case, the main point was that
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the issue could no longer be avoided. Pasteur’s observations highlighted immunity and led to a general understanding of the vaccination principles. His research was interrupted for a while and the suspensions containing the chicken cholera bacillus aged in the laboratory. When he resumed his research, it became apparent that the chickens injected with the bacillus taken from these old suspensions not only survived, but went on to resist a second injection of a fresh suspension of the virulent bacilli which resulted in the rapid death of untreated chickens. Pasteur understood that the chickens had been immunised and that the bacilli responsible for causing the disease could be attenuated. Attenuation was achieved by exposing the culture to the air for some time periods (and at a suitable temperature), as the same culture, which was attenuated when exposed to the air, retained all its virulence when enclosed in an airtight container. Virulence was therefore not a permanent characteristic but rather an unstable property that could be lost and even regained without affecting any of the other bacillus characteristics. Pasteur established a connection between this observation and the one made at the end of the 18th century by Jenner, an English country doctor, who succeeded in protecting humans against smallpox by inoculating them with the substance contained in “cowpox” pustules. Cowpox or vaccinia was a similar disease found in cows but which was harmless in humans. At that point, it was the only application known to man. In order to combine the two discoveries, the name “vaccination” was given to the phenomenon brought up to date by the chicken cholera experiments. Pasteur then became aware of the existence of a general rule, which he had already suspected: the possibility to make an animal less susceptible to the virulent form of a microorganism by putting it in contact with the attenuated form. This research would now have an impact on the wider issue of attenuating virulence in germs. While Pasteur was studying the problem of sheep anthrax, the disease was causing devastation on sheep, goat and cow farms. Koch, a young German doctor, had already won recognition through his work on the sheep anthrax bacillus. He proved, among other things, that the anthrax bacillus actually caused the disease and he also ascertained the complete life cycle of this bacillus: immobile rods which develop into long filaments and then into round granules—the spores (or the resistant form) which, in the right conditions, changed back into rods. It was at the beginning of his research into the anthrax bacillus that Pasteur discovered large amounts of another bacillus in the tissues of some of the animals infected by the disease. He named this second bacillus (which is rare in blood) “septic vibrio”. He showed that it was a strict anaerobic bacteria, which was responsible for causing both abnormal death and erratic results in animals which had purposely been contaminated with anthrax, using the blood from animals that had died from the disease. The vibrio septic bacteria killed the animals before the anthrax bacillus had a chance to grow. Pasteur also used the anthrax bacillus to prove that the presence of a pathogen in an organism did not necessarily mean that the disease was manifested itself,
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and that the environment could be crucial in influencing its development. Although chickens appeared to resist anthrax, when their usual temperature of 42 °C was lowered to 38 °C, they could be made almost as susceptible to the disease as rabbits or guinea pigs. The anthrax bacillus turned out to be extremely sensitive to temperatures of around 45 °C, which explains why it did not multiply or did not multiply well in chickens. If a chicken, which had been purposely infected with anthrax by artificially lowering its temperature, then had its usual temperature raised back to 42 °C, it was restored back to health. Pasteur also concluded that anthrax bacillus spores could survive over long periods of time in the earth around where the animal that had died from the disease was buried, and that worms in this earth were involved in bringing the spores back up to the surface. He injected earth taken from the worm’s intestines into guinea pigs, infecting them with anthrax. Pasteur pointed out that stubble left behind after harvest could injure the animal and thus provide the infection with a means of entry. This research into the etiology of anthrax has been widely acknowledged as exemplary work. The main incentive behind it was, of course, to develop a vaccine. However, the task turned out to be more difficult than for chicken cholera. At the most usual temperatures, the bacillus is present in its rod form (i.e. sensitive to virulence attenuation) but the spore form of the bacillus (i.e. resistant to attenuation process) is also detected. The problem was how to keep the bacillus alive while at the same time preventing the spores from developing. After much trial and error, the objective was achieved by storing the cultures at 42-43 °C inside shallow containers with oxygen present. Eight days in these conditions resulted in producing bacilli that were harmless to guinea-pigs, rabbits and sheep. Before reaching this final state, the bacilli went through various stages of attenuation in which they could be maintained. Pasteur discovered that it was best to vaccinate against anthrax in two steps: an initial inoculation using bacilli from a culture with very weak virulence followed by a second inoculation, 12 d later, using bacilli from a more virulent culture. This process protected the guinea-pigs, rabbits and sheep against the most virulent form of the bacillus. This led to a public experiment—a memorable event that took place at Pouilly-le-Fort in the Spring of 1881, in the presence of a crowd of journalists and inquisitive on-lookers. The challenge was to vaccinate 24 sheep, one goat and six cows and then to inject them with the virulent strain of the anthrax bacillus. At the same time, a similar group of unvaccinated, control animals was also injected with the same anthrax bacillus strain. The experiment was a complete success—only the vaccinated animals survived. Evidently, critics protested at the time against the principle of vaccination. Some of these criticisms came from Koch, the famous German microbiologist. He criticised Pasteur, who did not use his solid culture medium method, for working with impure bacillus cultures. Koch remained unfamiliar with this emerging new discipline—immunity— perhaps because he had apparently failed in his own attempt
to protect against the tuberculosis bacillus. He remained relatively sceptical to the possibility of attenuating pathogen virulence, even though his own work on the anthrax bacillus and his important discoveries (the tuberculosis bacillus and the human cholera vibrio) contributed a great deal to establishing the microbial theory of infectious diseases. Most of the ill feeling between Koch and Pasteur stemmed from the extreme sensitivity of scientists to anything related to their own personal work, to say nothing of the severe FrancoGerman bitterness that existed at the time. It would appear that a scientist’s sensitivity could be as acute as his contribution to progress was crucial. The German school of medical bacteriology attracted considerable international interest. It discovered various bacteriological agents of infectious diseases. It had an advantage over the French school because—so it is claimed—it was larger and better organised. However, through its research and particularly through Pasteur’s contribution, the French school can be credited with the discovery of streptococcus (1879) (which was causing damage in maternity wards under the name of puerperal fever), staphylococcus (1878), pneumococcus (1881) and septic vibrio (1877). This is what Roux said on the subject: “A small, round organism, made up of clusters, that cultivates easily in nutrient broth, can be found in the pus of warm abscesses and furuncles. It can also be found in infectious osteomyelitis in children. Pasteur affirmed that osteomyelitis and furuncle are two forms of the same disease and that osteomyelitis was really a furuncle of the bone. This assertion caused much amusement among surgeons in 1878. Pasteur never gave botanical names to the microbes he discovered, but named them according to their shape or culture. Thus, for him, the furuncle microbe was the “grain cluster microbe” and the puerperal fever microbe was the “grain chaplet microbe”. These same discoveries are better known in the world of bacteriology as Staphylococcus and Streptococcus pyogenes” [9]. And they also enabled certain people to claim them as their own discoveries. The discovery of the plague bacillus by the pasteurien, Yersin, could also be added to this never-ending list. As far as research into erysipelas or swine fever was concerned, the problem was of a different nature. If the swine fever bacillus is injected from rabbit to rabbit, the bacillus acclimatises to the rabbit. All the rabbits end up dying after a few days, whereas initially the rabbits always got sick but did not die with the same frequency. If pigs are then injected with the blood from this group of rabbits, the virulence for pigs gradually decreases over several successive rabbit inoculations and this injection no longer causes the pigs to die but just makes them sick. Once they recover from the disease, the pigs are immunised. This must have been enormously encouraging for everyone who worked alongside Pasteur in this amazing development, i.e. the application of essential principles: the use of attenuated, living bacilli in relation to a recognition system that was gradually being discovered and which ensured that
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the “superior” organism being studied was protected in some way. Two fundamental approaches, centred around a concern for public health, immediately set the Pasteur school apart from other bacteriology schools—firstly, research into microbial agents that cause infectious diseases and secondly, using this research (with the aim of attenuating virulence as soon as the disease agent is exposed) to prepare a vaccine for prophylactic use and afterwards, a serum for use as a cure. As far as virulence attenuation procedures for microorganisms were concerned, initial attempts dramatically showed how diverse they could be, as no less than three different attenuation methods were designed under Pasteur’s supervision for the first three bacterial vaccines. One of the main reservations at the time was based on the fact that people remained reluctant to the idea that these tiny and apparently insignificant beings—bacilli—could have such a devastating effect on “superior” organisms [1,10]. 7. Human vaccination against rabies Without a doubt, these experiments required an enormous amount of intellectual courage, of courage in every sense of the word. There has been much debate about the decision to carry out research into rabies. Clearly it was Pasteur’s choice. We can assume that the determining factor was the fact that rabies was a disease common to humans and animals and that it was possible to experiment on animals. The least that can be said is that the experiments involved several rather startling activities. Firstly, they involved a virus that could not be detected under an optical microscope. Secondly, the disease had a long incubation period—over a month from the moment of contamination to the appearance of the first symptoms. This period was used to try and obtain resistance by vaccination, even after the infectious bite. The first positive result was the discovery that the virulence was based in the nervous system and that the best inoculation route was via dura mater after trephination. By regularly transferring contaminated spinal cord via this route, from rabbit to rabbit (a process requiring around fifty passages), a fixed virus was obtained which had a regular incubation period of 7 d. The second positive result was that viral virulence could be attenuated and extinguished by exposing the spinal cord from contaminated rabbits to dry, sterile air (Fig. 4). It was not denied that this could have been due to a reduced amount of rabies virus rather than a reduction in virulence as, unlike bacteria, it was impossible to quantify the virus. It is easy to imagine the moral dilemma that Pasteur must have faced when 9-year-old Meister came to him on 6th July 1885 from an Alsatian village where he had been bitten on the 4th July. Imagine what a request for help from Alsace must have felt like in France in 1885. The child was suffering from 14 wounds and his death appeared unavoidable. The decision was made to apply the treatment that had been continually successful in dogs. Roux was working on the same problem and we can credit him for his idea of gradually drying out rabbit spinal cord in air inside sterile flasks.
Fig. 4. A painting by Edelfelt showing Louis Pasteur inspecting a rabbit spinal cord drying out in a sterile flask. Photo courtesy of Institut Pasteur.
Pasteur adapted this idea by adding potassium particles to speed up the dehydration process. Roux believed that the animal experiments had not been perfected enough for use on humans. He left the laboratory to mark his disapproval and pointedly got back to his seat when the diatribe against Pasteur reached its paroxysm. The method involved injecting subcutaneously suspensions of spinal cord via the peritoneal region. First non-virulent spinal cord was used, then more and more recent spinal cord until finally an extremely virulent spinal cord was administered. The treatment began on 6th July 1885 and continued until the 16th. This is Pasteur’s brief account: “For each of the different spinal cords used, we also inoculated two new rabbits by trephination in order to monitor the virulence of the spinal cords.... Spinal cords dated from the 6th, 7th, 8th, 9th and 10th July were not virulent as they did not cause rabies in the rabbits. All the spinal cords from 11th, 12th, 13th, 14th, 15th and 16th July were virulent and the virulent matter became progressively stronger and rabies broke out in the rabbits.... Joseph Meister therefore avoided not only the rabies that his bite wounds may have otherwise caused but also the rabies that I inoculated him with..., a more virulent form of rabies than the kind found in stray dogs...” [1,10]. This description, which remained true for all subsequent individuals treated for the disease, meant that there was no basis for the allegations accusing the treatment of being ineffective. Strangely enough, however, the same allegations still exist today.
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8. By way of conclusion What more can be said about this scientific career when all attempts at describing it are feeble in comparison. The most striking element is the exponential nature of every area of Pasteur’s research and the fact that—due to a lack of time owing to the choices made—he was not able to investigate further, or perfect every area of his work. He opened up fantastic opportunities for stereochemistry. He is recognised as one of the founders of microbiology. He discovered that life without oxygen is possible. His observation that chicken cholera bacillus, deadly for this species of animal yet harmless in guinea-pigs (which could carry the bacillus without becoming sick), provided a foundation for epidemiology. Lister, the English surgeon, revered as the founder of antiseptic techniques, always remained grateful to Pasteur for his intellectual contribution to medicine. Pasteur did not lay down any specific rules concerning hygiene but we can consider that he left it up to others to enforce such rules after he had brought the problem to light. He noticed that microorganisms in soil could affect the anthrax bacillus to such an extent that it no longer spread the disease. He realised that this discovery could lead to therapeutic applications. He played his part in the bacteriological struggle, offering, albeit unsuccessfully, to provide the Australian Government, whose territory was infested with rabbits, with the chicken cholera bacillus as he knew that rabbits were sensitive to the bacillus and that it could destroy them. He identified the principle of immunity and appreciated that all animals were indelibly marked when they came into contact with a bacillus. He also discovered that it was possible to attenuate the virulence of such bacilli. Even if we only consider that he showed medicine the microbial approach to infectious diseases—an approach marked by rigorous experiments—he would have been one of the very few who, during their lifetime, must have noticed the enormous repercussions their scientific work had on the improvement of the human condition. As well as being enormously generous in his contributions towards public health, Pasteur also gave rise to a long tradition of scientists who were uninterested in money. We have heard that he invested all the money from registered patents for the pasteurisation of beer, vinegar and wine back into the public domain, and he did not benefit personally from any of the industrial tools developed either. After the Pouilly-le-Fort experiment, he allocated to his laboratory the earnings from sales of sheep anthrax vaccinations in France, reserving only the proceeds from foreign markets for himself and his closest partners. He must have felt intensely nostalgic at not being able to travel to distant lands and study infectious diseases peculiar to different regions and contribute to their eradication. One of his major concerns was to send some of the most eminent students from his school instead. In this way Instituts Pasteur developed and prospered, and they still form a unique network in the world today. Some of them operate in the same way as the Parisian model, which Pasteur founded and devised policies for.
His life is a constant example of relentless demands of originality and quality, a permanent invitation to anxiety and cross-examination, a glorification of willpower. Pasteur’s fascinating self-assurance encourages us to ask ourselves what state of perfect equilibrium he must have reached and whether or not he belonged to those Plato enthusiasts for whom “to discover means to remember”. Better than anyone else, he demonstrated the presence of that strange energy that exhorts humans to surpass themselves and prevents them from sinking into indifference. Finally, he wished to see Bossuet’s philosophical principle engraved on the front wall of all laboratories: “the most unsettling thing for the spirit is to believe in things because we want to believe in them ...”. On his death at Villeneuve-l’Etang, in September 1895, he left his successors vast areas of research to investigate—all the different future prospects that he had laboured so hard at creating. It seems appropriate to apply to Pasteur those verses that Malraux borrowed from Hugo for the epigraph of his book on de Gaulle [11]. Oh! What a terrible sound from the dusk Oak trees felled for Hercules’ stake!
Acknowledgements This article was translated from the French by Victoria Skewes. My warmest thanks go to Geneviève Milon and David Ojcius for their help and encouragement during the writing of this article. I also wish to thank Chantal Brulé, Sylvie Delassus and the Publications Service of the Institut Pasteur for their assistance.
References [1] [2]
E. Duclaux, Pasteur, Histoire d’un esprit, Charaire, Sceaux, 1896. R. Dubos, Louis Pasteur, Franc-tireur de la science, La Découverte, Paris, 1995. [3] R. Vallery-Radot, Pasteur, Dessinateur et pastelliste (1836-1842), Emile Paul, Paris, 1912. [4] L. Pasteur Vallery-Radot, Œuvre de Pasteur. Volume 1: Dissymétrie moléculaire, Masson & Cie, Paris, 1933-1939. [5] L. Pasteur Vallery Radot, Œuvres de Pasteur. Volume 2: Fermentations et générations dites spontanées, Masson & Cie, Paris, 1933-1939. [6] L. Pasteur Vallery Radot, Œuvres de Pasteur. Volume 7: Mélanges scientifiques et littéraires, Masson & Cie, Paris, 1933-1939. [7] L. Pasteur Vallery Radot, Œuvres de Pasteur. Volume 3: Études sur le vinaigre et sur le vin. Volume 5: Etudes sur la bière, Masson & Cie, Paris, 1933-1939. [8] L. Pasteur Vallery Radot, Œuvres de Pasteur. Volume 4: Études sur la maladie des vers à soie, Masson & Cie, Paris, 1933-1939. [9] E. Roux, L’œuvre médicale de Pasteur, In Centième anniversaire de la naissance de Pasteur. Institut Pasteur, Hachette, Paris, 1922. [10] L. Pasteur Vallery Radot, Œuvres de Pasteur. Volume 6: Maladies virulentes, virus-vacccins et prophylaxie de la rage, Masson & Cie, Paris, 1933-1939. [11] A. Malraux, les chênes qu’on abat, Gallimard, NRF, Paris, 1971.