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The Life of Sir Alexander Fleming
Chapter VIII - First hope: the Lysozyme

... sometimes a man whose intelligence is arrested by things which do not strike others, who, knowing how to use his eyes, looks hard at what the others do not see. leriche

Never neglect any appearance or any happening which seems to be out of the ordinary: more often than not it is a false alarm, but it may be an important truth. Fleming

IN 1922', writes Dr Allison, 'I went to St Mary's to work in I the Inoculation Department with Fleming. From the very JL first he started to pull my leg about my excessive tidiness. Each evening I put my "bench" in order and threw away anything I had no further use for. Fleming told me that I was a great deal too careful. He, for his part, kept his cultures sometimes for two or three weeks and, before finally getting rid of them, looked very carefully to see whether by chance any unexpected or interesting phenomenon had appeared. The sequel was to prove how right he was and that, if he had been as neat as I am, he would probably have found out nothing new.

'About a month or two after I had started working with him, he was busy one evening cleaning up several Petri dishes which had been lying on the bench for perhaps ten days or a fortnight. As he took up one of the dishes in his hand, he looked at it for a long time, showed it to me, and said: "This is interesting." I had a good look at it. It was covered with large yellow colonies which appeared to me to be obvious contaminants. But the remarkable fact was that there was a wide area in which there were no organisms; and another, farther on, in which the organisms had become translucent and glassy. Beyond that, again, were organisms which were in a transitional stage of degradation, between the very glassy ones and those which were fully developed with their normal pigment.

'Fleming explained that this particular dish was one to which he had added a little of his own nasal mucus, when he had happened to have a cold. This mucus was in the middle of the zone containing no colony. The idea at once occurred to him that there must be something in the mucus which dissolved or killed the microbes in its immediate neighbourhood, and then became diffused in such a way that it progressively contaminated the already developed colonies. "Now, that really is interesting," he said again. "We must go into it more thoroughly.5' His first care was to pick off the organism and to stain it by gram. He found it was a large gram-positive coccus, not a pathogen, and not one of the known saprophytic organisms commonly met with, but obviously a contaminating organism which was more likely to have been in the atmosphere of the laboratory and may, of course, have blown in through the window, from the dust and air of Praed Street.

'The next step in the investigation was to try again the use of nasal mucus, and he tested some for its effect on this large gram-positive coccus, not on a plate but in a tube. He made a culture of the organism in broth and added nasal mucus to it. To his great surprise and mine, the opaque suspension of organisms became, in the space of a few minutes, completely clear — "clear as gin," he said at the time. Immediately afterwards, and in the same conditions, he tried the effect of tears. A single drop of tear-fluid dissolved the organisms in probably less than five seconds. It was astonishing and thrilling.

For the next five weeks, my tears and his were our main supply of material for experiment. Many were the lemons we had to buy to produce all those tears! We used to cut a small piece of lemon-peel and squeeze it into our eyes, looking into the mirror of the microscope. Then, with a Pasteur pipette, the point of which had been rounded in a flame, we collected the tears which we proceeded to put into a test-tube. In this way I often collected as much as J-J c.c. of tears for our experiments.'

Visitors, men and women alike, were put under contribution in this matter of tears. The St Mary's Hospital Gazette published a drawing which depicted children coming, for a few pennies, to the laboratory, where one attendant was administering a beating, while another collected their tears in a receptacle on which was written the word 'Antiseptics'. Even the laboratory attendants were condemned to undergo the 'ordeal by lemon', but they got threepence each time. They kept regular accounts and were paid for all their tears at the end of the month. Once, when Fleming noticed that the eyes of one of these men were very red, he remarked: Tf you cry often enough, you'll soon be able to retire!'

These experiments had proved that tears contain some substance which can dissolve certain microbes with surprising speed. Tt was possessed5, said Fleming, 'of extraordinary power. Up till then I used to wonder at the much slower action of the antiserum which, when added to an infected broth warmed in an incubator or in the water bath, takes some considerable time to dissolve the microbes, and then only incompletely. But when I studied this new substance, I put into a test-tube a thick, milky suspension of bacteria, added a drop of tear, and held the tube for a few seconds in the palm of my hand. The contents became perfectly clear. I had never seen anything like it.'

The phenomenon was indeed very impressive, and Fleming was the first person to observe it. The double piece of luck had been miraculous, for the mysterious substance had been brought in contact with the one microbe which was most sensitive to its action. All the same, though its power of dissolving (and so, killing) had been demonstrated in a more spectacular fashion in the case of the yellow 'coccus', which was inoffensive, the substance also dissolved, though more feebly, other microbes, some of which were pathogenic. In a series of experiments, Fleming showed that it had the properties of an enzyme (natural ferment).

What should this substance be called? As usual, the question was debated in the library, round the tea-table. Wright, as we have seen, delighted in constructing words from Greek roots. Since the new substance was a species of enzyme, its name must end with the syllable 'zyme'; and since it dissolved, or 'lysed', certain microbes, it was agreed to given it the name of 'lysozyme'. As to the microbe so easily 'lysed', Wright named it 'micrococcus lyso-deikticus' — from 'lysis' (dissolution) and 'deixeiri (to show): in other words — the organism which makes it possible to show, or note, a power to dissolve.

Fleming continued tenaciously with his investigation of the lysozyme. Since he had made the initial discovery, an idea had been taking form in his mind and becoming more and more insistent. How did it happen that a natural secretion of the body should possess such great strength as a bactericide? Obviously because it had a protective effect on exposed surfaces. This was a necessary provision of nature since, did it not exist, the human species would have died out long ago, or would never have developed at all, seeing that, from the moment of birth, human bodies are in contact with the innumerable germs which air, earth and water contain. At every moment of our lives microbes are being deposited on the surface of the skin, and are penetrating into the nose, the mouth and the alimentary canal. Many of these microbes are harmless, some are even useful and, for example, facilitate digestion. The organism tolerates them, but resists any attempt they may make to get beyond the mucous membrane or to multiply too rapidly.

The blood and its army of phagocytes provide one part of this system of natural defences. But there are certain sensitive and fragile areas, such as the conjunctiva of the eye, the membrane of the nose, and the mucous membrane of the respiratory channels, which are exposed to airborne microbes, and do not have the advantage of an abundant blood-flow. These parts of the body cannot be left without protection. It looked as though lysozyme might be one of the body's natural defences and, if the hypothesis could be verified, it seemed probable that this substance, or other substances of the same type, would be found distributed all over any animal body — whether of a man, a bird or a fish — and that this peculiarity would be present, too, in the vegetable world.

Fleming, therefore, organized a series of experiments with the object of showing that lysozyme would be found in other secretions and even in tissues. He discovered that a nail-paring, a scrap of skin, a drop of saliva or a few hairs, when introduced into a test-tube, exercised the same miraculous solvent action. He got into the habit, when speaking to his students about natural defences, of asking them to take a cutting from the edge of one of their finger-nails and place it in a microbial suspension. The instantaneous effect amazed them — 'especially as they had recently come from the hands of a physiologist who had taught them that fingernails consist of inert tissue.' Meanwhile, as he went on with the researches, he was finding more and more lysozyme everywhere: in the secretions of the buccal mucus; in the sperm of all animals; in the spawn of the pike; in a woman's milk; in the tip of a stalk; in leaves.

All the growing things in the garden were tested. Tulips and buttercups, nettles and peonies, were found to contain lysozyme. The turnip had an unusually large amount. But the richest store was egg-white. Fleming demonstrated that egg-white, when diluted in sixty million times as much water, was still capable of dissolving some microbes. The egg, therefore, possesses considerable power as a bactericide, and it needs to, for the white, and even the yolk, of an egg provide a marvellous culture medium for microbes. The shell of an egg is not impervious to them: consequently, if eggs can remain sterile'for several days in a dairyman's shop-window, where they are exposed to the attack of every kind of germ, the reason must be that they have some form of natural protection. 'It looks,5 said Fleming to his colleague, Ridley, 'as though the surfaces most exposed to infection are also the best protected. For instance, the slime secreted by an earth-worm is a highly potent bactericide.' He found lysozyme in the blood, especially inside the leucocytes and in the fibrin of clots. 'Would not this be,' he asked, 'a protective mechanism for open wounds, which rapidly become covered with a layer of fibrin and leucocytes, both of which are rich in lysozyme?'

Yes, lysozyme really did seem to be the body's natural antiseptic, the cells' first line of defence against microbic invasion. Fleming had every right to be proud of his work. He had discovered a new and very important aspect of those natural defences of the human body to which he had devoted so much study, in the worship of which he had been brought up by Wright. Not so very long ago, Metchnikoff had demonstrated the fact that certain special cells, the phagocytes, opposed the invasion of microbes. Fleming had found that these cells contained lysozyme. Was it not possible, therefore, to conclude that lysozyme was one of the weapons employed by the leucocytes in their battle against the microbes?

As to the skin and the mucous membranes, Metchnikoff had thought that they were protected only by mechanical means. 'Nature', he had said, 'does not use antiseptics to protect them. The fluids which bathe the surface of the mouth and other mucous membranes are not bactericidal, or only very imperfectly so.

Nature removes from the mucous membranes and the skin quantities of microbes by epithelial desquamation,1 and these are then expelled by the liquid secretions. Nature has chosen this mechanical procedure, just as the surgeon replaces the antisepsis of the mouth with a lavage of salt water.5 In 1921 most bacteriologists held this opinion.

Fleming had just proved that Metchnikoff's argument must, on this point, be modified. 'From the aforementioned experiments5, he said, 'it is clear that these secretions, and the greater part of the tissues, have, in a very high degree, the power to destroy microbes.5 This discovery was one of capital importance. But Fleming never used the word 'discovery5. It was one of those 'big words5 which he disliked. He always said 'my observation5. But, whether discovery or observation, this one gave him more satisfaction than any other. So great was his secret elation that in the first paper which he wrote on lysozyme he, as a rule so prudent, so reserved — he, who either from temperamental shyness, or in reaction from Wright's passion for vast abstractions, would never permit himself to talk of anything but facts — opened the flood-gates of his caution to a tide of wonderful hypotheses.

Not only was this discovery of his tremendous in its own right; it also brought to a head ideas which he had been pondering for a very long time. Somewhat later, in one of his rare moods of expansiveness, he said to Ridley: 'When I was a young doctor in the 514-'18 war, the Old Man was very much concerned with the power of the blood to kill bacteria by means of its own leucocytes and serums. But I realized that every living thing must, in all its parts, have an effective defence-mechanism; otherwise, no living organism could continue to exist. The bacteria would invade and destroy it.' Ridley adds: 'He left me in no doubt that he had disclosed to me in that simple sentence "every living thing must in all its parts be protected" something fundamental in his thinking. This, I believe, was the star that guided him all through his professional life.'

Against what microbes was lysozyme effective? Fleming devised an ingenious method by which to arrive at an answer to this question. He hollowed out in a gelatinous substance contained in a Petri dish either a hole or a gutter, in wjiich he placed some agar impregnated with lysozyme. Next, he 'planted5 certain microbes, some in streaks perpendicular to the gutter, others in lines forming the radii of a circumference of which the hole was the centre. Some of the microbes developed right up to the gutter or the hole. They were obviously insensitive to lysozyme. Others stopped short at a greater or less distance, and this distance marked the measure of their sensitiveness.

Unfortunately, lysozyme, which was so powerful against the inoffensive microbes, turned out to have a much weaker effect upon the dangerous germs, the pathogenic ones. Nothing, thought Fleming, could be more understandable. For what were the pathogenic germs if not those which could penetrate the defences of the organism, establish themselves and cause infection? Now, had they been as sensitive to the action of lysozyme as, for instance, the yellow 'coccus' (lysodeikticus), they would have been destroyed by the defenders, they would have been unable to establish themselves and do harm, and this in itself would be contrary to their own definition.

Does not all the difference between 'pathogenic' and 'nonpathogenic' lie precisely there? he reflected. Certain microbes can infect certain varieties of animal and not others; certain tissues, and not others. Is the solution to the problem of predilection to be found in a difference of the quantity or the quality of lysozyme in these animals or tissues? Starting from this hypothesis, Fleming conceived one of those experiments which were always so simple, but never failed to go straight to the heart of the problem.

He tried the effect of human tears on three groups of germs. The first was composed of one hundred and four inoffensive species, found in the air of the laboratory. The second contained eight germs, pathogenic to some animals, but not to human beings. The third was made up of germs which were pathogenic to human beings. The results were exactly what he expected them to be. The lysozyme exercised a very powerful action on seventy-five per cent of the first group, and on seven species (out of eight) of the second. Its action was weak in the third group, though not completely absent. Consequently, if the amount of lysozyme in the organism were increased, it might be possible in that way to stop the development of certain dangerous microbes. There was material here for investigation.

Fleming asked Dr Allison to join him in a programme of research along these lines. But before making further experiments, he read a paper on his discovery and on the conclusions he had drawn from it, in December 1921 to the Medical Research Club, a scientific body of respectable age (it had been founded in 1891) which was both exclusive and influential. The reception accorded to the paper was cold beyond belief. Not a single question was asked and no discussion followed the reading. Only utterly worthless papers were treated in this manner. Sir Henry Dale, who was among those present, has written: I very well remember his interesting paper, and the way in which we all of us said: "Charming, wasn't it? Just the sort of naturalist's observation Fleming would make" ' — and that was all.

This icy reception of so original a study hurt Fleming's feelings, for beneath his impenetrable mask he was extremely sensitive. But it did not stop him. He prepared another paper on the same subject which Wright presented to the Royal Society in February 1922.1 But, once again, it did not receive the attention it deserved. Fleming without being unduly upset continued with Allison's help to work on the substance, in the importance of which, despite the indifference of his peers, he persisted in believing. Between 1922 and 1927, they published a further five brilliant papers on lysozyme. They made an attempt to extract it in its pure state, but neither of the two men was a chemist (Fleming used to say that he would fail in an examination in elementary chemistry), and in the laboratories of the St Mary's Research Service there was no chemist or biochemist to be found. They could not isolate lysozyme, though they noted that alcohol could precipitate it, without destroying it.

Having observed that lysozyme found in egg-white was a hundred times more concentrated than that found in tears, they used it for their experiments and established conclusively that the substance, at a concentration double that to be found in tears, had a bactericidal action on almost all the pathogenic germs and, in particular, on the streptococci, the staphylococci, the meningococci, and the bacillus of diphtheria. They even tried the effect of egg-white, administered by mouth, on the streptococci of the intestine. Having made certain that lysozyme was not destroyed by the gastric juices, they chose a patient who had an abnormal quantity of streptococci in his intestine and made him swallow the white of four eggs every day. The streptococci returned to normal. Encouraged by this temporary success, they prescribed egg-white for several patients presenting the same anomaly, who complained of fatigue and 'migraine'. They obtained a change for the better in the symptoms. With prudence and honesty they concluded that: 'This may, of course, have been merely a psychological effect, or it may have been due to a temporary action of the lysozyme on the streptococci.'

Fleming, all this while, was going on with his general study of antiseptics. The purpose of it was the same: to conquer the infections. In 1923, the combined efforts of several research-workers in the lab. produced a new and effective technique for this type of investigation. Elliott Storer, who had thought it out, called it the slide-cell (a slide divided into cells) method, but the slides prepared by him gave disappointing results. Wright, who realized the value of this technique, perfected it, and Dyson added a further improvement. It had everything in it to please Fleming: it required skill in its manipulation; it cost nothing; and it could be worked with small quantities — a great advantage where human blood was concerned.

The slide-cell consisted of two slides of glass separated by five strips of Vaselined paper, placed at regular intervals at right-angles to the longest axis of the slides. The space between the slides was thus divided into four equal compartments, each one of which could contain a small quantity of blood. (Fleming had noticed that the paper on which a certain medical journal was printed had the ideal thickness required for the strips. When he was describing the method in his lectures, he would say, with perfect gravity, and much to the surprise of his students: 'For the Vaselined strips you should use the Journal ofExperimental Pathology')

The small compartments were then filled with defibrinated blood infected with the microbes to be studied, sealed at the two open ends with paraffin, after which the whole slide-cell was placed in the incubator. The microbes multiplied in colonies which, in this thin layer of blood, were easy to count. For instance, it was possible to observe that, if about one hundred staphylococci were put into a compartment containing normal blood, the leucocytes killed, on an average, ninety-eight per cent of them, so that only two of the colonies developed.

Fleming thought that this was an ideal technique for making a definitive study of the action of the antiseptics on the leucocytes. In the compartments of the slide-cell he mixed blood with more and more concentrated solutions of the antiseptic which he wanted to study. He noticed that the antiseptic killed the leucocytes at concentrations far below those required to kill the bacteria and so there were concentrations in which all the leucocytes, in other words all the defenders, were killed, while all the staphylococci flourished: a hundred microbial colonies were counted in each compartment instead of only two without antiseptics. His conclusion was as follows: 'These experiments show that there is little hope that any of the antiseptics in common use could be successfully introduced into the blood stream to destroy the circulating bacteria in cases of septicaemia.'1 By this beautiful and simple experiment, he had proved irrefutably that the antiseptics then in use destroyed the leucocytes in much weaker solutions than those which would have enabled them to act upon the microbes.

On the contrary, when the slide-cell was used to study the action of egg-white on the phagocytes, Fleming and Allison observed that 'whereas egg-white, in marked contrast to the chemical antiseptics, has no destructive effects on the leucocytes, it has considerable inhibitory or lethal effect on some of the bacteria'. They made the experiment of giving intravenous injections of an egg-white solution to a rabbit, and then measuring the bactericidal power of its blood. There were no unfortunate consequences. The anti-bacterial power was markedly enhanced. 'And it is possible', wrote Fleming, 'that in cases of generalized infection with a microbe susceptible to the bacteriolytic action of egg-white ... the intravenous injection of a solution of egg-white might be beneficial.. .' This was an important conclusion, for, with it, Fleming, the victorious adversary of antiseptics, was affirming that he had no prejudice against chemotherapy, provided the product employed did not destroy the natural defences of the blood.

But in order to make a series of intravenous injections without danger, it would have been necessary to rid the lysozyme of egg-white. Fleming and Allison, as we have seen, had attempted, without success, to extract lysozyme in its pure state. In 1926, a young doctor named Ridley came to do research work in Wright's laboratory. He was not a professional chemist, but he knew a great deal more about chemistry than did his colleagues. Fleming asked him to extract lysozyme in its pure state. Ridley tried, but unsuccessfully. Fleming was greatly disappointed. Tt is a pity,' he told Ridley, 'because if we had this substance pure, it ought to be possible to maintain in the body a concentration which would kill certain bacteria.'

Later, as we shall see, a biochemist did manage to purify and crystallize lysozyme.

Fleming was an obstinate man. He continued to make a study in vitro of the action of other products upon the bactericidal power of the blood. He wanted, for instance, to measure the action of salt. He found that every saline concentration which departed from that normally found in the human body weakened the phagocytes.

What would be the effect in vivo? In order to find out, he gave an intravenous injection of hypertonic salt (that is, a solution of greater concentration than the normal concentration in the organism) to a rabbit. The first injection was too strong. The rabbit had convulsions and for a few seconds seemed to be on the point of dying. Two minutes later, however, it had got over the shock. Fleming examined its blood. At first, and for as long as the concentration of salt in the animal's blood remained above the normal, the result was identical with that obtained in vitro. The bactericidal action of the blood was diminished. But, to his great astonishment, Fleming discovered that, after two hours, the concentration of salt in the blood having returned to normal, the bactericidal power of the blood was greatly enhanced, and this lasted for several hours.

Having perfected the experiment in such a way as to give a quantity of salt so little above the normal that it caused the animal no distress, Fleming tried his hypertonic salt on a patient. An intravenous injection produced an increase of the bactericidal power without causing the slightest discomfort.

He made further experiments on patients whenever his medical colleagues allowed him to do so. But, generally speaking, he was given only desperate cases, and even those very rarely. One or two other doctors made similar experiments and observed that the results were good. But they did not continue. Fleming was very fond of this little discovery and always regretted that it had been more or less ignored. He could not understand why greater advantage was not taken of a treatment which was wholly inoffensive and was probably more effective than the therapeutic vaccines. His sixth paper on lysozyme was written in 1927. It deals with an important phenomenon. By exposing microbes to increasing concentrations of lysozyme and picking up the few survivors, he had managed to create 'strains5 of the famous yellow coccus, or of the faecal coccus, eighty times more resistant than had been originally the case. Had these microbes, in developing greater resistance to lysozyme, also become more resistant to the action of the blood? Experiment showed that they had. Why? As we have seen, Fleming had found lysozyme in the phagocytes. Since the increase in resistance to lysozyme kept pari passu with resistance to phagocytosis, did it not look as though the action of the phagocytes was, in part, due — as he had already thought it was — to the lysozyme which they contained?

As in his first paper, he here found himself faced by problems of the greatest importance. The pathogenic germs are enemies dangerous to man, because they force the natural defences. Gould it be that lysozyme had in primeval times been an all-powerful weapon supplied to primitive man by nature to protect him against all germs? Might not the pathogenic germs be, in fact, descendants of the few germs which, having resisted lysozyme, had acquired an ever-increasing power of resistance, until they had become capable of overcoming nature's other defences? If that were so, could one not, by selection, transform an inoffensive into a virulent germ? This was the subject of his sixth paper.

Why was it that this series of superb studies, which had opened up new and vast horizons, attracted so little attention from British scientists? Was it due to the fact that Wright at that time was 'slowing down', and that the enemies of his school were now inclined to accept with a considerable degree of suspicion the work carried out in his laboratory? Fleming, loyal as ever, said that the fault was his, and that he should have presented his findings in the matter of lysozyme to an audience, not of doctors, but of physiologists, who would, most certainly, have been interested. However that may be, the indifference of his colleagues to work which, in spite of his modesty, he knew to be remarkable, made him more silent and reserved than ever, but also stronger. His judgment was not influenced by that of others. His momentum had by no means come to a halt. All through his life he never abandoned the search for a substance which would kill the microbes without weakening the phagocytes. He had looked for it, with his master, in the vaccines. He had hoped that he had found it in lysozyme, a substance which could have reconciled the physiological and the antiseptic schools, because it was an antiseptic enlisted in the service of the body's natural defenders. Being a tenacious scientist who was sure of his facts, he still looked forward to a future in which his lysozyme would play an important part.

Nor was he wrong. Even today much work is being done on lysozyme. It interests bacteriologists because it dissolves the mucins with which the microbes are coyered; industrialists because it protects foodstuffs from infection (the Russians use it for preserving caviare); doctors, because, when added to cow's milk, it reproduces the component structure of human milk, and also because they use it in the treatment of eye and intestinal affections. All this has come about because an attentive observer, when about to throw away a contaminated culture, looked at it carefully, and said: cThis is interesting.' The discovery which was received with icy silence in London in 1921, has in the space of thirty years become the subject of two thousand papers. Alexander Fleming always said: 'We shall hear more about lysozyme one day.' Who knows?

In the laboratory, those who held aloof from cliques realized its value. Martley, a charming bearded Irishman and one of nature's gentlemen, said to Pryce in 1927: 'Fleming's the really intelligent chap ... If the Old Man had been the author of those experiments with lysozyme and other things, what a sensation there'd have been! At the first International Congress of Microbiology, held in '93'? Jules Bordet, a Belgian scientist, a former pupil of Pasteur and President of the Congress, spoke in his opening address of Fleming's work on lysozyme in the highest terms. Fleming, completely taken by surprise, was tremendously pleased.

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