Launching The Antibiotic Era

Launching the Antibiotic Era is a commemorative book that memorializes a great scientist, Rene Dubos, who helped found the field of antibiotic discovery, and also recounts the early, heady days of discovery when antibiotics were just beginning to be isolated and harnessed for commercial purposes. The book is must-read for those of in the field, but I think it could have broad interest for biologists, chemists, and historians of science. The books is set of essays written by contemporaries of Rene Dubos, and they are really excellent account of research culture and of the remarkable rise of antibacterial compounds. In particular, I found Rollin Hotchkiss’ essay on isolating gramicidin to be a delight - in describing the isolation, and trying to figure out how it worked (a curiously complicated soap). The book is full of anecdotes and in the asides it is also possible to learn of the early days of big-pharma. It is also interesting that in the late 1940’s the entire scientific world was still intimate enough that it seemed everyone could know everyone else.

Multiple people comment on reading Dubos’ early books on bacterial cellular behavior and being struck by it. And although a number of groups are working on antibiotics its a small enough field to know who all your friends/rivals are (I suppose that hasn’t changed too much - the specialization has just increased). I also liked to learn that Joshua Lederberg, who discovered bacterial sex and did some great work on bacterial genetics, was critical of Rene Dubos approach which seemed to eschew the genetic approach in favor of what might be called the behavioral or phenotypic approach. In a conversation with Carol Moberg, I learned that later in life Lederberg, after decades of being critical of such an approach, said “you know, Dubos was right” (paraphrased). In this comment, we see a return of at least one genetecists to whole organism physiology and perhaps ecosystem-level understanding. This book shines a light on a fascinating era in science and focuses on a person who seemed to spark all those with whom he worked. Below are a few excerpts from the book but I would encourage anyone to read it in full.

Launching the Antibiotic Era#

Preface

Dubos’s discoveries of gramicidin, of an enzyme degrading pneumococcal capsular polysaccharide, of ribonuclease, and of a novel medium for the growth of the tubercle bacillus all stand as exciting milestones in his early career at The Rockefeller Institute for Medical Research. Although Dubos was trained first as an agronomist, then as a journalist, and later as a soil microbiologist, his interests always centered on biomedicine and human well-being. A practicality born of his Gallic background, a Pasteurian influence, and the independence afforded those working at the early Institute all combined to engender a career marked by marvelous versatility and productivity. Few scientists have maintained Dubos’s level of intensity and involvement over such a long career.

Introduction:

Joshua Lederberg

Science is often thought of as an automatic process. In fact it profits from a richness of styles, personalities, and approaches. Science benefits from accidents of personal history and functions within environments strongly shaped by cultural forces. Histories such as the development of antibiotics-and of Rene Dubos’s role in particular-are valuable because they remind us of science’s immense diversity.

My first experience with Rene Dubos was through The Bacterial Cell. It is a book that only Dubos could have written, because he brings into sharp focus what we now take for granted-namely, that bacteria are cells. The book is a lucid and accurate summary of the biology of bacteria known through the mid-194os. The structures and activities of bacteria, how they relate to problems of virulence, immunity, and chemotherapy, and the phenomena of bacterial variability are all impeccably presented. I know of no other work that had in it the seeds of its own obsolescence, since it inspired so many to pick up on his inspirations and challenges. In doing so, they brought about a rapid displacement of what Dubos said and substantially furthered the march of bacteriology.

The Bacterial Cell is the work from which I can say I learned most of the microbiology I know. The print in my copy is literally read off the pages and the covers are about to fall off. The book appeared in 1945 when I was a medical student working in Francis Ryan’s laboratory at Columbia University. I was just beginning to think about whether there was a bacterial genetics, and this work was the launching pad for my own investigations. Within a year, I was able to find evidence that genetic recombination did indeed occur and to find in Dubos a critical review of whether such contingencies could be logically imagined.

In 1954, at the dedication of the Waksman Institute at Rutgers University, I had the occasion to quote Dubos’s perspectives on bacteria. In 1945, he wrote To the biologist of the 19th century, bacteria appeared as the most primitive expression of cellular organization, the very limit of life …. In reality, it appears that it is only their small size and the absence of recognized sexual reproduction which has given the illusion that bacteria are “simple” cells.

Only a year later, he referred to the role of DNA in pneumococcal transformation and the beginning of biochemical genetics. He said:

Bacterial variation passes from the collector’s box of the naturalist to the sophisticated atmosphere of the biochemical laboratory. One may wonder whether the geneticist will not arrive too late to introduce his jargon into bacteriology.

Here was one of the very few insights where Dubos was wrong, as the science of bacterial genetics soon came into its own.

Dubos was far ahead of the times in so many ways that to this day we cannot quite catch up with him. He was one of the first hard scientists I encountered who had a sense of the need for a humanistic approach to the limits of science and to the consequences of scientific innovation. Consider, for example, all that is conveyed by Dubos’s philosophy in his phrase “mirage of health.” We may not be willing to go as far as he did to puncture the illusions about what science can do.

However, we needed his efforts to broaden our perspectives, which were often too narrow until he touched our lives so poignantly.

The Rockefeller University is proud to celebrate the fiftieth anniversary of Rene Dubos’s discovery of gramicidin and his role in launching the antibiotic era. The personal recollections that follow present yet another phase of his extraordinary personality and pioneering activities.

Joshua Lederberg President, 1978-1990 The Rockefeller University

From Microbes to Medicine: Gramicidin, Rene Dubos, and The Rockefeller

Rollin D. Hotchkiss

We contrived to commandeer some lab help and some large equipment for a summer of growing gallons of the soil organism Bacillus brevis and preparing the raw material Dubas had isolated. The crude brownish material was practically incompatible with water and, under organic solvents, congealed into a sticky mass as unpleasant as so much uncouth earwax. But it was powerful wax all right. Its amber alcohol solutions could be finessed into an acceptable suspension in water, and at great dilutions it would powerfully block bacterial growth, both in the test tube and in the peritoneal cavity of an infected mouse. Starting with some half a pound of the material, I set out that fall to find the nature of its powers.

We had high hopes that we had a powerful new curative and preventive medicine, and so did the directors of research at The Rockefeller Institute. I recall speculations that our preparations might eventually be added to fresh milk, and beer or other products, to obviate the need for pasteurization. One might ask, in a mercantile world, “What in the world was The Rockefeller Institute doing-in its Hospital!-letting a soil microbiologist and a footloose organic chemist follow their unorthodox dreams?” (Dubos has mused along this line also.) I can only say that the policy of taking such “calculated risks,” especially with ideas that come up “from below,” still seems to me one of the best features of an enlightened institution for research.

Significant scientific results also came from the work. We found tyrocidine and gramicidin to be small polypeptides, and as forerunners of cyclic peptides and of such antibacterial agents as polymyxin and bacitracin, they had some place in history. We showed that they contained some “unnatural” or D-amino acids (16); that is, inverted versions of the widespread usual components of proteins. This last work was done in collaboration with Fritz Lipmann, who thereby became interested and for some years involved with several aspects of the action and biosynthesis of these polypeptides. He and his co-workers established (17, 18) that these peptides are synthesized independently of the DNA/RNA-coding route, which is probably why they can include D-amino acids and can admit of some variations in sequence.

Tyrocidine: A “curiously complicated soap.”

Oxford, Howard Florey, and World War II

Edward P. Abraham

At this time I think that Howard Florey was waiting for further progress in the production of penicillin before undertaking experiments with laboratory animals. But two events may have persuaded him to bring these studies forward. In March 1940, J. M. Barnes, at Chain’s request, tested the toxicity of a very crude preparation of penicillin in a mouse. About forty milligrams of this material (almost certainly less than 0.2% pure) produced no ill effect when given intraperitoneally. Florey may well have regarded this experiment as an encroachment on his own field and it did not demonstrate the remarkable lack of toxicity of pure penicillin. However, it did indicate that future biological studies with crude penicillin were unlikely to be complicated by major toxic impurities. A second event in March was Heatley’s finding that penicillin with higher activity could be obtained by solvent transfer, although its purity was in fact still less than one percent.

By 1943 the study of the chemistry of penicillin had developed into a remark- able and confidential Anglo-American project in which similar findings were made independently on both sides of the Atlantic. Nevertheless, despite the combined efforts of about forty major academic and commercial organizations, only a trace of synthetic penicillin had been obtained by the end of the war and that by a process designed to synthesize the wrong structure. A few years later John Sheehan accomplished a notable rational synthesis of penicillin at MIT, after introducing a new reagent to close the ,6-lactam ring. By then the astonishing increase in the yields obtained from strains of Penicillium chrysogenum had made it un- likely that total synthesis would ever compete commercially with fermentation. But chemical studies bore fruit later, when clinically valuable semisynthetic penicillins and cephalosporins began to be produced from the penicillin nucleus and the cephalosporin nucleus, respectively.

Penicillin and Luck

Norman G. Heatley

In the summer of 1928 Fleming goes on holiday, unaware that he has been chosen by the Fates to take the first steps in introducing the antibiotics to man- kind. Having made a wise choice of their agent, the Fates also arranged that one of his plates, inoculated with staphylococci but not incubated, should be contaminated with a spore of Penicillium notatum, and that the weather during subsequent weeks should provide the sequence of rather narrow temperature ranges required to produce the penicillin effect. Fleming returns from his holiday and goes through the pile of used plates on his bench, looking at them and discarding them into the tray of disinfectant. The plates are many, and soon they are piled up, clear of the disinfectant. But what is this? Gracious Heavens, he has discarded the plate! All is not lost, for the Fates have a messenger on hand in the form of Fleming’s colleague, D. M. Pryce. Pryce makes his entrance, they chat about staphylococci and, to make a point, Fleming picks up some of the discarded plates. The Fates hold their breath. Yes! He picks up the plate, looks at it, and says “That’s funny . . . " How fortunate that trays rather than buckets were used for discarded cultures and that D. M. Pryce was on hand at the critical moment.

Even at this time Florey was aware that penicillin would have to be tested in human patients before its manufacture could be seriously considered, but as he pointed out, a man is three thousand times as big as a mouse.

Chloramphenicol, Kuala Lumpur, and the First Therapeutic Conquests of Scrub Typhus and Typhoid Fever

Theodore E. Woodward

During this post-WWII period, P.R. Burkholder, of Yale, noted in 1947 that the growth of adjacent inocula of both gram-positive and -negative bacteria was inhibited on agar streak cultures by a new actinomycete isolated from the soil of a mulched field near Caracas, Venezuela. From culture filtrates of this new soil organism, later named Streptomyces venezuelae, a crystalline antibiotic was iso- lated that was inhibitory for a wide range of gram-positive and -negative bacteria, rickettsiae, and the psittacosis virus. Its unusually broad range of antimicrobial action, low toxicity for animals, great stability, and effective absorption from the gastrointestinal tract suggested that chloramphenicol, as it was called, might be- come the first major antibiotic . for clinical use since the discovery of streptomycin.

Once, Rene called to ask if he might use a quote which I had expressed on the occasion of a dinner meeting held in his honor after one of his lectures. The quote was really about a Bushman of the Kalahari Desert in Southwest Africa. On being asked his age, the aged Bushman remarked, “I am as young as the most beautiful wish in my heart, and as old as all of the unfilled longings in my life.” This response also characterized Rene Dubos.

New Remedies for an Ancient Infection: Antibiotics and Tuberculosis

George B. Mackaness

Florey was an ideal mentor:strong willed, determined, and well informed. He was generous, not with praise but with advice, encouragement, wise guidance, and with his refusal to take any credit for the achievements of his students. When I asked him to share authorship of my first publication he said, in his curt manner of address, “That won’t do you any good, Mackaness.”

Until shortly before I began work on my new project, this could not be done with tubercle bacilli because of their high content of waxes which made them intensely hydrophobic. The unwettable bacilli adhere tenaciously to each other, forming tangled skeins that float on the surface of simple aqueous culture media. The resulting mass of bacilli resist the fiercest efforts to extricate even a few individual bacilli for experimental purposes. Fortunately, Dubos, at The Rockefeller Institute in New York, needed a highly dispersed suspension of tubercle bacilli and a reliable method of determining how many living tubercle bacilli it contained, so that he could inoculate animals with known numbers of them. He suggested to his colleague Bernard Davis how this might be done with the aid of a detergent. Bernie rendered the idea into a practical culture medium in which most strains of the tubercle bacillus would grow in a highly dispersed state from which single-celled suspensions could be readily prepared. The Davis-Dubos media (4), liquid and solid, became indispensable tools to all who would work quantitatively with the tubercle bacillus (see page 92).

Cornforth et al. (6) had shown that some surface-active agents, including the polyoxyethyler.c ethers, had antituberculous activity in vivo even though they actually facilitated growth of virulent tubercle bacilli in vitro. After learning from me that Triton had no effect on tubercle bacilli living in monocytes maintained in its presence, Dick Rees suggested that I should test monocytes obtained from rabbits that had been injected repeatedly with the detergent. Upon doing this, I was surprised to find that the monocytes from Triton-treated animals had under- gone a profound functional change which made them immune to challenge with tubercle bacilli in tissue culture. Not only were tubercle bacilli unable to multiply in monocytes from Triton-treated rabbits, but bacilli of some strains actually disappeared from the cytoplasm after a few days in culture. It is now suspected that the detergent, which is known to enter monocytes, acts to wet the bacilli and render them susceptible to the phagocytes’ normal antimicrobial and digestive mechanisms. It is regrettable, therefore, that Triton WR 13 39 and other surface-active agents cause severe disturbances of fat metabolism and are prohibitively toxic.

It was fortunate for me that the early investigation of isoniazid had been conducted in great secrecy, and also that Hens Pharmaceuticals had sent me some of it to test before anyone outside the two drug houses knew anything of its existence. Within four weeks I had found that isoniazid was powerfully and equally bactericidal for both intra- and extracellular tubercle bacilli. It combined well with streptomycin to produce an even more rapid and potent bactericidal effect. When tested in tissue culture it produced complete sterilization of infected monocytes at drug concentrations that were achievable in man. In short, it brought a dramatic change in outlook for those seeking to improve the management of tuberculosis. In my test system it behaved in a way that lent credence to the glowing newspaper reports of its apparent clinical efficacy. Although other good drugs have been added to the list (there are now eleven drugs in current use for the treatment of tuberculosis), isoniazid is still the one most commonly used. To convey a sense of how important it was in the battle against tuberculosis, I can tell you that five years after the introduction of streptomycin no reduction had occurred in the number of hospital beds being occupied by tuberculous patients in the United States. Five years after the discovery of isoniazid, eighty percent of the beds available in TB hospitals were unoccupied, and many sanatoria had closed their doors. A famous one was destined to become a jail; and a nearby neighbor became a research institute in the field of immunology. It would be wrong, however, to attribute all of this progress to isoniazid. No one drug by itself could have produced cure rates as spectacular as those being achieved by 1957. The real breakthrough in the management of tuberculosis came from the experiments and astute observations of the many clinicians, microbiologists, and pathologists whose work collectively established that the biggest barrier to a cure for tuberculosis was the development of drug resistance; and that simultaneous administration of two or more drugs that did not actively antagonize each other (as some drug combinations do) could be relied upon to prevent the emer-gence of drug resistance in most patients. The application of this therapeutic principle has changed the face of the disease that Dubos described so vividly in The White Plague (1952).

Two Perspectives: on Rene Dubos, and on Antibiotic Actions

Bernard D. Davis

Dubos was then finishing his biography of Louis Pasteur (5), and he invited me to read the manuscript. I could not escape the feeling that Louis Pasteur must have been very much a model for him: both liked to deal with problems that combined intellectual and practical challenges, and both had a talent for dramatizing their work. But though Dubos was a dedicated experimenter, I felt that he did not have quite Pasteur’s flair for the key experiment that would convincingly establish a profound principle, in one problem after another. On the other hand, Dubos had stronger literary, poetic, and philosophical interests and talents; his book on Pasteur, and his comments in conversation on other scientific developments, showed great critical acuity and sense of significance.

I would like to offer a somewhat different view of Waksman’s achievement. Many years later, when I had the occasion to make some remarks at his funeral in Woods Hole, I was struck by the thought that his really important discovery was not streptomycin: it was the principle that a patient, systematic search for useful antibiotics will eventually pay off. With this discovery, the search for antibiotics took off in industry, and the result has been several thousand new compounds, among which roughly a hundred are useful. Moreover, their uses have been ex- tended to areas other than antimicrobial action-for example, cyclosporin as a suppressor of autoimmune responses to organ transplants.

In a curious way, this infection turned out to be a boon. There is an exten- sive and romantic literature, discussed by Dubos and his wife Jean in their book The White Plague (1952), claiming that tuberculosis tends to afflict creative people. I doubt that the bacillus has any basis for such selectivity in its choice of victims. On the other hand, its major impact has been on young adults, and the traditional treatment gave the victims a prolonged opportunity to suspend their daily rou- tines and engage in reading and reflection. The lucky survivors may thus have had a chance to improve their perspective on their goals and hence sometimes to become more creative.

I would further suggest that Dubos’s great influence, on microbiology and later on a wider public, depended much on his intense personality and his flair for dramatizing ideas, as well as on his capacity for recognizing significant ideas. If we had in science people who played a role like that of drama or music or art critics I would say he was a great science critic. His biography of Pasteur shows marvelous capacity to bring out the most significant aspects of each phase of his work, and to make this old history fascinating today. In this connection, I recall Dubos’s view (against which I argued) that the personal aspects are of utterly no relevance in presenting the life of a scientist; all that counts, and should interest the audience, are his achievements and his influence.

This view was paralleled by the nature of Dubos’s interactions with col- leagues, at least so far as I experienced them. What we discussed were always ideas, and not personal matters; and though he had a great influence on my life the relationship remained impersonal, with few contacts. Beneath his showmanship was a great deal of reserve.

Friend of the Good Earth

Carol L. Moberg

Before resuming his studies, Dubos wanted to visit America. By 1924 he had earned money for passage to the United States on the steamship Rochambeau. On board, fate intervened when he met Selman Waksman, the American soil bacteriologist whom Dubos had recently guided around Rome during an international congress on soil science. When Waksman learned Dubos had ambitions but no definite plans to study bacteriology, he offered the young Frenchman a fellowship to study at Rutgers University. Dubos arrived in New York and accompanied Waksman that same evening to the Rutgers campus in New Jersey. Three years later, Dubos earned his doctorate. In the spirit of Winogradsky, Dubos showed that the environmental characteristics of the soil determine which microbes are activated to decompose cellulose, the main ingredient of wood. In 1927 two more chance events brought Dubos to The Rockefeller Institute (renamed The Rockefeller University in 1965). His application for a National Research Council fellowship was denied because he was not a citizen. The rejection letter contained a handwritten note suggesting he consult with fellow Frenchman Alexis Carrel at Rockefeller. Carrel was kind and considerate but

He waved a vial of the purified polysaccharide and portrayed the scientific drama that lay within. Dubos replied, rather brashly for a new Ph.D., “Well, I think I can find a microbe to decompose that capsule, and from it I can extract an enzyme.”

Dubos predicted that increasingly crowded, uniform societies would bring new diseases into being. Eminent scientist, physician, and author Lewis Thomas adds, “Dubos was quite certain that antibiotics in whatever abundance were not going to be the solution. He had a prescient mind and he was especially afraid of what the new viruses might do.”

When Dubos’s book So Human an Animal won the Pulitzer Prize in 1969, he was drawn into the mainstream of the environmental crusade. With amazing resilience and energy between his seventieth and eighty-first years, he devoted all his skills as speaker and author, coupled with his stature as a scientist, to formulate emerging environmental and social issues for an extensive public audience.