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Author: Kholhring Lalchhandama[a]ORCID iD.svg  , et al.

Kholhring Lalchhandama; et al., "History of penicillin", WikiJournal Preprints, Wikidata Q107303937




Abstract

The history of penicillin follows several observations and discoveries of apparent evidence of antibiotic activity of the mould Penicillium that led to the development of penicillins that became the most widely used of antibiotics. Following the identification of Penicillium rubens as the source of the compound in 1928 and with the production of a pure compound in 1942, penicillin became the first naturally derived antibiotic. There are anecdotes about ancient societies using moulds to treat infections, and in the following centuries many people observed the inhibition of bacterial growth by various moulds. However, it is unknown if the species involved were Penicillium species or if the antimicrobial substances produced were penicillin. While working at St Mary's Hospital in London, Scottish physician Alexander Fleming was the first experimentally to discover that a Penicillium mould secretes an antibacterial substance, and the first to concentrate the active substance involved, which he named penicillin in 1928. The mould was determined to be a rare variant of Penicillium notatum (now Penicillium rubens), a laboratory contaminant in his lab. For the next 16 years, he pursued better methods of production of penicillin, medicinal uses and clinical trial. His successful treatment of Harry Lambert who had fatal streptococcal meningitis in 1942 proved to be a critical moment in the medical use of penicillin. Many later scientists were involved in the stabilization and mass production of penicillin and in the search for more productive strains of Penicillium. Important contributors include Ernst Chain, Howard Florey, Norman Heatley and Edward Abraham. Fleming, Florey and Chain shared the 1945 Nobel Prize in Physiology or Medicine for the discovery and development of penicillin. Dorothy Hodgkin received the 1964 Nobel Prize in Chemistry for determining the structures of important biochemical substances including penicillin. Shortly after the discovery of penicillin, there were reports of penicillin resistance in many bacteria. Research that aims to circumvent and understand the mechanisms of antibiotic resistance continues today.


Early history[edit | edit source]

Penicillin core.svg

 The core structure of penicillin, where R is a variable group; the central "square" structure is the β-lactam ring, which is the key component for destruction of bacterial cell walls.


Yikrazuul, Public domain

Penicillin (Figure 1) is the second antibiotic and the first naturally-occurring antibiotic discovered.[1][2] The first antibiotic discovered was arsphenamine, marketed as Salvarsan, by German physician Paul Ehrlich and his Japanese assistant Sahachiro Hata in 1909.[3] It was a modified compound of a highly toxic chemical arsenic[4] that was used for the treatment of sexually transmitted bacterial (Treponema pallidum) infection, called syphilis, and became the most commonly prescribed drug in the early 20th century.[5] But it was overshadowed by penicillin as the safer and more effective antibiotic, as the new drug was effective against a wide range of Gram-positive bacteria,[6] as well as Gram-negative T. pallidum.[7]

But the discovery of penicillin from the mould Penicillium (from the Latin word penicillum, meaning "painter's brush") from which its name was derived, was preceded by many observations and traditional practices.[8] Many ancient cultures, including those in Egypt, Greece and India, independently discovered the useful properties of fungi and plants in treating bacterial infections.[9] In one of the earliest specific records, a Greek king in the 16th-century BCE reported the use of bread moulds by a women healer to treat wounded soldiers. Around the same time, Chinese traditional practitioners used moulds from soya bean for wound infections.[10]

In 17th-century Poland, wet bread was mixed with spider webs (which often contained fungal spores) to treat wounds. The technique was mentioned by Henryk Sienkiewicz in his 1884 book With Fire and Sword. In England in 1640, the idea of using mould as a form of medical treatment was recorded by apothecaries such as John Parkinson, King's Herbarian, who advocated the use of mould in his book on pharmacology.[11] One of the common practices for treating impetigo, an infection due to the bacterium Staphylococcus aureus, was mould therapy, the moulds being obtained from bread and porridge.[12] A Canadian biologist A.E. Cliffe made a vivid report, saying:

It was during a visit through central Europe in 1908 that I came across the fact that almost every farmhouse followed the practice of keeping a mouldy loaf on one of the beams in the kitchen. When I asked the reason for this I was told that this was an old custom and that when any member of the family received an injury such as a cut or bruise, a thin slice from the outside of the loaf was cut off, mixed into a paste with water and applied to the wound with a bandage. It was assumed that no infection would result from such a cut.[13]

One of the most detailed medical narratives was how Brenda Ward (née Whitnear) cured her daughter of facial impetigo in 1929. An eight-year-old Brenda Whitnear was treated by the family physician James Twomey with all possible medications available, but in vain. Twomey, based on traditional practice, suggested Brenda Ward to prepare a starch paste. The paste was was left in the pantry kept at the cellar head for several days until it became very mouldy. The mouldy paste was applied on the girl's face as an ointment regularly for over a week, and she was completely healed.[14] Unfortunately, there was no written record of the treatment other than the receipt of the consultation fee. Ward recalled that the mould initially appeared yellow in colour, grew into bronze colour, and finally turned into blue-green colonies. This description is considered as an indication of either Penicillium or Aspergillus. British microbiologist Milton Wainright experimented this assumption in 1989 and found that Penicillum was the more likely mould based on the growth pattern and antibacterial activty.[15]

These treatments often worked because many organisms, including many species of mould, naturally produce antibiotic substances. However, ancient practitioners could not precisely identify or isolate the active components in these organisms.

Early scientific evidence[edit | edit source]

The modern history of penicillin research began in earnest in the 1870s in the United Kingdom. Sir John Scott Burdon-Sanderson, who started out at St. Mary's Hospital (1852–1858) and later worked there as a lecturer (1854–1862), observed that culture fluid covered with mould would produce no bacterial growth. Burdon-Sanderson's discovery prompted Joseph Lister, an English surgeon and the father of modern antisepsis, to discover in 1871 that urine samples contaminated with mould also did not permit the growth of bacteria.[16] Lister also described the antibacterial action on human tissue of a species of mould he called Penicillium glaucum, and reportedly cured a patient in 1877.[17] A nurse at King's College Hospital whose wounds did not respond to any traditional antiseptic was then given another substance that cured him, and Lister's registrar informed him that it was called Penicillium.

In 1873, Welsh physician William Roberts, who later coined the term "enzyme", conducted experiments to test the hypothesis of spontaneous generation (abiogenesis) and observed glass tubes were easily contaminated by airborne bacteria and moulds.[18] In his report in the Philosophical Transactions of the Royal Society in 1874 he stated: "I have repeatedly observed that liquids in which the Penicillum glaucum was growing luxuriantly could with difficulty be artificially infected with Bacteria; it seemed, in fact, as if this fungus played the part of the plants in an aquarium, and held in check the growth of Bacteria, with their attendant putrefactive changes."[19] John Tyndall, professor of physics at the Royal Institution of Great Britain, followed up on Roberts's work on refutation of abiogenesis and demonstrated in 1875 the antibacterial action of the P. glaucum. His report was read before the Royal Society in 1876 (and published as a monograph in 1881),[20] in which he described:

[The] two most actively charged tubes were in part crowned by beautiful tufts of Penicilllum Glaucum. This expanded gradually until it covered the entire surface with thick tough layer, which must have seriously intercepted the oxygen necessary to the Bacterial life. The bacteria lost their translatory power, fell to the bottom, and left the liquid between them and the superficial layer clear.[21]

In 1876, German biologist Robert Koch discovered that Bacillus anthracis was the causative pathogen of anthrax,[22] which became the first demonstration that a specific bacterium caused a specific disease, and the first direct evidence of the germ theory of diseases.[23] In 1877, French biologists Louis Pasteur and Jules Francois Joubert observed that cultures of the anthrax bacilli, when contaminated with moulds, could be successfully inhibited.[24] Reporting in the Comptes Rendus de l'Académie des Sciences, they concluded:

Neutral or slightly alkaline urine is an excellent medium for the bacteria... But if when the urine is inoculated with these bacteria an aerobic organism, for example one of the "common bacteria," is sown at the same time, the anthrax bacterium makes little or no growth and sooner or later dies out altogether. It is a remarkable thing that the same phenomenon is seen in the body even of those animals most susceptible to anthrax, leading to the astonishing result that anthrax bacteria can be introduced in profusion into an animal, which yet does not develop the disease; it is only necessary to add some "common 'bacteria" at the same time to the liquid containing the suspension of anthrax bacteria. These facts perhaps justify the highest hopes for therapeutics.[25]

The phenomenon was described by Pasteur and Koch as antibacterial activity and was named as "antibiosis" by French biologist Jean Paul Vuillemin in 1877.[26][27] (The term antibiosis, meaning "against life", was adopted as "antibiotic" by American biologist and later Nobel laureate Selman Waksman in 1947.[28]) It has also been asserted that Pasteur identified the mould as Penicillium notatum. However, Paul de Kruif's 1926 Microbe Hunters describes this incident as contamination by other bacteria rather than by mould.[29] In 1887, Swiss physician Carl Alois Philipp Garré developed a test method using glass plate to see bacterial inhibition and found similar results.[27] Using his gelatin-based culture plate, he grew two different bacteria and found that their growths were inhibited differently, as he reported:

I inoculated on the untouched cooled [gelatin] plate alternate parallel strokes of B. fluorescens [Pseudomonas fluorescens] and Staph. pyogenes [Streptococcus pyogenes ]... B. fluorescens grew more quickly... [This] is not a question of overgrowth or crowding out of one by another quicker-growing species, as in a garden where luxuriantly growing weeds kill the delicate plants. Nor is it due to the utilization of the available foodstuff by the more quickly growing organisms, rather there is an antagonism caused by the secretion of specific, easily diffusible substances which are inhibitory to the growth of some species but completely ineffective against others.[25]

In 1895, Vincenzo Tiberio, an Italian physician at the University of Naples, published research about moulds initially found in a water well in Arzano; from his observations, he concluded that these moulds contained soluble substances having antibacterial action.[30][31][32]

Penicillium rubens (Fleming's strain).png

 Fleming's mould, Penicillium rubens CBS 205.57. A–C. Colonies 7 d old 25 °C. A. CYA. B. MEA. C. YES. D–H. Condiophores. I. Conidia. Bars = 10 µm.


Houbraken et al., 2011, CC-BY 4.0

French medical student Ernest Duchesne at École du Service de Santé Militaire (Military Service Health School) in Lyon independently discovered the healing properties of P. glaucum.[33] He was able to grow the mould on pieces of moist food. When he mixed the mould with the bacterium Escherichia coli, he found that the bacteria could never grow. When he injected the mould juice into guinea pigs which were experimentally inoculated with typhoid bacteria (Salmonella enterica), the guinea pigs never developed the disease.[34] He described his experiment in a doctoral dissertation titled Contribution à l'étude de la concurrence vitale chez les microorganismes (Contribution to the study of vital competition between microorganisms: antagonism between moulds and microbes) in 1897.[35][36] Pasteur Institute, to which the thesis was submitted, ignored the discovery and was forgotten.[37]

A librarian rediscovered the thesis 50 years later when penicillin was already discovered. Duchesne could not continue his experiments due to severe illness (believed to be tuberculosis) he contracted 5 years later and died in 1912 while serving in the French Army.[34] He was himself using a discovery made earlier by Arab stable boys, who used moulds to cure sores on horses. He did not claim that the mould contained any antibacterial substance, only that the mould somehow protected the animals.[24] His conclusion was nonetheless prognostic, stating that competition between bacteria and moulds could be useful in the medical management of infections.[38] Penicillin does not cure typhoid and so it remains unknown which substance might have been responsible for Duchesne's cure.[a] A Pasteur institute scientist, Costa Rican Clodomiro Picado Twight, similarly recorded the antibiotic effect of Penicillium in 1923. In these early stages of penicillin research, most species of Penicillium were non-specifically referred to as P. glaucum, so that it is impossible to know the exact species and that it was really penicillin that prevented bacterial growth.[24]

An Italian physician Bartolomeo Gosio was the first to discover and isolate an antibiotic compound from Penicillium in 1896.[39] Gosio investigated the case of pellagra, which at the time was a common disease in southern Europe and America. It was known that the staple food of people having the disease was corn, and fungal contamination of corn was regarded as the source. Gosio identified the mould Penicillium brevicompactum as one possible cause in 1893.[40][41] He developed a simple culture method with which he could make pure culture extract in crystalline form. In 1896, he tested the substance on anthrax bacillus and found that it was highly potent against the bacteria.[42][43] Gosio's discovery was largely forgotten as the substance was found not to be the cause of pellagra, and the medicinal potential was not obvious. American biochemist Conrad Elvehjem identified, in 1937, the aetiology as deficiency of niacin (vitamin B3). American scientists, Carl Alsberg and Otis Fisher Black resynthesized Gosio's substance in 1912 giving the name mycophenolic acid, which is now used as an immunosuppressant.[41][44]

Andre Gratia and Sara Dath at the Free University of Brussels, Belgium, were studying the effects of mould samples on bacteria. In 1924, they found that dead Staphylococcus aureus cultures were contaminated by a mould, a streptomycete. On further experimentation, they showed that the mould extract could kill not only S. aureus, but also Pseudomonas aeruginosa, Mycobacterium tuberculosis and Escherichia coli.[45] Gratia called the antibacterial agent "mycolysate" (killer mould). The next year they found another killer mould that could inhibit anthrax bacterium (B. anthracis). Reporting in Comptes Rendus Des Séances de La Société de Biologie et de Ses Filiales, they identified the mould as Penicillium glaucum.[46] In 1927, Gratia reported their medical use, saying:

A poor patient who during three years had suffered from furuncles [infection by S. aureus], inspite of all treatments, was sent to us in despair. Jaumain did not hesitate to continue the treatment by a series of injections of the mycolysat. The result was remarkable. Not only was the recovery rapid, but it is now three years that [sic] this recovery continues without the slightest relapse. Since that time we have given the mycolysat to a very large number of cases. It is the most effective treatment even of the most resistant types of staphylococcic diseases.[39]

But these findings received little attention as the antibacterial agent and its medical values were not fully understood; moreover, Gratia's samples were lost.[45]

The breakthrough discovery[edit | edit source]

Background[edit | edit source]

Professor Alexander Fleming at work in his laboratory at St Mary's Hospital, London, during the Second World War. D17801.jpg

 Alexander Fleming in his laboratory at St Mary's Hospital, London.


Ministry of Information Photo Division Photographer, Public domain

Penicillin as we know it today was discovered by a Scottish physician Alexander Fleming in 1928. While working at St Mary's Hospital, London, Fleming was investigating the pattern of variation in S. aureus (Figures 2 and 3).[47][48] He was inspired by the discovery of an Irish physician Joseph Warwick Bigger and his two students C.R. Boland and R.A.Q. O’Meara at the Trinity College, Dublin, Ireland, in 1927. Bigger and his students found that when they cultured a particular strain of S. aureus, which they designated "Y" that they isolated a year before from the pus of an axillary abscess from one individual, the bacterium grew into a variety of strains. They published their discovery as “Variant colonies of Staphylococcus aureus” in The Journal of Pathology and Bacteriology, by concluding:

We were surprised and rather disturbed to find, on a number of plates, various types of colonies which differed completely from the typical aureus colony. Some of these were quite white; some, either white or of the usual colour were rough on the surface and with crenated margins.[49]

Fleming and his research scholar Daniel Merlin Pryce pursued this experiment but Pryce was transferred to another laboratory in early 1928. After a few months of working alone, a new scholar, Stuart Craddock, joined Fleming. Their experiment was successful and Fleming was planning and agreed to write a report in A System of Bacteriology to be published by the Medical Research Council by the end of 1928.[48]

Initial discovery[edit | edit source]

In August, Fleming spent a vacation with his family at his country home The Dhoon at Barton Mills, Suffolk. Before leaving his laboratory (Figures 3 and 4), he inoculated several culture plates with S. aureus. He kept the plates aside on one corner of the table away from direct sunlight and to make space for Craddock to work in his absence. While on vacation, he was appointed Professor of Bacteriology at the St Mary's Hospital Medical School on 1 September 1928. He arrived at his laboratory on 3 September, where Pryce was waiting to greet him.[50] As he and Pryce examined the culture plates, they found one with an open lid and the culture contaminated with a blue-green mould. In the contaminated plate the bacteria around the mould did not grow, while those farther away grew normally, meaning that the mould killed the bacteria.[51] Fleming commented as he watched the plate: "That's funny".[50][52] Pryce remarked to Fleming: "That's how you discovered lysozyme."[53]

Experiment[edit | edit source]

Flemming laboratory (3).JPG

 St Mary's Hospital showing Fleming's lab (on the second floor) and Praed Street, from where Fleming alleged the mould came from.


Vera de Kok, CC-BY 4.0

Fleming went off to resume his vacation and returned for the experiments late in September.[48] He collected the original mould and grew them in culture plates. After four days he found that the plates developed large colonies of the mould. He repeated the experiment with the same bacteria-killing results. He later recounted his experience:

When I woke up just after dawn on September 28, 1928, I certainly didn't plan to revolutionize all medicine by discovering the world's first antibiotic, or bacteria killer. But I suppose that was exactly what I did.[54]

He concluded that the mould was releasing a substance that was inhibiting bacterial growth, and he produced a culture broth of the mould and subsequently concentrated the antibacterial component.[55] After testing against different bacteria, he found that the mould could kill only specific bacteria. For example, Staphylococcus, Streptococcus, and diphtheria bacillus (Corynebacterium diphtheriae) were easily killed; but there was no effect on typhoid bacterium (Salmonella typhimurium) and influenza bacillus (Haemophilus influenzae). He prepared a large-culture method from which he could obtain large amounts of the mould juice. He called this juice "penicillin", as he explained the reason was: "to avoid the repetition of the rather cumbersome phrase 'Mould broth filtrate,' the name 'penicillin' will be used."[56] He invented the name on 7 March 1929.[50] He later (in his Nobel lecture) gave a further explanation, saying:

I have been frequently asked why I invented the name "Penicillin". I simply followed perfectly orthodox lines and coined a word which explained that the substance penicillin was derived from a plant of the genus Penicillium just as many years ago the word "Digitalin" was invented for a substance derived from the plant Digitalis.[57]

Fleming had no training in chemistry so that he left all the chemical works to Craddock; he once remarked: "I am a bacteriologist, not a chemist."[48] In January 1929, he recruited Frederick Ridley, his former research scholar who had studied biochemistry, specifically to study the chemical properties of the mould.[52] But they could not isolate penicillin and before the experiments were over, Craddock and Ridley both left Fleming for other jobs.[50] It was due to their failure to isolate the compound that Fleming practically abandoned further research on the chemical aspects of penicillin,[58] although he did biological tests up to 1939.[50]

Identification of the mould[edit | edit source]

Penicillium rubens (type specimen).png

 Penicillium rubens (type specimen).


Houbraken et al., 2011, CC-BY 4.0

After structural comparison with different species of Penicillium, Fleming initially believed that his specimen was Penicillium chrysogenum, a species described by an American microbiologist Charles Thom in 1910. He was fortunate as Charles John Patrick La Touche, an Irish botanist, had just recently joined as a mycologist at St Mary's to investigate fungi as the cause of asthma. La Touche identified the specimen as Penicillium rubrum,[59][60] the identification used by Fleming in his publication.

In 1931, Thom re-examined different Penicillium including that of Fleming's specimen. He came to a confusing conclusion, stating: "Ad. 35 [Fleming's specimen] is P. notatum WESTLING. This is a member of the P. chrysogenum series with smaller conidia than P. chrysogenum itself."[61] P. notatum was described by Swedish chemist Richard Westling in 1811. From then on, Fleming's mould was synonymously referred to as P. notatum and P. chrysogenum. But Thom adopted and popularised the use of P. chrysogenum.[62] In addition to P. notatum, newly discovered species such as P. meleagrinum and P. cyaneofulvum were recognised as members of P. chrysogenum in 1977.[63] To resolve the confusion, the Seventeenth International Botanical Congress held in Vienna, Austria, in 2005 formally adopted the name P. chrysogenum as the conserved name (nomen conservandum).[64] Whole genome sequence and phylogenetic analysis in 2011 revealed that Fleming's mould belongs to P. rubens (Figure 5), a species described by Belgian microbiologist Philibert Biourge in 1923, and also that P. chrysogenum is a different species.[65][66]

The source of the fungal contamination in Fleming's experiment remained a speculation for several decades. The Royal Society of Chemistry initially was of the opinion that it was from a cup of coffee left by Fleming on the table.[37] Fleming himself suggested in 1945 that the fungal spores came through the window facing Praed Street. This story was regarded as a fact and was popularised in the literature,[67] starting with George Lacken's 1945 book The Story of Penicillin.[50] But it was later disputed by his co-workers including Pryce, who testified much later that Fleming's laboratory window was kept shut all the time.[68] Ronald Hare also agreed in 1970 that the window was most often locked because it was difficult to reach due to a large table with apparatus placed in front of it. In 1966, La Touche told Hare that he had given Fleming 13 specimens of fungi (10 from his lab) and only one from his lab was showing penicillin-like antibacterial activity.[67] It was from this point a consensus was made that Fleming's mould came from La Touche's lab, which was a floor below in the building, the spores having drifted in the air through the open doors.[69]

Reception and publication[edit | edit source]

Fleming's discovery was not regarded initially as an important discovery. Even as he showed his culture plates to his colleagues, all he received was an indifferent response. He described the discovery on 13 February 1929 before the Medical Research Club. His presentation titled "A medium for the isolation of Pfeiffer's bacillus" did not receive any particular attention.[48]

In 1929, Fleming reported his findings to the British Journal of Experimental Pathology on 10 May 1929, and was published in the next month's issue.[70][71] It failed to attract any serious attention. Fleming himself was quite unsure of the medical application and was more concerned about the application for bacterial isolation, as he concluded:

In addition to its possible use in the treatment of bacterial infections penicillin is certainly useful to the bacteriologist for its power of inhibiting unwanted microbes in bacterial cultures so that penicillin insensitive bacteria can readily be isolated. A notable instance of this is the very easy, isolation of Pfeiffers bacillus of influenza when penicillin is used...It is suggested that it may be an efficient antiseptic for application to, or injection into, areas infected with penicillin-sensitive microbes.[70]

G. E. Breen, a fellow member of the Chelsea Arts Club, once asked Fleming: "I just wanted you to tell me whether you think it will ever be possible to make practical use of the stuff [penicillin]. For instance, could I use it?" Fleming gazed vacantly for a moment and then replied: "I don't know. It's too unstable. It will have to be purified, and I can't do that by myself."[48] Even as late as in 1941, the British Medical Journal reported that: "the main facts emerging from a very comprehensive study [of penicillin] in which a large team of workers is engaged... does not appear to have been considered as possibly useful from any other point of view."[72][73][b]

Isolation[edit | edit source]

Ernst Boris Chain, a Jewish-German (later naturalised British) chemist, flee from Germany to England on 30 January 1933, as Nazi party formed the government, as Chain himself described as the day "Hitler acceded to power and Europe was temporarily plunged into a darkness in comparison with which the darkest Middle Ages now appear as a blaze of light."[74] After working at the University College Hospital in London, he joined Australian scientist Howard Florey (later Baron Florey) at the Sir William Dunn School of Pathology at the University of Oxford in 1936 to investigate antibiotics. Florey assigned him to investigate on lysozyme, an antibacterial enzyme discovered by Fleming in 1922.[75] While writing for the research findings, he came across Fleming's 1929 paper in 1938, and informed his supervisor of the potential medical benefits of penicillin.[76] A year before, Florey had thought of pyocyanase (a pigment from the bacterium Bacillus pycyaneus, now called Pseudomonas aeruginosa) as a lead substance to work on, but agreed that penicillin was medically more promising.[77] In 1939, Florey and Chain acquired a research grant of $25,000 from the Rockefeller Foundation to study antibiotics.[78][79] They assembled a research team that included Edward Abraham, Arthur Duncan Gardner, Norman Heatley, Margaret Jennings, J. Orr-Ewing and G. Sanders.[80][81]

The Oxford team prepared a concentrated extract of P. rubens as: "a brown powder" that: "has been obtained which is freely soluble in water".[82] They found that the powder was not only effective in vitro against bacterial cultures but also and in vivo against bacterial infection in mice. On 5 May 1939, they injected a group of eight mice with a virulent strain of S. aureus, and then injected four of them with the penicillin solution. After one day, all the untreated mice died while the penicillin-treated mice survived. Chain remarked it as "a miracle."[76] They published their findings in The Lancet in 1940.[82]

The team reported details of the isolation method in 1941 with a scheme for large-scale extraction. They also found that penicillin was most abundant as a yellow concentrate from the mould extract.[83] But they were able to produce only small quantities. By the early 1942, they could prepare highly purified compound,[84] and derived the empirical chemical formula as C24H32O10N2Ba.[85] In the June 1942 issue of the British Journal of Experimental Pathology, Chain, Abraham and E. R. Holiday reported the production of the pure compound, with a conclusion:

The penicillin preparation described in this paper is the most powerful antibacterial agent with predominantly bacteriostatic action so far known. Though it has not yet been obtained crystalline there are indications that it possesses a considerable degree of purity... The unusual biological properties of penicillin are linked with an exceptionally unstable chemical configuration. Inactivation by acid, alkali, and by boiling at any pH has been shown to be accompanied by definite chemical changes.[86]

First medical use[edit | edit source]

Fleming performed the first clinical trial with penicillin on Craddock. Craddock had developed severe infection of the nasal antrum (sinusitis) and had undergone surgery. Fleming made use of the surgical opening of the nasal passage and started injecting penicillin on 9 January 1929 but without any effect. It probably was due to the fact that the infection was with influenza bacillus (Haemophilus influenzae), the bacterium which he had found not susceptible to penicillin.[87] Fleming gave some of his original penicillin samples to his colleague-surgeon Arthur Dickson Wright for clinical test in 1928.[88][89] Although Wright reportedly said that it: "seemed to work satisfactorily,"[17] there are no records of its specific use.

Cecil George Paine, a pathologist at the Royal Infirmary in Sheffield, was the first, successfully, to use penicillin for medical treatment.[14] He was a former student of Fleming and when he learned of the discovery, requested a penicillin sample from Fleming.[90] He initially attempted to treat sycosis (eruptions in beard follicles) with penicillin but was unsuccessful, probably because the drug did not penetrate deeply enough. Moving on to ophthalmia neonatorum, a gonococcal infection in babies, he achieved the first cure on 25 November 1930, four patients (one adult, the others infants) with eye infections.[91][92]

Florey's team at Oxford showed that Penicillium extract killed different bacteria (Streptococcus pyogenes, Staphylococcus aureus, and Clostridium septique) in culture and effectively cured Streptococcus infection in mice.[76] They reported in the 24 August 1940 issue of The Lancet: "Penicillin as a chemotherapeutic agent" with a conclusion:

The results are clear cut, and show that penicillin is active in vivo against at least three of the organisms inhibited in vitro. It would seem a reasonable hope that all organisms in high dilution in vitro will be found to be dealt with in vivo. Penicillin does not appear to be related to any chemotherapeutic substance at present in use and is particularly remarkable for its activity against the anaerobic organisms associated with gas gangrene.[82]

In 1941, the Oxford team treated a policeman, Albert Alexander, who had a severe face infection; his condition improved, but he eventually died as they ran out of penicillin. Subsequently, several other patients were treated successfully.[93] In December 1942, survivors of the Cocoanut Grove fire in Boston were the first burn patients to be successfully treated with penicillin.[94]

The most important clinical test was in August 1942 when Fleming cured Harry Lambert of a fatal infection of the nervous system (streptococcal meningitis). Lambert was a work associate of Robert, Fleming's brother, who had requested Fleming for medical treatment.[95] Fleming asked Florey for apurified penicillin sample, which Fleming immediately used to inject into Lambert's spinal canal. Lambert showed signs of improvement the next day,[96] and completely recovered within a week.[97][98] Fleming reported his clinical trial in The Lancet in 1943.[99] It was on this medical evidence that the British War Cabinet set up the Penicillin Committee on 5 April 1943. The committee consisted of Cecil Weir, Director General of Equipment, as Chairman, Fleming, Florey, Sir Percival Hartley, Allison and representatives from pharmaceutical companies as members.[96] This led to mass production of penicillin by the next year.[100][101]

Mass production[edit | edit source]

Penicillium notatum.jpg

 The cantaloupe strain of Penicillum (P. chrysogenum or P. notatum) which is the best source of penicillins and was used in the first mass production in US.


Crulina 98, CC-BY 3.0

Knowing that large-scale production for medical use was futile in a confined laboratory, the Oxford team tried to convince war-torn British government and private companies to undertake mass production, but in vain.[102] Florey and Heatley travelled to the US in June 1941 to persuade United States (US) government and pharmaceutical companies there.[103] Knowing that by keeping the mould sample in vials it could be easily lost, they instead smeared their coat pockets with the mould.[76] They arrived in Washington D.C. in early July to discuss with Ross Granville Harrison, chairman of the National Research Council (NRC), and Charles Thom and Percy Wells of the United States Department of Agriculture. They were directed to approach the USDA Northern Regional Research Laboratory (NRRL, now the National Center for Agricultural Utilization Research) where large-scale fermentations were done.[104] They reached Peoria, Illinois, on 14 July to meet Andrew Jackson Moyer and Robert D. Coghill at the NRRL. The Americans quickly worked on the mould and were able to make a culture by the end of July.[102] But they realised that Fleming's mould was not efficient enough to produce large quantities of penicillin.

NRRL mycologist Kenneth Bryan Raper got the help of US Army Transport Command to search for similar mould in different parts of the world and the best moulds were found to be those from Chungkin (China), Bombay (Mumbai, India) and Cape Town (South Africa). But the single-best sample was from cantaloupe (a type of melon) sold in Peoria fruit market in 1943. The mould was identified to be P. chrysogenum and designated as "NRRL 1951" or "cantaloupe strain" (Figure 6).[104][105] There is a popular story that Mary K. Hunt (or Mary Hunt Stevens[106]), a staff member at Raper, collected the mould;[107] for which she had been popularised as "Mouldy Mary."[108][109] But Raper remarked this story as a "folklore" and that the fruit was delivered to the lab by a woman from the Peoria fruit market.[104]

Between 1941 and 1943, Moyer, Coghill and Kenneth Raper developed methods for industrialized penicillin production and isolated higher-yielding strains of the Penicillium fungus.[110] Simultaneous research by Jasper H. Kane and other Pfizer scientists in Brooklyn developed the practical, deep-tank fermentation method for production of large quantities of pharmaceutical-grade penicillin.[111]

PenicillinPSAedit.jpg

 Penicillin ad for World War II servicemen, c. 1944.


National Institute of Health, Public domain

When production first began, one-litre containers had a yield of less than 1%, but improved to a yield of 80–90% in 10,000 gallon containers. This increase in efficiency happened between 1939 and 1945 as the result of continuous process innovation (Figure 7 shows one of the first mass applications). Orvill May, director of the Agricultural Research Service, had Robert Coghill, who was the chief of the fermentation division, use his experience with fermentation to increase the efficiency of extracting penicillin from the mould. Shorty after beginning, Moyer replaced sucrose with lactose in the growth media, which resulted in an increased yield. An even larger increase occurred when Moyer added corn steep liquor.[103]

One major issue with the process that scientists faced was the inefficiency of growing the mould on the surface of their nutrient baths, rather than having it submerged. Although a submerged process of growing the mould would be more efficient, the strain used was not suitable for the conditions it would require. This led NRRL to a search for a strain that had already been adapted to work, and one was found in the mouldy cantaloupe acquired from a Peoria farmers' market.[112] To improve on that strain, researchers subjected it to X-rays to facilitate mutations in its genome and managed to increase production capabilities.[113][112]

Now that scientists had a mould that grew well submerged and produced an acceptable amount of penicillin, the next challenge was to provide the required air to the mould for it to grow. This was solved using an aerator, but aeration caused severe foaming as a result of the corn steep. The foaming problem was solved by the introduction of an anti-foaming agent known as glyceryl monoricinoleate.[113]

Chemical analysis[edit | edit source]

The chemical structure of penicillin was first proposed by Edward Abraham in 1942.[114] Dorothy Hodgkin determined the correct chemical structure of penicillin using X-ray crystallography at Oxford in 1945.[115][116][117] In 1945, the US Committee on Medical Research and the British Medical Research Council jointly published in Science a chemical analyses done at different universities, pharmaceutical companies and government research departments. The report announced the existence of different forms of penicillin compounds which all shared the same structural component called β-lactam.[118] The penicillins were given various names using Roman numerals in the United Kingdom (UK) (such as penicillin I, II, III, and IV) in order of their discoveries and letters (such as F, G, K, and X) in US referring to their origins or sources, as below:

UK nomenclature US nomenclature Chemical name
Penicillin I Penicillin F 2-Pentenylpenicillin
Penicillin II Penicillin G Benzylpenicillin
Penicillin III Penicillin X p-Hydroxybenzylpenicillin
Penicillin IV Penicillin K n-Heptylpenicillin

The use of two names for each penicillin started to cause problem in their identification and application.[119] As the chemical structures were known, the chemical names added more to the confusion. Scientists used any of the names as they wished so that penicillin literature became a mixture of three naming systems; chemists mostly adhered to the chemical names,[120][121] while biologists preferred the classic numbered or lettered names.[122][123] The chemical names were based on the side chains of the compounds.[121] To avoid the controversial names, Chain introduced in 1948 the chemical names as standard nomenclature, remarking: "To make the nomenclature as far as possible unambiguous it was decided to replace the system of numbers or letters by prefixes indicating the chemical nature of the side chain R."[124]

In Austria, Hans Margreiter and Ernst Brandl of Biochemie (now Sandoz) developed the first acid-stable penicillin for oral administration, penicillin V in 1952.[125] American chemist John C. Sheehan at the Massachusetts Institute of Technology (MIT) completed the first chemical synthesis of penicillin in 1957.[126][127][128] Sheehan had started his studies into penicillin synthesis in 1948, and during these investigations developed new methods for the synthesis of peptides, as well as new protecting groups—groups that mask the reactivity of certain functional groups.[128][129] Although the initial synthesis developed by Sheehan was not appropriate for mass production of penicillins, one of the intermediate compounds in Sheehan's synthesis was 6-aminopenicillanic acid (6-APA), the nucleus of penicillin.[130][131]

An important development was the discovery of 6-APA itself. In 1957, researchers at the Beecham Research Laboratories (now the Beecham Group) in Surrey isolated 6-APA from the culture media of P. chrysogenum. 6-APA was found to constitute the core 'nucleus' of penicillin (in fact, all β-lactam antibiotics) and was easily chemically modified by attaching side chains through chemical reactions.[132][133] The discovery was published in Nature in 1959.[134] This paved the way for new and improved drugs as all semi-synthetic penicillins are produced from chemical manipulation of 6-APA.[135]

The second-generation semi-synthetic β-lactam antibiotic methicillin, designed to counter first-generation-resistant penicillinases, were introduced in the United Kingdom in 1959. Methicillin-resistant forms of Staphylococcus aureus likely already existed at the time.[136][137]

Outcomes[edit | edit source]

Patents for the discovery became a matter of concern and conflicts. Chain had wanted to file a patent while Florey and his teammates objected to it arguing that it should be a benefit for all.[138] He sought advice from authorities. On 26 and 27 March 1941, Sir Henry Hallett Dale, then Chairman of the Wellcome Trust and member the Scientific Advisory Panel to the Cabinet of British government, and John William Trevan, then Director of the Wellcome Trust Research Laboratory, visited Sir William Dunn School of Pathology and deliberated the idea of patenting penicillin. Dale specifically advised that doing so would be unethical.[139] Chain then approached Sir Edward Mellanby, then Secretary of the Medical Research Council, who also objected on ethical grounds.[140] Chain later said that he had "many bitter fights" with Mellanby,[139] but Mellanby's decision was taken as final.[140]

Methods for production and isolation of penicillin were patented by Andrew Jackson Moyer in US in 1945.[141][142][143] Moyer could not obtain a patent in the US as an employee of the NRRL, and filed his patent at the British Patent Office (now the Intellectual Property Office). He gave the license to the US company, Commercial Solvents Corporation. Although there were no legal issues, his colleague Coghill opined that it was an injustice for outsiders to have the royalties for the "British discovery." In 1946, Moyer asked Coghill for permission to file another patent based on the use of phenylacetic acid that could increase penicillin production by 66%, but as the principal researcher, Coghill refused.[144]

When Fleming learned of the American patents on penicillin production, he was infuriated and commented:

I found penicillin and have given it free for the benefit of humanity. Why should it become a profit-making monopoly of manufacturers in another country?[145]

Fleming, Florey and Chain equally shared the 1945 Nobel Prize in Physiology or Medicine "for the discovery of penicillin and its curative effect in various infectious diseases."[146] Dorothy Hodgkin received the 1964 Nobel Prize in Chemistry "for her determinations by X-ray techniques of the structures of important biochemical substances."

Development of penicillin-derivatives[edit | edit source]

The narrow range of treatable diseases or "spectrum of activity" of the penicillins, along with the poor activity of the orally active phenoxymethylpenicillin, led to the search for derivatives of penicillin that could treat a wider range of infections. The isolation of 6-APA, the nucleus of penicillin, allowed for the preparation of semisynthetic penicillins, with various improvements over benzylpenicillin (bioavailability, spectrum, stability, tolerance). The first major development was ampicillin in 1961. It was produced by Beecham Research Laboratories in London.[147] It was more advantageous than the original penicillin as it offered a broader spectrum of activity against Gram-positive and Gram-negative bacteria.[147] Further development yielded β-lactamase-resistant penicillins, including flucloxacillin, dicloxacillin, and methicillin. These were significant for their activity against β-lactamase-producing bacterial species, but were ineffective against the methicillin-resistant Staphylococcus aureus strains that subsequently emerged.[148]

Another development of the line of true penicillins was the antipseudomonal penicillins, such as carbenicillin, ticarcillin, and piperacillin, useful for their activity against Gram-negative bacteria. However, the usefulness of the β-lactam ring was such that related antibiotics, including the mecillinams, the carbapenems and, most important, the cephalosporins, still retain it at the centre of their structures.[133][149]

The penicillins related to β-lactams have become the most widely used antibiotics in the world.[150] Amoxicillin, a semisynthetic penicillin developed by Beecham Research Laboratories in 1970,[151][152] is the most commonly used of all.[153][154]

Drug resistance[edit | edit source]

Fleming warned of the possibility of penicillin resistance in clinical conditions; in his Nobel Lecture, and said:

The time may come when penicillin can be bought by anyone in the shops. Then there is the danger that the ignorant man may easily underdose himself and by exposing his microbes to non-lethal quantities of the drug make them resistant.[155]

In 1940, Ernst Chain and Edward Abraham reported the first indication of antibiotic resistance to penicillin, an E. coli strain that produced the penicillinase enzyme, which was capable of breaking down penicillin and completely negating its antibacterial effect.[136][71][156] Chain and Abraham worked out the chemical nature of penicillinase which they reported in Nature:

The conclusion that the active substance is an enzyme is drawn from the fact that it is destroyed by heating at 90° for 5 minutes and by incubation with papain activated with potassium cyanide at pH 6, and that it is non-dialysable through 'Cellophane' membranes.[157]

In 1942, strains of Staphylococcus aureus had been documented to have developed a strong resistance to penicillin. Most of the strains were resistant to penicillin by the 1960s.[158] In 1967, Streptococcus pneumoniae was also reported to be penicillin resistant. Many strains of bacteria have eventually developed a resistance to penicillin.[117][71]

Notes[edit | edit source]

  1. At the time, the term Penicillium glaucum was used as a catch-all phrase for a variety of different fungi, though not for Penicillium notatum. Duchesne's specific mold was unfortunately not preserved, which makes it impossible to be certain today which fungus might have been responsible for the cure and, consequently, even less certain which specific antibacterial substance was responsible.
  2. The statement "does not appear to have been considered as possibly useful from any other point of view" seems to be later deleted, but is still apparent from Fleming's response (BMJ, 2 (4210): 386–386).

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