History of penicillin

Many ancient cultures, including those in Australia, China, Egypt, Greece and India, independently discovered the useful properties of fungi and plants in treating infection. 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.[1][2][3]

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.[4] 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.[5]

The modern history of penicillin research begins 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. Lister also described the antibacterial action on human tissue of a species of mould he called Penicillium glaucum.[6][7]

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 1874, the Welsh physician William Roberts, who later coined the term "enzyme", observed that bacterial contamination is generally absent in laboratory cultures of P. glaucum. John Tyndall followed up on Burdon-Sanderson's work and demonstrated to the Royal Society in 1875 the antibacterial action of the Penicillium fungus.[8]

In 1876, German biologist Robert Koch discovered that a bacterium (Bacillus anthracis) was the causative pathogen of anthrax,[9] which became the first demonstration that a specific bacterium caused a specific disease, and the first direct evidence of germ theory of diseases.[10] 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.[11] Reporting in the Comptes Rendus de l'Académie des Sciences, they concluded:

Neutral or slightly alkaline urine is an excellent medium for the bacteria. If the urine is sterile and the culture pure the bacteria multiply so fast that in the course of a few hours their filaments fill the fluid with a downy felt. 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.[12]

The phenomenon was described by Pasteur and Koch as antibacterial activity and was named as "antibiosis" by French biologist Jean Paul Vuillemin in 1877.[13][14] (The term antibiosis, meaning "against life", was adopted as "antibiotic" by American biologist and later Nobel laureate Selman Waksman in 1947.[15]) It has also been asserted that Pasteur identified the strain as Penicillium notatum. However, Paul de Kruif's 1926 Microbe Hunters describes this incident as contamination by other bacteria rather than by mould.[16] In 1887, Swiss physician Carl Alois Philipp Garré developed a test method using glass plate to see bacterial inhibition and found similar results.[14] 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.[17]

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.[18][19][20][21]

Two years later, Ernest Duchesne at École du Service de Santé Militaire in Lyon independently discovered the healing properties of a P. glaucum mould, even curing infected guinea pigs of typhoid. He published a dissertation in 1897,[22] but it was ignored by the Institut Pasteur. Duchesne was 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. Penicillin does not cure typhoid and so it remains unknown which substance might have been responsible for Duchesne's cure. 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.[11]

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. Upon further experimentation, they showed that the mould extract could kill not only S. aureus, but also Pseudomonas aeruginosa, Mycobacterium tuberculosis and Escherichia coli.[23] Gratia called the antibacterial agent as "mycolysate" (killer mould). The next year they found another killer mould that could inhibit B. anthracis. Reporting in Comptes Rendus Des Séances de La Société de Biologie et de Ses Filiales, they identified the mould as P. glaucum.[24] But these findings received little attention as the antibacterial agent and its medical value were not fully understood, and Gratia's samples were lost.[23]

Howard Florey in his office in 1944

In 1939, at the Sir William Dunn School of Pathology at the University of Oxford, Ernst Boris Chain found Fleming's largely forgotten 1929 paper, and suggested to the professor in charge of the school, the Australian scientist Howard Florey, that the study of antibacterial substances produced by micro-organisms might be a fruitful avenue of research.[64]^: 297 Florey led an interdisciplinary research team that also included Edward Abraham, Mary Ethel Florey, Arthur Duncan Gardner, Norman Heatley, Margaret Jennings, Jean Orr-Ewing and Gordon Sanders.[65][66] Each member of the team tackled a particular aspect of the problem in their own manner, with simultaneous research along different lines building up a complete picture. This sort of collaboration was practically unknown in the United Kingdom at the time.[67] Three sources were initially chosen for investigation: Bacillus subtilis, Trueperella pyogenes and penicillin.[68] "[The possibility] that penicillin could have practical use in clinical medicine", Chain later recalled, "did not enter our minds when we started our work on penicillin."[64]^: 111

The broad subject area was deliberately chosen to be one requiring long-term funding.[64]^: 297 Florey approached the Medical Research Council in September 1939, and the secretary of the council, Edward Mellanby authorized the project, allocating £250 (equivalent to £16,000 in 2021) to launch the project, with £300 for salaries and £100 for expenses per annum for three years. Florey felt that more would be required. On 1 November 1939, Henry M. "Dusty" Miller Jr from the Natural Sciences Division of the Rockefeller Foundation paid Florey a visit. Miller was enthusiastic about the project. He encouraged Florey to apply for funding from the Rockefeller Foundation and recommended to Foundation headquarters in New York that the request for financial support be given serious consideration.[69][70] "The work proposed", Florey wrote in the application letter, "in addition to its theoretical importance, may have practical value for therapeutic purposes."[71] His application was approved, with the Rockefeller Foundation allocating US$5,000 (£1,250) per annum for five years.[69][70]

The Sir William Dunn School of Pathology in Oxford

The Oxford team's first task was to obtain a sample of penicillin mould. This turned out to be easy. Margaret Campbell-Renton, who had worked with Georges Dreyer, Florey's predecessor, revealed that Dreyer had been given a sample of the mould by Fleming in 1930 for his work on bacteriophages. Dreyer had lost all interest in penicillin when he discovered that it was not a bacteriophage.[72][73] He had died in 1934, but Campbell-Renton had continued to culture the mould.[74] The next task was to grow sufficient mould to extract enough penicillin for laboratory experiments. The mould was cultured on a surface of liquid Czapek-Dox medium. Over the course of a few days it formed a yellow gelatinous skin covered in green spores. Beneath this the liquid became yellow and contained penicillin. The team determined that the maximum yield was achieved in ten to twenty days.[75]

Most laboratory containers did not provide a large, flat area, and so were an uneconomical use of incubator space, so glass bottles laid on their sides were used.[75] The bedpan was found to be practical, and was the basis for specially-made ceramic containers fabricated by J. Macintyre and Company in Burslem. The containers were rectangular in shape and could be stacked to save space.[76] The Medical Research Council agreed to Florey's request for £300 (equivalent to £17,000 in 2021) and £2 each per week (equivalent to £116 in 2021) for two (later) women factory hands. In 1943 Florey asked for their wages to be increased to £2 10s each per week (equivalent to £120 in 2021).[77] Heatley collected the first 174 of an order for 500 vessels on 22 December 1940, and they were seeded with spores three days later.[78]

Flasks used in the cultivation of penicillin mould for large-scale production. One of the first flasks (centre) was made from a biscuit tin. Ceramic flasks (rear) were later produced.

Efforts were made to coax the mould to produce more penicillin. Heatley tried adding various substances to the medium, including sugars, salts, malts, alcohol and even marmite, without success.[79] At the suggestion of Paul Fildes, he tried adding brewing yeast. This did not improve the yield either, but it did cut the incubation time by a third.[75] The team also discovered that if the penicillin-bearing fluid was removed and replaced by fresh fluid, a second batch of penicillin could be prepared,[75] but this practice was discontinued after eighteen months, due to the danger of contamination. The mould had to be grown under sterile conditions.[80] Abraham and Chain discovered that some airborne bacteria that produced penicillinase, an enzyme that destroys penicillin.[81] It was not known why the mould produced penicillin, as the bacteria penicillin kills are no threat to the mould; it was conjectured that it was a byproduct of metabolic processes for other purposes.[80]

The next stage of the process was to extract the penicillin. The liquid was filtered through parachute silk to remove the mycelium, spores and other solid debris.[82] The pH was lowered by the addition of phosphoric acid and cooled.[83] Chain determined that penicillin was stable only with a pH of between 5 and 8, but the process required one lower than that. By keeping the mixture at 0 °C, he could retard the breakdown process.[84] In this form the penicillin could be drawn off by a solvent. Initially ether was used, as it was the only solvent known to dissolve penicillin. At Chain's suggestion, they tried using the much less dangerous amyl nitrite instead, and found that it also worked.[82][85]

Ernst Chain in his laboratory

Heatley was able to develop a continuous extraction process. The penicillin-bearing solvent was easily separated from the liquid, as it floated on top, but now they encountered the problem that had stymied Craddock and Ridley: recovering the penicillin from the solvent. Heatley reasoned that if the penicillin could pass from water to solvent when the solution was acidic, maybe it would pass back again if the solution was alkaline. Florey told him to give it a try. Sodium hydroxide was added, and this method, which Heatley called "reverse extraction", was found to work.[82][85] The next problem was how to extract the penicillin from the water. The usual means of extracting something from water was through evaporation or boiling, but this would destroy the penicillin. Chain hit upon the idea of freeze drying, a technique recently developed in Sweden. This enabled the water to be removed, resulting in a dry, brown powder.[82][84]

Heatley developed a penicillin assay using agar nutrient plates in which bacteria were seeded. Short glass cylinders containing the penicillin-bearing fluid to be tested were then placed on them and incubated for 12 to 16 hours at 37 °C. By then the fluid would have disappeared and the cylinder surrounded by a bacteria-free ring. The diameter of the ring indicated the strength of the penicillin.[83] An Oxford unit was defined as the purity required to produce a 25 mm bacteria-free ring.[74] It was an arbitrary measurement, as the chemistry was not yet known; the first research was conducted with solutions containing four or five Oxford units per milligram. Later, when highly pure penicillin became available, it was found to have 2,000 Oxford units per milligram.[86] Yet in testing the impure substance, they found it effective against bacteria even at concentrations of one part per million. Penicillin was at least twenty times as active as the most powerful sulfonamide.[84] The Oxford unit turned out to be very small; treating a single case required about a million units.[87]

The Oxford team reported details of the isolation method in 1941 with a scheme for large-scale extraction, but they were able to produce only small quantities. By early 1942, they could prepare highly purified compound,[88] and had worked out the chemical formula as C₂₄H₃₂O₁₀N₂Ba.[89] In mid-1942, Chain, Abraham and E. R. Holiday reported the production of the pure compound.[90]

Florey's team at Oxford showed that Penicillium extract killed different bacteria. Gardner and Orr-Ewing tested it against gonococcus (against which it was most effective), meningococcus, Streptococcus, Staphylococcus, anthrax bacteria, Actinomyces, tetanus bacterium (Clostridium tetani) and gangrene bacteria. They observed bacteria attempting to grow in the presence of penicillin, and noted that it was not an enzyme that broke the bacteria down, nor an antiseptic that killed them; rather, it interfered with the process of cell division.[91][92] Jennings observed that it had no effect on white blood cells, and would therefore reinforce rather than hinder the body's natural defences against bacteria. She also found that unlike sulphonamides, it was not destroyed by pus. Medawar found that it did not affect the growth of tissue cells.[93]

Thousands of glass fermentation vessels like this one were used in Glaxo (now GlaxoSmithKline) laboratories to produce penicillin. The mould was grown on the surface of a liquid filled with nutrients. The stopper kept contaminants out while allowing the mould to breathe.

By March 1940 the Oxford team had sufficient impure penicillin to commence testing whether it was toxic. Over the next two months, Florey and Jennings conducted a series of experiments on rats, mice, rabbits and cats in which penicillin was administered in various ways. Their results showed that penicillin was destroyed in the stomach, but that all forms of injection were effective, as indicated by assay of the blood. It was found that penicillin was largely and rapidly excreted unchanged in their urine.[94] They found no evidence of toxicity in any of their animals. Had they tested against guinea pigs research might have halted at this point, for penicillin is toxic to guinea pigs.[95]

At 11:00 am on Saturday 25 May 1940, Florey injected eight mice with a virulent strain of streptococcus, and then injected four of them with the penicillin solution. These four were divided into two groups: two of them received 10 milligrams once, and the other two received 5 milligrams at regular intervals. By 3:30 am on Sunday all four of the untreated mice were dead. All of the treated ones were still alive, although one died two days later.[96][97] Florey described the result to Jennings as "a miracle."[98]

Jennings and Florey repeated the experiment on Monday with ten mice; this time, all six of the treated mice survived, as did one of the four controls. On Tuesday, they repeated it with sixteen mice, administering different does of penicillin. All six of the control mice died within 24 hours but the treated mice survived for several days, although they were all dead in nineteen days.[97] On 1 July, the experiment was performed with fifty mice, half of whom received penicillin. All fifty of the control mice died within sixteen hours while all but one of the treated mice were alive ten days later. Over the following weeks they performed experiments with batches of 50 or 75 mice, but using different bacteria. They found that penicillin was also effective against Staphylococcus and gas gangrene.[99] Florey reminded his staff that promising as their results were, a man weighed 3,000 times as much as a mouse.[100]

The Oxford team reported their results in the 24 August 1940 issue of The Lancet as "Penicillin as a Chemotherapeutic Agent" with names of the seven joint authors listed alphabetically. They concluded:

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.[96]

The publication of their results attracted little attention; Florey would spend much of the next two years attempting to convince people of its significance. One reader was Fleming, who paid them a visit on 2 September 1940. Florey and Chain gave him a tour of the production, extraction and testing laboratories, but he made no comment and did not even congratulate them on the work they had done. Some members of the Oxford team suspected that he was trying to claim some credit for it.[101][102]

Unbeknown to the Oxford team, their Lancet article was read by Martin Henry Dawson, Gladys Hobby and Karl Meyer at Columbia University, and they were inspired to replicate the Oxford team's results. They obtained a culture of penicillium mould from Roger Reid at Johns Hopkins Hospital, grown from a sample he had received from Fleming in 1935. They began growing the mould on 23 September, and on 30 September tested it against viridans streptococci, and confirmed the Oxford team's results. Meyer duplicated Chain's processes, and they obtained a small quantity of penicillin. On 15 October 1940, doses of penicillin were administered to two patients at the Presbyterian Hospital in New York City, Aaron Alston and Charles Aronson. They became the first persons to receive penicillin treatment in the United States.[103][104] The Columbia team presented the results of their penicillin treatment of four patients at the annual meeting of the American Society for Clinical Investigation in Atlantic City, New Jersey, on 5 May 1941. Their paper was reported in by William L. Laurence in The New York Times and generated great public interest.[104][105][106]

A laboratory worker sprays a solution containing penicillin mould into flasks of corn steep liquor medium, to encourage further penicillin growth.

At Oxford, Charles Fletcher volunteered to find test cases for human trials. Elva Akers, an Oxford woman dying from incurable cancer, agreed to be a test subject for the toxicity of penicillin. On 17 January 1941, he intravenously injected her with 100 mg of penicillin. Her temperature briefly rose, but otherwise she had no ill-effects. Florey reckoned that the fever was caused by pyrogens in the penicillin; these were removed with improved chromatography.[107] Fletcher next identified an Oxford policeman, Albert Alexander, who had had a small sore at the corner of his mouth, which then spread, leading to a severe facial infection involving streptococci and staphylococci. His whole face, eyes and scalp were swollen to the extent that he had had an eye removed to relieve the pain.[107][108]

On 12 February, Fletcher administered 200 mg of penicillin, following by 100 mg doses every three hours. Within a day of being given penicillin, Alexander started to recover; his temperature dropped and discharge from his suppurating wounds declined. By 17 February, his right eye had become normal. However, the researchers did not have enough penicillin to help him to a full recovery. Penicillin was recovered from his urine, but it was not enough. In early March he relapsed, and he died on 15 March. Because of this experience and the difficulty in producing penicillin, Florey changed the focus to treating children, who could be treated with smaller quantities of penicillin.[107][108]

Subsequently, several patients were treated successfully. The second was Arthur Jones, a 15-year-old boy with a streptococcal infection from a hip operation. He was given 100 mg every three hours for five days and recovered. Percy Hawkin, a 42-year-old labourer, had a 4-inch (100 mm) carbuncle on his back. He was given an initial 200 mg on 3 May followed by 100 mg every hour. The carbuncle completely disappeared. John Cox, a semi-comatose 4-year-old boy was treated starting on 16 May. He died on 31 May but the post-mortem indicated this was from a ruptured artery in the brain weakened by the disease, and there was no sign of infection. The fifth case, on 16 June, was a 14-year-old boy with an infection from a hip operation who made a full recovery.[109]

In addition to increased production at the Dunn School, commercial production from a pilot plant established by Imperial Chemical Industries became available in January 1942, and Kembel, Bishop and Company delivered its first batch of 200 imperial gallons (910 L) on 11 September. Florey decided that the time was ripe to conduct a second series of clinical trials. Ethel was placed in charge, but while Florey was a consulting pathologist at Oxford hospitals and therefore entitled to use their wards and services, Ethel, to his annoyance, was accredited merely as his assistant. Doctors tended to refer patients to the trial who were in desperate circumstances rather than the most suitable, but when penicillin did succeed, confidence in its efficacy rose.[110] Ethel and Howard Florey published the results of clinical trials of 187 cases of treatment with penicillin in The Lancet on 27 March 1943.[111]

Ethel and Howard Florey published the results of clinical trials of penicillin in The Lancet on 27 March 1943, reporting the treatment of 187 cases of sepsis with penicillin.[112] It was upon 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.[113] This led to mass production of penicillin by the next year.[114]

Knowing that large-scale production for medical use was futile in a confined laboratory, the Oxford team tried to convince the war-torn British government and private companies for mass production, but the initial response was muted. In April 1941, Warren Weaver met with Florey, and they discussed the difficulty of producing sufficient penicillin to conduct clinical trails. Weaver arranged for the Rockefeller Foundation to fund a three-month visit to the United States for Florey and a colleague to explore the possibility of production of penicillin there.[115] Florey and Heatley left for the United States by air on 27 June 1941.[116] Knowing that mould samples kept in vials could be easily lost, they smeared their coat pockets with the mould.[92]

Mary Hunt, believed to be on the left, with a fellow worker inside the USDA Northern Regional Research Laboratory, c. 1943

Florey met with John Fulton, who introduced him to Ross Harrison, the Chairman of the National Research Council (NRC). Harrison referred Florey to Thom, the chief mycologist at the Bureau of Plant Industry of the United States Department of Agriculture (UDSDA) in Beltsville, Maryland, and the man who had identified the mould reported by Fleming. On 9 July, Thom took Florey and Heatley to Washington, D.C., to meet Percy Wells, the acting assistant chief of the USDA Bureau of Agricultural and Industrial Chemistry and as such the head of the USDA's four laboratories. Wells sent an introductory telegram to Orville May, the director of the UDSA's Northern Regional Research Laboratory (NRRL) in Peoria, Illinois. They met with May on 14 July, and he arranged for them to meet Robert D. Coghill, the chief of the NRRL's fermentation division, who raised the possibility that fermentation in large vessels might be the key to large-scale production.[117][118][119]

On 17 August, Florey met with Alfred Newton Richards, the chairman of the Committee for Medical Research (CMR) of the Office of Scientific Research and Development (OSRD), who promised his support.[120] On 8 October, Richards held a meeting with representatives of four major pharmaceutical companies: Squibb, Merck, Pfizer and Lederle. Vannevar Bush, the director of OSRD was present, as was Thom, who represented the NRRL. Richards told them that antitrust laws would be suspended, allowing them to share information about penicillin. This was not legalized until 7 December 1943, and it covered only penicillin and no other drug.[121][122] OSRD arranged with the War Production Board (WPB) for them to have priority for equipment for laboratories and pilot plants.[123]

The USDA's penicillin research team. Back row, left to right: Dorothy Fennell Alexander, H. T. Herrick, F. H. Stodola, Kenneth B. Raper, Robert Coghill, George Ward and Andrew J. Moyer

Coghill made Andrew J. Moyer available to work on penicillin with Heatley, while Florey left to see if he could arrange for a pharmaceutical company to manufacture penicillin. As a first step to increasing yield, Moyer replaced sucrose in the growth media with lactose. An even larger increase occurred when Moyer added corn steep liquor, a byproduct of the corn industry that the NRRL routinely tried in the hope of finding more uses for it. The effect on penicillin was dramatic; Heatley and Moyer found that it increased the yield tenfold.[116]

At the Yale New Haven Hospital in March 1942, Anne Sheafe Miller, the wife of Yale University's athletics director, Ogden D. Miller, was losing a battle against streptococcal septicaemia contracted after a miscarriage. But her doctor, John Bumstead, was also treating John Fulton at the time. He knew that Fulton knew Florey, and that Florey's children were staying with him. He went to Fulton to plead for some penicillin. Florey had returned to the UK, but Heatley was still in the United States, working with Merck. A phone call to Richards released 5.5 grams of penicillin earmarked for a clinical trial, which was despatched from Washington, D. C., by air. The effect was dramatic; within 48 hours her 106 °F (41 °C) fever had abated and she was eating again. Her blood culture count had dropped 100 to 150 bacteria colonies per millilitre to just one. Bumstead suggested reducing the penicillin dose from 200 milligrams; Heatley told him not to. Heatley subsequently came to New Haven, where he collected her urine; about 3 grams of penicillin was recovered. Miller made a full recovery, and lived until 1999.[124][125][126]

A notebook page signed by Dorothy Fennell Alexander, an NRRL "penicillin survey" team member who worked to identify Penicillium mold strains that produced high amounts of penicillin under submerged culture conditions. The circled strain, PS46 (later known as NRRL 1951), was the top performer.

Until May 1943, almost all penicillin was produced using the shallow pan method pioneered by the Oxford team,[127] but NRRL mycologist Kenneth Bryan Raper experimented with deep vessel production. The initial results were disappointing; penicillin cultured in this manner yielded only three to four Oxford units per cubic centimetre, compared to twenty for surface cultures.[128] He got the help of U.S. Army's Air Transport Command to search for similar mould in different parts of the world. The best moulds were found to be those from Chungking, Bombay, and Cape Town. But the single-best sample was from a cantaloupe sold in a Peoria fruit market in 1943. The mould was identified as Penicillium chrysogenum and designated as NRRL 1951 or cantaloupe strain.[119][129] The spores may have escaped from the NRRL.[130] On 17 August 2021, Illinois Governor J. B. Pritzker signed a bill designating it as the official State Microbe of Illinois.[131] There is a popular story that Mary K. Hunt (or Mary Hunt Stevens),[132] a staff member of Raper's, collected the mould;[133] for which she had been popularised as "Mouldy Mary".[134][131] 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.[119]

Between 1941 and 1943, Moyer, Coghill and Kenneth Raper developed methods for industrialized penicillin production and isolated higher-yielding strains of the Penicillium fungus.[135] To improve upon that strain, researchers at the Carnegie Institution of Washington subjected NRRL 1951 to X-rays to produce mutant strain designated X-1612 that produced 300 per millilitre, twice as much as NRRL 1951. In turn, researchers at the University of Wisconsin used ultraviolet radiation to on X-1612 to produce a strain designated Q-176. This produced more than twice the penicillin that X-1612 produced, but in the form of the less desirable penicillin K. Phenylacetic acid was added to switch it to producing the highly potent penicillin G. This strain could produce up to 550 milligrams per litre.[136][137][129]

A 1957 fermentor used to grow Penicillium mould in the Science Museum, London

Pfizer was a small Brooklyn company that specialised in making citric acid, but it had experience in deep fermentation techniques. Its vice president, John L. Smith, whose daughter had died from an infection, put all of Pfizer's resources into the development of a practical deep submergence technique.[138] The company invested $2.98 million of its own money in penicillin in 1943 and 1944. (equivalent to $46 million in 2021) Pfizer scientists Jasper H. Kane, G. M. Shull, E. M. Weber, A. C. Finlay and E. J. Ratajak worked on the fermentation process while R. Pasternak, W. J. Smith, V. Bogert and P. Regna developed extraction techniques.[139]

Now that they 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 of the corn steep. The foaming problem was solved by the introduction of an anti-foaming agent, glyceryl monoricinoleate. The technique also involved cooling and mixing.[140]

Pfizer opened a pilot plant with a 2,000 US gallons (7,600 L) fermentor in August 1943 and Ratajak delivered the first penicillin liquor from it on 27 August. The one tank was soon producing half the company's output. Smith then decided to construct a full-scale production plant. The nearby Rubel Ice plant was acquired on 20 September 1943 and converted into the first deep submergence production plant, with fourteen 34,000 US gallons (130,000 L) tanks. The work was carried out in five months under the leadership of John E. McKeen and Edward J. Goett, and the plant opened on 1 March 1944.[138][139][141]

During the campaign in Western Europe in 1944–1945, penicillin was widely used both to treat infected wounds and as a prophylactic to prevent wounds from becoming infected. Gas gangrene had killed 150 out of every 1,000 casualties in the First World War, but the instance of this disease now disappeared almost completely. Open fractures now had a recovery rate of better than 94 per cent, and recovery from burns of one-fifth of the body or less was 100 per cent.[173]

1945 molecular model of Penicillin by Dorothy Hodgkin

The chemical structure of penicillin was first proposed by Abraham in 1942.[174] Dorothy Hodgkin determined the correct chemical structure of penicillin using X-ray crystallography at Oxford in 1945.[175][176][177][178][57] 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.[179] The penicillins were given various names such as using Roman numerals in UK (such as penicillin I, II, III) in order their discoveries and letters (such as F, G, K, and X) referring to their origins or sources, as below:

The chemical names were based on the side chains of the compounds. To avoid the controversial names, Chain introduced in 1948 the chemical names as standard nomenclature, remarking as: "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."[180]

In Kundl, Tyrol, Austria, in 1952, Hans Margreiter and Ernst Brandl of Biochemie (now Sandoz) developed the first acid-stable penicillin for oral administration, penicillin V.[181] American chemist John C. Sheehan at the Massachusetts Institute of Technology (MIT) completed the first chemical synthesis of penicillin in 1957.[182][183][184] 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.[184][185] 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.[186][187]

An important development was the discovery of 6-APA itself. In 1957, researchers at the Beecham Research Laboratories (now the Beechem 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.[188][189] The discovery was published Nature in 1959.[190] This paved the way for new and improved drugs as all semi-synthetic penicillins are produced from chemical manipulation of 6-APA.[191]

The second-generation semi-synthetic β-lactam antibiotic methicillin, designed to counter first-generation-resistant penicillinases, was introduced in the United Kingdom in 1959. Methicillin-resistant forms of S. aureus likely already existed at the time.[178][192]

Penicillin patents became a matter of concern and conflict. Chain had wanted to apply for a patent but Florey had objected, arguing that penicillin should benefit all.[193] He sought the advice of Sir Henry Hallett Dale (Chairman of the Wellcome Trust and member of the Scientific Advisory Panel to the Cabinet of British government) and John William Trevan (Director of the Wellcome Trust Research Laboratory). On 26 and 27 March 1941, Dale and Trevan met at Sir William Dunn School of Pathology to discuss the issue. Dale specifically advised that patenting penicillin would be unethical. Undeterred, Chain approached Sir Edward Mellanby, then Secretary of the Medical Research Council, who also objected on ethical grounds. As Chain later admitted, he had "many bitter fights" with Mellanby,[194][195] but Mellanby's decision was accepted as final.[196]

In 1945, Moyer patented the methods for production and isolation of penicillin.[197][198][199] He could not obtain patents in the US as an employee of the NRRL, but filed four patent at the British Patent Office (now the Intellectual Property Office). He gave the license to a US company, Commercial Solvents Corporation.[200] Although completely legal, Coghill felt it was an injustice for outsiders to have the royalties for the "British discovery." A year later, Moyer asked Coghill for permission to file another patent based on the use of phenylacetic acid that increased penicillin production by 66%, but as the principal researcher, Coghill refused.

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?[201]

The patenting of penicillin-related technologies by US companies gave rise to a myth in the UK that British scientists had done the work but American ones garnered the rewards.[200] When the Rockefeller Foundation published its annual report in 1944, The Evening News contrasted its generous support of the Oxford team's work with that of the parsimonious MRC.[202][203] In April 1945, the British firm Glaxo signed agreements with Squibb and Merck under which it paid 5 per cent royalties on its sales of penicillin for five years in return for the use of their deep submergence fermentation techniques. Glaxo paid almost £0.5 million (equivalent to 13 million in 2021) in royalties between 1946 and 1956.[196][200] The controversy over patents led to the establishment of the UK National Research Development Corporation (NRDC) in June 1948.[204]

After the news about the curative properties of penicillin broke, Fleming revelled in the publicity, but Florey did not. When the press arrived at the Sir Willim Dunn School, he told his secretary to send them packing.[205][206] Journalists could hardly be blamed for preferring being fibbed to by Fleming to being fobbed off by Florey,[207] but there was a larger issue: the story they wished to tell was the familiar one of the lone scientist and the serendiptous discovery. British medical historian Bill Bynum wrote:

The discovery and development of penicillin is an object lesson of modernity: the contrast between an alert individual (Fleming) making an isolated observation and the exploitation of the observation through teamwork and the scientific division of labour (Florey and his group). The discovery was old science, but the drug itself required new ways of doing science.[208]

In 1943, the Nobel committee received a single nomination for the Nobel Prize in Physiology or Medicine for Fleming and Florey from Rudolph Peters. The secretary of the Nobel committee, Göran Liljestrand made an assessment of Fleming and Florey in 1943, but little was known about penicillin in Sweden at the time, and he concluded that more information was required. The following year there was one nomination for Fleming alone and one for Fleming, Florey and Chain. Liljestrand and Nanna Svartz considered their work, and while both judged Fleming and Florey equally worthy of a Nobel Prize, the Nobel committee was divided, and decided to award the prize that year to Joseph Erlanger and Herbert S. Gasser instead. There was an avalanche of nominations for Florey and Fleming or both in 1945, and one for Chain, from Liljestrand, who nominated all three. Liljestrand noted that 13 of the 16 nominations that came in mentioned Fleming, but only three mentioned him alone. This time evaluations were made by Liljestrand, Sven Hellerström and Anders Kristenson, who endorsed all three.[209][210][211][212][213]

There were rumours that the committee would award the prize to Fleming alone, or half to Fleming and one-quarter each to Florey and Chain. Fulton and Sir Henry Dale lobbied for the award to be given to Florey.[210] The Nobel Assembly at the Karolinska Institute did consider awarding half to Fleming and one-quarter each to Florey and Chain, but in the end decided to divide it equally three ways.[209] On 25 October 1945, it announced that 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."[214][215] When The New York Times announced that "Fleming and Two Co-Workers" had won the prize, Fulton demanded – and received – a correction in an editorial the next day.[216][217][218]

Dorothy Hodgkin received the 1964 Nobel Prize in Chemistry "for her determinations by X-ray techniques of the structures of important biochemical substances."[219] She became only the third woman to receive the Nobel Prize in Chemistry after Marie Curie in 1911 and Irène Joliot-Curie in 1935.[219]

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.[220] It was more advantageous than the original penicillin as it offered a broader spectrum of activity against Gram-positive and Gram-negative bacteria.[220] 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 (MRSA) strains that subsequently emerged.[221]

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 center of their structures.[189][222]

The penicillins related β-lactams have become the most widely used antibiotics in the world.[223] Amoxicillin, a semisynthetic penicillin developed by Beecham Research Laboratories in 1970,[224][225] is the most commonly used of all.[226][227] Antibiotic preferences differed from country to country: in Europe, amoxicillin was widely used in the UK and Germany; France, Italy and Spain preferred broad-spectrum combinations like co-amoxiclav; and the Scandinavian countries relied on narrow-spectrum penicillin V.[228]

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.[178][57][229] Chain and Abraham worked out the chemical nature of penicillinase which they reported in Nature as:

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.[230]

In his Nobel lecture, Fleming warned of the possibility of penicillin resistance in clinical conditions:

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.[231]

At the time, only poisons required a doctor's prescription, and this represented a real possibility. Legislation was passed in the UK in 1947 to require a prescription for antibiotics. The United States followed in 1951.[232] Elsewhere in the world, the export of Western pharmaceuticals diffused faster than Western medical knowledge and practices, and penicillin was often dispensed by practitioners of traditional medicine.[233]

As late as 1999, a survey in the UK found that 39 per cent of respondents believed that antibiotics could cure colds and flus, and 12 per cent believed that they were the best treatment for them. The widespread belief that antibiotics could cure all ailments had serious consequences. It reduced the status of doctors to providers of pills. Many more people sought medical attention for ailments they would have ignored before, and they often demanded antibiotics. For their part, overworked doctors were increasingly willing to provide them even if not asked to do so.[234]

By 1942, some strains of Staphylococcus aureus had developed a strong resistance to penicillin and many strains were resistant to penicillin by the 1960s.[235] In 1946, bacteriologist Mary Barber began a study of penicillin resistance through natural selection at Hammersmith Hospital in London. She found that in 1946, seven out of eight bacterial infections were susceptible to penicillin, but two years later only three out of eight were. Nurses were exposed to both bacteria and penicillin and haboured and transmitted bacterial infections. Miller found that three out of ten student midwives were colonized by bacteria when they arrived; after three months, seven out of ten were. The problem was sloppy hygiene practices by health care workers, poor medical practices like prophylactic use of antibiotics, and slipshod administrative practices, such as taking babies from their mothers to large hospital nurseries.[236]

Antibiotic-resistant infections were reported in Australia in 1952.[236] During the 1957–1958 influenza pandemic there were 16,000 deaths in the UK and 80,000 in US from bacterial complications; 28 per cent of those who contracted pneumonia died. Most cases of pneumonia were contracted in hospitals, and many of these were antibiotic resistant strains that had been nurtured there.[237] In 1965, the first case of penicillin resistance in Streptococcus pneumoniae was reported from Boston.[238][239] Since then other strains and many other species of bacteria have now developed resistance.[240]

Research conducted by the American Cyanamid laboratories in the late 1940s and early 1950s demonstrated that adding penicillin to chicks' feed increased their weight gain by 10 per cent. The reasons for this were still subject to debate in the twenty-first century. Subsequent research indicated that adding penicillin to animal feed also improved feed-conversion efficiency, promoted more uniform growth and facilitated disease control. After the Food and Drug Administration improved the use of penicillin as feed additives for poultry and livestock in 1951, the pharmaceutical companies ramped up production to meet the demand.[241]

By 1954, the United States was producing 2 million pounds (910 t) of antibiotics each year, of which 490,000 pounds (220 t) was going into animal feed; in the 1990s, the United States was producing 50 million pounds (23,000 t) of antibiotics per year, of which half was going to livestock. The largest user remained the poultry industry, which consumed 10.5 million pounds (4,800 t) of antibiotics each year, compared to 10.3 million pounds (4,700 t) for hogs and 3.7 million pounds (1,700 t) for cattle. It was estimated in 1981 that banning their use in animal feed could cost American consumers up to $3.5 billion a year in increased food prices.[241] The story was similar in the UK, where 44 per cent of antibiotic production was consumed by animals by 1963.[242]

By the mid-1950s, there were reports in the United States that milk was not curdling to make cheese. The FDA found that the milk was contaminated with penicillin. In 1963 the World Health Organization reported high levels of penicillin in milk worldwide. People who were allergic to penicillin could now get a reaction from drinking milk.[243] A committee chaired by Lord Netherthorpe was established in the UK to inquire into the use of antibiotics in animal feed. The committee recommended that restrictions on the use of antibiotics in animals be relaxed. It contended that the benefits were substantial and that even if bacteria became resistant, new antibiotics would soon be developed, and there was no evidence that bacterial resistance in animals impacted human health.[244][245]

The Netherthorpe committee's conclusions were undermined by new research even before they were published, and the committee was recalled in 1965.[246] In 1967, a multiresistant strain of E. coli caused the deaths of fifteen children in the UK. The use of antibiotics in animals for nontherapeutic use was banned in the UK in 1971. Many other European countries soon followed.[247] When Sweden acceded to the European Union (EU) in 1995, there had been a total ban on antibiotic growth promoters (AGPs) in place there for ten years. This would be superseded by more relaxed EU rules unless Sweden could demonstrate scientific evidence in favour of a ban. Two Swedish and two Danish took up the fight. The odds seemed against them but this coincided with the United Kingdom BSE outbreak, which resulted in intense political pressure. In December 1996, the European Parliament's Standing Committee on Health and Welfare voted to ban the use of AGPs. The EU went further and recommended broad restrictions on the use of antibiotics.[248]

This article was submitted to WikiJournal of Medicine for external academic peer review in 2021 (reviewer reports). The updated content was reintegrated into the Wikipedia page under a CC-BY-SA-3.0 license (2021). The version of record as reviewed is: Kholhring Lalchhandama; et al. (22 October 2021), "History of penicillin" (PDF), WikiJournal of Medicine, 8 (1): 3, doi:10.15347/WJM/2021.003, ISSN 2002-4436, Wikidata Q107303937

• Debate in the House of Commons on the history and the future of the discovery.