History of penicillin

The history of penicillin follows a number of observations and discoveries of apparent evidence of antibiotic activity in moulds before the modern isolation of the chemical penicillin in 1928. 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 molds.[1] However, it is unknown if the species involved were Penicillium species or if the antimicrobial substances produced were penicillin.

The core structure of penicillin, where R is a variable group
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.

The Scottish physician Alexander Fleming was the first to suggest that a Penicillium mould must secrete an antibacterial substance, and the first to concentrate the active substance involved, which he named penicillin, the first modern antibiotic, in 1928.[2][3] During the next twelve years Fleming grew, distributed and studied the original mold, which was determined to be a rare variant of Penicillium notatum (now Penicillium rubens).[4]

Many later scientists were involved in the stabilization and mass production of penicillin and in the search for more productive strains of Penicillium.[5] Important contributors include Ernst Chain, Howard Florey, Norman Heatley and Edward Abraham.[2] Shortly after the discovery of penicillin, scientists found that some disease-causing pathogens display antibiotic resistance to penicillin. Research that aims to develop more effective strains and to study the causes and mechanisms of antibiotic resistance continues today.[6][7]

Early history

Many ancient cultures, including those in Egypt, Greece and India, independently discovered the useful properties of fungi and plants in treating infection.[8] 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.

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 mold as a form of medical treatment was recorded by apothecaries such as John Parkinson, King's Herbarian, who advocated the use of mold in his book on pharmacology. [9]

Early scientific evidence
NB
In the early stages of penicillin research, most species of Penicillium were generally referred to as Penicillium glaucum, so we cannot identify the actual strains used. Thus, it is difficult to tell whether it was really penicillin preventing bacterial growth.[10]

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 mold 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 mold also did not permit the growth of bacteria. Lister also described the antibacterial action on human tissue of a species of mold he called Penicillium glaucum.[11] A nurse at King's College Hospital whose wounds did not respond to any traditional antiseptic was then given another substance that cured her, and Lister's registrar informed her 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 Penicillium 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.[12]

By this time, Bacillus anthracis had been shown to cause anthrax, the first demonstration that a specific bacterium caused a specific disease. In 1877, French biologists Louis Pasteur and Jules Francois Joubert observed that cultures of the anthrax bacilli, when contaminated with molds, could be successfully inhibited. Some references say 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 mold.[13] In 1887, Garré found similar results. In 1895, Vincenzo Tiberio, an Italian physician at the University of Naples, published research about molds initially found in a water well in Arzano; from his observations, he concluded that these molds contained soluble substances having antibacterial action.[14][15][16][17]

Two years later, Ernest Duchesne at École du Service de Santé Militaire in Lyon independently discovered the healing properties of a Penicillium glaucum mold, even curing infected guinea pigs of typhoid. He published a dissertation[18][19][20] in 1897 but it was ignored by the Institut Pasteur. Duchesne was himself using a discovery made earlier by Arab stable boys, who used molds to cure sores on horses. He did not claim that the mold contained any antibacterial substance, only that the mold somehow protected the animals. The penicillin isolated by Fleming does not cure typhoid and so it remains unknown which substance might have been responsible for Duchesne's cure.[lower-alpha 1]

In Belgium in 1920, Andre Gratia and Sara Dath observed a fungal contamination in one of their Staphylococcus aureus cultures that was inhibiting the growth of the bacterium. They identified the fungus as a species of Penicillium and presented their observations as a paper, but it received little attention. An Institut Pasteur scientist, Costa Rican Clodomiro Picado Twight, similarly recorded the antibiotic effect of Penicillium in 1923.

The breakthrough discovery

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

Penicillin 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.[21] 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 a pus of 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.[22]

Fleming and his research scholar Daniel Merlin Pryce pursued this experiment but Price was transferred to another laboratory in the early 1928. After a few months of working alone, Fleming was joined by a new scholar Stuart Craddock. The 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.[21]

Initial discovery

In August, Fleming spent a vacation with his family at his country home The Dhoon at Barton Mills, Suffolk. Before leaving his laboratory, 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 in a 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.[23] 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 had bacteria around the mould did not grow, while those farther away grew normally, meaning that the mould killed the bacteria.[24] Fleming commented as he watched the plate: "That's funny".[23][25] Pryce remarked to Fleming: "That’s how you discovered lysozyme."[26]

Experiment
St Mary's Hospital showing Fleming's lab and Praed Street

Fleming went off to resume his vacation and returned for the experiments late in September.[21] 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.[27]

He concluded that the mold was releasing a substance that was inhibiting bacterial growth, he produced culture broth of the mold and subsequently concentrated the antibacterial component.[28] 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 large-culture method from which he could obtained large amounts of the mould juice. He called this juice "penicillin", as he explained the reason as "to avoid the repetition of the rather cumbersome phrase 'Mould broth filtrate,' the name 'penicillin' will be used."[29] He invented the name on 7 March 1929.[23] 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.[30]

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."[21] In January 1929, he recruited Frederick Ridley, his former research scholar who had studied biochemistry, specifically for the chemical properties of the mould.[25] But they could not isolate penicillin and before the experiments were over, they both left Fleming's lab for other jobs.[23] It was due to their failure to isolate the compound that Fleming practically abandoned further research on the chemical aspects of penicillin,[31] although he did biological tests up to 1939.[23]

Identification of the mould
Penicillium rubens (type specimen)

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,[32][33] the identification used be 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."[34] 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.[35] In addition to P. notatum, newly discovered species such as P. meleagrinum and P. cyaneofulvum were recognised as members of P. chrysogenum in 1977.[36] 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).[37] Whole genome sequence and phylogenetic analysis in 2011 revealed that Fleming's mould belongs to P. rubens, a species described by French microbiologist Philibert Melchior Joseph Ehi Biourge in 1923, and also that P. chrysogenum is a different species.[38][39]

The source of the fungal contamination in Fleming's experiment remained a speculation for several decades. 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 literature,[40] starting with George Lacken's 1945 book The Story of Penicillin.[23] 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.[41] 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 apparatuses placed in front of it. In 1966, La Touche told Hare that he had given Fleming 13 specimen of fungi (10 from his lab) and only one from his lab was showing penicillin-like antibacterial activity.[40] 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 being drifted in the air through the open doors.[42]

Reception and publication

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.[21]

In 1929, Fleming reported his findings to the British Journal of Experimental Pathology on 10 May 1929, and was published in the next month issue.[29][43] It failed to attract any serious attention. Fleming himself was quite unsure of the medical application and was more concerned on 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.[29]  

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."[21] 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."[44][45][lower-alpha 2]

First medical use

During the next twelve years, Fleming grew and distributed the original mould. He was unsuccessful in making a stable form of it for mass production.[46] Although Fleming did some research with penicillin directly on patients and greatly contributed to its medical use, he did not realize its revolutionary potential because of the impurity of the penicillin that he made and the difficulty in producing it in mass. Most of his further research with penicillin was focused mostly on the properties of penicillin rather than medical treatment with the drug.[28]

Cecil George Paine, a pathologist at the Royal Infirmary in Sheffield, was the first to use penicillin for medical treatment. He initially attempted to treat sycosis (eruptions in beard follicles) with penicillin but was unsuccessful, probably because the drug did not penetrate deep 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.[47]

In 1940, Australian scientist Howard Florey (later Baron Florey) and a team of researchers (Ernst Boris Chain, Edward Abraham, Arthur Duncan Gardner, Norman Heatley, Margaret Jennings, J. Orr-Ewing and G. Sanders) at the Sir William Dunn School of Pathology, University of Oxford made progress in isolating the chemical compound and experimenting for medical usage.[48][49] They showed that penicillin effectively cured bacterial infection in mice.[50][51] In 1941, they treated a policeman, Albert Alexander, with a severe face infection; his condition improved, but then supplies of penicillin ran out and he died. Subsequently, several other patients were treated successfully.[52] In December 1942, survivors of the Cocoanut Grove fire in Boston were the first burn patients to be successfully treated with penicillin.[53]

Isolation and mass production

The Oxford team were the first to isolate penicillin as "a brown powder" that "has been obtained [from Penicillium notatum culture broth] which is freely soluble in water"; and that this powder was effective in vitro and in vivo against bacteria. They published their findings in 24 August 1940 issue of The Lancet.[54] Chain and Abraham worked out the chemical nature of penicillin in December 1940, 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.[55]

As an enzyme, they gave a new name "penicillinase".[56] 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 yellow concentrate from the mould extract.[57] But they were able to produce only small quantities. In 1942, Chain, Abraham and E.R. Holiday produced the pure compound.[58]

Knowing that large-scale production for medical use was futile in England, Florey and Heatley travelled to the US in 1941 to persuade pharmaceutical companies for funding mass production.[59][60] Between 1941 and 1943, Moyer, Coghill and Raper at the USDA Northern Regional Research Laboratory (NRRL) in Peoria, Illinois, United States, developed methods for industrialized penicillin production and isolated higher-yielding strains of the Penicillium fungus.[61] In December 1942, survivors of the Cocoanut Grove fire in Boston were the first burn patients to be successfully treated with penicillin.[53] 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.[62]

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

When production first began, one-liter 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. Orvill May, the 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 mold. Shorty after beginning, Andrew 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.[59]

One major issue with the process that scientists faced was the inefficiency of growing the mold on the surface of their nutrient baths, rather than having it submerged. Even though a submerged process of growing the mold 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 a moldy cantaloupe acquired from a Peoria farmers' market.[63] To improve upon that strain, researchers subjected it to X-rays to facilitate mutations in its genome and managed to increase production capabilities even more.[64][63]

Now that scientists had a mold that grew well submerged and produced an acceptable amount of penicillin, the next challenge was to provide the required air to the mold 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.[65]

Structure determination

The chemical structure of penicillin was first proposed by Edward Abraham in 1942.[66] Dorothy Hodgkin determined the correct chemical structure of penicillin using X-ray crystallography at Oxford in 1945.[67][68][69][6] 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.[70] American chemist John C. Sheehan of the Massachusetts Institute of Technology completed the first chemical synthesis of penicillin in 1957.[71] 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 Staphylococcus aureus likely already existed at the time.[6][72]

American chemist John C. Sheehan at the Massachusetts Institute of Technology (MIT) completed the first chemical synthesis of penicillin in 1957.[73][74][75] 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.[75][76] 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.[77][78] Attaching different groups to the 6-APA 'nucleus' of penicillin allowed the creation of new forms of penicillin.[79][80]

Outcomes

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."[81]

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

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.[82] It was more advantageous than the original penicillin as it offered a broader spectrum of activity against Gram-positive and Gram-negative bacteria.[83] 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.[84]

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.[80][85]

Drug resistance

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.[6][7][86] 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.[87] In 1967, Streptococcus pneumoniae was also reported to be penicillin resistant. Many strains of bacteria have developed a resistance to penicillin.

Notes
  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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Further reading
  • Bud R (2007). Penicillin: Triumph and Tragedy. Oxford: Oxford University Press. ISBN 9780199254064.
  • Brown KW (2004). Penicillin man: Alexander Fleming and the antibiotic revolution. Scarborough, Ont: Sutton Pub. ISBN 978-0-7509-3152-6. (St Mary's Trust Archivist and Alexander Fleming Laboratory Museum Curator)
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