Penicillin

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For the Japanese band, see Penicillin (band).
Penicillin core structure, where "R" is the variable group.

Penicillin (sometimes abbreviated PCN or pen) is a group of antibiotics derived from Penicillium fungi,[1] including penicillin G (intravenous use), penicillin V (oral use), procaine penicillin, and benzathine penicillin (intramuscular use).

Penicillin antibiotics were among the first drugs to be effective against many previously serious diseases, such as bacterial infections caused by staphylococci and streptococci. Penicillins are still widely used today, though misuse has now made many types of bacteria resistant. All penicillins are β-lactam antibiotics and are used in the treatment of bacterial infections caused by susceptible, usually Gram-positive, organisms.

Several enhanced penicillin families also exist, effective against additional bacteria: these include the antistaphylococcal penicillins, aminopenicillins and the more-powerful antipseudomonal penicillins.

Medical uses[edit]

The term "penicillin" is often used generically to refer to benzylpenicillin (penicillin G), procaine benzylpenicillin (procaine penicillin), benzathine benzylpenicillin (benzathine penicillin), and phenoxymethylpenicillin (penicillin V). Procaine penicillin and benzathine penicillin have the same antibacterial activity as benzylpenicillin but act for a longer period of time. Phenoxymethylpenicillin is less active against gram-negative bacteria than benzylpenicillin.[2][3] Benzylpenicillin, procaine penicillin and benzathine penicillin are given by injection (parenterally), but phenoxymethylpenicillin is given orally.

Susceptibility[edit]

Despite the expanding number of penicillin resistant bacteria, penicillin can still be used to treat a wide range of infections caused by certain susceptible bacteria. Some of these bacteria include Streptococci, Staphylococci, Clostridium, and Listeria genera. The following list illustrates minimum inhibitory concentration susceptibility data for a few medically significant bacteria:[4][5]

  • Listeria monocytogenes: from less than or equal to 0.06 μg/ml to 0.25 μg/ml
  • Neisseria meningitidis: from less than or equal to 0.03 μg/ml to 0.5 μg/ml
  • Staphylococcus aureus: from less than or equal to 0.015 μg/ml to more than 32 μg/ml

Adverse effects[edit]

Common adverse drug reactions (≥ 1% of patients) associated with use of the penicillins include diarrhoea, hypersensitivity, nausea, rash, neurotoxicity, urticaria, and superinfection (including candidiasis). Infrequent adverse effects (0.1–1% of patients) include fever, vomiting, erythema, dermatitis, angioedema, seizures (especially in people with epilepsy), and pseudomembranous colitis.[6]

Mechanism of action[edit]

Bacteria that attempt to grow and divide in the presence of penicillin fail to do so, and instead end up shedding their cell walls.
Penicillin and other β-lactam antibiotics act by inhibiting penicillin-binding proteins, which normally catalyze cross-linking of bacterial cell walls.

Bacteria constantly remodel their peptidoglycan cell walls, simultaneously building and breaking down portions of the cell wall as they grow and divide. β-Lactam antibiotics inhibit the formation of peptidoglycan cross-links in the bacterial cell wall; this is achieved through binding of the four-membered β-lactam ring of penicillin to the enzyme DD-transpeptidase. As a consequence, DD-transpeptidase cannot catalyze formation of these cross-links, and an imbalance between cell wall production and degradation develops, causing the cell to rapidly die.

The enzymes that hydrolyze the peptidoglycan cross-links continue to function, even while those that form such cross-links do not. This weakens the cell wall of the bacterium, and osmotic pressure becomes increasingly uncompensated—eventually causing cell death (cytolysis). In addition, the build-up of peptidoglycan precursors triggers the activation of bacterial cell wall hydrolases and autolysins, which further digest the cell wall's peptidoglycans. The small size of the penicillins increases their potency, by allowing them to penetrate the entire depth of the cell wall. This is in contrast to the glycopeptide antibiotics vancomycin and teicoplanin, which are both much larger than the penicillins.[7]

Gram-positive bacteria are called protoplasts when they lose their cell walls. Gram-negative bacteria do not lose their cell walls completely and are called spheroplasts after treatment with penicillin.

Penicillin shows a synergistic effect with aminoglycosides, since the inhibition of peptidoglycan synthesis allows aminoglycosides to penetrate the bacterial cell wall more easily, allowing their disruption of bacterial protein synthesis within the cell. This results in a lowered MBC for susceptible organisms.

Penicillins, like other β-lactam antibiotics, block not only the division of bacteria, including cyanobacteria, but also the division of cyanelles, the photosynthetic organelles of the glaucophytes, and the division of chloroplasts of bryophytes. In contrast, they have no effect on the plastids of the highly developed vascular plants. This supports the endosymbiotic theory of the evolution of plastid division in land plants.[8]

The chemical structure of penicillin is triggered with a very precise, pH-dependent directed mechanism, effected by a unique spatial assembly of molecular components, which can activate by protonation. It can travel through bodily fluids, targeting and inactivating enzymes responsible for cell-wall synthesis in gram-positive bacteria, meanwhile avoiding the surrounding non-targets. Penicillin can protect itself from spontaneous hydrolysis in the body in its anionic form, while storing its potential as a strong acylating agent, activated only upon approach to the target transpeptidase enzyme and protonated in the active centre. This targeted protonation neutralizes the carboxylic acid moiety, which is weakening of the β-lactam ring N–C(=O) bond, resulting in a self-activation. Specific structural requirements are equated to constructing the perfect mouse trap for catching targeted prey.[9]

Structure[edit]

Chemical structure of Penicillin G. The sulfur and nitrogen of the five-membered thiazolidine ring are shown in yellow and blue respectively. The image shows that the thiazolidine ring and fused four-membered β-lactam are not in the same plane.

The term "penam" is used to describe the common core skeleton of a member of the penicillins. This core has the molecular formula R-C9H11N2O4S, where R is the variable side chain that differentiates the penicillins from one another. The penam core has a molecular weight of 243 g/mol, with larger penicillins having molecular weights near 450—for example, cloxacillin has a molecular weight of 436 g/mol. The key structural feature of the penicillins is the four-membered β-lactam ring; this structural moiety is essential for penicillin's antibacterial activity. The β-lactam ring is itself fused to a five-membered thiazolidine ring. The fusion of these two rings causes the β-lactam ring to be more reactive than monocyclic β-lactams because the two fused rings distort the β-lactam amide bond and therefore remove the resonance stabilisation normally found in these chemical bonds.[10]

Biosynthesis[edit]

Penicillin biosynthesis

Overall, there are three main and important steps to the biosynthesis of penicillin G (benzylpenicillin).

  • The first step is the condensation of three amino acids—L-α-aminoadipic acid, L-cysteine, L-valine into a tripeptide.[11][12][13] Before condensing into the tripeptide, the amino acid L-valine must undergo epimerization to become D-valine.[14][15] The condensed tripeptide is named δ-(L-α-aminoadipyl)-L-cysteine-D-valine (ACV). The condensation reaction and epimerization are both catalyzed by the enzyme δ-(L-α-aminoadipyl)-L-cysteine-D-valine synthetase (ACVS), a nonribosomal peptide synthetase or NRPS.
  • The second step in the biosynthesis of penicillin G is the oxidative conversion of linear ACV into the bicyclic intermediate isopenicillin N by isopenicillin N synthase (IPNS), which is encoded by the gene pcbC.[11][12] Isopenicillin N is a very weak intermediate, because it does not show strong antibiotic activity.[14]
  • The final step is a transamidation by isopenicillin N N-acyltransferase, in which the α-aminoadipyl side-chain of isopenicillin N is removed and exchanged for a phenylacetyl side-chain. This reaction is encoded by the gene penDE, which is unique in the process of obtaining penicillins.[11]

Production[edit]

Penicillin is a secondary metabolite of certain species of Penicillium and is produced when growth of the fungus is inhibited by stress. It is not produced during active growth. Production is also limited by feedback in the synthesis pathway of penicillin.

α-ketoglutarate + AcCoAhomocitrateL-α-aminoadipic acidL-lysine + β-lactam

The by-product, l-lysine, inhibits the production of homocitrate, so the presence of exogenous lysine should be avoided in penicillin production.

The Penicillium cells are grown using a technique called fed-batch culture, in which the cells are constantly subject to stress, which is required for induction of penicillin production. The available carbon sources are also important: Glucose inhibits penicillin production, whereas lactose does not. The pH and the levels of nitrogen, lysine, phosphate, and oxygen of the batches must also be carefully controlled.

The biotechnological method of directed evolution has been applied to produce by mutation a large number of Penicillium strains. These techniques include error-prone PCR, DNA shuffling, ITCHY, and strand-overlap PCR.

Semisynthetic penicillins are prepared starting from the penicillin nucleus 6-APA.

History[edit]

Discovery[edit]

Main article: History of penicillin
Alexander Fleming, who is credited with discovering penicillin in 1928.
Sample of penicillin mould presented by Alexander Fleming to Douglas Macleod, 1935

The discovery of penicillin is attributed to Scottish scientist and Nobel laureate Alexander Fleming in 1928.[16] He showed that, if Penicillium rubens[17] were grown in the appropriate substrate, it would exude a substance with antibiotic properties, which he dubbed penicillin. This serendipitous observation began the modern era of antibiotic discovery. The development of penicillin for use as a medicine is attributed to the Australian Nobel laureate Howard Walter Florey, together with the German Nobel laureate Ernst Chain and the English biochemist Norman Heatley.

There are studies that anticipated Fleming. The first published reference appears in the publication of the Royal Society in 1875, by John Tyndall.[18] Joaquim Monteiro Caminhoá, Professor of Botany and Zoology of the Faculty of Medicine of the Federal University of Rio de Janeiro in Brazil, also recognised the antibiotic activity of Penicillium and other fungi in 1877. In his book, Elements of General and Medical Botany (under a section titled "Useful fungi, harmful and curious"), he stated:

"The mould (Penicillium infestans, Penicillium glaucum, figure 1680, Ascophora and many others) is useful because it feeds on decaying organic matter and destroys putrifaction so that, as a rule, the odour of infection does not occur, or is produced in infinitely smaller amounts."[19]

In 1895, Vincenzo Tiberio, physician of the University of Naples published a research paper about a mold found in a water well that had an antibacterial action.[20][21][22] His findings were disregarded as coincidence. Ernest Duchesne documented it in an 1897 paper,[23] which was not accepted by the Institut Pasteur because of his youth. In March 2000, doctors at the San Juan de Dios Hospital in San José, Costa Rica, published the manuscripts of the Costa Rican scientist and medical doctor Clodomiro (Clorito) Picado Twight (1887–1944). They reported Picado's observations on the inhibitory actions of fungi of the genus Penicillium between 1915 and 1927. Picado reported his discovery to the Paris Academy of Sciences, yet did not patent it,[24] even though his investigations started years before Fleming's. Joseph Lister was experimenting with Penicillum in 1871 for his aseptic surgery. He found that it weakened the microbes, but then he dismissed the fungi.[citation needed]

These early investigations did not lead to the use of antibiotics to treat infection because they took place in obscure circumstances, and the idea that infections were caused by transmissible agents was not widely accepted at the time. Sterilization measures had been shown to limit the outbreak and spread of disease; however, the mechanism of transmission of disease by parasites, bacteria, viruses and other agents was unknown. In the late 19th century, knowledge was increasing of the mechanisms by which living organisms become infected, how they manage infection once it has begun and, most importantly in the case of penicillin, the effect that natural and man-made agents could have on the progress of infection.[citation needed]

Fleming recounted that the date of his discovery of penicillin was on the morning of Friday, September 28, 1928.[25] It was a fortuitous accident: in his laboratory in the basement of St. Mary's Hospital in London (now part of Imperial College), Fleming noticed a Petri dish containing Staphylococcus plate culture he mistakenly left open, was contaminated by blue-green mould, which formed a visible growth. There was a halo of inhibited bacterial growth around the mould. Fleming concluded the mould released a substance that repressed the growth and lysing the bacteria. He grew a pure culture and discovered it was a Penicillium mould, now known to be Penicillium notatum. Charles Thom, an American specialist working at the U.S. Department of Agriculture, was the acknowledged expert, and Fleming referred the matter to him. Fleming coined the term "penicillin" to describe the filtrate of a broth culture of the Penicillium mould. Even in these early stages, penicillin was found to be most effective against Gram-positive bacteria, and ineffective against Gram-negative organisms and fungi. He expressed initial optimism that penicillin would be a useful disinfectant, being highly potent with minimal toxicity compared to antiseptics of the day, and noted its laboratory value in the isolation of Bacillus influenzae (now Haemophilus influenzae).[26] After further experiments, Fleming was convinced penicillin could not last long enough in the human body to kill pathogenic bacteria, and stopped studying it after 1931. He restarted clinical trials in 1934, and continued to try to get someone to purify it until 1940.[27]

Medical application[edit]

Florey (pictured), Fleming and Chain shared a Nobel Prize in 1945 for their work on penicillin.

In 1930, Cecil George Paine, a pathologist at the Royal Infirmary in Sheffield, attempted to use penicillin to treat sycosis barbae, eruptions in beard follicles, but was unsuccessful, probably because the drug did not penetrate the skin deeply enough. Moving on to ophthalmia neonatorum, a gonococcal infection in infants, he achieved the first recorded cure with penicillin, on November 25, 1930. He then cured four additional patients (one adult and three infants) of eye infections, and failed to cure a fifth.[28]

In 1939, Australian scientist Howard Florey (later Baron Florey) and a team of researchers (Ernst Boris Chain, Arthur Duncan Gardner, Norman Heatley, M. Jennings, J. Orr-Ewing and G. Sanders) at the Sir William Dunn School of Pathology, University of Oxford made significant progress in showing the in vivo bactericidal action of penicillin. Their attempts to treat humans failed because of insufficient volumes of penicillin (the first patient treated was Reserve Constable Albert Alexander), but they proved it harmless and effective on mice.[29]

Some of the pioneering trials of penicillin took place at the Radcliffe Infirmary in Oxford, England. These trials continue to be cited by some sources as the first cures using penicillin, though the Paine trials took place earlier.[28] On March 14, 1942, John Bumstead and Orvan Hess saved a dying patient's life using penicillin.[30][31]

Survivors of November 28, 1942 Cocoanut Grove fire in Boston, which killed 492 people, were treated with penicillin. Merck and Company rushed a 32-liter supply of the drug, in the form of culture liquid in which the Penicillium mould had been grown, from New Jersey to Boston in early December. The drug was crucial in combating staphylococcus bacteria which typically infect skin grafts. As a result of the success of penicillin in preventing infections, the US Government decided to support the production and distribution of penicillin to the armed forces.[32]

Mass production[edit]

Dorothy Hodgkin determined the chemical structure of penicillin.
A technician preparing penicillin in 1943

The chemical structure of penicillin was determined by Dorothy Crowfoot Hodgkin in 1945.[33] Penicillin has since become the most widely used antibiotic to date, and is still used for many Gram-positive bacterial infections. A team of Oxford research scientists led by Australian Howard Florey and including Ernst Boris Chain and Norman Heatley devised a method of mass-producing the drug. Florey and Chain shared the 1945 Nobel Prize in Medicine with Fleming for their work. After World War II, Australia was the first country to make the drug available for civilian use.

The challenge of mass-producing this drug was daunting. On March 14, 1942, the first patient was treated for streptococcal septicemia with US-made penicillin produced by Merck & Co.[34] Half of the total supply produced at the time was used on that one patient. By June 1942, just enough US penicillin was available to treat ten patients.[35] In July 1943, the War Production Board drew up a plan for the mass distribution of penicillin stocks to Allied troops fighting in Europe.[36] The results of fermentation research on corn steep liquor at the Northern Regional Research Laboratory at Peoria, Illinois, allowed the United States to produce 2.3 million doses in time for the invasion of Normandy in the spring of 1944. After a worldwide search in 1943, a mouldy cantaloupe in a Peoria, Illinois market was found to contain the best strain of penicillin for production using the corn steep liquor process.[37] Large-scale production resulted from the development of deep-tank fermentation by chemical engineer Margaret Hutchinson Rousseau.[38] As a direct result of the war and the War Production Board, by June 1945, over 646 billion units per year were being produced.[36]

Penicillin was being mass-produced in 1944.

G. Raymond Rettew made a significant contribution to the American war effort by his techniques to produce commercial quantities of penicillin.[39] During World War II, penicillin made a major difference in the number of deaths and amputations caused by infected wounds among Allied forces, saving an estimated 12%–15% of lives.[citation needed] Availability was severely limited, however, by the difficulty of manufacturing large quantities of penicillin and by the rapid renal clearance of the drug, necessitating frequent dosing. Methods for mass production of penicillin were patented by Andrew Jackson Moyer.[40][41][42] Penicillin is actively excreted, and about 80% of a penicillin dose is cleared from the body within three to four hours of administration. Indeed, during the early penicillin era, the drug was so scarce and so highly valued that it became common to collect the urine from patients being treated, so that the penicillin in the urine could be isolated and reused.[43] This was not a satisfactory solution, so researchers looked for a way to slow penicillin excretion. They hoped to find a molecule that could compete with penicillin for the organic acid transporter responsible for excretion, such that the transporter would preferentially excrete the competing molecule and the penicillin would be retained. The uricosuric agent probenecid proved to be suitable. When probenecid and penicillin are administered together, probenecid competitively inhibits the excretion of penicillin, increasing penicillin's concentration and prolonging its activity. Eventually, the advent of mass-production techniques and semi-synthetic penicillins resolved the supply issues, so this use of probenecid declined.[43] Probenecid is still useful, however, for certain infections requiring particularly high concentrations of penicillins.[6]

Human experimentation[edit]

In a 1946 to 1948 study in Guatemala, U.S. researchers used prostitutes to infect prison inmates, insane asylum patients, and Guatemalan soldiers with syphilis and other sexually transmitted diseases (STDs), to test the effectiveness of penicillin in treating such diseases. They later tried infecting people with "direct inoculations made from syphilis bacteria poured into the men's penises and on forearms and faces that were slightly abraded ... or in a few cases through spinal punctures".[44] Approximately 1300 people were infected as part of the study. The study was sponsored by the Public Health Service, the National Institutes of Health and the Pan American Health Sanitary Bureau (now the World Health Organization's Pan American Health Organization) and the Guatemalan government. The team was led by John Charles Cutler, who later participated in the Tuskegee syphilis experiments. Cutler chose to do the study in Guatemala because he would not have been permitted to do it in the United States.[45][46][47][48] The Presidential Commission for the Study of Bioethical Issues determined that 83 people died; however, it was not possible to determine whether the experiments were the direct cause of death.[49]

Total synthesis[edit]

Chemist John C. Sheehan at the Massachusetts Institute of Technology (MIT) completed the first chemical synthesis of penicillin in 1957.[50][51][52] 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.[52][53] Although the 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.[52][54] Attaching different groups to the 6-APA 'nucleus' of penicillin allowed the creation of new forms of penicillin.

Developments from penicillin[edit]

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, which offered a broader spectrum of activity than either of the original penicillins. 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.

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.[55]

See also[edit]

Notes[edit]

  1. ^ Penicillin at the Free Dictionary
  2. ^ Garrod, L. P. (1960). "Relative Antibacterial Activity of Three Penicillins". British Medical Journal 1 (5172): 527–29. doi:10.1136/bmj.1.5172.527. 
  3. ^ Garrod, L. P. (1960). "The Relative Antibacterial Activity of Four Penicillins". British Medical Journal 2 (5214): 1695–6. doi:10.1136/bmj.2.5214.1695. PMC 2098302. PMID 13703756. 
  4. ^ "Penicillin (Benzylpenicillin, Penicillin G, Bicillin C-R/L-A, Pfizerpen, Wycellin)". The Antimicrobial Index. Knowledgebase. Retrieved 4 March 2014. 
  5. ^ "Penicillin G sodium salt Susceptibilty and Resistance Data". TOKU-E. Retrieved 4 March 2014. 
  6. ^ a b Rossi S, editor, ed. (2006). Australian Medicines Handbook. Adelaide: Australian Medicines Handbook. ISBN 0-9757919-2-3. 
  7. ^ http://www.facm.ucl.ac.be/Full-texts-FACM/Vanbambeke-1999-3.pdf
  8. ^ Kasten, Britta; Reski, Ralf (March 30, 1997). "β-lactam antibiotics inhibit chloroplast division in a moss (Physcomitrella patens) but not in tomato (Lycopersicon esculentum)". Journal of Plant Physiology 150 (1–2): 137–140. doi:10.1016/S0176-1617(97)80193-9. 
  9. ^ Mucsi, z; Chass, GA; Ábrányi-Balogh, P; Jójárt, B; Fang, DC; Ramirez-Cuesta, AJ; Viskolcz, B; Csizmadia, IG (2013). "Penicillin's catalytic mechanism revealed by inelastic neutrons and quantum chemical theory". Phys Chem Chem Phys 15 (47): 20447–20455. PMID 23760063. 
  10. ^ Nicolaou (1996), pg. 43.
  11. ^ a b c Al-Abdallah, Q., Brakhage, A. A., Gehrke, A., Plattner, H., Sprote, P., Tuncher, A. (2004). "Regulation of Penicillin Biosynthesis in Filamentous Fungi". In Brakhage AA. Molecular Biotechnolgy of Fungal beta-Lactam Antibiotics and Related Peptide Synthetases (88). pp. 45–90. doi:10.1007/b99257. ISBN 3-540-22032-1. 
  12. ^ a b Brakhage, A. A. (1998). "Molecular Regulation of β-Lactam Biosynthesis in Filamentous Fungi". Microbiol Mol Biol Rev. 62 (3): 547–85. PMC 98925. PMID 9729600. 
  13. ^ Baldwin, J. E., Byford, M. F., Clifton, I., Hajdu, J., Hensgens, C., Roach, P, Schofield, C. J. (1997). "Proteins of the Penicillin Biosynthesis Pathway". Current Opinion in Structural Biology (7): 857–64. 
  14. ^ a b Fernandez, F. J., Fierro, F., Gutierrez, S, Kosalkova, K . Marcos, A. T., Martin, J. F., Velasco, J. (September 1994). "Expression of Genes and Processing of Enzymes for the Biosynthesis of Penicillins and Cephalosporms". Anton Van Lee 65 (3): 227–43. doi:10.1007/BF00871951. PMID 7847890. 
  15. ^ Baker, W. L., Lonergan, G. T. "Chemistry of Some Fluorescamine-Amine Derivatives with Relevance to the Biosynthesis of Benzylpenicillin by Fermentation". J Chem Technol Biot. 2002, 77, pp1283-1288.
  16. ^ "Alexander Fleming – Time 100 People of the Century". Time. "It was a discovery that would change the course of history. The active ingredient in that mold, which Fleming named penicillin, turned out to be an infection-fighting agent of enormous potency. When it was finally recognized for what it is—the most efficacious life-saving drug in the world—penicillin would alter forever the treatment of bacterial infections." 
  17. ^ Houbraken J, Frisvad JC, Samson RA (2011). "Fleming's penicillin producing strain is not Penicillium chrysogenum but P. rubens". IMA Fungus 2 (1): 87–95. doi:10.5598/imafungus.2011.02.01.12. PMC 3317369. PMID 22679592. 
  18. ^ Phil. Trans., 1876, 166, pp. 27–74. Referred to at: Discoveries of anti-bacterial effects of penicillium moulds before Fleming
  19. ^ J. M. Caminhoá, "Cogumélos uteis, nocivos e curiosos." In: Elementos de Botânica geral e médica, 1877, p. 1718. Published in 5 vols, Typographia Nacional, Rio de Janeiro, Brazil. (Reported by Prof P. R. Chocair as held at the Biblioteca da Câmara Municipal de São Paulo, Brasil)
  20. ^ [1], 2011, Italian Journal of Public Health.
  21. ^ http://www.almanacco.rm.cnr.it/reader/cw_usr_view_recensione?id_articolo=1704&giornale=1679
  22. ^ http://festival2011.festivalscienza.it/site/home/programma-2011/eventi-per-tipo/conferenze/vincenzo-tiberio-vero-scopritore-degli-antibiotici.html
  23. ^ Duchesne 1897, Antagonism between molds and bacteria. An English translation by Michael Witty. Fort Myers, 2013. ASIN B00E0KRZ0E and B00DZVXPIK.
  24. ^ [2] Alexander Fleming (1881–1955)
  25. ^ Haven, Kendall F. (1994). Marvels of Science : 50 Fascinating 5-Minute Reads. Littleton, CO: Libraries Unlimited. p. 182. ISBN 1-56308-159-8. 
  26. ^ Fleming A. (1929). "On the antibacterial action of cultures of a penicillium, with special reference to their use in the isolation of B. influenzæ". British Journal of Experimental Pathology 10 (31): 226–36. 
  27. ^ Brown, Kevin. (2004). Penicillin Man: Alexander Fleming and the Antibiotic Revolution. Stroud: Sutton. ISBN 0-7509-3152-3. 
  28. ^ a b Wainwright M, Swan HT (January 1986). "C.G. Paine and the earliest surviving clinical records of penicillin therapy". Medical History 30 (1): 42–56. doi:10.1017/S0025727300045026. PMC 1139580. PMID 3511336. 
  29. ^ Drews, Jürgen (March 2000). "Drug Discovery: A Historical Perspective". Science 287 (5460): 1960–4. doi:10.1126/science.287.5460.1960. PMID 10720314. 
  30. ^ Saxon, W. (June 9, 1999). "Anne Miller, 90, first patient who was saved by penicillin". The New York Times. 
  31. ^ Krauss K, editor (1999). "Yale-New Haven Hospital Annual Report" (PDF). New Haven: Yale-New Haven Hospital. 
  32. ^ Stuart B. Levy, The Antibiotic Paradox: How the Misuse of Antibiotics Destroys Their Curative Powers, Da Capo Press, 2002: pp. 5–7. ISBN 0-7382-0440-4
  33. ^ The Nobel Prize in Chemistry 1964, Perspectives. Retrieved July 14, 2012.
  34. ^ The First Use of Penicillin in the United States, by Charles M. Grossman. Annals of Internal Medicine July 15, 2008: Volume 149, Issue 2, Pages 135–136.
  35. ^ John S. Mailer, Jr., and Barbara Mason. "Penicillin : Medicine's Wartime Wonder Drug and Its Production at Peoria, Illinois". lib.niu.edu. Retrieved February 11, 2008. 
  36. ^ a b John Parascandola (1980). The History of antibiotics: a symposium. American Institute of the History of Pharmacy No. 5. ISBN 0-931292-08-5. 
  37. ^ Mary Bellis. "The History of Penicillin". Inventors. About.com. Retrieved October 30, 2007. 
  38. ^ Chemical Heritage Manufacturing a Cure: Mass Producing Penicillin
  39. ^ "ExplorePAhistory.com". Retrieved May 11, 2009. 
  40. ^ Andrew Jackson Moyer, Method for Production of Penicillin, United States Patent Office, US Patent 2,442,141, filed 11 May 1945, issued 25 March 1948.
  41. ^ Andrew Jackson Moyer, Method for Production of Penicillin, United States Patent Office, US Patent 2,443,989, filed 11 May 1945, issued 22 June 1948.
  42. ^ Andrew Jackson Moyer, Method for Production of Penicillin, United States Patent Office, US Patent 2,476,107, filed 11 May 1945, issued 12 July 1949.
  43. ^ a b Silverthorn, DU. (2004). Human physiology: an integrated approach. (3rd ed.). Upper Saddle River (NJ): Pearson Education. ISBN 0-8053-5957-5. 
  44. ^ Michael Cook (October 2, 2010). "US apologizes for 1940s unethical research in Guatemala". BioEdge. 
  45. ^ "U.S. sorry for Guatemala syphilis experiment". CBC News. October 1, 2010. 
  46. ^ Rob Stein (October 1, 2010). "U.S. apologizes for newly revealed syphilis experiments done in Guatemala". Washington Post. 
  47. ^ "US sorry over deliberate sex infections in Guatemala". BBC News. October 1, 2010. 
  48. ^ Chris McGreal (October 1, 2010). "US says sorry for 'outrageous and abhorrent' Guatemalan syphilis tests". Guardian (London). 
  49. ^ Rob Stein (August 30, 2011). "U.S. scientists knew 1940s Guatemalan STD studies were unethical, panel finds". Washington Post (?). 
  50. ^ Sheehan, John C.; Henery-Logan, Kenneth R. (March 5, 1957). "The Total Synthesis of Penicillin V". Journal of the American Chemical Society 79 (5): 1262–1263. doi:10.1021/ja01562a063. 
  51. ^ Sheehan, John C.; Henery-Logan, Kenneth R. (June 20, 1959). "The Total Synthesis of Penicillin V". Journal of the American Chemical Society 81 (12): 3089–3094. doi:10.1021/ja01521a044. 
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  53. ^ Nicolaou, K.C.; Vourloumis, Dionisios; Winssinger, Nicolas; Baran, Phil S. (2000). "The Art and Science of Total Synthesis at the Dawn of the Twenty-First Century**". Angewandte Chemie International Edition 39 (1): 44–122. doi:10.1002/(SICI)1521-3773(20000103)39:1<44::AID-ANIE44>3.0.CO;2-L. PMID 10649349. 
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References[edit]

  • Nicolaou, K.C.; Sorensen, E.J. ; with a foreword by E.J. Corey (1996). Classics in Total Synthesis : Targets, Strategies, Methods (5. repr. ed.). Weinheim: VCH. ISBN 3-527-29284-5. 
  • Dürckheimer, Walter; Blumbach, Jürgen; Lattrell, Rudolf; Scheunemann, Karl Heinz (March 1, 1985). "Recent Developments in the Field of β-Lactam Antibiotics". Angewandte Chemie International Edition in English 24 (3): 180–202. doi:10.1002/anie.198501801. 
  • Hamed, Refaat B.; Gomez-Castellanos, J. Ruben; Henry, Luc; Ducho, Christian; McDonough, Michael A.; Schofield, Christopher J. (January 1, 2013). "The enzymes of β-lactam biosynthesis". Natural Product Reports 30 (1): 21–107. doi:10.1039/c2np20065a. PMID 23135477. 

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