Alexander Fleming: penicillin and so much more

Mention the name of the scientist Alexander Fleming, and many will say, "Oh, he's the guy who discovered penicillin!" That's only partly true, but it's the story or legend we have come to accept. What's the real story, and what else do we know before and after Fleming's discovery?

Sample of the fungus from Fleming's lab, donated to Douglas Macleod, St. Mary's Hospital (Science Museum London)

Born in southwestern Scotland on August 6, 1881, Alexander Fleming grew up on a farm with seven siblings. He moved to London at 13 to live with his older brother Thomas, who was an ophthalmologist. Alexander had to quit school, and he worked in a shipping company until his brother convinced him to use recently acquired inheritance money from an uncle to study medicine at St. Mary's Hospital Medical School. There, he got his bachelor's degree with distinction in 1906. He'd been a private in the London Scottish Regiment of the Territorial Army and was considered a good marksman, so the captain of the St. Mary's rifle club wanted him  to join, and to do that he introduced Fleming to Sir Almoth Wright (also a club member) to conduct research there on vaccine therapy in his newly established laboratory of the Inoculation Department. Otherwise, he'd have had to leave St. Mary's to pursue a career in surgery. Almoth developed the first British vaccine against typhoid, and other noted researchers worked there, including Augustus Desire Waller, who developed the first electrocardiogram in 1887.

The young researcher Fleming at St. Mary's

Fleming worked with Wright on several projects. Earlier, in 1905, German bacteriologist August von Wassermann had developed a diagnostic test for syphilis, which Fleming was tasked with simplifying in 1909. A year later, Paul Ehrlich, a German physician, discovered an arsenic compound that cured syphilis, and Fleming used his previously published work to develop a better method to administer this new drug.

Then, he served as a captain in the Royal Army Medical Corps in France during World War I, after which he returned to St. Mary's where he got a master's degree. It was while he worked in field hospitals during the war that he was exposed to the horrific results of battle and how poorly medicine at the time was combatting infections like gangrene, tetanus, and general septicemia. Fleming noticed how antiseptics were used even for deep wounds but to no avail, which resulted in loss of limb or life. He even published a paper on it in 1917 to show how gauze absorbed antiseptics so much that it made them useless for such injuries. But nobody paid much attention. Another paper he wrote that year showed how antiseptics kill the body's protective white blood cells more than they kill bacteria, so they are useless except on the surface of a wound. And, if there was a lot of pus in the wound, that tended to block the entry of the antiseptic anyway. Again, field doctors continued their regular practices.

Treating battle injuries in World War I

Back in London, he remained interested in how the body itself fought disease. In 1921, when he had a cold, he wondered whether mucus from his nose would have any antibacterial effect, so he applied it to one of his Petri dishes. Bacteria that had blown in randomly onto the plate grew in colored patches except 1 cm (half an inch) outside the drop of his mucus. "This is interesting", he calmly told a research assistant. Something had seeped from the mucus through the plate agar and killed the bacteria! 

He then tested other fluids like tears, saliva, sputum, serum, and more and found it in all of them. He named this material lysozyme, meaning that it causes the lysis (destruction) of bacterial cells like an enzyme. It was weak but present in many tissues, suggesting its importance in natural immunity. In the bacterial plates below, you can see the effect using tears. The left petri dish has whitish bacteria growing on the place except near a paper disk soaked in tears. The right dish shows how mixing bacteria with tears causes them to break apart and grow in a more disrupted way, less dense than without tears.

Figures from Fleming's 1922 paper on lysozyme

Fleming managed to find time to do research despite being promoted to Assistant Director in 1919. As AD, he acquired massive responsibilities for financial support over medical supplies, as well as salaries and housing of technicians, research workers, and office staff, most of whom lived at St. Mary's. He was also in charge of directing most of the research done there and coordinating commercial activities such as vaccine production and antitoxin testing in the hospital.

Enter the year 1928. Fleming was studying a common bacteria called Staphylococcus (colloquially called "Staph") because it is a very common one spread by the wind and feces and because it is therefore a common contaminant in wounds. His lab bench was very cluttered (see below), and he was running several experiments at once, whether in test tube cultures or petri dishes.

Fleming's lab bench (Wikipedia)

On September 3, 1928, at age 47, he came back to his lab after a vacation and began to examine and tidy up many of the cultures with junior researcher Merlin Pryce. On one petri dish that he'd inoculated with Staph before his break, he noticed a gray-greenish patch of mold. That plate is shown below. Notice that like the lysozyme culture with tears, there is a blank space of agar gel between the Penicillium mold and

the bacteria. Once again, this suggested something he called "mold juice" was excreted by the Penicillium species and flowed into the agar, not only stopping bacteria from growing in that zone, but obliterating what had already grown in Fleming's absence. Once again, he reacted with a curt, "That's funny". He repeated the experiment on September 28 with success.

Fleming examining a petri dish culture of Staph (steemit.com)

Where did the Penicillium mold in Fleming's petri dish come from? His lab window was sealed shut. The particular species on his plate is not a common airborne one, so two speculations have arisen. The Royal Society of Chemistry thought that it came from Fleming's messy lab and spilled coffee which would have killed other molds around, allowing this special one to breed. Another thought is more reasonable. There was a laboratory one floor below where Charles La Touche was studying molds and fungi, so it might have drifted in from there. Fleming's messiness might have actually helped in another way. It is reported that after his return from vacation, he glanced at his cultures and missed the one that was later deemed key. He put it on top of other plates in a bath of Lysol (in use since 1889) to disinfect things before throwing them away, and it was only when he and Pryce were reviewing the plates before disposal that he took notice of the reaction. This also explains why he felt the need to repeat the experiment; the one he saw was not covered in the Lysol, but he wanted to be sure it had not accidentally entered the plate.

But this is not where the story ends. Fleming didn't leap to purification and mass production of the active chemical agent he called penicillin and become world famous. In fact, he wasn't the first to notice that molds help combat bacteria. 

• Ancient physicians from Greece, Serbia, and India used molds even though they didn't know why they worked. Russian peasants did the same with soil containing mold. 
• England's Royal Botanist John Parkinson published a book Theatrum Botanicum (with a 157-word title!) in 1640 on herbalist remedies like Penicillium.

From 1850-1890, the world got used to the notion of bacteria causing illnesses, thanks to the work of Louis Pasteur and Robert Koch. Treating wounds and diseases was still not performed very consistently due to the lack of knowledge. 

• Researchers like Sir John Burdon-Sanderson (1870) and Joseph Lister (1871) had both noted that species of the Penicillium mold would inhibit growth of bacteria. Sanderson just grew mold on the top of test tubes and saw no bacteria underneath. No reason given. Lister had simply noted that urine samples did not grow bacteria if they had mold in them.
• Theodor Billroth, the German "father of abdominal surgery", noticed in 1874 that Penicillium but not bacteria grew in some test tube cultures. He supposed that the sterilizing process of the liquid media or the mold itself had somehow changed the chemical composition of the growth liquid to make it unsuitable for bacteria. 
• Physicist John Tyndall also noted Penicillium's antibacterial properties in 1875, but he ascribed them to choking the oxygen from the top of the test tube culture where they grew, not by producing any chemical.
• In 1895, Italian medical officer Vincenzo Tiberio noticed that people in his home became sick after the walls of the well were cleaned of mold (including a Penicillium species), so he thought it afforded some protection to the well water. He scraped it off and used it in culture, on animals, and eventually on humans with success, thinking the mold made some curative material. Italian medicine ignored his results as a coincidence.
• French physician Ernest Duchesne recorded the opposite effect, typhoid bacteria killing Penicillium mold in culture, but when he injected both together into guinea pigs in 1897, the animals didn't die of typhoid. So he thought the mold made something to weaken the bacteria. Unfortunately, his doctoral work was ignored by Louis Pasteur simply because Duchesne was an unknown researcher in his early 20s.
• Since then, papers around the world sporadically reported antibacterial effects of Penicillium--Sturli (1908), Lieske (1921), Twight (1923), and Gratia & Dath (1924)--but none of these  attracted great attention for medical purposes.

Gilbert Shama wrote an article in the 2017 Journal of Pharmaceutical Microbiology summarizing these and other instances which he labels it as "simultaneous discovery", something not uncommon in science. Fleming got the credit, probably because he tried to tie the lab results to potential usefulness in medical practice.

Fleming published his findings from the fateful petri dish in 1929. Although a modest sized paper (10 pages), it didn't inspire the medical community. That by itself is odd because it described the following properties of crude batches, all practical for potential mass production:

• it dissolves easily in water
• it can be filtered into a sterile solution
• it can survive moderate heating (56C/132F, or 80C/176F)
• it is most stable at body pH
• it was effective against many types of bacteria
• it was not toxic to rabbits or mice or white blood cells
• it did not irritate skin or corneas

When he read another paper on penicillin that year, he again suggested its use for treating human infections, but nobody was interested in this lab oddity. The same thing happened at a conference of renowned scientists shortly thereafter.

Being trained in surgery and doing research in bacteriology, Fleming was no chemist, so he and his assistants found it impossible to mass produce penicillin in a pure form, only as a crude extract from the bottom of flasks. A year later, he gave up.

Batch cultures of Penicillium (British Pathe)

Shortly thereafter in 1935, "sulfa drugs" were discovered. These were sulfonamides capable of curing diseases caused by Staph and streptococcus bacteria, and the race was on to produce even more. Penicillin took a back seat. And then World War II broke out.

Australian Howard Florey (pharmacologist) and German Ernst Chain (biochemist) worked at the University of Oxford at the time. Despite having little funding there, they set up a lab to study antibacterial products made by two bacteria and Penicillium based on reading Fleming's papers. They convinced the Rockefeller Foundation in the U.S. to fund them for 5 years. Like with Fleming, they had no problems in growing Penicillium. With more resources than him, Florey and Chain hired six women to mass product the mold.

Florey and Chaim's scaled up batch production (Wikipedia)

With their expertise, they solved the problem of extracting pure penicillin from the raw culture liquid. Florey was searching for a single chemical to kill all bacteria, but he settled on penicillin with its major effects. His comment on this altruistic venture show how the two of them were just ordinary scientists:

"People sometimes think that I and the others worked on penicillin because we were interested in suffering humanity. I don't think it ever crossed our minds about suffering humanity. This was an interesting scientific exercise, and because it was of some use in medicine is very gratifying, but this was not the reason that we started working on it." (Florey)

In March 1940, they tested their batches on rats, mice, rabbits, and cats. Their first human test was on a 43-year-old policeman on February 12, 1941. Britain's chemical industry then was devoted to wartime efforts, so Florey went to the U.S. to solicit help. A contact at the Department of Agriculture's Northern Regional Research Laboratory (NRRL) in Peoria, Illinois improved the growing process and purification methods. American pharmaceutical companies Merck, Squibb, and Lilly had done some penicillin research already, and Pfizer was about to. Collaborative agreements were made in 1942 between all but Lilly to help Florey, while his Oxford lab conducted clinical trials and published on 187 cases in 1943. Pfizer opened the first commercial penicillin production plant on March 1, 1944. Penicillin eventually became mass produced through concerted efforts by many players and was available without restrictions by early 1945. On D-Day, every Allied soldier carried a dose with them.

Advertisement from National World War II museum

Chain tried to convince Florey to file for a patent on penicillin production, but Florey said it should be free for all. An advisor to the Scientific Advisory Panel to the British Cabinet told Florey it would be unethical. Chain's result from speaking to the Secretary of the Medical Research Council was the same. Americans Robert D. Coghill and Andrew J. Moyer ignored the British situation and filed for a patent on penicillin production, unlike other researchers. This drove Fleming crazy, and he wrote about that:

"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?"

Despite penicillin's success, Fleming's further research was showing that many bacteria can develop resistance to drugs like penicillin. He cautioned that it should be used appropriately to avoid that problem.

In 1944, Fleming, Florey, and Chaim together won the Nobel Prize in Physiology or Medicine for their combined discoveries. Fleming was also knighted in that year. He died on March 11, 1955 of a heart attack.

Receiving the Nobel Prize (1945)

The history of developing penicillin for mass production can be found at this link.