Penicillin, The Wonder Drug

Introduction

Today, penicillin is a common treatment for many diseases, and it is one that has been developed recently. The idea of using fungal products, such as penicillin, to produce medicine is a relatively new one, but the use of fungi in many folk remedies have been utilized for a long period of time, even though the incorporation of fungi into the remedy was not specifically known. Over three thousand years ago, the Chinese had put moldy soybean curd on boils and other types of skin infections. Other cultures have placed warm earth, which contains molds and other fungi, as first aid in injuries. So, although the concept of antibodies is relatively recent, the indirect use of fungi for medicinal purposes is not.

The discovery of penicillin has often been described as a miracle drug, and that is exactly what it is. Prior to the discovery of medicine, death could occur in what would seem to be very trivial injuries and diseases. Death could occur from minor wounds that became infected or from diseases that are common place, today, such as Strep Throat and venereal diseases such as syphilis and gonorrhea.

You are all undoubtedly familiar with the story of penicillin. In all introductory text books, in the life science, the story always tells how penicillin was discovered accidentally, at St. Mary's Hospital, in London, by Dr. Alexander Fleming. Fleming was examining a culture of Staphylococcus aureus, a pathogenic bacterium on which he was doing some research, when he noticed that it had become contaminated by a species of Penicillium. Although, the species of the mold was unknown to Fleming, at the time, he did observe that it was inhibiting the bacterial growth. Fleming reported his findings in 1929 and the rest is history.

If life was only that simple. Such short summaries really do not tell you the entire story, and in this case,  saids that Fleming's discovery of penicillin was one of chance and does not credit other people, who were just as deserving or more so in the development of penicillin for medicinal use.

Some luck, surely was involved, as is true with many events. Alexander Fleming did not have any ambition to become a doctor throughout his life. His start into bacterialogical medicine came from an unlikely string of events. In 1900, when the Boer War broke out between England and colonies in southern Africa, Fleming and two brothers joined a Scottish regiment. This turned out not to be such a dangerous time for them as they had chanced upon a country club environment. They spent much of their time shooting, swimming, and even playing water polo. Following the war, Fleming returned home to discover that his uncle had died and left him and his brothers with a sizable inheritance. His older brother, Tom, who was a successful doctor by this time, advised him to invest his money on his career and suggested that he attend medical school. Fleming scored high on his examination and was able to select from three medical schools. He knew nothing of these schools and selected St. Mary's Hospital, in London, only because he had once played waterpolo against them. After graduation, Fleming had trained to be a surgeon for just as random a reason, but then found himself in a choice that was even more bizarre. He had the option of taking a position, as a surgeon and leaving St. Mary's or he could join the Inoculation Service and stay at St. Mary's. The major influence on Fleming staying was that the captain of St. Mary's rifle club knew of his option and was desperate to improve his team. Knowing that Fleming was a great shot he did all he could to keep him at St. Mary's. He convinced Fleming to join his department in order to work with its brilliant director -- and to join the rifle club. Fleming would stay at St. Mary's, where his discovery of penicillin was made, for the rest of his career .


The Discovery of Penicillin

That Alexander Fleming discovered penicillin by chance is a myth. Before Fleming, there were a series of observations that influenced his research, and allowed him to come to the correct conclusion when a chance contamination in his bacterial culture was observed. This same opportunity came to others as well, but their only response to the contamination was that it had ruined their experiment, discarded the cultures and thought nothing more of it.

Discoveries in science are rarely made by chance. Often, it involves knowledge that has been gained over a long period of time so that all discoveries, today, have come about because we have "stood on the shoulders of giants that have come before us." Another words, discoveries are based on previous discoveries from the past. In the case of of penicillin, it was based upon historical records that had originated as early as 1500 B.C. There was already records describing the use of molds and fermented materials in the treatment of diseases. Because such treatments were carried out without an understanding on the nature of the cure or an understanding of cellular and biochemical processes of the human body, there was a likelyhood of the patient dying from the treatment as well as being cured. It would not be until the late nineteenth century that Pasteur put forth the concept of the germ theory of disease. Not until then was there any concerted effort made that would destroy the microorganisms that were responsible for the actual causes of diseases. One of the most common problem that occured, and still occurs today, was the contamination of bacterial cultures by other micoorganisms, especially fungi. These contaminations led to a number of observations in the late 1800s:

Thus, a number of observations set the stage for Fleming's accidental discovery. As early as 1920, Fleming was searching for antibacterial agents, which was influenced by his war time experience. During the First World War, Fleming witnessed the deaths of many soldiers that died not from the wounds that were received during combat, but from septicemia or in layman's term, blood poisoning that was responsible for the death of the wounded soldiers following successful operations on those wounds. It was only recently that surgeons had adopted Lister's concept of antiseptic surgery. Lister argued that since bacterial infections was harmful to the human body, that antiseptics should be used on the wounds to kill the bacteria. However, Fleming while serving with a colleague, from St. Mary's Hospital,   argued that while this was true, the antiseptics also had an adverse effect on the patient. They reasoned that the leukocytes of the immune system was the first line of defense in preventing infection and that application of antiseptics to the wounds of patients would destroy the leukocytes more rapidly than the invading bacteria. They recommended the use of a saline solution to cleanse wounds. However, there was no one who wound follow this procedure since Lister's antiseptic theory was at that time a revolutionary, cutting edge science concept that experiments had proven to be true. So without a doubt, Fleming was searching for antibacterial agents. In 1922, Fleming discovered lysozyme, what can be thought of as the "little brother" of penicillin. Lysozymes are enzymes present in biological substances as varied as egg whites, tears and mucus, that causes bacteria to lyse or burst. The first biological substance that he tested was mucous from his nose (he was sick with a cold at the time). This would later be a major discovery, but at that time, lysozyme was seen as interesting, but not with a great deal of applications since only the less virulent bacteria would respond to these enzymes.

In 1928, he was researching the properties of the group of bacteria known as staphylococci and became another in the long line of scientists to benefit from a seemingly chance observation. His problem during this research was the frequent contamination of culture plates with airborne molds. However, he was also a sloppy scientist as well. Cultures that he worked on were constantly forgotten, in his lab, which was normally in a state of great disorder. One day he observed a contaminated culture plate and noted that the Staphylococci bacteria had burst in the area immediately surrounding an invading mold growth. He realized that something in the mold was inhibiting growth of the surrounding bacteria. Subsequently Fleming isolated an extract from the mold and he named it penicillin. Although his discovery was published, there was not a great deal of attention paid to this paper. Despite this success, further attempts by Fleming to produce a concentrated extract of penicillin failed and he was unable to prove its therapeutic value.

It could be argued here that Fleming did accidentally discover penicillin since he wasn't looking for it at that moment in time. However, he had been looking for several years prior to this and without his background in lysozyme research, Fleming may not have really ever considered further investigation of the contaminating mold. This was certainly the opinions of his colleagues at St. Mary's Hospital.

As further test continued, Fleming began to realize that he was on the verge of a great discovery. However, he still did not know the identity of the fungus. He had virtually no background with fungi and knew little about these organisms. Because of the lack of information on this fungus, the press had initially called this mold "yellow magic" because when it was cultured in a large vat of liquid nutrient, the liquid became a bright yellow color. Although, Fleming was not very knowledgeable about fungi, he was able to identify it as a species of Penicillium, and even gave it a tentative species name, P. rubra, which was incorrect. However, it was his identification to genus, that prompted Fleming to name this compound, that inhibited bacterial growth, penicillin. The fungus would eventually be identified by Charles Thom, who was the authority on the taxonomy of Penicillium, as Penicillium notatum.

Although very similar to lysozymes that he had tested, it was obvious that penicillin was far more powerful an antibacterial compound. His crude extracts could be diluted 1,000 times and still be effective in killing bacteria. Further test were done with various species of bacteria and also test were carried out with laboratory animals to be certain that penicillin would not be toxic to animals. These results were, again, very interesting, but how could they be translated to practical use? Fleming only continued to work on and off with with penicillin between 1928-1931, but was unable to produce it in the quantity necessary for testing or practical applications. In addition, there were problems in the production of penicillin. Fleming found that, under the conditions which he was growing his mold, penicillin was unstable and the culture stopped producing penicillin after eight days. However, this did not dampen Fleming's enthusiasm, he continued to work on penicillin and talk of its great potential value in medicine. Unfortunately, this had the opposite effect on his colleagues that he wanted. After several years of working on penicillin and not being able to make practical use of it, many of his colleagues grew tired of hearing his stories on what he was doing with penicillin. It was only his conversation with Howard Florey at a professional medical meetings that they attended that may have sparked an interest. So, Fleming did not become famous overnight, the newspaper did not come to interview him, representatives from pharmaceutical companies from near and far did not come to see him when his discovery was made public, and there were, in fact, many people that doubted that the discovery would be of any value.

It would not be Fleming, but one of his former student, Dr. Cecil Paine, that would be the first to demonstrate the value of penicillin in medicine. Unfortunately, he would become the forgotten man in the development of penicillin. Paine became inspired, to do research on penicillin, by Fleming's original penicillin paper and not because he was inspired by Fleming in one of his classes. Paine recalled, when interviewed about Fleming, that he was "a shocking lecturer, the worst you could possibly imagine,'" not because of inherent shyness, but because he never appeared enthusiastic about his subject. This was not only Paine's opinion, but was a general opinion of Fleming as a lecturer. This may possibly be one reason he failed to stimulate any enthusiasm for his penicillin work among his contemporaries.

Paine was, at that time, employed in a joint position in Sheffield, at the University Medical School and Royal Infirmary at the time he was working with penicillin. His first effort at using penicillin was in treating patients with a skin infection called sycosis barbae. This infection of the hair follicle, of the beard, is caused by Staphylococus, a very common infection, in pre-antibiotic days. Following the infection, pus would form in the follicle, and would become swollen and inflamed. Paine treated the infection by soaking gauze with the crude penicillin extract that he produced and then using the gauze to cover the patient's face. This proved to be a failure, but did not dampen his enthusiasm to continue working with penicillin.

Paine's next attempt, this time a dramatically successful one, at using penicillin, involved the lacerated eye of a local miner. The stone responsible for the laceration was still embedded in the eye and an infection had set in which was caused by Pneumococcus, a pathogenic bacterium whose presence normally would have required the removal of the eye. However, after Paine irrigated the eye with the crude penicillin extract, the miner's eye and eyesight was saved. Another case involved a different form of eye infection. This time occurring in the eyes of a baby. The baby had contracted gonorrhea, from his mother, during birth. At this time, gonorrhea was not treatable and babies often became blind through hereditary gonorrhea. Paine's irrigation of the babies eyes with his penicillin extract was again successful.

If these successful treatments with penicillin had become known at the time, it may have spurred the development of penicillin many years earlier. However, Paine never published his results concerning these patients nor did he ever present an oral paper on these patients even though he had written and presented papers on other subjects. When asked years later why he did not publish on this important event, he said that he felt that since he was using a crude extract and that there was not sufficient testing, he didn't feel it was worthy of publication. Furthermore, he had to stop working with penicillin because he had taken a position elsewhere which called for his research to take a different direction. Thus, Fleming was still floundering at this time to get people to carry out more research with penicillin. Fleming had accepted by this time that personally, he lacked the skills to advance the science of penicillin further. He believed now that he would at this point require the expertise of a chemist. He would be proved right later.

As an added note on Paine. Paine would have an influence on someone who would be important in penicillin research a few years later. While still at Sheffield University, in 1932, he discussed his penicillin work with a newly arrived Professor of Pathology. The professor said Paine "took not the slightest interest at that time." Yet six years later, he was to begin a program of research that would lead to the mass production of penicillin. The Professor's name was Howard Florey who we will have much more to say about later.

By the mid 1930's, several new discoveries came about in medicine that began to make Fleming doubt as to whether he should continue his work with penicillin. Sulfa drugs, a class of synthetic chemical substances, was developed in Germany and was effective in treating some bacterial infections. These drugs inhibited the action of para-aminobenzoic acid, a substance that bacteria need in order to reproduce. Another synthetic drug, simply known as M and B, after the English firm of May and Baker, where the drug was developed, was demonstrated to be effective against pneumonia. Fleming began working with both these drugs and had stopped working with penicillin by 1934. That he was still interested in penicillin would, in later years, be obvious.

During this same ten year period, between 1928-1938, Dr. Howard W. Florey, Professor of Pathology at Oxford's Sir William Dunn School of Pathology, also became interested in research on lysozymes, and later, in 1938, in antibiotic when he came across Fleming's paper on penicillin. Florey, however, did not work alone, he had a team of scientist that consisted of Drs. Ernst B. Chain, Leslie Falk, Norman G. Heatley and twenty other scientists and technicians. Where Fleming had a poorly equipped lab with no staff support, Florey's lab was well staffed and equipped.

There are many conflicting stories concerning the relationship between Florey and Chain. Chain is always credited as the person who actually began working with penicillin in Florey's lab and even located a culture that was sub-cultured from Fleming's original isolate of P. notatum. Florey, on the other hand, was the person in charge of the research program, but was often thought of as not being very knowledgeable about penicillin before Chain's rediscovery of Fleming's paper. This seems unlikely since Florey had been on the editorial board of the journal which published Fleming's first penicillin paper and there is every likelihood that he may have even helped in editing it. He also knew Fleming quite well from attending professional meetings and considering Fleming's obsession with penicillin, it seems highly unlikely that the subject would not have come around to penicillin. Finally, when Florey met Paine in 1932, Florey had heard about the potential for penicillin. However, when questioned about the latter, Florey denied that this had any influence on his direction of research and that he had forgotten that conversation by 1938, when he choose to work on penicillin. Nevertheless, one of his former students remembered quite distinctly that Florey mentioned Paine's work in a lecture, at Oxford, in 1936.

One thing that Florey and Chain did agree upon was that scientific interest rather than the desire to introduce a life-saving drug was the reason they devoted their efforts into penicillin research. This would be one of the few things that Chain and Florey would agree upon. As the years passed, there would be an ever widening rift between the two. Chain explained his motive for working with penicillin:

I became interested - immediately - in Fleming's paper, not because I hoped to discover a miraculous drug for the treatment of bacterial infection which for some reason had been overlooked, but because I thought it had great scientific interest. In fact, if I had been working at that time in aim-directed scientific surroundings, say in the laboratory of a pharmaceutical firm, it is my belief that I would never have obtained the agreement of my bosses to proceed with my project to work with penicillin.

I thought this would be a relevant passage to quote because in many universities, this one in particular, there are those politicians who wish to restrict apparently purely academic research because they see no value in its pursue. They should probably read this passage by Chain.

It was also Chain who began immediate work with penicillin by extracting and what he believed to purified penicillin. After a great deal of effort, enough was extracted for experimentation to begin. Tests began with laboratory animals. Initially only two mice were tested. Chain was anxious to begin, but being a biochemist by training, he was not qualified to inject laboratory animals. Rather than Florey, Chain asked another colleague, J.M. Barnes, to inject the two mice. The two mice were injected and were unharmed. Chain explained Barnes involvement was due to the absence of Florey who was away from Oxford and explained that he had approached Florey four times in several weeks about doing the injections. The last time he requested his assistance, Florey turned to Mrs. Margaret Jennings, who was in the lab, and said, pointing to Chain, "in one of my weak moments I promised this man to test his fractions and here he comes pestering me again." After what Chain described as "these humiliating remarks in front of others not involved in the project, it became clear to me that Florey was not really interested in penicillin…" This was the reason for asking Barnes rather than Florey to carry out the inoculations. As another digression, following this successful inoculation, Florey was not at all pleased with this test being done in his absence. Chain maintained that Florey was not interested in participating until after he had carried out this test successfully.

Following the successful injection of two white mice, fifty white mice were inoculated with deadly Streptococcus germs, followed by injection of penicillin in half the mice. The second group, did not receive penicillin. All of the latter group died the next day while the former mice all recovered.

The first real test for the Florey lab came when Albert Alexander, a 48 year old London policeman had nicked himself while shaving. It was only a minor cut and was ignored for several days, but this would have a major impact on medical history. After a time, the Alexander's face became swollen and infected and developed a temperature that rose to 105F. The minor cut had developed septicaemia. When he was rushed to Radcliffe Hospital, sulfa drugs were administered without success. When the doctors believed that the policeman only had several more hours of life remaining, Florey and Chain requested that they be given permission to treat this patient with their "purified" penicillin. This would be their first human subject to which penicillin was applied. Florey and Chain took charge of the case personally and injections of penicillin were begun. After five days, the impossible had happened, the patient was recovering. Unfortunately, because penicillin was in such short supply, that small amounts had to be extracted from the patient's urine. after five days, the supply of penicillin that was produced for experimentation had been used up. There was no more penicillin available, and the policeman had a relapse and died five days later. However, the experiment did demonstrate that penicillin was not harmful to humans and was effective. Penicillin had temporarily halted the infection, but in the end it had failed in its first test. Unfortunately, because the patient had died, there were a number of macabre jokes that came about, one that is well known, even to this day: "the treatment was a success, but the patient died."

The next two treatments, however, would be successful. The first was a 15 year old boy who had a form of blood poisoning from a badly inflamed hip joint and the second was a 48 year old laborer who had a large carbuncle on his back. In both cases, the penicillin treatment was a success. Encouraged with this success, they next tested penicillin on a 4 year old boy who was ill from an infection, following measles. Sulpha drugs were used unsuccessfully. The boy, by this time, had developed an infection in one eye socket which spread to the base of the skull and was semi-comatose and it was believed that he had contracted meningitis by this time. Penicillin was not tried until the infection had already progressed to this advanced state. On the third day of penicillin treatment, the swelling in the boy's eye had retreated and he was emerging from the coma. The infection had been defeated and the penicillin injections stopped in order to preserve the supply of penicillin. Then suddenly the boy had a sudden convulsion and he died four days later. Although the boy died, the autopsy revealed that he had died of a burst blood vessel in his brain, and that the infection had been cured by the penicillin injections.

During this period Fleming was not idle. Fleming contacted Florey to request penicillin for the treatment of an old friend Harry Lambert, who was in critical condition, suffering from meningitis. Sulfa drugs had already been used and without success. Florey thought the procedure planned by Fleming a risky one, but did supply him with the penicillin. Fleming injected the penicillin into his friend's spine and the treatment proved completely successful, with his friend making a miraculous recovery. However, Florey had, at the same time, injected penicillin into the spines of artificially infected animals, with the result that they had all died! Luckily, Fleming did not learn about Florey's result until after he had injected Lambert. He probably would have hestitated to give the injection resulting in the death of his friend. Although the relationship between Florey and Fleming seemed to be an amiable one. It would later turn sour.

By this time, it was now 1941, it was now acknowledged that penicillin was indeed a worthwhile drug and could save thousands of lives. Unfortunately, the process that Florey and Chain developed, for extracting penicillin, was producing only one part penicillin per million part of culture medium. Thus, their penicillin was not purified as they thought it had been.The amount was small indeed, and scientist could be heard saying, "you could get more gold out of ordinary seawater than penicillin out of the mold."

With World War II raging in Europe and the German bombardment of England, all of England's resources and efforts had to be placed into the war effort. Florey and Chain would be unable to utilize any factory resources in England to experiment further with producing penicillin in adequate amounts to be useful for large scale usage. Also, it was feared that even if a factory could be utilized, it was feared that all their efforts cold possibly be lost if the factory was bombed.

However, during the summer of 1941, Florey had negotiated an agreement with the Rockefeller Foundation, which had been funding his research, to fly he and one of his assistant to the United States to continue his work with penicillin. The United States, who was at that time a neutral country, would enter into World War II in another few months. This gave added incentive to the penicillin project which became declared a war project and was given top priority.

Despite the efforts and resources that were being given to producing large quantities of penicillin, it soon became obvious that Fleming's original culture would not be able to produce enough penicillin regardless of the environment in which the fungus was grown. By 1942, there was only enough penicillin produced to treat a few hundred people. There are many species of Penicillium, and a search was started to find other species that could be tested for penicillin production. Eventually, one was found, on a moldy cantaloupe in a market in Peoria, Illinois. This species would be identified as Penicillium chrysogeum, and would produce approximately 200 times as much penicillin than P. notatum. This is the species that is currently used to produce penicillin. However, even this amount would be inadequate to produce the amount that would be required.

Scientist then began to try to increase the amount of penicillin produced by P. chrysogenum, by irradiating it with X-rays and UV rays in order to induce mutations of this species. This eventually lead to a mutant that produced 1000 times the amount of penicillin than Fleming's original culture. In addition to the development of this mutant, a new means of growing the mold was also perfected. Previously, penicillin was grown in flask, the size of milk bottles, and hundreds of bottles of Penicillium notatum were needed to produce enough penicillin for only a single person. The new method involved growing the mold in large metal tanks, which held 25,000 gallons of nutrient, were aerated so that the mold could grow throughout the entire tank rather than on top. Aeration was the key to growing it in such large tanks. Previously, this had not been tried because it was known that the mold would only grow on the surface of the liquid medium. Thus, utilization of a large tank, under such circumstances would be highly inefficient in terms of cost, space and penicillin production.

With this new method, production quantity began to rise. In 1943, 29 pounds were produced, and with increases in the number of pharmaceutical companies producing penicillin, there was a tremendous increase. By the end of the war, enough penicillin was produced to treat seven million patients/year.

With respect to the war, the use of penicillin was immediately apparent. During World War I, death rate from pneumonia in the American Army totaled 18%. In World War II, it fell to less than 1%.

One illness after another, that was tested, was cured by penicillin, which was by this time dubbed a "wonder drug." In addition to pneumonia and blood poisoning, the major causes of death, in hospitals, during the war, strep throat, scarlet fever, diphtheria, syphilis, gonorrhea, meningitis, tonsillitis, rheumatic fever, and many other diseases were successfully treated with penicillin.

During this period of time when mass production of penicillin was being perfected, Fleming had little to do with penicillin. Florey did update him as to the progress being made and samples were often sent to him, but by this time Fleming was no longer part of penicillin research. Although, Fleming was the discoverer of penicillin, he was almost forgotten by the time penicillin was being mass produced. However, he would be rescued from oblivion, in 1943, when he was knighted along with Howard Florey, and two years later was awarded the Nobel Prize in Physiology and Medicine along with Florey and Chain.

So Fleming eventually did get credit for the discovery of penicillin, but it was certainly not an overnight success. Also, Fleming had little to do with penicillin while research was being done with regards to its application and mass production. Thus, Fleming's role in penicillin was not that of a lone researcher discovering a wonder drug, but rather one of many people that contributed to the successful exploitation of penicillin in medicine.

Penicillin brought about the biggest search in medical history. It was reasoned that if there was one antibiotic in nature, there must be many more, and many more would later be found. However, few would be fungal in origin. Most of the later antibiotic discovered would be derived from bacteria, specifically Actinomycetes. Yet, without the discovery of penicillin, all of these other antibiotics would possibly never been discovered.

As a postscript, Fleming would, unfortunately, make a prediction that would come true. That the use of penicillin would, in time, be of limited value because bacteria would eventually recombine genetically to resist the effects of penicillin. By as early as 1952, as much as three-fifths of all staph infections were penicillin resistant. Various steps were taken so as to continue the use of antibiotics. New antibiotics are constantly being sought for this reason. Other approaches include using combination of antibiotics and changing the chemical structure of antibiotics in the laboratory so that all of slightly different properties. These attempts have all been tried and have been successful, but unfortunately, the bacteria are still recombining genetically to become resistant to these new efforts.

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