Penicillin

PENICILLIN was still a laboratory curiosity in the summer of 1941. It might have remained so had it not been for certain fortuitous events and the urgency of developing new drugs during the war. Today the drug is being manufactured in large quantities. It is the drug of choice for the treatment of many infections and diseases, and it aided immeasurably in reducing the number of war casualties. To produce the drug in the quantities needed in war and peace, it was necessary to develop a new industry with buildings and equipment valued at more than 25 million dollars. New outlets for agricultural products were realized in the development of this industry. The production of lactose, or milk sugar, was almost doubled, and a new and important use was found for corn steep liquor, a byproduct of the wet corn milling industry.  Penicillin was discovered in 1928 at St. Mary’s Hospital in London by Alexander Fleming. He noted the presence of a contaminating blue-green Penicillium in plate cultures of Staphylococcus and observed that adjacent to this mold the colonies of bacteria were apparently being lysed, or dissolved. The phenomenon was investigated. When grown in pure culture, the mold, which was subsequently identified as Penicillium notatum Westling, was found to produce a substance that inhibited the growth of Staphylococcus and other disease-producing, gram-positive bacteria.  Professor Fleming published the results of his investigations in 1929 and to the active substance he applied the name “penicillin,” after the generic name of the mold that produced it. He determined that the substance was relatively nontoxic and he pointed out that it might have therapeutic value if it could be produced in quantity.

In the years that followed, penicillin was almost forgotten. It was not until 1940 that real interest in it was revived. Professors Florey, Chain, Heatley, and their collaborators at Oxford University demonstrated that a crude penicillin in the form of a brown powder (now known to have contained only 2 or 3 percent pure penicillin) possessed curative properties when injected into mice previously infected with Staphylococcus and other disease-producing bacteria. A year later they presented clinical data on six patients, who showed a favorable clinical response. The toxicity of the substance was found to be very low.

Because of conditions then prevailing in England, it was not feasible to produce there the amount of penicillin needed for further clinical trials. For that reason, aided by the Rockefeller Foundation, Drs. Florey and Heatley came to the United States. Here they were referred to the National Research Council and to Charles Thom, principal mycologist in the Department of Agriculture in Washington. They were advised to come to the Northern Regional Research Laboratory in Peoria, Ill., where members of the staff of the fermentation division had had experience in mold fermentations and a large collection of molds was maintained. Dr. O. E. May, then director of this laboratory, and Dr. R. D. Coghill, head of the Fermentation Division, realized the tremendous possibilities of the drug, and arrangements were made to begin work on the problem at once.

Research on penicillin at the Peoria laboratory was directed primarily along the following lines: To develop, if possible, a culture medium that would favor the production of a greater amount of penicillin; to investigate the possibility of producing penicillin in submerged culture; and to try to develop strains capable of producing increased yields.

Dr. Fleming had employed a nutrient broth as a culture medium, and Florey and his associates produced penicillin in a modified Czapek-Dox solution containing yeast extract and glucose. At the Northern Regional Research Laboratory, the nutrient solution was altered in many ways by A. J. Moyer, microbiologist, and many substances, known to promote the growth of micro-organisms, were investigated for their ability to increase penicillin production. Of such substances, corn-steep liquor, commonly referred to as “steep liquor,” was found to be outstanding. At levels below those that actually inhibited mold growth, yields of penicillin were found to increase with the addition of increased amounts of this product. It was recognized that the steep liquor constituted the principal source of nitrogen and apparently contributed other important nutrients necessary for the formation of penicillin.

At the same time various sources of carbon were investigated, including glucose, sucrose, lactose, corn dextrin, and corn starch. Of these different carbon sources, lactose, or milk sugar, was found to be slowly assimilable by the mold and most generally favorable for penicillin production.

The Northern Regional Research Laboratory therefore recommended for the production of penicillin a culture medium whose principal ingredients were corn-steep liquor and lactose. Through Dr. A. N. Richards, chairman of the Committee on Medical Research, Office of Scientific Research and Development, these discoveries were made available to all producers of penicillin in the United States and allied countries. With certain modifications in the proportions of the ingredients used, depending upon the particular mold culture employed and the method of production, this medium has remained in general use.

The pioneer work of Professor Fleming had been done with surface or still cultures, as had also the equally important work of Professor Florey and associates. It was natural, therefore, that the same method should have been followed in our early work, and it was from studies with surface cultures that the lactose-steep liquor medium was developed.  Experience with other fermentations, however, indicated that penicillin could, in all probability, be produced at a much lower cost, if a satisfactory tank or submerged fermentation could be developed. Attention was early directed toward this goal. Penicillin-producing molds were inoculated into lactose-steep liquor medium and subjected to continuous and vigorous agitation for several days, during which time the broth was assayed daily for penicillin content. When thus agitated, the mold grows submerged, usually assuming the form of small, rounded pellets.

Culture solutions whose principal ingredients are corn-steep liquor and lactose are most favorable for the production of penicillin in both surface and submerged culture. For submerged production, however, the concentration of these nutrients should be approximately one-half that employed for production in surface culture. When increased amounts of steep liquor are employed, the growth of the mold is excessively heavy and the yield of penicillin is markedly reduced. Standard solutions for surface and submerged production were recommended.

Some modifications in the composition of the culture solution for both surface and submerged cultures can be made without seriously affecting penicillin yields, and such alterations are often desirable when new equipment is employed or a new penicillin-producing mold is investigated.

The two methods of penicillin production worked out in the laboratory have their direct counterparts in industry. Penicillin has been made successfully on a commercial scale by both methods.

In the surface-culture method, the mold is grown upon the surface of a quiescent nutrient solution which is dispensed in flasks, bottles, or trays usually to a depth of 5 to 34 inch. A common practice is to use bottles of approximately 2-quart capacity and to incubate them on their sides to obtain the greatest possible amount of culture surface. This method entails a great deal of hand labor, and manufacturing costs are high. In some industrial plants as many as 30,000 bottles were inoculated each day and the plants were operated on a 6- to 10-day cycle. Maximum penicillin is produced at a temperature of about 24° to 25° C., and large incubating rooms had to be built to accommodate the 200,000 to 300,000 growing cultures.

The surface-culture method has now been supplanted by the submerged process. It was the process first developed on an industrial scale, however, and all the penicillin used in the clinical trials that first established the curative properties of the drug was produced by it.

All penicillin now made in the United States is produced by the submerged, or tank process. Inoculation with the mold may be in the form of spores or growing culture, commonly referred to as preformed inoculum. Fermentation periods vary somewhat, depending upon the mold employed and the equipment used, but commonly range from 2½ to 4 days. Large amounts of sterile air are required and the mold growth must be constantly stirred. The fermentation must be run at a favorable temperature of approximately 24° to 25° C., and measures must be taken to remove or dissipate the heat generated by the vigorously growing mold.

Other methods of producing penicillin have been recommended but have not succeeded in large-scale operations.

Different molds are employed for different methods of production.  The strain isolated by Fleming was employed for all of the early studies, and in early tests made at this laboratory it was found to produce higher yields than any other unimproved strain when grown in surface culture.  The Fleming strain was observed to be quite unstable in laboratory culture, and substrains possessing different cultural characteristics could be separated from it. Although most of the latter failed to equal the productivity of the parent strain, one of them, designated NRRL 1249.B21, produced yields approximately double those of the parent culture and raised the titre, or penicillin content, from about 75 to 100 units per milliliter to 150 to 200 units per milliliter. It was made available to producers and was thereafter generally employed for the production of penicillin by the surface-culture method.

More spectacular success has been achieved in developing improved cultures for the production of penicillin in submerged culture. As it became apparent that the submerged fermentation was industrially feasible, the need for developing higher-yielding submerged cultures was recognized. Strain NRRL 832, the culture employed for this type of production, was studied intensively. Efforts to obtain from it a natural variant characterized by substantially increased production were unsuccessful. Attention was then directed toward the isolation of new strains from nature. Previous work had shown that almost all members of the P. notatum-chrysogenum group produced some penicillin. It seemed probable, therefore, that new strains possessing greater productive capacity than NRRL 832 might be obtained if a large number of isolates were examined. Such a search was undertaken early in 1943.

New cultures were obtained from moldy food products, fruits and vegetables in early stages of spoilage, and from fertile soil collected from various stations in the United States and from many foreign countries.

The most important culture discovered, however, was isolated from a moldy cantaloupe in Peoria. The culture represented a strain of Penicillium chrysogenum Thom, a species closely allied to P. notatum, and was designated NRRL 1951 in our collection of cultures. When first studied, it produced penicillin in slightly greater yields than NRRL 832, but within a few months a natural variant, which more than doubled the amount, was developed from it. This substrain, designated NRRL 1951.B25, was studied intensively here, and was at the same time made available to the penicillin industry in 1944. It was soon generally adopted for submerged production.

Faced with the demand by the armed forces for ever-increasing amounts of penicillin, the Office of Production Research and Development of the War Production Board early in 1944 set up projects at the University of Wisconsin, Stanford University, and the Carnegie Institution of Washington to discover or develop more productive cultures.  Largely because of the work already done at the Northern Regional Research Laboratory, it then seemed probable that cultures capable of producing greatly increased yields of penicillin might be obtained by one or more of the following means: The isolation of new strains from nature; the selection of natural variants from such new stocks; and the production of induced mutations from known good producing strains by X-ray and ultraviolet radiation, or by other artificial means.

NRRL 1951 P. chrysogenum, isolated from a moldy cantaloupe, capable of producing approximately 100 u/ml. of penicillin in submerged culture.

NRRL 1951.B25 A naturally occurring variant from NRRL 1951, capable of producing up to 250 u/ml. of penicillin.

X-1612 An X-ray-induced mutation from NRRL 1951.B25, capable of producing more than 500 u/ml. of penicillin.

Wis. Q-176 An ultraviolet-induced mutation from X-1612, capable of producing more than 900 u/ml. of penicillin.

The importance of the foregoing developments to present penicillin production cannot be overemphasized, because current yields of 750 to 900 units per milliliter are obtained in nutrient solutions of approximately the same composition as those used to produce maximum yields of 75 to 100 units per milliliter with NRRL 832 just a short time ago.

Some difficulties were encountered when these high-yielding strains were first adopted for commercial production, for they were found to produce primarily penicillin K, a type that is rapidly destroyed in the animal body and hence is much less useful clinically. However, if phenylacetic acid or phenylacetamide is added to the production medium, these strains can be made to produce primarily the more useful penicillin G. This procedure has been adopted by industry.

Although we refer to the drug penicillin as a definite product, it should be noted that molds are known to produce at least six different penicillins. Four of these, commonly referred to in this country as F, G, X, and K, are produced under natural culture conditions by members of the P. notatum-chrysogenum group. They represent different chemical compounds and possess different physical and chemical properties. Two or more of these penicillins may be produced in the same culture solution and may, in varying proportions, be contained in the final dried product of commerce. Whereas penicillin F was the first penicillin to be studied and crystallized, experience soon showed that penicillin G was chemically more stable and hence more easily recovered. Furthermore, the culture (NRRL 832) first employed for the submerged production of the drug yielded penicillin mostly of type G. For these reasons, commercial penicillin soon came to represent primarily penicillin of this type.

The several penicillins differ in their inhibitory effect upon susceptible bacteria, some being relatively more effective against particular species than others. Besides, they differ markedly in their behavior within the animal body. These differences render some penicillins more effective than others in combating disease. For example, pure sodium penicillin K when tested in vitro against Staphylococcus aureus shows an activity of 2200-2300 u/mg. in contrast to pure sodium penicillin G, which contains 1667 u/mg. When tested in vivo, however, penicillin K is rapidly destroyed and adequate blood levels are difficult to maintain. It is not a satisfactory drug.

On the other hand, penicillin X, which is chloroform-insoluble, and hence can be obtained free from the other types without great difficulty, has been found to be more effective against streptococci, pneumococci, and gonococci than commercial penicillin containing mostly penicillin G.  Believing that the production of penicillin X might be of importance from a clinical point of view, we have successfully developed a mold that produces substantially increased amounts of this type of penicillin. From a culture that produced penicillin X in yields approximating 15 percent of the total, an ultraviolet-induced mutation has been developed that produces penicillin X in 50-percent yields. It remains to be seen whether penicillin X will attain significance as a distinct drug.

Before October 1944, penicillin was measured in terms of the Oxford unit—the amount of penicillin which, when dissolved in 1 cubic centimeter of water, gave the same inhibition as an arbitrary standard, estab- lished by Florey and associates, which produced zones of inhibition averaging 24 mm. in diameter. In October 1944 a conference was held in London under the auspices of the Health Organization of the League of Nations for the purpose of establishing an international standard and an international unit of penicillin. Pure sodium penicillin G was adopted as the international standard and the international unit was defined as the specific penicillin activity contained in 0.6 microgram of the international penicillin standard. Pure sodium penicillin G, therefore, by definition contained 1,667 units per milligram. An international standard was subsequently prepared by F. H. Stodola and J. L. Wachtel, chemists at the laboratory, by the recrystallization of pooled samples of sodium penicillin G contributed by manufacturers in the United States and Great Britain. Two strains of Staphylococcus aureus were designated as standard test organisms, namely the Food and Drug Administration No. 209P (NRRL B-313) and the strain employed by Heatley (NRRL B-314).

In the assay, or measurement, of penicillin potencies, the so-called cylinder plate method is generally used. Modifications and improvements have been made since 1941 by W. H. Schmidt, in charge of penicillin assays at the laboratory, and others, but the method remains basically the same as that originally developed by Dr. Heatley. A plate containing nutrient agar is warmed to 37° to 40° C., evenly flooded with a suspension of the test bacteria in warm agar (approximately 45° C.), and allowed to solidify. On the surface of this plate are placed a number of hollow cylinders, which make a water-tight seal. Into these are pipetted small amounts of the solutions to be tested. A part of the cylinders in every plate contains a standard of known penicillin content for purposes of comparison.

The plates are then incubated overnight at the optimum temperature for the test bacterial species. The penicillin contained in the cups diffuses into and through the underlying agar, inhibiting the growth of the bacteria in circular zones. The zones of inhibition for all samples are then measured in millimeters, and the potencies of the unknown are determined by comparing their average diameters with those produced by the samples of known penicillin content. The method is reasonably accurate for measuring aqueous solutions containing from 1 to 4 units per milliliter. To attain these levels of concentration, the samples to be tested are diluted with sterile phosphate buffer of pH 6.0.

A modification of the above method, now in common use, involves the use of paper disks of standard dimensions that are dipped into the penicillin solutions to be tested and placed on the surface of seeded agar plates. The plates are then incubated and zones of inhibition measured and evaluated as in the cylinder-cup technique. Two other methods of assay have been successfully used, the serial dilution method and the turbidimetric method.

When the fermenters contain maximum titres of penicillin, the mold mycelium is removed by filtration or centrifugation. In current practice the mycelium as well as the exhausted liquid residues are usually dis- carded. Feeding trials by the Bureau of Animal Industry, however, indicate that the dried mycelium and the culture-liquor residues may be successfully used in poultry feeds. The remaining clear broth is immediately chilled to prevent the development of bacterial contaminants which would otherwise quickly destroy the penicillin. Recovery practices differ, but a common procedure is to set the pH of the broth at pH 2.0 to 3.0 and then extract all of the free penicillin into a solvent such as amyl acetate, butyl alcohol, or chloroform. Penicillin is also subject to rapid decomposition in the presence of strong acids or alkali, and-this destruction is tremendously accelerated at elevated temperatures. The extraction step, therefore, should be carried out as rapidly as possible and at a low temperature. The solvent containing the penicillin is then extracted with aqueous sodium bicarbonate to yield a solution of the sodium salt. To concentrate the penicillin further, it can be transferred back and forth between various solvents and buffer solutions of appropriate pH. An alternative procedure is to adsorb the penicillin from the filtered broth on charcoal, then remove it with a solvent, and purify it.

The final concentrated solution of sodium penicillin is then vacuum dried. This may be done in the same containers in which it is subsequently marketed, or the penicillin may be dried in bulk and added to vials in weighed amounts. Generally these are small rubber-stoppered vials of 20 milliliter capacity. To use the penicillin it is only necessary to pierce the stopper with a sterile hypodermic needle and redissolve the penicillin salt in sterile saline or water. As marketed, penicillin is a white to pale yellow powder generally containing about 60 to 90 percent of sodium penicillin and assaying from 1,000 to 1,500 units per milligram. The remaining portion represents salts of organic acids contained in the broth and other nontoxic impurities carried over in the recovery operations.  While penicillin is usually recovered and marketed as the sodium salt, other salts such as potassium, calcium, or barium penicillin have been prepared to meet special needs. In recent months a few manufacturers have come out with crystalline products containing essentially pure penicillin. It seems probable that in the near future penicillin will be generally marketed in this form.

Before they are sold, samples of all commercial lots of penicillin are carefully tested by the Food and Drug Administration for toxicity and for the presence of pyrogens, or fever-producing substances.

The chemistry of penicillin has been thoroughly investigated by many workers in university, Government, and industrial laboratories collaborating under the joint supervision of the Committee on Medical Research, Washington, D. C., and the Medical Research Council, London. The intense interest in this chemistry was due in great part to the belief held during the early days of commercial production that the enormous military needs could be met only if chemical synthesis were accomplished.  A practical synthesis of penicillin, however, was not realized during the war; fortunately, the production by fermentation proved adequate.

So far, six naturally occurring penicillins have been isolated in the pure state and all have the same empirical chemical formula, C₉H₁₁O4SN₂—R. All of the penicillins show the same qualitative action against microorganisms. The quantitative differences in potency (from 900 to 2,300 units per mg.) between the penicillins depend on the nature of the R-group. Of the different penicillins, penicillin X, first isolated by Stodola, Wachtel, and Coghill of this laboratory, is of unusual interest in that new, highly active penicillins can be produced from it by substitution reactions.

Before January 1943, the commercial production of penicillin was negligible, although much valuable clinical information had been obtained. In the 5-month period ending May 1943, 400 million units were produced, or roughly enough penicillin to treat 400 hospitalized cases.  In the following month, 425 million units were produced and since June 1943 production has climbed steadily and rapidly. In the latter part of 1945, monthly production reached a level of approximately 800 billion units; during the 30 months after July 1943 it increased more than a thousandfold.  In May 1946 approximately 2,700 billion units were produced, with an estimated value of approximately $15,000,000. There is every reason to anticipate still greater production. During the period from June 1943 to July 1946, the price of penicillin dropped from $20 per 100,000 units (acknowledged to be less than cost) to about 55 cents per 100,000 units. Parallel with the increase in production and the decrease in price there has been a steady improvement in the quality of the drug manufactured. Penicillin, as marketed in 1943, commonly contained about 100 u/per mg. of pure sodium penicillin; now the average is very much higher, with 1,500 u/per mg. of material not uncommon.

Penicillin is now the drug of choice in the treatment of many types of bacterial infection. It is not a cure-all, however, and it is of little or no value in the treatment of many serious diseases. Generally speaking, its application can be correlated with the identity and character of the pathogen. It is particularly effective against the pyogenic cocci and the gram-positive, spore-forming bacilli, including the anaerobic forms belonging to the genus Clostridium.

Penicillin may be administered in a variety of ways, including intramuscular, intravenous, or local injection ; by continuous intravenous drip; and by topical application. The intramuscular method is most commonly used. A solution of penicillin of appropriate concentration is made with sterile isotonic saline, or distilled water. The dosage commonly ranges from 10,000 to 30,000 units and is administered every 3 hours, day and night. It is important that the injections be continued until the clinical picture clearly warrants their cessation.

The intravenous method is usually employed for the purpose of obtaining quickly a blood concentration of penicillin adequate to halt or reduce infection. Penicillin may be injected locally into abscesses, joint cavities, and so forth in varying doses, depending upon the extent of the infection. Penicillin may also be administered topically at localized sites of infection, where it is applied in the form of wet compresses or as a powder. As penicillin becomes more generally available it is probable that it will be administered orally on an increasingly greater scale. To obtain the proper blood levels to combat and eliminate infection, it is necessary to give four to five times the amount required if administered parenterally. The patient’s gain in comfort would more than compensate for the added cost of the drug.

The total amount of penicillin necessary to clear up an infection depends upon the nature and extent of the infection and the sensitivity of the causative organism. The gonococci are the most sensitive of all pathogens and most cases of gonorrhea can be cured by a series of 3 to 6 intramuscular injections of 30,000 units administered at 3-hour intervals. Cases of bacterial endocarditis, on the other hand, require much greater amounts of penicillin, often running to several million units administered at rates of 200 thousand to 1 million units a day. While specific hospital cases require varying amounts of the drug, an average figure would be approximately a million units administered over a period of 3 to 7 days.

Penicillin is usually marketed in the form of the sodium salt and the methods of administration cited above are based upon the use of solutions of the drug in this form. It may also be embodied in the form of ointments or creams that find their greatest usefulness for the treatment of second- or third-degree burns and localized surface infections. It may be suspended in a suitable vehicle for nebulization and used as a nasal or oral spray to combat infection of the throat and nasal passages. The use of penicillin in the form of troches and dental cones has been recommended to combat oral infections.

Penicillin has played a major role in military medicine. It was employed quite freely in forward areas as a preventative measure to forestall the development of gas gangrene and other serious infections. Behind the lines, it found wide application in the treatment of osteomyelitis and other deep-seated wound infections in addition to the multiple uses for which it is recommended in general practice. New uses for penicillin are constantly being discovered, new modes of administration are being developed, and clinical practice, therefore, is undergoing continual change.

In the field of veterinary medicine, the most successful application of penicillin has been made in the treatment of bovine mastitis in which the causative organisms were Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, and Streptococcus uberis. Streptococcic infections responded to smaller doses than those in which the invading organism’ was Staphylococcus aureus. The penicillin solution is administered through the teat canal immediately after a milking period and apparently has no adverse effects on the mammary glands or on the quality of the milk.

Penicillin has been used with positive results in the treatment of strangles of horses caused by Streptococcus equi, in hemorrhagic septicemia of cattle, “shipping fever of horses, swine erysipelas, peritonitis and osteomyelitis in dogs, and canine distemper. The method of admin- istration and the dosage varies with the size of the animal, the nature and severity of the infection, and the identity-of the causative organisms.

Some investigations have indicated the possible usefulness of penicillin to combat certain plant diseases. Such studies have been limited in number and would need to be performed on a much more extensive scale before any conclusions can be reached regarding the usefulness of the drug in this field.

The addition of penicillin to milk and other highly perishable food products has been recommended but its usefulness as a food preservative is questioned. Penicillin in low concentrations will inhibit the growth of many bacteria responsible for food spoilage. It is not effective, however, in preventing the growth of other forms often equally responsible. Gram-negative species are usually not affected, and among the gram-positive forms, which are generally penicillin sensitive, a number of spore formers, such as Bacillus cereus, produce enzymes, termed pencillinases, which rapidly destroy the drug. While the addition of penicillin may extend somewhat the useful life of a product under certain conditions, the varied microflora normally present precludes its continuing effectiveness.

THE AUTHOR
Kenneth B. Raper is senior microbiologist in charge of the Culture Collection Section, Fermentation Division, Northern Regional Research Laboratory, Peoria, Ill. He has been associated with the Bureau of Agricultural and Industrial Chemistry during most of the past 18 years. Dr. Raper holds degrees from the University of North Carolina, George Washington University, and Harvard University. At the Northern Regional Research Laboratory, Dr. Raper and his co-workers are building a large collection of cultures of fungi, yeasts, and bacteria, many of which are essential in the industrial fermentation processes used in the production of antibiotics and use full organic chemicals. During the period in which penicillin fermentation was being developed, he examined hundreds of cultures obtained from all parts of the world in efforts to discover strains of the Penicillium notatumchrysogenum group that would be more satisfactory than existing strains for the production of high yields of penicillin.