The Classic: Streptomycin, a Substance Exhibiting Antibiotic Activity against Gram-Positive and Gram-Negative Bacteria (2005)

Selman Waksman (Figs 1, 2) came to the United States from Imperial Russia in 1910 when he was in his early 20s. He entered Rutgers University in New Jersey a year later and became a soil microbiologist at the Agriculture College there. He observed that certain fungi in soil suppressed the growth of mycobacterium tuberculosis and eventually he and the workers in his laboratory (including his co-authors on this classic article, Albert Schatz and Elizabeth Bugie) isolated one that proved extremely effective and minimally toxic-streptomycin.Fig 1.: Selman Waksman (right) is shown with Albert Schatz. The two were co-discoverers of streptomycin.Fig 2.: Selman Waksman (left) is shown with Sir Albert Fleming, who discovered penicillin.Selman Waksman claimed (and received the credit for) the discovery of streptomycin in the mid 1940s). With the credit came fame and fortune, which included a Nobel Prize and medals and accolades from numerous nations, prestigious societies, and universities. It also brought a great deal of money. Elizabeth Bugie and Albert Schatz, Waksman’s putative collaborators, objected to Waksman’s one-sided winnings and eventually filed a lawsuit against him for what they considered to be their fair share. Waksman argued that he, as the director of the laboratory at Rutgers University, had spent 30 years perfecting the techniques that made the discovery possible. Schatz, as a PhD worker in the laboratory, had done the work but only under Waksman’s direction and using Waksman’s methods. Elizabeth Bugie, another worker in the laboratory, had only been asked to corroborate Schatz’s work, and she eventually withdrew from the civil action. In the end Waksman got 10% of the money derived from the sale of the streptomycin patent, Schatz got 3%, and all others employed in the laboratory (and they were many) split 7%. Rutgers University got 80% and with it built the Waksman Institute of Microbiology, which became a major research institution in that field. The accompanying paper by Bosworth et al was published previously in CORR as a Classic Article. It documents the effectiveness of streptomycin in treating the draining sinuses of tuberculosis. In it Bosworth et al note that the use of streptomycin resulted in “dramatic healing of sinuses and a marked improvement in general physical condition” in about 80% of the 95 patients in their review. The patients who did not have such a good outcome had sequestra, thick walled abscesses, or uncontrolled dead spaces, all of which were amenable to surgical intervention because of the effect of streptomycin. With the exception of streptothricin,1 most of the antibiotic substances known at the present time, including penicillin and other mold products as well as gramicidin and actinomycin, act largely upon gram-positive bacteria. The activity of these substances upon gram-negative organisms is highly selective as in the case of penicillin, which affects the Neisseria group and has little activity upon Escherichia coli and other gram-negative bacteria,2 or else much larger quantities are required to bring about the inhibition of these bacteria, as in the case of actinomycin.3 Among the antibiotic agents that act selectively alike against both gram-positive and gram-negative bacteria, streptothricin occupies a prominent place; since this substance is water-soluble and possesses limited toxicity to animals, it is of particular interest from a chemotherapeutic point of view. Unfortunately, streptothricin has very little activity against a number of bacteria found among both the gram-negative (Pseudomonas fluorescens, Ps. aeruginosa) and the gram-positive (Bacillus mycoides and B. cereus) groups. In a search for antagonistic organisms that are active against gram-negative bacteria, and from which antibiotic substances could be isolated, the actinomycetes were found4 to offer extensive potentialities. Although most of the antibacterial agents produced by these organisms are also active against gram-positive bacteria, certain few of them exert a marked selective activity against many of the gram-negative types of bacteria. Actinomyces lavendulæ, which produces streptothricin is such an organism. After detailed examination of a large number of cultures, either isolated at random from different natural and enriched soils and composts, or selected from the culture collection, another oganism was found that produces an antibiotic substance which apparently combines many of the desirable antibacterial properties. This organism is similar, in most of its cultural characteristics as well as in its morphology, to A. griseus isolated from the soil some 28 years ago.5 The active substance is in many respects similar to streptothricin, although it differs from it in its greater activity against various gram-negative bacteria, notably the Ps. aeruginosa group, as well as against those aerobic spore-forming bacteria, such as B. mycoides, which are resistant to streptothricin. Because of its similarity to streptothricin, this substance may be designated as streptomycin, derived from the generic name that has recently been given to the aerial-mycelium producing and sporulating group of actinomycetes, namely Streptomyces.6 Two strains of the organism producing streptomycin were obtained, one (No. 18-16) from a heavily manured field soil and the other (D-1), a somewhat less active form, from a smear plate of the throat of a chicken; it is doubtful, however, whether this organism is a normal inhabitant of the animal system. In order to compare the antibacterial properties of the culture filtrate of the two strains of this organism with those of other actinomycetes producing antibiotic substances, the results of a typical experiment are reported in Table I. The filtrates of A. antibioticus and of Micromonospora sp. gave no activity against E. coli; however, A. lavendulæ and the 2 strains of A. griseus exerted an appreciable effect upon this bacterium. Results obtained by the agar diffusion method were found to be comparable to the dilution units for B. subtilis and Staphylococcus aureus. The nature of the medium in which the organisms were grown is of considerable importance in the production of the different antibiotic agents: streptothricin is produced most abundantly in a tryptone-starch medium, and streptomycin in ordinary nutrient broth, namely, a peptone-meat extract medium.Table I: Production of Antibiotic Substances by 5 Actinomycetes*A further study of the influence of the composition of the medium upon the production of the active agent by A. griseus brought out the fact that, whereas streptothricin is formed abundantly in a simple medium, streptomycin requires the presence of a specific growth-promoting substance supplied by meat extract. Corn steep liquor can take the place of the meat extract. Addition of glucose further increases the yield of the substance. The nature of the protein hydrolyzate is apparently immaterial, since tryptone gave about the same degree of activity as peptone. A medium was finally adopted, consisting of 1% glucose, 0.5% peptone, 0.3% meat extract or 1.2% corn steep, and 0.5% NaCl. The course of production of streptomycin under submerged and stationary conditions is brought out in Table II. The organism did not form any acid either in the submerged or in stationary cultures, as was found to be the case of A. lavendulæ,7 the reaction of the medium becoming alkaline even in the presence of glucose. Growth was much more rapid in shaken cultures, although very good activity was also obtained in a stationary condition.Table II: Metabolism of A. griseus and Course of Production of StreptomycinThe growth of the organism was allowed to proceed for 5 to 12 days and the streptomycin isolated from the culture filtrate, using a method similar to that developed previously for the isolation of streptothricin.1 The antibacterial behavior of streptomycin, as compared with that of streptothricin, can best be illustrated by an examination of the respective bacteriostatic spectra for the two substances, as presented in Table III. Concentrated preparations, although not of the same degree of purificaton, of the materials were used. Taking the activity against E. coli as a standard, streptomycin was found to have the same activity as streptothricin against B. subtilis, A. aerogenes, and P. vulgaris; it was less active against S. aureus and certain strains of Salmonella; it was much more active against B. mycoides, B. cereus, Mycobacterium phlei, Serratia marcescens, Ps. aeruginosa, Ps. fluorescens, and Cl. butylicum.Table III: Comparative Bacteriostatic Spectra of Streptomycin and Streptothricin. On Basis of Crude, Ash-Free Dry MateralStreptomycin, like streptothricin, possesses strong bactericidal properties, and preliminary experiments tended to indicate that the two substances are also comparable in their low toxicity to animals and in their in vivo activity. The various chemical and biological properties of streptomycin tend to point to this compound as one closely related to streptothricin; the fact that it differs from the latter in the nature of its antibacterial activity may indicate a closely related but not the same type of molecule. Summary. A new antibacterial substance, designated as streptomycin, was isolated from two strains of an actinomyces related to an organism described as Actinomyces griseus. This substance resembles streptothricin in its solubility in water, mode of isolation and concentration from culture medium, its selective activity against gram-negative bacteria, and its limited toxicity to animals. However, the two substances differ in the nature of their respective bacteriostatic spectra as well as in their quantitative action upon different bacteria. It is suggested that one is dealing here with two closely related chemical compounds.