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Neomycin—Production and Antibiotic Properties

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Neomycin—Production and Antibiotic Properties NEOMYCIN—PRODUCTION AND ANTIBIOTIC PROPERTIES Selman A. Waksman, … , Hubert A. Lechevalier, Dale A. Harris J Clin Invest. 1949;28(5):934-939. https://doi.org/10.1172/JCI102182. Research Article Find the latest version: https://jci.me/102182/pdf NEOMYCIN-PRODUCTION AND ANTIBIOTIC PROPERTIES 1, 2 3 By SELMAN A. WAKSMAN, HUBERT A. LECHEVALIER, AND DALE A. HARRIS (From the Department of Microbiology, New Jersey Agricultural Experiment Station, Rutgers University, New Brunswick, N. J.) ANTIBIOTIC SURVEYS All these cultures proved to be highly active against mycobacteria. of During the last 10 years, a large number anti- Only few of the cultures, however, that gave biotics which are active against Gram-negative good activity by the agar-streak method yielded and Gram-positive bacteria, mycobacteria, rickett- filtrates which possessed corresponding potency. siae, and certain of the larger viruses were iso- This may be due to a variety of factors, such as lated (1) from various species and strains of the the formation by a single organism of more than genus Streptomyces. This served to focus atten- one antibiotic or the production of different anti- tion on the actinomycetes as potential producers of biotics under different conditions of culture. One antimicrobial agents that might possess promising of these cultures proved to be highly promising chemotherapeutic properties. The fact that nearly and was selected for more detailed investigations. 20 to 50%o of these organisms possess antimicrobial This culture was entered into the Collection as No. activities served to heighten this interest. Numer- 3535. The nature of its antimicrobial spectrum, ous surveys have been conducted. Particular as compared to corresponding spectra of known attention has been paid to the formation and iso- antibiotic-producing organisms and measured by lation of antibiotics that would possess the follow- the agar-cross-streak method, is shown in Table I. ing characteristics: (a) High activity against The spectrum of 3535 was found to be quite di- Gram-negative bacteria and mycobacteria; (b) stinct from those of the streptomycin-producing antibiotic action against streptomycin-resistant Streptomyces griseus and streptothricin-producing bacteria; (c) low toxicity to animals; (d) other Streptomyces lavendulae. The streptomycin-re- desirable properties, such as activity against rick- sistant strain of Escherichia coli and the Boden- ettsiae, viruses, tumors and phages. heimer organism were found to be sensitive to the In our own laboratories, large numbers of ac- antibiotic activity of 3535; further, the antibiotic tinomycetes were isolated from various natural substrates and tested for their antimicrobial activi- TABLE I ties. The agar-cross-streak method, frequently Antibiotic spectrum of culture No. 3535 as compared was to spectra of streptothricin-producing (S. Iavendulae) supplemented by other procedures, commonly and streptomycin-producing (S. griseus) organisms used for screening purposes. Those cultures that Agar-cross-streak method. Zone of inhibition, in mm. proved to be most active were selected and grown in liquid media. Only a small number of these Culture S. griseus S. lavendulac were found of rise to active anti- Test organism* No. capable giving 3535 biotics. The most promising were selected for 3463 3516 Escherichia coli SS 18 Active 20 further studies. For example, of some 300 freshly E. coli RS 25 None Active isolated cultures tested by the above method, only E. coli DS NG GG NG Bacillus subtiis 21 Active 22 10 exhibited activities that justified further study. B. mycoides 20 Active 3 B. cereus 18 Active 5 1 Presented at the Second National Symposium on Staphylococcus aureus 19 Active 19 Recent Advances in Antibiotics Research held in Wash- Bodenheimer's culture 19 None Active Pseudomonas aeruginosa 3 LA LA ington, D. C., April 11-12, 1949, under the auspices of the Proteus vulgaris 22 Active Active Antibiotics Study Section, National Institutes of Health, Mycobacterium 607 24 25 25 Public Health Service, Federal Security Agency. M. avium 20 20 23 2 Paper in the Journal Series, New Jersey Experiment M. ranac 15 25 23 Station, Rutgers University-The State University of M.phli 28 27 26 New Jersey, Department of Microbiology. * RS = re- 8 These investigations were supported by a grant from SS- streptomycin-sensitive; streptomycin- sistant; DS - streptomycin-dependent; NG - no growth; the Rutgers Research and Endowment Foundation. GG = good growth; LA = limited activity. 934 NEOMYCIN-PRODUCTION AND ANTIBIOTIC PROPERTIES 935 TABLE II culture. This effect was overcome by addition of CaCO,, Effect of CaCO: upon the production of antibiotic 3535 as brought out in Table II. This could also be accom- plished by increasing the peptone content of the medium Incubation, No CaCOs CaCOa. 1% to 1 or even 2%, particularly when vegetable peptones, days such as soya peptone, were used, or by reducing the sugar pH u./ml. pH U./ml. content. If sufficient peptone were used, the glucose con- 2 5.6 25 6.8 35 tent could also be increased, with a delaying but favorable 3 5.8 47 6.7 88 effect on the production of the antibiotic. The results of 5 6.1 68 7.8 172 6 7.7 156 8.0 200 a typical experiment are given in Table III. The addi- tion of a small amount of zinc (1 mg. to 10 mg. ZnSO4, 7HO per liter) was later found to exert a favorable substance produced by 3535 cannot replace strep- tomycin in making possible the growth of the TABLE IV Influence of composition of improved media upon the streptomycin-dependent strain of E. coli. The ac- production of antibiotic 3535 tivity of 3535 upon Bacillus mycoides and Bacillus Dilution units per 1 ml. of medium; cup readings made cereus serves to differentiate this antibiotic from against a neomycin standard streptothricin. The various mycobacteria were Nature B. Cup found to be sensitive alike to the antibiotic pro- Of pH E. coli S. aurens B. subtliis readings medium mciemycides duced by 3535 and tb the other two antibiotics. three days incubation METHODS N2* 7.8 50 240 150 750 35 Formation and isolation of antibiotic 3535 N4t 7.6 50 300 300 > 1,000 24 Ns 7.5 25 100 75 500 28 The next steps in the study of the new antibiotic com- No 7.4 150 > 1,000 > 1,000 > 1,000 154 prised the development of suitable media for its maximum N. 6.5 <30 <30 <30 240 <5 production. Fortunately, media previously found to be best for the production of streptomycin proved suitable. four days incubation A medium containing 5 gm. peptone, 5 gm. meat extract, N, 8.3 50 300 240 1,000 56 5 gm. glucose, 5 gm. NaCl, 1,000 ml. tap water gave good N4 7.8 150 300 300 2,400 85 results. Various modifications were later introduced. N, 8.3 75 240 300 > 1,000 68 The reaction of the medium tended to become acid during N, 8.0 500 2,400 1,500 > 10,000 316 the early stage of growth; this favored early lysis of the N7. 6.0 <10 30 10 >100 <5 TABLE III six days incubation Influence of composition of medium on the production N. 8.7 100 3007' 240 1,000 56 of antibiotic 3535 N4 8.7 200 1,000 900 >3,000 128 N, 8.7 90 240 240 1,000 74 Constituents of medium, N Ni N| Ns No. 3 N9 8.7 000 7,500 3,000 > 10,000 407 gm./liter N7. 8.4 30 150 100 >300 24 Meat extract 5 5 5 5 Soya peptone 10 10 20 10 N2 = Soya peptone-2%, glucose-1%, NaCl-0.5%, Bacto peptone 5 meat extract-0.5%, tap H2O-1000 ml. NaCl 5 5 5 5 5 t N4- N. minus meat extract; N, = N2 minus NaCl; Glucose 10 10 10 20 10 N, - N. + 10 mg./liter ZnSO4*7H20; N7. = N2 + 2% Tap water 1000 1000 1000 1000 1000 glucose. Incubation, days effect, yielding culture filtrates with an activity of 500 to Medium | 1,000 u./ml., as shown in Table IV. Larger amounts of 13 1 4 1 6 zinc proved to be injurious, however. Meat extract could be replaced by yeast extract. Glucose could be IH* u./ml.t pH U./ml. pH u./ml. N 8.3 100 8.4 240 l 8.7 240 replaced by starch in shaken cultures. When distilled Ni 8.2 <10 8.2 10 l 8.4 75 water was used, no activity was obtained unless zinc was 8.6 150 8.6 240 9.0 150 added. N,Ns 7.4 150 7.8 240 7.9 100 Aeration proved to be an important factor in the pro- No. 3 8.4 <10 8.5 10 8.8 10 duction of the antibiotic, the amount of air required being apparently less than for the production of streptomycin * Initial pH of medium 6.5-6.8. - and streptothricin. The reduced aeration could be accom- t u./ml. E. coli dilution units, as measured by agar streak dilution method. plished by increasing the volume of the medium in the 936 SELMAN A. WAKSMAN, HUBERT A. LECHEVALIER, AND DALE A. HARRIS shaken flasks or by adding a small amount of agar to the TABLE V medium. A higher temperature (350 C.) was found to Comparative antibacterial spectra of be more favorable for rapid formation of neomycin than neomycin and streptomycin a lower temperature (26-280 C.); however, a higher level Amounts required to inhibit growth of organism of activity was reached upon prolonged incubation at in 1 ml. of culture 280 C. In measuring the potency or concentration of the new Organism Neomycin* Streptomycint antibiotic, the agar plate dilution method was first used, u./ml.
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