US3794721A

US3794721A - Antibiotic steffimycin b and process for the preparation thereof

Info

Publication number: US3794721A
Application number: US00283908A
Authority: US
Inventors: T Brodasky; F Reusser
Original assignee: Upjohn Co
Current assignee: Pharmacia and Upjohn Co
Priority date: 1972-08-28
Filing date: 1972-08-28
Publication date: 1974-02-26
Anticipated expiration: 1991-02-26
Also published as: DE2339136A1; JPS5641231B2; FR2197574A1; JPS4942895A; FR2197574B1; NL7311551A; GB1387080A

Abstract

New antibiotic, steffimycin B produced by the controlled fermentation of the new microorganism Streptomyces elgreteus Dietz sp. n. in an aqueous nutrient medium. Steffimycin B is active against Gram-positive bacteria and can be used in various environments to eradicate or control such bacteria.

Description

United States Patent [191 Brodasky et al.

451 Feb. 26, 1974 ANTIBIOTIC STEFFIMYCIN B ANl) PROCESS FOR THE PREPARATION THEREOF [75] Inventors: Thomas F. Brodasky, Kalamazoo;

Fritz Reusser, Portage, both of Mich.

[73] Assignee: The Upjohn Company, Kalamazoo,

Mich.

22 Filed: Aug. 28, 1972 211 App]. N0.: 283,908

52 us. Cl. 424/120 [5]] Int. Cl A6lk 21/00 [58] Field of Search 424/120 Primary Examiner-Albert T. Meyers Assistant ExaminerDaren M. Stephens Attorney, Agent, or Firm-Roman Saliwanchik [57] ABSTRACT New antibiotic, steffimycin B produced by the controlled fermentation of the new microorganism Slreptomyces elgreteus Dietz sp. n. in an aqueous nutrient medium. Steffimycin B is active against Grampositive bacteria and can be used'in various environments to eradicate or control such bacteria.

4 Claims, 3 Drawing Figures PAH-INTEDFms x974 SHEET 1 BF 3 lllllllllllll llllllllllllllllll F ucaui NOISSIWSNVHJ.

PATENTEDFEBZB 1974 3.794.721 sum 2 [IF 3 BRIEF SUMMARY OF THE INVENTION DETAILED DESCRIPTION OF THE INVENTION CHEMICAL AND PHYSICAL PROPERTIES OF STEFFIMYCIN B v CI-IL,1 Elemental Analysis: Calcd. for C I-I c, 59.18; H, 5.48; 0,- 3534 1 Found: C, 58.68; H, 5.58; O, 35.74 (diff) Molecular Weightf588 (determinedby mass spectrometry). Specific Rotation: [(110 94 (c, 2, CHC1 Ultraviolet Absorption Spectra:

:A max. at 234 p. (a 47.3) sh at 254 1.(11 36.9)

sh at 274 p. (a 33.3)

:A max. at 223 p. (a 53.7) sh at 259 u (a 45.0)

:A max. at 234 p. (a 46.7) sh at 254 p. (a 36.6)

sh at 274 p. (a 33.0)

In ethanol In O.lN alcoholic KOH mam alcoholic HCl Infrared Absorption Spectra: Infrared absorption spectrum of steffimycin B suspended in a mineral oil mull is reproduced in FIG. I of the drawings. Steffimycin B shows peaks at the following wave lengths expressed in reciprocal centimeters:

Band Frequency I Band Frequency (cm Intensity (cm Intensity 3610 (sh) W 1230 S 3550 (sh) M I200 S 3530 S I185 S 3410 M 1165 S 3070 (sh) W 1 I35 S 2930 (oil) s 1110 s 2880 (Oil) S I087 S I717 (sh) .M I077 S 1710 S 1056 S I674 M 1040 S I625 S 1015 S I603 S 1004 S I568 (sh) M 995 M 1560 M 963 S 1470 (sh) S 927 M I460 S 918 M I445 (sh) S 880 (sh) W 1440 (sh) S 870 M 4 l4l0 S 865 M I390 S 845 M 1375 S 831 W 1345 M 827 W 1320 (21h) S 818 W 1312 S 803 M 1287 S 790 M 1270 S 761 S Band Frequency Band Frequency (cm' Intensity (cm Intensity Following is a tabulation.of the infrared absorption spectrum wave lengths when the compound is pressed into a KBr pellet:

Band Frequency Band Frequency (cm Intensity (cm Intensity 3520 v M I275 (sh) S 3480 (sh) M I240 S 3070 W 4 1230(sh) S 2980 M 1200 S 2940 5 I (III!) S 29IOUth) M I164 S I7I7 (ah) M I I35 5 1710 S I I I0 S I675 M 1095 (sh) S 1620 S 1077 (sh) S 1605 (sh) S 1055 S 1560 M 1035 S 1475 (sh) S IOI5 S I458 S I005 (sh) S 1450 S 990 S 1408 S 960 S I389 S 925 M I375 (sh) S 917 M 1320 (sh) S 880 (sh) W 1310 S 865 M 1288 S 845 M 830 M 8.18 W 803 M 788 M 761 S 742 M 730 M 715 M 700 M 687 M Infrared band intensities, throughout this disclosure,

are indicated as S, M, and W", respectively, and 1 and, W bands are less than /a as intense as the strongest band. These estimates are made on the basis of a percent transmission scale. sh denotes a shoulder.

Nuclear Magnetic Resonance (NMR): Steffimycin B has a characteristic NMR spectrum as shown in FIG. 2 of the accompanying drawings. The NMR spectrum was recorded on a Varian A-60 spectrometer on a solution (ca. 0.5 ml., ca. 15 percent concentration) of the sample of steffimycin B in deutero-chloroform (CDCI The spectrum was calibrated against internal tetramethylsilane and v the precision of the Av was i l c.p.s. Frequencies were recorded in c.p.s. downfield from tetramethylsilane.

Papergram: Steffimycin B is distinguished from steffimycin by a characteristic papergram R, as shown in FIG. 3 of the accompanying drawings.

Mobile phase: Benzene 1 part; methanol 1 part;

water 2 parts (use only upper phase for development) Matrix: Schleicher and Scheull 589 Blue Ribbon paper Detecting Organism: S. lutea Antibacterial Activity Of Stcffimycin B Minimum Inhibitory Con Inhibitory in mcg./ml.

Test Microorganism Staphylococcus aureus 4.0 Streptococcus hemolyricus 7.8 i 4.0 Streptococcus faecalis 7.8 Escherichia coli 500 Proteus vulgaris 500 Klebsiella pneumoniae 500 Pseudomonas aeruginosa 500 Diplococcus pneumoniae The tube dilution assay procedure was conducted with the medium BHI (Brain Heart Infusion Broth, Difco, Detroit, Mich.). Assay tubes (13 mm. X 100 mm.) were prepared in the customary manner as set out in Snell, E. E., Vitamin Methods, Volume 1, Academic Press, Inc., New York 1950, p. 327. Test organisms grown for 18 hours at 37 C. were used to inoculate the test medium. The assays were read at 17 hours.

THE MICROORGANISM DESCRIPTION OF THE MICROORGANISM Taxonomy: Streptomyces elgreteus Dietz sp. n.

Color characteristics: Aerial growth lavender-gray to gray-pink. Melanin-negative. Appearance on Ektachrome is given in Table 1. Reference color characteristics are given in Table 2. The culture may be placed in the Gray (GY) or Red (R) color groups of Tresner and Backus (Tresner, H. D., and E. J. Backus. 1963. System of color wheels for streptomycete taxonomy. Appl. Micro-biol. 11:335-338).

Microscopic characteristics: Sporophores long,

straight (RF) in the sense of Pridham et a1. (Dridham, T. G., C. W. Hesseltine, and R. G. Benedict. 1958. A guide for the classification of streptomycetes according to selected groups. Placement of strains in morphological sections. Appl. Microbiol. 6:52-79). Spores, examined with the transmission electron microscope, appeared long (often rectangular) with flattened ends and smooth surface by direct examination; by examination of carbon replica preparations, the spore surface appears to have peeled and looks curly. The spore surface by direct examination with the scanning electron microscope was hairy with a wooly appearance. Electron microscope procedures were those of Dietz and Mathews (Dietz, A., and J. Mathews. 1962. Taxonomy by carbon replication. I. An examination of Streptomyces hygroscopicus. Appl. Microbiol. 102258-263; see also Appl. Microbiol. 21:526-533).

Cultural and biochemical characteristics: Cultural and biochemical characteristics are described in Table 3.

Carbon utilization: In the synthetic medium of Pridham and Gottlieb (Pridham, T. G., and D. Gottlieb. 1948. The utilization of carbon compounds by some Actinomycetales as an aid for species determination. J. Bacteriol. 56:107-114), S. elgreteus had poor growth on the control (basal medium without added carbon compound), D-xylose, L- arabinose, lactose, raffinose, inulin, dulcitol, D- mannitol, D-sorbitol, salicin, sodium acetate, and sodium citrate; moderate growth on rhamnose, D- fructose, maltose, sucrose, dextrin, and sodium succinate; good growth on D-galac-tose, D- glucose, D-mannose, cellobiose, soluble starch, glycerol, and inositol; no growth on phenol, cresol, sodium formate, sodium oxalate, sodium tartrate, and sodium salicylate. In the synthetic medium of Shirling and Gottlieb (Shirling, E. B., and D. Gottlieb. 1966. Methods for characterization of Streptomyces species. Int. J. Syst. Bacteriol. 16:313-340), the culture grew poorly on the negative control (synthetic medium without added carbon compound) and on D-xylose and D-mannitol; well on the positive control (synthetic medium with D-glucose), and inositol, and moderately well on D-fructose and rhamnose. The culture did not grow on O-arabinose, sucrose, raffinose, or cellulose.

Temperature: S. elgreteus had good aerial growth on Bennetts agar at 18C-28C and on maltosetryptone agar at 24C28C. The culture had fair vegetative growth on these media at 37C and no growth in 24 hours at 45C and 55C.

Antibiotic-producing properties: The culture produces steffimycin B and some steffisburgensimycin (U. S. Pat No. 3,309,273), also known as steffimycin.

Source: Soil.

Type culture: Streptomyces elgreteus sp. n. UC 5453.

DISCUSSION A new soil isolate capable of producing the antibiotic steffimycin has been characterized and determined to be a new streptomycete species. The new culture is distinctly different from the steffimycin-producer, Streptomyces steffisburgensis (Dietz, A. 1967. Streptomyces steffisburgensis sp. n. J. Bacteriol.

9420222026). The culture has lavender-gray to graypink aerial growth, is melanin negative, solubilizes tyrosine, does not solubilize xanthine, and has straight sporophores bearing long spores with smooth to curly to hairy surface appearance. S. stefflsburgensis has gray aerial growth, is melanin positive, does not solubilize tyrosine, solubilizes xanthine, and has short, straight, open spiral to spiral sporophores bearing short spiny (Dietz, A. 1967. Streptomyces steflisburgensis sp. n. J. Bacteriol. 94:2022-2026) or warty (Dietz, A., and J. Mathews. 1970. Classification of Streptomyces spore surfaces into five groups. Appl, Microbiol. 21:527-533) spores. The new soil isolate differs from cultures characterized in Waksman (Waksman, S. A. 1961. The Actinomycetes, Vol. 2. Classification, Identificatiomand Descriptions of Genera and Species. The Williams & Wilkins Co., Baltimore), Hutter (l-Iutter, R. l967. Systematik der Streptomyceten. S. Karger, Basel. 382p), and Shirling and Gottlieb (Shirling, E. B., and D. Gottlieb. 1968. Cooperative description of type cultures of Streptomyces. II. Species descriptions from first study. Int. J. Syst. Bacteriol. 18169-189. Shirling, E. B., and D. Gottlieb. I968. Cooperative description of type cultures of Streptomyces. Ill. Additional species de-- scriptions from first and second studies. Int. J. Syst. Bacteriol. 18:279-399. Shirling, E. B., and D. Gottlieb. I969. Cooperative description of type cultures of Streptomyces. IV. Species descriptions from the second, third and fourth studies. Int. J. Syst. Bacteriol. 192391-512) and from cultures in The Upjohn Culture Collection. Therefore, it is proposed that the culture characterized herein be designated Streptomyces elgreteus Dietz sp. n. It is understood that this type species is also to becqnsigered the type variety in accordance with Rule 7 of the International Code of Nomenclature of Bacteria (INTERNATIONAL CODE OF NOMENCLATURE OF BACTERIA. 1966. Edited by the Editorial Board of the Judicial Commission of the TABLE "2 International Committee on Nomenclature of Bacteria. Intern. J. System. Bacteriol. 162459-490).

The characteristics of Streptomyces elgreteus NRRL 5634, are given in the following tables:

Dietz, A. I954. Ektachrome transparencies as aids in actinom ycele classific Ann. N.Y. Acad. Sci. 6(l2l52-l54.

Reference Color Characteristics of Srrepromyces elgreteus Color Harmony Manual Agar Medium 3rd Ed. l948* NBS Circular 553. 1955" Bennett's S 2fe' covert gray 94g light olive brown I I2 gm light olive gray R 31;: beige brown, mist hrown 80m grayish yellowish hrown 95g moderate olive hrown P Zge covert tan, gricge 94m light olive brown 109 gm light grayish olive Czapek's sucrose S 3:10 natural R 3ge beige, camel 79m light grayish yellowish brown 7 94m light olive brown P 4ge light fawn, rose beige 60gm light grayish brown Maltose-tryptone S Sdc pussywillow gray lOgm pinkish gray R 4ie cork tan 57gm light brown P 4ge light-fawn, rose beige 60gm light grayish brown Yeast extract- S 3drnatural malt extract (|SP-2) R 5 lg cocqaQ-gygn, rosewood Alg rmderate reddish brown r tan, russel tan 46m grayish reddish brown P 61;: dark redwood V l9m g rayish red 46gm moderate reddish brown Oatmeal S 3dc natural (lSP-3) R 3dr natural P inorganic salts S 517a shell pink 9m pinkish white starch (lSP-A) R Sha shell pink 9m pinkish White F Glycerol-asparagine S 3fe silver gray 63gm light brownish gray (lSP-S) I R Zig slate'tan l 10g grayish olive l l2m light olive gray p S surface R reverse P pigment .Jacohson. E.. W. C. Granville. and C. E. Foss. I948. Color harmony manual.

"Kelly. K. L. and D. B. Judd. I955. The ISCC-NBS' method of designating colors and a dictionary of color names. US. Dept. of Comm. Circ. 553. Washington. D.(..

3rd ed. Container Corporation of America. Chicago.

FEE 3 alion.

Cultural and Biochemical Characteristics of S. elgrereus Medium Surface Reverse Other Characteristics Agar Peptone-iron Tan Pale Tan I Melanin negative Calcium malate Gray-pink aerial Gray-pink Pink pigment growth Ma-late not solubilized Glycerol asparagine Gray-pink Pink-tan Pink pigment TABLE 3 Continued Cultural and Biochemical Characteristics of S. elgreu-us ring Synthetic nitrate Nutrient nitrate The new compound of the invention is produced when the elaborating organism is grown in an aqueous nutrient medium under submerged aerobic conditions. It is to be understood also that for the preparation of limited amounts surface cultures and bottles can be employed. The organism is grown in a nutrient medium containing a carbon source, for example, an assimilable carbohydrate, and a nitrogen source, for example, an assimilable nitrogen compound or proteinaceous material. Preferred carbon sources include glucose, brown sugar, sucrose, glycerol, starch, cornstarch, lactose, dextrin, molasses, and the like. Preferred nitrogen sources include cornsteep liquor, yeast, autolyzed brewers yeast with milk solids, soybean meal, cottonseed meal, cornmeal, milk solids, pancreatic digest of casein, distillers solids, animal peptone liquors, meat and bone scraps, and the like. Combinations of these carbon and nitrogen sources can be used advantageously. Trace metals, for example, zinc, magnesium, cobalt, iron, and the like, need not be added to the fermentation media since tap water and unpurified ingredients are used as components of the medium.

Production of the compounds of the invention can be effected at any temperature conducive to satisfactory growth of the microorganism, for example, between about 18 and 40 C., and preferably between 20 and 32 C. Ordinarily, optimum production of the compounds is obtained in about 2 to 10 days. The medium normally remains basic during the fermentation. The final pH is dependent, in part, on the buffers present, if any, and in part on the initial pH of the culture medium.

When growth is carried out in large vessels and tanks, it is preferable to use the vegetative form, rather than the spore form, of the microorganism for inoculation to avoid a pronounced lag in the production of the new" compounds and the attendant inefficient utilization of Medium Surface Reverse Other Characteristics Skim milk Yellow-tan Yellow-tan pigment Casein not solubilized Tyrosine Pale tan Pale tan pigment Tyrosine solubilized Xanthine Cream Cream pigment Xanthine not solubilized Nutrient starch Pale gray aerial Pink-tan Pinktan pigment growth Starch not hydrolyzed Yeast extract- Pale gray-pink Red-tan Deep pink pigment malt extract aerial growth Peptone-yeast Colorless Melanin negative extract-iron (lSP-6) Tyrosine Gray aerial growth Yellowtan Yellow-tan (lSP-7) Melanin negative Gelatin Plain Colorless to tan Yellow-red pigment red surface ring Liquefaction, A Nutrient Colorless surface Yellow pigment ring Liquefaction, Ax-V2 Broth Litmus milk Brown surface pH 6.7

Flocculent colorless bottom growth Nitrate not reduced to nitrite Cloudy throughout Compact bottom growth Nitrate not reduced to nitrite the equipment. Accordingly, it is desirable to produce a vegetative inoculum in a nutrient broth culture by inoculating this broth culture with an aliquot from a soil or a slant culture. When a young, active vegetative inoculum has thus been secured it is transferred aseptically to large vessels or tanks. The medium in which the vegetative inoculum is produced can be the same as, or different from, that utilized for the production of the new compound, as long as it is such that a good growth of the microorganism is obtained. 7

Steffimycin B forms salts with alkali metals, alkaline earth metals, and amines. Metal salts can be prepared by dissolving Steffimycin B in methanol, adding a dilute metal base until the pH of the solution is about 7 to 8, and freeze drying the solution to provide a dried resi- .due consisting of the Steffimycin B metal salt. Steffimycin B metal salts include the sodium, potassium, and calcium salts. Amine salts of Steffimycin B, including those with organic bases, such as primary, secondary, and tertiary, mono-, di-, and polyamines also can be formed using the above-described rather commonly employed procedures. Other salts are obtained with therapeutic effective bases which impart additional therapeutic effects thereto. Such bases are, for example, the purine bases such as theophyllin, theobromin, caffeine, or derivatives of such purine bases; antihistaminic bases which are capable of forming salts with weak acids; pyridine compounds such as nicotinic acid amide, isonicotinic acid hydrazide, and the like; phenylalkylamines such as adrenaline, ephedrine, and the like; choline, and others.

Steffimycin B and its salts are active against Staphylococcus aureus and Streptococcus faecalis and used as disinfectants on various dental and medical equipment contaminated with Staphylococcus aureus. Further, since steffimycin B and its salts are active against Streptococcus hemolyticus, they can be used to disinfect instruments, utensils or surfaces where the inactivation of this microorganism is desirable.

The new compound of the invention is an acidic chemical compound. it is soluble in halogenated hydrocarbons and lower alcohols. it is relatively insoluble in hydrocarbons and water.

A variety of procedures can be employed in the isolation and purification of steffimycin B, for example, solvent extraction, partition chromatography, silica gel chromatography, liquid-liquid distribution in a Craig apparatus, absorption on resins, and crystallization from solvents. Solvent extraction procedures are preferred for commercial recovery inasmuch as they are less time consuming and less expensive.

In a preferred recovery process, steffimycin B is recovered from its culture medium by separation of the mycelia and undissolved solids by conventional means, such as by filtration or centrifugation. The antibiotic is then removed from the filtered or centrifuged broth by extraction. For the extraction of steffimycin B from the filtered broth, water-immiscible organic solvents in which it is soluble, for example, l-butanol, methyl ethyl ketone, benzene, and methylene chloride (preferred) can be used. Advantageously, the extraction is carried on after the filtered beer is adjusted to a pH of about 2 to 4 with a mineral acid. The methylene chloride extractions are combined and evaporated to dryness under vacuum.

Steffimycin B can be recovered in the pure form from a methylene chloride extract, as described above, by use of silica gel chromatography.

Alternatively, steffimycin B can be recovered from fermentation filtered broth by use of a resin comprising a non-ionic macro porous copolymer of styrene crosslinked with divinylbenzene. Suitable resins are Amberlite XAD-l and XAD-2 disclosed in U. S. Pat. No. 3,515,717. The resin is eluted with an organic or aqueous organic solvent in which the sorbed antibiotic is soluble.

An alternative purification procedure involves subjecting a crude preparation of steffimycin B to successive crystallizations from lower alcohols, for example, methanol, ethanol, propanol, and the like; chlorinated hydrocarbons, for example, chloroform, methylene chloride, and the like; hydrocarbon solvents, for example, pentane, hexane, 'heptane, and the like; or admixtures of any of these.

it is to be understood that the microbiological process disclosed herein, though described in detail with particular references to the novel microorganism Streptomyces elgreteus sp. n., NRRL 5634, is not limited to this particular microorganism fully described by the cultural charcteristics disclosed herein. It is intended that this invention also include other strains or mutantsof the said microorganism which can be produced by procedures well known in the art, for example, by subjecting the novel microorganism to x-ray or ultraviolet radiation, nitrogen mustard, phage exposure, and the like.

Hereinafter are described non-limiting examples of the process and products of the subject invention. All percentages are'by weight and all solvent mixture proportions are by volume unless otherwise noted.

Example 1 Part A. Fermentation Mycelium and/or spores from a slant of Streptomyces elgreteus sp. n., NRRL 5634, are used to inoculate a Series of SOO-ml. Erlenmeyer flasks each containing 100 ml. of seed medium consisting of the following ingredi-. ents:

2 5 gm./l. 25 gm./l. l liter Glucose monohydrate Pharmamedia Tap water q.s.

Pharmamedia is an industrial grade of cottonseed flour produced by Trader's Oil Co., Ft. Worth, Texas.

The presterilization of the seed medium is 7.2 The seed is grown for three days at 28 C. on a Gump rotary shaker operating at 250 r.p.m.

- The fermentation proper is carried out in the medium of the following composition:

Blackstrap molasses 25 gm./l. Yellow cornmeal 20 gm./l. Pharmamedia l5 gm./l.

Day Assay (BU/ml.)

l 0 2 4 3 7 4 l6 5 l3 The assay was an agar disc-plate assay against the microorganism Sarcina lutea. The assay against Sarcina lutea is conducted on agar buffered to pH 7.4 with pH 7.4 phosphate buffer. A unit volume (0.08 ml.) of solution containing the substance to be assayed is placed on a 12.7 mm. paper disc which is then placed on a Penassay Seed Agar (Difco), l mm.'deep, plate seeded with the assay organism. The agar plate is then incubated for 16-18 hours at 32 C. A biounit (BU) is defined as the concentration of the antibiotic which gives a 20 mm. zone of inhibition under the standard assay conditions. Thus, if for example a fermentation beer-has to be diluted 1/100 to give a 20 mm. zone of inhibition, the potency of such beer is BU per ml. Part B. Recovery 7 Whole fermentation beer, obtained as described above, is filtered using diatomaceous earth as a filter aid. The filter cake is washed with water. The wash is combined with the clear beer and the combined solution is extracted twice with an equal volume of methylene chloride. The methylene chloride extracts are combined and evaporated to dryness at less than 40 C. under vacuum. The potencies of the clear broth, spent broth and extracts are determined by dip spotting (12 mm. discs) on S. lutea seeded agar. (The potency of the preparation is determined by preparing a 50; meg/ml.-

solution in CH Cland making serial dilutions to y/ml., 80 mcl. are applied to 12 mm. paper discs and spotted on S. lutea seeded agar. The dilution giving a 20 mm. zone of inhibition determines the number of biounits.) The composition of the preparation is determined by thin layer chromotography (tlc) on silica gel using a mobile phase consisting of the following solvents:

CH2Cl2-30 parts Acetoneparts Hexane-- 3 parts Methanol-Z parts 1 The chromatography column is wet packed (using the mobile phase described above) with 50 to 100 gm. of silica gel H(Merck/gm. of steffimycin B preparation to be processed. After the column has settled, a solution of the steffimycin B preparation in a minimum volume of CH Cl is charged onto the column. The column is eluted with the mobile phase, described above, at a flow rate of approximately 10 ml./min. During the elution, a number of colored bands develop. The fraction containing the first faint yellow band is discarded and the fraction containing the first deep orange band is collected. This fraction is cut so that none of the second red-orange band which contains steffimycin is collected. The purity of steffimycin B is checked by tlc on silica gel using the above mobile phase. The purified steffimycin B, thus obtained, does not contain any other colored (or fluorescent) material by tlc.

Steffimycin B is obtained in its pure crystalline form by subjecting the fraction containing steffimycin B, as described above, to successive crystallization procedures from methanol.

We claim: 1. Steffimycin B, a compound which: a. is effective in inhibiting the growth of various Gram-positive bacteria;

7 b. has an optical rotation [a]p h4 kjijiilgx c. has a characteristic ultraviolet absorption spectrum as follows:

In 0.1N alcoholic KOH ln 0.1N alcoholic HCl d. has a characteristic infrared absorption spectrum in mineral oil mull as reproduced in FIG. 1 of the drawings;

e. has the following elemental analyses: C, 58.68; H, I

f. has a characteristic nuclear magnetic resonance spectrum as shown in FIG. 2 of the drawings;

g. has a characteristic R; in the chromatographic system, as shown in FIG. 3, which differentiates it from steffimycin; and

h. as a molecular weight of 588 as determined by mass spectrometry, or non-toxic base addition salts thereof.

2. A process for making steffimycin B, the compound of claim 1, which comprises cultivating Streptomyces elgreteus sp. n., having the identifying characteristics of NRRL 5634, in an aqueous nutrient medium under aerobic conditions until substantial antibiotic activity is imparted to said medium by the production of steffimycin B.

3. A process, according to claim 2, wherein said aqueous nutrient medium contains a source of assimilable carbohydrate and assimilable nitrogen.

4. A process, according to claim 2, wherein said steffimycin B is isolated from the fermentation broth.

UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent No. 3,79 ,7 1 Dated February 26, 1974 Inventofls) Thomas r y, Fritz RGU SSG-l' It is' certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:

Column 1, l ine 2 l, For "'CHL, 1 Elemental" read Elemental Column 2, line 65, for "Scheull" read Schuel l Column 5, l ines 5-6, For "Con- Inh ibitory" read Concentration Column 3, line-52, for "Dridham" read Pridham Column l, line 18, For "D-galac-tose" read D-galactose Column l, line 52, for O-arabinose read L-arabinose Column 1, line 65, for "Appl,"

read Appl. Column 5,. table2, for "P tan, russet tan" read tan, ru'sset 'tan Column 11', line 1 1, for

"(Merck/gm. read (Merck)/gm.

Signed and sealed this 10th day of Septemb er 1971 (SEAL) Attest:

McCOY M. GIBSON, JR. f Y c. MARSHALL DANN Attesting Officer Commissioner of Patents

Claims

2. A process for making steffimycin B, the compound of claim 1, which comprises cultivating Streptomyces elgreteus sp. n., having the identifying characteristics of NRRL 5634, in an aqueous nutrient medium under aerobic conditions until substantial antibiotic activity is imparted to said medium by the production of steffimycin B.
3. A process, according to claim 2, wherein said aqueous nutrient medium contains a source of assimilable carbohydrate and assimilable nitrogen.
4. A process, according to claim 2, wherein said steffimycin B is isolated from the fermentation broth.