Composition of matter and process

Abstract

Novel antibiotic U-64,815 producible in a fermentation under controlled conditions using a biologically pure culture of the microorganism Streptomyces microspinus, NRRL 12524. This antibiotic is active against various Gram-positive bacteria, for example, Staphylococcus aureus. It is also active against Haemophilus influenzae. Thus, antibiotic U-64,815 can be used in various environments to eradicate or control such bacteria.

Description

BACKGROUND OF THE INVENTION

Antibiotic U-64,815 can be differentiated from the known related antibiotics kijanimicin [A. K. Mallams, et al., J. Amer. Chem. Soc. 103, 3938, 3940 (1981)], antlermicins [K. Kobinata, et al., J. Antibiotics 33, 244 (1980); K. Isono, et al., ibid., 33, 772 (1980)] and tetrocarcins [F. Tomita, et al., J. Antibiotics 33, 668 (1980); T. Tamaoki, et al., ibid., 33, 946 (1980)] on the basis of observed .sup.13 C magnetic resonance spectra. Compound 12 disclosed in the J.A.C.S. article, appears to be identical to antibiotic U-64,815. Final resolution of this identity is in progress.

BRIEF SUMMARY OF THE INVENTION

Antibiotic U-64,815 is producible in a fermentation under controlled conditions using a biologically pure culture of the new microorganism Streptomyces microspinus, NRRL 12524.

Antibiotic U-64,815 has moderate to excellent in vitro activity against all strains of Haemophilus influenzae tested. It is also active against various Gram-positive bacteria. Further, the base addition salts of antibiotic U-64,815 are also active against these bacteria. Thus, antibiotic U-64,815 and its salts can be used to disinfect washed and stacked food utensils contaminated with S. aureus. They can also be used as disinfectants on various dental and medical equipment contaminated with S. aureus. Still further, antibiotic U-64,815 and its salts can be used as a bacteriostatic rinse for laundered clothes, and for impregnating papers and fabrics; and, they are also useful for suppressing the growth of sensitive organisms in plate assays and other microbiological media.

DETAILED DESCRIPTION OF THE INVENTION

Chemical and Physical Properties of Antibiotic U-64,815:

Molecular Weight Range: 1,104-1,324 (based on molecular formula).

Molecular Formula: C.sub.60-65 H.sub.82-102 N.sub.1-3 O.sub.18-25.

Ultraviolet Absorption Spectrum:

The UV spectrum of antibiotic U-64,815 is shown in FIG. 2 of the drawings. The solution of antibiotic U-64,815 in ethanol displayed absorption as follows:

______________________________________ Solvent .lambda. max______________________________________ Ethanol 215 240 267 278 (sh) .1 N HCl 213 in Ethanol 261______________________________________

Melting Point: 185-187 (dec)

Infrared Absorption Spectrum:

Antibiotic U-64,815 has a characteristic infrared absorption spectrum in a mineral oil mull as shown in FIG. 1 of the drawings. Peaks are observed at the following wave lengths:

______________________________________Band BandFreq. Inten. Type Freq. Inten. Type______________________________________3480.0 40 SH * 1196.9 43 BRD3442.3 40 BRD 1170.9 18 AVG3280.0 56 SH * 1149.7 18 SH3058.5 67 SH 1141.9 17 AVG3025.7 56 AVG 1127.5 10 AVG2956.2 0 BRD M 1095.6 8 AVG2931.1 0 BRD M 1059.0 6 AVG2870.4 3 BRD M 1014.6 10 AVG2854.0 2 AVG M 1000.2 14 AVG2726.7 70 BRD M 981.8 14 AVG2661.1 71 AVG 936.5 51 SH2617.7 76 SH 927.8 43 SHP1760.2 16 AVG 914.3 56 AVG1729.3 19 AVG 897.0 56 SHP1629.0 31 AVG 865.1 37 AVG1575.0 37 SH * 831.4 55 AVG1544.2 10 AVG 800.5 70 AVG1510.4 43 AVG 779.3 55 AVG1455.4 9 AVG M 730.1 51 AVG1413.9 37 AVG 704.1 62 AVG1378.3 12 AVG M 667.4 57 SH1366.7 20 AVG 656.8 54 AVG1346.4 26 AVG 619.2 54 SH1311.7 25 AVG 607.6 50 AVG1250.9 26 AVG1220.1 31 AVG______________________________________ Band Freq.: Band Frequencies in Wavenumbers (CM.sup.-1) Inten.: Intensity in Percent Transmittance (% T) Date Type in Local Peak Region: BRD = Broad; AVG = Average; SHP = Sharp; SH = Shoulder Peak List Edited. * Indicates Peaks Added. M: Possible Interference from Mineral Oil.

Antibiotic U-64,815 has a characteristic infrared absorption spectrum when passed into a KBr pellet. Peaks are observed at the following wave lengths:

______________________________________Band BandFreq. Inten. Type Freq. Inten. Type______________________________________3480.0 32 SH * 1170.9 27 AVG3441.4 30 BRD 1141.0 26 AVG3259.1 52 SH 1126.5 18 AVG3029.5 55 AVG 1096.6 14 AVG2971.6 31 AVG 1058.0 11 AVG2931.1 16 AVG 1014.6 16 AVG2911.8 24 SH 999.2 20 AVG2852.0 36 SHP 981.8 21 AVG2799.0 66 SH 937.5 55 AVG2662.0 67 AVG 927.8 50 AVG1761.2 25 AVG 913.4 60 SH1734.2 27 AVG 897.0 59 AVG1631.0 33 AVG 865.1 46 AVG1577.9 41 SH 843.9 60 AVG1545.1 18 SHP 831.4 59 AVG1508.5 43 AVG 800.5 67 AVG1451.6 28 AVG 778.3 59 AVG1434.2 39 SH 732.0 59 AVG1413.9 42 AVG 706.0 65 AVG1384.0 31 AVG 667.4 60 SH1382.1 31 SH 657.8 59 AVG1368.6 34 AVG 618.2 61 SH1346.4 36 AVG 607.6 58 AVG1310.7 36 AVG 507.3 60 AVG1249.0 39 BRD 489.0 59 AVG1235.5 40 BRD 430.0 52 AVG *1221.0 37 AVG______________________________________ Band Freq.: Band Frequencies in Wavenumbers (CM.sup.-1) Inten.: Intensity in Percent Transmittance (% T) Date Type in Local Peak Region: BRD = Broad; AVG = Average; SHP = Sharp; SH = shoulder Peak List Edited. * Indicates Peaks Added.

.sup.13 C-Nuclear Magnetic Resonance (NMR) Spectrum:

The .sup.13 C-NMR spectrum of antibiotic U-64,815 is shown in FIG. 3 of the drawings. The .sup.13 C-NMR spectrum was observed on a Varian XL-200 Spectrometer (50 mHz) on a solution (ca. 0.5 ml., ca. 40 mg/ml) of the sample of the antibiotic in deutero-methanol. The spectrum was calibrated against internal tetramethylsilane and frequencies were recorded in ppm downfield from tetramethylsilane.

Solubilities:

Antibiotic U-64,815 is soluble in methanol acetone, methylene chloride, ethanol and dimethylsulfoxide. It is insoluble in water and hexane.

Antimicrobial Spectrum of Antibiotic U-64,815:

Antibiotic U-64,815 is active against various Gram-positive bacteria and H. influenzae as shown in the following table.

Assay:

The antibacterial assay is a standard disc plate assay. Ten 6.35 mm absorbent discs are saturated with a 1000 .mu.g/ml solution prepared in methanol (solvent) and air-dried prior to spotting on organism-seeded agar plates. The results are as follows:

______________________________________ Zone Size (mm)______________________________________Haemophilus influenzae UC 6482 10(Ampicillin-susceptible)Haemophilus influenzae UC 6483 9(Ampicillin-resistant)Streptococcus pyogenes UC 152 13Streptococcus pneumoniae UC 41 16 - outer ring 10 - inner ringStaphylococcus aureus UC 6685 8(Multiple antibiotic-resistant)Staphylococcus aureus UC 6690 8 - light(Multiple antibiotic-resistant)N. gonorrhoeae UC 3065 16______________________________________

"UC" is a registered trademark of The Upjohn Company Culture Collection. These cultures can be obtained from The Upjohn Company in Kalamazoo, Mich., upon request.

THE MICROORGANISM

The microorganism used for the production of antibiotic U-64,815 is a biologically pure culture of Streptomyces microspinus, NRRL 12524.

A subculture of this microorganism can be obtained from the permanent collection of the Northern Regional Research Laboratory, U.S. Department of Agriculture, Peoria, Ill., U.S.A. Its accession number in this depository is NRRL 12524. It should be understood that the availability of the culture does not constitute a license to practice the subject invention in derogation of patent rights granted with the subject instrument by governmental action.

The microorganism of this invention was studied and characterized by Alma Dietz of The Upjohn Research Laboratories.

Streptomyces microspinus, Dietz sp. nov. ["microspinus" denotes the small spines found on the surface of the spores], appeared most similar on Ektachrome (Table 1) to Streptomyces impexus NRRL 3809, which is described in Goldstein and Prokop [Goldstein, A. W. and J. F. Prokop. 1972. Antibiotic effective against gram-positive bacteria and method of preparation. U.S. Pat. No. 3,651,217], and Streptomyes fungicidicus ATCC.RTM.21013, which is described in Shibata et al [Shibata, M., E. Higashide, K. Hatano, K. Mizuno, M. Asai, and M. Muroi. 1974. Antibiotic enduracidin and process for the production thereof. U.S. Pat. No. 3,786,142]. All the cultures have open spiral chains of spores with spiny surfaces. S. impexus and S. fungicidicus have abundant chains of flexuous spore chains as well as open-spiral spore chains. The spines on spores of these cultures are more readily apparent than those on spores of Streptomyces microspinus. All cultures are melanin-negative. All have gray aerial growth when the surface color is matched with the NBS color chips [SRM 2106. ISCC-NBS Centroid Color Charts. Office of Standard Reference Material, Room B311, Chem. Building, National Bureau of Standards, Washington, D.C. 20234], but only Streptomyces microspinus belongs to the Gray series of Tresner and Backus [Tresner, H. D., and E. J. Backus. 1963. System of color wheels for streptomycete taxonomy. Appl. Microbiol. 11:335-338]. Streptomyces microspinus is further distinguished from S. impexus and S. fungicidicus by its growth on carbon compounds (Table 3) 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] and by its general cultural characteristics (Table 4). The new culture grows well in the temperature range of 18.degree.-55.degree. C.; the other cultures do not grow at 45.degree.-55.degree. C. The new culture produces the antibiotic U-64815; S. impexus produces M-411 and S. fungicidicus produces Enduracidin. It can also be differentiated from cultures in the Gray spiral, melanin-negative, spiny spore group in Bergey's Manual, 8th ed. [Pridham, T. G., and H. D. Tresner. 1974. Part 17. Actinomycetes and related organisms. Family VII. Streptomycetaceae Waksman and Henrici 1943. Genus I. Streptomyces Waksman and Henrici 1943. Table 17.42h of the Gray series. Pages 782-785 in Buchanan and Gibbons, eds., Bergey's Manual of Determinative Bacteriology, 8th ed. The Williams and Wilkins Co., Baltimore]. Therefore, it is proposed that Streptomyces microspinus be considered a new species of the actinomycete genus Streptomyces. It is understood that in accordance with the Rules of Nomenclature of Bacteria [Lapage, S. P., P. H. A. Sneath, E. F. Lessel, V. B. D. Skerman, H. P. R. Seeliger, and W. A. Clark, ed. 1975. International code of nomenclature of bacteria, 1976 Revision. American Society for Microbiology, Washington, D.C.], this is the type strain and that, should another strain be found, the type strain would also be the type subspecies.

Streptomyces microspinus Dietz sp. nov. NRRL 12524.

Color Characteristics: Aerial mycelium prednominantly gray. Melanin-negative. The color pattern of the culture on Ektachrome is given in Table 1. Reference color characteristics are given in Table 2. The culture may be placed in the Gray (GY) color series of Tresner and Backus [Tresner, H. D., and E. J. Backus, Ibid.].

Microscopic Characteristics: Spore chains are predominantly open-spiral. Spores are oval, 1.1.times.0.76 .mu.m. Many spores have depressions like those of red blood cells; others have a double collapsed appearance which gives a ridged appearance. The surface of the spores is covered with blunt spines. Spore surface detail is best determined at magnifications of 10,000 to 20,000 with the scanning electron microscope. Fourteen day cover glass preparations were used for the microscopic examinations.

Growth on Carbon Compounds: See Table 3.

Whole Cell Analysis: L-diaminopimelic acid was detected.

Culture Characteristics-General: See Table 4.

Temperature: Growth was good at 18.degree.-55.degree. C. on Czapek's sucrose and Maltose-tryptone agars and at 18.degree.-45.degree. C. on Bennett's agar.

Source: Kansas soil.

Type Strain: Streptomyces microspinus sp. nov. NRRL 12524.

TABLE 1__________________________________________________________________________Color Characteristics* on Ektachrome** Deter- S. microspinus S. impexus S. fungicidicus mina- NRRL 12524 NRRL 3809 ATCC 21013Agar Medium tion Chip Color Chip Color Chip Color__________________________________________________________________________Bennett's S 233 pale gray 32 grayish 70 light yellowish orange pink yellow R 72 dark orange 70 light 70 light yellow orange orange yellow yellowCzapek's S 233 pale gray 32 grayish 9 pink whitesucrose yellowish pink R 32 grayish 32 grayish 9 pink white Yellowish yellowish pink pinkMaltose- S 233 pale gray 31 pale yellow 7 pale pinktryptone pink R 72 dark orange 67 brilliant 68 strong yellow orange orange yellow yellowPeptone- S 71 moderate 32 grayish 71 moderateiron orange yellowish orange yellow pink yellow R 68 strong 68 strong 68 strong orange orange orange yellow yellow yellow0.1% S 32 grayish 32 grayish 7 pale pinkTyrosine yellowish yellowish pink pink R 38 dark 38 dark 38 dark reddish reddish reddish orange orange orangeCasein S 233 pale gray 32 grayish 10 pinkishstarch yellowish gray pink R 93 yellowish 76 light 93 yellowish gray yellowish gray brown__________________________________________________________________________ S = Surface R = Reverse *Growth on media in tubes was photographed after seven days incubation at 28.degree. C. Color was determined by comparison with NBS color chips [SP 440. Color: Universal Language and Dictionary of Name. U.S. Government Printing Office, Washington, D.C. 20402; SRM 2106. Ibid.]. **Dietz, A. 1954. Ektachrome transparencies as aids in actinomycete classification. Ann. N.Y. Acad. Sci. 60:152-154 and Dietz, A. and D. W. Thayer (ed.). 1980. Actinomycete Taxonomy (Procedures for Studying Aerobi Actinomycetes with Emphasis on the Streptomycetes). SIM Special Publication Number 6. Soc. for Ind. Microbiol. Arlington, VA. TABLE 2__________________________________________________________________________Reference Color Characteristics* Deter- S. microspinus S. impexus S. fungicidius mina- NRRL 12524 NRRL 3809 ATCC 21013Agar Medium tion Chip Color Chip Color Chip Color__________________________________________________________________________Bennett's S 266 dark gray 90 grayish 90 grayish yellow yellow R 58 moderate 90 grayish 70 light orange brown yellow yellow P 71 moderate -- -- -- -- orange yellowCzapek's S 45 light gray- 63 light 63 light brown-sucrose ish red- brownish ish gray dish brown gray R 93 yellowish 73 pale orange 73 pale orange gray yellow yellow P -- -- -- -- -- --Maltose- S 266 dark gray 93 yellowish 93 yellowishtryptone gray gray R 57 light brown 71 moderate 71 moderate orange orange yellow yellow P 87 moderate 87 moderate 87 moderate yellow yellow yellowYeast S 23 dark red- 22 reddish 63 light brown-extract- dish gray gray ish graymalt R 57 light brown 71 moderate 71 moderateextract orange orange(ISP-2) yellow yellow P 71 moderate 87 moderate 87 moderate orange yellow yellow yellowOatmeal S 45 light gray- 63 light brown- 63 light brown-(ISP-3) ish red- ish gray ish gray dish brown R 29 moderate 71 moderate 71 moderate yellowish orange orange pink yellow yellow P 30 dark yel- 87 moderate 87 moderate lowish pink yellow yellowInorganic S 23 dark red- 63 light brown- 22 reddishSalts dish gray ish gray grayStarch R 73 pale orange 71 moderate 71 moderate(ISP-4) yellow yellow yellow P 79 light gray- 80 grayish 80 grayish ish yel- yellowish yellowish lowish brown brown brownGlycerol- S 266 dark gray 93 yellow gray 93 yellow grayasparagine R 57 light brown 71 moderate 71 moderate(ISP-5) orange orange yellow yellow P -- -- 87 moderate 87 moderate yellow yellow__________________________________________________________________________ S = Surface R = Reverse P = Pigment *Color determination was made on growth on plates incubated 14 days at 28.degree. C. Color was determined by comparison with NBS color chips [SP 440, Ibid. and SRM 2106, Ibid. TABLE 3______________________________________Growth on Carbon Compounds in the SyntheticMedium of Shirling and Gottlieb*Synthetic Medium S. microspinus S. impexus S. fungicidicus(ISP-9) NRRL 12524 NRRL 3089 ATCC 21013______________________________________Negative Control - - -(No carbon cpd.)Positive Control + ++ +(D-glucose)L-arabinose ++ ++ ++Sucrose - .+-. .+-.D-xylose ++ ++ ++Inositol ++ ++ ++D-mannitol ++ ++ ++D-fructose + ++ ++Rhamnose ++ ++ ++Raffinose - - -Cellulose - - -______________________________________ ++ = Strong utilization + = Positive utilization .+-. = Doubtful utilization - = No utilization *Shirling, E. B. et al., Ibid. TABLE 4__________________________________________________________________________Culture Characteristics - General Deter- mina- S. microspinus S. impexus S. fungicidicusAgar Medium tion NRRL 12524 NRRL 3809 ATCC 21013__________________________________________________________________________AgarPeptone- S pale gray white center; gray center;iron pink-tan edge white edge R tan light yellow- yellow tan P -- -- -- O melanin melanin melanin negative negative negativeCalcium S gray gray graymaleate R pale gray gray gray-yellow P -- -- -- O malate not malate not malate not solubilized solubilized solubilizedGlucose- S gray-pink yellow vegeta- cream-grayasparagine growth R pink-tan deep yellow deep yellow P pale pink -- --Skim milk S brown vegeta- gray center; trace gray tive growth cream edge R tan orange orange P tan orange orange O casein not casein solu- casein solu- solubilized bilized bilizedTyrosine S pale gray gray center; cream center; cream edge gray-white edge R orange-tan orange-yellow orange-tan P orange-tan pale orange orange-tan O tyrosine tyrosine tyrosine solubilized solubilized solubilizedXanthine S pale gray on gray cream cream edge; tan vegetative center R yellow-cream orange cream P pale yellow -- pale yellow O xanthine xanthine not xanthine not solubilized solubilized solubilizedNutrient S blue-gray gray center; creamstarch cream edge R yellow cream yellow yellow P pale yellow pale yellow pale yellow O starch solu- starch par- starch par- bilized tially solu- tially solu- bilized bilizedYeast S lavender-gray gray-cream gray with creamextract- edgemalt R orange-tan yellow-orange yellowextract tan P pale orange- pale yellow pale yellow tanPeptone- S light gray- light gray- colorless vegeta-yeast pink white tive with veryextract- slight traceiron gray-white(ISP-6) R yellow-tan yellow-tan yellow-tan P yellow-tan yellow-tan yellow-tan O melanin melanin melanin negative negative negativeTyrosine S steel gray mottled gray- light steel gray(ISP-7) yellow with yellow exudate R light gray yellow-tan light yellow-gray P -- trace yellow- -- tan O melanin melanin melanin negative negative negativeBrothSynthetic S trace white trace white slight whitenitrate aerial on aerial on aerial on surface surface surface pellicle pellicle pellicle P -- -- -- O fine growth trace growth slight growth throughout throughout throughout flocculent flocculent flocculent growth at base growth at base growth at base growth color- growth color- growth color- less less less nitrates not nitrates nitrates reduced reduced reducedNutrient S gray white white aerial white aerialnitrate aerial on on surface on surface surface pellicle pellicle pellicle P -- -- -- O flocculent flocculent flocculent yellowish yellowish yellowish bottom growth bottom growth bottom growth nitrates not nitrates nitrates reduced reduced reducedLitmus S trace gray- pale gray- pale gray-milk white aerial white aerial white aerial on blue- on surface on surface gray surface pellicle pellicle ring blue-gray-tan blue-gray-tan surface ring surface ring P -- -- -- O litmus reduced litmus reduced litmus reduced pH 7.7 pH 7.6 pH 7.4GelatinPlain S trace tan trace tan trace tan vegetative vegetative vegetative growth at growth at growth at surface surface surface P trace olive trace yellow trace yellow O liquefaction liquefaction liquefaction complete complete completeNutrient S tan vegetative tan vegetative tan vegetative growth at growth at growth at surface surface surface P -- tan yellow O liquefaction liquefaction liquefaction complete complete complete__________________________________________________________________________ S = Surface (aerial growth unless otherwise noted) R = Reverse P = Pigment O = Other Characteristics

The compound of the invention process 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 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 brewer's yeast with milk solids, soybean meal, cottonseed meal, cornmeal, milk solids, pancreatic digest of casein, fish meal, distiller's 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, manganese, cobalt, iron, and the like, need not be added to the fermentation medium since tap water and unpurified ingredients are used as components of the medium prior to sterilization of the medium.

Production of the compound by the invention process can be effected at any temperature conducive to satisfactory growth of the microorganism, for example, between about 18.degree. and 40.degree. C., and preferably between about 20.degree. and 28.degree. C. Ordinarily, optimum production of the compound is obtained in about 3 to 15 days. The medium normally remains alkaline 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 compound and the attendant inefficient utilization of 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, liquid N.sub.2 agar plug, 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 compound, so long as a good growth of the microorganism is obtained.

A variety of procedures can be employed in the isolation and purification of the compound produced by the subject invention from fermentation beers. Isolation can be accomplished by extraction with solvents such as methylene chloride, acetone, butanol, ethyl acetate and the like; and silica gel chromatography can be used to purify crude preparations of the antibiotic.

In a preferred recovery process, the compound produced by the subject process is recovered from the culture medium by separation of the mycelia and undissolved solids by conventional means, such as by filtration or centrifugation and solvent extraction of the filtered broth. The filtrate can be extracted with a solvent for antibiotic U-64,815, for example, methylene chloride, and the extract evaporated under reduced pressure to an aqueous concentrate. This preparation can be purified by chromatography on silica gel. The solvent system used for the chromatography is methylene chloride:methanol (95:5) (v/v) and (90:10). Antibiotic U-64,185 is shown at R.sub.f =0.50-0.55 silica gel G plates in MeCl.sub.2 /MeOH 9:1 using standard bioautography on Bacillus subtilis.

The antibiotic of the subject invention also can be recovered from the fermentation broth by resin sorption on a resin comprising a non-ionic macro porous copolymer of styrene cross linked with divinylbenzene. Suitable resins are Amberlite XAD-2; XAD-4, and XAD-7, according to the procedure disclosed in U.S. Pat. No. 3,515,717. (Amberlite resins are available from Rohm and Haas, Philadelphia, PA). The antibiotic can be eluted from said resins by using acetone.

Salts of antibiotic U-64,815 also can be formed with inorganic or organic bases. Such salts can be prepared, as for example, by suspending antibiotic U-64,815 in water, adding a dilute base until the pH of the suspension is about 10.0 to 11.0, and freeze-drying to provide a dried residue consisting of the U-64,815 salt. Antibiotic U-64,815 salts with inorganic cations which can be formed include the sodium, potassium, and calcium salts. Other salts of U-64,815, including those with inorganic bases such as primary, secondary, and tertiary monoamines as well as with polyamines, also can be formed using the above-described or other commonly employed procedures. Other valuable salts are obtained with therapeutically effective bases which impart additional therapeutic effects thereto. Such bases are, for example the purine bases such as theophyllin, theobromine, 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. Salts of U-64,815 can be used for the same biological purposes as the parent compound.

The following examples are illustrative of the process and product of the invention, but are not to be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.

EXAMPLE 1

A. Fermentation

A biologically pure culture of Streptomyces microspinus, NRRL 12524, is used to inoculate 500-ml Erlenmeyer seed flasks containing 100 ml of sterile medium consisting of the following ingredients:

______________________________________ g/liter______________________________________Cerelose 25Pharmamedia* 25Deionized water q.s. 1 liter______________________________________ *Pharmamedia is an industrial grade of cottonseed flour produced by Traders Oil Co, Fort Worth, TX.

The seed medium post-sterilization pH is .about.6.5. The seed inoculum is grown for three days at 28.degree. C. on a Gump rotary shaker operating at 250 rpm and having a 21/2 inch stroke.

Seed inoculum (100 ml), prepared as described above, is used to inoculate 500-ml fermentation flasks (Erlenmeyer) containing 100 ml of sterile fermentation medium consisting of the following ingredients:

______________________________________ g/liter______________________________________Cerelose 10Bacto-Peptone (Difco) 5Black Strap Molasses* 20Tap Water Balance______________________________________ *Supplied by Knappen Milling Co., Augusta, MI.

The pH is adjusted to 7.2 with NaOH before autoclaving.

The inoculated fermentation medium is incubated at a temperature of 28.degree. C. for 5 days on a Gump rotary shaker operating at 250 rpm and having a 21/2 inch stroke.

A typical five-day fermentation has the following titers of antibiotic in the fermentation broth:

______________________________________ Day Assay, BU/ml______________________________________ 2 8.0 3 >16 4 >16 5 >16______________________________________

The assay is a Bacillus subtilis disc plate assay using 0.1 M potassium phosphate buffer, pH 5.0 as diluent.

B. Recovery

Whole beer, from a fermentation as described above, is filtered and adjusted to neutral pH with HCl and filtered with 5% diatomaceous earth (wt/v). The clear beer is then defatted with two half volumes of Skellysolve B (isomeric hexanes). The defatted beer is then extracted with two half volumes of methylene chloride and these extracts are combined and reduced in volume.

______________________________________1-liter whole beer adj. to pH 7 with HCl ##STR1##Clear Beer(1/2" dipped disc bioassay) Whole Beer Clear BeerOrganism Zone (mm) Zone (mm)______________________________________B. subtilis 29 29M. luteus 25 24C. perfringens 28 28______________________________________ 1st 2nd CombinedOrganism Extract Extract Extracts Spent Beer______________________________________Skellysolve BB. subtilis 0 0 0 28M. luteus 0 0 0 23C. perfringens 0 0 0 27Me.sub.2 Cl.sub.2B. subtilis 27 19 27 21S. lutea 23 0 21 16C. perfringens 27 18 27 20______________________________________

C. Purification

A 21.5 g sample of a CH.sub.2 Cl.sub.2 extract, prepared as described above, is dissolved in a minimal volume of CH.sub.2 Cl.sub.2 and passed onto the head of a 5' column packed with 1400 g of Merck Silica Gel 60 slurried in CH.sub.2 Cl.sub.2. A flow rate of ca. 7 ml/min is established with a solvent of CH.sub.2 Cl.sub.2 /MeOH; 90/5; 20 ml fractions are collected. After 8.7 liters, the solvent is changed to CH.sub.2 Cl.sub.2 /MeOH; 90/10.

Fractions are analyzed by thin layer chromatography (TLC) using Analtech Silica gel GF plates run in CH.sub.2 Cl.sub.2 /MeOH, 9/1. The component of interest is first detected by quenching under U.V. 254 nm light followed by spraying with a p-anisaldehyde reagent (EtOH:H.sub.2 SO.sub.4 :Acetic Acid:p-anisaldehyde, 90:5:1:5) and heating. U-64,815 runs in this system with R.sub.f =0.50-0.55 and develops a deep purple coloration when processed with this reagent.

Fractions 436-570 are pooled to give 4.25 g of a brown solid which has .about.32 bu/mg when assayed with 1/2" discs on B. subtilis by a dilution bioassay. (One bu is defined as the amount of antibiotic needed to produce a 20 mm zone).

The 4.25 g solid is dissolved in a minimal volume of MeOH/H.sub.2 O, 80/20 and loaded onto the head of a 25 mm I.D. column packed with 42.5 g of Whatman LRP-1 reverse phase equilibrated with the solvent. A flow rate of 1.5 ml/min is established with MeOH/H.sub.2 O, 80/20 while 50 ml fractions are collected. After the seventh fraction, the solvent is changed to 100% MeOH. Fractions are analyzed in the identical manner as before and also with dipped discs plated against B. subtilis. The active fractions containing the best material as seen by TLC are pooled and reduced in volume to give 2.75 g of an off-white powder of essentially pure antibiotic U-64,815.

Claims

1. A process for preparing antibiotic U-64,815, which in its essentially pure form has the following characteristics:

(a) molecular weight range of 1,104-1,324;

(b) molecular formula: C.sub.60-65 H.sub.82-102 N.sub.1-3 O.sub.18-25 ;

(c) is insoluble in water and hexane, soluble in methanol, acetone, methylene chloride, ethanol, and dimethylsulfoxide;

(d) a characteristic .sup.13 C-NMR spectrum as shown in FIG. 3 of the drawings;

(e) a characteristic UV spectrum as shown in FIG. 2 of the drawings; and,

(f) a characteristic infrared absorption spectrum when dissolved in a mineral oil mull as shown in FIG. 1 of the drawings;

which comprises cultivating Streptomyces microspinus, having the identifying characteristics of NRRL 12524, in an aqueous nutrient medium under aerobic conditions until substantial antibiotic U-64,815 activity is imparted to said medium.

2. A process, according to claim 1, wherein said aqueous nutrient medium contains a source of assimilable carbohydrate and assimilable nitrogen.