Abstract:
Novel antibiotic U-58,431 producible in a fermentation under controlled conditions using a biologically pure culture of the microorganism Streptomyces helicus Dietz and Li sp. n., NRRL 11461. This antibiotic is active against various Gram-positive bacteria, for example, Staphylococcus aureus, Streptococcus hemolyticus, and Streptococcus faecalis. It is also active against various Gram-negative bacteria, for example, Escherichia coli, Proteus vulgaris, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Salmonella schottmuelleri. It has strong activity against Diplococcus pneumoniae. Thus, antibiotic U-58,431 can be used in various environments to eradicate or control such bacteria.

Description:
CROSS REFERENCE TO RELATED APPLICATION 
     This is a continuation of application Ser. No. 34,247, filed Apr. 30, 1979, now abandoned. 
    
    
     BRIEF SUMMARY OF THE INVENTION 
     Antibiotic U-58,431 is producible in a fermentation under controlled conditions using a biologically pure culture of the new microorganism Streptomyces helicus Dietz and Li sp.n., NRRL 11461. 
     Antibiotic U-58,431 is active against various Gram-positive and Gram-negative bacteria. Further, the base addition salts of antibiotic U-58,431 are also active against these bacteria. Thus, antibiotic U-58,431 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. Since antibiotic U-58,431 and its salts are active against P. vulgaris, they can be used as an oil preservative to inhibit this bacterium which is known to cause spoilage in oil. Still further, antibiotic U-58,431 and its salts can be used as a bacteriostatic rinse for laundered cloths, 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-58,431: 
     Molecular Weight: 294.0843 (high resolution spectrometry). 
     Molecular Formula: C 13  H 14  N 2  O 6   
     Elemental Analysis: C, 52.70; H, 5.40; N, 9.45. 
     Ultraviolet Absorption Spectrum: 
     The solution of antibiotic U-58,431 in methanol displayed absorption as follows: 
     
         ______________________________________λ max     Absorbance    Absorptivity (ε)______________________________________261       44.80         13,250472        5.16          1,550______________________________________ 
    
     No shifts in acid or base. 
     Melting point: 155.9° C. to 157.3° C. with decomposition. 
     Infrared Absorption Spectrum: 
     Antibiotic U-58,431 has a characteristic infrared absorption spectrum in a mineral oil mull as shown in the drawing. Peaks are observed at the following wave lengths expressed in reciprocal centimeters. 
     
         ______________________________________Band Frequency(Wave Numbers)      Intensity______________________________________3355                S3280                S3190                S2960                S2925                S2850                S2730                W1685                M1650                S1635                S1614                S1587                S1577                  S, sh1487                  M, sh1463                S1428                  M, sh1402                M, sh1377                S1364                S1331                M1318                M1290                S1228                M1195                S1180                M1168                M1125                M1095                S1073                S1020                W1003                M990                 M967                 W915                 W888                 W869                 W843                 M828                 M805                 W791                 M767                 W734                 M720                 M700                 M642                 M______________________________________ Key: S = Strong M = Medium W = Weak sh = shoulder 
    
     Solubilities 
     Antibiotic U-58,431 is soluble in water, methanol, and ethanol, and slightly soluble in cold methylene chloride and chloroform. 
     Antimicrobial Spectrum Of Antibiotic U-58,431 
     Antibiotic U-58,431 is active against various Gram-positive and Gram-negative bacteria and fungi as shown in the following tables. 
     Assay 
     The antibacterial assay is a standard microbiological broth assay. The MIC is determined by standard methods using two-fold dilutions of the antibiotic in Brian Heart Infusion Broth (Difco Lab., Detroit, Michigan). The inocula are overnight cultures of the test organisms, diluted so that the final population contains approximately 10 5  cells/ml. The tubes are incubated at 28° to 37° C. for 42 hours. The lowest antibiotic concentration which allows no growth=MIC or minimum inhibitory concentration. 
     
         ______________________________________               Minimum InhibitoryMicroorganism       Concentration (mcg/ml)______________________________________Staphylococcus aureus 284 UC 76               125Staphylococcus aureus UC 570               125Staphylococcus aureus UC 746               125Streptococcus hemolyticus UC 152               125Streptococcus faecalis UC 694               250Escherichia coli UC 45               250Proteus vulgaris UC 93               250Klebsiella pneumoniae UC 58               125Salmonella schottmuelleri UC 126               62.5Pseudomonas aeruginosa UC 95               125Diplococcus pneumoniae UC 41               1.0______________________________________ 
    
     The antifungal spectrum for antibiotic U-58,431 was conducted on a standard agar difusion assay. The results are as follows: 
     
         ______________________________________               Results At Indicated               Concentration               (mcg/ml)Test Organisms        1000    100    10  1______________________________________Nocardia asteroides UC 2052                 -       +      +   +Blastomyces dermatidis UC 1466                 +       +      +   +Geotrichum sp. UC 1207                 +       +      +   +Hormodendrum compactum UC 1222                 +       +      +   +Cryptococcus neoformans UC 4869                 +       +      +   +Cryptococcus neoformans UC 1139                 +       +      +   +Sporotrichum schenckii UC 1364                 +       +      +   +Candida albicans UC 7163                 +       +      +   +Candida albicans UC 7164                 +       +      +   +Trichophyton rubrum UC 1458                 -       +      +   +Trichophyton violaceum UC 1459                 +       +      +   +Trichophyton asteroides UC 4775                 +       +      +   +Trichophyton mentagrophytes UC 4797                 -       +      +   +Trichophyton mentagrophytes UC 4860                 +       +      +   +______________________________________ Key: - = inhibition + = no inhibition 
    
     &#34;UC&#34; is a registered trademark of The Upjohn Company Culture Collection. These cultures can be obtained from The Upjohn Company in Kalamazoo, Michigan, upon request. 
     Antifungal in vitro Method 
     Method for Antifungal Testing 
     An agar dilution method is used. 100 mg amounts of material is solubilized in 1 ml of dimethylformamide (DMF) and diluted to 10 ml with mycophil Broth (BBL labs.) unless completely soluble in water. The material to be tested is incorporated in melted (48° C.) mycophil Agar at concentrations of 1000, 100, 10 and 1 mcg/ml. Fifteen ml of agar is pipetted into a petri dish. After solidification, the surface is streaked with agar spore suspensions of human fungal pathogens. The suspensions are made by adding 10 ml sterile distilled water to agar slants of the test organisms. The slant surface is scraped with a sterile inoculating needle, and 0.5 ml of this suspension is added to 9.5 ml melted (48° C.) mycophil Agar in a stamp streak well. (Each stamp streak base has 6 wells.) 
     The streaked plates are incubated at 28° C. for 72 hours at which time results are recorded. Readings are by visual observation. 
     THE MICROORGANISM 
     The microorganism used for the production of antibiotic U-58,431 is a biologically pure culture of Streptomyces helicus Dietz and Li sp. n., NRRL 11461. 
     A subculture of this microorganism can be obtained from the permanent collection of the Northern Regional Research Laboratory, U.S. Department of Agriculture. Peoria, Illinois, U.S.A. Its accession number in this depository is NRRL 11461. It should be understood that the availability of the culture does not constituted a license to practice the subject invention in derogation of patient rights granted with the subject instrument by governmental action. 
     The microorganism of this invention was studied and characterized by Alma Dietz and Grace P. Li of The Upjohn Research Laboratories. 
     The culture characterized is considered to be a new species of Streptomyces for which the designation proposed is Streptomyces helicus Dietz and Li sp. n. It is understood that this new type species is to be designated the type subspeices, should a variant be found. This is in accordance with the rules set forth in the International Code of Nomenclature of Bacteria [Lapage, S.P., et al. eds. 1975. International Code of Nomenclature of Bacteria (Bacteriological Code, 1976 Revision). ASM, Wash., D.C.]. 
     The culture is differentiated from Streptomyces olivaceus ATCC 3335 and Streptomyces griseoroseus ATCC 12125. 
     The methods used in characterizing the culture were modifications of those cited in Dietz [Dietz, A. 1954. Ektachrome transparencies as aids in actinomycete classification. Ann. N.Y. Acad. Sci. 60: 152-154.][Dietz, A. 1967. Streptomyces steffisburgensis sp. n. J. Bacteriol. 94: 2022-2026.], Dietz and Mathews [Dietz, A., and J. Matthews. 1970. Classification of Streptomyces spore surfaces into five groups. Appl. Microbiol. 21: 527-533.], and Shirling and Gottlieb [Shirling, E. B., and D. Gottlieb. 1966. Methods for characterization of Streptomyces species. International Journal of Systematic Bacteriology. 16: 313-340]. 
     Streptomyces helicus Dietz and Li sp.n., NRRL 11461, a new soil isolate, appeared similar to Streptomyces olivaceus ATCC 3335 [Shirling, E. B., and D. Gottlieb. 1968. Cooperative description of type cultures of Streptomyces. II. Species descriptions from first study. Int. J. Syst. Bacteriol. 18: 69-189.] [Waksman, S. A. 1961. The actinomycetes. Vol. II. Classification identification and descriptions of genera and species. The Williams &amp; Wilkins Co., Baltimore, MD, 363 pp.] and Streptomyces griseoroseus ATCC 12125 [Hutter, R. 1967. Systematik der Streptomycetan unter besonderer Berucksichtigung der von ihnen gebildeten Antibiotic. S. Karger, Basel and New York, 382 pp.] [Walker, J. E., M. Bodanszky, and D. Perlman. 1970. The biogenetic origin of the N-methyl-γ-methyl-L-isoleucine residue of etamycin. J. Antibiotics. XXIII: 255-256.] by comparison on Ektachrome [Dietz, A. 1954. Ektachrome transparencies as aids in actinomycete classification. Ann. N.Y. Acad. Sci. 60: 152-154.]. All showed long straight to open spiral to spiral spore chains when observed by light microscopy and scanning electron microscopy (SEM). All had oval-shaped smooth spores with a depressed or ridged surface. (This was determined by SEM). The new culture had a greater number of spore chains with helical tips than the cultures with which it was compared. Other differences are given in Tables 2-5. The most distinctive differences are in reverse color, gelatin liquefaction, nitrate reduction, reactions in litmus milk and growth on carbon compounds in synthetic medium. 
     The cultures also differ in their antibiotic-producing properties. S. olivaceus shows little or no antagonistic properties in our media. The production of etamycin [Lapage, S. P. spura] [Walker, J. E. surpra] by S. griseoroseus has been confirmed. The new culture produces the antibiotic U-58,431. 
     The new culture may be placed in the Red series of Pridham and Tresner in Bergey&#39;s Manual, 8th ed. [Buchanan, R. E., and N. E. Gibbons, eds. 1974. Bergey&#39;s manual of determinative bacteriology, 8th Ed. The Williams &amp; Wilkins Co., Baltimore, MD. 1246 pp.] based on surface color reading with the color chip system of Tresner and Backus [Tresner, H. D., and E. J. Backus, 1963. System of color wheels for streptomycete taxonomy. Applied Microbiol. 11: 335-338.]. The new culture is distinguished from the cultures cited for the Red series. The Gray series may also be considered since S. olivaceus which was characterized in the International Streptomyces Project [Shirling, E. B., and D. Gottlieb. 1968. Cooperative description of type culture of Streptomyces. II. Species descriptions from first study. Int. J. Syst. Bacteriol. 18: 69-189.] is placed in this series. The new culture differs from cultures in this series. This conclusion is based on comparison of our results with those in Bergey&#39;s Manual. 
     Color characteristics: Aerial growth lavender gray. Melanin negative. The appearance of the culture on Ektachrome [Buchanan, R. E. supra] is given in Table 1. Reference color characteristics are given in Table 2. The culture may be placed in the (R) Red color series of Tresner and Backus [supra]. 
     Microscopic characteristics: The spore chains, observed with the light microscope and the scanning electron microscope, are long, flexuous to open spiral to spiral. The spore surface, as observed with the scanning electron microscope, is smooth with ridges or depressions. 
     Cultural and biochemical characteristics: These are given in Table 3. 
     Carbon utilization: See Tables 4 and 5. 
     Temperature: The culture grew well at 18°-37° C. on Bennett&#39;s, Czapek&#39;s sucrose, and maltose-tryptone agars. 
     
                       TABLE 1______________________________________Ektachrome Comparison Of S. helicus, S. olivaceusATCC 3335, And S. griseoroseus ATCC 12125Agar     S.          S.          S.Medium   helicus     olivaceus   griseoroseus______________________________________Bennett&#39;s  S     Trace gray  Trace gray                              Very slight                              trace gray  R     Pale yellow Yellow-tan                              TanCzapek&#39;s  S     Lavender-gray                    Pale gray Palesucrose                            lavender-gray  R     Pale gray   Colorless ColorlessMaltose-  S     Lavender-gray                    Lavender-gray                              Gray-salmontryptone  R     Light       Gray-tan  Red-brown        red-brownPeptone-  S     Colorless   Colorless Colorlessiron   R     Tan         Tan       Tan0.1%   S     Colorless   Colorless ColorlessTryosine  R     Red         Pale red  RedCasein S     Colorless   Colorless Colorlessstarch R     Colorless   Colorless Light tan______________________________________ S = Surface R = Reverse 
    
     
                                           TABLE 2__________________________________________________________________________Reference Color Characteristics [NBS Color Chips*]Agar    Deter-  S.          S.          S.Medium  mination           helicus     olivaceus   griseoroseus__________________________________________________________________________Bennett&#39;s   S    60 l.gy.Br = light                     31                       p.y. Pink = pale                                 32                                   gy.y Pink = Grayish           grayish brown                       yellowish pink                                   yellowish pink   R    109           1.gy.Ol = light                     77                       m.y. Br = moderate                                 57                                   l.Br = light brown           grayish olive                       yellowish brown   P    106           l.Ol = light                     --          57                                   l.Br = light brown           oliveCzapek&#39;s   S    33 br Pink = brownish                     31                       p.y. Pink = pale                                 31                                   p.y. Pink = palesucrose         pink        yellowish pink                                   yellowish pink   R    68 s. OY = strong                     73                       p. OY = pale                                 73                                   p. OY = pale           orange yellow                       orange yellow                                   orange yellow   P    --           --          --Maltose-   S    32 gy.y Pink = grayish                     31                       p.y. Pink = pale                                 33                                   br Pink = brownishtryptone        yellowish pink                       yellowish pink                                   pink   R    87 m.Y = moderate                     77                       m.y Br = moderate                                 65                                   br Black =           yellow      yellowish brown                                   brownish black   P    106           l.Ol = light                     --          59                                   d. Br = dark           olive                   brownYeast extract-   S    33 br Pink = brownish                     60                       l.gy.Br = light                                 31                                   p.y Pink = palemalt extract    pink        grayish brown                                   yellowish pink(ISP-2) R    114           Ol Black = olive                     77                       m.y Br = moderate                                 58                                   m. Br = moderate           black       yellowish brown                                   brown   P    151           d.gy.G = dark                     --          77                                   m.y Br = moderate           grayish green           yellowish brownOatmeal S    33 br Pink = brownish                     60                       l.gy. Br = light                                 31                                   p.y Pink = pale(ISP-3)         pink        grayish brown                                   yellowish pink   R    67 brill. OY (center) =                     77                       m.y. Br = moderate                                 73                                   p. OY = pale           brilliant orange                       yellowish brown                                   orange yellow           yellow        16 d.R. (edge) =           dark red   P    8  gy. Pink = grayish                     --          73                                   p. OY = pale           pink                    orange yellowInorganic-salts   S    60 l.gy. Br = light                     60                       l.gy. Br = light                                 31                                   p.y. Pink = palestarch (ISP-4)  grayish brown                       grayish brown                                   yellowish pink   R    75 deep y Br = deep                     70                       l. OY = light                                 65                                   br Black =           yellowish brown                       orange yellow                                   brownish black   P    8  gy. Pink = grayish                     --          57                                   l. Br = light           pink                    brownGlycerol-   S    60 l.gy. Br = light                     60                       l.gy. Br = light                                 31                                   p.y Pink = paleasparagine      grayish brown                       grayish brown                                   yellowish pink(ISP-5) R    188           bk blue = 70                       l. OY = light                                 58                                   m. Br = moderate           blackish blue                       orange yellow                                   brown   P    187           d.gy. blue = dark                     --          57                                   l.Br = light           grayish blue            brown__________________________________________________________________________ S = Surface R = Reverse P = Pigment? *SP 440. Color: Universal Language and Dictionary of Names. U.S. Government Printing Office, Washington, D.C. 20402. SRM 2106. ISCCNBS Centroid Color Charts. Office of Standard Reference Material, Room B311, Chem. Bldg., National Bureau of Standards, Washington, D.C. 20234. 
    
     
                                           TABLE 3__________________________________________________________________________Cultural and Biochemical CharacteristicsMedium  Deter-        S.          S.          S.Agar    mination        helicus     olivaceus   griseoroseus__________________________________________________________________________Peptone-iron   S    Greenish tan vegetative                    Greenish tan vegetative                                Tan vegetative growth        growth      growth   R    Greenish tan                    Yellowish tan                                Tan   P    --          Yellow      Tan   O    Melanin negative                    Melanin negative                                Melanin negativeCalcium malate   S    Gray-white  Gray-white  Pink-orange   R    Gray-white  Gray-white  White   P    --          --          --   O    Malate solubilized                    Malate solubilized                                Malate partially                                solubilizedGlucose S    Gray-tan with                    Gray        Pink-orangeasparagine   yellow edge   R    Yellow      Light yellow-tan with                                Brown                    dark green-tan edge   P    --          --          --Skim milk   S    Gray-white with                    Gray        Pink-orange        yellow edge   R    Orange-yellow                    Yellow-tan  Orange-tan   P    Yellow-tan  Yellow-tan  Orange-tan   O    Casein solubilized                    Casein solubilized                                Casein solubilizedTyrosine   S    Gray-white  Gray        Gray-pink   R    Red-tan     Red-tan     Dark red-brown   P    Red-tan     Red-tan     Dark red-tan   O    Tyrosine solubilized                    Tyrosine solubilized                                Tyrosine solubilizedXanthine   S    Gray-white  Gray-beige  Gray-pink   R    Light yellow                    Yellow-tan  Yellow-tan   P    Light yellow                    --          --   O    Xanthine solubilized                    Xanthine solubilized                                Xanthine solubilizedNutrient   S    Gray-white  Gray-beige  Gray-pinkstarch  R    Light yellow                    Yellow-tan  Tan   P    --          --          --   O    Starch solubilized                    Starch partially                                Starch partially                    solubilized solubilizedYeast extract-   S    Gray        Gray-beige  Gray-pinkmalt extract   R    Dark gray-tan                    Tan         Red-brown   P    Yellow      --          Red-brownPeptone-yeast   S    Pale greenish-tan                    Yellow-tan vegetative                                Pale yellow-tanextract-iron vegetative growth                    growth      vegetative growth(ISP-6) R    Pale greenish-tan                    Yellow-tan  Pale yellow-tan   P    Pale greenish-tan                    Yellow-tan  Pale yellow-tan   O    Melanin negative                    Melanin negative                                Melanin negativeTyrosine   S    Pale lavender-pink                    Pale lavender-gray                                Heavy cream-pink(ISP-7) R    Pale yellow-pink                    Pale cream-gray                                Maroon-tan        maroon   P    Trace pale pink-tan                    --          Pale maroon-tan   O    Melanin negative                    Melanin negative                                Melanin negativeGelatinPlain   S    Colorless vegetative                    Colorless vegetative                                Colorless vegetative   P    --          --          --   O    None to very slight                    No liquefaction                                Complete liquefaction        liquefactionNutrient   S    Colorless vegetative                    Colorless vegetative                                Trace cream aerial                                on surface growth   P    --          --          --   O    Liquefaction complete                    No liquefaction                                Liquefaction completeNitrate BrothSynthetic   S    --          Partial pellicle                                Partial pellicle   P    --          --          --   O    Nitrates reduced to                    Nitrates not reduced                                Nitrates not reduced        nitritesNutrient   S    Light gray-cream aerial                    Light gray-cream aerial                                Cream-pink aerial on        on surface ring and                    on surface ring                                surface pellicle        pellicle   P    --          --          --   O    Poor, compact bottom                    Compact bottom growth                                Compact to flocculent        growth                  bottom growth        Nitrates reduced to                    Nitrates not reduced                                Nitrates not reduced        nitritesLitmus milk   S    Orange-tan surface ring                    Blue-gray aerial growth                                Maroon surface ring                    on blue-gray surface                    ring   P    --          Blue-gray   Deep purple   O    No peptonization                    Peptonization                                Slight peptonization        pH 5.9      pH 7.5      pH 6.4__________________________________________________________________________ S = Surface R = Reverse P = Pigment O = Other Characteristics 
    
     
                       TABLE 4.______________________________________Growth On Carbon Compounds In TheSynthetic Medium Of Pridham And Gottlieb*         S.       S.        S.         helicus  olivaceus griseoroseusCONTROL       (+)      (+)       (+)______________________________________1.   D-xylose      +       +       +2.   L-arabinose  +        +       +3.   Rhamnose     +        +       +4.   D-fructose   +        +       +5.   D-galactose  +        +       +6.   D-glucose    +        +       +7.   D-mannose    +        +       +8.   Maltose      +        +       +9.   Sucrose      (+)      +       (+)10.  Lactose      +        +       +11.  Cellobiose   +        +       +12.  Raffinose    (+)      (+)     (+)13.  Dextrin      +        +       +14.  Inulin       (+)      +       (+)15.  Soluble starch             +        +       +16.  Glycerol     +        +       +17.  Dulcitol     (+)      (+)     (+)18.  D-Mannitol   +        +       +19.  D-Sorbitol (-)             (-)      (+)20.  Inositol     +        +       (+)21.  Salicin      (+)      (+)     +22.  Phenol       -        -       -23.  Cresol       -        -       -24.  Na formate   (-)      (-)     (-)25.  Na oxalate   (+)      (-)     (+)26.  Na tartrate  (+)      (+)     (+)27.  Na salicylate             -        -       -28.  Na acetate   (+)      (+)     +29.  Na citrate   (+)      (+)     (+)30.  Na succinate (+)      (+)     (+)______________________________________ + = Good growth? (+) = Moderate growth? (-) = Poor growth? - = No growth. *Pridham, T.G., and D. Gottlieb. 1948. The utilization of carbon compound by some Actinomycetales as an aid for species determination. J. Bacteriol 56: 107-114. 
    
     
                       TABLE 5______________________________________Utilization Of Carbon Compounds In TheSynthetic Medium Of Shirling And Gottlieb*         S.       S.        S.ISP #9        helicus  olivaceus griseoroseus______________________________________CONTROLNegative (No carbon         ±     -         -compound added)Positive      +        +         +(D-glucose)Carbon CompoundsL-Arabinose   +        +         ++Sucrose       ±     -         -D-xylose      ++       ++        ++Inositol      +        ++        ±D-Mannitol    ++       ++        ++D-Fructose    ++       ++        ++Rhamnose      ++       ++        ++Raffinose     ±     -         -Cellulose     ±     -         -______________________________________ ++ Strong utilization ± Doubtful utilization + Positive utilization - No utilization *Shirling, E.B., and D. Gottlieb. 1966. Methods for characterization of Streptomyces species. Int. J. Syst. Bacteriol. 16: 313-340. 
    
     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 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 brewer&#39;s yeast with milk solids, soybean meal, cottonseed meal, cornmeal, milk solids, pancreatic digest of casein, fish meal, distillers&#39; 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 media 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 conductive to satisfactory growth of the microorganism, for example, between about 18° and 40° C., and preferably between about 20° and 28° 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 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 both mycelial cake and clarified broth. The mycelial cake can be extracted with a lower alcohol (1 to 4 carbon atoms, inclusive), methanol (preferred) and the extract evaporated under reduced pressure to an aqueous concentrate. The aqueous concentrate is added to the filtered broth, which then can be extracted three times with a half volume of a lower alcohol, 1-butanol is preferred. The combined preparations are purified by chromatography on silica gel. The active fractions from the column after removal of the solvent yields a red material which is crystallized from boiling chloroform to yield antibiotic U-58,431. The solvent system used for the chromatography is chloroform:methanol (9:1). 
     The antibiotic of the subject invention also can be recovered from 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 and XAD-4, 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 resin by a water-lower alcohol (preferably methanol) mixture or a water-acetone mixture. 
     Salts of antibiotic U-58,431 also can be formed with inorganic or organic bases. Such salts can be prepared, as for example, by dissolving antibiotic U-58,431 in water, adding a dilute base until the pH of the solution is about 10.0 to 11.0, and freeze-drying the solution to provide a dried residue consisting of the U-58,431 salt. Antibiotic U-58,431 salts with inorganic cations which can be formed include the sodium, potassium, and calcium salts. Other salts of U-58,431, including those with 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, 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. Salts of U-58,431 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 helicus Dietz and Li sp.n., NRRL 11461, is used to inoculate 500-ml Erlenmeyer seed flasks containing 100 ml of sterile medium consisting of the following ingredients: 
     
         ______________________________________ Glucose monohydrate   25 g/lPharmamedia*           25 g/lTap water q.s.         1 liter______________________________________ *Pharmamedia is an industrial grade of cottonseed flour produced by Traders Oil Mill Company, Fort Worth, Texas. 
    
     The preseed medium presterilization pH is 7.2. The preseed inoculum is grown for three days at 28° C. on a Gump rotary shaker operating at 250 rpm and having a 21/2 inch stroke. 
     Preseed inoculum (300 ml), prepared as described above, is used to inoculate a seed tank containing 20 liters of sterile seed medium consisting of the following ingredients: 
     
         ______________________________________ Glucose monohydrate   25 g/lPharmamedia            25 g/lTap water              Balance______________________________________ 
    
     The inoculated seed medium is incubated at a temperature of 28° C. for 2 days while being agitated at a rate of 400 rpm and aerated at a rate of 10 standard liters per minute with a back pressure of 10 psig. 
     After 2 days incubation, the seed medium is used to inoculate (the inoculation rate is 5 liters of seed inoculum per 100 liters of fermentation medium) a 250 liter tank fermentation containing sterile fermentation medium consisting of the following ingredients: 
     
         ______________________________________ Glucose monohydrate   15 g/lDextrin                25 g/lCorn gluten meal       20 g/lOatmeal                15 g/lCalcium carbonate       8 g/lUcon*                  0 3 ml/l______________________________________ pH  7.2 (presterilization) *Ucon is a synthetic defoamer supplied by Union Carbide, New York, New York. 
    
     The fermentation tank is incubated at a temperature of 28° C. with agitation of 280 rpm and aeration at 185 standard liters per minute. Harvest is usually after about five days of fermentation. A typical five-day fermentation has the following titers of antibiotic in the fermentation broth: 
     
         ______________________________________Day             Assay, U/ml______________________________________1               02               03               04               85               32______________________________________ 
    
     The assay is a Bacillus subtilis disc plate assay using 0.1 M Tris buffer, pH 7.0 as diluent. 
     B. Recovery 
     The whole beer (ca. 3 l) from a fermentation, as described above, is adjusted to pH 5.0 with concentrated HCl and the mycelium removed by filtration through diatomaceous earth (dicalite). To the clear broth (2.7 l) are added 1.2 kg solid (NH 4 ) 2  SO 4 . After dissolution of the salt, the broth is extracted with 1600 ml 1-butanol. The mycelium cake is mixed with 600 ml of methanol for 1 hour. The suspension is filtered through a dicalite pad; yield 600 ml clear filtrate. The extracts from the clear broth and the mycelial cake are combined at this point and reduced to an oil under vacuum. The oily residue is triturated with 150 ml warm methylene chloride. Insolubles are removed by filtration and the methylene chloride extract is subjected to column chromatography on silica gel. Two hundred grams of silica gel (Merck) is suspended in methylene chloride-methanol (9:1 v/v) and a column 35×3.8 cm is poured. The above methylene chloride extract is taken up in 5 g of dry silica gel and the material is applied to the column. The column is eluted with a methanol-methylene chloride mixture (1:9 v/v). Fractions of 100 ml are collected and assayed vs. Sarcina lutea on a standard disc assay. Fraction 7 contains most of the activity and is reduced to approximately 4 ml under vacuum. The concentrated solution is allowed to stand at 4° C. overnight and the crystals of antibiotic U-58,431 formed are recovered by filtration; yield=26 mg. The concentrated crystals are recrystallized from boiling chloroform; yield=10 mg of antibiotic U-58,431. 
     Initial analysis of antibiotic U-58,431 by X-ray crystallography gives the following structure: ##STR1## 
     Antibiotic U-58,431 can be acylated at the two hydroxyl and two amino groups. By using a minimum amount of acylating agent the two amino groups are acylated first to give di-N-acylates. Di-O-acylates are obtained by methods known in the art, e.g., protecting the amino groups, acylating the two hydroxyls, then removing the amino protective groups. Tetra acylates are formed by acylating U-58,431 with an excess of acylating agent. 
     U-58,431 can be acylated under standard acylating conditions with an appropriate acid halide or anhydride to give the acylated compounds. The acylation is carried out in the presence of an acid-binding agent. Suitable acid-binding agents include: amines such as pyridine, quinoline, and isoquinoline, and buffer salts such as sodium acetate. The preferred base is pyridine. Carboxylic acids suitable for acylation include (a) saturated or unsaturated, straight or branched chain aliphatic carboxylic acids, for example, acetic, propionic, butyric, isobutyric, terbutylacetic, valeric, isovaleric, caproic, caprylic, decanoic, dodecanoic, lauric, tridecanoic, myristic, pentadecanoic, palmitic, margaric, stearic, acrylic, crotonic, undecylenic, oleic, hexynoic, heptynoic, octynoic acids, and the like; (b) saturated or unsaturated, alicyclic carboxylic acids, for example, cyclobutanecarboxylic acid, cyclopentanecarboxylic acid, cyclopentenecarboxylic acid, methylcyclopentenecarboxylic acid, cyclohexanecarboxylic acid, dimethylcyclohexanecarboxylic acid, dipropylcyclohexanecarboxylic acid, and the like; (c) saturated or unsaturated, alicyclic aliphatic carboxylic acids, for example, cyclopentaneacetic acid, cyclopentanepropionic acid, cyclohexaneacetic acid, cyclohexanebutyric acid, methylcyclohexaneacetic acid, and the like; (d) aromatic carboxylic acids, for example, benzoic acid, toluic acid, naphthoic acid, ethylbenzoic acid, isobutylbenzoic acid, methylbutylbenzoic acid, and the like; and (e) aromatic aliphatic carboxylic acids, for example, phenylacetic acid, phenylpropionic acid, phenylvaleric acid, cinnamic acid, phenylpropiolic acid, and naphthylacetic acid, and the like. Also, suitable halo-, nitro-, hydroxy-, amino-, cyano-, thiocyano-, and lower alkoxy- hydrocarbon carboxylic acids include hydrocarboncarboxylic acids as given above which are substituted by one or more of halogen, nitro, hydroxy, amino, cyano, or thiocyano, or lower alkoxy, advantageously lower alkoxy of not more than six carbon atoms, for example, methoxy, ethoxy, propoxy, butoxy, amyloxy, hexyloxy groups and isomeric forms thereof. Examples of such substituted hydrocarbon carboxylic acids are: 
     mono-, di- and trichloroacetic acid; 
     α- and β-chloropropionic acid; 
     α- and γ-bromobutyric acid; 
     α- and δ-iodovaleric acid; 
     mevalonic acid; 
     2- and 4-chlorocyclohexanecarboxylic acid; 
     shikimic acid; 
     2-nitro-1-methyl-cyclobutanecarboxylic acid; 
     1,2,3,4,5,6-hexachlorocyclohexanecarboxylic acid; 
     3-bromo-2-methylcyclohexanecarboxylic acid; 
     4- and 5-bromo-2-methylcyclohexanecarboxylic acid; 
     5- and 6-bromo-2-methylcyclohexanecarboxylic acid; 
     2,3-dibromo-2-methylcyclohexanecarboxylic acid; 
     2,5-dibromo-2-methylcyclohexanecarboxylic acid; 
     4,5-dibromo-2-methylcyclohexanecarboxylic acid; 
     5,6-dibromo-2-methylcyclohexanecarboxylic acid; 
     3-bromo-3-methylcyclohexanecarboxylic acid; 
     6-bromo-3-methylcyclohexanecarboxylic acid; 
     1,6-dibromo-3-methylcyclohexanecarboxylic acid; 
     2-bromo-4-methylcyclohexanecarboxylic acid; 
     1,2-dibromo-4-methylcyclohexanecarboxylic acid; 
     3-bromo-2,2,3-trimethylcyclopentanecarboxylic acid; 
     1-bromo-3,5-dimethylcyclohexanecarboxylic acid; 
     homogentisic acid, o-, m-, and p-chlorobenzoic acid; 
     anisic acid; 
     salicyclic acid; 
     p-hydroxybenzoic acid; 
     β-resorcyclic acid; 
     gallic acid; 
     veratric acid; 
     trimethoxybenzoic acid; 
     trimethoxycinnamic acid; 
     4,4&#39;-dichlorobenzilic acid; 
     o-, m-, and p-nitrobenzoic acid; 
     cyanoacetic acid; 
     3,4- and 3,5-dinitrobenzoic acid; 
     2,4,6-trinitrobenzoic acid; 
     thiocyanoacetic acid; 
     cyanopropionic acid; 
     lactic acid; 
     ethoxyformic acid (ethyl hydrogen carbonate); and the like. 
     The acylated compounds, as described above, can be used in animals for the same biological purposes as disclosed above for U-58,431. For example, the acylated compounds can be given in oral form to an animal possessing the necessary enzyme to remove the acyl group, thus freeing the parent antibiotic compound which then inhibits susceptible bacteria.