Abstract:
This disclosure describes a new antibacterial agent designated LL-C23201δ, produced in a microbiological fermentation under controlled conditions using a new strain of Streptomyces olivaceo-griseus, sp. nov. and mutants thereof. This new antibacterial agent is active against a variety of microorganisms and thus is useful in inhibiting the growth of such bacteria wherever they may be found.

Description:
BRIEF SUMMARY OF THE INVENTION 
     This invention relates to a new antibacterial agent designated LL-C23201δ, to its production by fermentation, to methods for its recovery and concentration from crude solutions and to processes for its purification. The present invention includes within its scope the antibacterial agent in dilute form, as a crude concentrate and in pure crystalline form. The effects of this new antibacterial agent on specific microorganisms, together with its chemical and physical properties, differentiate it from previously described antibacterial agents. 
     The molecular structure of LL-C23024δ was determined by X-ray crystallography of the rubidium salt, and it has the following formula: ##STR1## 
    
    
     LL-C23201δ is a new polyether antibiotic useful as an antibacterial and anticoccidial agent. Field desorption mass spectroscopy indicates that the sodium salt has a molecular weight of 907 and the potassium salt a molecular weight of 923. Further characterization of LL-C23201δ is detailed in the Examples and is shown in the accompanying FIGS. I-IV: 
     FIG. I: IR spectrum of LL-C23201δ sodium salt in KBr. 
     FIG. II: PMR spectrum 79.5 MHz in CDCl 3  of LL-C23201δ sodium salt. 
     FIG. III: IR spectrum of LL-C23201δ potassium salt in KBr. 
     FIG. IV: PMR spectrum 79.5 MHz in CDCl 3  of LL-C23201δ potassium salt. 
    
    
     The  13  CNMR spectrum of the sodium salt of LL-C23201δ was obtained using a Varian FT80 instrument at 20 MHz in CDCl 3 . The chemical shifts for both the sodium salt of the antibiotic 6016 [J. Antibiotics, 32, 244-246 (1979)] [Otake, N., et al., J. Chem. Soc. Chem. Comm., 875-876 (1978)] which is a polyther antibiotic and the sodium salt of LL-C23201δ are given in Table I. 
     
                       TABLE I______________________________________.sup.13 CNMR Spectra of Sodium Salt, LL-C23201δ, andAntibiotic 6016LL-C23201δ    Antibiotic 6016C.sub.47 H.sub.80 O.sub.15    C.sub.46 H.sub.78 O.sub.16                 LL-C23201δ                            Antibiotic 6016______________________________________ 5.0      5.0         39.7       39.411.5     --           46.0       --11.7     11.7         55.9       55.812.6     12.5         56.8       56.713.1     13.0         58.8       58.816.9     16.8         61.1       60.917.3     17.3         64.3       64.818.3     18.2         --         72.124.3     24.3         74.3       73.926.4     26.5         74.6       74.427.0     26.9         74.9       74.628.1     28.0         78.7       78.629.1     29.1         78.9       79.030.6     30.5         79.8       79.630.8     30.6         80.2       79.931.5     31.2         80.5       80.732.8     32.7         82.5       82.334.3     33.4         82.8       82.635.3     34.0         90.0       89.936.9     36.5         96.8       96.636.9     36.7         99.2       98.237.5     36.9         100.1      99.239.1     38.9         108.1      107.939.4     39.3         180.5      178.4                 47 × C                            46 × C______________________________________ 
    
     The above spectra show that LL-C23201δ has 47 carbons and the structure shown herein. 
     The antibiotic 6016 has 46 carbons and its structure is: ##STR2## 
     Antibiotic LL-C23201δ is an organic carboxylic acid and thus is capable of forming salts with non-toxic pharmaceutically acceptable cations. Thus, salts formed by admixture of the antibiotic free acid with stoichiometric amounts of cations, suitably in a neutral solvent, may be formed with cations such as sodium, potassium, calcium, magnesium and ammonium, as well as organic amine cations such as tri(lower alkyl)amine (e.g., triethylamine, triethanolamine), procaine and the like. The cationic salts of antibiotic LL-C23024δ are, in general, crystalline solids, relatively insoluble in water but are soluble in most common organic solvents such as methanol, ethyl acetate, acetone, chloroform, heptane, ether and benzene. 
     DETAILED DESCRIPTION OF THE INVENTION 
     The new antibacterial agent LL-C23201δ is formed during the cultivation under controlled conditions of a new species of Streptomyces named Streptomyces olivaceo-griseus sp. nov. 
     This new antibiotic strain was isolated as an aerial contaminant at the Medical Research Division, American Cyanamid Company, Pearl River, N.Y. and is maintained in the culture collection of the aforesaid Medical Research Division as culture member LL-C23201. A viable culture of this new microorganism has been deposited with the Culture Collection Laboratory, North Central Region, Northern Regional Center, U.S. Department of Agriculture, Peoria, Ill. and has been added to its permanent collection. It is freely available to the public from this depository under its accession number NRRL 15357. 
     TAXONOMIC CHARACTERIZATION OF CULTURE LL-C23201 
     The culture LL-C23201 was taxonomically characterized and identified as a new species of the gray-spored Streptomyces to be known as Streptomyces olivaceo-griseus, sp. nov. 
     Observations were made of the cultural, physiological, and morphological features of the culture in accordance with the methods detailed by Shirling, E. B. and Gottlieb, D., Internat. J. Syst. Bacteriol. 16, 313-340 (1966). Media used in this study were selected from those recommended by Pridham, T. G., et. al., Antibiotics Annual, pp. 947-953 (1956/57) and Gordon R. E., et. al., Internat. J. Syst. Bacteriol. 24, 54-63 (1974) for the taxonomic study of actinomycetes and soil bacteria, respectively. Chemical composition of the cell walls of the culture was determined using the method of Lechevalier, et. al., Adv. Appl. Microbiol. 14, 47-72 (1971). Details are recorded in Tables I-V, and a general description of the culture is given below. Underscored descriptive colors are taken from Kelly, K. L. and Judd, D. B., Nat. Bur. Stand., Spec. Publ. 440 (1976) and the accompanying Inter-Society Color Council, National Bureau of Standards, Centroid Color Charts. 
     Isolate LL-C23201 was compared to an appropriate reference strain, Streptomyces ravidus NRRL 11300, the culture which produce ravidomycin. A comparison of 14-day growth of each of these cultures on Hickey-Tresner agar is shown below: 
     
         ______________________________________    SPORE MASS   SOLUBLE    REVERSECULTURE  COLOR        PIGMENTS   COLOR______________________________________S. ravidus    Light Gray   Red        GrayishNRRL 11300                       Reddish BrownS. olivaceo-    Light Gray   None       Blackish GreengriseusLL-C23201______________________________________ 
    
     The gross colonial morphology of LL-C23201 does not resemble S. ravidus, and significant differences were also observed in physiology. S. ravidus reduces nitrates and utilizes arabinose but does not utilize mannitol or sucrose. A search of the current streptomycete literature failed to reveal any described species which resembled LL-C23201; therefore, a new species is designated to be known as Streptomyces olivaceo-griseus, sp. nov. 
     MICROMOPHOLOGY 
     Spores are formed in coiled chains (Spira) on aerial sporophores. The spores are ovoid (1.5-1.8 microns by 2.0-2.5 microns), and the surface of the mature spores is smooth when observed by scanning electron microscopy. 
     CELL WALL COMPOSITION 
     Whole cell hydrolysates of this culture contain the L,L-isomer of diaminopimelic acid, placing it in the Type I cell wall group of Lechevalier, et al. (vide supra). This is typical of all Streptomyces species. 
     AMOUNT OF GROWTH 
     Good growth is observed on most media; moderate growth is observed on glycerol-asparagine agar; poor growth is observed on oatmeal agar; no growth is observed on tomato paste-oatmeal agar. 
     AERIAL MYCELIUM AND SPORE COLOR 
     Aerial mycelium is white on most media but becomes tinged with pink or inorganic salts-starch agar; spore mases are 264. light gray in color. 
     SOLUBLE PIGMENTS 
     Absent on many media; brownish shades where produced. 
     REVERSE COLOR 
     Greenish black to olive black shades on all media. 
     PHYSIOLOGICAL REACTIONS 
     Nitrates not reduced to nitrites in 14 days; no liquifaction of gelatin in 14 days; no black pigment (melanin) produced on either peptone-yeast extract-iron agar or tyrosine agar; strong peptonization of litmus milk in 14 days. Carbohydrate utilization as per the method of Pridham, T. G., and Gottlieb, D. J., Bacteriol., 56, 107-144 (1948), good utilization of glucose; moderate utilization of fructose and inositol; poor utilization of galactose, mannitol, rhamnose, or salicin. Several organic acids were tested as sole carbon sources: citrate, malate, and succinate were strongly utilized; lactate was weakly utilized; and benzoate, mucate, and oxalate were not utilized. Adenine, hypoxanthine, and tyrosine were hydrolysed in 14 days, while guanine and xanthine were not. 
     
                                           TABLE I__________________________________________________________________________Cultural Characteristics of Streptomyces olivaceo-griseus LL-C23201Incubation 14 days   Temperature: 28 ° C.    Amount of               SolubleMedium   Growth Aerial Mycelium and/or Spores                            Pigment                                 Reverse Color__________________________________________________________________________Glycerol-    Moderate           Relatively flat, waxy growth with                            Brownish                                 GreenishAsparagine Agar no aerial mycelia; growth 77.                                 black           moderate yellowish brownHickey-Tresner    Good   Raised waxy colonies with plicate                            none BlackishAgar            centers; vegetative growth 152,                                 green           blackish green; very little           aerial mycelia or spore           production; spores 264. light           grayInorganic    Moderate to           Slightly raised waxy colonies;                            None --Salt-Starch    Good   110 grayish olive to 114, oliveAgar            black, becoming powdery in sporu-           lating areas; aerial mycelia           white to 28. light grayish pink           in areas; spores 264. light grayNz-amine Good   Raised ridged growth; vegetative                            Brownish                                 GreenishGlucose Starch  mycelia 157. greenish black;                                 blackAgar            heavy production of aerial           mycelia and spores; spores 264.           light grayOatmeal Agar    Poor   Flat, dull growth with no aerial                            None --           mycelia; vegetative mycelia 90.           grayish yellow to 112. light           olive grayYeast Extract    Good   Raised, ridged colonies with                            Brown                                 Olive BlackMalt Extract    heavy sporulation; vegetativeAgar            mycelia 114. olive black; spore           mass 264. light gray__________________________________________________________________________ 
    
     
                                           TABLE II__________________________________________________________________________Micromorphology of Streptomyces olivaceo-griseus LL-23201  Aerial Mycelium and/or Sporiferous                   Spore                       Spore   SporeMedium Structures       Shape                       Size    Surface__________________________________________________________________________Inorganic  Spore chains arise as coiled chains                   Ovoid                       0.6-0.7 micron                               SmoothSalts-Starch  from aerial (Spira)  XAgar                        0.6-0.8 micron__________________________________________________________________________ 
    
     
                       TABLE III______________________________________Physiological Reaction of Streptomycesolivaceo-griseus LL-C23201       Incubation Amount of PhysiologicalMedium      Period (days)                  Growth    Reaction______________________________________Peptone-Iron Agar        7         Good      No Blackening       14         Good      No blackeningTyrosine Agar        7         Good      No blackening(ISP-7)     14         Good      Greenish                            Black pigmentLitmus Milk  7         Good      Slight                            proteolysis       14         Good      Strong                            proteolysisNutrient Gelatin        7         Slight    No proteolysis       14         Slight    No ProteolysisNitrate Broth        7         Good      No reduction       14         Good      No reductionAdenine Agar        7         Good      Strong hydro-                            lysis       14         Good      Strong hydro-                            lysisGuanine Agar        7         Good      No hydrolysis       14         Good      No hydrolysisHypoxanthine        7         Good      No hydrolysisAgar        14                   Strong hydro-                            lysisTyrosine Agar        7         Good      No hydrolysis       14         Good      Weak hydro-                            lysisXanthine Agar        7         Good      No hydrolysis       14         Good      No hydrolysis______________________________________ 
    
     
                       TABLE IV______________________________________Carbon Source Utilizationof Streptomyces olivaceo-griseus LL-C23201Incubation: 14 Days  Temperature: 28° C.Carbon Source   Utilization*______________________________________l-Arabinose     0Fructose        2d-Galactose     1d-Glucose       3l-Inositol      2d-Mannitol      1d-Raffinose     0l-Rhamnose      0Salicin         0Sucrose         1Xylose          1Negative control           0______________________________________ *3 = Good utilization 2 = Fair Utilization 1 = Poor Utilization 0 = No Utilization 
    
     
                       TABLE V______________________________________Utilization of organic acidsStreptomyces olivaceo-griseusLL-C23201 on Gordon&#39;s modificationof Kosher&#39;s basal agar(Koser&#39;s citrate agar)Incubation: 14 Days  Temperature: 28° C.Carbon Source   Utilization______________________________________Benzoate        -Citrate         +Lactate         ±Malate          +Mucic Acid      -Oxalate         -Succinate       +______________________________________ 
    
     It is to be understood that for the production of this antibacterial agent, the present invention is not limited to this particular organism or to organisms fully answering the above growth and microscopic characteristics, which are given for illustrative purposes only. In fact, it is desired and intended to include the use of mutants produced from this organism by various means such as exposure to X-radiation, ultraviolet radiation, nitrogen mustard, actinophages, and the like. 
     This antibactrial agent is active in vitro against gram-positive bacteria (Table VI) and against gram-positive and gram-negative anaerobes (Table VII). 
     
                       TABLE VI______________________________________Antibacterial Activity of LL-C23201δAgar Dilution Method, Meuller-Hinton Agar                Minimal InhibitoryGram-Positive Organism                Concentration (mcg/ml)______________________________________Staphylococcus aureus Smith                0.5Staphylococcus aureus SSC80-11                1Staphylococcus aureus SSC80-32                1Staphylococcus aureus SSC80-38                1Staphylococcus aureus LL-14                0.5Staphylococcus aureus LL-45                0.5Staphylococcus aureus LL-27                2Staphylococcus aureus ATCC25923                0.5Streptococcus pyogenes C203                ≦0.12Streptococcus β-hemdytic Keller T623                ≦0.12Streptococcus pneumoniae 78-1                0.25Enterococcus SSC80-62                0.5Enterococcus SSC80-63                0.5______________________________________ 
    
     
                       TABLE VII______________________________________Activity of LL-C23201δ Against AnaerobicBacteria Agar-Dilution, Wilkins-Chalgren Agar,Incubated in a Gas-Pac at 37° C. for 48 hours           No. of  Minimal Inhibitory           Strains ConcentrationOrganism        Tested  mcg/ml Range______________________________________Bacteroides fragilis           13       8-256Bacteroides thetaiotaomicron           5       16-256Bacteroides vulgatus           2       8Bacteroides distasonis           1       ≦0.12Clostridium perfringens           2       ≦0.12Clostridium innocuum           1       ≦0.12Clostridium bifermentans           1       ≦0.12Clostridium difficile           1       ≦0.12Peptococcus anaerobius           1       ≦0.12Peptococcus magnus           1       ≦0.12______________________________________ 
    
     In addition, LL-C23201δ is active in vitro and in vivo as an anticoccidial agent. In tissue culture it is active against Eimeria tenella at 0.001 to 0.1 ppm. It is active in chicks against Eimeria tenella and Eimeria acervulina in the range of 10-125 ppm. 
     GENERAL PROCEDURE FOR THE PRODUCTION OF LL-C23201δ 
     Cultivation of Streptomyces olivaceo-griseus NRRL 15357 may be carried out in a wide variety of liquid culture media. Media which are useful for the production of this novel antibacterial agent include an assimilable source of carbon such as starch, sugar, molasses, glycerol, etc., an assimilable source of nitrogen such as protein, protein hydrolysate, polypeptides, amino acids, corn steep liquor, etc., and inorganic anions and cations, such as potassium, sodium, ammonium, calcium, sulfate, carbonate, phosphate, chloride, etc. Trace elements such as boron, molybdenum, copper, etc., are supplied as impurities of other constituents of the media. Aeration in tanks and bottles is provided by forcing sterile air through or onto the surface of the fermenting medium. Further agitation in tanks is provided by a mechanical impeller. An antifoaming agent such as lard oil may be added as needed. 
     INOCULUM PREPARATION 
     Shaker flask inoculum of Streptomyces olivaceo-griseus NRRL 15357 is prepared by inoculating 100 ml of sterile liquid medium in 500 ml flasks with scrapings or washings of spores from an agar slant of the culture. The following is an example of a suitable medium: 
     
         ______________________________________Beef extract    0.3%Bacto tryptone  0.5%Glucose         1.0%Yeast extract   0.5%Bacto agar      0.15%Water qs        100%______________________________________ 
    
     The flasks are incubated at a temperature of 25°-35° C., preferably 32° C. and agitated vigorously on a rotary shaker for 24-28 hours. 
     Portions of the above flask inoculum are then used to inoculate multi-liter portions of the same sterile medium in bottles which are aerated with sterile air while growth is continued for 24-48 hours. This bottle inoculum is then used to inoculate a seed tank fermentor containing 30-300 liters of the same sterile medium which is grown for 18-24 hours at 32° C. with aeration and then used to inoculate tank fermentors. 
     TANK FERMENTATION 
     For the production of LL-C23201δ in tank fermentors the following sterilized medium may be used: 
     
         ______________________________________Glucose                1.5%Glycerol               1.5%Soy flour              1.5%Calcium carbonate      0.1%Sodium chloride        0.3%Water qs               100%______________________________________ pH adjusted to 7.0 with 6N sodium hydroxide 
    
     Each tank is inoculated with 3-10% of inoculum prepared as described above. Aeration is supplied at the rate of 0.5 to 2.0 liter of sterile air per liter of broth per minute and the fermenting medium is agitated by an impeller driven at 100-400 rpm. The temperature is maintained at 25°-35° C., preferably at 32° C. The fermentation is usually continued for 100-150 hours, at which time the mash is harvested. 
     EXAMPLE 1 
     Inoculum Preparation 
     A typical medium used to grown the primary inoculum was prepared according to the following formula: 
     
         ______________________________________Beef extract    0.3%Bacto tryptone  0.5%Glucose         1.0%Yeast extract   0.5%Bacto agar      0.15%Water qs        100%______________________________________ 
    
     Scraped spores from an agar slant of Streptomyces olivaceo-griseus NRRL 15357 were used to inoculate two 500 ml flasks each containing 100 ml of the above sterilized medium. The flasks were placed on a rotary shaker and agitated vigorously for 24 hours at 32° C. The resulting flask inoculum (200 ml) was used to inoculate 12 liters of the same sterile medium in a 15 liter bottle. This bottle inoculum was aerated with sterile air while growth was continued for 48 hours at 28° C. This bottle inoculum (12 liters) was then used to inoculate 300 liters of the same sterile medium in a seed fermentation tank. This seed inoculum was grown, with aeration, for 18 hours at 32° C. 
     EXAMPLE 2 
     Fermentation 
     A fermentation medium was prepared according to the following formulation: 
     
         ______________________________________Glucose                1.5%Glycerol               1.5%Soy flour              1.5%Calcium carbonate      0.1%Sodium chloride        0.3%Water qs to            100%______________________________________ 
    
     The pH was adjusted to 7.0 with 6N sodium hydroxide prior to sterilization. A 300 liter portion of the seed inoculum (Example 1) was used to inoculate 2600 liters of the above medium in a large tank fermentor. Sterile aeration was supplied to the mash which was agitated by an impeller driven at 100 rpm. The fermentation was carried out at 32° C. for 116 hours at which time the mash was harvested. 
     EXAMPLE 3 
     Preliminary Isolation of LL-C23201δ 
     A total of 3000 liters of harvest mash (Example 2) was adjusted to pH 4.0, using 6N hydrochloric acid and was then stirred with 1/2 of its volume of methylene chloride. The mixture was filtered through diatomaceous earth, then the organic solvent layer was drawn off, washed with aqueous sodium bicarbonate solution and concentrated in vacuo to 6.5 liters of a viscous oily residue. 
     A glass column with a diameter of 8.5 cm was packed dry to a height of 97 cm with Woelm silica gel TSC. An 1100 ml portion of the above oily residue was diluted with 2 liters of methylene chloride and allowed to seep into the column. This column was developed first with 11 liters of methylene chloride:ethyl acetate (3:1) and then with 4 liters of ethyl acetate. Fractions of 60 ml each were collected and checked for antibacterial activity by the bioautography of impregnated paper discs on large agar plates at pH 6.0 seeded with Bacillus subtilis Stansley R-78. Fractions 124-136 were combined and concentrated in vacuo, giving 1765 mg of active residue. 
     A glass column with a diameter of 4.5 cm was packed dry to a height of 84 cm with Woelm silica gel TSC. The above active residue (1765 mg) was dissolved in 50 ml of methylene chloride and allowed to seep into the column which was then developed with methylene chloride:ethyl acetate (1:1) collecting fractions of 45 ml each. The fractions were again checked by bioautography and the active fractions were combined and desolventized in vacuo, giving LL-C23201δ as a viscous residue. 
     EXAMPLE 4 
     Preparation of the Crystalline Sodium Salt of LL-C23201δ 
     The purified residue, prepared in Example 3, was dissolved in 200 ml of hexane:ether (1:1) and then placed over dilute hydrochloride acid (7 ml of 0.1N hydrochloric acid and 150 ml of water). The two phases were thoroughly mixed, allowed to settle and the acidic aqueous portion discarded. The organic layer was washed twice with 200 ml volumes of water. The washed organic layer was transferred over 150 ml of fresh water and dilute sodium hydroxide was added dropwise until the pH stabilized between 10.5 and 11.0 after shaking and settling. The organic layer was again washed twice with 200 ml volumes of water and was then dried over sodium sulfate, concentrated in vacuo to about 30 ml and then allowed to stand at 4° C. for 2 hours. The crystals that formed were collected, washed with cold hexane and air dried, giving 1223 mg of the sodium salt of LL-C23201δ, having the following characteristics: 
     Elemental analysis: C, 57.29; H, 8.59; ash, 5.34. 
     Molecular weight (F.D. mass spectroscopy): 907. 
     [α] D   22  =-28° (0.49% in methanol). 
     MP=164°-168° C. 
     Infra red spectrum in KBr as shown in FIG. I; 
     UV spectra shows only weak end absorption; 
     Proton magnetic resonance spectrum 79.5 MHz in CDCl 3  using a Varian FT80 as shown in FIG. II. 
     EXAMPLE 5 
     Preparation of the Crystalline Potassium Salt of LL-C23201δ 
     A 540 mg portion of purified LL-C23201δ, prepared as described in Example 3 was treated as described in Example 4, substituting potassium hydroxide for sodium hydroxide. The yield was 332 mg of the white crystalline potassium salt of LL-C23201δ, having the following characteristics: 
     Elemental analysis: C, 60.83; H, 8.60; ash, 4.20. 
     Melting point 167° C. 
     Molecular weight (F.D. mass spectroscopy)=923. 
     [α] D   22  =-28° (0.49% in methanol). 
     Infrared spectrum in KBr as shown in FIG. IV. 
     Proton magnetic resonance spectrum 79.5 MHz in CDCl 3  using a Varian FT80 as shown in FIG. IV. 
     EXAMPLE 6 
     High Performance Liquid Chromatography of LL-C23201δ 
     LL-C23201δ using the following conditions for analytical high performance chromatography: 
     Instrument: Waters ALC/GPC 244 
     Column: Ultrasphere ODS, pH 5.0/methanol (10/90) 
     Detector: Refractive index, 4×setting 
     LL-C23201δ was homogeneous and had a retention time of 22.3 minutes.