Abstract:
Cultivation of a strain of the microorganism Pseudomonas cepacia SC 11,783, A.T.C.C. No. 39356 yields a novel antibiotic substance which is made up of two compounds, i.e., 5-[3-[3-(hepta-1,2-dien-4,6-diynyl)oxiran-2-yl]-3-hydroxy-1-propanyl]dihydro-2(3H)-furanone and 5-[[3-[3-(hepta-1,2-dien-4,6-diynyl)-2-oxiranyl]-2-oxiranyl]hydroxymethyl]dihydro-2(3H)-furanone.

Description:
SUMMARY OF THE INVENTION 
     Cultivation of a strain of the microorganism Pseudomonas cepacia SC11,783, which has been deposited in the American Type Culture Collection as A.T.C.C. No. 39356, yields a novel antibiotic substance cepacin. 
     Cepacin has been analyzed and found to be made up of two compounds; i.e., 5-[3-[3-(hepta-1,2-dien-4,6-diynyl)oxiran-2-yl]-3-hydroxy-1-propenyl]dihydro-2(3H)-furanone, a compound of the formula ##STR1## referred to as cepacin A, and 5-[[3-[3-(hepta-1,2-dien-4,6-diynyl)-2-oxiranyl]-2-oxiranyl]hydroxymethyl]dihydro-2(3H-furanone, a compound having the formula ##STR2## referred to as cepacin B. 
     Each of the above compounds exhibits activity against gram-positive bacteria. Cepacin A shows activity against some gram-negative bacteria and cepacin B shows activity against a wider range of gram-negative bacteria. 
     DETAILED DESCRIPTION OF THE INVENTION 
     The Microorganism 
     The microorganism used for the production of cepacin is Pseudomonas cepacia, SC 11,783. A subculture of the microorganism can be obtained from the permanent collection of the American Type Culture Collection, Rockville, Md. Its accession number in the repository is A.T.C.C. No. 39356. In addition to the specific microorganism described and characterized herein, it should be understood that mutants of the microorganism (e.g., mutants produced through the use of x-rays, ultraviolet radiation or nitrogen mustards) can also be cultivated to produce cepacin. 
     Isolation of Pseudomonas cepacia SC 11,783 from a soil sample (obtained in West Windsor, N.J.) in which it is present can be accomplished by plating the soil onto an agar of the following composition: 
       1  Synthetic Sea Salts--1.0 grams 
     Aspartic Acid--0.05 grams 
     Asparagine--0.05 grams 
     Yeast Extract--1.0 grams 
     KH 2  PO 4  --0.02 grams 
     K 2  HPO 4  --0.02 grams 
       2  Compost Extract--300.0 ml 
     Distilled Water--700.0 ml 
     Glycerol--5.0 ml 
     The medium is adjusted to pH about 6.5 and sterilized in an autoclave at 121° C. for 30 minutes. After cooling to room temperature, the sterile and cooled medium is supplemented with the following: 
       3  Biotin (0.1% solution)--1.0 ml 
       3  Thiamin (0.2% solution)--1.0 ml 
       3  Actidione (1.0% solution)--10.0 ml 
    
    
    
     After 24-72 hours incubation at 28° C., the colonies of Pseudomanas cepacia SC 11,783 are isolated from the plated soil. The isolated colonies are picked off and maintained on an agar medium composed of: 
     Yeast Extract--1 gram 
     Beef Extract--1 gram 
     NZ Amine A--2 grams 
     Glucose--10 grams 
     Agar--15 grams 
     Distilled Water to --1000 ml 
     The medium is adjusted to pH 7.3 and sterilized in an autoclave at 121° C. for 30 minutes. 
     Pseudomonas cepacia SC 11,783 is a gram negative rod, motile by means of multitrichous polar flagella. It is non-fluorescent, oxidase positive, accumulates poly β-hydroxybutyrate as intracellular reserve material. Arginine dihydrolase is lacking, nitrate is reduced to nitrate, and growth occurs at 41° C. 
     The following compounds are utilized as the sole carbon source on the basal medium described in Stanier et al., J. Gen. Microbiol, 43:159 (1966): glucose, xylose, arabinose, fructose, sucrose, ribose, mannitol, sorbitol, salicin, acetate, citrate, d-tartrate, and putrescine. Rhamnose, maltose, lactose, erythritol are not utilized. 
     Pseudomonas cepacia SC 11,783 is identical to Pseudomonas cepacia A.T.C.C. No. 17759 and matches the published description of this species (Ballard et al., J. Gen. Microbiol, 60:199 (1970)) except for the failure to produce a yellow intracellular pigment. 
     Production of the Antibiotic 
     Pseudomonas cepacia SC 11,783 produces cepacin. To form cepacin according to the preferred methodology, the microorganism is grown at, or near, room temperature (25° C.) under submerged aerobic conditions in an aqueous nutrient medium containing an assimilable carbohydrate and nitrogen source. The fermentation is carried out until substantially activity is imparted to the medium, usually about 12 to 40 hours, preferably about 18 to 20 hours. 
     At harvest, cells can be removed by centrifugation. Cepacin can be extracted from the supernate into moderately polar organic solvents (e.g., n-butanol, ethyl acetate, chloroform, and dichloromethane) and can then be purified by normal-phase chromatography on silica gel or silicic acid or by partition chromatography on Sephadex LH-20 in methanol-chloroform-heptane (1:3:6) to give cepacin A and cepacin B in approximately equal amounts. These antibiotics can also be separated by reverse-phase chromatography on a Waters C 18  μBondapak column eluting with acetonitrile-water (3:7), and monitoring the effluent at 261 nm. 
     Cepacin A and cepacin B are very unstable when all the solvent is removed and decompose rapidly to give a dark brown insoluble solid. They should, therefore, be stored in dilute solution. They are also very base labile, rearranging to a triyne-ene system. This rearrangement has been observed in ethanol-water (1:9) without added base, presumably catalyzed by traces of base on the glassware. The γ-lactone ring is subject to methanolysis so storage in methanol should be avoided. 
    
    
     The following examples further illustrate the preparation and isolation of cepacin A and cepacin B. 
     EXAMPLE OF FERMENTATION OF PSEUDOMONAS CEPACIA 
     SC 11,783 
     Pseudomonas cepacia SC 11,783 was maintained on the following sterilized agar medium (A): 
     
         ______________________________________            Grams______________________________________Yeast Extract      1.0Beef Extract       1.0NZ Amine A         2.0Glucose            10.0Agar               15.0Distilled H.sub.2 O to 1 liter______________________________________ 
    
     The pH was adjusted to 7.3 before sterilization at 121° C. for 30 minutes. A loopful of surface growth from an agar slant (Medium A) of Pseudomonas cepacia SC 11,783 was used to inoculate each of four 500 ml Erlenmeyer flasks containing 100 ml each of the following sterilized medium (B): 
     
         ______________________________________            Grams______________________________________Yeast Extract      4.0Malt Extract       10.0Dextrose           4.0Distilled H.sub.2 O to 1 liter______________________________________ 
    
     The pH was adjusted to 7.3 before sterilization at 121° C. for 15 minutes. After inoculation, the flasks were then incubated at 25° C. on a rotary shaker (300 rpm; 2 inch stroke) for approximately 24 hours. After incubation as described above, 1% (vol/vol) transfers were made from the grown culture flasks to thirty 500 ml Erlenmeyer flasks each containing 100 ml of sterilized medium (B), as described above. After inoculation, the flasks were once again incubated at 25° C. on a rotary shaker (300 rpm; 2 inch stroke) for approximately 24 hours. After incubation as described above, a 1% transfer (vol/vol) was made to a 380-liter stainless steel fermentation tank containing 250 liters of sterilized medium (B). After inoculation the fermentation was continued under the following conditions: temperature 25° C.; pressure 10 psig; aeration--10 CFM; agitation 155 rpm. Ucon LB-625 (polypropylene glycol; Union Carbide) was added as needed as an antifoam agent. After approximately 18-20 hours, the fermentation was completed. The fermentation broth was then adjusted to pH 5.0 using hydrochloric acid and the broth contents of the tank was centrifuged yielding approximately 240 liters of supernatant broth. 
     EXAMPLE OF ISOLATION OF CEPACIN FROM A 50 LITER FERMENTATION 
     The broth supernate (pH 6) from a 50 liter fermentation of Pseudomonas cepacia SC 11,783 was extracted with 16 liters of dichloromethane and the extract was concentrated in vacuo to 700 ml. The concentrate was dried (sodium sulfate), taken to dryness, and the residue immediately redissolved in chloroform-heptane-methanol-water (2:3:4:1) (system I). The lower (water rich) phase was separated, washed once with an equal volume of the upper phase of system I, and then concentrated to 50 ml in vacuo. The concentrate was extracted with 25 ml of chloroform, giving 45 ml of lower phase, LP-1, that was stored in the freezer until further purification.sup.(1). The solution contained 180 mg of non-volatile material, ca. 50 mg. of which was cepacin A and cepacin B judging from the UV absorbance at 263 nm. 
    
     LP-1 was concentrated in vacuo and the residue immediately dissolved in 5 ml of methanolchloroform-heptane (1:3:6) (system II). The solution was chromatographed on a 2.5×90-cm (450 ml) column of Sephadex LH-20 in system II, eluting at 2 ml/minute and collecting 20 ml fractions. Cepacin A and cepacin B were located by UV absorbance at 263 nm. Fractions 95 to 103 were combined, concentrated in vacuo to 5 ml and diluted with chloroform to give 35 ml of solution which contained 6.8 mg of cepacin A: UV max in CHCl 3  (E 1% ) 248.5 (613), 262.0 (827), 276.8 nm (672); nmr in CDCl 3  +CD 3  OD δ2.05 (1H, m), 2.47 (1H, m), 2.50 (1H, m. J&lt;1 Hz), 2.58 (1H, m), 3.02 (1H, J=4.6, 2.1 Hz, Δδ=0.01 ppm), 3.47 (1H, J=7.8, 2.0, 0.6 Hz), 4.17 (1H, m), 5.03 (1H, m), 5.33 (1H, J=7.9, 6.8, 0.6 Hz), 5.68 (1H, J=6.7 Hz), 5.91 ppm, (2H, m); ir in CHCl 3  3291, 2218, 1944, 1770, 1177 cm -1  ; [α] D  (c=0.23 in CHCl 3 ) -129°. Similarly, fractions 108 to 119 gave 35 ml of solution which contained 10.7 mg of cepacin B: UV max in CHCl 3  (E 1% ) 250.5 (399), 263.7 (575), 279.0 (465); nmr in CDCl 3  +CD 3  OD δ2.31 (1H, m), 2.52 (1H, m, J&lt;1 Hz), 2.55 (1H, J=17.9, 9.6, 8.2 Hz), 2.66 (1H, J=17.9, 9.8, 5.7 Hz), 3.01 (1H, J=4.3, 1.8 Hz), 3.15 (1H, J=4.3, 2.1 Hz), 3.21 (1H, J=4.0, 2.1 Hz), 3.46 (1H, J=7.9, 1.9, 0.6 Hz), 3.69 (1H, J=4.0, 4.0 Hz), 4.61 (1H, J= 7.0, 7.0, 3.6 Hz), 5.31 (1H, J=7.9, 6.7, 0.6 Hz), 5.70 ppm (1H, J=6.7, 0.9, 0.9 Hz); ir in CHCl 3  3295, 2217, 2197, 1946, 1773, 1180 cm -1 . 
     EXAMPLE OF ISOLATION OF CEPACIN A FROM A 250 LITER FERMENTATION 
     Cepacin was extracted with dichloromethane from 200 liters of broth supernate, partitioned in chloroform-heptane-methanol-water (2:3:4:1), and extracted into chloroform as described in the previous example. The chloroform extract was applied to a 1 liter column of Whatman LPS-1 silica gel. The column was washed with 1 liter of chloroform and then eluted with chloroform-ethyl acetate (3:1), collecting 250 ml fractions. The effluent was monitored by TLC on silica gel (chloroform-ethyl acetate (1:1), detection, with phosphomolybdic acid; cepacin A, R f  0.19; cepacin B, R f  0.13). Fractions 6 and 7 contained cepacin A and fractions 9 to 12 contained cepacin B. Both of the compounds were about 50% pure at this stage and were further purified by partition chromatography on Sephadex LH-20 in methanol-chloroform-heptane (1:3:6) as described in the previous example. 
     Biological Activity 
     The following methodology is used to determine the minimum inhibitory concentration (hereinafter referred to as MIC) of the compounds of this invention. 
     The test organisms are grown in approximately 15-20 ml of Antibiotic Assay broth (Difco) by inoculating (in tubes) the broth with a loopful of the organism from a BHI (Difco) agar slant. The inoculated tubes are incubated at 37° C. for 18 to 20 hours. These cultures are assumed to contain 10 9  colony forming units (hereinafter CFU) per milliliter. The cultures are diluted 1:100 to give a final inoculum level of 10 7  CFU; dilutions were made with K-10 broth*. 
     The compounds are dissolved in the appropriate diluent at a concentration of 1000 μg/ml. Two-fold dilutions are made in K-10 broth resulting in a range from 1000 μg/ml to 0.5 μg/ml. 1.5 ml of each dilution is placed into individual square petri dishes to which 13.5 ml of K-10 agar** is added. The final drug concentration in the agar ranges from 100 μg/ml to 0.05 μg/ml. Organism growth control plates containing agar only are prepared and inoculated before and after the test plates. The organisms are applied to the agar surface of each plate with the Denley Multipoint Inoculator (which delivers approximately 0.001 ml of each organism) resulting in a final innoculum level of 10 4  CFU on the agar surface. 
    
     Beef extract--1.5 g 
     Yeast extract--3.0 g 
     Peptone--6.0 g 
     Dextrose--1.0 g 
     Distilled water q.s. 1 liter 
    
     Beef extract--1.5 g 
     Yeast extract--3.0 g 
     Peptone--6.0 g 
     Dextrose--1.0 g 
     Agar--15.0 g 
     Distilled water q.s. 1 liter 
     The plates are incubated at 37° C. for 18 hours and the MIC&#39;s are determined. The MIC is the lowest concentration of compound inhibiting growth of the organism. 
     The tables that follow are tabulated results obtained when the compounds of this invention were tested against various organisms. The number following each organism refers to the number of the organism in the collection of E. R. Squibb &amp; Sons, Inc., Princeton, N.J. A dash (-) in the tables means that the compound tested did not show activity against the particular organism at 50 μg/ml. 
     
         ______________________________________        M.I.C. (μg/ml)Organism       SC#*     Cepacin A Cepacin B______________________________________Staphylococcus aureus          1276     0.2       &lt;0.05Staphylococcus aureus          2399     0.1       &lt;0.05Staphylococcus aureus          2400     0.2       &lt;0.05Staphylococcus aureus           10165   0.2       &lt;0.05Streptococcus faecalis          9011     50        --Streptococcus agalactiae          9287     50        --Micrococcus luteus          2495     0.2       3.13Escherichia coli          8294     50        0.78Escherichia coli           10857   1.6       0.1Escherichia coli           10896   12.5      0.4Escherichia coli           10909   6.3       0.2Klebsiella aerogenes           10440   --        0.78Klebsiella pneumoniae          9527     --        --Proteus mirabilis          3855     --        6.3Proteus rettgeri          8479     &gt;25       6.3Proteus vulgaris          9416     1.6       &lt;0.05Salmonella typhosa          1195     25        0.4Shigella sonnei          8449     50        0.8Enterobacter aleacae          8236     --        25Enterobacter aerogenes           10078   --        3.1Citrobacter freundii          9518     50        0.8Serratia marcescens          9783     --        --Pseudomonas aeruginosa          9545     --        --Pseudomonas aeruginosa          8329     --        --Acinetobacter calcoacetieus          8333     --        --______________________________________ *Organisms from the culture collection of E. R. Squibb &amp; Sons, Inc.