GB2174385A - Antibiotic from streptomyces - Google Patents

Abstract

Antibiotic material is produced by culturing the novel microorganism Streptomyces sp A61-7(ASA) (NCIB 12066), or a variant or mutant thereof, and optionally purifying the resultant fermentation broth. The resulting antibiotics Ab650a1, 650a2, 650c, and 650c2 contain betalactamase inhibitory activity.

Description

SPECIFICATION Antibiotics from streptomyces The present invention relates to the production of antibiotic material by a strain of Streptomyces.

In accordance with the present invention there is provided the new and useful strain of Streptomyces designated Streptomyces sp A61-7(ASA) (NCIB 12066).

Antibiotic material can readily be obtained from the fermentation broth produced by culture of this strain or a variant or mutant thereof. The antibiotic material can be purified, and gives several substantially pure compounds, as assessed by HPLC. The compounds, which include known and novel compounds, seem mainly to be carboxylic acids, and accordingly the purification can include an extraction as a salt with a suitable organic or inorganic cation.

We have used the designations AB650a1, Ab650a2, Ab650b, Ab650c,, Ab650c2, and Ab651 to identify the compounds of this invention which we have successfully isolated and characterized. The compound Ab650b appears to be the known compound asparenomycin A, and the compound Ab651 appears to be the known compound carpetimycin A.

In view of their biological activity, the fermentation products provided by this invention, including the compounds Ab650a" Ab650a2, Ab650b, Ab650c,, Ab650c2 and Ab651, can be formulated with a pharmaceutically acceptable carrier in order to give a pharmaceutical composition. The compounds of this invention include compounds with betalactamase inhibitory activity. Such compounds can be used to prepare pharmaceutical compositions which further contain a betalactam antibiotic, for example a penicillin, cephalosporin, or other betalactam antibiotic.

STREPTOMYCES SP. A61-7(ASA) PRODUCER OF ANTIBIOTIC SUBSTANCES THE ORGANISM The organism has been isolated from a soil sample from the outskirts of Le6n (Spain) and has been deposited in the Collection of Antibi6ticos, S.A. under the name Streptomyces sp.

A61-7(ASA) and in the National Collection of Industrial and Marine Bacteria Ltd. (Torry Research Station, Aberdeen, Scotland) under the number NCIB 12066 with restrictions, on 8 April 1985.

The characteristics of the microbe are described as follows: 1. Whole cell hydrolyzate shows that L,L-diaminopimelic acid is present (Type I cell wall).

Also are present the sugars ribose and glucose, being doubtful galactose, rhamose, mannose, xylose and arabinose are absent.

2. The strain has aerial mycelium with straight flexuous sporophores with chains of up to 30 conidia, which spore surface has not been studies yet. The sporophores are not verticil late.

3. In Table I some morphological and cultural characteristics are described, where it is demonstrated that its aerial mycelium belongs to the Red color series of Tresnor and Backus (Appl. Microbiol, 11:335, 1963) and that melanoid pigments are not produced at least in ISP 1 medium (Shirling and Gottlieb. Int. J. Syst. Bacteriol. 16:313, 1960.

4. In Table II some physiological characteristics are shown.

The microorganism, as other Actinomycetes, can mutate spontaneously or by inducing agents such as UV, X rays, chemicals, etc., therefore, it mutants can be used in the process so long they have the ability to produce all or individually any of the substances described here.

If we compare with our organism the known Streptomyces species producers of asparenomycin A and/or carpetimycin A, St. tokunonensis ATCC 31569, Streptomyces sp ATCC 31493, S.

argenteolus ATCC 31589 and S. griseus subsp. cryophilus ATCC 31486; the main differences observed are: -Streptomyces tokunonensis ATCC 31569 (US Patent 4,387,052) pertains to the spirales group: it utilizes l-inositol for growth, but not D-xylose, D-fructose, L-rhamnose or D-raffinose. It does not produce carpetimycins.

-Streptomyces sp. ATCC 31493 (European Patent Application 0050961) has an aerial mycelium that belongs to the White color series, the substrate mycelium is pigmentless, L-arabinose and l-inositol are utilized for growth but not D-raffinose.

-Streptomyces argenteolus ATCC 31589 (US Patent 4,387,053) has an aerial mycelium that belongs to the Grey series, with sporophores of the Spirales group. Grows in L-arabinose and l-inositol as sole carbon source and does not utilize D-raffinose. It does not produce carpetimy cins.

-Streptomyces griseus subsp. cryophilus ATCC 31486 (Imada et. al. J. Antibiotic 33:1417, 1930) has an aerial mycelium of the Yellow color series and a substrate mycelium colorless. L arabinose is utilized for growth and D-raffinose is not. It does not produce asparenomycins.

All the above differences demonstrate that Streptomyces sp A61-7(ASA) is different from the other known producers of asparenomycins and carpetmycins.

THE FERMENTATION The antibiotic substances described herein may be produced in solid or liquid media, although the latter are more convenient. In the liquid media the production can be carried out by surface or submerged culture, being the latter most useful for preparing large amounts of the substances.

The production medium comprises nutrients commonly used in the cultivation of aerobic Actinomycetes and may have: (a) An assimilable carbon source such as glucose, maltose, dextrins, starch, glycerol or fruc tose, alone or in combination.

(b) An assimilable nitrogen source such as vegetable meals (soybean), cottonseed, peanut, etc.) corn steep liquor, vegetal or animal peptones, yeast extract, urea, ammonium salts or nitrates, alone or in combination.

(c) Mineral salts, acids or bases may be added to provide the medium with a pH buffering capacity. Also the salts may be used to supply the medium with mineral elements.

(d) Vegetal or animal oils as foam suppresors and also as carbon source. Also the polyglycols and silicons.can be used as antifoam agents.

The production media must be seeded with the organism Streptomyces sp. A61-7(ASA) or its variant or mutants, in the form of spores or vegetative mycelium, which have been prepared in previous liquid or solid media with a nutrient composition that permits its growth or sporulation.

The liquid media (production and seed) should be aerated and agitated as is usual in many other antibiotic fermentation methods, pH is optimum in the range of 6-7.5 and the temperature between 25 and 32"C. Sterile conditions are needed to avoid contamination with extraneous organisms.

The antibiotic substances are produced during the fermentation, reaching its maximum between the second and the fifth day. In Table Ill a typical fermentation data are shown.

BROTH BIOLOGICAL ACTIVITIES The broth as it is shown in Table IV has antibacterial and betalactamase inhibitory activities.

These activities can be separated by HPLC using two different systems: system 1 with a mobile phase composed of 0.05M ammonium phosphate buffer pH 7/acetonitrile 97:3, and system 2 with 0.05M ammonium phosphate buffer pH 7. For apparatus and conditions see ASSAYS.

In Table V the chromatographic results for systems 1 and 2 are shown.

For the detection of the activity against Micrococcus luteus it is necessary to concentrate the filtered broth by adsorption in a column of MP-20 and elution with an aqueous solution of 30% acetone, with subsequent lyophilization of the active fractions. 100 ,ul of 10mg/ml solution of the lyophilized powder were injected using as a mobile system 3: 0.05M ammonium phosphate buffer pH 7/0/acetonitrile 90:10. The results are shown in Fig. 1.

A 10 Ill sample of a culture filtrate was loaded onto silica gel plates which were developed as indicated in ASSAYS, appearing two activities at R, 0.43 and 0.23 which correspond respectively to a mixture of the Ab-650 a2, Ab-650 b, Ab-650 c2 and Ab-650 a1, Ab-650 cl, Ab-651.

EXTRACTION AND PURIFICATIONS The active substances which are contained in the broth are predominantly produced extracellularly and are water-soluble acids being therefore, mainly in the liquid part of the broth.

Therefore, it is an advantage to separate the solids by centrifugation or filtration using, in this case, a filter aid as, for example, Dicalite (trade mark), collecting the filtrate and isolating the actives substances from it.

Assay of the potency of the broth filtrate can be performed using the microbiological method for bectalactamase inhibitory activity with Klebsiella aerogenes ATCC 29655 or Enterobacter cloacae P-99 and the biochemical test (see ASSAYS).

The preparation and the purification of the active substances from the broth filtrate can be carried out by any of the procedures generally used for isolation of metabolites produced by microorganisms, for example, extraction in organic solvents, ion-exchange chromatography, adsorptions in resins and another adsorbents, molecular sieve chromatography, precipitation, concentration under reduced pressure and freeze-dried which may be used alone or in any combinations, in any order and/or in repetition. Different adsorbents may be used as, for example, high porous resins, anion-exchange resins, activated carbon, reverse phase packings, cellulose and silica gel, and various eluting solvents depending the type of the carrier as mixtures of watersoluble organic solvents, for example, acetone, methanol, ethanol or acetonitrile, aqueous solutions of acids or alcalis, buffers and aqueous solutions or organic or inorganic salts.

All the betalactamase inhibitory substances found in the broth are extremely unstable at higher temperatures than 25"C and in acidic or alkaline solutions. Therefore, during the isolation process care should be taken to avoid the solutions to reach higher temperatures and pH far from neutral. The operations described should be performed, preferably, at temperatures near 10 C and at pH neutral. Because its acidic instability it is preferably to isolate the salts of each compound instead of the free acid.

The first extraction stage is preferably carried out departing from the broth filtrate, adjusted at pH 7, by adsorption in a Diaon HP-20 column and elution with a mixture of water and soluble organic solvents as methanol, ethanol or acetone. Other adsorbants can be used evenly as Amberlite XAD-2, XAD-4 or active carbon. Most of the betalactamase inhibitory activity is found in the aqueous eluate. The active fractions are concentrated under reduced pressure to evaporate the organic solvent, adsorbed in a column of Diaon HP-20 and eluted with deionized water.

The first eluted fractions principally contained the compounds named Ab-650 mixture and the active fractions eluted later contained Ab-651. These fractions are pooled, concentrated under reduced pressure and freeze-dried to obtain crude powders. Since the active substances of the present invention are acidic in nature, anionic-exchange resin in Cl- form, for example, Amberlite IRA-400, 402, 458, 410, Dowex 1 or Diaon PA-306 may be used in the first steps of isolation. The active substances are eluted with sodium chloride in buffered solution desalted by adsorption in Diaon HP-20, eluted with deionized water and freeze-dried to give crude powders of the Ab-650 mixture and Ab-651.

For the separation of the compounds named Ab-650 (Ab-650 ai, a2, b, c, and c2) the crude powder obtained as mentioned above is chromatographed using an anion-exchange carrier, such as OAE Sephadex A-25 or DEAE Sephadex A-25, being preferably an strongly base anion exchange resin as QAE Sephadex A-25. This separation is performed by adsorption of the crude powder in a column of QAE Sephadex A-25 and elution with a concentration gradient method between 0.01M phosphate buffer solution pH 7.0 and 3% sodium chloride in the same buffer. Firstly, Ab-650 c2 is eluted and then Ab-650 b, Ab-650 a1 and Ab-650 c, in the order indicated, being eluted finally Ab-650 a2.

The active fractions containing the same compound are pooled, desalted using Diaon HP-20SS and freeze-dried to obtain powders of each compound.

For further purification of these compounds they are repeatedly chromatographed using anionexchange carriers, such as Amberlite CG-400, QAE Sephadex A-25 and/or DEAE SEPHADEX A-25, gel filter media such Biogel P-2 or Sephadex G-10, porous macroreticular resins as HP-20 SS or subjected to reverse phase chromatography using a column packed with Bondapak Cis (55-105 microns).

Another step of purification is necessary in some cases, being used then a preparative high performance liquid chromatography in a column of z-Bondapak 10 microns (7.8X300mm) and 0.05M ammonium phosphate buffer pH 7.0 as mobile phase. The active fractions are analyzed by analytical HPLC and those showing a single peak are pooled, desalted and freeze-dried.

Following this method, it is possible to isolate in its purified form the individual substances Ab-650 a1, a2, b, cl, c2 and Ab-651 as a salt, being the type of salt determined by the cation used in the purification process, preferably Na or K.

PHYSICO-CHEMICAL PROPERTIES OF THE SODIUM SALT OF SUBSTANCE A-650 a, 1. Appearance : Colorless powder.

2. Ultraviolet absorption spectrum: The spectrum of the substance taken in an aqueous solution is substantially as shown in Fig. 2 with maximum absorptipn at 313 nm.

3. Solubility in solvents: Soluble in water but insoluble in organic solvents, such as acetone, chloroform, ethyl acetate and pethroleum ether.

4. Color reaction: Positive Ehrlich 5. Thin layer chromatography: Plate: Silica gel F254 (Merck & Co. Inc.).

Solvent CHCI3/MeOH/0.01M phosphate buffer pH 7.0 75/65/10 0.35 n-BuOH/EtOH//CHCI3/H20 5/5/1/2 0.43 CH2CN/H2O 7/3 0.39 n-PrOH/H2O 7/3 0.35 CH3CN/CH3-COOH 0.75% 6/4 0.49 EtOH/H20 7/3 0.78 n-PrOH/0.1M phosphate buffer pH 7.0 7/3 0.43 6. High performance liquid chromatography: Column: Spherisorb ODS-2 10 microns (3,9X300 mm) Flow rate: 1.0 ml/min Retention Solvent time 0.05M ammonium phosphate buffer pH 7.0/CH2CN 97:3 4 min 0.05M ammonium phosphate buffer pH 7.0 11 min PHYSCICO-CHEMICAL PROPERTIES OF THE SODIUM SALT OF SUBSTANCE Ab-650 a2 1. Appearance: Colorless powder.

2. Ultraviolet absorption spectrum: The spectrum of the substance taken in an aqueous solution is substantially as shown in Fig. 3 with maximum absorption at 306 nm and 222 nm.

3. Solubility in solvents: Soluble in water but insoluble in organic solvents, such as acetone, chloroform, ethyl acetate and pethroleum ether.

4. Color reaction: Positive Ehrlich 5. Thin layer chromatography: Plate: Silica gel F254.

Solvent CHCl2/MeOH/O.01M phosphate buffer pH 7.0 75/65/10 0.44 N-BuOH/EtOH/CHCl3/H2O 5/5/1/2 0.57 CH2CN/H2O 7/3 0.54 nPrOH/H20 7/3 0.63 CH3CN/CH3COOH 0.75% 7/3 0.63 EtOH/H20 7/3 0.73 n-PrOH/0.1M phosphate buffer pH 7.0 7/3 0.66 6. High performance liquid chromatography: Column: Spherisorb ODS-2 10 microns 3,9X300 mm) Flow rate: 1.0 ml/min Retention Solvent time 0.05M ammonium phosphate buffer pH 7.0 CH3CN 97:3 4 min 0.05M ammonium phosphate buffer pH 7.0 11 min PHYSICO-CHEMICAL PROPERTIES OF THE SODIUM SALT OF SUBSTANCE Ab-650 b.

1. Appearance: Pale yellow powder.

2. Ultraviolet absorption spectrum: The spectrum of the substance taken in an aqueous solution substantially as shown in Fig. 4 with maximum absorption at 310 nm, 270 mn and 289 nm.

3. Molecular formula: C14H16N2O5S 4. 'H-NMR spectrum: On measurement in D2O, the spectrum of the substance is substantially as shown in Fig. 5 Main peaks a D2O (ppm): 1.73 (3H,s,C-CH3); 1.85(3H,s,COCH,) 2.88 (1 H,d,J= 1 OHz),4.00(2H,s),4.76(m),6.09(1 H,d,J= 1 4Hz(7.3 (1 H,d,J= 14Hz).

5. Solubility in solvents: Soluble in water methanol but insoluble in organic solvents, such as acetone, chloroform, ethyl acetate and pethroleum ether.

6. Color reactions: Positive Ehrlich.

7. Thin layer chromatography: Plate: Silica gel F254.

Solvent R, CHCI3/MeOH/0.01M phosphate buffer pH 7.0 75/65/10 0.49 n-BuOH/EtOH/CHCl2/H2O 5/5/1/2 0.55 CH2CN/H2O 7/3 0.55 n-PrOH/H20 7/3 0.63 CH3CN/CH3COOH 0.75% 7/3 0.44 EtOH/H2O 7/3 0.72 n-PrOH/O.1M phosphate buffer pH 7.0 7/3 8. High performance liquid chromatography Column: Spherisorb ODS-2 10 microns (3,9X300 mm).

Flow rate: 1.0 ml/min.

Retention Solvent time 0.05M ammonium phosphate buffer pH 7.0/CH2CN 7/3 5 min 0.05M ammonium phosphate buffer pH 7.0 17 min From the above characteristic properties, the substance Ab-650 b has been identified as asparenomycin A (K. Tanaka: J. Antibiotics 34, 909-911 (1981) and N. Tsuji: J. Antibiotics 35, 10-45 (1982)).

PHYSICO-CHEMICAL PROPERTIES OF THE SODIUM SALT OF SUBSTANCE Ab-650 c,.

1. Appearance: Colorless poder.

2. Ultraviolet absorption spectrum: The spectrum of the substance taken in an aqueous solution substantially as shown in Fig. 6 with maximum absorption at 284 nm and 239 nm.

3. Solubility in solvents: Soluble in water but insoluble in organic solvents, such as acetone, chloroform, ethyl acetate and pethroleum ether.

4. Color reaction: Positive Ehrlich.

5. Thin layer chromatography: Plate: silica gel F254.

Solvent R, CHCI3/MeOH/0.01M phosphate buffer pH 7.0 75/65/10 0.37 n-BuOH/EtOH/CHCl2/H20 5/5/1/2 0.41 CH3CN/H20 7/3 0.52 n-PrOH/H2O 7/3 0.56 CH3CN/CH3-COOH 0.75% 7/3 0.45 EtOH/H20 7/3 0.75 n-PrOH/0.1M phosphate buffer pH 7.0 0.53 6. High performance liquid chromatography Column: Spherisorb ODS-2 10 microns (3,9X300 nm) Flow rate: 1.0 ml/min Retention Solvent time 0.05M ammonium phosphate buffer pH 7.0/CH3CN 97:3 8 min 0.05M ammonium phosphate buffer pH 7.0 21 min PHYSICO-CHEMICAL PROPERTIES OF THE SODIUM SALT OF SUBSTANCE Ab-65O C2.

1. Appearance: Colorless powder.

2. Ultraviolet absorption spectrum: The spectrum of the substance taken in an aqueous solution substantially as shown in Fig. 7 with maximum absorption at 300 nm and 257 nm.

3. Solubility in solvents: Soluble in water but insoluble in organic solvents, such as acetone, chloroform, ethyl acetate and pethroleum ether.

4. Color reaction: Positive Ehrlich.

5. Thin layer chromatography: Plate: silica gel F254.

Solvent R, CHCl2/MeOH/0.O1M phosphate pH 7.0 75/65/10 0.45 n-BuOH/EtOH/CHCl2/H2O 5/5/1/2 0.50 CH3CN/H2O 7/3 0.52 n-PrOHfH2O 7/3 0.53 CH3CN/CH3COOH 0.75% 7/3 0.60 EtOH/H2O 7/3 0.68 n-PrOH/O.1M phosphate buffer pH 7.0 0.57 6. High performance liquid chromatography Column: Spherisorb ODS-2 10 microns (3,9X300 nm) Flow rate: 1.0 ml/min Retention Solvent time 0.05 ammonium phosphate buffer pH 7.0/CH3CN 97:3 8 min 0.05M ammonium phosphate buffer pH 7.0 27 min PHYSICO-CHEMICAL PROPERTIES OF THE SODIUM SALT OF SUBSTANCE Ab-651.

1. Appearance: Pale yellow powder.

2. Ultraviolet absorption spectrum: The spectrum of the substance taken in an aqueous solution substantially as shown in Fig. 2 with maximum absorption at 287 nm and 241 nm.

3. Infrared absorption spectrum: The spectrum of the substance taken in KBr is substantially as shown in Fig. 9.

4. Molecular formula: C14H15N2O6S.

5. 1H-NMR spectrum.

On measurement in D2O, the spectrum of the substance is substantially, as shown in Fig.

10.

Main peaks J: D2O (ppm) 1.32 (3H,s,C-CH2), 1,40 (3H,s,C-CH2) 2,15 (3H,s,CO-CH2), 3.03 (1 H,dd,J= 10.6,1 7.2Hz,CH2), 3.81 (1 H,d,J=6.6Hz,6-CH), 3.92 (1 H,dd,J=8, 1 7.2Hz,CH2) 4.47 (1 H,m,5-CH),6.37(1 H,d,J= 1 3.9Hz,S-CH=), 7.61(1 H,d,J= 1 3.9Hz,NH-CH=).

6. Solubility in solvents: Soluble in water and methanol but insoluble in organic solvents, such as acetone, chloroform, ethyl acetate and pethroleum ether.

7. Color reaction: Positive Ehrlich and negative ninhydrin.

8. Thin layer chromatography: Plate Silica gel F254 (Merck & Co. Inc.).

Solvent R, CHCI3/MeOH/0.01M phosphate buffer pH 7.0 75/65/10 0.42 n-BuOH/EtOH/CHCl3/H2O 5/5/1/2 0.48 CH2CN/H2O 7/3 0.48 n-PrOH/H2O 7/3 0.57 CH3CN/CH3-COOH 0.75% 7/3 0.58 EtOH/H2O 7/3 0.74 n-PrOH/0.1M phosphate buffer pH 7.0 7/3 0.55 9. High performance liquid chromatography Column: Spherisorb ODS-2 10 microns (3,9X300 mm).

Flow rate: 1.0 1 mI/min.

Retention Solvent time 0.05M ammonium phosphate buffer pH 7.0/CH3CN 97:3 12 min 0.05M ammonium phosphate buffer pH 7.0 51 min 10. Other analytical assays: The betalactamase inhibitory disappear when Ab-651 is incubated with solutions of cystein (40mg/ml), hydroxylamine (1OmM) and dehydropeptidase renal at 37"C during an hour.

From the above characteristic properties, the substance Ab-651 has been identified as carpetimycin A (Masahito Wakayama: J. Antibiotics 33, 1388-1390 (1980)).

BIOLOGICAL ACTIVITIES The antimicrobial and betalactamase inhibitory activities of the different compounds appear in Table VI. The (M.I.C.) minimum inhibitory concentration, was studied in plates of Antibiotic, no 1 medium, incubated 24 h at 28 C.-ln these plates was studied also the synergy with penicillin G, which indicates a betalactamase inhibitory activity. The inhibition 50 (Iso), concentration of compound inhibiting 50% of penicillin G hydrolysis by Difco Penase, was determined by the hydroxamic method.

ASSAYS (a) Betalactamase inhibitory activity. This activity was measured following two methods, the first is a biochemical test employing penicillinase (Difco bacto-penase) and benzylpenicillin, measuring the residual penicillin by the hydroxyilamine method of Ford (Anal. Chem.

19:1004:1947) and studying the percentage of protection given by the inhibitor. The sec ond is a microbiological method by which the differences in the inhibition zone diameter between plates with a microorganism bearing betalactamase (Klebsiella aerogenes ATCC 29665 or Enterobacter cloacae P99) with and without a betalactam (benzylpenicillin or cephalosporin C respectively) are measured. In the biochemical method 140 LU and 3147 U of the Difco enzyme and of benzylpenicillin respectively were employed and the reaction was carryed out at 37"C during 30 minutes. In the microbiological method the Difco medium Antibiotic no 1, 50 ml in 15 cm Petri plates, was used, the incubation was overnight at 25"C for both organisms. The antibiotic concentration in the plates was 10 micrograms per ml in both cases.

(b) Antibiotic activity. For this activity Micrococcus luteus ATCC 9341a was employed as test organism in plates of Antibiotic no 1 medium incubated overnight at 25"C. The quantifica tion of the activity was done by the minimum inhibitory concentration method.

(c) Renal dehydropeptidase activity. For this assay fresh kidney tissue homogenates were em ployed and the activity was measured following the Kropop et al. paper (Antimicrob. Agents and Chemother, 22:62, 1982).

Apparatus: Pump Waters, mod. 6000 Injector Waters, Mod. U6K Detector Hewlett Packard, mod. 1040 Column: Spherisorb ODS-2 (3,9X300 nm) Flow rate: 1.0 ml/min (e) Thin layer chromatography. The samples were loaded onto silica-gel thin layer chromato graphy sheets (Merck Co.), developed with n-BuOH/EtoH/CHCI3/H20 (5:5:1:2) for 5 hours at 4"C. The betalactamase inhibitors were detected by bioautography on Klebsiella aerogenes in Antibiotic no 1 plates with and without benzylpenicillin (10 ,ug/ml) incubated 18 hours at 25"C.

EXAMPLE 1 A frozen mycellium vial of Streptomyces sp. A61-7(ASA) was employed to seed 40 ml of a previously sterilized liquid medium (C2) with 1% yeast extract, 0.5% soybean meal, 2% soluble starch and 0.2% dextrose in a 250 ml Erlenmeyer flask. The seeded medium was incubated during 48 h at 28"C in an orbital shaker at 250 rpm with 5 cm eccentricity.

20 ml of medium thus incubated were utilized as inoculum for 500 ml of medium Cl previously sterilized, composed of 0.5% dry baker yeast, 1% soybean meal, 0.2% NaCI, 0.05% K2HPO4, 0.00013% CoCI2 6H2O, 0.4% CaCO2, 1% dextrose and 3% corn dextrin in a two liter Erlenmeyer flask. The incubation was carried out in the same orbital shaker with the same temperature and length of time as before. Two liter of the inoculum thus prepared were used to seed 40 liter of Cl sterile medium in a fermentation tank of 75 I total capacity. The fermentation was carried out at 28"C during 66 h. The fermenter was agitated at a speed of 200 rpm and oxygenated with 20 liters of sterile air per minute with an overpressure of 0.2 atm.

At this point, the fermentation was stopped and the broth showed an activity against Micrococcus luteus ATCC 9341a and against the betalactamase producing strain Klebsiella pneumoniae ATCC 29665. Also it had a betalactamase inhibitory activity against the Difco Penase.

EXAMPLE 2 A frozen vial of Streptomyces sp. A61-7(ASA) was employed to seed 40 ml of medium C2 previously sterilized. Once seeded the incubation was carried out at 28"C during 48 h in orbital shaker at 250 rpm with 5 cms eccentricity.

20 ml of the medium thus incubated were utilized for seeding 500 ml of medium Cl previously sterilized. The incubation being carried out at 28"C during 48 h in the same orbital shaker as medium C2. 1.5 liter of the inoculum thus prepared were used to seed 100 liter of medium Cl in a 100 liter fermentation tank. During the fermentation the medium was agitated at a speed of 150 rpm and aerated with 50 liter of air per minute. The temperature of the medium was maintained at 28"C during the length of the incubation time 24 h. 20 liter of this broth were utilized to seed 400 liter of the same medium Cl in a 800 liter fermentation tank. During the fermentation period, 48-72 h, the tank was agitated at 150 rpm and aerated with 200 liter of air per minute, the temperature being held at 28"C.

EXAMPLE 3 200 1 of culture broth obtained by the procedure described in Examples 1 or 2 were filtrated through Dicalite (trade mark). The filtrate (150 I) was cooled to 10 C, adjusted at pH 7.0, added sodium chloride 5% (W/v) and passed through a column of 20 I Diaon HP-20 (Mitsubishi Chemical Industries Ltd.) previously washed with a 5% aqueous solution of NaCI. The column was washed again with 20 1 of 5% NaCI and eluted with 70 1 of a 30% aqueous solution of acetone, collecting 1 liter fractions. The fractions with ss-lactamase inhibitory activity (20 I) were pooled concentrated under reduced pressure to evaporate the acetone added sodium chloride to 5% and applied to a column with 6 1 of Diaon HP-20, previously washed with a 5% NaCI aqueous solution to separate the Ab-650 mixture of Ab-651.The column was eluted with deionized water, collecting 20 ml fractions. The first eluted active fractions (2.5 1) were freezedried to yield 32 gr of a crude powder of the compounds named Ab-650 mixture. The last eluted active fractions (2 1) were freeze-dried to give 9.8 gr of active material of Ab-651.

EXAMPLE 4 To 100 1 of the culture broth obtained as in Example 1 or 2, Dicalite was added as a filter aid to the culture broth and the broth was filtered to give a filtrate of 80 I. This filtrate was cooled to 10"C, adjusted at pH 7.0 and applied to a 3 1 column of Diaon PA-306 (Cl form) (Mitsubishi Chemical Industries Ltd.) equilibrated with a phosphate buffer solution pH 7.0. The column was washed with a 0.01M phosphate buffer solution (pH 7.0) and eluted with 20 1 of a 30% sodium chloride sblution in phosphate buffer. The fractions were subjected to the biochemical test with Difco Penase and benzylpenicillin and pooled the active fractions (7 I) which were adsorbed in a 3 1 Diaon HP-20 column, previously treated with 5% sodium chloride and eluted with a 30% aqueous solution of acetone.The active fractions (3 1) were concentrated under reduced pressure to evaporate the acetone and freeze-dried to give 32 gr of active material as crude powder.

The powder with betalactamase inhibitory activity was dissolved in 500 ml of phosphate buffer (pH 7.0) applied to a column of Amberlite CG-400 (Cl form) (Rohm and Haas), equilibrated with a phosphate buffer solution pH 7.0 and eluted by a concentration gradient method between 2 1 of 0.01M phosphate buffer solution (pH 7.0) and 2 1 of a 0.01M phosphate buffer solution (pH 7.0) with 30% sodium chloride.

4.3 1 of an active fraction were collected, followed by passage for adsorption through a column of 1 I Diaon HP-20 which had been previously washed with an aqueous solution of 5% sodium chloride. The column was washed with 1 I of a 5% NaCI solution and then eluted with deionized water.

The first active fractions eluted (1,3 1) contain the Ab-650 mixture which were freeze-dried to give 3.82 gr of crude powder. The active fractions (8.5 I) eluted later were pooled and freezedried to provide 6.7 gr of crude powder of the substance Ab-651.

EXAMPLE 5 9.8 gr of the Ab-651 crude powder obtained in Example 3 were dissolved in 120 ml of phosphate buffer (pH 7) and applied to a column with 11 of Amberlite CG-400 (Cl- form) previously equilibrated with a phosphate buffer solution (pH 7). The column was washed with a 0.01M phosphate buffer solution and eluted by a concentration gradient method between 3 1 of a 0.01M phosphate buffer (pH 7.0) and 3 1 of a 0.01M phosphate buffer solution (pH 7.0) with 30% sodium chloride, collecting 20 ml fractions.

Fractions 111-276 (3 I) with betalactamase inhibitory activity were pooled, desalted in a column of 1 I Diaon HP-20 by elution with deionized water and concentrated under reduced pressure. The active fractions were applied to 500 ml QAE Sephadex A-25 column (Cl- form) (Pharmacia Fine Chemicals) washed with a 0.01M phosphate buffer solution (pH 7.0) and eluted by a concentration gradient method using 1 I of 0.01M phosphate buffer (pH7.0) and 11 of 0.01M phosphate buffer (pH7.0) with 5% sodium chloride. Fractions 73-96 (500 ml) were pooled, desalted in a column with 500 ml of Diaon HP-20SS and freeze-dried to give 51 mg of a pure powder of the sodium salt of the substance Ab-651.

EXAMPLE 6 21 gr of the crude powder of the Ab-650 mixture obtained as in Example 3 were dissolved in 300 ml of phosphate buffer (pH 7.0) and applied to a 400 ml QAE Sephadex A-25 column (Cl- form) equilibrated with a 0.01M phosphate buffer solution(pH 7.0). The column was washed with 11 of 0.01M phosphate buffer 0.01M (pH 7.0) and eluted by a concentration gradient method between 3 1 of 0.01M phosphate buffer solution (pH 7.0) and 3 1 of 0.01M phosphate buffer solution (pH 7.0) with 3% sodium chloride, collecting 20 ml fractions.

The fractions with betalactamase inhibitory activity were subjected to analysis by liquid chromatography using 0.05M phosphate buffer (pH 7.0) as mobile phase. These fractions, with a betalactamase inhibitory activity with the same retention time, were desalted using Diaon HP-20SS and freeze-dried to give powders of each compound as it is shown below.

Fractions (OAE Sephadex A-25) Weight (mg) Active compound 48-65 286 Ab-650 c2 68-79 148 Ab-650 b 116-141 467 Ab-650 a, 152-170 285 Ab-650 c, 195-213 108 Ab-650 a2 EXAMPLE 7 851 mg of a crude powder of Ab-650 a, obtained as in Example 6 were dissolved in 20 ml of 0.05M ammonium phosphate buffer (pH 7.0) and applied to a column packed with 200 ml of Bondapak C,8 (55-105 microns) (Waters Associates) previously equilibrated with 0.05M ammonium phosphate buffer (pH 7.0). The column was eluted with 0.05M ammonium phosphate buffer (pH 7.0), collecting 20 ml fractions. The fractions with betalactamase inhibitory activity were subjected to the analysis of liquid chromatography as Example 6.The active fractions 11-36 (500 ml) were pooled, desalted with Diaon HP-20SS and freeze-dried to give 100 mg of the sodium salt of Ab-650 a,.

EXAMPLE 8 157 mg of crude powder of Ab-650 a2 obtained as in Example 6 were dissolved in 5 ml of 0.05M ammonium phosphate buffer (pH 7.0) and applied to a column packed with 60 ml of Bondapak C,8 (55-105 microns) equilibrated with 0.05M ammonium phosphate buffer (pH 7.0).

The column was eluted with 0.05M ammonium phosphate buffer (pH 7.0) and 10 ml fractions were collected. The active fractions nos. 15-30 (150 ml) were pooled, desalted and freeze-dried to give 16 mg of the sodium salt of Ab-650 a2.

EXAMPLE 9 350 mg of the crude powder of Ab-650 b obtained as in Example 6 were dissolved in 20 ml of deionized water, the solution being passed through a column (250 ml) of Biogel P-2 (Bio-Rad) and eluted with deionized water. The active fractions (300 ml) were applied to a column (400 ml) of QAE Sephadex A-25 (Cl-- form) equilibrated with phosphate buffer (pH 7.0) the column being eluted by a concentration gradient method employing 3 1 of a 0.01M phosphate buffer solution (pH 7.0) and 3% sodium chloride in 0.01M phosphate buffer solution (pH 7.0). The active fractions were desalted in a column (50 ml) of Diaon HP-20SS and freeze-dried to give 40 mg of a colorless solid. The solid thus obtained was purified by preparative high performance liquid chromatography using a ,uBondapak C,8 column (7.8X300 mm) (Waters Associates) with 0.05M ammonium phosphate buffer pH 7.0. The active fractions were passed through a column packed with 50 ml of Diaon HP-20SS and eluted with water. Each of the fractions having betalactamase inhibitory activity were subjected to analysis by liquid chromatography using 0.05M phosphate buffer pH 7.0 as mobile phase. The fractions which showed a single peak with the same retention time were pooled and freeze-dried to obtain 2 mg of a pale yellow powder of Ab650 b.

EXAMPLE 10 851 mg of crude powder of Ab-650 c1 obtained as in Example 6 were dissolved in 25 ml of 0.05M ammonium phosphate buffer (pH 7.0) and applied to a column packed with 200 ml of Bondapak C,8 (55-105 microns) equilibrated with 0.05M ammonium phosphate buffer (pH 7.0).

The column were eluted with 0.05M ammonium phosphate buffer (pH 7.0) and 10 ml fractions were collected. The active fractions nos. 50-140 (900 ml) were pooled, desalted and freezedried to give 150 mg of the sodium salt of Ab-650 C1.

EXAMPLE ii 925 mg of the crude powder of Ab-650 c2 obtained as in Example 6 were dissolved in 25 ml of 0.05M ammonium phosphate buffer (pH 7.0) and applied to a column packed with 200 ml of Bondapak C,8 (55-105 microns) equilibrated with 0.05M ammonium phosphate buffer (pH 7.0) and 10 ml fractions were collected. The active fractions 90-150 (600 ml) were pooled, desalted and freeze-dried to give 50 mg of the sodium salt of Ab-650 c2.

TABLE I MORPHOLOGICAL AND CULTURAL CHARACTERISTICS OF STREPTOMYCES sp. A61-7(ASA).

Time Aerial Substrate Medium (days) Growth mycelium mycelium** Soluble pigment Yeast malt 14 Good Moderate Orange 191 None agar Orange 178 Fawn Tryptone yeast 2 Good None extract broth Inorganic salts 14 Poor Scant starch agar Colorless Colorless None Czapek 14 Fair Moderate Colorless Colorless None Emerson 14 Good Scant Orange 199 Orange 193 None Naples Yellow Ocher Yeast extract Scant dextrose agar 14 Good Orange 199 Yellow 250 None Naples yellow Chamois Shirling and Gottlieb Int. J. Syst. Bacteriol. 16:313, 1966.

** Color were designated first after the color plates of the "Code Universel des Couleurs" by E.

Seguy, Ed. P. Lechevalier (Paris), 1936 and second subjectively using the regular names for these colors.

TABLE II PHYSIOLOGICAL CHARACTERISTICS OF STREPTOMYCES SP. A61-7(ASA).

1. Carbon utilization Pridham and Gottlieb medium, 21 days at 28"C.

Utilized: D-glucose, D-fructose, maltose, D-melibiose, L rhamnose, D-xylose, D-raffinose, D-galactose, glycerol D-trehalose, D-mannose and lactose.

Not utilized: L-arabinose, l-inositol, sucrose and D-mannitol 2. Hydrolisis of starch is positive.

3. NaCI tolerance: Growth occurs with 4% but no with 7% NaCI.

TABLE III TIME COURSE OF A 50-LITER FERMENTATION*.

MIC MIC MIC Time(hr) PCV** pH %Glucose %Dextrin 1150*** (M.luteus) (Klebsiella) (K+PGK) 0 7 6.5 1 2.6 0 0 NT NT 18 19 6.8 1 2.5 1/2 1/2 NT NT 42 27 6.8 0.7 1.9 1/250 1/10 NT NT 66 32 6.9 0.3 1.6 1/500 1/64 1/16 1/1024 * Cl medium 280C ** Packed cell volume Broth dilution to give a 504/0 inhibition of Difco penicillinase against benzylpenicillin TABLE IV ANTIMICROBIAL AND BETALACTAMASE INHIBITORY ACTIVITIES AS STREPTOMYCES sp A-6 1-7(ASA) DIAMETER OF INHIBITION ZONE (mm) Broth Dilution M.luteus K. aerogenes K. aer.+PGK*' E.cloacae E.cloacae+CC** 1 28 32 37 25 35 1/5 19 23 29 21 28 1/20 13 17 25 18 25 1/100 0 0 20 0 18 Broth 1:1 renal DHP*** NT 0 27 NT NT Broth 1::1 water NT 23 31 NT NT * Fermentation in 250 ml Erlenmeyer flasks with 30 ml Cl medium at 28C during 72 hours.

** Potasium benzylpenicillin (PGK). Cephalosporin C (CC).

Renal dehydropeptidase TABLE V SEPARATION BY HPLC SYSTEM 1 AND 2 OF THE BETALACTAMASE INHIBITORY ACTIVITIES OF STREPTOMYCES sp A61-7(ASA) BROTH.

SYSTEM 1 SYSTEM 2 SAMPLE (RETENTION TIMES) (RETENTION TIMES) Ab-650-a, 4 min. 11 min.

Ab-650-a2 4 min. 11 min.

Ab-650 b 5 min. 15 min.

Ab-650 c1 7 min. 21 min.

Ab-650 c2 7 min. 27 min.

Ab-651 12 min. 51 min.

TABLE VI COMPARATIVE IN VITRO ANTIMICROBIAL AND BETALACTAMASE INHIBITORY ACTIVITIES OF COMPOUNDS Ab-650 a1, Ab-650 a2, Ab-650 b, Ab-650 c1, Ab-650 c2 and Ab-651.

MIC Kp MIC PG Iso Ab-650 a, 50 50 25 Ab-650 a, +30 PG* 12 - Ab-650 a2 3 0.75 0.5 Ab-650 a2 +30 PG 0.2 - Ab-650 b 12.5 6 6 Ab-650 b +30 PG 1.5 - Ab-650 c, > 6 0.4 0.2 Ab-650 c, +30 PG 0.2 - - Ab-650 c2 100 50 25 Ab-650 c2 +30 PG 12 - Ab-651 50 25 6 Ab-651 +30 PG 6 - - Penicillin G > 50 > 50 > 50 Kp: Klebsiella pneumoniae ATCC 2966S.

PG: The same plates as Kp with benzylpenicillin 15 mcg/ml.

MIC: Minimum inhibitory concentration in mcg/ml.

Difco Penase 15o in mcg/ml.

* 0.1 ml of a mixture of the inhibitor and 30 mcg/ml of benzylpenicillin were placed in the same well.

Claims (13)

1. Streptomyces sp A61-7(ASA) (NCIB 12066).

2. A fermentation broth produced by culture of Streptomyces sp A61-7(ASA) (NCIB 12066) or a variant or mutant of this strain.

3. An antibiotic material obtained by purification of a fermentation broth as defined in claim 2.

4. An antibiotic material according to claim 3, in the form of a substantially pure compound, as assessed by HPLC.

5. An antibiotic material according to claim 3 or 4, when extracted as a salt.

6. An antibiotic material according to claim 3, 4 or 5, with betalactamase inhibitory activity.

7. Antibiotic compound Ab-650a1, as herein defined.

8. Antibiotic compound Ab-650a2, as herein defined.

9. Antibiotic compound Ab-650c1, as herein defined.

10. Antibiotic compound Ab-650c2, as herein defined.

11. A pharmaceutical composition which comprises an antibiotic material according to any of claims 3 to 10, together with a pharmaceutically acceptable carrier.

12. A pharmaceutical composition according to claim 11, which further contains a betalactam antibiotic.

13. A process for producing antibiotic material which process comprises culturing Strept & myces sp A61-7(ASA) (NCIB 12066), or a variant or mutant thereof, and optionally purifying the resultant fermentation broth.