GB2042501A - Antibiotic AX-127B-1 - Google Patents

Abstract

A new antibiotic AX-127B-1 is produced by fermentation of a microorganism belonging to the species Micromonospora pilosospora. The antibiotic is accumulated in the culture medium and is isolated by conventional methods of extraction. The antibiotic is effective against Gram-positive and Gram-negative organisms, and may be incorporated in pharmaceutical compositions for treatment of bacterial infections.

Description

SPECIFICATION Antibiotic AX-127B-1 This invention relates to antibiotics.

A new antibiotic AX-127B-1 is produced by a microorganism belonging to the genus Micromonospora.

This antibiotic is produced by growing the culture in a nutrient medium until substantial antibacterial activity is accumulated. The antibiotic is then isolated from the culture medium by an ion exchange resin treatment.

Figure 1 illustrates the infrared absorption spectrum of antibiotic AX-1 27B-1 free base, Figure 2 illustrates the mass spectrum of antibiotic AX-127B-1 free base, Figure 3 illustrates the carbon magnetic resonance spectrum of antibiotic AX-1 27B-1 free base.

AX-1 27B-1 is produced by fermentation of the microorganism belong to the genus Micromonospora. A particularly suitable microorganism is Micromonospora pilisospora sp. nov. This type strain, originally designated AB-127B-46, was isolated from a soil sample collected in Sevilla, Columbia. This strain has been deposited without restriction in the ARS Culture Collection at Northern Region Research Center, 1815 North University Street, Peoria, Illinois 61604. It has been given the accession number NRRL 11415.

Morphology Strain AB-1 27B-46 has the following properties. Micromonospora pilosospora AB-1 27B-46 is Grampositive and non-acid fast. True aerial mycelia are not produced on solid media. A lustrous, waxy, greenish-black layer of spores is observed on the colonial surface on agar media favouring sporulation.

Scanning electron microscopic observation of 21 day growth on sporulation agar (consisting of 0.1% yeast extract, 0.1% beef extract, 0.2% tryptose, 1% glucose, 2% starch, 0.1% CaCO3, trace minerals and 1.5% agar), revealed vegetative mycelia about 0.3 um in diameter. Single spores are formed at the end of sporophores which arise along the length of the substrate mycelia. Sporophores are from 0.1 um to 1 um in length; spores are spherical (0.75 um in diameter) to ellipsoidal (0.9 um x 0.75 um) and have a hairy surface.

Culture Characteristics The degree of growth, surface state of the colony and production of soluble pigments during cultivation of M. pilosospora AB-1 27B-46 on various media are shown in Table 1. Growth was observed after 14 days incubation at 30"C. Color indications are given according to the classification in the National Bureau of Standards Special Publication No. 440, December 1976, USA.

Physiological properties The carbon source utilization data for M. pilosospora AB-1 27B-46 are shown in Table 2. The method used was that of Luedmann and Brodsky, Taxonomy of gentimicin producing Micromonospora, Antimicrobial Agents and Chemotherapy, p. 116-124, (1964) except that 0.1% yeast extract was used in the basal medium rather than 0.5%. Observations were made after 7 and 14 days of incubation at 30"C.

The physiological properties of M. pilosospora AB-127B-46 are shown in Table 3. All tests were incubated for 14 days at 30or. except for gelatin hydrolysis, which were incubated for 28 days at 300C., and the optimum temperature determination in which cultures were incubated 7 days.

TABLE1 Growth Characteristics on Various Media Soluble Medium Growth Color Pigment ATCC medium #172 Good grayish yellow None raised brown ridged (80) Sporulation Good greenish black None agar raised (157) ridged Yeast extract- Good greenish black None malt extract agar raised (157) (ISP-2) ridged Oatmeal agar Poor pale orange yellow None (ISP-3) flat (73) waxy greenish black (157) Inorganic salts Good pale orange yellow None starch agar flat (73) (ISP-4) waxy greenish black (157) Glycerol-apara- Poor colorless None gine agar (ISP-5) flat Czapek-sucrose Moderate colorless None agar flat blackish green waxy (152) to greenish black (157) Czapek-starch Moderate colorless None agar flat blackish green waxy (152) Gause No.1 agar Moderate colorless None flat blackish green waxy (152) L-arginine-glu- Moderate colorless to None cose agar flat greenish black waxy (157) TABLE 2 Utilization of Carbon Sources Carbon Source Utilization D-arabinose L-arabinose ++ cellobiose ++ D-glucose ++ D-galactose ++ 1 ss-lactose D-fructose + d(+)-mannose ++ D-melezitose a-melibiose raffinose L-rhamnose D-ribose + sucrose ++ D-xylose ++ glycerol soluble starch ++ inositol mannitol TABLE 3 Physiological Tests Test Reaction gelatin hydrolysis hydrolysis action on milk slow peptonization decomposition of cellulose negative hydrolysis of starch positive NaCI tolerance 0-1.5% pH range 6.0-7.4 temperature range 20" - 37"C.

reduction of nitrates negative formation of melanoid negative to weak pigments positive The characteristics of this microorganism are different enough from known species to consider it a new species of the genus Micromonospora. It has been named Micromonospora pilisospora. The name refers to the hairy ornamentation on the spores.

Two variant strains of Micromonospora pilosospora have also been isolated which have the ability to produce AX-127B-1. These variants, AB-127B-44 and AB-127B-48, are different from Micromonospora pilosospora AB-1 27 B-46, in that they exhibit weak melanin production on tyrosine media. They also produce a dark orange yellow (7?) soluble pigment on ATCC medium #172 and sporulation agar. They are both likewise slightly more salt and acid tolerant than M. pilosospora AB-1 27B-46. Colonial morphology of all three strains on various media is similar, but AB-127B-48 sporulates very slowly, if at all, on most media.

Neither AB-1 27B-44 nor AB-1 27B-48 attack milk, and AB-127B-44 does not hydrolyze gelatin. In other respects the variants closely resemble the type strain. AB-127B-44 and AB-127B-48 have also been deposited at Northern Regional Research Center where they have been accorded the accession numbers NRRL 11416 and NRRL 11417 respectively. Cultures represented by NRRL 11415, NRRL 11416 and NRRL 11417 are freely available to the public.

As is the case with other strains of Actinomy-cetales, the microorganism useful for carrying out the present invention can undergo mutation by artificial means such as ultraviolet irradiation, Co60 irradiation, X-ray irradiation and various mutation-inducing chemicals. Accordingly, any strain even if thus mutated, is appropriate for the present invention, insofar as it has the ability to produce AX-1 278-1.

Fermentation Cultivation of cultures AB-1278-46, AB-127B-44 or AB-1 278-48 for the production of this novel antibiotic may be carried out in a variety of liquid media. Thus, assimilable carbon sources such as glucose, sucrose, fructose, starch, molasses and dextrin either alone or in combination may be used. Organic and inorganic nitrogen sources such as soybean meal or flour, peptone, meat extract, corn steep liquor, amino acids, dried yeast, yeast extract, urea, ammonium sulfate, ammonium chloride, ammonium nitrate and sodium nitrate alone or in combination may be used. Inorganic salts such as potassium chloride, sodium chloride, calcium carbonate and phosphates may also be added to the medium. Other organic or inorganic ingredients may be added to stimulate production of the antibiotic.

A liquid, submerged, stirred culture process is preferred for the production of this antibiotic.

Fermentations are carried out at a temperature range of 25 to 37"C and the pH of the fermentation is preferably maintained between 6 and 9. Antibiotic is produced and accumulated between 3 and 12 days after inoculation of the fermentation.

Isolation Isoiation and purification of antibiotic AX-127B-1 is accomplished by methods ordinarily employed for recovery of water soluble basic antibiotics from fermentation broths. These methods include, but are not restricted to, adsorption on and desorption from cation exchange resins or active carbon, chromatography on the above materials or on silica gel, cellulose, cross-linked dextran, etc., and extraction from water into a water immiscible solvent by ion-pair formation with a strongly acidic, highly lipophilic agent such as lauryl sulfonic acid.

It has been found most convenient to separate the crude antibiotic from the fermentation broth by contacting the broth with a cation exchange resin, such as co-polymer of acrylic acid and divinyl benzene, or methacrylic acid and divinyl benzene, (such products are sold by Rohm & Haas Company under the brand name AMBERLITE, specifically AMBERLITE IRC 84, IRC 72, or IRC 50) and, after washing the resin with water, eluting with aqueous ammonia, concentrating the resulting solution under vacuum until the ammonia is gone, and adjusting the pH of the concentrated solution to about 6.5 with aqueous sulfuric or hydrochloric acid. The antibiotic salt thus formed may be obtained as a powder by freeze drying or spray-drying or in the case of the sulfate salt, by adding its concentrated aqueous solution with stirring to a water miscible organic solvent, such as methanol.

The crude product obtained as described may be purified considerably by chromatography on a column packed with a cation exchange resin, using a fine mesh product such as pulverized AMBERLITE IRC 50 or a spherical product of similar composition sold by the Fisher Scientific Company under the tradename REXYN 102, developing with a gradient of increasing concentration of ammonia in water, collecting fractions, locating the anitbacterial activity by dipping filter paper discs in the fractions, placing the discs on nutrient agar seeded with a test organism sensitive to the antibiotic, incubating the agar plates until the organism has grown out, observing the zones of inhibition around the paper discs, pooling the active fractions, evaporating to remove ammonia and either neutralizing to form a salt or evaporating the solution to dryness to obtain the free base as a solid.

An alternative purification method consists of adsorbing the crude antibiotic on active carbon from an aqueous solution at pH 8 + 0.5, washing the carbon with water, and eluting the antibiotic with dilute acid, e.g., 0.05N sulfuric acid.

This process is preferably conducted in a column and fractions of the eluate are tested for antibacterial activity before combining. To obtain the sulfate salt of the purified antibiotic, the excess acid is removed by stirring the solution with an anion exchange resin in the basic cycle until the pH is raised above 4.5. The solution is then separated from the resin and evaporated to dryness, or the concentrated solution may be poured with stirring into a water miscible solvent such as methanol to precipitate the salt of the antibiotic. A suitable anion exchange resin, containing tertiary amine groups on a cross linked acrylic polymer, is AMBERLITE IRA-68, but a great variety of anion exchange resins may be used.

If the free base of the antibiotic is to be isolated, the biologically active acidic eluate from the carbon column is passed through a column of a strong base anion exchange resin (such as a styrene-divinyl benzene co-polymer of a low degree of crosslinkage and bearing quaternary ammonium functional groups) in the hydroxide form. Suitable resins for this application are DOWEX 1 -X2 or DOWEX 2-X4 manufactured by the Dow Chemical Company, although a number of other brands are available.

Identification Antibiotic AX-1 27B-1 sulfate is a nearly white powder soluble in water and virtually insoluble in all the common organic solvents.

Antibiotic AX-1 278-1 is distinguished from other water soluble basic antibiotics produced by members of the genus Micromonospora by chromatography in a number of solvent systems.

System 1 Chloroform, methanol and 17% aqueous ammonia, 2:1:1 by volume respectively, are equilibrated in a separatory funnel. The lower phase is used to develop paper strips suspended in a one-liter glass stoppered cylinder. The upper end of the strip is held between and extends beyond the ground glass surfaces of the cylinder and the stopper. The solvent reaches the stopper a distance of 36 centimeters above the surface, in about 6.5 hours, after which it evaporates from the upper end of the strip while development is continued for another 10 hours. The antibiotics are detected on the strips by bioautography against a sensitive bacterial strain.

System 2 A single phase system consisting of normal butanol, pyridine, acetic acid and water 3:2:1:2 by volume is used to develop sheets of filter paper (Whatman No. 1) for 72 hours by the downflow technique. After removal from the developing tank, the sheets are hung in an ammonia atmosphere for 20-30 minutes to neutralize the acetic acid, then bioautographed.

System3 Thin layer chromatography on silica gel developed with the solvent mixture obtained by equilibrating equal volumes of chloroform, methanol, and concentrated ammonium hydroxide, in a separatoryfunnel, transferring the lower phase to the chromatography tank, and adding enough methanol to prevent the separation of a small amount of a water-rich phase (about 1% of the solvent volume in the tank). Antibiotics are detected on the developed plate by spraying with ninhydrin or other reagents commonly used for detection of amino groups.

Comparison of AX-127B-1 with other antibiotics System 1 System 2 System 3 Antibiotic (cm) (cm) (Rf) AX-127B-1 9.7 10.7 .20 gentamicin Cia 7.9 11.0 .23 gentamicin C2 13.6 14.2 .28 gentamicin C1 24.5 .32 sisomicin 6.3 14.3 .25 fortimicinA 16.0 .35 verdamicin 13.6 Antibiotic AX-127B-1 free base is a white powder, soluble in water, very slightly soluble in methanol, and virtually insoluble in other common organic solvents. On a Fisher Johns Melting point block, it darkens around 160"C. and melts with further darkening from 220-222"C. Its optical rotation is [cc] 26 = + 49" (c 1, H20).

The infrared spectrum of the antibiotic free base (KBr pellet) is shown in Figure 1. The observed absorption bans are consistent with the hypothesis that the antibiotic is an aminoglycoside antibiotic. The characteristic bands are tabulated in wave numbers, 3410 cm~' (Strong-Broad) 2935 (Weak) 1635 (Strong) 1565 (Strong) 1473 (Medium) 1380 (Shoulder) 1332 (Medium) 1140 (Weak) 1072 (Medium) 1005 (Weak) 810 (Weak) The mass spectrum is shown in Figure 2. By peak matching at high resolution, a value of 460.2993 was obtained for the parent ion. This value is compatible with a molecular formula of C20H40N606 and not with any other 20 carbon compound containing only carbon, hydrogen, nitrogen and oxygen.

Figure 3 shows the carbon magnetic resonance spectrum of antibiotic AX-127B-1 free base in deuterium oxide (D20). Twenty carbon resonances are seen, as tabulated below. (Numbers refer to parts per million downfield from tetramethylsilane. The reference standard used was p-dioxane, which is taken at 67.4).

23.1 41.4 48.9 72.5 96.2 29.1 43.4 56.0 73.5 98.5 34.4 44.2 57.8 81.4 150.9 38.5 46.8 60.4 82.6 175.3 The data are consistent with the hypothesis that antibiotic AX-127B-1 is a glycosidic antibiotic with six nitrogen atoms.

Since AX-1278-1 contains basic groups, it can exist in the form of acid addition salts. Accordingly, the present invention contemplates the pharmaceutically acceptable nontoxic acid addition salts of the antibiotic including the mineral acid addition salts such as hydrochloride, hydrobromide, hydroiodide, sulfate, sulfamate and phosphate and the organic addition salts such as maleate, acetate, citrate, oxalate, succinate, benzoate, tartrate, fumarate, malate, mandelate, ascorbate and the like.

Antibacterial properties The antibacterial activity of AX-1278-1 against a variety of microorganisms is illustrated in table 4.

Minimum inhibitory concentrations were determined by the agar dilution method in BBL streptomycin assay agar with yeast extract (pH 7.9).

TABLE 4 Minimum Inhibitory Aminogylcoside Concentration Test Organism Resistnce* mcg/ml Staphylococcus auereus ATCC 6538 P 0.05 Acinetobacter sp.

A4008 0.4 Enterobacter agglo merans A 4020 SM 0.05 Enterobacter cloacae A4014 KM,GM 0.1 Escherichia coli ATCC 26 0.2 Escherichia coli A4006 SM,KM,GM,TOB,AMK 1.6 Escherichia coli A4007 SM,KM,GM,TOB,AMK 12.5 Escherichia coli A4013 SM,GM 0.1 Escherichia coli A4017 SM,KM 0.2 Escherichia coli A4018 SM,KM,GM,TOB 0.1 Escherichia coli A4019 SM,KM,GM,TOB,AMK 6.3 Klebsiella pneumoniae ATCC 8045 0.05 Klebsiella pneumoniae A4002 SM,KM 0.1 Klebsiella pneumoniae A4023 SM,KM,GM,TOB 0.2 Proteus mirabilis A4003 SM 0.8 Proteus rettgeri A4021 SM,KM,GM,TOB 0.2 Proteus rettgeri A4022 SM,KM,GM,TOB,AMK 25.0 Proteus vulgaris ATCC 6897 0.39 Proteus vulgaris A4001 SM,KM,GM,TOB,AMK 6.3 Proteus vulgaris A 4005 KM 0.8 Providencia stuartii ATCC25825 0.1 Providencia stuartii A4011 SM,GM,TOB 0.8 Pseudomonas aeruginosa A4009 SM,KM 1.6 Pseudomonas aeruginosa A4010 SM,KM,GM,TOB 25.

Pseudomonas aeruginosa A4015 SM,KM,GM 12.5 Pseudomonas aeruginosa A4016 KM,TOB 6.3 Salmonella typhi ATCC 9992 0.1 Serratia marcescens ATCC 4003 0.1 Serratia marcescens A4004 SM,KM,TOB,AMK 0.8 Shigella sonnei ATCC 9290 0.4 *SM - streptomycin TOB - tobramycin KM - kanamycin AMK - amikacin GM - gentamicin It is evident from the above table that AX-127B-1 has broad activity against Gram-positive and Gram-negative bacteria. AX-127B-1 is further characterized by showing strong antibacterial activity against strains of Escherichia coli, Klebsiella pneumoniae, Proteus sp. and Serratia marcescens which are resistant to other amonoglycoside antibiotics.

In view of the excellent broad spectrum anti-bacterial activity, this substance can be used alone or in combination with other antibacterial agents to prevent the growth of, or reduce the number of, organisms present in various enviroments. For example, it can be used in papermill systems to inhibit the growth of cultures which are known to produce slime in such systems. It is also useful as an oil preservative, for example, as a bacteriostatic agent for inhibiting the growth of Proteus vulgaris which is known to cause spoilage in cutting oils.Also, it is useful in wash solutions for sanitation purposes, as in the washing of hands and the cleaning of equipment, floors, orfurnishings of contaminated rooms or laboratories; it is also useful as an industrial preservative, for example, as a bacteriostatic rinse for laundered clothes and for impregnating paper and fabrics, and it is useful for suppressing the growth of sensitive organisms in plate assays and other microbiological media. This antibiotic can be used in treating infections caused by susceptible organisms in warm-blooded animals.

Example 1 Culture AB-127B-46 was maintained on ATCC medium #172 agar slants consisting of 1% glucose, 2% soluble starch, 0.5% Difco yeast extract, 0.5% N-Z amine type A (Sheffield Chemical Co.), 0.1% CaCO3, 1.5% agar, and distilled water OS to 1 liter.

First passage inoculum seed tubes (25 x 150 mm) containing 10 ml. of sterile S-3 seed medium (Table 5) and closed with Bellco stainless steel caps were inoculated with a sterile loop from ATCC medium #172 agar slant cultures of AB-127B-46. Seed tubes were incubated on a rotary shaker (250 r.p.m.) at 30 C for 96 hours.

At that time 5% vegetative inoculum from the first passage seed tube was transferred aseptically to 500 ml.

Erlenmeyer flasks containing 100 ml. of sterile S-3 seed medium and closed with cotton plugs. Inoculated second passage seed flask were then incubated on a rotary shaker (250 r.p.m.) at 30"C for 72 hours. Antibiotic production fermentation flasks (500 ml. Erlenmeyer) containing 100 ml. of sterile AF1 b medium (Table 5) and closed with cotton plugs were inoculated with 5% vegetable inoculum from the second passage seed flasks.

The inoculated AF1 b antibiotic production medium flasks were then incubated on a rotary shaker (250 r.p.m.) at 300C for 5 to 7 days and then harvested.

The harvested whole culture fermentation beer from a series of flasks was pooled (30 liters), adjusted to pH 2 with sulfuric acid and clarified by centrifugation or by filtration through celite. The clarified fermentation liquor was then poured into a 6.5cm. diameter glass column containing 0.7 liters of AMBERLITE IRC 84 cation exchange resin (ammonia form). The active antibiotic was adsorbed on the resin and the effluent beer was discarded. The resin column was washed thoroughly with water. Antibiotic activity was then eluted with a N aqueous ammonia. Active fractions were determined by dipping paper discs in eluate fractions and testing for activity on agar plates seeded with Staphylococcus aureus ATCC 6538P.Active fractions were combined and concentrated to remove excess ammonia and were then neutralized to pH 6.5 with sulfuric acid. The concentrate was then passed through a glass column containing REXYN 102 (NH4+) 2 cm.

diameter x 6 cm. in height or 18 ml. of resin. The column was washed with water and then eluted by stepwise gradient with aqueous ammonia starting with 0.05 N and increasing to 1 N ammonia.

Active fractions were again located by the paper disc method and further examined by both paper chromatography and thin-layer chromatography as previously described. Active fractions containing the antibiotic described in this invention were combined, concentrated to remove excess ammonia, neutralized to pH 6.5 with sulfuric acid and reduced to dryness under vacuum. The sulfate salt of the antibiotic was dissolved in distilled water and converted to free base by passing through a small glass column containing DOWEX-1-X2 (OH-).

TABLES TABLE5 S-3 Seed Medium Ingredient gm/liter Staclipse J soluble starch (Staley) 24 glucose monohydrate 1 yeast extract (Difco) 5 tryptone (Difco) 5 beef extract (Wilson) 3 CaCO3 4 tap water QS to 1.0 liter sterilization: 30 min., 121"C. at 15-16 Ib. pressure AF1b Fermentation Medium Ingredient gm/liter glucose monohydrate 10 peptone (Difco) 5 yeast extract (Difco) 5 CaC03 1 pH 7.3 tap water QS to 1.0 liter sterilization: 30 min., 121"C. at 15-16 lb. pressure Example 2 Culture AB-127B-46 was inoculated into first passage 500 ml. Erlenmeyer seed flasks containing 100 ml. of sterile S-3 seed medium and closed with cotton plugs.Inoculated flasks were incubated on a rotary shaker (250 r.p.m.) at 30 C. for 96 hours, At that time, 5% vegetative inoculum was transferred into similar 500 ml.

Erlenmeyer flasks containing 100 ml. of sterile S-3 seed medium. Inoculated second passage seed flasks were inoculated on a rotary shaker at 30"C. for 72 hours. Second passage seed flasks were used to inoculate a series of 30 liter stainless steel fermentors at a level of 5% Fermentation conditions for 30 literfermentors were as follows: Fermentation Medium: AFlb (see Table 5) FermentorVolume: 12 liters Sterilization Time: 1 hr., 121"C., 15-16lb pressure Antifoam: .01% P-2000 polyethylene glycol (Dow Chemical Co.) Incubation Temp.: 30"C Agitation: 250 r.p.m.

Impeller Blade Angle: 45" Air Rate: 1 volume/volume/min.

Fermentors were incubated for 5 days and then harvested. The desired antibiotic described in this invention was isolated and purified as described in Example 1.

Claims (7)

1. The antibiotic AX-1 278-1 free base which comprises a white powder characterized as follows: a) soluble in water, very slightly soluble in methanol and essentially insoluble in other common organic solvents; b) a molecularweightof460; c) the empirical formula of C20H40N6O6; d) a characteristic infrared absorption spectrum as shown in Figure 1; e) a characteristic mass spectrum as shown in accompanying Figure 2; f) a characteristic carbon magnetic resonance spectrum as shown in accompanying Figure 3; or a pharmaceutically acceptable acid addition salt thereof.

2. A process for producing the antibiotic AX-127B-1 which comprises culturing a microorganism belonging to the species Micromonospora pilosospora having the ability to produce antibiotic AX-127B-1 in a nutrient medium and accumulating the antibiotic in said medium,

3. A process according to Claim 2 wherein said microorganism is selected from the group consisting of Micromonospora pilosospora NRRL 11415, Micromonospora pilosospora NRRL 11416, and Micromonos pora pilosospora NRRL 11417, and mutations thereof.

4. A process according to Claim 3 wherein said antibiotic is isolated from said culture medium.

5. A process according to Claim 3 wherein said microorganism is cultured at a temperature of about 25-37"C and at a pH of about 6-9.

6. A process for producing the antibiotic AX-127B-1 which comprises culturing a microorganism selected from the group consisting of Micromonospora pilosorpora NRRL 11415, Micromonospora pilosospora NRRL 11417, in a nutrient medium at a temperature of about 25-37"C. and a pH of about 6-9, accumulating the said antibiotic in said medium, and recovering said antibiotic therefrom.

7. A biologically pure culture of the microorganism Micromonospora pilosospora NRRL 11415, NRRL 11416 and NRRL 11417 and mutations thereof, said culture being capable of producing the antibiotic AX-127B-1 upon culturing of said microorganism in a nutrient medium.