GB2243610A - Cyclic glycopeptides - Google Patents

Abstract

Antibiotic compound of formula (I): <IMAGE> is produced by fermentation of novel-organism Saccharothrix aerocolonigenes ATCC 53829. It is active against Gram- positive bacteria, and may be used as an animal feed supplement.

Description

"Glyccpeptide Antibiotic" Background of the Invention This invention is concerned with a new no per of the glycopeptide group of antibiotics, a class of compounds characterised biologically by their Gram-positive antibacterial action exerted by the inhibition of bacterial cell wall biosynthesis. This family of antibiotics includes such well known agents as avorarcin; actaplanin; teichoplanin; A41030 complex and the aridicins. The subject has been reviewed by Williams and Barna. "Structure and mode of action of glycopeptide antibiotics of the vancomycin group". Annual Rev. Microbial., 38, 339, 1984.

In the search for new antibiotics, structural modification of knawn antibiotics is attempted whenever possible. This approach is limited, however, to modifications which retain the desired activity. Many antibiotics, including the glycopqptides, have such complex structures that even small changes can be difficult to make by chemical means. The discovery of new antibiotics produced by fermentation processes continues, therefore, to be of great importance even in cases where the antibiotic, once recognised, is white similar to a previously) know antibiotic.

The glyccpeptide antibiotics listed above are active against Gramrpositive bacteria. They have therefore been employed with varying degrees of success for administration to poultry and other farm animals, including the ruminants and pigs, to control infection or to promote growth or milk production.

Among a number of conditions which can be treated with these agents is enteritis, a disease which can cause severe economic losses to livestock producers. Enteritis occurs in chickens, swine, cattle and sheep and is attributed mainly to anaerobic bacteria, particularly Clostridium perfrinqens, and viruses.

Er1terotceiia in ruminants, an example of which is "overeating disease" in sheep, is a condition caused by C. perfringens infection.

Performance enhancement (increased rate of growth and/or increased efficiency of feed utilization) in ruminants such as cattle, and in mlonogastric animals such as swine, is another econcoically desirable objective of veterinary science. Of particular interest is iitraved performance achieved by increasing the efficiency of feed-utilization. The mechanism for utilization of the major nutritive portion of ruminant feeds is well known.

Microorganisms in the rumen of the animal degrade carbohydrates to produce monosaccharides and then convert these monosaccharides to pyruvate compounds. Pyruvates are metabolized by microbiological processes to form acetates, butyrates or propionates, collectively knawn as volatile fatty acids. Although acetates and butyrates are utilized, propionates are utilized with greater efficiency.

Furthermore, when too little propionate is available, animals may develop ketosis. A beneficial compound, therefore, stimulates animals to produce a higher proportion of propionates from carbohydrates, thereby increasing carbohydrate utilization efficiency and also reducing the incidence of ketosis.

Detailed Description of the invention this invention is concerned with a new glycopeptide antibiotic designated UK-69542, produced by the submerged aerobic propagation in aqueous nutrient media of a microorganism isolated froan a soil sample fran Ueno City, Mie Prefecture, Japan. The antibiotic is active against a variety of microorganisms and is effective in promoting growth and increasing efficiency of feed utilisation in poultry, swine and ruminants. The microorganism is designated herein as Saccharothrix aerocolonigenes, strain N706-5.

It was recognised to be a species of Saccharothrix because of its randomly fragtnerrted hyphae, white aerial mycelium, cream or pale yellow to brown substrate mycelium and the whole-cell amino acid, sugar and phospholipid compositions.

A culture thereof, designated herein as N706-5 was planted fran a slant onto ATOC no. 172 broth and grown for four days at 280C on a shaker. It was then centrifuged for 20 minutes, washed three times with sterile distilled water and planted on media commonly used for identification of members of the Actinomycetales as hereinafter described. The culture was incubated at 28 C and the results were read at varying times but most were commonly taken at 14 days. The colours are described in common terminology but exact colours were determined by comparisons with colour chips fran the Colour Harmony Manual, fourth edition.The methods of whole-cell amino acid and sugar analyses are those described in Becker, B et al, Appl. Microbiol.

12.421-423, 1964; and in lechevalier, M.P., J. Lab. Clin. Med., 71,934-944, 1968.

Identification media used for the characterisation of the culture and references for their composition are as follows: 1. Tryptone-Yeast Extract Broth - (ISP medium mo. 1, Difco).

2. Yeast Extract-Malt Extract Agar - (ISP medium no. 2, Difco).

3. Oatmeal Agar - (ISP medium no. 3, Difco) 4. Inorganic Salts-Starch Agar - ISP medium no. 4, Difco).

5. Glycerol-Asparagine Agar - (ISP medium no 5, Difco).

6. Peptone-Yeast Extract Iron Agar - (ISP medium no. 6, Difco).

7. Czapek-Sucrose Agar - S.A. Waksman, The Actinomycetes, Vol.

2, medium no 1, p. 328, 1961.

8. Glucose-Asparagine Agar - Ibid, medium no. 2, p. 328.

9. Bennett's Agar - Ibid, medium no. 30, p. 331.

10. flnerson's Agar - mid, medium no 28, p. 331.

11. Nutrient Agar - Ibid, medium no 14, p. 330.

12. Glucose-Yeast Extract Agar - Ibid, medium no 29, p. 331.

13. Peptone-Czapek Agar - J. N. Couch, J. Elisha Mitchell Soc., 79.53-70, 1963.

14. Hickey and Tresner's Agar - R. J. Hickey and H. D. tresner, J. Bacteriol., 64,891-892, 1952.

15. Gordon and Smith's Tyrosine Agar - R. E. Gordon and M. M.

Smith, J. Bact., 69,147-150, 1955.

16. Casein Agar - Ibid.

17. Calcium Malate Agar - S. A. Waksman, Bact. Rev., 21,1-29, 1957.

18. Gelatin Agar-R. E. Gordon and J. M. Mihm, J. Bact., 73, 15-27. 1957.

19. Starch Agar - Ibid.

20. Organic Nitrate Broth - Ibid.

21. Potato Carrot Agar - M. P. Lechevalier, J. Lab. and Clin.

Med. 71,934,944, 1968, but use only 30 g potatoes, 2.5 g carrots and 20 g agar.

22. 2% Tap Water Agar.

23. Dextrose Nitrate Broth - S. A. Waksman, The Actinomycetes, Vol. 2, medium no. 1, p. 328, 1961, with 3 g dextrose substituted for 30 g sucrose and agar anitted.

24. Cellulose Utilization a) H. L. Jenson, Proc. Linn. Soc. N.S.W., 55,231-248, 1930.

b) M. Levine and H. W. Schoenlein, A Compilation of Culture media, medium no. 2511, 1930.

25. SkSmmed MLIk -Difco.

26. Carbohydrates - G. M. Luedemann and B. C. Brodsky, Antimicrob. Agents Chemother., 1964,47, 1965, ISP medium no. 9.

Difco.

27. Temperature Range - ATCC medium 172 in ATCC Media Handkook, 1st ed., p. 10, 1984.

The observations of growth and appearance of the organism were as follows: Yeast Extract-Malt Extract Agar - Growth good, brown to dark brown (3 ie, 3 lg), raised, wrinkled, no aerial mycelium; reverse brown (3 ie, 3 lg); soluble pigment yellowish brown (3 lc).

Oatmeal Agar - Growth moderate to good; white, cream to pale gray (2 ca, near gray series 2 ba); raised, smooth, with white to cream (2 ca) aerial mycelium; reverse cream (2 ca); soluble pigment cream (2 ca).

Inorganic Salts-Starch Asar - Growth moderate to good, white, moderately raised, smooth, with white aerial mycelium; reverse cream to pale yellowish (2 ca, 2 ea); soluble pigment cream (2 ca).

Glycerol-Asparasgine Agar - Growth moderate, white to cream (2 ca), slightly to moderately raised, smooth, with white aerial mycelium; reverse colorless to cream (2 ca); soluble pigment cream (2 ca).

Czapek-Sucrose Agar - Growth good, white, moderately raised, smooth, aerial mycelium white; reverse colorless to cream (2 ca); no soluble pigment.

Glucose-Asparagine Agar - Growth good, white to cream (2 ca), moderately raised, smooth but wrinkled in scme areas, aerial mycelium white; reverse cream to pale yellowish (2 ca, 2 ea); no soluble pigment.

Bennett's Agar - Growth good, tan (2 gc) but pale gray to gray (near gray serie 3 dc, 3 fe) in some areas, raised, wrinkled; aerial mycelium sparse, white; reverse tan to pale yellowish (2 gc, 2 ea); soluble pigment pale yellowish (2 ea).

Emerson's Agar - Growth good, yellowish to brown (2 ea, 2 ga, 3 ie), raised, wrinkled, no aerial mycelium; reverse yellowish brown (2 ic); soluble pigment yellowish brown to orange brown (3 Ic, 4 lc).

Nutrient Agar - Growth moderate, pale yellowish to yellowish tan (2 ea, near 2 gc), slightly raised, smooth, no aerial mycelium; reverse pale yellowish (2 ea); no soluble pigment.

Gordon and Smith's Tyrosine Aqar - Growth good, brown to red brown (3 ie, 4 ng, moderately raised, smooth to wrinkled, no aerial mycelium; reverse brown (3 ne); soluble pigment red-brown (5 pi).

Casein Agar - Grcwth good, brown (4 ie), raised, wrinkled, no aerial mycelium; reverse yellowish brown (3 ic); soluble pigment brown (4 le).

Calcium Malate Agar - Growth scant, colourless to cream (2 ca), thin, smooth, no aerial mycelium; reverse colourless to cream (2 ca); no soluble pigment.

Gelatin Agar - Growth good, cream (2 ca), moderately raised, smooth to wrinkled, no aerial mycelium; reverse cream to pale yellowish (2 ca, 2 ea); no soluble pigment.

Starch Agar - Growth good, cream to pale yellowish (2 ea, 2 ca), raised, wrinkled, no aerial mycelium; reverse cream to pale yellowish (2 ca, 2 ea); no soluble pigment.

Potato Carrot Agar - Growth moderate, white to pale gray (near gray series 3 ba), slightly raised, smooth, aerial mycelium white; reverse colourless to cream (2 ca); no soluble pigment.

Tan Water Agar - Growth moderate, white, slightly riased, smooth, aerial mycelium white; reverse colorless; no soluble pigment.

Morphological Properties - Morphological properties were observed after two weeks of incubation on inorganic salts-starch agar: substrate mycelium branched, 0.4 - 1.0 um wide; aerial mycelium white, may fragment into rods of varying lengths, 1.2 - 3.0 um or more than 3 um long and 0.4 - 0.8 um wide.

Biochemical Properties - Melanin not produced in tryptone-yeast extract broth; hydrogen sulfide produced on peptone-yeast extract iron agar; gelatin liquefied; starch hydrolyzed; nitrate reduced to nitrite in both organic nitrate broth and dextrose nitrate broth; good growth but no disintegration on both cellulose broths; coagulation and clearing on milk; casein digestion positive; digestion of calcium malate negative; digestion of tyrosine positive; digestion of xanthine and hypoxanthine negative; digestion of adenine negative.The organism did not survive at 500C. Carbohydrate utilization: glucose, arabinose, fructcse, inositol, mannitol, raffinose, sucrose, xylose, adonitol, cellobiose, erthritol, galactose, glycerol, lactose, maltose, nannose, melezitose, melibiose, alpha-methyl-O-glucoside, ribose, salicin, soluble starch, sorbitol and trehalose utilized: rhamnose, dulcitol and sorbose not utilized.

Temperature Relations: 21 C 28 C 37 C 45 C Excellent Excellent Good to No Growth Grorth Growth Excellent Growth Cell Wall Analvses - The whole-cell hydrolysates contained mesodiaminopimelic acid, galactose, glucose, and ribose.

Mycolate Analysis - The cell wall contained no mycolates.

Phospholinid Analysis - The extracts of the cell membrane contained phosphatidylethanolamine, phosphatidylglycerol, and phosphatidylinositol.

Culture N706-5 is characterised by the white aerial mycelium; the cream, pale yellowish, yellowish brown to brown substrate mycelium; narrow substrate hyphae, sparse aerial hyphae and the randan fragmentation of the hyphae. Attempts to induce spore or sporangium production have failed. The whole cell hydrolysates contained meso-diaminopimelic acid, galactose, glucose, and ribose. There is a lack of mycolic acids in the cell walls. The absence of arabinose in the whole-cell hydrolysates indicates that it has type III cell walls rather than type IV cell walls. The phospholipid composition of this culture included phosphatidylethanolamine as the characteristic phospholipid, placing it in phospholipid pattern group PII.It fits into the description of the genus Saccharothrix except that there is no rhamnose in the whole-cell hydrolysates.

Culture N706-5 closely resembles Saccharothrix aerocolonigenes (Labeda, Int. J. Syst. Bacteriol. 36: 109-110.

1986) in most of the biochemical properties. However, it differs fran the latter in the decomposition of xanthine; and the failure to produce acid from -methyl-D-glucoside, melezitose, and sorbitol. From the data mentioned above, culture N706-5 is considered as a new strain of Saccharathrix aerocoloniaenes (Shinobu & Kawato) Labeda.

It has been deposited at the American Type Culture Collection, 12301 Parklawn Drive, Rcckville, Maryland 2d852, USA under the provision of the Budapest Treaty and under the accession number AICC 53829 on 28th November, 1988.

Cultivation and isolation UK-69542 may be conducted under conditions similar to those generally employed to produce antibiotics by fermentation. Cultivation preferably takes place in aqueous nutrient media under submerged aerobic conditions with agitation at a temperature of 240C to 3600. Nutrient media useful for cultivation include a source of assimilable carbon such as sugars, starches and glycerol; a source of organic nitrogen such as casein, enzymatic digest of casein, soybean meal, cotton seed meal, peanut meal, wheat gluten, soy flour, meat meal and fish meal.A source of growth substances such as grain solubles, fishmeal, cotton seed meal, and yeast extract as well as mineral salts such as sodium chloride and calcium carbonate and trace elements such as iron, magnesium, cceper, zinc, cobalt and manganese may also be utilized with advantageous results. If excessive foaming is encountered during fermentation, antifoam agents such as polypropylene glycols or silicones may be added to the fermentation medium. Aeration of the medium in tanks for submerged growth is preferably maintained at the rate of about 1/2 to 2 volumes of sterile air per volume of fermentation broth per minute forced into the broth through a sparger.Agitation may be maintained by means of agitators generally familiar to those skilled in the fermentation art. The rate of agitation depends on the type of agitator employed. A shake flask is usually run at 150 to 200 cycles per minute whereas a fermentor is usually run at 300 to 1700 revolutions per minute. Aseptic conditions must, of course, be maintained through the transfer of the organism and throughout its growth.

Inoculum for the preparation of the antibiotics according to this invention may be obtained by employing growth fran a slant of the culture or Roux bottles inoculated with the culture. A solid medium suitable for initial growth of the organism on slants and in Roux bottles is ATOC medium no. 172. The growth may be used to inoculate either shake flasks or inoculum tanks or the inoculum tanks may be seeded fram the shake flasks. Growth in shaken flasks will generally have reached its maximum in 4 to 5 days whereas inoculum in submerged inoculum tanks will usually be in the most favourable period in 3 to 6 days.

The progress of antibiotic production during fermentation and the bioactivity of the fermentation broth can be monitored by biological assay of the broth employing a sensitive strain of Staphylococcus aureus or Bacillus subtillus. Strain ATCC 6633 is a suitable strain for this purpose. Standard plate assay technique is employed in which the zone of inhibition surrounding a filter paper disc saturated with the broth is used as a measure of antibiotic potency.Also, thin-layer chromatography employing silica gel is a useful tool for detecting the antibiotics produced in fermentation media and analyzing the composition of crude and purified materials extracted fran the fermentation broths. The chromatograms are developed with acetonitrile, water, ammonium hydroxide (3:1:7) and the developed plate is overlayed with agar seeded with either S. aureus or B. subtilis and incubated at 37 C for 16 hours to visualize the antibiotics.

Antibiotic UK-69542 produced by fermentation of Saccharathrix aerocolonigenes ATOC 53829 may be separated and removed by filtering the broth to remove mycelium.

The antiobiotic can be further purified by a series of adsorptions on suitable adsorbents, such as ion exchange resins, chemically modified hydrcphobic inorganic supports as used in high performance reverse-phase liquid chromatography, or high porosity polymers, eluting the antibiotic in each case with a suitable solvent.

Antibiotic UK-69542 can be characterised by virtue of the following physiochemical properties, A) Ultraviolet absorption spectrum. The compound exhibits the following absorption maxima: Solvent max(nm) a) water 218, 280 b) NaOH 218, 292 0,01M B) Infrared absorption spectrum. The compound exhibits the following absorption maxima (cm-1): 3500-3100; 1670; 1610; 1515; 1395; 1250; 1150; 1050; 1010.

C) Retention time (Rt) of 12.3 minutes when analysed by reverse phase HPLC under the following conditions: Column: C-18 Nicro-Bondapak, 3.9 mm (i.d.) x 150 mm (waters) Eluent: CH3CN 14%, 0.1S aqueous ammonium formate 86%, adjusted to pH 7.3.

Flow rate: 2 ml/min.

Detection: U.V. at 225 nm.

D) Molecular weight deduced groan a fast atom bombardment mass spectrum showing the MB peak at mZe 1489.

On the basis of nuclear magnetic resonance spectroscopy in combination with mass spectrometry UK-69542 has been assigned the following structure: The value of animal feed has generally been determined directly by feeding the animal. British Patent Specification No 1,197,826 details in an in vitro rumen technique whereby the changes occurring in feeds brought about by microorganisms are measured more readily and with great accuracy in the evaluation of animal feeds. This technique Involves the use of an apparatus in which the digestive processes of the amimals are conducted and studied in vitro.The animal feed, rumen inoculum and various growth promotants are introduced into and withdrawn from a laboratory unit under carefully controlled conditions and the changes taken place are studied critically and progressively during the consumption of the feed by the microorganisms. An increase in the prop ionic acid content of the rumen fluid indicates that a desirable response in overall ruminant performance has been brought about by the growth promotant in the feed composition. The change in propionic acid content is expressed as percent of the prop ionic acid content found in the control rumen fluid. Long term in vivo feeding studies are used to show a reliable correlation between propionic acid increase in the rumen fluid and improved animal performance.

Rumen fluid is collected fratt a fistulated cow which is fed on a commercial fattening ration plus hay. The rumen fluid is immediately filtered through cheese cloth, and 10 ml added to a 50 ml conical flask containing 400 mg of standard substrate (68% corn starch + 17% cellulose + 15% extracted soybean meal), 10 ml of a pH 6.8 buffer and the test compound. The flasks are gassed with oxygen free nitrogen for about two minutes, and incubated in a shaking water bath at 390C for about 16 hours. All tests are conducted in triplicate.

After incubation 5 ml of the sample is mixed with 1 ml of 25% metaphosphoric acid. After 10 minutes 0.25 ml of formic acid is added and the mixture centrifuged at 1500 rpa for 10 minutes.

Samples are then analysed by gas-liquid chromatography by the method of D W Kellog, J. Dairv Science, 52, 1690, 1969. Peak heights for acetic, propionic and butyric acids are determined for samples frapn untreated and treated incubation flasks.

When tested by this in vitro procedure, Antibiotic UK-69542 at the level of 10 microorgrams per milliliter gave rise to an increase of about 63% in the production of propionic acid aver that produced in the control solution without added Antibiotic UK-69542. By comparison the commercially available compound salinomycin (a polyether antibiotic) at 10 mog/ml produced about an 99% increase of propionic acid aver the control.

These data shows that Antibiotic UK 69542 will improve feed utilization by ruminants such as cattle and sheep. The compOunds will also have a similar effect in monogastric animals such as pigs and poultry.

In particular, as shown in Table 1, Antibiotic UK-69542 is of benefit for improving weight gain and feed utilisation in poultry.

The compound was added to the feed which is provided to four day old broiler chicks housed in tierbrooders on a free-access basis. After ten days the birds are weighed and the live weight gain compared with an untreated control grcup to give a percentage improvement in live weight gain. The amount of feed consumed is divided by the live weight of the animals in the group at the completion of the trial to give a feed conversion ratio (which gives a measure of the amount of feed required to produce 1 kg increse in body weight), and this is also compared with the control group and the improvement in the feed conversion ratio calculated as a percentage.The results show a significant improvement in both live weight gain and in food conversion efficiency when Antibiotic UK-69542 is added to feed at a level of 20 ppm: TABLE 1 PERFORMANCE ENCHANCING ACTIVITY IN POULTRY liveweight gain Food conversion rate Ppm (% improvement) (% improvement) 20 9.9 5.0 Antibiotic UK-69542 may be incorporated in feed composition as the pure compound. Alternatively crude forms of Antibiotic UK-69542 or dried fermentation broth containing the antibiotic may be used by incorporation in feed canpositions to give the desired potency concentration of antibiotic.

The invention is further illustrated by the following Examples.

EXAMPLE 1 1. PrEparation of Inoculum A sterile aqueous medium having the following composition was prepared.

Ingredient Grams/litre Glucose 1 Starch 24 Peptone 5 Yeast extract 5 Meat extract 3 Calcium carbonate 4 One litre of medium was distributed into 2.8 litre conical flasks and sterilised at 1200C and 15 p.s.i. for 30 minutes.

After cooling, the medium was inoculated with a vegetative cell suspension from a slant culture of Saccharothrix aerocolonigens ATCC 53829.

The flasks are shaken at at 2800 on a rotary shaker having a displacaant of 4 to 7 cm and 150 to 200 cycles per minute for three to five days.

2. Fermentation and inoculation of Antibiotic UK-69542 2 litres of the inoculum medium described above was used to inoculate a 100 litre fermentation tank containing 20 litres of sterile medium of the following composition to which 70 ml of polypropylene glycol antifoaming agent had been added Incredients Grarrrs/litre Oerelose 27.5 Soya flour 12.5 Molasses 1.5 Dipotassium hydrogen phosphate 0.125 Calcium carbonate 1.25 pH 6.9-7.0 Fermentation was carried out at 280C with stirring at 350 revolutions per minte and aeration at one volume air per volume of broth per minute until substantial activity was observed abased on antibiotic disc assay versus B. subtilus ATCC 6633) usually after 4-6 days.The bioactivity of the broth, and of subsequent recovery streams was followed by using a sensitive strain of Bacillus subtilis ATCC 6633 or Staphylococcus aureus ATCC 6538.

The antibiotic component in the broth and recovery streams was detected after chromatographic separation using silica gel plates developed with acetonitrile, water, ammonium hydroxide (3:1:0.7). The plates were visaalised with W light at 254nm and then overplayed with agar, seeded with either S. aureus or B.

subtilis, to which 1.0 ml of a 1% solution of 2,3,5-tripheeyl-2Htetrazolium chloride had been added, and incubated at 370C for 16 hours to visualise the antibiotic as a white area against a pink background.

At the end of the fermentation production stage, the broth was adjusted to pH 10 and filtered to remove mycelium. The antibiotic UK-69542 was absorbed onto an affinity column comprising a D-alanyl-D-alanine ligand immobilised on an agarose matrix (as described in EP 0132117) and eluted with water.

Further purification was achieved using silanised silica gel as a support and acetonitrile: 0.1 M aqueous ammonium formate (pH 7.3) (15:85) as eluent to give UK-69542 in pure form.

It will be appreciated from the foregoing that what we will claim may include the following:- 1. Antibiotic UK-69542 2. A process for producing UK-69542 by fermentation of the microorganism Saccharothrix aerocolonipenes. ATCC 53829 3. Animal feed compositions containing Antibiotic UK-69542.

4. A method for promoting growth and/or increasing the efficency of food utilisation in poultry, swine, cattle or sheep by administering Antibiotic UK-69542.

5. A biologically pure culture of the microorganism Saccharothrix aerccoloniaenes ATCC 53829

Claims (9)

1. Claims 1. A camposition having antibiotic properties obtainable by fermentation of micro-organism ATCC 53829 or a mutant or recombinant form thereof having the ability to produce said composition.
2. 2. A process for producing a composition having antibiotic properties which comprises fermenting micro-organism mIOC 53829 or a mutant or recombinant form thereof having the ability to produce said composition in a fermentation medium, and isolating said composition from the medium.
3. 3. A conpoand of the formula (I):
4. 4. A compound obtainable by fermentation of micro-organism ATCC 53829 having the following characteristics (a) to (d): (a) Ultraviolet absorption spectrum: Solvent max (nm) water 218,280 0.01M NaOH 218,292 (b) Infrared absorption spectrum: absorption maxima (cm-1) at 3500-3100; 1670; 1610; 1515; 1395; 1250; 1150; 1010.

   (c) Retention time (Rt) of 12.3 minutes when analysed by reverse phase HPLC under the following conditions: Column: C-18 Micro-Bondapak, 3.9mm (i.d.) x 150mm (Waters) Eluent: CH3CN 14%, 0.1M aqueous ammonium formate, 86%, adjusted to pH 7.3.

   Flow rate: 2ml/min Detection: Ultraviolet at 225nm.

   (d) Molecular weight: Mass spectrum (FAB) m/e = 1489 at M+ peak.
5. 5. An antibiotic composition, comprising a compound according to claim 3 or 4.
6. 6. A compound according to claim 3 or 4, for human or veterinary medicine.
7. 7. Use of a compound according to claim 3 or 4, for making a medicament for treatment of bacterial infections or to promote growth and/or milk production in animals.
8. 8. An antibiotic composition or compound, substantially as hereinbefore described with reference to the Examples.
9. 9. Micro-organism Saccccrothrix aerocolonigenes ATCC 53829.