Rifamycin R and microorganisms useful in the production of rifamycins

Abstract

Novel rifamycin compounds, designated P, Q, R and U, produced by the fermentation of mutant strains of Streptomyces mediterranei.

Description

This invention is directed to novel rifamycin compounds which are produced by fermentation of mutant strains of Streptomyces mediterranei in an aqueous nutrient medium under aerobic conditions. These novel rifamycins are hereinafter referred to as rifamycin P, rifamycin Q, rifamycin R and rifamycin U.

It has previously been reported that during fermentation in normal growth media, Streptomyces mediterranei synthesizes a family of antibiotics collectively referred to as the rifamycin complex (P. Sensi, et al., Antibiotics Annual 1959-1960, page 262). Subsequent work revealed that the addition of sodium diethyl barbiturate to the culture medium resulted essentially in the formation of a single fermentation product, rifamycin B (Margalith P. and Pagani H., Applied Microbiology, 9, 325, 1961). According to a further discovery, a mutant strain of Streptomyces mediterranei was found which elaborates essentially rifamycin B only, irrespective of the presence or absence of sodium diethyl barbiturate in the fermentation medium, so that an improved process for making rifamycin B results. The rifamycin B producing strain was identified as Streptomyces Mediterranei ATCC 21789. See U.S. Pat. No. 3,871,965.

The new antibiotic substances which are the object of this invention are produced by mutant strains derived from Streptomyces mediterranei ATCC 13685. The new strains are obtained by treating strain ATCC 13685 with common chemical mutagenic agents such as nitrous acid or nitroso guanidine derivatives or with physical mutagenic agents such as X rays and U.V. radiation. The new mutant strains rather than to the genus Streptomyces mediterranei are assigned to the genus Nocardia in accordance with the proposals by J. E. Thiemann et al., Arch. Mikrobiol., 67, 147-155 (1969).

ISOLATION OF THE MUTATED STRAINS PRODUCING NOVEL RIFAMYCINS

A suspension of spores of Streptomyces mediterranei ATCC 13685 was treated with N-methyl-N'-nitro-N-nitrosoguanidine at 1 mg/ml in a pH 9.0 tris(hydroxymethyl)aminomethane buffer for 60 minutes at 28.degree. C. The mutagen-treated spores were then washed and plated onto Petri dishes containing Bennett agar. After 14 days incubation at 28.degree. C., the surviving colonies were picked off and examined for their ability to inhibit the growth of Bacillus subtilis in the following manner: a disc of agar (diameter 4-8 mm) carrying a single colony was transferred onto a Petri dish containing Penassay agar at pH 7.2 previously seeded at 2% (v/v) with Bacillus subtilis.

Under these conditions rifamycin B is practically inactive and a normal colony producing rifamycin B gives no inhibition of the growth of Bacillus subtilis in the underlying agar, whereas any colony producing a novel rifamycin with antimicrobial activity will cause a clear cut zone of inhibition of growth around the disc of agar. Three strains were isolated in this way and were originally given our internal code D-2, MM18-6 and M-36. Samples of these microorganisms were deposited with the ATCC where they are identified respectively as Nocardia mediterranea ATCC 31064 Nocardia mediterranea ATCC 31065 and Nocardia mediterranea ATCC 31066.

DESCRIPTION OF THE NEW NOCARDIA MEDITERRANEA STRAINS

Table 1 reports the results of the macroscopic and mycroscopic examination of the Nocardia mediterranea strains identified respectively with the ATCC No. 31064, 31065 and 31066, and Table 2 reports the cultural characteristics of the same Nocardia mediterranea strains.

The characteristics of the parent strain Streptomyces mediterranei ATCC 13685 (ME/83) are also reported:

TABLE 1__________________________________________________________________________ Colonies Aerial mycelium Spores__________________________________________________________________________N. mediterranea Colonies conical-crateriform and Greyish to pinkish, CylindricalATCC 31064 convolute, 2-3 mm. of diameter, branched, 0.6-0.8 (0.8-1 .mu.m .times. 3.0-5.0.mu.) reddish-brown with ochre yellow .mu.m of diameter(D-2) soluble pigmentN. mediterranea Colonies conical-crateriform and Pink to light orange CylindricalATCC 31065 convulate, 2-3 mm. of diameter, 1 .mu.0.6-0.8 (0.8-.mu.m .times. 3.0-5.0.mu.) deep orange with deep orange- .mu.m of diameter(MM18-6) yellow soluble pigmentN. mediterranea Colonies conical-crateriform and Pink to pink orange CylindricalATCC 31066 convolate, 2-3 mm. of diameter, branched, 0.6-0.8 (0.8-1 .mu.m .times. 3.0-5.0.mu. ) orange brown with amber soluble .mu.m of diameter(M-36) pigmentStreptomyces medi- Colonies conical-crateriform and Pinkish, branched, Cylindricalterranei ATCC 13685 convolate, 2-3 mm of diameter, 0.6-0.8 .mu.m of dia- (0.8-1 .mu.m .times. 3.0-5.0.mu. ) orange-yellow with light amber meterME/83 soluble pigment__________________________________________________________________________ TABLE 2__________________________________________________________________________ N. mediterranea ATCC 31064 N. mediterranea ATCC 31065 N. mediterranea ATCC 31066 Streptomyces mediterraneiMedia (D-2) (MM 18-6) (M-36) ATCC 13685__________________________________________________________________________ (ME/83)Oat meal Abundant growth with Abundant growth with Abundant with smooth sur- Fair growth with smoothagar slightly wrinkled sur- smooth surface, orange. face, deep amber. surface, yellowish with face, hazel-brown with Traces of pinkish aerial Traces of pink aerial pinkish reverse. Whitish orange edges. Pink-beige mycelium. Some spores. mycelium. Burnt amber aerial mycelium with pink soluble pigment. Pink-beige soluble pig- soluble pigment. tinge. Traces of yellow- ment. ish soluble pigment.Yeast Abundant growth with Abundant growth with Abundant growth with Abundant growth withextract glu- very wrinkled surface, very wrinkled surface, wrinkled surface. Vege- rough surface yellowishcose agar amber. Greyish aerial brick red. Deep rusty tative mycelium, burnt to pink. Scanty aerial(Medium mycelium, deep rusty soluble pigment. amber. Burnt amber so- mycelium.No. 2 soluble pigment. luble pigment.ShirlingandGottlieb)Emerson's Abundant growth with Abundant growth with Abundant growth with Abundant growth withglucose very wrinkled and cru- very wrinkled and cru- wrinkled surface, burnt rough surface, yellowishagar sty surface, brick red sty surface, brick red, amber. Burnt amber solu- to pink orange. Pinkish Amber soluble pigment. Amber soluble pigment. ble pigment. aerial mycelium. Pale amber soluble pigment.Bennett's Abundant growth, very Abundant growth with Abundant growth with Good growth, yellowishagar wrinkled, reddish brown very wrinkled surface, wrinkled surface, orange turning orange-yellow. Traces of greyish aerial deep orange. Deep yellow brown. Amber soluble Pinkish aerial mycelium mycelium. Ochre yellow soluble pigment. pigment. Light amber soluble pig- soluble pigment. ment.Penassay Moderate growth, slight- Moderate growth, slight- Moderate growth with Poor growthagar ly wrinkled light ly wrinkled light oran- smooth and thin surface, orange. No soluble pig- ge. No soluble pigment. light orange. No soluble ment. pigment.Yeast Abundant growth Abundant growth with Abundant growth with cru- Abundant growth with roughextract very wrinkled, brick red very wrinkled surface, sty surface, burnt amber surface, colorless tomolasse Deep rusty soluble pig- brick red. Deep rusty Burnt amber soluble pig- yellowish. Whitish aerialagar ment. soluble pigment. ment. mycelium. Deep amber solu- ble pigment.Czapek- Moderate growth with Good growth with smooth Abundant growth with Poor growth with thinDox smooth and thin surface surface, orange. Light smooth surface, salmon. surface, colorless tosaccharose light orange. Pale pink orange aerial mycelium Abundant aerial mycelium light melon. Traces ofagar aerial mycelium. Some pink orange. Traces of pinkish white aerial spores. Deep lemon yellow light yellow soluble mycelium soluble pigment. pigment.Potato agar Scarce growth,light oran- No growth Moderate growth with Poor growth with thin ge. Light hazel brown smooth surface. Light surface, colorless. soluble pigment. orange. No soluble pig- Traces of whitish aerial ment. mycelium. No soluble pigment.Glucose Abundant growth with Scarce growth with smooth Fair growth, gold. Fair growth with smoothasparagine smooth surface orange. surface, light orange. No soluble pigment. and thin surface, lightagar Light yellow soluble No soluble pigment. orange pink. Some light pigment. yellow soluble pigment.Glycerolasparagine Abundant growth with Scarce growth, deep Moderate growth with Fair growth with smoothagar smooth surface deep orange. slightly wrinkled sur- and thin surface. Light(medium orange. Traces of No soluble pigment. face, deep orange. Traces orange pink. Some lightn.degree. 5 Shir- yellowish soluble pig- of yellow soluble pig- yellow soluble pigment.ling and ment. ment.Gottlieb)Nutrient Moderate growth with Moderate growth with Fair growth with thin Moderate growth with smoothagar thin and smooth sur- wrinkled and thin sur- and smooth surface, surface, melon to orange. face, pale orange. face, pale orange. orange. No soluble Pinkish white aerial my- No soluble pigment. No soluble pigment. pigment. celium.Pridham's Moderate growth with Abundant growth with Abundant growth, slight- Moderate growth withagar smooth and thin surface, smooth surface, brick ly crusty, amber brown. smooth surface, colorless amber. Light pinkish- red. Pink aerial myce- Traces of pinkish aerial with lobster red spots. orange aerial mycelium. lium. Yellow soluble mycelium. Pink aerial mycelium. Pinkish soluble pigment. pigment. Amber brown soluble pig- ment.Starch Moderate growth with Moderate growth with Abundant growth with Poor growth, colorless toagar smooth surface orange. smooth surface, deep smooth surface, orange. light orange pink.(medium Abundant aerial myce- orange. Traces of pinkish aerial Scarce white aerial myce-n.degree. 4 Shir- lium, pink. Abundant Traces of straw soluble mycelium. Light yellow lium. Starch hydrolysis:ling and production of spores. pigment. Starch hydro- soluble pigment. Starch doubtful.Gottlieb) Yellow soluble pigment. lysis: negative. hydrolysis: negative. Starch hydrolysis: nega- tive.Dextrose Abundant growth with Moderate growth, wrin- Abundant growth with Abundant growth pinktrip- wrinkled surface melon. kled, orange. Orange wrinkled surface, rose- orange. Pinkish aerialtone agar Orange melon soluble yellow soluble pigment. melon. Rose melon solu- mycelium. Light golden pigment. ble pigment. yellow soluble pigment.Hickey's Moderate growth with Abundant growth with Moderate growth with Moderate growth, lightand smooth and thin surface, smooth surface, rose slightly wrinkled sur- pinkish orange. SomeTresner's colorless. Whitish aerial melon. Light beige-rose face, rose-melon. Light pinkish aerial mycelium.cobalt agar mycelium. Beige soluble soluble pigment. beige soluble pigment. Yellowish soluble pigment. pigment.Tyrosine Abundant growth, very Abundant growth, very Abundant growth with Poor growthagar wrinkled and crusty wrinkled and crusty. crusty surface, deep(Medium brick red. Deep yellow Deep orange. Pinkish- burnt orange. Yellown.degree. 7 Shir- soluble pigment. Tyrosi- tan soluble pigment. ochre soluble pigment.ling and ne reaction: positive Tyrosine reaction: Tyrosine reaction:Gottlieb) (good). strongly positive. strongly positive.Ca-malate Scarce growth with Scarce growth, light Poor growth, light Fair growth colorlessagar smooth and thin surface, orange. Hydrolysis of orange. No soluble pig- Whitish pink aerial light orange yellow. Ca-malate: negative. ment. Ca-malate dige- mycelium. Partial dige- Chrome lemon soluble stion: negative. stion of Ca-malate. pigment near the growth and coral pink in the medium. Hydrolysis: po- sitive.Egg Abundant growth with Abundant growth with Good growth with smooth Fair growth, pink.albumin smooth surface, light smooth surface, orange. surface orange-yellow.agar orange. Light yellow No soluble pigment. Light yellow soluble pig- soluble pigment. ment.Peptone Abundant growth, slight- Abundant growth, wrin- Abundant growth withglucose ly wrinkled, deep oran- kled, deep orange. wrinkled surface, burntagar ge. Pinkish aerial myce- Gold yellow soluble amber. Burnt amber so- lium. Yellow soluble pig- pigment. luble pigment. ment.Gelatine hydrolysis: positive hydrolysis: positive hydrolysis: positive hydrolysis: positiveNitrate reduction: negative reduction: negative reduction: negative reduction: negativebrothLitmus no coagulation no coagulation no coagulation no coagulationmilk no peptonization no peptonization no peptonization no peptonizationPeptone Moderate growth with Moderate growth with Poor growth withyeast smooth surface, pale smooth surface, pale smooth surface, color-extract orange. H.sub.2 S production: orange. H.sub.2 S production: less. No soluble pig-iron agar negative negative. ment. H.sub.2 S production:(Medium negative.n.degree. 6 Shir-ling andGottlieb)Potato plug Scarce growth, orange. Very scarce growth, Poor growth, light or- Poor growth, colorless. orange. ange.Loeffler No growth No growth No growthbloodserumH.sub.2 O + Scarce growth colorless. Scarce growth, colorless. Scarce growth, colorlessagar Traces of white aerial Traces of white aerial Traces of white aerialDifco 2% mycelium. Abundant pro- mycelium. Abundant pro- mycelium. Abundant pro- duction of spores. duction of spores. duction of spores.Skim milk Abundant growth with Good growth, slightly Abundant growth withagar smooth surface, orange. wrinkled, orange with wrinkled surface, oran- Cadmium yellow soluble brown shadow. ge. Gold brown soluble pigment. Hydrolysis of Hazel brown soluble pigment. Casein hydroly- casein: good. pigment. Hydrolysis of sis: strongly positive. casein: good.__________________________________________________________________________ Shirling and Gottlieb: Methods for characterization of Streptomyces species, Intern. J. Syst.Bact., 16,313-338 (1966).

The following table 3 reports the physiological characteristics of the new Nocardia Strains compared with those of the parent strain.

TABLE 3__________________________________________________________________________ N.mediterranea N.mediterranea N.mediterranea Streptomyces mediterranei (D-2) (MM 18-6) (M-36) (ME/83)__________________________________________________________________________Strach hydro- - - - .+-.lysisTyrosin hydro- ++ +++ +++ -lysisCasein hydro- ++ ++ +++lysisCa-malate ++ - - +hydrolysisNitrate re- - - - -ductionLitmus milk no coagulation no coagulation no coagulation no coagulation no peptonization no peptonization no peptonization no peptonizationGelatin ++ ++ ++ +liquefaction__________________________________________________________________________

Following Table 4 reports the utilization of carbon sources examined according to the method of Pridham and Gottlieb (J.Bact. 56, 107, 1948).

TABLE 4__________________________________________________________________________ N.mediterranea N.mediterranea N.mediterranea Streptomyces mediterranei (D-2) (MM 18-6) (M-36) (ME/83)__________________________________________________________________________Arabinose + +++ ++ +++Xylose + + ++ ++Glucose ++ +++ ++ ++Mannose + ++ ++ ++Fructose ++ +++ ++ ++Lactose + +++ ++ ++Sucrose + .+-. ++ ++Inositol + + + + ++Rhamnose + + ++ ++Raffinose - - - -Salicin .+-. + + +Mannitol ++ +++ ++ ++Glicine + .+-. + -Sorbitol - - - -Ma-succinate - - - +Na-citrate - - - -Na-acetate - - - -Inulin .+-. + - -Dulcitol - - - -Cellulose - - - -Maltose ++ +++ ++ +Galactose + ++ ++ +++Glicerol ++ +++ ++ +++__________________________________________________________________________

FERMENTATION

The procedure for the fermentation essentially consists in cultivating one of said mutants of Streptomyces mediterranei in a nutrient medium containing assimilable carbon and nitrogen sources and essential mineral salts, until a substantial antibiotic activity is imparted to said medium and in extracting the rifamycins from the medium. More particularly these mutants are cultivated under stirred and aerated submerged conditions at a temperature ranging from 25.degree. to 37.degree. C., and preferably at 28.degree. C. As the sources of carbon the following carbohydrates and carbon derivatives can be used: glucose, galactose, lactose, sucrose, maltose, glycerol, mannitol, etc. Useful nitrogen sources are for instance amino-acids and their mixtures, peptides, proteins and their hydrolisates, as peptone, yeast extract, soybean meal, corn steep liquor, fish soluble, meet extracts, aqueous fractions from cereal seeds. The fermentation can be carried out for 180-220 hours. The starting pH, generally adjusted at about 6.4-6.6 increases at the end of the fermentation to 7.0-8.5. Generally the best results are observed at the 200 hours of the fermentation. At the end of the fermentation the rifamycins can be isolated by the following procedure. The fermentation medium is filtered at the final pH of 7.0-8.5. The filtrate is quickly acidified, preferably to a pH lower than about 5, to ensure the best stability to the antibiotic substance. The activity is extracted with water immiscible solvents such as chloroform, butanol, ethyl, propyl, butyl or amyl acetate. The ratio between the volume of the medium and that of the solvent changes dependently on the choosen solvent: generally a ratio ranging from 2:1 to 10:1 is used.

The mycelium still retains a microbiological activity, which is extracted from the mycelium by means of a water immiscible solvent, and then combined with the organic phase, already containing most of the rifamycins. Alternatively, the extraction of the activity from the mycelium can be effected by means of a solvent miscible with water such as acetone. In this case, the liquid is filtered, the acetone is evaporated in vacuo, the rifamycins are extracted with a water immiscible solvent and the procedure is carried on as said above.

Once most of the antibiotic activity has been transferred into the solvent, this is distilled in vacuo to dryness, preferably at a temperature lower than 30.degree. C.

PURIFICATION

The crude extract of rifamycins can be purified chromatographically on a column of silica-gel. Prior to chromatography it is convenient to dissolve the crude extract in a phosphate buffer pH 7.0-8.0 and to treat with a mild oxidizing agent. The buffer solution is then extracted with a water-immiscible solvent, this organic extract contains a new rifamycin denominated rifamycin P. The extracted buffer solution is acidified to pH 2-4 and then again extracted with a water immiscible solvent. This organic extract contains three new compounds of the rifamycin family denominated rifamycin P, Q and U.

The further purification of rifamycin R is effected by chromatography on a column of suitable adsorbing material such as silica-gel and eluting with an appropriate mixture of organic solvents.

The second organic extract containing rifamycins P, Q and U is purified in a similar way to that described for rifamycin R.

The following examples are given for the purpose of better illutrating the subject of the invention.

EXAMPLE 1

The mutant strains identified as Nocardia mediterranea ATCC 31064 is propagated for 6-8 days on Bennett's agar and incubated at 28.degree. C.

With the culture obtained from the agar slant, two 500 ml. Erlenmeyer flasks are inoculated under sterile conditions. The flasks contain 100 ml. of the vegetative medium of the following composition:

______________________________________Beef extract 5 g.Yeast extract 5 g.Peptone 5 g.Casein hydrolyzate 3 g.Glucose 20 g.NaCl 1.5 g.H.sub.2 O to 1 liter.______________________________________ The pH is adjusted to 7.3 with NaOH.

The flasks so inoculated are placed on an alternative shaker at 28.degree. C. for 72 hours. The content of the two Erlenmeyer flasks is used as inoculum by pouring it in a 10 liters prefermenter, containing 4 liters of the above mentioned vegetative medium. The incubation is carried out at 28.degree. C. with an agitation of 300 r.p.m. and 1 v/v/m aeration. After 48 hours of growth a volume of 7-10% of packed cells is obtained. In the next stage a 10 liter glass fermenter containing 4 liters of the hereinafter mentioned fermentation medium is used:

______________________________________Peanut flour 25 g.Soybean flour 5 g.(NH.sub.4).sub.2 SO.sub.4 9.5 g.MgSO.sub.4 . 7H.sub.2 O 0.85 g.Glucose 95 g.Glycerol 40 g.KH.sub.2 PO.sub.4 1 g.Propylene glycol 5 g.CaCO.sub.3 8.5 g.Na diethylbarbiturate 1.7 g.CuSO.sub.4 . 5H.sub.2 O 2.8 mg.FeSO.sub.4 . 7H.sub.2 O 8.5 mg.ZnSO.sub.4 . 7H.sub.2 O 42.5 mg.MnSO.sub.4 . 4H.sub.2 O 3.4 mg.CaCl.sub.2 . 6H.sub.2 O 1.7 mg.(NH.sub.4).sub.6 Mo.sub.7 O.sub.24 . 4H.sub.2 O 0.85 mg.H.sub.2 O to 1 liter______________________________________ The pH is adjusted to 7.8 with NaOH. Sterilization for 60 minutes at 120.degree. C. After sterilization the pH is 6.4. An amount of the prefermenter content equal to 5% of the fermenter content is used as inoculum.

The fermentation is carried out at 28.degree. C. with a 750 r.p.m. agitation and aerating at a rate of 1 v/v/m. Silicone A is used as antifoam. The culture broth turns to a characteristic red-brown color during the fermentation. After 200 hours of growth a volume of packed cells is obtained. The pH of the broth is 7.5 and at this moment the broth is harvested.

EXAMPLE 2

A culture of Nocardia mediterranea ATCC 31064 obtained as disclosed in example 1, is prepared in a flask, under stirring, as described in example 1. For the preculture it is poured into a 10 liters glass fermenter, containing 4 liters of the following medium:

______________________________________Glucose 5 g.Peanut flour 7.5 g.CaCO.sub.3 1.65 g.MgSO.sub.4 . 7H.sub.2 O 0.33 g.KH.sub.2 PO.sub.4 0.33 g.FeSO.sub.4 . 7H.sub.2 O 3.3 mg.ZnSO.sub.4 . 7H.sub.2 O 16.5 mg.MnSO.sub.4 . 4H.sub.2 O 1.3 mg.H.sub.2 O to 1 liter______________________________________ The pH is adjusted to 7.5. Sterilization 50 minutes at 120.degree. C. After sterilization the pH value is 6.4. After 48 hours of growth the volume of packed cells is 6-8% of total volume. An inoculum equal to 10% is used for a 20 liters glass fermenter, containing 10 liters of the following fermentation medium: ______________________________________Corn steep liquor 20 g.Soybean flour 15 g.(NH.sub.4).sub.2 SO.sub.4 6 g.MgSO.sub.4 . 7H.sub.2 O 0.85 g.Glucose 100 g.KH.sub.2 PO.sub.4 1 g.CaCO.sub.3 6 g.FeSO.sub.4 . 7H.sub.2 O 8.5 mg.ZnSO.sub.4 . 7H.sub.2 O 42.5 mg.MnSO.sub.4 . 4H.sub.2 O 3.4 mg.CuSO.sub.4 . 5H.sub.2 O 2.8 mg.CoCl.sub.2 . 6H.sub.2 O 1.7 mg.H.sub.2 O to 1 liter______________________________________ The pH is adjusted to 7.8 with NaOH. Sterilization for 50 minutes at 120.degree. C. After sterilization the pH is 6.4. The fermentation is carried out at 28.degree. C. for 200 hours. The pH of the fermentation broth at the harvest is 7.5.

The fermentation broths obtained in examples 1 and 2 are purified in the following way. The mycelium is removed by filtration and discarded, the filtrate is adjusted to pH 2.0 with 10% (v/v) hydrochloric acid and extracted three times with an equal volume of ethyl acetate. This organic extract is concentrated to dryness under vacuum at 35.degree. C. and the residue (4 g. from example 1 and 11 g. from example 2) is dissolved in 0.05 M sodium phosphate buffer pH 7.5 and sodium nitrite added to give a final concentration of 0.2% (w/v). After stirring for 30 min. at room temperature the buffer solution is extracted three times with an equal volume of ethyl acetate. The combined organic extracts are concentrated to dryness under vacuum at 35.degree. C. (1.sup.st ethyl acetate extract). The exhausted buffer solution is adjusted to pH 2.0 with 10% hydrochloric acid and extracted with an equal volume of ethyl acetate three times. The combined organic extracts are concentrated to dryness under vacuum at 35.degree. C. (2.sup.nd ethyl acetate extract).

The dry powder from the 1.sup.st ethyl acetate extract (1.4 g example 1 and 4.2 g. example 2) is dissolved in chloroform and chromatographed on a column of silica-gel (70-230 Mesh ASTM) using chloroform containing 2% (v/v) methyl alcohol as an eluent. Rifamycin R is the first major product to elute from the column recognizable by its orange-brown colour.

On thin layer chromatography using silica-gel plates (merck 60 F.sub.254) with chloroform: methyl alcohol as solvent system (95:5) rifamycin R has an Rf of 0.59. The fractions containing rifamycin R are combined, taken to dryness under vacuum at 35.degree. C., redissolved in ethyl acetate which is then washed with 0.01 N hydrochloric acid and finally with water. The organic extract is concentrated to a small volume and rifamycin R crystallizes from solution at 4.degree. C. (obtained 600 mg. from example 1 and 1.6 g. from example 2). The second ethyl acetate extract is purified by column chromatography on silica-gel in a similar way to rifamycin R. The dry residue (2.4 g. from example 1 and 4.2 g. from example 2) is dissolved in chloroform and applied to the silica gel.

The column is eluted with a mixture of chloroform and methyl alcohol (98:2). The first compound to emerge is rifamycin U, then rifamycin P and finally rifamycin Q. All three of these compounds have a yellow orange colour in the eluent solution and can be identified on the bases of their mobility on thin layer chromatography. On silica gel plates (Merck 60 F.sub.254) using chloroform: methanol (95:5) as solvent system, the Rf values are the following:

______________________________________ rifamycin U Rf = 0.63 rifamycin P Rf = 0.57 rifamycin Q Rf = 0.32______________________________________

The appropriate fractions containing rifamycins U, P and Q are combined taken to dryness under vacuum and crystallized from ethyl acetate. From the first example are obtained rifamycin U 80 mg., rifamycin P 400 mg., rifamycin Q 280 mg: from the second example rifamycin U 190 mg., rifamycin P 900 mg., rifamycin Q 750 mg.

By following essentially the same procedure of the above examples but using respectively Nocardia mediterranea ATCC 31065 or Nocardia mediterranea ATCC 31066 as the producing strains, yields of the same order as above are obtained.

PHYSICO-CHEMICAL CHARACTERISTICS OF THE NOVEL RIFAMYCINS

Rifamycin P

(1) Elemental analysis (%): Found, C=60.6; H=6.2; N=3.8; O=25.2; S=4.1.

(2) U.V. and visible absorption bands:

The compound shows the following values:

______________________________________methanol 0.1 N HCl______________________________________.lambda..sub.max(m.mu.) E.sub.1.sup.1% cm .lambda..sub.max (m.mu.) E.sub.1.sup.1% cm406 197 416 183350 (shoulder) 300 319297 344 225 521257 423224 550______________________________________

The complete figure of the spectrum is given in FIG. 1. The spectrum was recorded with Perkin Elmer Spectracord 4000 A instrument.

(3) Infrared Spectrum: The most significant absorption peaks in Nujol occur at the following frequencies (cm.sup.-1): 3700-3150 (m,br); 3100 (w); 3060-2800 (vs); 1465 (s); 1380 (b): Nujol; 1722 (m); 1645 (m,br); 1580 (m); 1510 (m); 1325 (m); 1250 (s br); 1160 (m); 1130 (w); 1070 (m, br) 1030 (w); 982 (m); 960 (m); 925 (w); 890 (m); 818 (w); 770 (w); 735 (w).

A complete figure of the I.R. spectrum is given in FIG. 2. The spectrum was recorded with a Perkin Elmer Mod. 421 instrument.

(4) Mass spectrum: The mass spectrum obtained at 70 eV shows the molecular ion peak M.sup.+ at the following m/e value: 738. The spectrum was recorded with an Hitachi Perkin Elmer RMU-6L instrument.

(5) Nuclear Magentic Resonance spectrum: The complete figure of N.M.R. spectrum at 100 MH.sub.z in CDCl.sub.3 is given in FIG. 3. This compound does not show the characteristic polarographic behavior of the rifamycins possessing a chromophoric moiety with quinonic structure.

Rifamycin O

(1) Elemental analysis: Found, C=60.7; H=6.3; N=3.60; O=25.3; S=4.2

(2) U.V. and visible absorption bands:

The compound shows the following values in methanol:

______________________________________.lambda..sub.max (m.mu.) E.sub.1.sup.1% cm406 178350 (shoulder)297 305257 405224 518______________________________________

(3) Infrared Spectrum: The most significant absorption peaks in Nujol occur at the following frequencies (cm.sup.-1): 3700-3300 (s,br); 3300-3080 (m br); 3040-2780 (vs); 1460 (s); 1378 (s): Nujol; 1740 (m); 1700 (m); 1650 (s, br); 1605 (s, br); 1555 (s); 1510 (m, br); 1315 (w); 1275 (m); 1240 (m); 1220 (m); 1160 (m); 1090 (m); 1050 (m,br); 1020 (w); 970 (m); 945 (w); 910 (m); 808 (m); 765 (w); 720 (w).

A complete figure of the I.R. spectrum is given in FIG. 4.

(4) Nuclear Magnetic Resonance Spectrum: The complete figure of the N.M.R. spectrum at 60 MH.sub.z in CDCl.sub.3 is given in FIG. 7.

Rifamycin R

(1) Elemental analysis: Found: C=62.0; H=6.4; N=2.2; O=29.6.

(2) U.V. and visible absorption bands:

The compound shows the following values in which 0.1 N HCl in methanol:

______________________________________ .lambda..sub.max (m.mu.) E.sub.1.sup.1% cm 219 444 281 415 340 114 410 72______________________________________

(3) Infrared Spectrum: The most significant absorption peaks in Nujol occur at the following frequencies (cm.sup.-1): 3700-3100 (s br); 3040-2780 (vs); 1465 (s); 1380 (s): Nujol; 1745 (s); 1710 (m); 1640 (s); 1600 (s); 1505 (s); 1415 (m); 1325 (s); 1260 (s, br); 1220-1130 (s, br); 1120 (w); 1075 (s); 1020 (w); 975 (s); 950 (w); 920 (w); 888 (m); 823 (m); 785 (w, br); 735 (w, br); 650 (w, br).

A complete figure of the I.R. spectrum is given in FIG. 5.

(4) Mass spectrum: The mass spectrum obtained at 70 eV shows the molecular ion peak M.sup.+ at the following m/e value: 711.

(5) Nuclear Magnetic Resonance spectrum: The complete figure of the N.M.R. spectrum at 60 MH.sub.z in CDCl.sub.3 is given in FIG. 6.

The compound shows the characteristic polorographic behavior of the rifamycins possessing a chromophoric moiety with quinonic structure.

The above data support the following structure for rifamycin R. ##STR1##

Rifamycin U

(1) U.V. and visible absorption bands:

The compound shows the following values in methanol:

______________________________________.lambda..sub.max (m.mu.) E.sub.1.sup.1% cm412 163353 (shoulder)299 265258 389220 451______________________________________

(2) Infrared spectrum: The most significant absorption peaks in Nujol occur the following frequencies (cm.sup.-1): 3700-3100 (m,br); 3060 (vw); 3040-2740 (vs), 2720 (vw), 1460 (s), 1378 (m): Nujol; 1750-1705 (m,br); 1680-1620 (m,br); 1600 (s); 1558 (s); 1490 (w, shoulder); 1308 (w); 1270 (w, shoulder); 1245 (m, shoulder); 1225 (m); 1160 (m); 1120 (w); 1090 (vw); 1070 (w); 1030 (vw); 1020 (vw); 1000 (vw); 975 (w); 960 (w, shoulder); 905 (m); 850 (m); 802 (w); 800 (vw, shoulder); 777 (vw); 760 (vw); 720 (vw); 685 (vw); 670 (vw).

BIOLOGICAL ACTIVITY OF THE COMPOUNDS

The in vitro activity spectrum of rifamycins P, Q and R is reported in the following table:

__________________________________________________________________________ Minimal inhibition concentration (.mu.g/ml)Strains Rifamycin P Rifamycin Q Rifamycin R Rifamycin M__________________________________________________________________________Staphylococcus aureus ATCC 6538 0.00235 0.039 0.019 0.05-0.1Staphylococcus aureus ATCC 6538 0.00235 0.078 0.0047 --(20% bovine serum)Staphylococcus aureus Tour 0.0035 0.06 0.00235 --Staphylococcus aureus Tourrifampicin-resistant 25-50 >100 100 --Streptococcus hemolyticus C 203 0.0047 0.019 0.00312 --Streptococcus faecalis ATCC 10541 0.0035 0.312 0.19 --Diplococcus pneumoniae UC 41 0.0047 0.039 0.00625 --Proteus vulgaris X 19 N ATCC 881 0.78 12.5 12.5 --Escherichia coli ATCC 10536 3.12 12.5 50 >50Klebsiella pneumoniaeATCC 10031 12.5 50 -- --Pseudomonas aeruginosaATCC 10145 12.5 50 100 --Mycobacterium tub. H37Rv ATCC 9360 1 2.5 0.1 2.5__________________________________________________________________________ --: Activity not determined? The novel compounds are active also in the laboratory animals injected with Staphylococcus aureus, Escherichia coli. The following table reports the results of representative experiments on mice in comparison with a known natural rifamycin, i.e. rifamycin SV. ______________________________________ ED.sub.50 mg/kg.Compound Infecting Strain s.c. o.s.______________________________________rifamycin P Staphylococcus aureus 0.3 0.8rifamycin P Escherichia coli 40 --rifamycin Q Staphylococcus aureus 8 --rifamycin SV Staphylococcus aureus 17 74______________________________________ Rifamycin P which results to be the most active natural rifamycin so far isolated has also low toxicity since its LD.sub.50 value in mice is higher than 500 mg/kg. i.p.

Claims

1. A biologically pure culture of the microorgansim Nocardia mediterranea, having the identifying characteristics of ATCC 31064, said culture being capable of producing an antibiotic selected from the group consisting of rifamycin P, Q, R, and U in a recoverable quantity upon aerobic fermentation in an aqueous nutrient medium containing assimilable sources of carbon, nitrogen, and inorganic minerals.

2. A biologically pure culture of the microorganism Nocardia mediterranea, having the identifying characteristics of ATCC 31065, said culture being capable of producing an antibiotic selected from the group consisting of rifamycin P, Q, R, and U in a recoverable quantity upon aerobic fermentation in an aqueous nutrient medium containing assimilable sources of carbon, nitrogen, and inorganic minerals.

3. A biologically pure culture of the microorganism Nocardia mediterranea, having the identifying characteristics of ATCC 31066, said culture being capable of producing an antibiotic selected from the group consisting of rifamycin P, Q, R, and U in a recoverable quantity upon aerobic fermentation in an aqueous nutrient medium containing assimilable sources of carbon, nitrogen, and inorganic minerals.

4. A rifamycin compound defined as rifamycin R having the following formula: ##STR2##