IL43295A - Antibiotic bm123 and production thereof - Google Patents

Abstract

1439668 Antibiotic BM123 AMERICAN CYANAMID CO 10 Sept 1973 [12 Oct 1972] 42535/73 Heading C2A Antibiotic BM123 is prepared by cultivating a microorganism having the characteristics of Nocardia sp. NRRL 5646 or mutants thereof in an aqueous nutrient medium containing assimilable sources of carbon, nitrogen and inorganic salts under submerged aerobic conditions until substantial antibacterial activity is imparted to the medium, and then recovering BM123 therefrom. The antibiotic exists in three forms, designated BM123α, BM123# and BM123#. Antibiotic BM123α is characterized by (a) having an optical rotation (C= 1.00 in water); (b) having the following elemental analysis (per cent); C, 33.89; H, 5.71; N, 11.88; O, 35.40; and (c) the infra-red absorption spectrum indicated in Fig. 1 (not shown). Antibiotic BM123# is characterized by (a) having an optical rotation (C=1.25 in water); (b) having the following elemental analysis: C, 47.53; H, 7.39; N, 16.54; ash, 1.04; and (c) the infra-red absorption spectrum indicated in Fig. 2 (not shown). Antibiotic BM123 # is characterized by (a) having an optical rotation (C= 1.004 in water); (b) having the following elemental analysis: C, 46.13; H, 6.65; N, 17.00; O, 24.96; ash, 0.90; and (c) the infra-red absorption spectrum indicated in Fig. 3 (not shown). These antibiotics may be incorporated with suitable carriers into various pharmaceutical forms. [GB1439668A]

Description

ANTIBIOTIC BM123 AND PRODUCTION THEREOF "Πϊ»·1 BM123 *B1*a»03« ΤδΙΠ This Invention relates to three new antibacterial agents, to their production by fermentation, to methods for their recovery and concentration from crude solutions, and to processes for their purification. The • present invention includes within its scope the bacter- ial agents in dilute forms, as crude concentrates, and in pure crystalline forms. The effects of the new antibacterial agents on specific microorganisms, together with their chemical and physical properties, differentiate them from previously described antibacterial agents The new antibacterial agents which we have designated BM123 The following is a general description of the microorganism Nocardia sp . , NRRL 5646, based on diagnostic characteristics observed. Observations were made of the cultural, physiological, and morphological features of the organism in accordance with the methods detailed by Shirling and Gottlieb, Internat . Journ. of Syst., Bacteriol. 16:313-240 (1966). The chemical compositions of the culture was determined by the procedures given by Lechevalier ejt al . , Advan. Appl. Microbiol. 14:47-72 (1971). The underscored descriptive colors and color chip designations are taken from Jacobson e_t al . , Color Harmony Manual, 3rd edition (1948), Container Corpor. of America, Chicago, Illinois. Descriptive details are recorded in Tables I through V below.

Amount of Growth Moderate on yeast extract, asparagine dextrose, Benedict's Bennett's, potato dextrose and Weinstein's agars; light on Hickey and Tresner's, tomato paste oatmeal and pablum agars; and only a trace of growth on inorganic salts-starch, Kuster's oatflake, Czapek's solution and rice agars.

Aerial Mycelium Aerial Mycelium whitish when present; produced only on yeast extract, asparagine dextrose, Benedict's, Bennett's and potato dextrose agars.

Soluble Pigments No soluble pigments produced.

Reverse Color Colorless to yellowish shades.

Miscellaneous Physiological Reactions No liquefaction of gelatin; nitrates reduced to nitrites in 7 days; melanoid pigments not formed on peptone-iron agar; no peponization or curd formation in purple milk; NaCl tolerance in yeast extract agar but < %; optimal growth temperature 32°C.

Carbon source utilization, according to the Pridham and Gottlieb method [J. Bacteriol. 56:107-114 (19^8)] as fol lows: Good utilization of glycerol, salicin, d-trehalose and dextrose; fair utilization of i-ino- sitol; and poor "to non-utilization of d-fructose, mal tose, adonitol, 1-arabinose, lactose, d-mannitol, d- -mellbiose, d-raffinose, l-rhamnose, sucrose and d- -xylose .

Chemical Compositions The organism belongs to cell wall type IV, i.e., contains meso-2, 6-dlamlnopimelic acid and has a type A whole-cell sugar pattern, i.e., contains arabinose and galactose. Methylated whole cell extracts, when subjected to gas chromatography, showed fatty acid patterns similar to those produced by Nocardia aster- oides, ATCC 3308. ^ Mlcromorphology Aerial mycelium arises from substrate mycelium as sparingly branched moderately long flexuous elements that commonly terminate in elongated primitive spirals. The flexous elements are irregularly segmented into short elliptical to cylindrical sections (spores?) which disarticulate readily. The spiral terminal portions are less conspicuously segmented. Segments generally range 0.8-1.7 m x 0.3-0.5 Jim, averaging Diagnosis The morphological characteristics of Culture No. BM123 are difficult to observe and interpret because of the poor development of aerial mycelium on most media.

Hence, considerable importance is attached, out of necessity, to the chemical analysis in determining the generic relationship of the organism. On the basis of the system proposed by Lechevalier et al . , Culture BM123 contains meso-2 , 6-diaminopimelic acid in its whole cells and sugar analysis shows arabinose and galactose to be present. Therefore, the culture belongs to cell wall type IV. A comparison of the gas chromatography pattern of culture BM123 with that of Nocardla asteroides ATCC 3308 showed the two to be remarkably similar. Other characteristics of culture BM123 that are in keeping with the Nocardia concept, are its fragmenting aerial growth on some media and the total absence of aerial growth on most media. In view of the lack of adequate criteria for the charac- terization of Nocardias to the species level, no '^j attempt has been made to make this determination. Therefore, culture BM123 will be considered an undetermined species of Nocardia until such a diagnosis is feasible.

Cultural Characteristics of Nocardia sp. NRRL Incubation: 1 days Temperature: 32 °C .MEDIUM AMOUNT OF GROWTH AE R IAL MYCE LIUM AND/OR SPORE S SOLUBLE PIGM Aerial mycelium whitish, Yeast Extract Moderate light . None Agar Kickey and Tresner's Light No aerial mycelium None Agar Aspar gine dextrose Moderate Trace of whitish aerial None .Agar mycelium.

Benedict's Agar Moderate Aerial mycelium whitish, None light.

Bennett's Agar Moderate Trace of whitish aerial None mycelium. ' Inorganic Salts- No aerial mycelium None. starch Agar Trace :-Custer *s. Oatflake No aerial mycelium Trace N Agar Czapek's Solution Irace No aerial mycelium N Agar Aerial iriycelium vhitish, No Potato dextrose Moderate light.

Agar " ' Tomato Paste Light No aerial mycelium No Catneal Agar Pahlurn A.gar Light No aerial mycelium No Rice Agar Trace No aerial mycelium No tfeinstein's A.gar Moderate No aerial mycelium No Kuster's. Oatflake Trace No aerial iyceliiua Agar N TABLE II Micromorphology of Nocardia sp NRRL Aerial Mycelium and/or Sporiferous Medium Structures Yeast Extract Aerial mycelium arises from substrate, Agar mycelium as sparingly branched, flexuous elements that commonly terminate in elongated primitive spirals.

The flexuous elements are Irregularly segmented into short sections (spores?) which disarticulate readily. The spiral terminal portions are less conspicuously segmented. Segments generally range 0.8-1.7 ja x Ο.5-Ο.5 jm, averaging 0Λ jam x 1.2 jam.

TABLE III Miscellaneous Physiological Reaction of Nocardia sp.

MEDIUM INCUBATION P ERIOD AMOUNT OF GROWTH PH Gelatin 7 days Light No lique Gelatin Ik days Good No lique Organic . itrate 7 days Good Nitrates Broth Organic Nitrate lk days Good Nitrates Broth Peptone-iron 2 - 8 hours Good No melan Agar Purple Milk 7 days Good No pepto Yeast extract 7 days Moderate NaCl tol Anar plus (¾, 7, 10 iuvl 13-') I!¾C1 • TABLE IV Carbon Source Utilization Pattern of Nocarrtia sp. NRRL L) G O 3-Good Utilization 1-Poor Utilization 2-Fair Utilization 0-No Utilization Table V "γ~ Chemical Compositions of Nocardia sp. NRRL 5646 Cell Wall Type Major Constituents Type IV meso-DAP, arabinose, galactose It is to be understood that for the production of these new antibacterlals the present Invention is not limited to this particular organism or to organisms fully answering the above growth and microscopic character-istics which are given for illustrative purposes. In fact, it is desired and intended to include the use of mutants produced from this organism by various means such as exposure to X-radlation, ultraviolet radiation, nitrogen mustard, actinophages , and the like.

Preliminary isolation, thin layer chromatography, and paper chromatography experiments have shown that at least three antibiotics are produced during the aerobic fermentation of Nocardia sp. NRRL 5646 as heretofore designatred. Nutrient media studies resulted in two types of mashes, a gamma type which produced primarily BM123' along with lesser amounts of BM123 Fermentation Process Selected to Produce Prlmari/Ly BM123a /cultivation ίΐ Nocardia sp/. NRRL 6 6 /may be carried out in a wide/ ariety of liquid culture/ media .

Media wlich are useful for the production of t¾(is novel antibacterial inclufie an assimilable source of| carbon . ) an as-similable source/of nitrogen suclfi as protein, protein hy- 43295/2 I The specification (page 12, lines 15-23) clearly specify that three types of BM123 are produced simultaenously during the aerobic fermentation of Nocardia sp, NRRL 5646 regardless of the nutrient mash employed. However, by selecting a particular type of nutrient medium more or less of one of the types of BM123 is produced and which can be separated by methods well known by one skilled in the art. It should be emphasized/ however, that all three types of BM123 are produced simultaneously}' regardless of the nutrient medium.

In general, therefore, our position is that only one antibiotic is prepared i.e; BM123 which is composed of a number of elements. This fact should, however, not result in a holding that 3 separate inventions are involved.

Fermentation Process Selected to Produce Primarily BM123¾< Cultivation of Nocardia sp. NRRL 5646 may be carried out in a wide variety of liquid culture media.

Media which are useful for theproduction of this novel antibacterial include an assimilable source of carbon such as starch, sugar, molasses, glycerol, etc. an as-similable source ofnitrogen such as protein, protein hy- drolysate, polypeptides, amino acids, corn steep liqu^, etc.; and inorganic anions and cations, such as potassium, sodium, calcium, sulfate, phosphate, chloride, etc. Trace elements such as boron, molybdenum, copper, etc.; are supplied as impurities of other constituents of the media. Aeration in tanks and bottles is provided by forcing sterile air through or onto the surface of the fermenting medium. Further agitation in tanks is provided by a mechanical impeller. An antifoaming agent such as lard oil may be added as needed.

Inoculum Preparation for BM123a Shaker flask inoculum of Nocardia sp . NRR1 ^6^6 is prepared by inoculating 100 ml. of sterile liquid medium in 500 ml. flasks with scrapings or washings of spores from an agar slant of the culture. The following is an example of a suitable medium: Beef extract 3.0 gm.

Bacto-tryptone 5.0 gm.

Yeast extract 5·0 gm.

Starch 2*1.0 gm.

Dextrose 1.0 gm.

Water to 1,000 ml.

Adjust medium pH to 7.0 with NaOH The flasks are incubated at a temperature from 25°-29°C., preferably 28°C, and agitated vigorously on a rotary shaker for 30-48 hours. These 100 ml. portions of inoculum are then used to inoculate one liter and 12 liter batches of the same medium in 2 liter and 20 liter glass fermentors . The inoculated mash is aerated with sterile air while growth is continued for 40-55 hours.

These batches of inoculum are used to inoculate tank fermentors.

Tank Fermentation for BM123a For the production of BM123a in tank ferment-ors the following medium is regularly used: Bacto-peptone 10.0 gm.

Dextrose 10.0 gm.

Molasses 20.0 gm.

Ferric ammonium citrate 0.1 gm.

Calcium carbonate 1.0 gm.

Water to 1,000 ml.

Each tank is inoculated with 3 to 10% of inoculum made as described above. Aeration is supplied at the rate of 0.2-0.8 liter of sterile air per liter of broth per minute and the fermenting mixture is agitated by an impeller driven at 50-200 r.p.m. The temperature is maintained at 25°-29°C, usually at 28°C. The fermentation is ordinarily continued for 180-2*10 hours, at which time the mash is harvested.

Isolation of B 123a After the fermentation is completed, the fermented mash containing BM123a is filtered, preferably with the aid of dlatomaceous earth or some other conven-tional filter aid. Normally the mycelial filter cake pad is washed with water and the filtrate and washings are combined. An average filtrate may constitute about 30 liters. The pH is adjusted to 6.5. Sodium fluoride is added and the mixture is stirred for about 1 hours. The resulting suspension is filtered through a filter aid such as dlatomaceous earth. The filtrate is allowed to percolate through a column of IRC® -50(Na+) (50-100 mesh), a weakly acidic resin manufactured by Rohm and Haas Co., Philadelphia, Pennsylvania, with a bed volume of 1 liter. The column of resin is washed with liters of water. The B 123a is eluted by passing 0.3N HgSO^ through the column. The eluate is collected in fractions totaling 500 ml. Fractions 1 to 7, inclusive, which possess the active component BM123a are pooled and adjusted to pH 7:2, using Ba(0H)2. The resulting barium sulfate is removed by filtration giving a clear filtrate. The clear filtrate is passed through a column of -50 (H+) with a bed volume of 6 0 ml. The column is washed with water and eluted with 0.3N H^O^ as describ-ed above. The active fractions (1 to 7) were similarly adjusted to pH 7.0 with Ba(0H)2 and filtered to give a clear solution. This solution is concentrated In vacuo for subsequent chromatography on carbon. Granular Darco® G-60 (20-^0 mesh) is suspended in water, trans-ferred to a glass column and allowed to settle. The excess water is allowed to drain away and the BM123a concentrate is allowed to seep slowly into the column. The charged column is washed with water and then developed with 50% aqueous methanol with the collection of frac-tions of 0 ml. volume. The active fractions (18 to 68) are combined, concentrated to an aqueous layer in vacuo and lyophilized to white, solid B 123<*. Additional BM123a may be recovered by further developing the carbon column with 0% acidic aqueous methanol (pH 1.8, Η,,βΟ^ ) for an additional 16 fractions. The acidic fractions are combined, concentrated in vacuo to an aqueous phase, adjusted to pH 7.2 with Ba(0H)2, filtered and lyophilized. The physical characteristics for BM123a are given below in Example 3· Fermentation Process Selected to Produce Primarily ~f BM1233 and ΒΜ123τ/ Same as for BM123a.

Inoculum Preparation for BM1233 and BM123* Same as for BM123a.

Tank Fermentation for BM1233 and BM123 For the preparation of BM123o in tank ferment-ors the following fermentation medium is regularly used: Dextrose 10.0 gm.

Beef extract 4.0 gm.

Bacto-peptone 4.0 gm.

Sodium chloride 2.5 gm.

Yeast extract 1.0 gm.

Water to 1,000 ml.

Adjust medium to pH 7.0 with NaOH Each tank is inoculated with 3 to 10% of inoculum made as described above. Aeration is supplied at the rate of 0.2-0.8 liters of sterile air per liter of broth per minute and the fermenting mixture is agitated by an impeller driven at 50-200 r.p.m. The temperature is maintained at 25°-290C, usually at 28°C. The fermentation is ordinarily continued for 45-85 hours, at which time the mash is harvested.

Isolation of BM1233 and BM123p/ " The fermented mash containing BM1233 and BM123 is filtered using 1% diatomaceous earth as a filter aid. The cake is washed with water. The combined filtrate and washing is then passed through a 10 liter bed volume of IRCR 50 (Na+) resin by gravity with a flow rate of 330 ml. per minute. The charged column is then washed with water. The activity is then eluted from the column using 0.3N I^SOjj . The eluate is collected in two portions. The first portion is composed of the eluate from the start (pH 7.0) to the eluate pH of 5-0. Th^. first portion is discarded because of its high salt content. The second portion has a pH of about 1.4.

This acidic portion is adjusted to pH 6.0 using Ba(0H)2« The barium sulfate, which forms, is removed by filtration through diatomaceous earth. The pad is washed with water and the combined filtrate and washings are concentrated in vacuo . The concentrate is warmed on a steam bath to 50°C. and added to a hot solution of Reinecke salt (150 gm. per 1500 ml. at 75°C). The hot solution is allowed to stand for about ¾8 hours and then filtered to give a red-purple solid. This solid is washed with water. The wet solid is then stirred with a mixture of water and- 1-butanol (2:5) and adjusted to pH 1.5 with 0.25N ^SO^ . The mixture is filtered and the aqueous phase from the filtrate is adjusted to pH 6 to 8, filtered and the second filtrate is concentrated in vacuo. The concentrate is passed through a 100 ml. column of Dowex® l-xM(Cl~) and lyophilized.

Chromatographic Separation of Components of BM1233 and B 123,Z/ Cellulose powder is mixed thoroughly with the upper phase obtained by mixing 90% phenol : chloroform: -pyridine:acetic acid: water ( 1500 : 300 : ^5 : ^5 : 750 ) . The wetted cellulose powder is packed into a glass column in increments. The charge is applied to the top of the column as a mixture of the above described lyophilized component mixture dissolved in upper phase and cellulose powder (1:2 v/w). The column is then developed using air pressure on a reservoir attached to the top of the glass column. A total of 75 fractions (25 ml . fractions ) are collected using an automatic fraction collector.

The activity is located by bioautography of dipped paper discs, air dried, and washed with ether before applica-tion to a large agar plate seeded with K. pneumoniae strain AD. Two separate active bands are observed. One of these (BM123i is composed of fractions 3 to 18 and the second (BM1238) is composed of fractions 31 to 52. Fractions 3 to 18 are combined and added to chloroform creating a two phase system. The lower phase is discarded. The upper aqueous phase (pH .5) is extracted twice with an equal volume of ether to remove any residual phenol. The aqueous phase is adjusted to pH 6.5 using IRR 45(OH~) and lyophilized to yield B 123/V .

Fractions 31 to 52 are combined and treated in the same manner to yield BM1233.

The three antibacterials were compared in vitro using a variety of gram positive and gram negative bacteria as well as . smegmatis by the standard agar dilu-tion procedure. The results are reported as minimal inhibitory concentrations (meg. /ml.) in Table VI. Gen-tamicin sulfate was run as a comparison.

TABLE VI Minimal Inhibitory Conce Gentamicin Organism Sulfate Mycobacterium smegmatis No. 607 0.1 0.25 Staphylococcus aureus Rose ATCC 14154 0.1 0.25 Staphylococcus aureus Smith ATCC 13709 0.05 0.25 Staphylococcus aureus No. 3 050B122-3 0.25 0.25 Bacillus cereus ATCC 96 4 0.25 1 Bacillus globigii 0.025 0.25 Bacillus subtilis No. 17 Stansly R-78 0.025 0.25 Bacillus subtilis No. 18 Stansly R-76 0.1 0.5 Corynebacterium xerosis NRRL B-1397 0.025 0.25 Enterococcus GK 2.5 5 > Sarcina lutea ATCC 9341 0.25 1 Enterobacter aerogenes No. 75 0.1 0.25 Escherichia coli U311 0.05 0.25 Table VT continued Minimal Inhibitory Co Gentamicin Organism Sulfate BM12V Escherichia coli No. 29 0.05 0.25 Klebsiella pneumoniae, Type A, Strain D 0.05 0.1 Proteus mirabilis ATCC 4671 0.25 0.25 Proteus organii ATCC 8019 0.1 0.25 Proteus vulgaris ATCC 948 0.1 0.25 Pseudomonas aeruginosa ATCC 10145 2.5 5 Pseudomonas aeruginosa PA7 5 10 > SaL-nonella gallinarum Lederle 605 0.05 0.1 1 Salmonella ty—p—ho—sa ATCC 6539 0.1 0.1 .Shigella shiga 0.25 0.25 The three antibacterial components BM123 The usefulness of these new antibacterial agents is demonstrated by their ability to control systemic lethal infections in mice. These new substances show high in vivo antibacterial activity in mice against Proteus mirabills ATCC Wl, Klebsiella pneumoniae AD, and Escherichia coll UC3H when administered by a single subcutaneous dose to groups of Carworth Farms CF-1 mice, weight about 20 gm., infected intraperitoneally with a lethal dose of these bacteria in 10-1'^, 10"^ and 10"^ trypticase soy broth TSP dilutions, respectively, of a 5 hour TSP blood culture.

Table VII, below, Illustrates the in vivo antibacterial activity of ΒΜ123α, BM1230, and B 123V against these three bacteria.

TABLE VII Single Subcutaneous Alive/Total Mice Tested, 7 Days fter Infection Dose m ./k . BM123 Example 1 Inoculum Preparation A typical medium used to grow the primary inoculum was prepared according to the following formula: Beef extract 3.0 gm.

Bacto-tryptone 5·0 gm.

Yeast extract 5·0 gm.

Starch 24.0 gm.

Dextrose 1.0 gm.

Water to 1,000 ml.

Adjust medium to pH 7.0 with NaOH Washed or scraped spores from an agar slant of Nocardia sp. NRRL 5646 were used to inoculate two 500 ml. flasks containing 100 ml. each of the above sterile medium. The flasks were placed on a rotary shaker and agitated vigorously for 48 hours at 28°C. The resulting flask inoculum was transferred to a 5 gallon glass fer-mentor containing 12 liters of sterile medium. The mash was aerated with sterile air while growth was carried out for about 48 hours, after which the contents were used to seed a 300 liter tank fermentor containing 150 liters of sterile inoculum medium. This final state of inoculum mash was aerated with sterile air at 1 liter of air per liter of mash per minute, introduced at the bottom of the fermentor. No additional agitation was provided. The mash was maintained at 28°C. Lard oil was used as a defoaming agent. After 53 hours of growth, this inoculum mash was used to seed a 2,000 liter fermentor containing 1,350 liters of sterile fermentation medium.

Example 2 Fermentation Employing Nocardia sp. NRRL 5646 and Medium Favoring the Production of BM123 Dextrose 10.0 gm.

Molasses 20.0 gm.

Ferric ammonium citrate 0.1 gm .

Calcium carbonate 1.0 gm .

Water to 1,000 ml.

The fermentation medium was sterilized at 120°C. with steam at 20 lbs. pressure for 60 minutes. The pH of the medium after sterilization was 6.7. Thir-teen hundred and 50 liters of sterile medium in a 2000 liter tank fermentor was inoculated with 150 liters of inoculum prepared as described in Example 1. The fermentation was carried out at 28°C. using lard oil as a defoaming agent. Aeration was supplied at the rate of 0.6-0.7 liter of sterile air per liter of mash per minute. The mash was agitated by an impeller driven at 50 r.p.m. At the end of approximately 230 hours of fermentation time the mash was harvested.

Example 3 Isolation of BM123a A fermentation was carried out as described in Example 2. A 30 liter portion of the fermented mash having a pH of 7.7 was filtered using 300 gm. of diato-maceous earth as a filter aid. The cake was washed with water to give a combined filtrate and wash of 31 liters and the pH was adjusted to 6.5 with hydrochloric acid. Sodium fluoride (62 gm.) was added and the mixture was stirred for 1 hour. The resulting suspension was filtered using 310 gm. of diatomaceous earth as a filter aid to give a filtrate of 31 liters. This filtrate was allowed to percolate through a column of IRCr^ 50(Na^ (50-100 mesh) with a bed volume of 1 liter. The column of resin was washed with 4 liters of water. The BM123a was eluted by passing 0.3N H^O^ through the column and collecting fractions with a volume of 500 ml. Fractions 1 to 7 inclusive, possessing the active component were pooled and adjusted to pH 7.2 using solid Ba(0K)5. The barium sulfate was removed by filtration giving a clear filtrate. The clear filtrate was run overnight through a column of IRC^ 50(H+) with a bed volume of 650 ml. The column was washed with liters of water and eluted with 0.3N H2S0jj as described above. The active fractions (1-7) were similarly adjusted to pH 7.0 with Ba(OH)2 and filtered to give a clear solution of 2.9 liters.

This was concentrated in vacuo to 75 ml. for subsequent chromatography on carbon.

One liter of granular Darco® G-60 (20-*J0 mesh) was suspended in water, transferred to a glass column and allowed to settle. The excess water was allowed to drain away and the 75 ml. concentrate was allowed to seep slowly into the column. The charged column was washed with *J liters of water and then developed with 3.5 liters of 50% aqueous methanol with the collection of fractions of 50 ml. volume. The active fractions (18 to 68) were combined, concentrated to an aqueous phase in vacuo, and lyophilized to 6 grams of white, solid BM123a. The carbon column was then developed with 0% acidic aqueous methanol (pH 1.8 with I^SO^) for an additional 16 fractions. The acidic fractions were combined, concentrated In vacuo to an aqueous phase, adjusted to pH 7.2 with Ba(OH)2, filtered and lyophil z-ed to give an additional 1.5 gm. of BM123a. Both solids were a mixture of and oc^ by thin layer chromatography. This BM123a was dried in an Abderhalden drying apparatus over boiling ethanol for 16 hours prior to microanalysis.

The BM123a does not possess a definite melting point, gradual decomposition starting in the vicinity of 200°C. The antibiotic gave the following microanalytical data: C, 33-89; H, 5-71; N, 11.88; O(direct), 35.40; ash, 0. The BM123 The B 123x exhibited characteristic absorption in the infrared region of the spectrum at the following wavelengths: 780, 815, 950, 1050, 1110, 1250, 1340, 1395, 156O, I670, 1705, 2 0, and 3330 cm"1. A standard infrared absorption spectrum of B 123a prepared in a KBr pellet is shown in Figure 1 of the accompanying drawings.

Example 4 Fermentation Employing Nocardia sp. NRRL 5646 and Medium Flavoring the Production of B 123 Beef extract 4.0 gm.

Bacto-peptone 4.0 gm.

Sodium chloride 2.5 gm.

Yeast extract 1.0 gm.

Water to 1,000 ml.

Adjust medium to pH 7.0 with NaOH The fermentation medium was sterilized at 120°C with steam at 20 lb. pressure for 60 minutes. The pH^f the sterilized medium was 6.7. Thirteen hundred and 50 liters of sterile medium in a 2000 liter tank fermentor was inoculated with 150 liters of inoculum prepared as described in Example 1. The fermentation was carried out at 28°C. using lard oil as a defoaming agent. Aeration was supplied at the rate of 0. liter of air per liter of mash per minute. The mash was agitated by an impeller driven at 50 r.p.m. At the end of approximately 85 hours of fermentation time the mash was harvested.

Example 5 Isolation of BM1233 and BM123 A 13 0 liter portion of fermentation mash, prepared as described in Example 4 , was filtered using 1% diatomaceous earth as a filter aid. The cake was washed with 50 liters of water and discarded. The combined filtrate and wash was then passed through a 10 liter bed volume of 50(Na+) by gravity with a flow rate of 330 ml. /minute. The charged column was then washed with 30 liters of water. The activity was eluted from the column using 170 liters of 0.3N HjSO^. The eluate was collected in two portions. The first portion was composed of the eluate from the start (pH 7.0) to an effluent pH of 5.0. This first 45 liter portion was dis-carded because of its high salt content. The second portion (120 liters) had a pH of 1.4. This acid portion was adjusted to pH 6.0 using solid Ba(0H)2. The barium sulfate which formed was filtered off using 1 kg. of diatomaceous earth as filter aid. The pad was washed with 5 liters of water and the combined filtrate and wash was concentrated in vacuo to 2 liters. This concentrate warmed on a steam bath to 50°C. and added to a hot solution of Reinecke salt (150 gm. in 1500 ml. of water at 75°C). The hot solution was allowed to stand at room temperature for 48 hours and then filtered to give a red--purple solid. This solid was washed with 600 ml. of water. The wet solid was stirred with 2 liters of water and 5 liters of 1-butanol and adjusted to pH 1.5 with 0.25N I-^SO^. The mixture was filtered and the aqueous phase from the filtrate was adjusted to pH 6.5 using solid Ba(0H)2. The mixture was filtered and concentrated in vacuo to 350 ml. The concentrate was passed through a 100 ml. column of Dowex® l-x4(Cl~) and lyophilized to give 50 gm. of product.

A 250 gm. portion of cellulose powder was mixed with 200 ml. of the upper phase obtained by mixing 90% phenol:chloroform:pyridine:acetic acidrwater (1500:-300:45:45:750 by volume). The wetted cellulose powder was packed into a glass column of 1 1/2" diameter in increments. The charge was applied to the top of the column as a mixture of.10 gm. of the above lyophilized product in l6 ml. of upper phase and 20 gm. of cellulose powder. The column was then developed using air pres- 2 sure at 3 lb./in. on a reservoir attached securely to the top of the glass column. A total of seventy-five 25 ml. fractions were collected using an automatic fraction collector. The activity was located by bioauto-graphy of dipped paper discs, air dried and washed with ether before application to a large agar plate seeded with K. pneumoniae strain AD. Two separated active bands were observed. One of these bands (ΒΜ123τ waj^■-· composed of fractions 3 to 18 and the second band (BM1233) was composed of fractions 31 to 52. Fractions 3-18 were combined and 1*100 ml. of chloroform was added, resulting in a two phase system. After separation the upper aqueous phase (pH 4.5) was extracted twice with an equal volume of ether to remove any residual phenol. The aqueous phase was adjusted to pH 6.5 using 45(OH~) any lyophilized to yield BM123 (1.62 gm.).

Fractions 31 to were combined and treated as described above to give BM1233 (1.05 gm.).

The BM123'i (l gm.) was stirred with 20 ml. of absolute methanol for 15 minutes. The suspension was filtered to give "305 mg. of solid and a filtrate. A 60 ml. portion of ether was added to the filtrate to give a suspension of a white flocculant material in the ether-methanol . This was filtered to give a white solid and an off-white filtrate. The solid was washed on the funnel with ether and air dried to give 602 mg. of BM123'] The BM123 was dried in vacuo in an Abderhalden drying apparatus over boiling ethanol for 16 hours prior to microanalytical and spectrophotometric determinations.

The BM123V/'does not possess a definite melting point, gradual decomposition starting at 200°C. Micro-analysis gave C, 46.13; H, 6.65; N, 17.00; 0 (direct), 24,9 ; ash, 0.90. In 90¾ methanol the compound gave a If U.V. absorption maximum at 286 n (E, Icm'= 200). Specific rotation = [a]p5° = +53.8 (C = 1.004 in H20).

The B 123' exhibited characteristic absorption in the infrared region of the spectrum at the following wavelengths: 765, 870, 980, 10 5, 1112, 1175, 1400, 1465, 1510, 1570, 1610, 1660, 2910, and A standard infrared absorption spectrum of BM123' 'prepared in a KBr pellet is shown in Figure 3 of the accomp-anying drawings .

A 670 mg. portion of BM1233 was stirred with 12 ml. of methanol for 15 minutes and filtered. A 36 ml. portion of ether was added to the filtrate to give a white precipitate. The precipitate was collected on a filter, washed with ether and air dried to give 466 mg. of BMI233 which was. then dried in vacuo over boiling ethanol for 4 hours prior to microanalysis.

The BM1233 does not possess a definite melting point, but gradual decomposition starts in the vicinity of 200°C. Microanalysis gave C, 47.53; H, 7-39; N, 16.54; ash, 1.04. In 98% methanol BM1233 gave a U.V. 1¾ absorption maximum at 286 nm with E,lcm = 200. The po-sition of this maximum did not change with pH. BM1233 had a specific rotation of [ ]^5°= +53.2 (C = 1.250 in H20).

The BM1233 exhibited characteristic absorption in the infrared region of the spectrum at the following wavelengths: 826, 870, 922, 980, 1035, 1105, 1170, 1220, 1340, 1390, 1510, 1560, 1600, 1670, 2950, 3080, and 3350 cm"1. A standard infrared absorption spectrum of BM1233 prepared in a KBr pellet is shown in Figure 2 of the accompanying drawings .

Example 6 Paper Partition and Thin Layer Chromatography of Active Components The antibacterial agents can be distinguish^, by paper chromatography. For this purpose, Whatman No. 1 strips were spotted with a water or methanol solution of the substances and equilibrated for 1 to 2 hours in the presence of both upper and lower phases. The strips were developed overnight with the lower (organic) phase obtained from mixing 90% phenol:m-cresol:acetic acid:-pyridine: water (100 : 25 : k : H : 75 by volume). The strips were removed from the chromatographic chamber, air dried for 1 to 2 hours, washed with ether to remove residual phenol and bioautographed on large agar plates seeded with K. pneumoniae . Representative Rf values are listed in Table VIII.

Table VIII Component Rf .

BM1230 0.62, 0.71 B 123 0.20, 0.^7 Both the a and 3 components were a mixture of two antibiotics. The 3 component was composed of a major antibiotic (Rf = 0.62) called BM123 J and a minor component B 123 2 (Rf = 0.71). The most polar component of BM123a (Rf = 0.20) was named BM123a1 and the less polar component (Rf = 0.47) was called ΒΜ123α2· ΒΜ123α was separated into two components, alternatively, using a method based on thin layer chroma-tography on Polygram^ Cel UV254, a form of thin layer cellulose, sold by Brinkmann Instruments Ltd., Westbury, New York. The spotted thin layer plates were developed with water containing 1% trisodium citrate. The zones were detected by bioautography on agar plates as described above. Two zones were evident, BM123a1 with an Rf » 0.90 and BM123a with an Rf = 0.65.

Claims (7)

43295/2

1. Antibacterial BM123 which is effective in inhibiting the growth of bacteria; mixture of antibiotics designated as B 123o< , BM123ji, and B 123Y ; BM123o has an optical 25° rotation [>= ]p a +52.0 (C = 1.00 in water); has the following elemental analysis (percent); C, 33.89; H, 5.71; N, 11.88; 0,35.40; and has the characteristic infrared absorption spectrum as shown in Figure 1 of the accompanying drawings; BM123^ has an optical rota- 25° tion [es ]D =53.2 (C = 1.250 in water) ; has the following elemental analysis, (percent) : C, 47.53; H, 7.39; N, 16,54; ash, 1.04; and has a characteristic infrared absorption spectrum as shown in Figure 2 of the accompanying drawings: BM123V has an optical rotation [e^]D B53 (C <=> 1.004 in water); has the following elemental analysis (percent): C, 46.13; H, 6.65; N, 17.00; 0, 24*96; Ash, 0.90; and has a characteristic infrared absorption spectrum as shown in Figure 3 of the accompanying drawings.

2. A compound as defined in claim 1, antibacterial BM123<-t , inits essentially pure form.

3. A compound as defined in claim 1, antibacterial B 12Sj3, in its essentially pure ,form.

4. A compound as defined in claim 1, antibacterial BM123V, in its essentially pure form.

5. process for the production of antibac terial BM123a which comprises cultivating a microorganism having the characteristics of Nocardla sp. NRRL 5646 or mutants thereof in an aqueous nutrient medium containing assimilable sources of carbohydrate, nitrogen, and inorganic salts under submerged aerobic conditions until substantial antibacterial activity is imparted to said medium, and then recovering antibacterial BM123a therefrom .

6. A process for the production of antibacterial BM1233 which comprises cultivating a microorganism having the characteristics of Nocardla sp. NRRL 5646 or mutants thereof in an aqueous nutrient medium containing assimilable sources of carbohydrate, nitrogen, and inorganic salts under submerged aerobic conditions until substantial antibacterial activity is imparted to said medium, and then recovering antibacteriaif B 1233 therefrom.

7. -9- A process for the production of antibacterial B I23 which comprises cultivating a microorganism having the characteristics of Nocardla sp. NRRL 5646 or mutants thereof in an aqueous nutrient medium containing assimilable sources of carbohydrate, nitrogen, and inorganic salts under submerged aerobic conditions until substantial antibacterial activity is imparted to said medium, and then recovering antibacterial BM123 V^there- from. S. HOROWITZ & CO. AGENTS FOR APPLICANTS