Antibiotic A-32887 and process for production thereof

Antibiotic A-32887 is produced by submerged aerobic fermentation of Streptomyces albus NRRL 11109. A-32887 is an antibacterial, antiprotozoal, anticoccidial, and insecticidal agent. A-32887 also increases feed-utilization efficiency in ruminants.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
New, improved antibiotics are continually in demand. In addition to 
antibiotics which are useful for human diseases, improved antibiotics are 
also needed in the veterinary field. Improved growth promotion in animals 
is one important goal for these antibiotics. Growth promotion is achieved, 
for example, by reducing disease and by increasing feed-utilization 
efficiency. 
Coccidiosis is one disease important to veterinary science, especially to 
the poultry industry. Coccidiosis results from infection by one or more 
species of Eimeria or Isopora (for a summary see Lund and Farr in 
"Diseases of Poultry," 5th ed., Biester and Schwarte, Eds., Iowa State 
University Press, Ames, Iowa, 1965, pp 1056-1096). Economic losses due to 
coccidiosis are great, and known anticoccidial agents have many 
disadvantages. Improved anticoccidial agents continue to be needed. 
Promotion of growth in ruminants, such as cattle, is another 
economically-desirable objective of veterinary science. Of particular 
interest is growth promotion which is achieved by increasing 
feed-utilization efficiency. The mechanism for utilization of the major 
nutritive portion (carbohydrates) 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 known as volatile fatty 
acids (VFA). For a more detailed discussion, see Leng in "Physiology of 
Digestion and Metabolism in the Ruminant," Phillipson et al., Eds., Oriel 
Press, Newcastle-upon-Tyne, England, 1970, pp 408-410. 
The relative efficiency of VFA utilization is discussed by McCullough in 
Feedstuffs, June 19, 1971, page 19; Eskeland et al. in J. An. Sci. 33, 282 
(1971); and Church et al. in "Digestive Physiology and Nutrition of 
Ruminants," Vol. 2, 1971, pp 622 and 625. Although acetates and butyrates 
are utilized, propionates are utilized with greater efficiency. A 
beneficial compound, therefore, stimulates animals to produce a higher 
proportion of propionates from carbohydrates, thereby increasing 
carbohydrate-utilization efficiency. 
2. The Prior Art 
A-32887 is a new member of the group of polyether antibiotics. Examples of 
members of this group include monensin (U.S. Pat. No. 3,501,568); 
dianemycin [R. L. Hamill, M. M. Hoehn, G. E. Pittenger, J. Chamberlin, and 
M. Gorman, J. Antibiotics 22, 161 (1969)]; nigericin [L. K. Steinrauf, 
Mary Pinkerton, and J. W. Chamberlin, Biochem. Biophys. Res. Comm. 33, 29 
(1968)]; salinomycin (U.S. Pat. No. 3,857,948); A-130-A (U.S. Pat. No. 
3,903,264); A-28695 A and B (U.S. Pat. No. 3,839,558); grisorixin [Chem. 
Commun., p. 1421 (1970)]; A-218 and K-41 [J. Antibiotics 29 (1), 10-14 
(1976)]. 
Of this group of antibiotics, A-32887 is most closely related to K-41. A 
convenient method for distinquishing A-32887 from K-41 is by 
chromatography. A-32887 can be separated from K-41, for example, by 
silica-gel thin-layer chromatography in the following two systems (R.sub.f 
values are approximate): 
______________________________________ 
Antibiotic Solvent System 
R.sub.f Value 
______________________________________ 
A-32887 chloroform:methanol 
0.78 
(92:8) 
K-41 chloroform:methanol 
0.84 
(92:8) 
A-32887 ethyl acetate:ethanol 
0.76 
(1:4) 
K-41 ethyl acetate:ethanol 
0.70 
(1:4) 
______________________________________ 
SUMMARY OF THE INVENTION 
This invention relates to an antibiotic substance which is produced by 
culturing a hitherto undescribed strain of the organism Streptomyces albus 
NRRL 11109. 
The antibiotic substance of this invention is arbitrarily designated herein 
as A-32887. The C.sub.2 -C.sub.6 -acyl ester derivatives of antibiotic 
A-32887, the methyl ether derivative of antibiotic A-32887, and the 
pharmaceutically-acceptable salts of antibiotic A-32887 and of said ester 
and ether derivatives are also part of this invention. To simplify 
discussions of utility, the term "A-32887 compound" is used and refers to 
antibiotic A-32887, a specified acyl ester derivative of A-32887, the 
methyl ether derivative of A-32887 or a pharmaceutically-acceptable salt 
of A-32887 or of said ester or ether derivatives. 
A-32887 is produced by culturing a novel strain of Streptomyces albus, NRRL 
11109, under submerged aerobic fermentation conditions until a substantial 
level of antibiotic activity is produced. A-32887 is extracted from 
basified broth filtrate with polar organic solvents. The extracted A-32887 
is purified by absorption chromatography. 
A-32887 inhibits the growth of organisms which are pathogenic to animal and 
plant life. More specifically, A-32887 is an antibacterial, antifungal, 
antiprotozoal, anticoccidial, antiviral and insecticidal agent. In 
addition, A-32887 increases feed-utilization efficiency in ruminants, 
inhibits the enzyme ATPase, and is a blood-pressure-lowering agent.

DETAILED DESCRIPTION 
The following paragraphs describe the properties of antibiotic A-32887. 
A-32887 is a white, amorphous powder which melts at approximately 
90.degree. C. Elemental analysis of A-32887 indicates that it has the 
following approximate percentage composition (average): Carbon, 61.61%; 
Hydrogen, 8.56%; Oxygen, 28.63%. A-32887 has an approximate empirical 
formula of C.sub.48-49 H.sub.80-86 O.sub.17-18 and a preferred empirical 
formula of C.sub.48 H.sub.82 O.sub.18. 
A-32887 has a molecular weight of about 946, as determined by mass 
spectrometry. 
The infrared absorption spectrum of A-32887 (free acid) in chloroform is 
shown in FIG. 1 of the accompanying drawings. Significant absorption 
maxima occur at the following frequencies (cm.sup.-1): 3540 (shoulder), 
3420 (medium), 2945 (shoulder), 2905 (strong), 2860 (shoulder), 2805 
(shoulder), 1710 (weak), 1450 (medium), 1370 (medium), 1350 (shoulder), 
1300 (shoulder), 1275 (weak), 1170 (shoulder), 1155 (medium), 1100 
(shoulder), 1085 (strong), 1060 (strong), 1010 (weak), 990 (weak), 980 
(shoulder), 948 (medium), 890 (weak), and 850 (weak). 
The ultraviolet spectrum of A-32887 shows no significant absorption. 
The proton-magnetic-resonance spectrum of A-32887 indicates the presence of 
five methoxyl groups. 
A-32887 (free acid) has the following specific rotation: 
[.alpha.].sub.D.sup.25 + 15.9.degree. (c 1, CHCl.sub.3). 
A-32887 mixed sodium-potassium salt crystallizes from acetone-water and has 
a melting point of about 187-190.degree. C. Elemental analysis of A-32887 
Na-K salt indicates that it has the following approximate percentage 
composition (average): 
Carbon, 60.14; Hydrogen, 8.11%; Oxygen, 29.64%; Sodium, 2.31%; Potassium, 
0.46%. 
The infrared absorption spectrum of A-32887 Na-K salt in chloroform is 
shown in FIG. 2 of the accompanying drawings. Significant absorption 
maxima are observed at the following frequencies (cm.sup.-1): 3400 
(shoulder), 3210 (medium), 2970 (strong), 2925 (strong), 2870 (weak), 2820 
(weak), 1605 (medium), 1455 (medium), 1375 (shoulder), 1358 (medium), 1310 
(weak), 1285 (weak), 1183 (medium), 1160 (medium), 1110 (shoulder), 1090 
(strong), 1060 (strong), 1012 (weak), 980 (medium), 942 (medium), 910 
(weak), 875 (shoulder), and 858 (weak). 
A-32887 Na-K salt, crystallized from acetone-water, has the following 
characteristic X-ray powder diffraction pattern (CuNi, 1.5405 .lambda., d 
= interplanar spacing in angstroms): 
______________________________________ 
Relative 
d Intensity 
______________________________________ 
12.53 50 
10.10 100 
9.5 40 
9.05 40 
8.07 70 
7.16 100 
6.77 100 
6.48 70 
6.16 60 
5.58 50 
5.35 50 
5.10 30 
4.86 60 
4.70 50 
4.44 50 
4.25 50 
4.06 40 
3.80 30 
3.68 40 
3.58 30 
3.42 60 
3.22 10 
3.06 10 
2.97 10 
2.85 10 
2.76 02 
2.61 15 
2.49 05 
2.47 05 
2.44 15 
2.36 10 
2.29 10 
2.20 02 
2.12 02 
2.03 05 
1.96 05 
1.90 02 
1.81 02 
______________________________________ 
A-32887 Na-K salt has the following specific rotation: 
[.alpha.].sub.D.sup.25 + 9.6.degree. (c 1, CHCl.sub.3). 
Electrometric titration of A-32887 in 80% aqueous dimethylformamide 
indicates the presence of a titratable group with a pK.sub.a value of 
4.60. 
A-32887 is soluble in a variety of organic solvents such as methanol, 
ethanol, dimethylformamide, dimethyl sulfoxide, ethyl acetate, chloroform, 
acetone, and benzene; is slightly soluble in non-polar organic solvents 
such as hexane and heptane; and is insoluble in water. 
A-32887 is stable in aqueous solutions having a pH of from about 3 to about 
11, but is unstable in solutions having a pH lower than about 3. 
A-32887 sodium salt has a molecular weight of about 968, and A-32887 
potassium salt has a molecular weight of about 984, both as determined by 
field-desorption (FD) mass spectrometry. The ion at m/e 969 (M + H) in the 
FD spectrum of A-32887 sodium salt was peak matched with a field-ionized 
mass standard. The found mass was 969.5373; the theoretical mass for 
C.sub.48 H.sub.82 O.sub.18 Na is 969.5399. This finding supports a 
molecular formula of C.sub.48 H.sub.82 O.sub.18 for A-32887 free acid. 
In the following paper-chromatographic systems, using Bacillus subtilis 
ATCC 6633 bioautography for detection, A-32887 has these approximate 
R.sub.f values: 
______________________________________ 
Solvent System R.sub.f Value 
______________________________________ 
Water saturated with methyl 
isobutyl ketone (MIBK) 0.58 
Water:methanol:acetone 
(12:3:1). This solution 
is adjusted to pH 10.5 with 
NH.sub.4 OH and then lowered 
to pH 7.5 with H.sub.3 PO.sub.4 
0.32 
Propanol:water (1:9) 0.85 
Methanol:propanol:water (6:2:1). 
Paper buffered with 0.75 M KH.sub.2 PO.sub.4, 
pH 4.0. 0.79 
Methanol:0.05 M sodium citrate at 
pH 5.7 (7:3). Paper buffered with 
0.05 M sodium citrate at pH 5.7. 
0.85 
Propanol:water (7:3) 0.91 
______________________________________ 
In the following silica-gel TLC systems, using either vanillin spray 
reagent or Bacillus subtilis ATCC 6633 bioautography for detection, 
A-32887 has the these approximate R.sub.f values: 
______________________________________ 
Solvent System R.sub.f Value 
______________________________________ 
Methanol 0.80 
Ethyl acetate:ethanol (1:4) 
0.65 
Ethyl acetate:chloroform (1:1) 
0.36 
Ethyl acetate:chloroform (6:1) 
0.67 
Benzene:ethyl acetate:methanol 
(6:4:0.2) 0.58 
______________________________________ 
A-32887 has an acid function capable of forming salts and ester derivatives 
and has at least one hydroxyl group capable of esterification. The C.sub.2 
-C.sub.6 -acyl ester derivatives of A-32887 and the 
pharmaceutically-acceptable salts of these ester derivatives are also 
useful as antibiotics and as agents which increase feed-utilization 
efficiency. 
The A-32887 acyl ester derivatives are typically prepared by reacting 
A-32887 with the corresponding C.sub.2 -C.sub.6 -acid anhydride or acid 
chloride at room temperature. 
The following paragraphs describe characteristics of typical A-32887 acyl 
ester derivatives. 
A-32887 acetyl ester derivative (Na-K salt) is a white amorphous powder 
which has a molecular weight of about 988 and a melting point of about 
127-129.degree. C. A-32887 acetyl ester derivative has an approximate 
empirical formula of C.sub.50-51 H.sub.82-88 O.sub.18-19. 
The infrared absorption spectrum of A-32887 acetyl ester derivative (Na-K 
salt) in chloroform is shown in FIG. 4 of the accompanying drawings. 
Significant absorption maxima occur at the following frequencies 
(cm.sup.-1): 3000, 2975, 2932, 2875, 2830, 1733, 1634, 1620, 1610, 1453, 
1372, 1305, 1158, 1110, 1092, 1062, 1014, 996, 978, 948, 909, 893, and 
853. 
A-32887 n-butyryl ester derivative (Na-K salt) is a white amorphous powder 
which has a molecular weight of about 1016 and a melting point of about 
59-62.degree. C. A-32887 n-butyryl ester derivative has an approximate 
empirical formula of C.sub.52-53 H.sub.86-92 O.sub.18-19. 
The infrared absorption spectrum of A-32887 n-butyryl ester derivative 
(Na-K salt) in chloroform is shown in FIG. 5 of the accompanying drawings. 
Significant absorption maxima occur at the following frequencies 
(cm.sup.-1): 3000, 2968, 2932, 2875, 2830, 1724, 1630, 1620, 1610, 1592, 
1452, 1372, 1355, 1179, 1155, 1110, 1090, 1060, 1012, 977, 946, and 892. 
The C.sub.2 -C.sub.6 -acyl ester derivatives of A-32887 are soluble in a 
variety of organic solvents such as methanol, ethanol, dimethylformamide, 
dimethyl sulfoxide, ethyl acetate, chloroform, acetone, and benzene; are 
slightly soluble in non-polar organic solvents such as hexane and heptane, 
and are insoluble in water. 
A-32887 can be distinguished from its C.sub.2 -C.sub.6 acyl ester 
derivatives by TLC. For example, the acetyl and n-butyryl ester 
derivatives can be separated from A-32887 by silica-gel TLC using a 
benzene:ethyl acetate (1:1) solvent system. A sulfuric acid spray reagent 
can be used for detection. In this system A-32887 and its acetyl and 
n-butyryl ester derivatives have the following approximate R.sub.f values: 
______________________________________ 
R.sub.f value 
______________________________________ 
A-32887 (Na-K) 0.47 
A-32887 acetyl ester 
derivative (Na-K) 0.40 
A-32887 n-butyryl ester 
derivative (Na-K) 0.64 
______________________________________ 
The A-32887 hydroxyl group can react with lower alkanols, lower-alkyl 
thiols, and glycols to form ether derivatives using procedures similar to 
those described for the preparation of A204I derivatives in U.S. Pat. No. 
3,907,832. The A-32887 C.sub.1 -C.sub.4 -alkyl ether derivatives and their 
pharmaceutically-acceptable salts are especially useful as antibiotics and 
as agents which increase feed-utilization efficiency. Of the C.sub.1 
-C.sub.4 -alkyl ether derivatives, the methyl ether derivative of A-32887 
and its pharmaceutically-acceptable salts are preferred. 
A-32887 methyl ether derivative has an approximate empirical formula of 
C.sub.49-50 H.sub.82-88 O.sub.17-18. The sodium salt of A-32887 methyl 
ether derivative is a white crystalline (n-hexane:ethyl acetate) compound 
having a melting point of about 214-216.degree. C. 
The molecular weight of A-32887 methyl ether derivative sodium salt is 
about 982; the molecular weight of A-32887 methyl ether derivative free 
acid is about 960 (both as determined by FD mass spectrometry). 
The infrared absorption spectrum of A-32887 methyl ether derivative (Na 
salt) in chloroform is shown in FIG. 6 of the accompanying drawings. 
Significant absorption maxima occur at the following frequencies 
(cm.sup.-1): 3400 (broad), 2990, 2960, 2930, 2870, 2820, 1725, 1610, 1455, 
1407, 1370, 1309, 1282, 1240, 1180, 1158, 1110, 1093, 1082, 1057, 1010, 
980, 945, 900, 868, 858, 827, 802, 700, and 653. 
The proton-magnetic-resonance spectrum of A-32887 methyl ether derivative 
(Na salt) indicates the presence of six methoxyl groups. 
A-32887 methyl ether derivative (Na salt) has the following specific 
rotation: [.alpha.].sub.D.sup.25 - 5.1.degree. (c 1, CHCl.sub.3) 
A-32887 methyl ether derivative (Na salt), crystallized from n-hexane:ethyl 
acetate, has the following characteristic X-ray powder diffraction pattern 
(CuNi, 1.5405 .lambda., d = interplanar spacing in angstroms): 
______________________________________ 
Relative 
d Intensity 
______________________________________ 
13.18 50 
12.01 50 
9.02 100 
8.26 100 
7.19 80 
6.46 50 
5.78 20 
5.46 20 
5.00 30 
4.24 20 
3.72 20 
3.36 05 
______________________________________ 
Electrometric titration of A-32887 methyl ether derivative (Na salt) in 80% 
aqueous dimethylformamide indicates the presence of a titratable group 
with a pK.sub.a value of about 5.4. 
A-32887 methyl ether derivative is soluble in a variety of organic solvents 
such as methanol, ethanol, dimethylformamide, dimethyl sulfoxide, ethyl 
acetate, chloroform, acetone, and benzene; is slightly soluble in 
non-polar organic solvents such as hexane and heptane; and is insoluble in 
water. 
A-32887 methyl ether derivative (Na salt) can be separated from A-32887 
(Na-K salt) by silica-gel TLC, using a benzene:ethyl acetate (1:1) solvent 
system and sulfuric acid spray reagent for detection. In this system, 
A-32887 and its methyl ether derivative have the following R.sub.f values: 
______________________________________ 
R.sub.f value 
______________________________________ 
A-32887 (Na-K) 0.425 
A-32887 methyl ether 0.22 
derivative (Na) 
______________________________________ 
A-32887, the C.sub.2 -C.sub.6 -acyl ester derivatives of A-32887, and 
A-32887 methyl ether derivative are capable of forming salts. The 
pharmaceutically-acceptable alkali-metal, alkaline-earth-metal and amine 
salts of A-32887, the C.sub.2 -C.sub.6 -acyl ester derivatives of A-32887, 
and A-32887 methyl ether derivative are also part of this invention. 
"Pharmaceutically-acceptable" salts are those in which the toxicity of the 
compound as a whole toward warm-blooded animals is not increased relative 
to the non-salt form. Representative and suitable alkali-metal and 
alkaline-earth metal salts of A-32887 include the sodium, potassium, 
lithium, cesium, rubidium, barium, calcium, and magnesium salts. Suitable 
amine salts of A-32887 include the ammonium and the primary, secondary, 
and tertiary C.sub.1 -C.sub.4 -alkylammonium and hydroxy-C.sub.2 -C.sub.4 
-alkylammonium salts. Illustrative amine salts include those formed by 
reaction of A-32887 with ammonium hydroxide, methylamine, sec-butylamine, 
isopropylamine, diethylamine, di-isopropylamine, ethanolamine, 
triethylamine, 3-amino-1-propanol and the like. 
The alkali-metal and alkaline-earth-metal cationic salts of A-32887 are 
prepared according to procedures commonly used for the preparation of 
cationic salts. For example, the free-acid form of A-32887 is dissolved in 
a suitable solvent such as acetone; a solution containing the 
stoichiometric quantity of the desired inorganic base in aqueous acetone 
is added to this solution. The salt thus formed can be isolated by routine 
methods, such as filtration or evaporation of the solvent. 
The salts formed with organic amines can be prepared in a similar manner. 
For example, the gaseous or liquid amine can be added to a solution of 
A-32887 in a suitable solvent such as acetone; the solvent and excess 
amine can be removed by evaporation. 
It is well known in the veterinary pharmaceutical art that the form of an 
antibiotic is not ordinarily of great significance when treating an animal 
with the antibiotic. In most cases, conditions within the animal change 
the drug to a form other than that in which it was administered. The salt 
form in which it may be administered is, therefore, not of great 
significance. The salt form may, however, be chosen for reasons of 
economy, convenience, and toxicity. 
A-32887 is produced by culturing an A-32887-producing strain of 
Streptomyces albus under submerged aerobic conditions in a suitable 
culture medium until substantial antibiotic activity is produced. A-32887 
is separated from the culture medium by the use of various isolation and 
purification procedures understood in the art. 
The new microorganism useful for the preparation of antibiotic A-32887 was 
isolated from a soil sample collected in Curacao in Dutch Antilles. This 
organism is classified as a strain of Streptomyces albus (Rossi-Doria) 
Waksman and Henrici. This classification is based upon a comparison with 
the published description of the neotype strain ATCC 3004 [A. J. Lyons, 
Jr., and T. G. Pridham, J. Bacteriol. 83, 370-380 (1962)] and the 
Streptomyces albus strain IMRU 3005 [S. A. Waksman, "The Actinomycetes, 
Vol. II, Classification, Identification and Descriptions of Genera and 
Species," The Williams and Wilkins Co., Baltimore, 1961]. 
This classification is based on methods recommended by the International 
Streptomyces Project [E. B. Shirling and D. Gottlieb, "Methods for 
Characterization of Streptomyces Species," Intern. Bull. Systematic 
Bacteriol. 16, 313-340 (1966)] along with certain supplementary tests. 
Color names were assigned according to the ISCC-NBS method (K. L. Kelly and 
D. B. Judd, "The ISCC-NBS Methods of Designating Colors and a Dictionary 
of Color Names," U.S. Dept. of Commerce Circ. 553, 1955, Washington, 
D.C.). Figures in parentheses refer to the Tresner and Backus color series 
[H. D. Tresner and S. J. Backus, "System of Color Wheels for Streptomycete 
Taxonomy," Appl. Microbiol. 11, 335-338 (1963)]. Color tab designations 
are underlined. Maerz and Paul color blocks (A. Maerz and M. R. Paul, 
"Dictionary of Color," McGraw-Hill Book Co., Inc., New York, N.Y., 1950) 
are enclosed in brackets. 
Cell walls were prepared by a modified method of Heymann et al., "Structure 
of Streptococcal Cell Walls. I. Methylation Study of C-Polysaccharide," J. 
Biol. Chem. 238(2):502-509 (1963). Amino acids were determined by 
automatic amino-acid analysis by the modified method of Spackman et al., 
Anal. Chem. 30:1190-1206. Cultures were grown at 30.degree. C. unless 
otherwise noted. 
CHARACTERIZATION OF A-32887-PRODUCING STRAIN 
Morphology 
Spiralled sporophores are produced. These are usually short. Although some 
chains have 3-10 spores per chain, there are usually more than 10 spores 
per chain. Spores are spherical to slightly oval and measure 0.78 .mu. 
.times. 0.56 .mu. with a range in size of 0.625 .mu. .times. 0.5 .mu. to 
0.625 .mu.. The spores are smooth, as observed by electron micrographs. 
Morphologically, culture A-32887 could be confused with a verticillate 
type of morphology in which the branches are equidistant; however, the 
morphology actually is of a type in which the branching is very irregular. 
TABLE I 
__________________________________________________________________________ 
CULTURAL CHARACTERISTICS ON VARIOUS MEDIA 
Medium Characteristics 
__________________________________________________________________________ 
Czapek's-Solution Agar. 
Abundant growth; reverse moderate yellow 
[11K3]; abundant sporulation and aerial 
mycelium; (W) a white; no soluble pigment. 
ISP Medium #2 Abundant growth; reverse moderate yellow 
(Yeast-Extract--Malt-Extract 
[11K3]; abundant sporulation and aerial 
Agar) mycelium; (W) a white; no soluble pigment. 
Tryptone--Yeast Agar. 
Scant growth; reverse pale yellow [11Cl]; 
scant sporulation and aerial mycelium; (W) 
a white; no soluble pigment. 
Nutrient Agar Good growth; reverse pale yellow [11Cl]; 
good aerial mycelium and sporulation; (W) 
a white; no soluble pigment. 
V-8 Juice--Dextrose Agar 
Abundant growth; reverse moderate yellowish 
brown [14E7]; abundant aerial mycelium and 
sporulation; (W) a white; no soluble pigment. 
Glucose--Asparagine Agar 
Good growth; reverse pale yellow [1102]; good 
aerial mycelium and sporulation; (W) a white; 
no soluble pigment. 
Tomato-Paste--Oatmeal Agar 
Abundant growth; reverse grayish yellow [12D3]; 
abundant aerial mycelium and sporulation; (W) 
a white to (Y) 2 ba pale yellow; no soluble 
pigment. 
Emerson's Agar Abundant growth; reverse light yellow brown 
[1217]; abundant aerial mycelium and sporula- 
tion; (W) a white to (GY) 2dc yellowish gray; 
no soluble pigment. 
ISP Medium #5 Scant growth; no color assignment due to poor 
(Glycerol--Asparagine Agar) 
growth. 
Salts--Starch Agar 
Good growth; reverse pale yellow [10B2]; good 
aerial mycelium and sporulation; (W) a white; 
no soluble pigment. 
ISP Medium #4 Scant-to-fair growth; reverse pale yellow 
(Inorganic-Salts--Starch Agar) 
[9D2]; scant aerial mycelium and sporulation; 
(W) a white; some clearing by area where inocu- 
lated; no soluble pigment. Growth not confluent 
but principally as isolated colonies. 
ISP Medium #3 Abundant growth; reverse pale yellow [10F2]; 
(Oatmeal Agar) abundant aerial mycelium and sporulation; (W) 
a white; no soluble pigment. 
Bennett's Modified(--COCl.sub.2) Agar 
Good-to-abundant growth; reverse pale yellow 
[11B2]; abundant aerial mycelium and sporula- 
lation; (W) a white to (GY) 2dc yellow-gray; 
no soluble pigment. 
Glycerol--Glycine Agar 
Good growth; reverse pale yellow [11B2]; scant 
aerial mycelium and sporulation; (W) a white; 
no soluble pigment. 
Tyrosine Agar Fair-to-good growth; reverse grayish yellow 
[12B3]; fair-to-good aerial mycelium and 
sporulation; (W) a white; no soluble pigment. 
Calcium-Malate Agar 
Good growth; reverse pale orange yellow; good 
aerial mycelium and sporulation; (W) a white; 
no soluble pigment. 
__________________________________________________________________________ 
The organism was studied for selected physiologically properties in 
accordance with standard procedures. The properties observed and 
characteristics found are given in Table II: 
TABLE II 
______________________________________ 
Property Observed 
Characteristics 
______________________________________ 
Action on Skim Milk 
No change after 14 days. A soft curd 
is formed after 10 days incubation, but 
milk is not cleared. 
Starch Hydrolysis 
Starch hydrolyzed 
Nitrate Reduction 
Negative 
Gelatin Liquefaction 
None at 14 days 
Melanin Pigment 
Production on: 
1. Tryptone-Yeast- 
Extract Broth. 
Negative 
2. Peptone-Yeast- 
Extract Iron Agar. 
Negative 
3. Tyrosine Agar 
Negative 
Growth on: 
Carrot slice 
Scant vegetative growth. 
Potato slice 
Abundant growth and sporulation; aerial 
and spores off-white to brownish gray. 
Temperature Require- 
No growth. 20.degree. 
Good growth; white aerial-reverse 
(Bennett's-agar slants; 
yellow brown; no soluble pigment. 
incubated 9 days) 
Good growth; white aerial-reverse 
yellow brown; no soluble pigment. 
Good growth; white aerial-reverse 
yellow brown; no soluble pigment. 
Fair growth; scant white aerial- 
reverse color darker than at 25.degree., 30.degree. 
brown; brown soluble pigment. 
No growth. 49.degree. 
No growth. 55.degree. 
______________________________________ 
The response of the culture to varying levels of sodium chloride, using 
Bennett's modified agar, is summarized in Table III. 
TABLE III 
______________________________________ 
Percent NaCl 
in Medium Characteristics 
______________________________________ 
1 Good-to-abundant growth; aerial 
mycelium and spores; white aerial. 
2 Abundant growth; abundant aerial 
mycelium and spores; white aerial. 
3 Abundant growth; abundant aerial 
mycelium and spores; white aerial. 
4 Abundant growth; abundant aerial 
mycelium and spores; white aerial. 
6 Good-to-abundant growth; abundant 
aerial mycelium and spores; white 
aerial. 
8 Good veg. growth; no aerial 
mycelium. 
10 Fair-to-good veg. growth; no 
aerial mycelium. 
12 Scant growth; no aerial myce- 
lium. 
14 Scant-to-no growth; no aerial 
mycelium. 
______________________________________ 
The results of carbon-utilization tests carried out with the organism are 
set forth in Table IV. The following symbols are used: 
+ = Positive utilization 
(+) = Probable utilization 
(-) = Questionable utilization 
- = No utilization 
TABLE IV 
______________________________________ 
Carbon Source Response 
______________________________________ 
L-arabinose + 
D-ribose (+) 
D-xylose (+) 
D-galactose (+) 
D-glucose (+) 
D-mannose (+) 
D-fructose (+) 
L-sorbose - 
cellobiose (+) 
lactose (+) 
maltose + 
melibiose (-) 
sucrose (+) 
turanose (-) to (+) 
trehalose + 
melezitose (-) 
raffinose (-) to (+) 
dextrin + 
salicin - 
starch (soluble) + 
fucose (+) 
rhamnose + 
glucosamine (+) 
.alpha.-methylglucoside 
- 
.alpha.-methylxyloside 
- 
adonitol (+) 
dulcitol (+) to (-) 
i-erythritol (+) 
glycerol (+) 
i-inositol + 
mannitol + 
sorbitol + 
-Carbon (Negative 
Control) - 
______________________________________ 
Cell Wall Studies 
Using hydrolyzed whole cells of the organism, the presence of certain 
diagnostic sugars was determined. Isolated cell walls were used to 
determine the isomers of diaminopimelic acid and the amino-acid content. 
The results of these cell-wall studies are set forth below: 
______________________________________ 
Test Result Observed 
______________________________________ 
Isomers of diamino- 
pimelic acid LL-isomer 
Diagnostic sugars No characteristic 
pattern 
Amino-acid content Major amounts of 
glutamic acid, glycine, 
alanine, and tyrosine. 
______________________________________ 
Certain characteristics of the A-32887-producing S. albus strain differ 
from those in the published description of S. albus (Rossi-Doria) Waksman 
and Henrici, supra. A comparison of the characteristics of the 
A-32887-producing strain (NRRL 11109) with those in the published 
description is given in Table V; differing characteristics are highlighted 
by an asterisk. 
TABLE V 
__________________________________________________________________________ 
Reaction of A-32887 
Published Description of 
Medium; Condition 
S. albus NRRL 11109 
Streptomyces albus 
__________________________________________________________________________ 
ISP Medium #2 White aerial; moderate 
White or yellow aerial; yellow 
Yeast--Malt-Extract Agar) 
yellow reverse 
brown reverse 
ISP Medium #3 White aerial; pale yel- 
White aerial; yellow brown reverse 
(Oatmeal Agar) low reverse 
ISP Medium #4 White aerial; pale yel- 
White aerial; yellow brown reverse 
(Inorganic-Salts--Starch Agar) 
low reverse 
ISP Medium #5 Scant growth; no color 
White or yellow aerial; yellow 
(Glycerol Asparagine Agar) 
assignment brown reverse 
Reaction to various levels of 
Tolerates levels of up 
Tolerates levels greater than 13%. 
NaCl to 12%. No growth at 
No growth at 15% 
14%. 
Temperature Requirements 
Optimum growth at 25.degree. - 
Optimum growth at 25.degree. - 44.degree. C 
43.degree. C 
Reaction to the following 
carbon sources: 
D-Glucose (+) + 
D-Mannitol + + 
D-Galactose (+) + 
L-Arabinose* + - 
Rhamnose* + - 
D-Xylose (+) + 
i-Inositol* + - 
D-Fructose (+) .+-. 
Salicin* - + 
Raffinose (-) - 
Whole-cell Hydrolysates 
LL-diaminopimelic acid 
LL-diaminopimelic acid 
Nitrate Reduction* 
Negative Positive 
Gelatin Liquefaction* 
None at 14 days 
Strong liquefaction 
Melanin-pigment Production 
Negative Negative 
Action on Skim Milk* 
No change after 14 days. 
Rapid peptonization 
Soft curd is formed after 
10 days, but milk is not 
cleared. 
Morphology Spiralled Spiralled 
Spore ornamentation 
Smooth Smooth 
__________________________________________________________________________ 
The Streptomyces albus culture useful for the production of antibiotic 
A-32887 has been deposited and made a part of the stock culture collection 
of the Northern Regional Research Center, Agricultural Research Service, 
U.S. Department of Agriculture, Peoria, Ill. 61604, from which it is 
available to the public under the number NRRL 11109. 
As is the case with other organisms, the characteristics of the 
A-32887-producing culture, Streptomyces albus NRRL 11109, are subject to 
variation. For example, artificial variants and mutants of the NRRL 11109 
strain may be obtained by treatment with various known mutagens such as 
ultraviolet rays, X-rays, high-frequency waves, radioactive rays, and 
chemicals. All natural and artificial variants and mutants which have the 
essential identifying characteristics of Streptomyces albus and produce 
A-32887 may be used in this invention. "Essential identifying 
characteristics" are those characteristics which are sufficient to 
classify an organism as Streptomyces albus NRRL 11109. One of these 
characteristics, of course, is the ability of the organism to produce 
A-32887. It will be understood by those skilled in the art that certain 
non-critical differences between the characteristics exhibited by a given 
organism and those identifying a reference organism can exist without 
affecting the classification of both such organisms as belonging to the 
same genus, species and strain. 
The culture medium employed to grow Streptomyces albus NRRL 11109 can be 
any one of a number of media. For economy in production, optimal yield, 
and ease of product isolation, however, certain culture media are 
preferred. Thus, for example, a preferred carbohydrate source in 
large-scale fermentation is glucose, although dextrin, starch, maltose, 
and the like can also be used. A preferred nitrogen source is meat 
peptone, although other peptones, enzyme-hydrolyzed casein, soybean meal, 
amino acids and the like are also useful. Among the nutrient inorganic 
salts which can be incorporated in the culture media are the customary 
soluble salts capable of yielding iron, potassium, sodium, magnesium, 
calcium, ammonium chloride, carbonate, sulfate, nitrate, and like ions. 
Essential trace elements necessary for the growth and development of the 
organism should also be included in the culture medium. Such trace 
elements commonly occur as impurities in other substituents of the medium 
in amounts sufficient to meet the growth requirements of the organism. It 
may be necessary to add small amounts (i.e. 0.2 ml/l.) of an antifoam 
agent such as polypropylene glycol (M.W. about 2000) to large-scale 
fermentation media if foaming becomes a problem. 
For production of substantial quantities of antibiotic A-32887, submerged 
aerobic fermentation in tanks is preferred. Small quantities of antibiotic 
A-32887 may be obtained by shake-flask culture. Because of the time lag in 
antibiotic production commonly associated with inoculation of large tanks 
with the spore form of the organism, it is preferable to use a vegetative 
inoculum. The vegetative inoculum is prepared by inoculating a small 
volume of culture medium with the spore form or mycelial fragments of the 
organism to obtain a fresh, actively growing culture of the organism. The 
vegetative inoculum is then transferred to a larger tank. The medium used 
for the vegetative inoculum can be the same as that employed for larger 
fermentations, but other media can also be employed. 
The A-32887-producing organism can be grown at temperatures between about 
22.degree. and about 45.degree. C. Optimum A-32887 production appears to 
occur at temperatures of about 30.degree. C. 
As is customary in aerobic submerged culture processes, sterile air is 
blown through the culture medium. For efficient production of antibiotic 
A-32887 the volume of air employed in tank productions is preferably about 
0.25-0.5 volume of air per volume of culture medium per minute (V/V/M). 
Production of antibiotic A-32887 can be followed during the fermentation by 
testing samples of the broth or of extracts of the mycelial solids for 
antibiotic activity against organisms known to be sensitive to this 
antibiotic. One assay organism useful in testing this antibiotic is 
Bacillus subtilis ATCC 6633. The bioassay is conveniently performed by 
paper-disc assay on agar plates. 
Following its production under submerged aerobic fermentation conditions, 
antibiotic A-32887 can be recovered from the fermentation medium by 
methods employed in the fermentation art. Although the antibiotic activity 
produced during fermentation of the A-32887-producing organism occurs in 
both the broth and in the mycelial mass, the major part of the activity is 
in the filtered broth. Maximum recovery of antibiotic A-32887 is 
accomplished, therefore, by an initial filtration to separate the broth 
from the mycelial mass. The filtered broth can then be further purified to 
give antibiotic A-32887. A variety of techniques may be used in this 
purification. A preferred technique for purification of the filtered broth 
involves adjusting the broth to about pH 9 and extracting with a suitable 
solvent such as ethyl acetate. The extracting solvent can then be 
evaporated under vacumm to give partially-purified antibiotic A-32887. 
Further purification of A-32887 involves the use of chromatography. A 
preferred adsorbent for this purification is silica gel. 
Alternatively, the culture solids, including medium constituents and 
mycelium can be used without extraction or separation, but preferably 
after removal of water, as a source of antibiotic A-32887. For example, 
after production of A-32887 antibiotic activity, the whole fermentation 
broth or the broth filtrate can be dried by lyophilization, by 
drum-drying, or by azeotropic distillation and drying. The dried whole 
broth or dried broth filtrate can then be mixed directly into feed premix. 
Antibiotic A-32887 inhibits the growth of pathogenic bacteria, especially 
gram-positive bacteria. Table VI summarizes the minimal inhibitory 
concentrations (MIC), as measured by standard agar-dilution assays, at 
which A-32887 (Na-K salt) inhibits certain bacteria. 
TABLE VI 
______________________________________ 
Test Organism MIC (mcg/ml 
______________________________________ 
Staphylococcus aureus 3055 
6.25 
Streptococcus faecalis 6.25 
Staphylococcus sp. 1.56 
Streptococcus sp. 3.12 
Pasteurella multocida (bovine) 
50.00 
Pseudomonas sp. 3.12 
______________________________________ 
The activity of A-32887 (Na-K salt) against illustrative bacteria, as 
measured by the conventional disc-diffusion method, is summarized in Table 
VI. 
TABLE VII 
______________________________________ 
Zone of 
Test Organism mcg/disc Inhibition (mm) 
______________________________________ 
Staphylococcus aureus 3055 
300 15.6 
Staphylococcus aureus 3055 
30 12.0 
Staphylococcus aureus 3074* 
300 16.0 
Staphylococcus aureus 3074* 
30 12.7 
Staphylococcus aureus 3130** 
300 15.2 
Staphylococcus aureus 3130** 
30 15.0 
Streptococcus Pyogenes (Group A) 
300 17.5 
Streptococcus pyogenes (Group A) 
30 14.0 
Streptococcus sp. (Group D) 
300 14.7 
Streptococcus sp. (Group D) 
30 13.7 
Diplococcus pneumoniae 
300 18.0 
Diplococcus pneumoniae 
30 16.0 
______________________________________ 
*Penicillin G-resistant 
**Methicillin-resistant 
The antimicrobial activity of two typical A-32887 acyl ester derivatives is 
compared with that of A-32887 (each as Na-K salts) in Table VIII. Activity 
against illustrative bacteria is measured by the conventional 
disc-diffusion method. In addition, the results of a conventional 
paper-disc agar-diffusion assay system (Plate Assay) against Bacillus 
subtilis ATCC 6633 are reported. The activity in this test is quantitated 
and uses a dried-broth reference standard which is assigned an arbitrary 
potency of 100 units/ml. The samples were assayed at 1 mg/ml. 
TABLE VIII 
__________________________________________________________________________ 
Plate Assay 
Staphylococcus 
Bacillus 
Micrococcus 
Bacillus 
Compound units/ml 
aureus subtilis 
lutea subtilis* 
__________________________________________________________________________ 
A-32887 1350 16 18 16 30 
A-32887 Acetyl 
Ester Derivative 
238 trace 12 trace 22 
A-32887 n-Butyryl 
Ester Derivative 
1109 14 16 12 27 
__________________________________________________________________________ 
*Minimal media 
In one important aspect, the A-32887 compounds inhibit the growth of 
anaerobic bacteria. Table IX summarizes the MIC's at which A-32887 (Na-K 
salt) inhibits various anaerobic bacteria, as determined by standard 
agar-dilution assay. End points were read after 24-hour incubation. 
TABLE IX 
______________________________________ 
Test Organism MIC (mcg/ml) 
______________________________________ 
Actinomyces israelii .ltoreq. 0.5 
Clostridium perfringens 
.ltoreq. 0.5 
Clostridium septicum .ltoreq. 0.5 
Eubacterium aerofaciens 
.ltoreq. 0.5 
Peptococcus asaccharolyticus 
.ltoreq. 0.5 
Peptococcus prevoti .ltoreq. 0.5 
Peptostreptococcus anaerobius 
.ltoreq. 0.5 
Peptostreptococcus intermedius 
1.0 
Propionibacterium acnes 
.ltoreq. 0.5 
Bacteriodes fragilis ssp 
fragilis 111 2 
Bacteriodes fragilis ssp 
fragilis 1877 2 
Bacteriodes fragilis ssp 
fragilis 1936B 2 
Bacteriodes fragilis ssp 
thetaiotaomicron 2 
Bacteriodes melaninogenicus 
1856/28 32 
Bacteriodes melaninogenicus 
2736 2 
Bacteriodes vulgatis 4 
Bacteriodes corrodens 2 
Fusobacterium symbiosum 
32 
Fusobacterium necrophorum 
32 
______________________________________ 
The activity of the A-32887 compounds against anaerobic bacteria, 
especially against Clostridium perfringens, suggests that the A-32887 
compounds would be beneficial in the treatment or prevention of enteritis 
in chickens, swine, cattle, sheep, and goats and in the treatment or 
prevention of enterotoxemia in ruminants. 
Activity against mycoplasma is another useful aspect of the antimicrobial 
activity of the A-32887 compounds. Mycoplasma species, also known as 
pleuropneumonia-like (PPLO) organisms, are pathogenic to man and various 
animals. Anti-mycoplasma agents are especially needed by the poultry 
industry. The MIC's of A-32887 (Na-K salt) against typical mycoplasma 
species, as determined by in vitro broth-dilution studies, are summarized 
in Table X: 
TABLE X 
______________________________________ 
Test Organism MIC (mcg/ml) 
______________________________________ 
Mycoplasma gallisepticum 
12.5 
Mycoplasma synoviae 3.12 
Mycoplasma hyorhinis 25.0 
Mycoplasma hyopneumoniae 
6.25 
______________________________________ 
The A-32887 compounds are also antiviral agents. For example, A-32887 is 
active against type B influenza virus (Maryland, dog kidney), 
Transmissible Gastroenteritis virus and Infectious Canine Hepatitis virus, 
as demonstrated by in vitro plaque suppression tests, similar to that 
described by Siminoff, Applied Microbiology 9 [1], 66-72 (1961). 
The acute toxicity of A-32887 (Na-K salt), when administered 
intraperitoneally to mice and expressed as LD.sub.50, is 37.5 mg/kg 
.times. 1. 
A most important property of the A-32887 compounds is their anticoccidial 
activity. For example, feeding experiments show that A-32887 (Na-K salt), 
when present in the feed of young chickens at levels as low as 40 ppm, 
decreases mortality and the number of lesions in chicks which have been 
challenged with coccidia. Tables XI through XIII summarize the results of 
tests with A-32887 (Na-K salt) in chicks challenged with various Eimeria 
species. 
TABLE XI 
__________________________________________________________________________ 
ACTIVITY OF A-32887 AGAINST E. tenella AND E. maxima.sup.1 
Average Average Lesion Score 
Treatment.sup. 2,3 
PPM Mortality.sup.4 
Weight Gain (g).sup.5 
Feed/Gain.sup.6 
Total Intestinal 
Cecal 
__________________________________________________________________________ 
Normal Controls 
0 0 195 1.65 0 0 
Infected Controls 
0 40 66 -- 10.8 3.7 
A-32887 (Na-K salt) 
100 0 159 1.87 1.6 0 
A-32887 (Na-K salt) 
80 0 182 1.83 3.0 0.3 
A-32887 (Na-K salt) 
60 0 155 1.83 5.9 0.6 
A-32887 (Na-K salt) 
40 0 176 1.89 4.9 1.6 
__________________________________________________________________________ 
.sup.1 70,000 oocysts/bird of each of E. tenella and E. maxima 
.sup.2 Infected 48 hours postmedication; terminated seven days 
postinoculation 
.sup.3 Four replicates; five birds/replicate 
.sup.4 Due to coccidiosis 
.sup.5 Per survivor 
.sup.6 Pens without deaths only 
TABLE XII 
__________________________________________________________________________ 
ACTIVITY OF A-32887 AGAINST E. tenella AND E. acervulina 
Oocysts 
Percent 
Percent Average Lesion Score 
Per Bird 
Treatment.sup.1,2 
ppm 
Mortality.sup.3 
Weight Gain.sup.4 
Intestinal 
Cecal 
(1.times.10.sup.6).sup.5 
__________________________________________________________________________ 
Infected Controls 
-- 12.5 63 1.2 3.6 29.37 
A-32887 (Na-K salt) 
121 
0 73 0.3 0 0.4 
A-32887 (Na-K salt) 
100 
0 85 0.6 0.1 1.28 
A-32887 (Na-K salt) 
77 
0 90 0.7 0 5.48 
A-32887 (Na-K salt) 
62 
0 97 0.9 0.1 16.40 
__________________________________________________________________________ 
.sup.1 6 replicates; 5 birds each 
.sup.2 Infection inoculum 48 hours post-medication onset 
.sup.3 Due to coccidiosis 
.sup.4 Normal controls = 100% 
.sup.5 Days 7-9 
TABLE XIII 
__________________________________________________________________________ 
ACTIVITY OF A-32887 AGAINST E. tenella, E. maxima and E. acervulina 
Percent 
Percent Average Lesion Score 
Oocysts/Bird.sup.6 
Treatment.sup.1,2 
ppm 
Mortality.sup.3 
Weight Gain.sup.4 
Feed/Gain.sup.5 
Intestinal 
Cecal 
1.times.10.sup.6 
Normal Controls 
-- 0 100 1.46 -- -- -- 
Infected Controls 
-- 32.5 40 -- 1.2 3.6 63.54 
A-32887 (Na-K salt) 
100 
0 93 1.50 0 0.1 6.49 
A-32887 (Na-K salt) 
75 0 90 1.49 0.9 2.0 24.35 
A-32887 (Na-K salt) 
50 0 79 1.67 1.8 3.8 35.78 
A-32887 (Na-K salt) 
25 35 57 -- 1.1 3.9 64.83 
__________________________________________________________________________ 
.sup.1 7 days post-inoculation; infection inoculum 24 hrs. post-medicatio 
onset 
.sup.2 Six replicates; 5 birds/each 
.sup.3 Due to coccidiosis 
.sup.4 Per survivor 
.sup.5 Pens without deaths, only 
.sup.6 Total days 5, 6 and 7 post-inoculation 
For the prevention or treatment of coccidiosis in poultry, a non-toxic 
anticoccidial amount of an A-32887 compound is administered to birds, 
preferably orally on a daily basis. The A-32887 compound can be supplied 
in many ways, but is is most conveniently supplied with a 
pharmaceutically-acceptable carrier, preferably the feed ingested by the 
birds. Although a variety of factors must be considered in determining an 
appropriate concentration of A-32887 compound, the rates of administration 
are generally in the range of about 30 to about 180 ppm in the feed and 
are preferably in the range of about 50 to about 120 ppm of feed ration. 
This invention further relates to feed compositions adapted to protect 
poultry from coccidiosis and containing from about 45 to about 110 pounds 
of A-32887 compound per ton of poultry feed. 
Another important property of the A-32887 compounds is their ability to 
improve feed-utilization efficiency in ruminants which have a developed 
rumen function. It is known that the efficiency of carbohydrate 
utilization in ruminants is increased by treatments which stimulate the 
animals' rumen flora to produce propionate compounds rather than acetate 
or butyrate compounds (for a more complete discussion see Church et al. in 
"Digestive Physiology and Nutrition of Ruminants", Vol. 2, 1971, pp 622 
and 625). 
The efficiency of feed use can be monitored by observing the production and 
concentration of propionate compounds in the rumen using the method 
described by Arthur P. Raun in U.S. Pat. No. 3,839,557 (see especially 
Example 6). 
Table XIV shows the ratio of volatile-fatty-acid (VFA) concentrations in 
A-32887-treated flasks to concentrations in control flasks in this test. 
TABLE XIV 
__________________________________________________________________________ 
Ratio of Treated to Control 
Molar% 
Molar % 
Molar % 
Total VFA 
Compound Dose(mcg/ml) 
Propionate 
Acetate 
Butyrate 
mM/1. 
__________________________________________________________________________ 
A-32887 (Na-K) 
1 1.4191* 
0.5744 
1.1116 
0.7498 
A-32887 (Na-K) 
10 1.2919* 
0.8857 
0.7012 
1.0086 
A-32887 Methyl 
Ether Derivative 
(Na-K) 1 1.1219* 
0.9451 
0.8166 
1.1286 
A-32887 Methyl 
Ether Derivative 
(Na-K) 5 1.2638* 
0.8997 
0.7404 
1.0693 
__________________________________________________________________________ 
*Statistically significant (P&lt;0.01) by the two-tailed LSD test (R.G.D. 
Steel and J. H. Torrie, "Principles and Procedures of Statistics", 
McGraw-Hill, New york, N.Y., 1960, p. 106) 
Carbohydrate-utilization efficiency is further measured by in vivo tests 
performed in animals which have had a fistula installed in the rumen, 
making it possible to withdraw specimens of the rumen contents. The 
procedure used in testing cattle in this manner is also described in 
Raun's U.S. Pat. No. 3,839,557 (see Example 9). Table XV summarizes the 
results of such a test with A-32887 (Na-K salt) wherein five feed-lot 
cattle weighing approximately 425 kg. were in each group and the mean 
percent increases in ruminal propionate concentration were averaged over 
four analyses in a 24-day treatment period. 
TABLE XV 
______________________________________ 
Molar % Molar% Molar % Total VFA 
Treatment 
Propionate 
Acetate Butyrate 
(mM/1.) 
______________________________________ 
Control 17.4 74.5 8.1 79.9 
A-32887 
(Na-K salt) 
27.1* 63.3* 9.6 79.6 
30 g/ton 
______________________________________ 
Significantly different (P&lt;0.01) from control by the two-tailed LSD test. 
Table XVI summarizes the results of a similar test in sheep with A-32887 
(Na-K salt) wherein four sheep were in each group and the mean percent 
increases in ruminal propionate concentration were averaged over four 
analyses in a 19-day treatment period. 
TABLE XVI 
______________________________________ 
Molar % 
Treatment Propionate 
______________________________________ 
Control 26.5 
A-32887 (Na-K salt) 29.5 
15 g/ton 
______________________________________ 
The A-32887 compounds are typically effective in increasing propionates 
and, thereby, the efficiency of feed-utilization efficiency when 
administered to ruminants orally at rates of from about 0.07 mg/kg/day to 
about 4.0 mg/kg/day. Most beneficial results are achieved at rates of from 
about 0.2 mg/kg/day to about 2.0 mg/kg/day. 
A preferred method of administration is to mix the A-32887 compound with 
the animals' feed; however, it can be administered in other ways, for 
example, tablets, drenches, sustained-release boluses, or capsules. 
Formulation of these various dosage forms can be accomplished by methods 
well known in the veterinary pharmaceutical art. Each individual dosage 
unit should contain a quantity of A-32887 compound directly related to the 
proper daily dose for the animal to be treated. 
This invention further relates to feed compositions adapted to fatten 
cattle comprising cattle feed and from 1 to 25 grams per ton of an A-32887 
compound. 
In another aspect the A-32887 compounds are useful in the prevention and 
control of swine dysentery. A preferred method of administration to swine 
is by incorporation of an appropriate amount of an A-32887 compound into 
the feed ration. The results of tests with A-32887 (Na-K salt) when 
administered to pigs infected with acute swine dysentery are reported in 
Table XVII. In this test, groups of four pigs were challenged orally with 
5.0 ml of a colon-content/tissue suspension prepared from pigs suffering 
from acute swine dysentery. Treated pigs were challenged 24 hours after 
initiating feed treatment. The test was carried out over a period of 26 
days, observing pigs daily, and weighing them weekly and on day 26. 
TABLE XVII 
______________________________________ 
Final- 
Average No. Died No. with Colon 
Wt. per per No. Diarrhea 
Lesions/No. in 
Treatment 
Pig (lbs) 
in Group Index* Group 
______________________________________ 
A-32887 
(Na-K salt) 
100 g/ton 
30.3 0/4 22 2/4 
Infected Non- 
medicated 
14.3 3/4 53 4/4 
Controls 
Infected Non- 
medicated 
14.8 2/4 52 4/4 
Controls 
______________________________________ 
*Fecal material for each group was rated daily with 0=normal, 1=slight 
blood or mucus, 2=moderate blood or mucus, 3=marked blood or mucus. Index 
is the total score per treatment for 25 days. 
The A-32887 compounds are also useful in the treatment of certain plant 
diseases. For example, A-32887 (Na-K salt), when applied at 400 ppm as a 
spray, inhibits powdery mildew disease in bean plants. 
In another aspect, the A-32887 compounds are useful as insecticides. For 
example, A-32887 (Na-K salt) is active against insects such as Mexican 
bean beetle, Southern armyworm and housefly when applied at a rate of 1000 
ppm. 
Antibiotic A-32887 exhibits ion-binding and iontransport properties and is, 
therefore, an ionophore (ion-bearer) (see B. C. Pressman, Alkali metal 
chelators-- the ionophores, in "Inorganic Biochemistry," Volume 1, G. L. 
Eichhorn, Elsevier, 1973). A-32887 can be used when the selective removal 
of particular cations is desired. Examples of such uses include the 
removal and recovery of silver ions from solutions in photography, the 
removal of toxic cations from industrial waste streams before such streams 
are discharged to the environment, and desalinization of sea water. 
A-32887 can be used as one component of an ion-specific electrode (O. 
Kedem, et al., U.S. Pat. No. 3,753,887, Aug. 21, 1973, Alkali metal 
specific measuring electrode). A-32887 alters the cation permeability of 
both natural and artificial membranes. A-32887 can be used, therefore, as 
a component in a membrane used for the selective transport of cations 
against a concentration gradient. One potential application of this 
property is in recovery of heavy and precious metals on a commercial basis 
[see E. L. Cussler, D. F. Evans, and Sister M. A. Matesick, Science 172, 
377 (1971)]. 
In yet another aspect, the A-32887 compounds are active as inhibitors of 
the enzyme ATPase. ATPase, an alkali-metal-sensitive enzyme found in cell 
membranes, is involved in the energy necessary for active transport. 
"Active transport" refers to the energy-requiring series of operations 
whereby intracellular and extracellular fluids maintain their 
compositions. Inhibitors of ATPase reduce the energy required for active 
transport. A-32887 (Na-K salt) has been shown to inhibit ATPase in in 
vitro tests using NaCl. 
The A-32887 compounds are potential cardiotonic agents. In tests using 
guinea pig left atria, A-32887 (Na-K salt) increased cardiac 
contractility. Response to this test is expressed as a percentage of the 
maximal force that could be elicited by a challenge dose of 
norepinephrine. A-32887 (Na-K salt), at a 10.sup.-5 molar concentration, 
increased cardiac contractility by 28.0 .+-. 7.1 percent. 
In order to illustrate more fully the operation of this invention, the 
following examples are provided 
EXAMPLE 1 
A. Shake-flask Fermentation of A-32887 
A lyophilized pellet of Streptomyces albus NRRL 11109 was dissolved in 1-2 
ml of sterilized water. This solution was used to inoculate an agar slant 
having the following composition: 
______________________________________ 
Ingredient Amount 
______________________________________ 
Agar 20 g 
Dextrin 10 g 
Yeast extract 1 g 
Beef extract 1 g 
Enzymatic hydrolysate 
of casein* 2 g 
CoCl.sub.2 . 6H.sub.2 O 
0.01 g 
Deionized water q.s. 1 liter 
______________________________________ 
NaOH was added to raise the pH of the medium from about 6.2 to about 7.0, 
before sterilizing; pH after sterilization about 6.9. 
*NZ Amine A, Humko Sheffield Chemical, Lyndhurst, N.J. 
The inoculated slant was incubated at 30.degree. C. for about 7 days. The 
mature slant culture was scraped with a sterile pipette or loop to loosen 
the spores. About one-fourth of the loosened spores were used to inoculate 
50 ml of a vegetative medium having the following composition: 
______________________________________ 
Ingredient Amount 
______________________________________ 
Glucose 15 g 
Soybean meal 15 g 
Corn steep liquor 10 g 
NaCl 5 g 
CaCO.sub.3 2 g 
Cold tap water q.s. 1 liter 
______________________________________ 
The pH of this medium was adjusted from approximately 5.8 to about 6.5 by 
the addition of NaOH; post-sterilization pH about 6.5. 
The inoculated vegetative medium was incubated in a 250-ml Erlenmeyer flask 
at 30.degree. C. for about 48 hours on a shaker rotating through an arc 
two inches in diameter at 250 RPM. 
This incubated vegetative medium (0.5-2.5 ml; 1-5%) was used to inoculate 
50 ml of a production medium having the following composition: 
______________________________________ 
Ingredient Amount (g/l.) 
______________________________________ 
Glucose 25.0 
Starch 10.0 
Peptone* 10.0 
Enzymatic-hydrolysate of 
casein 4.0 
Blackstrap molasses 5.0 
MgSO.sub.4 . 7H.sub.2 O 
0.5 
CaCO.sub.3 2.0 
Czapek's mineral stock*** 
Deionized water q.s. 1 liter 
______________________________________ 
*Wilson's Peptone 159, Wilsons' Protein Technology 
**NZ Amine A, Humko Sheffield Chemical, Lyndhurst, N.J. 
***Czapek's mineral stock has the following composition: 100 g KCl; 100 g 
MgSO.sub.4 . H.sub.2 O; 2 g FeSO.sub.4 . 7H.sub.2 O; q.s. to 1 liter with 
deionized water 
The inoculated production medium was incubated in a 250-ml Erlenmeyer flask 
at 30.degree. C. for about 2 to 3 days on a shaker rotating through an arc 
two inches in diameter at 250 RPM. 
B. tank Fermentation of A-32887 
In order to provide a larger volume of inoculum, 20 ml of incubated 
vegetative medium, prepared as described in Section A, was used to 
inoculate 400 ml of a second-stage vegetative-growth medium having the 
same composition as that of the vegetative medium. This second-stage 
vegetative medium was incubated in a 2-liter flask for about 24 hours at 
30.degree. C. on a shaker rotating through an arc two inches in diameter 
at 250 RPM. 
Incubated second-stage medium (800 ml) thus prepared was used to inoculate 
100 liters of sterile production medium, prepared as described in Section 
A. The inoculated production medium was allowed to ferment in a 165-liter 
fermentation tank for 3 to 4 days at a temperature of 30.degree. C. The 
fermentation medium was aerated with sterile air at the rate of 0.25 V/V/M 
and was stirred with conventional agitators at 250 RPM. 
EXAMPLE 2 
Separation of A-32887 
Whole fermentation broth (925 l.), obtained by the method described in 
Example 1, was adjusted to pH 8.5 by the addition of NaOH, stirred for 45 
minutes, and filtered with a filter aid (Hyflo Super-cel, a diatomaceous 
earth, Johns-Manville Products Corp.). The filtered cake was washed with 
water, and the water wash was added to the filtered broth. The 
filtered-broth solution was then extracted twice with ethyl acetate (2/3 
volumes). The ethyl acetate extracts were combined and concentrated under 
vacuum to give an oily residue. The residue was dissolved in benzene (4 
l.); the benzene solution was filtered; and the filtrate was applied to a 
9.5- .times. 162-cm silica-gel column (Grace, grade 62), prepared in 
benzene. After washing the column with benzene (24.1.), the eluting 
solvent was changed to benzene:ethyl acetate (3:2), collecting 37 liters 
consisting of fractions of 1 liter each. Elution was monitored by 
silica-gel thin-layer chromatography, using a benzene:ethyl acetate (1:1) 
solvent system and Bacillus subtilis bioautography for detection. The 
active fractions which contained A-32887 (11 1.) were combined and 
evaporated to dryness under vacuum. In order to remove color and other 
impurities, the residue thus obtained was dissolved in chloroform and 
chromatographed on a 2.2- .times. 40-cm column of carbon (Pittsburgh 12 
.times. 40), prepared in chloroform. The column was washed with chloroform 
(3 1.); the chloroform eluate was concentrated to dryness under vacuum. 
The residue thus obtained was dissolved in diethyl ether (200 ml). The 
resulting solution was evaporated slowly under vacuum to give a thick 
syrup. The syrup was slowly warmed, and n-hexane (500 ml) was added with 
stirring. This solution was allowed to stand at room temperature until the 
A-32887 had crystallized. The crystalline A-32887 was separated by 
filtration and dried. The A-32887 was recrystallized by dissolving in 
acetone (500 ml), slowly adding water (200 ml), and allowing the resulting 
solution to stand at room temperature. The recrystallized A-32887 was 
separated by filtration, washed with water and dried to give 59 g of 
A-32887 Na-K salt. Further recrystallization gave additional A-32287 (Na-K 
salt) in the following amounts: 8.4 g in the second crop, and 5.2 g in the 
third crop (mp 158-160.degree.). 
EXAMPLE 3 
Preparation of A-32887 Free Acid 
A-32887 Na-K salt (1 g), obtained as described in Example 2, was dissolved 
in dioxane (200 ml). Water (25 ml) was added to this solution; the 
resulting solution was adjusted to pH 3 by the addition of dilute HC1. The 
acidified solution was stirred, and maintained at pH 3 with HC1, as water 
(100 ml) was slowly added. The resulting solution was evaporated under 
vacuum to remove the dioxane; the resulting aqueous suspension was 
extracted twice with ethyl acetate (equal volumes). The combined ethyl 
acetate extract was evaporated under vacuum to give an oily residue. This 
residue was dissolved in chloroform and re-evaporated under vacuum to give 
A-32887 free acid as a white amorphous powder (524 mg; mp about 90.degree. 
C.) 
EXAMPLE 4 
Preparation of A-32887 Silver Salt 
A-32887 Na-K salt (200 mg), obtained as described in Example 2, was 
dissolved in methanol (10 ml). An aqueous solution of silver nitrate (2 
ml; 50 mg/ml) was added slowly. The resulting solution was placed in a 
beaker wrapped with aluminum foil to prevent degradation (reduction) of 
the silver. The solution was kept at 5.degree. C. until crystallization 
was complete. The crystals were separated by filtration and were 
recrystallized from n-hexane to give A-32887 silver salt as very fine 
white needles (mp 166-168.degree. C.). 
EXAMPLE 5 
Preparation of the Sodium Salt of A-32887 
A-32887 free acid (500 mg), prepared as described in Example 3, was 
dissolved in acetone (150 ml); water (20 ml) was added. The resulting 
mixture was adjusted to pH 9 with NaOH. Water (200 ml) was then added, and 
the resulting solution was stirred for one-half hour. The solution was 
concentrated under vacuum to remove acetone. The resulting suspension was 
extracted with an equal volume of ethyl acetate. The ethyl acetate extract 
was concentrated under vacuum to dryness. The residue was dissolved in 
warm acetone (20 ml); water was added until the solution was turbid; and 
the solution was then allowed to crystallize. The crystals were removed by 
filtration, washed with water, and dried to give 306 mg of A-32887 sodium 
salt (mp 130.degree.-133.degree. C.). 
EXAMPLE 6 
Preparation of the Methyl Ether Derivative of A-32887 
A-32887 free acid (3 g), prepared as described in Example 3, was dissolved 
in methanol (300 ml) and allowed to stand at room temperature for 12 
hours. The conversion was monitored by silica-gel TLC, using a 
benzene:ethyl acetate (1:1) solvent system and H.sub.2 SO.sub.4 spray for 
detection. The solution was evaporated to dryness under vacuum. The 
residue obtained was dissolved in benzene (40 ml). This solution was 
applied to a 3.2- .times.95-cm column of silica gel (Grace, grade 62), 
packed in benzene. The column was eluted with benzene:ethyl acetate (3:2), 
collecting 25-ml fractions, and monitoring elution by TLC. Fractions 
90-220, which contained most of the desired product, were combined and 
evaporated under vacuum to dryness. The residue, dissolved in benzene (15 
ml) was rechromatographed on a 1.8- .times. 112-cm column of silica gel 
(Grace, grade 62), prepared in benzene. The column was eluted with 
benzene:ethyl acetate (9:1), collecting 25-ml fractions and monitoring 
elution by TLC. At fraction 383, the eluting solvent was changed to 
benzene:ethyl acetate (4:1); and at fraction 780 the solvent was changed 
to benzene:ethyl acetate (7:3). Fractions 450-700 contained A-32887; 
fractions 702-760 contained a mixture of A-32887 and A-32887 methyl ether 
derivative; and fractions 761-1130 contained A-32887 methyl ether 
derivative. Fractions 761-1130 were combined and evaporated to dryness 
under vacuum. The residue was dissolved in ethyl acetate (20 ml); n-hexane 
(80 ml) was added; and the solution was allowed to crystallize. The 
crystals were removed by filtration and dried to give 242 mg of A-32887 
methyl ether derivative as the sodium salt (mp 214.degree.-216.degree. 
C.). 
EXAMPLE 7 
Preparation of the Acetyl Ester Derivative of A-32887 
A-32887 Na-K salt (200 mg), prepared as described in Example 2, was 
dissolved in pyridine (8 ml); acetic anhydride (3.2 ml) was added. The 
mixture was allowed to stand overnight and then was evaporated under 
vacuum to dryness. The residue was dissolved in t-butanol and lyophilized 
to give 231 mg of the acetyl ester derivative of A-32887 (Na-K salt) as a 
white amorphous powder, mp 127.degree.-129.degree. C. 
EXAMPLE 8 
Preparation of the n-Butyryl Ester Derivative of A-32887 
A-32887 (Na-K salt; 200 mg) was dissolved in pyridine (14 ml), and 
n-butyric anhydride (14 ml) was added. The mixture was allowed to stand 
for 17 hours at room temperature; water (14 ml) was added; and the 
solution was then concentrated to an oil is vacuo. The oily residue was 
lyophilized from dioxane-water several times to give 240 mg of the 
n-butyryl ester derivative of A-32887 (Na-K salt) as a white amorphous 
powder, mp 59.degree.-62.degree. C. 
EXAMPLE 9 
A-32887-modified Chick Ration for Coccidiosis Control 
A balanced, high-energy ration adapted to feed chicks for rapid weight gain 
is prepared by the following recipe: 
______________________________________ 
Ingredient % lbs 
______________________________________ 
Ground yellow corn 50 1,000 
Soybean meal, solvent- 
extracted dehulled, finely 
ground, 50 percent protein 
31.09 621.8 
Animal fat (beef tallow) 
6.5 130 
Dried fish meal, with 
solubles (60% protein) 
5.0 100 
Distillers' solubles 
from corn 4.0 80 
Dicalcium phosphate, 
feed grade 1.8 36 
Calcium carbonate 0.8 16 
Vitamin premix 
(representing vitamins A, 
D, E, K, and B.sub.12, choline, 
niacin, pantothenic acid, 
riboflavin, biotin, with 
glucose bulking agent) 
0.5 10 
Trace mineral premix 
(representing MnSO.sub.4, ZnO, 
KI, FeSO.sub.4, CaCO.sub.3) 
0.2 4 
2-Amino-4-hydroxybutyric 
acid 
(hydroxy analog of 
methionine) 0.1 2 
A-32887 (Na-K Salt) 
0.01 0.2 
______________________________________ 
These substances are mixed in accordance with standard feed-mixing 
techniques. Chicks fed such a ration, with water ad libitum, are protected 
against exposure to coccidiosis; weight gains are comparable to those of 
coccidiosis-free chicks fed a similar, unmedicated diet. 
EXAMPLE 10 
A-32887-improved Beef-Cattle Ration 
A balanced high-grain beef-cattle ration is prepared as follows: 
______________________________________ 
Ingredient % lbs 
______________________________________ 
Finely ground corn 67.8 1356 
Ground corn cob 10 200 
Dehydrated alfalfa meal, 
17 percent protein 5 100 
Dehulled soybean meal, 
solvent extracted, 
50 percent protein 9.9956 199.912 
Cane molasses 5 100.0 
Urea 0.6 12.0 
A-32887 (Na-K salt) 
0.0044 0.088 
Dicalcium phosphate, 
feed grade 0.5 10.0 
Calcium carbonate 0.5 10.0 
Sodium chloride 0.3 6.0 
Trace mineral premix 
0.03 0.6 
Vitamin A and D.sub.2 premix* 
0.07 1.4 
Vitamin E premix** 0.05 1.0 
Calcium propionate 0.15 3.0 
______________________________________ 
*Containing per pound: 2,000,000 I.U. of vitamin A; 227,200 I.U. of 
vitamin D.sub.2 and 385.7 g. of soybean feed with 1% oil added 
**Corn distillers dried grains with solubles containing 20,000 I.U. of 
d-alpha-tocopheryl acetate per pound 
The mixed feed is compressed into pellets. At an average daily ingestion 
rate of 15 pounds of feed per animal, this feed supplies approximately 300 
mg. of A-32887 (Na-K salt) per animal per day. 
EXAMPLE 11 
A-32887-improved Swine Ration 
A balanced swine ration is prepared as follows: 
______________________________________ 
Ingredient % lbs./ton 
______________________________________ 
Corn, Yellow, Ground 73.15 1463 
Soybean Oil Meal, Solvent 
Extracted, Dehulled, 50% 
12.30 246 
Alfalfa Meal, Dehydrated, 17% 
2.50 50 
Meat Scraps, 55% 2.50 50 
Fish Meal 2.50 50 
Distiller Dried Solubles (Corn) 
2.50 50 
Animal Fat 2.00 40 
Calcium Carbonate 0.70 14 
Dicalcium Phosphate, Feed Grade 
0.50 10 
NaCl 0.50 10 
Swine Vitamin Premix.sup.1 
0.50 10 
Methionine Hydroxy Analog, 93% 
0.20 4 
Trace Mineral Premix.sup.2 
0.10 2 
Selenium Premix.sup.3 
0.05 1 
Total 100.00 2000 
______________________________________ 
.sup.1 Each kg of premix contains the following: 77,161 IU Vitamin D.sub. 
; 2,205 IU Vitamin E; 411 mg riboflavin; 1,620 mg panthothenic acid; 2,20 
mg niacin; 4.4 mg Vitamin B.sub.12 ; 441 mg Vitamin K; 19,180 mg choline; 
110 mg folic acid; 165 mg pyridoxine; 110 mg thiamine; 22 mg biotin. 
.sup.2 Each kg of premix contains the following: 50 g manganese as 
manganese sulfate; 100 g zinc as zinc carbonate; 50 g iron as ferrous 
sulfate; 5 g copper as copper oxide; 1.5 g iodine as potassium iodide and 
150 g maximum and 130 g minimum calcium as calcium carbonate. 
.sup.3 Each kg of premix contains 200 mg of selenium as sodium selenite. 
A-32887 (Na-K salt; 100 g) is mixed with from about 5 to about 10 lbs. of 
soybean mill run to give a feed premix. From 5-10 lbs of this A-32887 
premix is thoroughly mixed with a sufficient amount of the above-described 
swine ration to give a concentration of 100 g of A-32887 per ton of 
ration. Swine fed such a ration, with water ad libitum, are protected 
against the lethal effects of swine dysentery.