Preparation of an antibiotic selectively effective against staphylococcus infections

Claim 2

A biologically pure culture of the microorganism Rothia dentocariosa, having the identifying characteristics of ATCC 31918, said culture being capable of producing the antibiotic staphylocidin, having selective antimicrobial activity against staphylococci including S. aureus and S. epidermidis, in a recoverable quantity upon fermentation in an aqueous nutrient medium containing assimilable sources of carbon, nitrogen and inorganic substances.

Claim 3

The biologically pure culture of claim 1, characterized as follows: (a) Morphology after 72 hours growth: colonies are smooth, creamy, off-white with an entire border, while the individual cells within each colony are gram-positive and cocco-bacillary in shape, and not incorporating any filamentous forms or being acid fast: (b) Biochemical reactions after 48 hours growth: ______________________________________ Positive (+) or Negative (-) Reaction ______________________________________ Hemolysis of sheep blood - Indole - Voges-Proskauer - Gelatinase + Nitrate reduction + Catalase + Urease - Lecithinase - Lipase - H.sub.2 S production - Hippurate hydrolysis - Aesculin hydrolysis + Starch hydrolyis - ______________________________________ (c) Acid-forming reactions with: ______________________________________ Positive (+) or Negative (-) Reaction ______________________________________ Adonitol - Amygdalin - L-Arabinose - Cellobiose - Dulcitol - Erythritol - D-Fructose + D-Galactose - D-Glucose + Glycerol - Glycogen - i-Inositol - Inulin - Lactose - Maltose + D-Mannitol - D-Mannose + D-Melezitose + Melibiose - Raffinose - L-Rhamnose - D-Ribose - Salicin + D-Sorbitol - L-Sorbose - Sucrose + Trehalose + D-Xylose - ______________________________________ (d) Whole-cell hydrolyzate: galactose as the sole carbohydrate, no DAP and no arabinose; and (e) Carbohydrate fermentation end-products: lactic acid and succinic acid, but not propionic acid.

Claim 4

The biologically pure culture of claim 1, capable of producing upon fermentation a cell murein: (a) comprising a peptidoglycan polymer having the following approximate amino acid and amino sugar contents: ______________________________________ Contents by Weight Amino Acid Relative to Alanine ______________________________________ Asp 0.16 Thr 0.09 Ser 0.07 Glu 0.43 Gly 0.16 Ala 1.00 Val 0.16 Ile 0.09 Leu 0.20 Tyr 0.06 Phe 0.08 Lys 0.34 His 0.04 Arg. 0.10 Muramic Acid 0.47 Glucosamine 1.04; and ______________________________________ (b) exhibiting the following R f values upon thin layer chromatography in an isopropanol/acetic acid/water system on silica gel G plates, after digestion with lysozymen and lysostaphin: ______________________________________ Fraction R.sub.f Value ______________________________________ (1) 0.66 ± 0.01 [n-acetylglucosamine = 0.65] (2) 0.58 ± 0.01 (3) 0.59 ± 0.01 (4) 0.74 ± 0.01 [n-acetylmuramic acid = 0.73]; and ______________________________________ (c) having bonded thereto the narrow-spectrum antibiotic staphylocidin, which is selectively effective against staphylococci including S. aureus and S. epidermidis; the murein forming a staphylocidin-containing extract upon treatment with a glacial acetic acid or n-butanol solvent and evaporation of the solvent from the supernate, which extract exhibits the following characteristics: (1) a biologically active component with an R f value of 0.98-0.99 upon thin layer chromatography in a methanol/acetic acid/phosphate-buffered saline system on a microcrystalline cellulose plate and yellow-green fluorescence in long wave ultraviolet light; (2) a biologically active component with an R f value of 0.04 upon thin layer chromatography in a butanol/acetic acid/phosphate-buffered saline system on a microcrystalline cellulose plate and yellow-green fluorescence in long wave ultraviolet light; (3) a biologically active component with an R f value of 0.04 upon thin layer chromatography in an ethyl acetate/pyridine/water system on a microcrystalline cellulose plate and yellow-green fluorescence in long wave ultraviolet light; (4) a component with an R f value of 0.88 upon thin layer chromatography in a methanol/acetic acid/phosphate-buffered saline system on a silica gel plate and yellow-green fluorescence in long wave ultraviolet light; and (5) the R f 0.98-0.99 component containing staphylocidin and exhibiting the HPLC peaks plotted in FIG. 1 of the accompanying drawings.

Claim 5

A staphylocidin-containing product, which comprises the cell murein recovered from the fermentation product of the biologically pure culture of either of claims 1 or 2: (a) comprising a peptidoglycan polymer having the following approximate amino acid and amino sugar contents: ______________________________________ Contents by Weight Amino Acid Relative to Alanine ______________________________________ Asp 0.16 Thr 0.09 Ser 0.07 Glu 0.43 Gly 0.16 Ala 1.00 Val 0.16 Ile 0.09 Leu 0.20 Tyr 0.06 Phe 0.08 Lys 0.34 His 0.04 Arg. 0.10 Muramic Acid 0.47 Glucosamine 1.04; and ______________________________________ (b) exhibiting the following R f values upon thin layer chromatography in an isopropanol/acetic acid/water system on silica gel G plates, after digestion with lysozymen and lysostaphin: ______________________________________ Fraction R.sub.f Value ______________________________________ (1) 0.66 ± 0.01 [n-acetylglucosamine = 0.65] (2) 0.58 ± 0.01 (3) 0.59 ± 0.01 (4) 0.74 ± 0.01 [n-acetylmuramic acid = 0.73]; and ______________________________________ (c) having bonded thereto the narrow-spectrum antibiotic staphylocidin, which is selectively effective against staphylococci including S. aureus and S. epidermidis; the murein forming a staphylocidin-containing extract upon treatment with a glacial acetic acid or n-butanol solvent and evaporation of the solvent from the supernate, which extract exhibits the following characteristics: (1) a biologically active component with an R f value of 0.98-0.99 upon thin layer chromatography in a methanol/acetic acid/phosphate-buffered saline system on a microcrystalline cellulose plate and yellow-green fluorescence in long wave ultraviolet light; (2) a biologically active component with an R f value of 0.04 upon thin layer chromatography in a butanol/acetic acid/phosphate-buffered saline system on a microcrystalline cellulose plate and yellow-green fluorescence in long wave ultraviolet light; (3) a biologically active component with an R f value of 0.04 upon thin layer chromatography in an ethyl acetate/pyridine/water system on a microcrystalline cellulose plate and yellow-green fluorescence in long wave ultraviolet light; (4) a component with an R f value of 0.88 upon thin layer chromatography in a methanol/acetic acid/phosphate-buffered saline system on a silica gel plate and yellow-green fluorescence in long wave ultraviolet light; and (5) the R f 0.98-0.99 component containing staphylocidin and exhibiting the HPLC peaks plotted in FIG. 1 of the accompanying drawings.

Claim 6

A staphylocidin-containing product, which comprises the cell murein recovered from the fermentation product of the biologically pure culture of claim 3.

Claim 7

A process for producing the narrow-spectrum antibiotic staphylocidin having selective antimicrobial activity against staphylococci including S. aureus and S. epidermidis, which comprises cultivating the bacterium of claim 1 in an aqueous nutrient medium containing assimilable sources of carbon, nitrogen and inorganic salts under submerged aerobic conditions until substantial antibacterial activity due to staphylocidin is produced.

Claim 8

The process of claim 6, including recovering a staphylocidin-containing product by separating the cell murein having the staphylocidin bonded thereto from the fermentation broth.

Claim 9

A process for producing the narrow-spectrum antibiotic staphylocidin having selective antimicrobial activity against staphylococci including S. aureus and S. epidermidis, which comprises cultivating the bacterium of claim 2 in an aqueous nutrient medium containing assimilable sources of carbon, nitrogen and inorganic salts under submerged aerobic conditions until substantial antibacterial activity due to staphylocidin is produced.

Claim 10

The process of claim 8, including recovering a staphylocidin-containing product by separating the cell murein having the staphylocidin bonded thereto from the fermentation broth.

Claim 11

A process for producing the narrow-spectrum antibiotic staphylocidin having selective antimicrobial activity against staphylococci including S. aureus and S. epidermidis, which comprises cultivating the bacterium of claim 3 in an aqueous nutrient medium containing assimilable sources of carbon, nitrogen and inorganic salts under submerged aerobic conditions until substantial antibacterial activity due to staphylocidin is produced.

Claim 12

The process of claim 10, including recovering a staphylocidin-containing product by separating the cell murein having the staphylocidin bonded thereto from the fermentation broth.

Claim 13

A staphylocidin-containing product, prepared by the process of any of claims 6-11.

Claim 14

A product containing the narrow spectrum antibiotic staphylocidin which is selectively effective against staphylococci including S. aureus and S. epidermidis, said product comprising a peptidoglycan polymer obtained from the microorganism Rothia dentocariosa, having the identifying characteristics of ATCC 31918, said polymer having the following approximate amino acid and amino sugar contents: ______________________________________ Contents by Weight Amino Acid Relative to Alanine ______________________________________ Asp 0.16 Thr 0.09 Ser 0.07 Glu 0.43 Gly 0.16 Ala 1.00 Val 0.16 Ile 0.09 Leu 0.20 Tyr 0.06 Phe 0.08 Lys 0.34 His 0.04 Arg 0.10 Muramic Acid 0.47 Glucosamine 1.04 ______________________________________ and which product: (a) exhibits the following R f values upon thin layer chromatography in an isopropanol/acetic acid/water system on silica gel G plates, after digestion with lysozyme and lysostaphin: ______________________________________ Fraction R.sub.f Value ______________________________________ (1) 0.66 ± 0.01 [n-acetylglucosamine = 0.65] (2) 0.58 ± 0.01 (3) 0.59 ± 0.01 (4) 0.74 ± 0.01 [n-acetylmuramic acid = 0.73]; and ______________________________________ (b) is so bonded to the staphylocidin as to be non-dissociable therefrom after treatment with 8 molar urea or boiling with sodium dodecyl sulfate; (c) upon treatment with a glacial acetic acid or n-butanol solvent and evaporation of the solvent from the supernate, leaves a staphylocidin-containing extract exhibiting the following characteristics: (1) a biologically active component with an R f value of 0.98-0.99 upon thin layer chromatography in a methanol/acetic acid/phosphate-buffered saline system on a microcrystalline cellulose plate and yellow-green fluorescence in long wave ultraviolet light; (2) a biologically active component with an R f value of 0.04 upon thin layer chromatography in a butanol/acetic acid/phosphate-buffered saline system on a microcrystalline cellulose plate and yellow-green fluorescence in long wave ultraviolet light; (3) a biologically active component with an R f value of 0.4 upon thin layer chromatography in an ethyl acetate/pyridine/water system on a microcrystalline cellulose plate and yellow-green fluorescence in long wave ultraviolet light; and (4) a component with an R f value of 0.88 upon thin layer chromatography in a methanol/acetic acid/phosphate-buffered saline system on a silica gel plate and yellow-green fluorescence in long wave ultraviolet light; (5) the R f 0.98-0.99 component containing staphylocidin and exhibiting the HPLC peaks plotted in FIG. 1 of the accompanying drawings.

Claim 15

A product containing the narrow spectrum antibiotic staphylocidin which is selectively effective against staphylococci including S. aureus and S. epidermidis, said product comprising a peptidoglycan polymer obtained from the microorganism Rothia dentocariosa, having the identifying characteristics of ATCC 31918, said polymer having the following approximate amino acid and amino sugar contents: ______________________________________ Contents by Weight Amino Acid Relative to Alanine ______________________________________ Asp 0.16 Thr 0.09 Ser 0.07 Glu 0.43 Gly 0.16 Ala 1.00 Val 0.16 Ile 0.09 Leu 0.20 Tyr 0.06 Phe 0.08 Lys 0.34 His 0.04 Arg 0.10 Muramic Acid 0.47 Glucosamine 1.04 ______________________________________ and which product: (a) exhibits the following R f values upon thin layer chromatography in an isopropanol/acetic acid/water system on silica gel G plates, after digestion with lysozyme and lysostaphin: ______________________________________ Fraction R.sub.f Value ______________________________________ (1) 0.66 ± 0.01 [n-acetylglucosamine = 0.65] (2) 0.58 ± 0.01 (3) 0.59 ± 0.01 (4) 0.74 ± 0.01 [n-acetylmuramic acid = 0.73]; and ______________________________________ (b) is so bonded to the staphylocidin as to be non-dissociable therefrom after treatment with 8 molar urea or boiling with sodium dodecyl sulfate; (c) upon treatment with a glacial acetic acid or n-butanol solvent and evaporation of the solvent from the supernate, leaves a staphylocidin-containing extract exhibiting the following characteristics: (1) a biologically active component with an R f value of 0.98-0.99 upon thin layer chromatography in a methanol/acetic acid/phosphate-buffered saline system on a microcrystalline cellulose plate and yellow-green fluorescence in long wave ultraviolet light; (2) a biologically active component with an R f value of 0.04 upon thin layer chromatography in a butanol/acetic acid/phosphate-buffered saline system on a microcrystalline cellulose plate and yellow-green fluorescence in long wave ultraviolet light; (3) a biologically active component with an R f value of 0.4 upon thin layer chromatography in an ethyl acetate/pyridine/water system on a microcrystalline cellulose plate and yellow-green fluorescence in long wave ultraviolet light; and (4) a component with an R f value of 0.88 upon thin layer chromatography in a methanol/acetic acid/phosphate-buffered saline system on a silica gel plate and yellow-green fluorescence in long wave ultraviolet light; (5) the R f 0.98-0.99 component containing staphylocidin and exhibiting the HPLC peaks plotted in FIG. 1 of the accompanying drawings.