Kalafungin, a new antibiotic produced by Streptomyces tanashiensis strain Kala.

APPuED MICROBIOLOGY, Dec. 1968, P. 1815-1821 Copyright @ 1968 American Society for Microbiology Vol. 16, No. 12 Printed in U.S.A. Kalafungin, a New Antibiotic Produced by Streptomyces tanashiensis Strain Kala LEROY E. JOHNSON AND ALMA DIETZ Department ofMicrobiology, The Upjohn Company, Kalamazoo, Michigan 49001 Received for publication 21 August 1968 Kalafungin is a new antimicrobial agent obtained from the culture broth of a soil isolate of Streptomyces tanashiensis, designated Streptomyces tanashiensis strain Kala UC-5063. Kalafungin is a chemically stable, nonpolyene agent which is exextremely inhibitory in vitro against a variety of pathogenic fungi, yeasts, protozoa, gram-positive bacteria, and, to a lesser extent, gram-negative bacteria. fermentation medium consisting of (per liter) 25 g of glucose monohydrate, 5 g of liquid peptone (Wilson Laboratories, Chicago, Ill.), 5 g of calcium carbonate, and tap water to 1 liter (pH adjusted to 7.2 before sterilization). Shaken-flask fermentations were run in 500-ml Erlenmeyer flasks containing 100 ml of medium, and were incubated at 32 C for optimal yields on a Gump rotary shaker operating at 250 rev/min with a 2.5-inch (6.4 cm) stroke. Antibiotic assay procedure. The antibiotic concentrations were determined with a standard disc-plate agar diffusion assay. Samples (0.08 ml) in 0.1 M phosphate buffer (pH 7.0) were placed on 12.7-mm paper discs and were assayed against Saccharomyces pastorianus. The zone of inhibition of growth was measured after incubation for 18 hr at 28 C. The assay procedure can also be used with the more sensitive S. cerevisiae. The activity was expressed in terms of biounits; a biounit is defined as the amount of antibiotic necessary to give a 20-mm zone of inhibition after 18 hr of incubation under these standard conditions. The antibacterial in vitro spectrum was determined by twofold dilution end points in Brain Heart Infusion MATERIALS AND METHODS (Difco) broth. Readings were made after 20 hr of Culture. Kalafungin is produced by a soil isolate, incubation at 37 C. The antifungal in vitro spectrum designated S. tanashiensis strain Kala UC-5063. The was determined by the agar dilution plate assay of organism was identified by color pattern, microscopic Whiffen (11), with readings made after 72 hr at 28 C. appearance, cultural characteristics, and comparison The antiprotozoal in vitro activity was determined by with known cultures of S. tanashiensis. Subsequent spotting paper discs on agar trays seeded with the test studies using the fermentation conditions described organism; the trays were incubated at room temperaherein showed kalafungin was also produced by ture (24 C) and read after 48 hr. S. tanashiensis NRRL B-1692. The production of Kalafungin was differentiated from other antikalafungin by S. tanashiensis (10) has not been pre- biotics by its antibacterial, antifungal, and antiprotoviously reported. zoal spectra, chemical properties, and by paper Medium. Seed flasks were inoculated with spore chromatography. For chromatography, the antipreprarations of the culture which were maintained biotic was spotted on Whatman no. 1 filter paper and in soil. The culture was incubated at 28 C for 72 hr developed without prior equilibration by use of the in a seed medium consisting of 25 g of glucose mono- descending method and the solvent systems listed in hydrate (Cerelose) and 25 g of Pharmamedia (Traders Fig. 3. Antibiotic activity was located by plating the Oil Mill Co., Fort Worth, Tex.) per liter. The vege- developed strips on trays of agar seeded with S. tative seed was used at a rate of 5% to inoculate a pastorianus subsp. arbignensis (ATCC 2366). 1815 Streptomyces tanashiensis strain Kala produces a new antibiotic designated as kalafungin. [Referred to as kalamycin (U-19,718) in U.S. Patent 3,300,382]. Bergy (1) characterized kalafungin as a chemically stable, nonpolyene agent with a molecular formula CirH1203 and a molecular weight of 300. The compound is weakly acidic, pKa = 10.0, orange-colored, and detrarotatory in chloroform, [a] 2D5 + 159. It exhibits ultraviolet and visible absorption maxima at 212, 256, and 425 nm in methanol. The material melts at about 163 to 166 C, is slightly soluble in water, but readily soluble in ethyl acetate, chloroform, and acetone. Reusser (8) reported that kalafungin inhibited respiration and phosphorylation in rat liver mitochondria. This paper describes the organism, fermentation conditions, paper chromatogram characterization, and some of the biological properties of the antibiotic. JOHNSON AND DIETZ APPL. MICROBIOL. STREPTOMYCES TANASHIENSIS STRAIN KALA 2 3 4 5 ii I STREPTQMYCES TANASHIENSIS NRRL B - 1692 2 3 4 5 FIG. 1. Ektacolor photographs of Streptomyces tanashiensis. (A) S. tanashiensis strain Kala; (B) S. tanashiensis NRRL B-i1692. VOL. 16, 1968 _ .. :. KALAFUNGIN, A NEW ANTIBIOTIC _ 1817 . * 2_ _ __ :; .... :: .S :::: B C _ A D - FiG. 2. Electron micrographs of spores of Streptomyces tanashiensis. Each index mark equals I micrometer. (A) S. tanashiensis strain Kala, whole spore mount; (B) S. tanashiensis strain Kala, carbon repligraph; (C) S. tanashiensis NRRL B-1692, whole spore mount; (D) S. tanashiensis NRRL B-1692, carbon repligraph. 1818 JOHNSON AND DIETZ APPL. MICROBIOL.- TABLE 1. General cultural characteristics of Streptomyces tanashiensis Medium S. tanashiensis strain Kala No aerial growth. Brown vegetative growth. Melanin-positive. Pale pink aerial growth. Cream-pink reverse. Trace cream-pink pigment. Malate solubilized. Glucose-aspara- No aerial growth. Pale cream vegetative growth. No pigment. gine agar No aerial growth. Colorless vegetative Skim milk agar growth. Yellow pigment. Casein solubilized. Pale gray aerial growth. Olive-cream Xanthine agar reverse. Olive-cream pigment. Xanthine solubilized. Pale gray aerial growth. Tan reverse. Tyrosine agar Tan pigment. Tyrosine solubilized. Casein starch agar Gray aerial growth. Gray-pink reverse. No pigment. Yeast extract-malt Gray-pink aerial growth. Red-tan reverse. Red-tan pigment. extract agar Heavy gray-white aerial growth. TanBennett's agar 18C brown reverse. 24C Heavy gray-white aerial growth. Tanbrown reverse. 28C Heavy gray aerial growth. Tan-brown reverse. Tan pigment. 37C Fair gray aerial growth. Tan-brown reverse. Tan pigment. Czapek's sucrose Poor gray-white aerial growth. Gray reverse. No pigment. 18C agar 24C Poor gray-white aerial growth. Gray reverse. No pigment. 28C Fair gray aerial growth. Gray reverse. No pigment. 37C Fair gray aerial growth. Gray reverse. No pigment. Maltose tryptone 18C Heavy gray-white aerial growth. agar Brown reverse. Pale tan pigment. 24C Heavy gray-white aerial growth. Brown reverse. Pale tan pigment. 37C Fair gray aerial growth. Tan-brown reverse. Tan pigment. Brown surface ring. Peptonization. pH Litmus milk 8.0. Brown pigment in upper one-quarter Plain gelatin of medium. Liquefaction complete. Nutrien (...truncated)