streptomyces tanashiensis strain kala

APPuED MICROBIOLOGY, Dec. 1968, P. 1815-1821Copyright @ 1968 American Society for Microbiology

Vol. 16, No. 12Printed in U.S.A.

Kalafungin, a New Antibiotic Produced byStreptomyces tanashiensis Strain Kala

LEROY E. JOHNSON AND ALMA DIETZDepartment 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 asoil isolate of Streptomyces tanashiensis, designated Streptomyces tanashiensis strainKala UC-5063. Kalafungin is a chemically stable, nonpolyene agent which is ex-extremely inhibitory in vitro against a variety of pathogenic fungi, yeasts, protozoa,gram-positive bacteria, and, to a lesser extent, gram-negative bacteria.

Streptomyces tanashiensis strain Kala producesa new antibiotic designated as kalafungin. [Re-ferred to as kalamycin (U-19,718) in U.S.Patent 3,300,382]. Bergy (1) characterized kala-fungin as a chemically stable, nonpolyene agentwith a molecular formula CirH1203 and a molecu-lar weight of 300. The compound is weakly acidic,pKa = 10.0, orange-colored, and detrarotatory inchloroform, [a] 2D5 + 159. It exhibits ultravioletand visible absorption maxima at 212, 256, and425 nm in methanol. The material melts at about163 to 166 C, is slightly soluble in water, butreadily soluble in ethyl acetate, chloroform, andacetone.

Reusser (8) reported that kalafungin inhibitedrespiration and phosphorylation in rat liver mito-chondria. This paper describes the organism, fer-mentation conditions, paper chromatogram char-acterization, and some of the biological propertiesof the antibiotic.

MATERIALS AND METHODS

Culture. Kalafungin is produced by a soil isolate,designated S. tanashiensis strain Kala UC-5063. Theorganism was identified by color pattern, microscopicappearance, cultural characteristics, and comparisonwith known cultures of S. tanashiensis. Subsequentstudies using the fermentation conditions describedherein showed kalafungin was also produced byS. tanashiensis NRRL B-1692. The production ofkalafungin by S. tanashiensis (10) has not been pre-viously reported.

Medium. Seed flasks were inoculated with sporepreprarations of the culture which were maintainedin soil. The culture was incubated at 28 C for 72 hrin a seed medium consisting of 25 g of glucose mono-hydrate (Cerelose) and 25 g of Pharmamedia (TradersOil Mill Co., Fort Worth, Tex.) per liter. The vege-tative seed was used at a rate of 5% to inoculate a

fermentation medium consisting of (per liter) 25 g ofglucose monohydrate, 5 g of liquid peptone (WilsonLaboratories, Chicago, Ill.), 5 g of calcium carbonate,and tap water to 1 liter (pH adjusted to 7.2 beforesterilization). Shaken-flask fermentations were run in500-ml Erlenmeyer flasks containing 100 ml of me-dium, and were incubated at 32 C for optimal yieldson a Gump rotary shaker operating at 250 rev/minwith a 2.5-inch (6.4 cm) stroke.

Antibiotic assay procedure. The antibiotic concen-trations were determined with a standard disc-plateagar diffusion assay. Samples (0.08 ml) in 0.1 M phos-phate buffer (pH 7.0) were placed on 12.7-mm paperdiscs and were assayed against Saccharomycespastorianus. The zone of inhibition of growth wasmeasured after incubation for 18 hr at 28 C. Theassay procedure can also be used with the more sensi-tive S. cerevisiae. The activity was expressed in termsof biounits; a biounit is defined as the amount ofantibiotic necessary to give a 20-mm zone of inhibitionafter 18 hr of incubation under these standardconditions.The antibacterial in vitro spectrum was determined

by twofold dilution end points in Brain Heart Infusion(Difco) broth. Readings were made after 20 hr ofincubation at 37 C. The antifungal in vitro spectrumwas determined by the agar dilution plate assay ofWhiffen (11), with readings made after 72 hr at 28 C.The antiprotozoal in vitro activity was determined byspotting paper discs on agar trays seeded with the testorganism; the trays were incubated at room tempera-ture (24 C) and read after 48 hr.

Kalafungin was differentiated from other anti-biotics by its antibacterial, antifungal, and antiproto-zoal spectra, chemical properties, and by paperchromatography. For chromatography, the anti-biotic was spotted on Whatman no. 1 filter paper anddeveloped without prior equilibration by use of thedescending method and the solvent systems listed inFig. 3. Antibiotic activity was located by plating thedeveloped strips on trays of agar seeded with S.pastorianus subsp. arbignensis (ATCC 2366).

1815

on April 12, 2018 by guest

http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

JOHNSON AND DIETZ

STREPTOMYCES TANASHIENSIS STRAIN KALA2 3 4 5

iiI

STREPTQMYCES TANASHIENSIS NRRL B - 16922 3 4 5

FIG. 1. Ektacolor photographs ofStreptomyces tanashiensis. (A) S. tanashiensis strain Kala; (B) S. tanashiensisNRRL B-i1692.

APPL. MICROBIOL.


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

KALAFUNGIN, A NEW ANTIBIOTIC_ .

*_ :; ....2_

__ ::.S

::::

_ .. :.B

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.

1817VOL. 16, 1968

C _


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

JOHNSON AND DIETZ

TABLE 1. General cultural characteristics of Streptomyces tanashiensis

Medium S. tanashiensis strain Kala S. tanaskiensis NRRL B-1692

Peptone-ironagar

Calcium malateagar

Glucose-aspara-gine agar

Skim milk agar

Xanthine agar

Tyrosine agar

Casein starch agar

Yeast extract-maltextract agar

Bennett's agar18C24C

28C

37C

Czapek's sucroseagar 18C

24C

28C

37C

Maltose tryptoneagar 18C

24C

37C

Litmus milk

Plain gelatin

Nutrient nitratebroth

Synthetic nitratebroth

No aerial growth. Brown vegetativegrowth. Melanin-positive.

Pale pink aerial growth. Cream-pinkreverse. Trace cream-pink pigment.Malate solubilized.

No aerial growth. Pale cream vegeta-tive growth. No pigment.

No aerial growth. Colorless vegetativegrowth. Yellow pigment. Caseinsolubilized.

Pale gray aerial growth. Olive-creamreverse. Olive-cream pigment. Xan-thine solubilized.

Pale gray aerial growth. Tan reverse.Tan pigment. Tyrosine solubilized.

Gray aerial growth. Gray-pink reverse.No pigment.

Gray-pink aerial growth. Red-tan re-verse. Red-tan pigment.

Heavy gray-white aerial growth. Tan-brown reverse.Heavy gray-white aerial growth. Tan-brown reverse.

Heavy gray aerial growth. Tan-brownreverse. Tan pigment.

Fair gray aerial growth. Tan-brownreverse. Tan pigment.

Poor gray-white aerial growth. Grayreverse. No pigment.

Poor gray-white aerial growth. Grayreverse. No pigment.

Fair gray aerial growth. Gray reverse.No pigment.


Heavy gray-white aerial growth.Brown reverse. Pale tan pigment.

Heavy gray-white aerial growth.Brown reverse. Pale tan pigment.

Fair gray aerial growth. Tan-brownreverse. Tan pigment.

Brown surface ring. Peptonization. pH8.0.

Brown pigment in upper one-quarterof medium. Liquefaction complete.

Trace white aerial growth on colorlesssurface growth. Yellow-tan pigment.Nitrates reduced.

Trace colorless surface growth. Paleyellow-tan pigment. Nitrates not re-duced.

Very slight trace gray aerial growth.Brown reverse. Melanin-positive.

Pale pink aerial growth. Cream-pinkreverse. Trace cream-pink pigment.Malate solubilized.

Pale pink aerial growth. Cream-pink re-verse. Pale yellow-pink pigment.

No aerial growth. Colorless vegetativegrowth. Yellow pigment. Casein solu-bilized.

Gray aerial growth. Olive-cream re-verse. Olive-cream pigment. Xanthinesolubilized.

Gray aerial growth. Tan reverse. Tanpigment. Tyrosine solubilized.

Gray aerial growth. Gray reverse. Nopigment.

Gray aerial growth. Red-tan reverse.Red-tan pigment.

Good gray aerial growth. Tan-brownreverse. Tan pigment.



Fair gray aerial growth. Tan reverse.Tan pigment.





Good gray aerial growth. Brown re-verse. Yellow-tan pigment.

Good gray aerial growth. Brown re-verse. Yellow-tan pigment.

Fair gray aerial growth. Brown reverse.Tan pigment.

Colorless to brown surface ring. Pep-tonization. pH 8.0.

Brown pigment in upper one-quarter ofmedium. Liquefaction complete.

Trace white aerial growth on colorlesssurface growth. Yellow-tan pigment.Nitrates reduced.

No surface growth. Colorless bottomgrowth. Nitrates not reduced.

1818 APPL. MICROBIOL.-


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

KALAFUNGIN, A NEW ANTIBIOTIC

TABLE 2. Utilization of carbon compounds insynthetic medium by Streptomyces tanashiensisa

Carbon Compound

NoneD-XyloseL-ArabinoseRhamnoseD-FructoseD-GalactoseD-GlucoseD-MannoseMaltoseSucroseLactoseCellobioseRaffinoseDextrinInulinSoluble starchGlycerolDulcitolD-MannitolD-SorbitolInositolSalicinPhenolCresolNa formateNa oxalateNa tartrateNa salicylateNa acetateNa citrateNa succinate

S. tanashiensis

strain Kala

(-)

+

+

(+)

(+)

+

+

+

+

(- )

+

+

(±)

+

(- )

+

(- )

(- )

(- )

(±)

(+)

(- )

(- )

(- )

S. tanashiensisNRRL B-1692

(-)

(+)

(+)

(+)

(+)

+

+

+

(- )

+

(+)

+

(- )

+

+

(- )

(- )

(- )

(+)

+

(- )

(- )

(- )

+

+

a + = positive utilization; = negative utili-zation; (-) = slight growth, no utilization;(+) = positive utilization, only slight growth.

RESULTS AND DISCUSSIONTaxonomy. The kalafungin-producing culture,

isolated from a soil sample screened in The Up-john Research Laboratories, was identified asS. tanashiensis.The S. tanashiensis cultures are melanin-positive,

have gray to gray-pink aerial growth when photo-graphed on Ektacolor (2, 3; Fig. 1) and belong tothe Red (R) Series of Tresner and Backus (9). Thecultures, when examined by the light microscope,have straight to flexuous sporophores arising fromthe aerial mycelium and may be placed in the Rec-tus Flexibilis (RF) group of Pridham et al. (7).Spores as observed in the electron microscope, ac-

cording to the method of Dietz and Mathews (3),are long, some with dark central portion; some are

balloon-like, hyaline with striations in the centerand dark at the poles (Fig. 2). Optimal aerialgrowth occurs at 18-28 C. S. tanashiensis strain

S PASTORlANUS02 0.4 0.6 0.6

IVIII

VI

FIG. 3. Paper chromatographic patterns of kalafun-gin. Solvent systems: (1) 1-butyl alcohol-water (84:16)developed 16 hr; (IH) 1-butyl alcohol-water (84:16) plus0.25% p-toluenesulfonic acid, developed 16 hr; (III)1-butyl alcohol-acetic acid-water (2:1: 1), developed16 hr; (IV) 1-butyl alcohol-water (84:16) plus 2%piperidine, developed 16 hr; (V) 1-butyl alcohol-water(4:96), developed 5 hr; (VI) 1-butyl alcohol-water(4:96) plus 0.25% p-toluenesulfonic acid, developed5 hr.

Kala is compared with S. tanashiensis NRRL B-1692 in Table 1 for general cultural characteristics,in Table 2 for utilization of carbon compoundsin synthetic medium according to Pridham andGottlieb (6), and in Table 3 for reference colorcharacteristics. S. tanashiensis strain Kala hasbeen deposited with the Northern Utilization Re-search and Development Division, U.S. Depart-ment of Agriculture, as NRRL 3215 (U.S. Patent3,300,382, 1967).

Fermentation studies. A typical 32 C fermenta-tion pattern is given in Table 4, and it comparessensitivity of S. cerevisiae assay and the S. pas-torianus assay.

Paper chromatography. Kalafungin is distin-quishable from all available similar antibiotics bypaper chromatography. Its paper chromatogrampattern in six solvent systems is shown in Fig. 3.

In vitro spectra. Table 5 shows the in vitroantibacterial spectrum with inhibition of gram-positive microorganisms from 1.0 to 16.0 ,g/mland gram-negative microorganisms from <100to 250 ,ug/ml. Kalafungin inhibits a wide spectrumof human pathogenic fungi at concentrations of0.5 to 20 ,ug/ml (Table 6). The three protozoatested were inhibited between 10 and 100 jAg/ml(Table 7).

ACKNOWLEDGMENTSWe express our appreciation to various members

of The Upjohn Company who contributed to this in-vestigation. In particular, we thank Kurt E. Weinkeand Clarence DeBoer for the in vitro antifungal andantiprotozoal testing, respectively, Norman A. Drakefor the color prints, John Mathews for the electronmicrographs, and John A. Fox for the paper chroma-tography.

VOL. 16, 1968 1819


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

TABLE 3. Reference color characteristics of Streptomyces tanashiensis

Agar medium S. lanashiensis strain Kala S. tanashiensis NRRL B-1692

Bennett'sSurface 5 dc (pussywillow gray; 4) = 10 g 5 dc (pussywillow gray) = 10 g

(pinkish-gray; 5) (pinkish-gray)Reverse 4 pi (oak brown, russet brown) = 5 pi (copper brown, russet brown) =

55 g (strong brown); 58 m 43 m (moderate reddish brown); 55 g(moderate brown) (strong brown)

5 pl (deep brown) = 46 m (grayishreddish brown)

Pigment 4 ng (light brown, saddle tan, maple) 4 ni (chestnut brown, spice brown) == 55 g (strong brown); 57 m (light 58 g (moderate brown); 64 m (brown-brown); 58 g (moderate brown) ish-gray)

Czapek's sucroseSurface e (gray) e (gray)Reverse e (gray) e (gray)Pigment None None

Maltose tryptoneSurface 3 dc (natural) 5 ba (shell pink) = 9 m (pinkish-white)Reverse 3 ng (yellow maple) = 77 m 4 pi (oak brown, russet brown) = 55 g

(moderate yellowish brown) (strong brown); 58 m (moderate3 lg (adobe brown, cinnamon brown, brown)

light brown) = 77 g (moderate 4 ng (light brown, saddle tan, maple) =yellowish brown) 55 g (strong brown); 57 m (light

brown); 58 g (moderate brown)Pigment 3 ie (camel, maple, sugar, tan) = 4 ni (chestnut brown, spice brown) =

76 m (light yellowish brown); 77 g 58 g (moderate brown); 64 m(moderate yellowish brown) (brownish gray)

TABLE 4. Fermentation titer and pH pattern ofkalafungin production by Streptomyces

tanashiensis strain Kala

Age S. pastorianus S. cerevisiae HAge (biounits/m 1) (biounits/ml) P

hr

0 0 0 7.124 1.3 1.8 7.848 2.8 6.9 8.372 4.5 9.6 7.796 3.6 8.0 7.8120 3.1 5.7 8.0

TABLE 5. Antibacterial spectrum of kalafungin inBrain Heart Infusion broth

MinimalOrganism inhibitory

concentration

Ag/mlStaphylococcus aureus UC-76. 16S. aureus UC-552... 2Streptococcus hemolyticus UC-152... 16S. faecalis UC-3235.. 4Escherichia coli UC-51 .... 250Proteus vulgaris UC-93.... 125Klebsiella pneumoniae UC-57.... 250Salmonella schottmuelleri UC-126... 125Pseudomonas aeruginosa UC-95.... 125Bacillus subtilis UC-564... IDiplococcus pneumoniae UC-41 .... 1Pseudomonas phaseolica UC-621... <100Xanthomonas pruni UC-3334.. <100Erwinia amylovora UC-3397.. <100

TABLE 6. Antifungal spectrum of kalafungin inWhiffen's agar medium


concen-tration

Ag/mlNocardia asteroides UC-2052.. 5.0Blastomyces dermatitidis UC-1911.. 0.5Coccidioides immitis UC-1119.. 2.0Geotrichum sp. UC-1207.. 20.0Hormodendrum compactum UC-1222.. 2.0Phialophora verrucosa UC-1807.. 0.5Cryptoccus neoformans UC-1139.. 2.0Histoplasma capsulatum UC-1220.. 2.0Sporotrichum schenckii UC-1364.. 10.0Monosporium apiospermum UC-1248.. 2.0Trichophyton rubrum UC-1458.. 0.2Trichophyton interdigitale UC-1399.. 10.0Candida albicans Ab UC-1077.. 20.0Trichophyton violaceum UC-1459.. 0.5Trichophyton mentagrophytes UC-4797.. 1.0Microsporum canis UC-1395.. 0.5Trichophyton asteroides UC-4775.. 2.0Trichophyton mentagrophytes UC-4860.. 2.0

TABLE 7. Antiprotozoal activity of kalafunginby agar diffusion technique


concentration

g/milOchromonas danica ............... 10.0Crithidia fasciculata ......... ....... 10.0Tetrahymena pyriformis ............ 100.0

1820


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

KALAFUNGIN, A NEW ANTIBIOTIC

LITERATURE CITED1. Bergy, M. E. 1968. Kalafungin, a new broad

spectrum antibiotic. Isolation and characteriza-tion. J. Antibiotics (Tokyo) 21:454-457.

2. Dietz, A. 1954. Ektachrome transparencies as aidsin actinomycete classification. Ann. N.Y. Acad.Sci. 60:152-154.

3. Dietz, A., and J. Mathews, 1962. Taxonomy bycarbon replication. I. An examination ofStreptomyces hygroscopicus. Appl. Microbiol.10:258-263.

4. Jacobsen, R., W. C. Granville, and C. E. Foss.1948. Color harmony manual, 3rd ed. Con-tainer Corporation of America, Chicago.

5. Kelly, K. L., and D. B. Judd. 1955. The ISCC-NBS method of designating colors and a dic-tionary of color names. U.S. Dept. Comm.Circ. 553, Washington, D.C.

6. Pridham, T. G., and D. Gottlieb. 1948. The utili-zation of carbon compounds by some actino-

mycetales as an aid for species determination.J. Bacteriol. 56:107-114.

7. Pridham, T. G., C. W. Heseltine, and R. G.Benedict. 1958. A guide for the classificationof streptomycetes according to selected groups.Appl. Microbiol. 6:52-79.

8. Reusser, F. 1968. On the mechanism of action ofantibiotic U-19,718 in rat liver mitochondria.Biochemistry 7:293-299.

9. Tresner, H. D., and E. G. Backus. 1963. Systemof color wheels for streptomycete taxonomy.Appl. Microbiol. 11:335-338.

10. Waksman, S. A. 1961. The actinomycetes, vol.2. Classification, identification and descriptionsof genera and species. The Williams & WilkinsCo., Baltimore.

11. Whiffen, A. J. 1948. The production, assay, andantibiotic activity of actidione, an antibioticfrom Streptomyces griseus. J. Bacteriol. 56:283-291.

VOL. 16, 1968 1821


http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

streptomyces tanashiensis strain kala

Documents