RIFOMYCIN. XI. TAXONOMIC STUDY ON STREPTO- MYCES MEDITERRANEI NOV. SP.

by

P. MARGALITH & G. BERETTA

(Research Laboratories, Lepetit S.jb.A., Milan, Italy)

(7.VII.1960)

During the course of a screening program for new antibiotics, we came across a Streptomyces that was isolated from a soil sample from a pine arboretrum at an altitude of ca. 200 m and about 50 m off the sea shore of St. Raphael (France). Isolation was performed by plating the soil suspension on Benett 's agar. The particular morphological appearance of the strain (receiving our collection number ME/83) as well as the antibiotic activity against Gram-posi- tive bacteria found in shake flask broth called our attention for further work on this Streptomyces, which appears to be a new spe- cies called by us Strcbtomyees mediterranei.

From the fermentation broths of this Streptomyces a mixture of several antibiotics (rifomycin-complex) was isolated(l). One of the components of rifomycin-complex, rifomycin B, has promising the- rapeutical applications (2, 3, 4, 5, 6).

In this paper we would like to describe some of the morphologi- cal and biochemical characters of Streptomyces mediterranei.

TAXONOMY

Culture ME/83 was compared with other species of Streptomyces as described by WAKSMAN & LECHEVALIE~ (7) and others. A detailed account of the morphology is given in the following description of ME]83 grown on various media, in slant cultures, for up to 20 days at 28 ° C. Unless stated otherwise, culture media were prepared ac- cording to WAKSMAN(8). Color determinations were performed ac- cording to }¢[AERZ • PAUL(9).

Oatmeal agar: Fair growth with smooth surface. Vegetative mycelium hyaline to yellowish with pinkish reverse. ~rhffish serial mycelium with pink tinge. Traces of yellowish soluble pigment.

Mycopathol. et Mycol. Appl. XIII, 4 21

322 P. MARGALITH AND G. BERETTA

Yeast-extract glucose agar:

Emerson glucose agar:

Oatmeal mix agar (1):

Benett's agar:

Penassay agar (Difco): Yeast extract molasse agar 2):

Czapek-Dox sucrose agar:

Potato agar:

Potato wedge:

Glucose-asparagine agar:

Glycerol-asparagine agar: Nutrient agar:

Abundant growth yellowish to pink with rough surface. Scanty aerial mycelium. No pigmentation of m e d i a . Abundant growth, yellowish to pink orange with rough surface. Aerial mycelium becomes pinkish (2/B/8). Pale amber soluble pigment Good growth, smooth, yellowish with light orange tinge. Aerial my- celium whitish to pink. Amber so- luble pigment. Good growth, yellowish turning orange yellow. Aerial mycelium becoming pinkish. Light amber pigment. Poor growth. Abundant, rough growth, colorless to yellowish. Whitish aerial my- celium. Deep amber soluble pigment (12/H/10). Poor growth, thin and colorless to light melon (ll/A/8). Traces of pinkish white aerial mycelium. No soluble pigment. Poor growth thin and colorless. Traces of whitish aerial mycelium. No soluble pigment. Poor development, hyaline. Color of wedge not changed. Fair growth with smooth surface. Thin vegetative mycelium of light orange pink color (10/B/12) and yellowish reverse. No aerial my- celium. Some light yellow soluble pigment. As glucose-asparagine agar. Moderate growth with smooth sur- face; melon to orange (2/B/10) with yellowish orange reverse. Aerialmy- celium pinkish white. Soluble pig- ment absent.

x) Oatmeal-mix agar contained in addit ion to oatmeal (20 g cooked ou t flakes fil trate): Enzymat ic casein hydrolyzate 5 g; Beef extract 3 g; Sucrose i0 g; Agar agar 40 g in 1000 ml H20 dist. p H corrected to 6.9.

~) Yeas t ex t rac t molasse agar contained: Glycerol 7.5 g; Molasse 7.5 g; Yeas t ex t rac t 5 g; NaC1 4 g; Agar agar 30 g; H~O dist. 1000 ml; p H corrected to 6.0.

S T R E P T O M Y C E S M E D I T E R R A N E I N. SP. 323

Pfidham's agar:

Starch agar:

Dextrose-Tryptone agar:

Hickey's and Tresner's cobalt agar:

Tyrosine agar: Ca-malate agar:

Dorset's albumin agar:

Gelatine:

Nitrates:

Litmus milk:

Cellulose:

Temperature:

Morphology of colonies:

Moderate growth, smooth, color- less with lobster red (2/G/11) spots. Pink aerial mycelium. No pigment- ation of medium. Poor growth, colorless to light orange pink. Starch hydrolysis doubtful. Scarce white aerial my- celium. Abundant growth, orange pink with golden yellow to orange re- verse. Pinkish aerial mycelium. Light golden yellow (9/J/6) soluble pigment. Moderate growth, hyaline to light pinkish orange. Some pinkish aerial mycelium. Some yellowish soluble pigment. Poor growth. Fair growth, colorless. Aerial my- celium whitish with pink tinge. No soluble pigment. Partial digestion of Ca-malate. Fair growth, pink. No aerial my- celium or pigmentation of medium. No pigmentation. Liquefaction slow and uncomplete. Surface growth with pinkish aerial mycelium. No reduction to nitrites. Broth becomes yellowish. No peptonization or coagulation. Slight alkaline reaction. No growth on filter paper disks placed on carbohydrate free miner- al base agar. Optimum 28 ° C. At 37 ° C, growth takes place but characters are less distinct. In Benett's agar, this strain forms small (usually not more than 2 ram) colonies with well defined contours, compact but rough surface; covered with whitish aerial mycelium. A great number of colonies plated on agar do not develop aerial myceli- um and remain in their orange-red vegetative mycelium that grows to considerable thickness until they assume a rock-like appearance.

2 1 "

3 2 ~ P. MARGALITH AND G. BERETTA

Morphology of aerial mycel ium

When sporulation takes place, spores appear in long chains, somewhat twisted; spirals are not visible. Spores are ellipsoidal to oblong (0.8 - - 1,0 # × 3.0 - - 5.0 #). Fig. 1 presents the aerial mycelium of ME/83 when grown on cellophane squares placed upon inositol-base medium (see below) for 4 days at 28 ° C.

Fig. 1. Aerial mycel ium of S. mediterranei grown on inositol-base med ium ( × 1000)

Test for utilization of carbon sources

These were performed according to PRIDt~A~ & GOTTLIEB (11). Results are given in table I. It can be seen that of all the compounds tested: raffinose, inulin, sorbitol, dulcitot, citrate, acetate and glycine were not utilized by S. mediterranei.

Classification

Streptomycetes with similar characters have been named: S. roseoflavus (12), S. microflavus (12), S. aurantiacus (7) and S. parvus (7). Table I compares these strains with Streptomyces ME/83. I t has to be pointed out, however, that S. microflavus, WAKSMAN strain obtained from C.B.S., does not correspond to the description given by TADASHI ARAI (12) in comparing the flavomycin produc- ing strains. Also the original description given by I(RASSlLNIKOV for S. aurantiacus as brought in WAKSMA~ (7) is insufficient and an a t tempt is made herewith to complete it, employing a similar strain in our laboratory.

STttEPTOMYCE$ MEDITERRANI~I N. SP. ~ 5

T A B L E I .

Assimila¢ion o] Carbon Compounds by S. mediferranei and similar strains on solid media 1).

S. roseo- S. micro- S. mediler- S. S. auran- Carbon source /lavus ~) /lavus s) ranei parvus tiacus

N.D. 4) Arabinose Rhamnose Xylose Glucose Galactose Fructose Mannose Lactose Maltose Sucrose Raffinose Dextrin Inulin Dulcitol Glicerol 3/Iannitol Inositol Sorbitol Na succinate Na citrate Na acetate Glycine Salicin

+

4- + + +

+ + +

+ +

+ + +

N.D.

+ + +

+ + + +

+ + +

+ +

+ + + +

+ + + +

+

+ + + + + + + + +

+ + + + + +

+ + + +

+ + +

+ + + + + + +

+

+

4-

+ +

+

m

4-

±

1) Degrees of growtll after 5 days incubation are indicated by +, + + or + + +. No growth is indicated by --.

~) Description from (12). 8) WAI<SMAN strain obtained from C.B.S. 4) No data available.

T h e m o s t i m p o r t a n t cha rac te r s of S. ME/83 can be s u m m a r i z e d as follows:

a) I t does n o t be long to the c h r o m o g e n i c group. b) On m o s t m e d i a v e g e t a t i v e g r o w t h assumes a ye l lowish t o o range

color, b u t neve r an in tense o r ange r ed t o c h e r r y o r l a v e n d e I i n tona t ion .

c) P o o r sporu la t ion on m o s t media . d) Charac te r i s t i c u t i l i za t ion of c a r b o n sources.

Since s t r e p t o m y c e t e s s imi lar to M E / 8 3 are d i f fe ren t in m a n y m a j o r charac te r i s t i cs , we feel jus t i f ied in n a m i n g this isola te S. m e d i t e r r a n e i nov. sp. Most p r o b a b l y th is species shou ld be g r o u p e d t o g e t h e r w i t h t he fo rmer ones in a S . r o s e u s series or g roup , t h o u g h n o t in t he sense of WAKSMAN (7) where also species l ike S . f r a d i a e are inc luded.

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3 2 8 P. MARGALITH AND G. B E R E T T A

Sporulation S. mediterranei produces very few spores when grown on the

media used for classification purposes. Any developing studies of a new streptomycete such as work with mutagens for the isolation of higher yielding strains, requires the preparation of fairly abundant spore suspensions. Efforts were therefore directed towards the find- ing of a solid medium more suitable for spore production. None of the proteic media tried would promote sporulation. Better results were obtained when mineral base media supplemented with various carbon sources were used. Sporulation took place on media con- raining 1 % glucose, lactose, glycerol and inositol (11). Separate sterilization of carbohydrates before addition to media did not prove advantageous. Best results were obtained on mineral base-lactose agar (ML) where the aerial mycelium assumed a rather mealy texture.

Fig. 2.

a

a) Normal culture;

b

b. Degenerate culture with actinophage like spots.

The following procedure was used: S. mediterranei was grown for 48 hrs. in shake flask (500 ml with 100 ml seed medium) at 28 ° C. The mycelium was then separated by centrifugation, resuspended in the original volume of sterile water and a 10 ml aliquot was inocul- ated into the surface layer of a 250 ml. ML agarin Roux flasks. After the

STREPTOMYCES MEDIT:ERRANEI N. SP. 329

setting of the mycelium the excess of liquid was removed aseptic- ally. Cultures were incubated at 28 ° C for 5--7 days. On washing the surface with 20 ml of a tween solution (1 : 104) and filtering through a glass filter to remove mycetial and spore conglomerates, a suspension of about 2 × 107 spores per ml was obtained. The ability of S. mediterranei to sporulate is one of the few macroscopic characters which are considerably variable due to mutagenic treatments. Thus we came across a strain ME/83/4008 which is a heavy sporulator, yielding under similar conditions up to 2 X 109 spores per ml.

Degeneration

S. mediterranei easily looses some of its characteristic morpholo- gical features when transplanted continuously on protein rich media. The aerial mycelium rapidly ceases to develop. Frequently the surface of a slant reveals transparent spots surrounded by whitish aerial mycelium reminding actinophage activity (see Fig. 2). When subculturing mycelium that lost the capacity to produce aerial hyphae, fresh cultures do not develop normally, the bare vegetative mycelium showing a strong tendency to tyse. Complete lysis was obtained when inoculating these cultures into shake flasks con- taining seed medium. In order to prevent the run down of this strep- tomycete, we used, exclusively, mineral base lactose sporulating medium on which the typical appearance of the organism could be maintained without difficulty.

SUMMARY

A new, antibiotic producing, Stre~tomyces has been isolated from a soil sample. A detailed description of its morphological and some of its biochemical characters are given. The name S. mediterranei nov. sp. is suggested for the new isolate. Sporulation of the new Streptomyces could be induced by cultivation on certain chemically defined media.

Bibliography I. S~I~sI, P., P. MARGALITH, ~ ~ . T. TIMBAL - - Farmaco (Ed. Sci.) 14, 146 (59). 2. SENSI, P., A. M. GRECO ~; R. BALLOTTA - - AIltibiotics Annual 1959--1960

New York, Medical Encyclopedia, Inc. 1960 - - p. 262. 3. TIMBAL, M. T. - - Antibiotics Annum 1959--1960, New York, Medical Ency-

clopedia, Inc. 1 9 6 0 - p. 271. 4. MAFFII, G. & M. T. TIMBAL - - Antibiotics Annual 1959.--1960, New York,

Medical Encyclopedia, Inc. 1960 - - p. 277. 5. FURESZ, S. & R. ScoTTI - - Anfibiotics Annual 1959---1960, New York,

Medical Encyclopedia, Inc. 1960 - - p. 285. 6. Fu~Esz, S. - - Antibiotic Medicine and Clinical Therapy. (in press). 7. ~VAKSMAN, S. A. & H. A. LECHEVALIER -- Actinomycetes and their antibiotics.

Williams and Wilkins Co., Baltimore, 1953. 8, WAKSMAN, S. A, -- The species concept among the actinomycetes with special

reference to the genus streptomyces. Bact. Reviews 21, 1 (1957).

330 P. M A R G A L I T H A N D G. BER:ETT2~

9. MAERZ, A. & M. R . PALTL - - A d ic t ionary of color - - 2nd edi t ion McGraw-Hi l l Book Co. 1953.

10. HICK~¥, 1R. J . & H. D. T ~ S N E R - - A coba l t con ta in ing m e d i u m for spo ru la t ion of s t r e p t o m y c e s species. J. Bact . 64, 891 (1952).

11. P R I D H A M , T. G, & D. GOTTLIEB - - T h e u t i l i za t ion of ca rbon c o m p o u n d s b y s o m e ac t inomyce ta l e s as a n a id for species de t e rmina t ion . J . Bac t . 86, 107 (1948).

12. TADASHI 2~RAI - - S tud ies of F l a v o m y c i n . J. Ant ib . 4, 215 (1951).