15.3. 1976

ployed, as well as t h e c h a r a c t e r i z a t i o n of all a c t i n o m y c e t e s of o u r collection w h i c h h a v e b e e n i n v e s t i g a t e d , will b e p r e s e n t e d elsewhere. Results and discussion. T h e r e s u l t s o b t a i n e d w i t h 62 s t r a i n s of 41 species a n d 16 g e n e r a of Actinomycetales are listed in t h e Table. F o r t h e p u r p o s e of classification, i t suffices t o i n d i c a t e t h e p r e s e n c e or a b s e n c e of a c e r t a i n class of f a t t y acids. F u r t h e r , o n l y f a t t y acids w i t h m o r e

Det. Signal

Nocardia

corollino

16,0

IEYl

~b

2'o

Det. Si~al MS:0 of-15:0 '

319

Speeialia

OI

i-16:0 A

10

3'o

Streptomyces

t (='~ol

rimosus

tinomyces, 2. + -- + + : Mycobacterium, Nocardiaceae, 3. + + -- -- : Streptomycetaceae, Actinoplanes, Amorphosporangium, Thermoactinomyces, Saccharomonospora, 4. + + + -- : Planobispora, Streptosporangium, Micromonospora, Microbispora, a n d 5. + + + + : Aetinomadura. T h e d i s t r i b u t i o n of t h e s e p a t t e r n s shows t h a t v a r i a t i o n exists in some of t h e p r e s e n t families: A c t i n o planaceae and Thermomonosporaceae. The 3 biochemical m a r k e r s discussed in t h i s p a p e r , as well as t h e d i s t r i b u t i o n of m y c o l i c acids of v a r i o u s t y p e s a m o n g A ctinomycetales ~~ will b e of g r e a t h e l p t o t h e a c t i n o m y c e t e taxonomist.

(:ti-17:0

2'0

t h a n 2 % of t o t a l acids were c o n s i d e r e d (for q u a n t i t a t i v e d a t a of t h e i n d i v i d u a l acids, see t h e f o r t h c o m i n g p u b l i c a tion). T h e D A P s e p a r a t e s t h e Streptomycetaceae (Table, No. 5, 6 a n d 7) w i t h LL-DAP f r o m all o t h e r Actinomycetales, m o s t of w h i c h c o n t a i n n L - D A P ; in Actinomyces t h e D A P of t h e p e p t i d o g l y c a n is r e p l a c e d b y lysine. N u m e r o u s a c t i n o m y c e t e s w i t h DL-DAP c a n b e different i a t e d b y t h e i r s u g a r a n d or f a t t y acid s p e c t r u m . H o w e v e r , Mycobacterium a n d t h e Nocardiaceae (No. 3 a n d 4) e x h i b i t b o t h t h e s u g a r t y p e A w i t h a r a b i n o s e a n d galacrose a n d t h e s a m e f a t t y acid s p e c t r u m . T h e s e o r g a n i s m s c a n b e d i f f e r e n t i a t e d b y t h e i r mycolic acid t y p e 10,11. As a l r e a d y s h o w n in t h e e a r l y studies3, 4 t h e o c c u r r e n c e of xylose a n d m a d u r o s e (sugar t y p e D a n d B respectively) are of d i a g n o s t i c value. H e r e we w a n t to a d d t o t h e s e wellk n o w n f e a t u r e s t h e f a t t y acid s p e c t r u m . If o n l y t h e 4 classes of acids 1. s a t u r a t e d , 2. iso- a n d a n t e i s o b r a n c h e d , 3. 1 0 - m e t h y l a n d 4. u n s a t u r a t e d , are t a k e n i n t o consideration, t h e following p a t t e r n s e m e r g e : 1. + -- -- + : Ac-

3'0

i (mini

Gaschromatographic resolution of fatty acid esters from Nocardia corallina and Streptomyces rimosus. (Parameters: Inj. port: 250~

oven: 155 ~ detector 300 ~ carrier gas N~: 30 Ilfl/min; hydrogen: 30 ml/min; air 240 ml/min; sample 1 [zl.)

Emericid*, a N e w Polyether Antibiotic from

10 1Vf. P. LECt~EVALIER, A. C. HogAN and H. A. LECHEVALIER, J. Bact. 705, 313 (1971). 11 D. E. )]~INNIKIN, J. ALSHAI~iAONYand lVf. GOODFELLOW, J. gen. lX{ierobiol. 88, 200 (1975).

Streptomyces hygroscopicus (DS 24 367)

L. NINET, F. BENAZET, I t . DEPAIRE, J. FLORENT, J. LUNEL, D. 1V[ANCY, A. ABRAHAM, J. R. CARTIER, N. LES, C. GODARD, M. MOREAU, 1~. TISSIER a n d J. Y. LALLEMAND

DE

CI-IEZEL-

Rhdne-Poulenc Industries, Centre Nicolas Grillet, 13, quai Jules Guesde, F-9d400 Vitry-sur-Seine (France)," and Ecole Normale Supdrieure, Service de Chimie, 24 rue Lhomond, F-75005 Paris (France), 7 November 7975. Summary. E m e r i c i d is a n e w p01yether polycyclic i o n o p h o r e a n t i b i o t i c e x c r e t e d b y Streptomyces hygroscopicus (DS 24 367). A c t i v e in v i t r o a g a i n s t G r a m - p o s i t i v e b a c t e r i a , it is ineffective in vivo. A t a 0 . 0 0 6 - 0 . 0 2 % level in t h e d i e t i t p r o t e c t s c h i c k e n s a n d r a b b i t s a g a i n s t coccidiosis. R e c e n t r e s e a r c h o n t h e p r o d u c t i o n of a n t i m i c r o b i a l a g e n t s b y m i c r o o r g a n i s m s h a v e led t o t h e d i s c o v e r y of e m e r i c i d (31 559 R.P.), a n e w a n t i b i o t i c i s o l a t e d f r o m t h e c u l t u r e b r o t h s of Slreptomyces hygroscopicus D S 24 367. T h e s t r a i n , w h i c h was i s o l a t e d f r o m a s a m p l e of soil collected a b r o a d a n d selected b y classical t e s t s of a n t i biosis 1, e x h i b i t s all tile m a i n m o r p h o l o g i c a l a n d b i o c h e m i cal f e a t u r e s of t h e species Streptorayces hygroscopicus, as d e s c r i b e d b y s e v e r a l a u t h o r s 2, ~. I t is s t o r e d as a d r y spores a n d sterile soil m i x t u r e . E m e r i c i d is p r o d u c e d b y s u b m e r g e d c u l t u r e as follows : t h e s t r a i n is first g r o w n f r o m t h e spores o n BENNETT'S a g a r m e d i u m 4 ill t e s t t u b e s for 15 d a y s a t 26~ A suitable i n o c u l u m is o b t a i n e d b y successive t r a n s f e r s of t h e t u b e c u l t u r e ; first i n t o 250 m l of a l i q u i d m e d i u m /corn-

p o s i t i o n i n g/l: y e a s t e x t r a c t 15, a n h y d r o u s glucose 10, a g a r 2) in a 2 1 f l a s k i n c u b a t e d for 48 h a t 260C o n a r o t a r y shaker, t h e n i n t o 40 1 of a n o t h e r m e d i u m (in g/l: p e p t o n e 10, y e a s t e x t r a c t 5, glucose m o n o h y d r a t e 10, a g a r 2, p H before s t e r i l i z a t i o n 7,3) c o n t a i n e d in a s t i r r e d a n d a e r a t e d 75 1 f e r m e n t e r . A f t e r 25 h a t 27 ~ t h i s l a s t * From a recent paper by N. OTAKE (Tetrahedron Lett. 1970, 4147) emericid and lonomycin would be identical. 1 S. A. WAKSMAN,MicrobialAntagonisms and Antibiotic Substances (The Commonnwealth Fund, New York 1954). 2 H. D. TRESNER and E. J. BACI4:US,Appl. Microbioi. 4, 243 (1956). S. A. WAKSMAN, The Actinomyces II (The Williams aad Wilkin~ Company, Baltimore 1961), p. 230, See%p. 331,

320

Speciatia

culture is poured as a whole into a 800 1 fermenter containing 400 1 of the following production medium (i'ii g/l) : dried kidney beans 25, distillers' solubles 5, glucose monohydrate (separately sterilized) 10, glycerine 15, sodium chloride 3, hydrated cobalt chloride 0,02, pH before sterilization being 7,5. The culture is aerated, stirred and kept at 27 ~ The production of emericid reaches 500 Fg/ ml at 120 h. Practically the whole antibiotic is retained by the mycelium. The cell mass, collected by filtration at pH 3 of the broth including Clarcel DIC (CD) as an adjuvant, is dispersed into 300 1 of methanol at pH i0. After filtration, the organic solution is reduced to 4% of its

EXPERIEN'TIA32/3

initial volume by distillation under low pressure. By this way, a crude product containing 80% of the sodium salt of emericid is precipitated. Sodium salt of pure emericid is obtained'by dissolving the crude product in acetone, dimethylformamide or methanol and diluting with water. I t is a fine colourless crystalline solid, m.p. 220~ with decomposition (KOFFLER); [C~]~) ~ = -/51, 2~ 4- 1,0 ~ (C = I in methanol). I t is practically insoluble in water, sparingly soluble in nhexane, soluble in alcohols, ketones, dimethylformamide and ethylacetate, easily soluble in chlorinated solvents. There is no UV-absorption peak as far as 210 nm. The IR-spectrum (KBr pellet) is given in the Figure 1.

Fig. 1. IR-absorption-spectrum of emerieid (IZBrpellet).

Fig. 2. 13C NMR-speetrum of emericid.

?CH3 C,H30ICH3 CH3-..-j/~CH~/~_JLH HO'7~ N/~-,. /-1k..^/J~ ~ cHr-cH 'HI COOH CH$

~

CH30CH3 CH3QCH 3 ~ I H ~ -~ ~ )'-CH3 / b-V-OH OH3

Fig. 3. Planar structure of emericid,

15.3. 1976 Bacteriostatic activity of Emericid Test organism

Staphylococcus aureus (straiu 2,09 P, ATCC 6 538 P) Staphylococcus aureus (strain Smith) Sarcina lutea (ATCC 9341) Streptococcus faecalis (ATCC 8043) Streptococcus pyogenes hemolyticus (strain Dig 7, Institut Pasteur) Diplococcus pneumoniae (strain Til, Institut Pasteur) Neisseria catarrhalis (A 152, Institut Pasteur) Bacillus subtilis (ATCC 6633) Bacillus cereus (ATCC 6630) Mycobacterium species (ATCC 607) Escherichia colt (ATCC 9637) Shigella dysenteriae (Shiga L, Institut Pasteur) Salmonella paratyphi A (strain Laeasse, Institut Pasteur Salmonella schottmuelIeri (paratyphi 13; strain Fougene, Institut Pasteur) Proteus vulgaris Pseudomo~as aeruginosa (strain Bass, Institut Pasteur

321

Specialia

Minimum inhibitory concentration ([zg/ml) 0.8 2.5 1.9 0.5 0.8 0.2 50 0.8 0.5 25 > 150 > 150 > 150 > 150 > 150 > 150

E l e m e n t a l c o m p o s i t i o n : C 63.2%, H 8.8%, O 24.3%, N a 2.9% is in a g r e e m e n t w i t h t h e h y p o t h e t i c m o l e c u l a r f o r m u l a C44H~5014 N a ; m . W . : 851.06; n e u t r a l e q u i v a l e n t w i t h p e r c h l o r i c acid in acetic acid: 840. A c c o r d i n g to its phTfsicochemical p r o p e r t i e s , m a i n l y t h e p e c u l i a r s o l u b i l i t y of its s o d i u m salt, emericid seems to b e l o n g to t h e f a m i l y of i o n o p h o r e cyclic p o l y e t h e r s K As a m a t t e r of fact, p r o t o n a n d 13 C N M R - s p e c t r o m e t r y (Figure 2) h a s s h o w n t h a t e m e r i c i d owns t h e s a m e c h a i n of cyclic p o l y e t h e r s as n i g e r i e i n 6 a n d grisorixin 7 w i t h o n l y m i n o r differences for t h e s i d e - s u b s t i t u e n t s . Moreover, a t t e m p t s t o i d e n t i f y e m e r i c i d w i t h some o t h e r p r o d u c t s of t h e family, especially a l b o r i x i n e s, d i a n e m y c i n 9, lasalocid 10, lysocellin 11, m o n e n s i n 1~-, A 20418, A 28 695 A a n d B 14, b y t h i n l a y e r c h r o m a t o g r a p h y h a v e b e e n u n successful ( c o n d i t i o n s : s p o t 100 ~g in m e t h y l e n e chloride o n a Silicagel M e r c k F 254 p l a t e ; d e v e l o p w i t h t h e l i g h t p h a s i s of a c y c l o h e x a n e , e t h y l a c e t a t e , w a t e r a n d b u t a n o l 50-50-25-5 m i x t u r e ; s p r a y w i t h a v a n i l l i n 3 g, m e t h a n o l 100 m l a n d c o n c e n t r a t e d sulfuric acid 0,5 m l r e a g e n t a n d h e a t a t 105 ~ F i n a l l y , t h e u n i q u e n e s s of t h i s c o m p o u n d was s u b s t a n t i a t e d b y m e a n s of X - r a y - c r y s t a l l o g r a p h y

of t h e silver salt, u n r a v e l i n g its s t r u c t u r a l f o r m u l a as s h o w n b y F i g u r e 315. T h e LDa0 of e m e r i c i d is 150 m g / k g for t h e chicken, w i t h a single p.o. a d m i n i s t r a t i o n . T h e b a c t e r i o s t a t i c a c t i v i t y of e m e r i c i d a g a i n s t some m i c r o o r g a n i s m s is s h o w n in t h e Table. T h e m i n i m u m i n h i b i t o r y c o n c e n t r a t i o n d e t e r m i n a t i o n s were c a r r i e d o u t b y t h e d i l u t i o n m e t h o d in a p p r o p r i a t e m e d i u m for e a c h o r g a n i s m a n d a f t e r i n c u b a t i o n for 18 h a t 37 ~ E m e r i c i d is o n l y a c t i v e in v i t r o a g a i n s t g r a m - p o s i t i v e b a c t e r i a a n d p r a c t i c a l l y i n a c t i v e a g a i n s t a n y o t h e r t y p e of b a c t e r i a , y e a s t s a n d fungi. I n vivo, e m e r i c i d is i n a c t i v e a g a i n s t s t a p h y l o c o c c a l a n d p n e u m o c o c c a l i n f e c t i o n s of mice b y t h e oral or s.c. route. As p r e v i o u s l y s h o w n ~*, e m e r i c i d is a n e x c e l l e n t coccidi o s t a t in c h i c k e n s ; m i x e d w i t h t h e feed a t 0 . 0 0 6 - 0 . 0 2 % levels a c c o r d i n g t o t h e i n f e c t i n g species of E i m e r i a , i t suppresses m o r t a l i t y a n d i n t e s t i n a l lesions, r e d u c e s oocysts e x c r e t i o n a n d allows a n o r m a l g r o w t h of t h e i n f e c t e d b i r d s ; t h e r e is n o e m e r g e n c e of r e s i s t a n t E i m e r i a s t r a i n d u r i n g t h e t r e a t m e n t . Similar r e s u l t s h a v e b e e n o b t a i n e d in r a b b i t s . I n conclusion, e m e r i c i d is a n e w i o n o p h o r e polycyclic p o l y e t h e r , a c t i v e in v i t r o a g a i n s t g r a m - p o s i t i v e b a c t e r i a b u t d e v o i d of a n y in v i v o a n t i b a c t e r i a l a c t i v i t y . I t s m a i n i n t e r e s t lies in t h e e r a d i c a t i o n of coccidiosis in c h i c k e n s a n d r a b b i t s a t t h e 0 . 0 0 6 - 0 . 0 2 % level in t h e diet.

5 j. ASSELIN`EAU and J. P. ZALTA, Les Antibiotiques, Structure et Mode d'action (Hermann, Paris 1973), p. 82. 6 g. K. STEINRAUF,~V[.PINKERTONand J. W. CHAMBERLIN,Biochem.

biophys. Res. Commun. 33, 29 (1968), 7 p. GAeHON, A. S'rARON`, Chem. 8 M. ALLEAUIKE, GO,lARD and T.

I~ERGOMARD, I~. VESCIIAMBRE, C. ]~STEVE and T. C o m m u n . 1970~ 1421. B. BUSETTA, C. FARGES, P. GACHON, A, KERSTARON`, Chem. C o m m u n . 7975, 411. 9 E. %V. CZERWINSKI and L. K. STEINRAUF, Biochem. biophys.

Res. Commun. 45, 1284 (1971). x0 j . W. WESTLEY, R. M. EVANS JR., T. WILLIAMSand A. STE~PEL, Chem. Commun. 7970, 71. ii N. OTAKE, M. KOENUMA, H. KINASKI, S. SATO and Y. SATO, Chem. C o m m u n . 1975, 92.

I2 A. AGTARAP, J. W: CHAMBERLIN, ]Vf. PINKERTON and L. STEINRAUF, J. Am. chem. Soc. 89, 5737 (1967). is N. D. JONES, lV[. O. CHANEY, J. W. CHAMBERLIN, R. L. HAMILL and SuE CHEN, J. Am. chem. Soc. 95, 3399 (1973). 1~ Eli Lilly and Co, U.S. Patent 3839559, 23/12/71. 15 C, RICHE and C. PASCARD-][3ILLY,Chem. Commun., 7975, 951. 16 F. BEICAZET, ~. R. CARTIER, J. FLORENT, C. JOHNSON', J. LU~EL,

D. MANCY, 9th International Congress of Chemotherapy, London 1975, Abst., IV[432.

Effect of Cyclic AMP and Theophylline on Phagocytotic Activity of

Tetrahymena pyriformis

G. CSABA a n d T. LANTOS Department o / B i o l o g y , Semmelweis University o / M e d i c i n e , P.O. B o x 95, H - T d 5 0 B u d a p e s t (Hungary), 3 October 1975. S u m m a r y . D i b u t y r i l c A M P a n d t h e c P D E - i n h i b i t o r t h e o p h y l l i n e b o t h e n h a n c e t h e p h a g o c y t o t i c a c t i v i t y of Tetrahymena. T h e o p h y l l i n e a n d c A M P - a c t i v a t i n g h i s t a m i n e are synergistic. I t follows t h a t t h e c A M P - a d e n y l c y c l a s e s y s t e m f u n c t i o n s in t h e u n i c e l l u l a r a n i m a l Tetrahymena. C e r t a i n f o r m s of h o r m o n a l r e g u l a t i o n h a v e b e e n s h o w n t o b e o p e r a t i v e in u n i c e l l u l a r o r g a n i s m s , a l t h o u g h t h e y are s e e m i n g l y of l i t t l e c o n s e q u e n c e a t t h i s stage of p h y l o genesis. E p i n e p h r i n e 1,~ a n d s e r o t o n i n a h a v e b e e n s h o w n to b e p r e s e n t in T e t r a h y m e n a a t c h a n g i n g c o n c e n t r a t i o n s , d e p e n d i n g o n t h e f u n c t i o n a l s t a g e of t h e p r o t o z o o n . Also, T e t r a h y m e n a was f o u n d t o r e s p o n d t o c e r t a i n h o r m o n e s n o t p r e s e n t in it a n d n o t e n c o u n t e r e d b y i t u n d e r n a t u r a l

c o n d i t i o n s . H i s t a m i n e ~ p r o d u c e d a c o n s i d e r a b l e increase in t h e p h a g o c y t o t i c a c t i v i t y of t h e p r o t o z o o n , w h e r e a s 1 j. j. BLuM, Proc. natn. Aead. Sci., USA 58, 81 (1967). 2 D. L. HILL, The Biochemistry and Physiology o/ Tetrahymena (Academic Press, New York 1972). 3 j. JANAKIVEDI, J. C. DEVEY and W. KIDDER, J. biol. Chem. 2d7, 2576 (1966). 4 G. CSABAand T. LANTOS~Cytobiologie 7, 361 (1973).