Bull. Environ. Contam. Toxicol. (1992) 49:743--749 9 1992 Springer-Verlag New York Inc.
i Environmental Contamination 3and T o x i c o l o g y
Production of Antibacterial Substances by Macroalgae of the New York/New Jersey Coast, USA Bonnie Lustigman, Lee H. Lee, Nancy Thees, and John Masucci Biology Department, Montclair State College, Upper Montclair, New Jersey 07043, USA Marine algae are known to produce biochemicals which show a high degree of toxicity. The neurotoxin produced by the dinoflagellate Gonyaulax polyedra is responsible for red tides in tropical waters (Atlas 1984). Research has revealed that several of these substances have antibacterial, antiviral and/or antineoplastic effects (Scheuer 1990). Kainic acid, derived from the red algae Digenia simplex is an active antihelminthic (Grant and Mackie 1977). The toxic nature of these substances, which include acrylic acid, phenols, terpenoids and halogenated compounds, has made many of them unfit for commercial exploitation (Burkholder and Sharma 1969). They are produced by the algae to aid in plant protection by acting as deterrents for herbivorous fish (Horn et ai.1985) and to prevent surface fouling by bacteria and other epibionts (Norris and Fenical 1982). These substances may be contained in different parts of the algal thallus. In some species the new areas of growth have higher concentrations, perhaps to better protect these more fragile regions ( Hornsey and Hide 1976; Lustigman and Brown 1990). Different phases of the life cycle of the algae are also associated with varying amounts of antibiotic production ( Hornsey and Hide 1985). Previous research has shown production of antibiotic substances by marine macroalgae which were effective in preventing the growth of human clinical bacteria (Lustigman and Brown 1990). Many marine bacteria however, live in or on the algal thallus (Barbeyron and Berger 1989) and may have greater resistance to these substances. In this study we report the effect of antibacterial substances produced by 35 seaweed species as challenged by three species of marine bacteria. A preliminary extraction of the inhibitory substances produced was also undertaken. Send reprint requests to Dr. Lustigman address.
743
at the above
MATERIALS
AND M E T H O D S
S a m p l e s of 35 s e a w e e d s p e c i e s w e r e c o l l e c t e d f r o m S a n d y Hook, N e w J e r s e y and M o n t a u k Point, N e w York f r o m M a r c h to September, 1 9 8 9 ( T a b l e i). A f t e r c o l l e c t i o n the s a m p l e s w e r e p l a c e d on ice for t r a n s p o r t to the laboratory. T h e y w e r e w a s h e d in 10% Chlorox, r i n s e d t w i c e in d i s t i l l e d w a t e r and the d i f f e r e n t s p e c i e s w e r e i d e n t i f i e d b a s e d on the m o n o g r a p h by H i l l s o n (1977). Samples w e r e t h e n frozen until use. S a m p l e s of 3 cm w e r e cut f r o m the algal thallus, i n c l u d i n g the g r o w i n g tip. T h e s e w e r e s u r f a c e s t e r i l i z e d u s i n g 10% C h l o r o x and r i n s e d in s t e r i l e d i s t i l l e d water. Each p i e c e was t h e n p l a c e d in a Petri d i s h w i t h m o l t e n Difco M a r i n e A g a r in o r d e r to p r o d u c e u n i f o r m c o n t a c t of the algal s a m p l e and the agar. A f t e r the agar hardened, e a c h d i s h was s t r e a k e d w i t h 0.i mL from a 6-8 hr b a c t e r i a l c u l t u r e of one of the three b a c t e r i a l species, V i b r i o m a r i n o f u l v i s , M i c r o c o c c u s imfimus, P s e u d o m o n a s atlantica. D u p l i c a t e s of e a c h of the s a m p l e s w e r e prepared. C o n t r o l s w e r e p r e p a r e d u s i n g m a r i n e agar and c u l t u r e s of b a c t e r i a to d e t e r m i n e c u l t u r e viability. A f t e r 18-24 hrs c u l t u r e s w e r e o b s e r v e d and zones of inhibition were measured. If the zone w a s not uniform, four m e a s u r e m e n t s from d i f f e r e n t areas w e r e taken. The m e a n was c a l c u l a t e d and this f i g u r e was used. R a w e x t r a c t s w e r e p r e p a r e d from the s e a w e e d s a m p l e s w h i c h h a d p r e v i o u s l y d e m o n s t r a t e d a c t i v i t y a g a i n s t the m a r i n e bacteria. Individual samples were separated into i0 g p o r t i o n s and b l e n d e d w i t h one of the f o l l o w i n g solvents: methanol, c h l o r o f o r m or w a t e r in a W a r i n g b l e n d e r for i0 minutes. Samples were c e n t r i f u g e d for i0 m i n u t e s at lOXg. The s u p e r n a t a n t was f i l t e r e d and a l l o w e d to e v a p o r a t e until dryness. The total dry w e i g h t was obtained. The e x t r a c t was r e s u s p e n d e d in the a p p r o p r i a t e s o l v e n t to give a final c o n c e n t r a t i o n of 40ug/uL. S t e r i l e f i l t e r paper discs w e r e i m p r e g n a t e d w i t h 100uL of r e s u s p e n d e d e x t r a c t (4mg) and air dried. P l a t e s c o n t a i n i n g m a r i n e a g a r w e r e i n o c u l a t e d w i t h one of the t h r e e b a c t e r i a l species. The discs w e r e p l a c e d on the b a c t e r i a l lawn. A f t e r 18-24 hours the zones of i n h i b i t i o n w e r e n o t e d and measured. RESULTS
AND DISCUSSION
The data s h o w t h a t s u b s t a n c e s are p r o d u c e d by s e a w e e d s of the N e w Y o r k / N e w J e r s e y coast, w h i c h i n h i b i t the g r o w t h of m a r i n e bacteria, at least to some e x t e n t (Table i). P s e u d o m o n a s a t l a n t i c a was not s u s c e p t i b l e to t h e s e substances. V i b r i o m a r i n o f l u v u s s h o w e d l i m i t e d inhibition, a zone of i n h i b i t i o n from 3 s e a w e e d species. A g r e a t e r e f f e c t was s e e n w i t h M i c r o c o c c u s imfimus; i n h i b i t i o n o c c u r r e d w i t h 8 species. W h i l e the first two s p e c i e s are g r a m negative, the last is g r a m positive. G r a m p o s i t i v e b a c t e r i a are k n o w n to be m o r e s e n s i t i v e to c e r t a i n a n t i b i o t i c s , due to d i f f e r e n c e s in cell wall structure. The g r a m n e g a t i v e b a c t e r i a are 744
Table
i. Z o n e s
of
inhibition Zones
Seaweed
of b a c t e r i a of
Vibrio (Gram -)
inhibition Micro (Gram+)
(mm) Pseudo (Gram -)
CHLOROPHYTA 6 0 0 0 0 7.5 0
ChaetomorDha linum CladoDhora gracilis Codium fragile Codium isthmocladium EnteromorDha linza SpongomorDha lanosa Ulva lactuca
0 3.5 0 5 7.5 0 0
0 0 0 0 0 0 0
0 0 0 0 4.5 0 0 0 4.25
0 0 0 0 0 0 0 0 0 0 0 0 0
PHAEOPHYTA Ascophyllum nodosum Chordaria flagelliformis Dictyosiphon foeniculaceus Ectocarpus tomentosus Fucus edentatus Fucus evanescens Fucus filiformis Fucus serratus Fucus spiralis Fucus vesiculosus Laminaria agardhii Laminaria saccharina Pilayella littoralis
0 0 0 0 0 0 0 3 0 0 0 0 0
RHODOPHYTA Ahnfeltia plicata Ceramium rubrum Chondrus crispus Cystoclonium purpureum Gracilaria verrucosa Grinnellia americana L a u r e n c i a s_p PhvlloDhora brodiaei Poiysiphonia denudata Polysiphonia fibrillosa Polysiphonia harveyi Polysiponia lanosa Porphyra leucosticta Porphyra umbilicalis Rhodymenia palmata
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
a
Vibrio Micro Pseudo
= Vibrio marinofulvis = Micrococcus im~imus = Pseudomonas atlantica
745
4
3 0 0
Figure i. Zone of inhibition produced by Laminaria aqardhii against Micrococcus imfimus. surrounded by a complex cellular envelope and therefore have a greater resistance to many antibiotics. Among the seaweeds, the chlorophyta displayed the most activity, 4 of 7 species (57%). The phaeophyta showed activity in 5 of 13 species (38%), mainly among the Fucales. The rhodophyta seem to be the least effective in the production of these substances, with only one active species. Not all species within a genus showed activity. Among the six Fucus species tested most were active, except for F__~.evanescenus and F_~. filiformis. Comparison of these results with previous work (Lustigman and Brown,1990), shows that human clinical bacteria displayed far greater susceptibility to the substances produced by the algae than did the marine species. With marine bacteria, the zones of inhibition were much smaller and the number of seaweeds producing b i o l o g i c a l l y active substances was fewer. Marine bacteria, which might have been exposed to these substances in the natural environment, would be more likely to have developed resistance to them than would clinical bacteria.
746
Table
2. Zones
Seaweed
of i n h i b i t i o n u s i n g algal e x t r a c t s Zones of i n h i b i t i o n ( m m ) Vibrio Micro Pseudo
Chordaria flagelliformis Codium isthmocladium Fucus e n d e n t a t u s Fucus s e r r a t u s Fucus s p i r a l i s Fucus v e s i c u l o s i s Laminaria agardhii S p o n g o m o r p h a lanosa
Vibrio Micro Pseudo
M
C
W
0 0 0 0 0 0 0 0
0 0 0 15 0 0 0 0
0 0 0 0 0 0 0 0
M
C
W
0 0 0 0 0 0 19 0 19 0 0 0 20 15 20 0 0 0 20 19 20 0 0 0
M
C
W
0 0 0 0 0 0 0 16 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0
= Vibrio marinofulvis = M i c r o c o c c u s imfimus = Pseudomonas atlantica
b M = methanol C = chloroform W = water
Table 2 gives the data o b t a i n e d for the e x t r a c t s p r e p a r e d from those algae w h i c h s h o w e d p r o m i s e of b a c t e r i c i d a l activity. The e x t r a c t s w e r e p r e p a r e d u s i n g e i t h e r methanol, c h l o r o f o r m or w a t e r as solvents. The r e s u l t s i n d i c a t e that for half of the s e a w e e d s tested, at least one of the e x t r a c t s was active. The zones of i n h i b i t i o n w e r e also s o m e w h a t larger t h a n w i t h the s e a w e e d t h a l l u s samples, p r o b a b l y due to g r e a t e r c o n c e n t r a t i o n of the s u b s t a n c e s in the discs. D i f f e r e n t a n t i m i c r o b i a l s u b s t a n c e s are p r o d u c e d by the seaweeds, as is seen by the d i f f e r e n c e in a c t i v i t y d e p e n d e n t u p o n the s o l v e n t used. M i c r o c o c c u s was m o r e s u s c e p t i b l e t h a n the g r a m n e g a t i v e species, a l t h o u g h the e x t r a c t of F . s e r r a t u s in c h l o r o f o r m d i s p l a y e d a c t i v i t y a g a i n s t V i b r i o and P s e u d o m o n a s , b u t not M i c r o c o c c u s . The Fucales were the m o s t active of the s e a w e e d s tested. R e s u l t s w e r e s i m i l a r to those a c h i e v e d with h u m a n c l i n i c a l bacteria, w i t h the c l i n i c a l b a c t e r i a again m o r e s u s c e p t i b l e to the a n t i b i o t i c s u b s t a n c e s ( L u s t i g m a n and Brown 1990). M o s t of the m e m b e r s of the F u c a l e s d i s p l a y e d b a c t e r i c i d a l activity. P o l y p h e n o l s are k n o w n to be e x c r e t e d by phaeophyta, such as F . s p i r a l i s (Bhakhuni and Silva 1974). These c h e m i c a l s cause cell lysis t h r o u g h m e m b r a n e damage. T e r p e n o i d s p r o d u c e d by the p h a e o p h y t a h a v e also shown to be a n t i m i c r o b i a l and i c h t h y o t o x i c (Schlenk and G e r w i c k 1987; Fenical 1982). The r e s u l t s for the c h l o r o p h y t a are s i m i l a r to those a c h i e v e d in p r i o r reports, w i t h a c t i v i t y seen w i t h the E n t e r o m o r p h a linza and S p o n g o m o r p h a k u t z u n g (Lustigman and Brown, 1990). C h l o r o p h y l l i d e s are among the active s u b s t a n c e s i s o l a t e d from c h l o r o p h y t e s and p h a e o p h y t e s ( B u r k h o l d e r and Sharma 1969). T e r p e n o i d s are also 747
produced by chlorophyta and rhodophyta (Fenical 1982). Acrylic acid is widely produced by the green, brown and red algae (Burkholder and Sharma, 1969). Many of the compounds produced are brominated, causing them to be highly toxic (Caccamese et al. 1981). Further research is underway to determine the chemical nature of the substances produced by the seaweeds studied here. Substances produced by macroalgae of the New York/New Jersey coast can be toxic to marine bacteria. There is a high survival value for algae producing secondary metabolites that are detrimental to organisms susceptible to those chemicals (Scheuer 1990). Marine bacteria showed more resistance to these substances than bacteria normally associated with humans, since the former have adapted over the generations to the presence of such substances produced and released in seawater. REFERENCES Atlas R (1984) Microbiology. Macmillan Publ Co, New York 10022, p 432. Barbeyron T, Berger Y (1989) Commensal bacteria living with two multicellular marine algae Antithamnion plumula and CladoDhora rupestris. Cahiers Biol Mar 30:361- 374. Bhakhuni DS, Silva M (1974) Biodynamic substances from marine flora. Bot Mar 17:40-51. Burkholder PR, Sharma GM (1969) Antimicrobial agents from the sea. Lloydia 32:466-483. Caccamese S, Azzolina R,Furnari G, Cormaci M, Grosso S (1981) Antimicrobial and antiviral activities of some marine algae from eastern Sicily. Bot Mar 24:365-367. Fenical W (1982) Natural products chemistry in the marine environment. Science 215:923-928. Grant PT, Mackie AM (1977) Drugs from the sea-fact or fantasy? Nature, 267:786-787. Hillson CJ (1977) Seaweeds. Pennsylvania State University Press, University Park, 16802 Horn MH, Neighbors MA, Rosenberg MJ, Murray SN (1985) Assimilation of carbon from dietary and nondietary macroalgae by a temperate-zone intertidal fish Cebidichthys-violaceus telopstei stachaeida. J Exp Mar Biol Ecol 86:241-254. Hornsey IS, Hide D (1976) The production of antimicrobial compounds by British marine algae III. Distribution of antimicrobial activity within the algal thallus. Brit Phycol J 11:175-181. Hornsey IS, Hide D (1985) The production of antimicrobial compounds by British marine algae IV. variation of anti-microbial activity with algal generation. Brit Phycol J 20:21-25. Lustigman B, Brown C (1990) Antibiotic production by marine algae isolated from the New York/New Jersey coast USA. Bull Environ Contam Toxicol 46:329-335. Norris JN, Fenical W (1982) Chemical defense in tropical marine algae. Science 218:417-432. 748
Scheuer PJ (1990) Some marine ecological phenomena: chemical basis and biomedical potential. Science 248:173-177. Schlenk D, Gerwick WH (1987) Dilophic acid a diterpenoid from the tropical brown seaweed DiloDhus guineensis. Phytochemistry 26:1081-1084. Received Received January 13, 1992; accepted April 30, 1992.
749
i Environmental Contamination 3and T o x i c o l o g y
Production of Antibacterial Substances by Macroalgae of the New York/New Jersey Coast, USA Bonnie Lustigman, Lee H. Lee, Nancy Thees, and John Masucci Biology Department, Montclair State College, Upper Montclair, New Jersey 07043, USA Marine algae are known to produce biochemicals which show a high degree of toxicity. The neurotoxin produced by the dinoflagellate Gonyaulax polyedra is responsible for red tides in tropical waters (Atlas 1984). Research has revealed that several of these substances have antibacterial, antiviral and/or antineoplastic effects (Scheuer 1990). Kainic acid, derived from the red algae Digenia simplex is an active antihelminthic (Grant and Mackie 1977). The toxic nature of these substances, which include acrylic acid, phenols, terpenoids and halogenated compounds, has made many of them unfit for commercial exploitation (Burkholder and Sharma 1969). They are produced by the algae to aid in plant protection by acting as deterrents for herbivorous fish (Horn et ai.1985) and to prevent surface fouling by bacteria and other epibionts (Norris and Fenical 1982). These substances may be contained in different parts of the algal thallus. In some species the new areas of growth have higher concentrations, perhaps to better protect these more fragile regions ( Hornsey and Hide 1976; Lustigman and Brown 1990). Different phases of the life cycle of the algae are also associated with varying amounts of antibiotic production ( Hornsey and Hide 1985). Previous research has shown production of antibiotic substances by marine macroalgae which were effective in preventing the growth of human clinical bacteria (Lustigman and Brown 1990). Many marine bacteria however, live in or on the algal thallus (Barbeyron and Berger 1989) and may have greater resistance to these substances. In this study we report the effect of antibacterial substances produced by 35 seaweed species as challenged by three species of marine bacteria. A preliminary extraction of the inhibitory substances produced was also undertaken. Send reprint requests to Dr. Lustigman address.
743
at the above
MATERIALS
AND M E T H O D S
S a m p l e s of 35 s e a w e e d s p e c i e s w e r e c o l l e c t e d f r o m S a n d y Hook, N e w J e r s e y and M o n t a u k Point, N e w York f r o m M a r c h to September, 1 9 8 9 ( T a b l e i). A f t e r c o l l e c t i o n the s a m p l e s w e r e p l a c e d on ice for t r a n s p o r t to the laboratory. T h e y w e r e w a s h e d in 10% Chlorox, r i n s e d t w i c e in d i s t i l l e d w a t e r and the d i f f e r e n t s p e c i e s w e r e i d e n t i f i e d b a s e d on the m o n o g r a p h by H i l l s o n (1977). Samples w e r e t h e n frozen until use. S a m p l e s of 3 cm w e r e cut f r o m the algal thallus, i n c l u d i n g the g r o w i n g tip. T h e s e w e r e s u r f a c e s t e r i l i z e d u s i n g 10% C h l o r o x and r i n s e d in s t e r i l e d i s t i l l e d water. Each p i e c e was t h e n p l a c e d in a Petri d i s h w i t h m o l t e n Difco M a r i n e A g a r in o r d e r to p r o d u c e u n i f o r m c o n t a c t of the algal s a m p l e and the agar. A f t e r the agar hardened, e a c h d i s h was s t r e a k e d w i t h 0.i mL from a 6-8 hr b a c t e r i a l c u l t u r e of one of the three b a c t e r i a l species, V i b r i o m a r i n o f u l v i s , M i c r o c o c c u s imfimus, P s e u d o m o n a s atlantica. D u p l i c a t e s of e a c h of the s a m p l e s w e r e prepared. C o n t r o l s w e r e p r e p a r e d u s i n g m a r i n e agar and c u l t u r e s of b a c t e r i a to d e t e r m i n e c u l t u r e viability. A f t e r 18-24 hrs c u l t u r e s w e r e o b s e r v e d and zones of inhibition were measured. If the zone w a s not uniform, four m e a s u r e m e n t s from d i f f e r e n t areas w e r e taken. The m e a n was c a l c u l a t e d and this f i g u r e was used. R a w e x t r a c t s w e r e p r e p a r e d from the s e a w e e d s a m p l e s w h i c h h a d p r e v i o u s l y d e m o n s t r a t e d a c t i v i t y a g a i n s t the m a r i n e bacteria. Individual samples were separated into i0 g p o r t i o n s and b l e n d e d w i t h one of the f o l l o w i n g solvents: methanol, c h l o r o f o r m or w a t e r in a W a r i n g b l e n d e r for i0 minutes. Samples were c e n t r i f u g e d for i0 m i n u t e s at lOXg. The s u p e r n a t a n t was f i l t e r e d and a l l o w e d to e v a p o r a t e until dryness. The total dry w e i g h t was obtained. The e x t r a c t was r e s u s p e n d e d in the a p p r o p r i a t e s o l v e n t to give a final c o n c e n t r a t i o n of 40ug/uL. S t e r i l e f i l t e r paper discs w e r e i m p r e g n a t e d w i t h 100uL of r e s u s p e n d e d e x t r a c t (4mg) and air dried. P l a t e s c o n t a i n i n g m a r i n e a g a r w e r e i n o c u l a t e d w i t h one of the t h r e e b a c t e r i a l species. The discs w e r e p l a c e d on the b a c t e r i a l lawn. A f t e r 18-24 hours the zones of i n h i b i t i o n w e r e n o t e d and measured. RESULTS
AND DISCUSSION
The data s h o w t h a t s u b s t a n c e s are p r o d u c e d by s e a w e e d s of the N e w Y o r k / N e w J e r s e y coast, w h i c h i n h i b i t the g r o w t h of m a r i n e bacteria, at least to some e x t e n t (Table i). P s e u d o m o n a s a t l a n t i c a was not s u s c e p t i b l e to t h e s e substances. V i b r i o m a r i n o f l u v u s s h o w e d l i m i t e d inhibition, a zone of i n h i b i t i o n from 3 s e a w e e d species. A g r e a t e r e f f e c t was s e e n w i t h M i c r o c o c c u s imfimus; i n h i b i t i o n o c c u r r e d w i t h 8 species. W h i l e the first two s p e c i e s are g r a m negative, the last is g r a m positive. G r a m p o s i t i v e b a c t e r i a are k n o w n to be m o r e s e n s i t i v e to c e r t a i n a n t i b i o t i c s , due to d i f f e r e n c e s in cell wall structure. The g r a m n e g a t i v e b a c t e r i a are 744
Table
i. Z o n e s
of
inhibition Zones
Seaweed
of b a c t e r i a of
Vibrio (Gram -)
inhibition Micro (Gram+)
(mm) Pseudo (Gram -)
CHLOROPHYTA 6 0 0 0 0 7.5 0
ChaetomorDha linum CladoDhora gracilis Codium fragile Codium isthmocladium EnteromorDha linza SpongomorDha lanosa Ulva lactuca
0 3.5 0 5 7.5 0 0
0 0 0 0 0 0 0
0 0 0 0 4.5 0 0 0 4.25
0 0 0 0 0 0 0 0 0 0 0 0 0
PHAEOPHYTA Ascophyllum nodosum Chordaria flagelliformis Dictyosiphon foeniculaceus Ectocarpus tomentosus Fucus edentatus Fucus evanescens Fucus filiformis Fucus serratus Fucus spiralis Fucus vesiculosus Laminaria agardhii Laminaria saccharina Pilayella littoralis
0 0 0 0 0 0 0 3 0 0 0 0 0
RHODOPHYTA Ahnfeltia plicata Ceramium rubrum Chondrus crispus Cystoclonium purpureum Gracilaria verrucosa Grinnellia americana L a u r e n c i a s_p PhvlloDhora brodiaei Poiysiphonia denudata Polysiphonia fibrillosa Polysiphonia harveyi Polysiponia lanosa Porphyra leucosticta Porphyra umbilicalis Rhodymenia palmata
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
a
Vibrio Micro Pseudo
= Vibrio marinofulvis = Micrococcus im~imus = Pseudomonas atlantica
745
4
3 0 0
Figure i. Zone of inhibition produced by Laminaria aqardhii against Micrococcus imfimus. surrounded by a complex cellular envelope and therefore have a greater resistance to many antibiotics. Among the seaweeds, the chlorophyta displayed the most activity, 4 of 7 species (57%). The phaeophyta showed activity in 5 of 13 species (38%), mainly among the Fucales. The rhodophyta seem to be the least effective in the production of these substances, with only one active species. Not all species within a genus showed activity. Among the six Fucus species tested most were active, except for F__~.evanescenus and F_~. filiformis. Comparison of these results with previous work (Lustigman and Brown,1990), shows that human clinical bacteria displayed far greater susceptibility to the substances produced by the algae than did the marine species. With marine bacteria, the zones of inhibition were much smaller and the number of seaweeds producing b i o l o g i c a l l y active substances was fewer. Marine bacteria, which might have been exposed to these substances in the natural environment, would be more likely to have developed resistance to them than would clinical bacteria.
746
Table
2. Zones
Seaweed
of i n h i b i t i o n u s i n g algal e x t r a c t s Zones of i n h i b i t i o n ( m m ) Vibrio Micro Pseudo
Chordaria flagelliformis Codium isthmocladium Fucus e n d e n t a t u s Fucus s e r r a t u s Fucus s p i r a l i s Fucus v e s i c u l o s i s Laminaria agardhii S p o n g o m o r p h a lanosa
Vibrio Micro Pseudo
M
C
W
0 0 0 0 0 0 0 0
0 0 0 15 0 0 0 0
0 0 0 0 0 0 0 0
M
C
W
0 0 0 0 0 0 19 0 19 0 0 0 20 15 20 0 0 0 20 19 20 0 0 0
M
C
W
0 0 0 0 0 0 0 16 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0
= Vibrio marinofulvis = M i c r o c o c c u s imfimus = Pseudomonas atlantica
b M = methanol C = chloroform W = water
Table 2 gives the data o b t a i n e d for the e x t r a c t s p r e p a r e d from those algae w h i c h s h o w e d p r o m i s e of b a c t e r i c i d a l activity. The e x t r a c t s w e r e p r e p a r e d u s i n g e i t h e r methanol, c h l o r o f o r m or w a t e r as solvents. The r e s u l t s i n d i c a t e that for half of the s e a w e e d s tested, at least one of the e x t r a c t s was active. The zones of i n h i b i t i o n w e r e also s o m e w h a t larger t h a n w i t h the s e a w e e d t h a l l u s samples, p r o b a b l y due to g r e a t e r c o n c e n t r a t i o n of the s u b s t a n c e s in the discs. D i f f e r e n t a n t i m i c r o b i a l s u b s t a n c e s are p r o d u c e d by the seaweeds, as is seen by the d i f f e r e n c e in a c t i v i t y d e p e n d e n t u p o n the s o l v e n t used. M i c r o c o c c u s was m o r e s u s c e p t i b l e t h a n the g r a m n e g a t i v e species, a l t h o u g h the e x t r a c t of F . s e r r a t u s in c h l o r o f o r m d i s p l a y e d a c t i v i t y a g a i n s t V i b r i o and P s e u d o m o n a s , b u t not M i c r o c o c c u s . The Fucales were the m o s t active of the s e a w e e d s tested. R e s u l t s w e r e s i m i l a r to those a c h i e v e d with h u m a n c l i n i c a l bacteria, w i t h the c l i n i c a l b a c t e r i a again m o r e s u s c e p t i b l e to the a n t i b i o t i c s u b s t a n c e s ( L u s t i g m a n and Brown 1990). M o s t of the m e m b e r s of the F u c a l e s d i s p l a y e d b a c t e r i c i d a l activity. P o l y p h e n o l s are k n o w n to be e x c r e t e d by phaeophyta, such as F . s p i r a l i s (Bhakhuni and Silva 1974). These c h e m i c a l s cause cell lysis t h r o u g h m e m b r a n e damage. T e r p e n o i d s p r o d u c e d by the p h a e o p h y t a h a v e also shown to be a n t i m i c r o b i a l and i c h t h y o t o x i c (Schlenk and G e r w i c k 1987; Fenical 1982). The r e s u l t s for the c h l o r o p h y t a are s i m i l a r to those a c h i e v e d in p r i o r reports, w i t h a c t i v i t y seen w i t h the E n t e r o m o r p h a linza and S p o n g o m o r p h a k u t z u n g (Lustigman and Brown, 1990). C h l o r o p h y l l i d e s are among the active s u b s t a n c e s i s o l a t e d from c h l o r o p h y t e s and p h a e o p h y t e s ( B u r k h o l d e r and Sharma 1969). T e r p e n o i d s are also 747
produced by chlorophyta and rhodophyta (Fenical 1982). Acrylic acid is widely produced by the green, brown and red algae (Burkholder and Sharma, 1969). Many of the compounds produced are brominated, causing them to be highly toxic (Caccamese et al. 1981). Further research is underway to determine the chemical nature of the substances produced by the seaweeds studied here. Substances produced by macroalgae of the New York/New Jersey coast can be toxic to marine bacteria. There is a high survival value for algae producing secondary metabolites that are detrimental to organisms susceptible to those chemicals (Scheuer 1990). Marine bacteria showed more resistance to these substances than bacteria normally associated with humans, since the former have adapted over the generations to the presence of such substances produced and released in seawater. REFERENCES Atlas R (1984) Microbiology. Macmillan Publ Co, New York 10022, p 432. Barbeyron T, Berger Y (1989) Commensal bacteria living with two multicellular marine algae Antithamnion plumula and CladoDhora rupestris. Cahiers Biol Mar 30:361- 374. Bhakhuni DS, Silva M (1974) Biodynamic substances from marine flora. Bot Mar 17:40-51. Burkholder PR, Sharma GM (1969) Antimicrobial agents from the sea. Lloydia 32:466-483. Caccamese S, Azzolina R,Furnari G, Cormaci M, Grosso S (1981) Antimicrobial and antiviral activities of some marine algae from eastern Sicily. Bot Mar 24:365-367. Fenical W (1982) Natural products chemistry in the marine environment. Science 215:923-928. Grant PT, Mackie AM (1977) Drugs from the sea-fact or fantasy? Nature, 267:786-787. Hillson CJ (1977) Seaweeds. Pennsylvania State University Press, University Park, 16802 Horn MH, Neighbors MA, Rosenberg MJ, Murray SN (1985) Assimilation of carbon from dietary and nondietary macroalgae by a temperate-zone intertidal fish Cebidichthys-violaceus telopstei stachaeida. J Exp Mar Biol Ecol 86:241-254. Hornsey IS, Hide D (1976) The production of antimicrobial compounds by British marine algae III. Distribution of antimicrobial activity within the algal thallus. Brit Phycol J 11:175-181. Hornsey IS, Hide D (1985) The production of antimicrobial compounds by British marine algae IV. variation of anti-microbial activity with algal generation. Brit Phycol J 20:21-25. Lustigman B, Brown C (1990) Antibiotic production by marine algae isolated from the New York/New Jersey coast USA. Bull Environ Contam Toxicol 46:329-335. Norris JN, Fenical W (1982) Chemical defense in tropical marine algae. Science 218:417-432. 748
Scheuer PJ (1990) Some marine ecological phenomena: chemical basis and biomedical potential. Science 248:173-177. Schlenk D, Gerwick WH (1987) Dilophic acid a diterpenoid from the tropical brown seaweed DiloDhus guineensis. Phytochemistry 26:1081-1084. Received Received January 13, 1992; accepted April 30, 1992.
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