The production of antimicrobial compounds by British marine algae I. Antibiotic-producing marine algae

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1 British Phycological Journal ISSN: (Print) (Online) Journal homepage: The production of antimicrobial compounds by British marine algae I. Antibiotic-producing marine algae I.S. Hornsey & D. Hide To cite this article: I.S. Hornsey & D. Hide (1974) The production of antimicrobial compounds by British marine algae I. Antibiotic-producing marine algae, British Phycological Journal, 9:4, , DOI: / To link to this article: Published online: 17 Feb Submit your article to this journal Article views: 1255 Citing articles: 82 View citing articles Full Terms & Conditions of access and use can be found at

2 Br. phycol J. 9: December 1974 I. THE PRODUCTION OF ANTIMICROBIAL COMPOUNDS BY BRITISH MARINE ALGAE ANT/BIOTIC-PRODUCING MARINE ALGAE By I. S. HORNSEY* and D. HIDE Department of Applied Biology, North East London Polytechnic, Romford Road, London E15 4LZ Using five species of bacteria as the test organisms, 151 species of British marine algae have been screened for the production of antibiotics. Of these, Asparagopsis armata, Bonnemaisonia asparagoides, Bonnemaisonia hamifera, Chondrus crispus, Dilsea carnosa, Gloiosiphonia capillaris, Sphondylothamnion multifidum, Desmarestia aculeata, Desmarestia ligulata, Laminaria digitata, Dictyopteris mcmbranacea, Dictyota dichotoma, Halidrys siliquosa and most members of the family Rhodomelaceae appear to possess outstanding antibacterial properties. Although the production of antibiotics would appear to be a characteristic of several families, it has not been possible to establish any major correlation between taxonomy and antibiotic production. In the case of two closely related and morphologically similar species, Chondrus crispus and Gigartina stellata, the former possesses considerable degrees of antimicrobial activity whilst the latter exhibits no such activity. The results also indicate that the production of antibiotics by the algae is affected by the season of the year. To date the only account of the antimicrobial activity of British marine algae has been given by Chesters & Stott (1956) who examined ethereal extracts of some common species. Considerable attention has been paid to the American and Japanese floras and, to a lesser extent, the German flora, and review articles summarising the results for these have been given by Sieburth (1964) and Burkholder & Sharma (1969). The present work was undertaken in order to provide a detailed survey of the British antibiotic-producing multicellular marine algae. Screening experiments were carried out at three-monthly intervals so that some indication of any seasonal variation in antibiotic activity could be detected. MATERIALS AND METHODS The following collection sites were used, material of any species examined at three-monthly intervals being taken from a single site: Kimmeridge, Portland Bill, Lulworth Cove and Lyme Regis, Dorset; Beer, Sidmouth and Exmouth, Devon; Dale, Pembrokeshire; Trearddur Bay, Rhosneigr, Porth Trecastell and Menai Bridge, Anglesey; and Beadnell, Northumberland. On collection from the sea, the algae were placed in wide-mouthed vacuum flasks containing Cardice and then screened for antibacterial activity within a period of 3 h. It is known that many seaweeds undergo enzymic degradation upon removal from the sea (Haas, 1935; Challenger & Simpson, 1948) and so only algae in the fresh condition were screened. The collection times were as follows: last week in March--first week in April (Spring); last week in June--first week in July (Summer); last two weeks in September (Autumn); last two weeks in January (Winter); all during t970. The algal nomenclature used is that recommended by Parke & Dixon (1968). The following bacteria were used as test microorganisms: Staphylococcus aureus (Polytechnic Culture Number 2), Escherichia coli (NCTC 4144), Bacillus subtilis (NCTC 8236), Streptococcus pyogenes (NCIB 8884) and Proteus morganii (Polytechnic Culture Number 84). * Present address: Department of Science, Cambridgeshire College of Arts and Technology, Collier Road, Cambridge, CB1 2AJ. 353

3 16'0 354 I. S. HORNSEY AND D. HIDE The bacteria were grown on a medium specially designed for this work and which consisted of dextrose, 20 g; peptone, 10 g; agar, 15 g and distilled water to ml aliquots of this agar were introduced into sterile Petri dishes and seeded with 0.1 ml of an 18 h-old nutrient broth culture of the test bacterium. Samples of the algal thallus, approximately 3 cm in length, were introduced into the seeded agar while it was in the molten condition. This ensured adequate contact between the algal thallus and the agar. For the larger macro-algae, a sample of thallus 24 mm in diameter was cut from the plant with the aid of a sterile cork-borer (such species are indicated by an asterisk in Table I). Samples of thallus were first surface sterilised by immersion in 10% sodium hypochlorite solution followed by immersion in sterile physiological saline. The algal sample was then rinsed in sterile distilled water. Control experiments carried out on known non-active algae and inert material indicated that no activity could be attributed to the surface sterilisation process. Each species was screened in triplicate and all assay plates were incubated for 24 h at 37 C. Those seaweeds which produced antibacterial substances showed a distinct zone of inhibition around the thallus. When the disc samples of thallus were examined for antibacterial activity the measurement of any zones of inhibition was relatively simple, but when uneven pieces of thalli were examined the zones of inhibition were more difficult to measure. In such cases a pair of dividers was used to take six separate measurements of the width of the inhibition zones extending from the edge of the algal thallus. A mean was then taken. OBSERVATIONS The algae that exhibited significant antibacterial activity at one or more of the quarterly mass-screenings are shown in Table I. TABLE I. Antimicrobially active British seaweeds Algal species Test microorganism Season of screening Spring Summer Autumn Winter Asparagopsis armata Bonnemaisonia asparagoides Bonnemaisonia hamifera Gracilariaverrucosa Phyllophora crispa Phyllophora membranifolia - Staph. aureus E. coil - 9"0 - - B. subtilis - 11"0 - - Prot. morganii - I Strep. pyogenes Staph. aureus - - E. coli B. subtilis Prot. morganii Strep. pyogenes Staph. aureus E. coil B. subtilis Prot. morganii - 14"0 - - Strep. pyogenes - 16"0 - - Staph. aureus 0" "0 4"0 E. coil "0 1.0 B. subtilis Prot. morganh 0" "0 2.0 Strep. pyogenes 0-0 0"0 0'0 2'5 Staph. aureus E. coli 1"0 0'0 0"0 0"0 B. subtilis 2.0 0'0 0'0 0"0 Prot. morganii Strep. pyogenes "0 Staph. aureus "5 0"0 0"0 E. coli B. subtilis "0 0'0 Prot. morganii 2" Strep. pyogenes 3"0 0'0 0"0 0'0

4 Antibiotic-producing marine algae 355 TABLE I. Continued. Algal species Test microorganism Season of screening Spring Summer Autumn Winter Chondrus erispus Dilsea carnosa Gloiosiphonia capillaris Callocolax neglectus Antithamnion cruciatum Antithamnion plumula Callithamnion arbuseula Callithamnion tetragonum Callithamnion tetricum Sphondylothamnion multifidum Delesseria sanguinea Staph. aureus 14' , E. coli B. subtilis Prot. rnorganff " Strep. pyogenes Staph aureus 12'5 12" E. coli B, subtilis 9.0 8"0 5'0 2.5 Prot. morganii "0 2.0 Strep. pyogenes Staph. aureus x - E. coli x - B. subtilis x - Prot. morganii - 24'0 x - Strep. pyogenes x - Staph. aureus 0' E. coli B. subtilis '5 x Prot. morganii 0.0 1"0 0.5 x Strep. pyogenes x Staph. aureus 4.0 2'5 1'0 1-0 E. coli B. subtilis Prot. morganii 2" Strep. pyogenes Staph. aureus E. coli I' B. subtilis "0 0.0 Prot. morganii "0 0,0 Strep. pyogenes Staph. aureus "5 2.0 E. coli ,0 B. subtilis 1' "0 Prot. morganff 1' "0 1-0 Strep. pyogenes 2.0 1' Staph. aureus 5" ,0 4'0 E. coli 2.5 3"0 2"5 2.0 B. subtilis 3.0 3" ,0 Prot. morganii 2" ,5 2"0 Strep. pyogenes Staph. aureus E. coli '0 B. subtilis ,5 Prot. morganii Strep. pyogenes Staph. aureus E. coli B. subtilis - 16, Prot. morganii - 15' Strep, pyogenes Staph. aureus 3-5 1" E. coli "0 0.0 B. subtilis Prot. morganii "0 0-0 Strep. pyogenes 2"

5 356 I. S. HORNSEY AND D. HIDE TABLE I. Continued. Algal species Test microorganism Season of screening Spring Summer Autumn Winter Membranoptera alata Bostrychia scorpioides Brongniartella byssoides Chondria dasyphylla Halopitys incurvus Laurencia hybrida Laurencia obtusa Laurencia pinnatifida Odonthalia dentata Polysiphonia elongata Polysiphonia lanosa Staph. aureus E. coil B. subtilis Prot. morganii 1" '0 0'0 Strep, pyogenes Staph. aureus "0 2.0 E. coli 1' "5 B. subtilis Prot. morganii Strep. pyogenes 1"5 0'5 0'5 1.0 Staph. aureus " E. coli "0 - B. subtilis '0 - Prot. morganff Strep. pyogenes 9" Staph. aureus 4-0 1' E. coli 2-5 0" "5 B, subtilis 3' "5 1"5 Prot. morganii Strep. pyogenes "5 2.0 Staph. aureus E. coli '0 B. subtilis 3" '5 1'0 Prot. morganff 2.0 1" Strep. pyogenes ,5 Staph. aureus "0 E. coli B. subtilis Prot. morganii "0 4.0 Strep. pyogenes 5.0 5,0 4,5 5-0 Staph. aureus 6.5 7" E. coli B. subtilis 4.5 4' "0 Prot. morganii 3-0 3" Slrep, pyogenes Staph. aureus '0 I0.0 11'0 E. coli 6"0 5" B. subtilis Prot. morganii 6, ,0 7.0 Strep. pyogenes '0 8.5 Staph. aureus ,0 E. coli 9, B. subtilis 10, '5 11'0 Prot. morganii Strep. p yogenes " Staph. aureus E. coli B. subtilis Prot. morganii "5 Strep. pyogenes Staph. aureus ' E. coli ' B. subtilis , Prot, morganii ' '0 Strep. pyogenes 15" '0

6 Antibiotic-producing marine algae 357 TABLE I. Continued. Algal species Test Season of screening microorganism Spring Summer Autumn Winter Polysiphonia nigra Staph. aureus "0 11" E. coli 7-0 7'0 7'5 7"0 B. subtilis '0 9-0 Prot. morganii Strep. pyogenes '0 9"5 Polysiphonia nigrescens Staph. aureus E. eoli B. subtilis "5 9'0 Prot. morganii Strep. pyogenes "5 9"0 9-5 Polysiphonia urceolata Staph. aureus " E. coli B. subtilis 8-0 8'5 8'0 8"5 Prot. morganii '0 6"0 Strep. pyogenes Pterosiphonia parasitica Staph. aureus x 3'5 x x E. coli x 2.0 x x B. subtilis x 2.0 x x Prot. morganii x 2-0 x x Strep. pyogenes x 2.5 x x Pterosiphonia thuyoides Staph. aureus '0 5"0 E. eoli B. subtilis Prot. morganii 0.5 3" Strep. pyogenes " Rhodornela confervoides Staph. aureus E. eoli "0 0.0 B. subtilis Prot. morganff '0 Strep. pyogenes "0 1.5 Ulva lactuca* Staph. aureus E. eoli '0 B. subtilis '0 Prot. morganii 0.0 0' Strep. pyogenes '5 Cladophora pellucida Staph. aureus "0 0.0 E. coli B. subtilis Prot. morganff "5 0"0 Strep. pyogenes "0 0.0 Bryopsis plumosa Staph. aureus E. eoli B. subtilis Prot. morganii 1.0 0" Strep. pyogenes Codium fragile Staph. aureus E. coli "5 1.5 B. subtilis Prot. morganii 3.0 3' "5 Strep. pyogenes Codium tomentosum Staph. aureus 6.0 5" "0 E. coli B. subtilis Prot. morganii "5 Strep. pyogenes

7 358 I. S. HORNSEY AND D. HIDE TABLE 1. Continued. Algal species Test microorganism Season of screening Spring Summer Autumn Winter Cylindroearpus berkeleyi Chordaria flagelliformis Eudesme virescens Mesogloia vermiculata Desmarestia aculeata Desmarestia ligulata Laminar& digitata* Laminaria saccharina* Alaria esculenta* Dictyopteris membranacea Dictyota diehotoma Staph. aureus E. coli B. subtilis - 1' Prot, morganii Strep. pyogenes Staph. aureus E. eoli B. subtilis Prot. morganii Strep. pyogenes Staph. aureus - 3' E. coli B. subtilis Prot. morganii - 1"5 1'0 - Strep. pyogenes Staph. aureus E. coli B. subtilis Prot. morganii - 1"0 - - Strep. pyogenes Staph. aureus E~ eoli 7"5 5" B. subtilis 8"5 7" '5 Prot. morganii Strep. pyogenes Staph. aureus E. coli B. subtilis - 20' Prot. morganii Strep, pyogenes Staph. aureus 9.0 7' E. eoli 6.0 2" "0 B. subtilis "5 Prot. morganii Strep. pyogenes 6' "5 Staph. aureus 8" E. coli 4-0 0' ,5 B. subtilis 5" "5 Prot. morganii "0 Strep. pyogenes 5.0 0" Staph. aureus 0' '0 E. eoli B. subtilis 0.0 2' Prot. morganii Strep. pyogenes 0"0 I' Staph. aureus E. coli 2.0 2' B. subtilis 4" Prot. morganii Strep. pyogenes 3"5 4" Staph. aureus 2.0 8' '0 E. coli '0 B. subtilis Prot. morganii Strep. pyogenes 0" '0

8 Antibiotic-producing marine algae 359 TABLE [. Continued. Algal species Bifurcar&bifurcata Cystoseira baccata Cystoseira tamariscifolia Halidrys siliquosa Test Season of screening microorganism Spring Summer Autumn Winter Staph. aureus 3" E. coli 0.0 0"0 0"0 - B. subtilis 1 '5 2" Prot. morganii 0" Strep. pyogenes 1.0 1" Staph. aureus 4.0 4' E. coli 2'5 2"0 2"0 0-0 B. subtilis "0 Prot. morganii '0 Strep. pyogenes 2" "5 0'0 Staph. aureus 5' "0 2-5 E. coli '0 B. subtilis "5 0"5 Prot. morganff 2' Strep. pyogenes Staph. aureus 6'5 5"0 4"0 2"5 E. coli B. subtilis Prot. morganii 3" "0 2'0 Strep. pyogenes "0 All figures represent zones of inhibition (in ram) as measured from the edge of the algal thallus to the edge of the region of normal bacterial growth; all represent the average of six individual measurements. Species marked with an asterisk were sampled by means of discs which were cut with a sterile cork-borer. Such disc samples were 24 mm in diameter. At certain times of the year, some species disappear from the environment and in such cases - appears in the relevant column. Where a species could not be located (although in theory should have been present) the absence of a test is denoted by x. The following algae exhibited either doubtful activity or no activity: Rhodochorton floridulum, Rhodochorton purpureum, Gelidium corneum, Gelidium latifolium, Nemalion helminthoides, Naccaria wiggii, Schizymenia dubyi, Furcellaria fastigiata, Halarachnion ligulatum, Catenella repens, Calliblepharis ciliata, Calliblepharis jubata, Cystoclonium purpureum, Rhodophyllis divaricata, Plocamium cartilagineum, Sphaerococcus coronopifolius, Ahnfeltia plicata, Gymnogongrus norvegicus, Stenogramme interrupta, Gigartina stellata, Hildenbrandia prototypus, Corallina officinalis, Jania rubens, Lithothamnion sp., Mesophyllum lichenoides, Dumontia incrassata, Polyides rotundus, Grateloupia filicina, Callophyllis laciniata, Kallymenia reniformis, Meredithia microphylla, Chylocladia verticillata, Gastroclonium ovatum, Lomentaria articulata, Lomentaria clavellosa, Rhodymenia palmata, Ceramium rubrum, Ceramium shuttleworthianum, Griffithsia flosculosa, Halurus equisetifolius, Plumaria elegans, Ptilota plumosa, Spyridia filamentosa, Cryptopleura ramosa, Hypoglossum woodwardii, Nitophyllum punctatum, Phycodrys rubens, P olyneura gmelinii, Heterosiphonia plumosa, Bangia fuscopurpurea, Porphyra purpurea, Porphyra umbilicalis, Prasiola stipitata, Ulothrix sp., Monostroma grevillei, Enteromorpha clathrata, Enteromorpha compressa, Enteromorpha intestinalis, Enteromorpha prolifera, Spongomorpha

9 360 I. S. HORNSEY AND D. HIDE aeruginosa, Chaetomorpha linum, Chaetomorpha melagonium, Cladophora rupestris, Derbesia tenuissima, Ectocarpus siliculosus, Giffordia hincksiae, Giffordia secunda, Pilayella littoralis, Ralfsia verruco~a, Elachista fucicola, Leathesia difformis, Asperococcus compressus, Asperococcus turneri, Litosiphon laminariae, Punctaria latifolia, Colpomenia peregrina, Petalonia fascia, Scytosiphon lomentaria, Sporochnus pedunculatus, Chorda ilium, Laminaria hyperborea, Saccorhiza polyschides, Sphacelaria bipinnata, Sphacelaria cirrosa, Halopteris illicina, Halopteris scoparia, Cladostephus spongiosus, Cladostephus verticillatus, Padina pavonia, Taonia atomaria, Ascophyllum nodosum, Fucus ceranoides, Fucus serratus, Fucus spiralis, Fucus vesiculosus, Pelvetia canaliculata and Himanthalia elongata. DISCUSSION Chesters & Stott (1956) state that some of their seaweed assays gave doubtful results. Unfortunately no information as to the nature of the doubts was included. During the course of the present screening experiments some algae yielded very small zones of inhibition (2 mm and less). Such values are at the lower limit of feasible measurement for the method employed and such algae are considered here to be doubtfully active. The results show that antibacterial activity is widespread throughout the Chlorophyceae, Phaeophyceae and Rhodophyceae, the three major seaweed classes. In cases where a number of species was examined from a single order, it was often found that one species is active while a closely related one is inactive. Probably the most striking examples of this are afforded by Chondrus crispus (active) and Gigartina stellata (non-active) and Laminara digitata (active) and Laminaria hyperborea (non-active). At present there is no explanation for this phenomenon. In some cases the presence or absence of antimicrobial activity appears to be of taxonomic significance. For example, all members of the Rhodomelaceae examined exhibit considerable activity, while the Fucaceae examined are inactive. Apart from these instances, taxonomic conclusions concerning the production of antimicrobial substances seem difficult to draw. However, we envisage that the considerable antimicrobial activity of Chondrus crispus and the lack of that characteristic in Gigartina stellata could be used as a chemotaxonomic factor in the identification of these two species. Several of the results presented here support the findings of Chesters & Stott (1956). The main anomaly concerns Pelvetia canaliculata, which Chesters & Stott found exhibited considerable antimicrobial activity but which was found to be non-active in our work. A possible explanation could be the fact that Chesters & Stott assayed ethereal extracts of their algae whereas we used samples of thallus. Chesters & Stott also state that each alga may have a period of maximum antimicrobial activity and/or a period of absence of activity in the year and this is indicated by some of the results in Table I. This factor could also contribute to the anomalous results obtained with Pelvetia canaliculata. The possible causes of other conflicting results obtained by various workers have been discussed previously (Hornsey, 1972).

10 Antibiotic-producing marine algae 361 The patterns of antimicrobial activity exhibited throughout the year by the algae studied in this work fall into three main categories: (a) uniform activity throughout the year (e.g. Polysiphonia lanosa); (b) conspicuous period of inactivity (e.g. Laminaria saccharina); (c) conspicuous peak of activity at one period of the year (e.g. Laminaria digitata). Several algae exhibited a spring peak of activity and this could be associated with their maximum growth period. However, until the compounds responsible for antimicrobial activity have been identified their possible function can only be a matter for speculation. In subsequent communications, the seasonal variation in production of antimicrobial activity will be further discussed and the isolation and identification of some of the active components will be described. ACKNOWLEDGEMENTS We wish to thank Dr W. Eifion Jones for making facilities available at The Marine Science Laboratories, Menai Bridge, Anglesey and Roger M. Smith for helpful advice. REFERENCES BURKHOLDER, P. R. SHARMA, G. M., Antimicrobial agents from the sea. Lloydia, 32: 46~483. CHALLENGER, F. & SIMPSON, M. I., Studies on biological methylation. XII. A precursor of dimethyl sulphide evolved by Polysiphonia fastigiata. J. chem. Soc., 3: CnESTEgS, C. G. C. & STo~r, J. A., Production of antibiotic substances by seaweeds. Proc. 2nd Int. Seaweed Syrup., Pergamon Press, New York. HAAS, P., Liberation of methyl sulphide by seaweed. Biochem. J., 29: HORNSEY, I. S., The production of antimicrobial substances by British marine algae. Ph.D. Thesis. University of London. PARKE, M. & DIXON, P. S., Check-list of British marine algae--second revision. J. mar. biol Ass. U.K., 48: SIEBURTH, J. McN., Antibacterial substances produced by marine algae. Devs ind. MicrobioL, 5: