Production of Axenic Gonyaulax Cultures by Treatment with Antibiotics

Transcription

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, July 12, p /82/ $02.00/0 Vol. 44, No. 1 Production of Axenic Gonyaulax Cultures by Treatment with Antibiotics CHARLES L. DIVAN* AND HEINRICH K. SCHNOES Department of Biochemistry, College of Agriculture and Life Sciences, University of Wisconsin, Madison, Wisconsin Received 27 January 12/Accepted 8 April 12 The effects of amphotericin B, chloramphenicol, dihydrostreptomycin sulfate, neomycin sulfate, polymyxin B sulfate, potassium penicillin G, and streptomycin sulfate (used singularly and in various combinations at different concentrations) on the growth and development of four marine dinoflagellates of the genus Gonyaulax and associated bacteria were studied. The combination of amphotericin B, dihydrostreptomycin, neomycin, and penicillin G was highly effective in eliminating bacteria and fungi without reducing dinoflagellate growth and provided a useful method for obtaining axenic cultures of two Gonyaulax species, G. catenella and G. excavata. In connection with our work on neurotoxins produced by marine dinoflagellates (2, 14), we have been studying various species of Gonyaulax. Of the four species examined, only G. tamarensis Plymouth is nontoxic. The other three organisms, G. catenella, G. excavata, and G. tamarensis subsp. excavata, produce potent neurotoxins, of which saxitoxin is the most widely known (2, 5, 9, 10, 14). These algae have been isolated from a variety of northern marine locations during "red tide" outbreaks. G. catenella was isolated from Washington coastal waters, G. excavata was isolated from Massachusetts coastal waters, G. tamarensis subsp. excavata was isolated from the coastal waters of British Columbia, and G. tamarensis Plymouth was isolated from the coastal waters of England. Axenic cultures are required for an understanding of nutritional requirements, environmental parameters affecting dinoflagellate growth, and toxin biosynthesis (6-8, 11, 12, 15). Previous studies have shown that treatment with antibiotics is an effective means of obtaining axenic algal cultures (1, 3, 6-8, 11-13, 15). However, the literature contains little quantitative data on the effect of antibiotics on the growth and development of Gonyaulax species, and two methods reported to yield axenic Gonyaulax cultures proved to be ineffective with our cultures (11, 15). Therefore, we undertook an examination of a series of antibiotics to determine their potential to eliminate the associated bacteria and not affect the dinoflagellates. This study used liquid culture media; methods involving plating of Gonyaulax species on artificial seawater-0.5% agar medium containing various antibiotics are not suitable in obtaining axenic cultures because these dinoflagellates will not 250 grow on 0.5% agar medium (11). Nonaxenic unialgal cultures were obtained from the following sources: G. catenella (L. Norris, Friday Harbor, Wash.), G. excavata (C. M. Yentsch, Bigelow Laboratory for Ocean Sciences, West Boothbay Harbor, Maine), G. tamarensis subsp. excavata (F. J. R. Taylor, University of British Columbia, Vancouver), and G. tamarensis Plymouth (Culture Centre of Algae and Protozoa, Cambridge, England). All of the algal cultures were grown in cottonplugged flasks containing 250 ml of natural seawater (Marine Biological Laboratory, Woods Hole, Mass.), sterilized by autoclaving, supplemented with salts and vitamins containing (total volume, 5 ml): NaNO3 (35 mg), Na24-glycerophosphate (5 mg), and H3BO3 (2.85 mg)-fec13 (24.5 p.g)-mnso4 (0.41 mg)-cocl2 (10.1,ug)- ZnSO4 (55 p.g)-fe(nh4)2(so4)2 (1.76 mg) chelated with disodium EDTA (1 M:1 M), thiaminehydrochloride (50,ug), vitamin B-12 (1,ug), biotin (0.5,ug), and 0.83 mm Tris-hydrochloride, ph 8.1 (F. J. R. Taylor, personal communication). The antibiotics (Sigma Chemical Co., St. Louis, Mo.) and the salt and vitamin supplement were filter sterilized through a Gelman TCM-200 Metricel membrane filter. This medium was inoculated with 20 ml of an actively growing algal culture (ca. 2 x 105 cells). The cultures were grown under constant illumination (4 Im/ cm2) for 16 to 17 days, at C. Cultures were not aerated. At the end of the growth period, 10 ml of the culture was centrifuged (International Clinical Centrifuge, Boston, Mass.) in a graduated conical tube at slow speed for 7 min. All but 0.5 ml of the supernatant was removed with a Pasteur pipette. The cells were resuspended in the remaining 0.5 ml of medium.

2 VOL. 44,12 NOTES 251 TABLE 1. Effect of antibiotics on growth of Gonyaulax sp. Antibiotic Antibiotic' (% of control)b Gonyaulax species concn (,ug/ml) A' C DiS N P PG S G. catenella Lysisd NDe Lysis ND ,000 ND ND G. excavata Lysis ND Lysis Lysis ,000 ND ND G. tamarensis subsp. excavata Lysis ND Lysis 0 0 ND ND ,000 ND ND 0 0 ND 0 0 G. tamarensis Plymouth Lysis ND Lysis 0 0 ND ND ,000 ND ND 0 0 ND 0 0 a Antibiotics: (A) amphotericin B, (C) chloramphenicol, (DiS) dihydrostreptomycin sulfate, (N) neomycin sulfate, (P) polymyxin B sulfate, (PG) potassium penicillin G, and (S) streptomycin sulfate. bcell counts: Control flasks, 16 days; antibiotic flasks, 16 or 17 days. c Concentration for amphotericin B, micrograms per 10 ml. d Free chlorophyll in media after 24 h. ' ND, Not determined. Cells were observed with an AO Microstar light microscope under a 1Ox objective (American Optical Corp., Buffalo, N.Y.) for changes in size, morphology, or motility. Cell numbers were determined by triplicate counts, using a hemacytometer ([American Optical Corp.]; number of cells counted per 10 squares x concentration factor-' x 103 = cells ml-1). Bacterial cell numbers were determined by a five-tube most probable number series (4). Samples were removed from each flask after 1 to 3 days of contact with the antibiotic(s). These samples were diluted in sterile seawater and used as inoculum for the most probable number reading. The most probable number tubes had media identical to those for algal cultures, except for the addition of either 0.1% yeast extract or 0.1% peptone (Difco Laboratories, Detroit, Mich. [7]). Both the yeast extract and the peptone were used to ensure that negative growth in the most probable number series was the result of exposure to antibiotics and not due to lack of nutrients in the media. Tubes were incubated in the dark at C and scored for turbidity after 3 days. Negative tubes were incubated for up to 4 weeks (7) and scored at that time. All transfers were performed aseptically in a sterile hood (Labconco Corp., Kansas City, Mo.). Wet mounts and Gram stains showed small motile gram-negative rods in all nonaxenic cultures. In addition, G. excavata cultures contained a gramnegative organism which formed rosettes and filaments. The effect of seven common antibiotics on the growth offour Gonyaulax species is documented in Table 1. The results in Table 1 show that G. catenella and G. excavata tolerated dihydrostreptomycin, neomycin, penicillin G, and strep-

3 252 NOTES APPL. ENVIRON. MICROBIOL. TABLE 2. Bactericidal activity of single antibiotics Antibiotica G. catenella G. excavata G. tamarensis subsp. G. tamarensis Plymouth excavata concn (pg/ml) 0.1% Yeast 0.1% 0.1% Yeast 0.1% 0.1% Yeast 0.1% 0.1% Yeast 0.1% extract Peptone extract Peptone extract Peptone extract Peptone Control 1.6 x 107b 1.6 x X X X X i X X 106 DiS 50 NDc ND ND ND 1.1 x 103 ND ND x x x x x x 102 ND ND x x 105 ND ND ND ND X i X x x 105 ND ND ND ND 1, x X X X 104 ND ND ND ND N 50 ND ND ND ND ND ND 6.0 x x x x x x 106 ND ND 1.7 x x X x 106 ND ND ND ND X X x x 106 ND ND ND ND 1, x x x x 105 ND ND ND ND p x x 106 ND ND ND ND ND ND 100 ND ND 8.0 x x 106 ND ND ND ND PG 100 ND ND ND ND 8.0 x x x x x x x x x x x x x x x x 105 ND ND ND ND S 10 ND ND ND ND ND ND x x x x 106 ND ND ND ND x x x x 106 ND ND ND ND x x x 106 ND ND ND ND 1, x x x x 106 ND ND ND ND a Antibiotics: (DiS) dihydrostreptomycin sulfate; (N) neomycin sulfate; (P) polymyxin B sulfate; (S) streptomycin sulfate. b Bacterial cells ml-'. c ND, Not determined. tomycin over a wide range of concentrations, whereas G. tamarensis subsp. excavata and G. tamarensis Plymouth were susceptible to all of these antibiotics. Both amphotericin B and chloramphenicol caused inhibition of dinoflagellate growth. Amphotericin B, a polyene antibiotic, is effective against organisms with high steroid contents in their membranes, such as fungi. G. catenella and G. excavata showed some resistance to amphotericin B, but inhibition occuffed at concentrations higher than 2.5,ug/ml. Chloramphenicol caused inhibition of all species examined; moreover, cell lysis occurred at concentrations greater than 50,ug/ml (Table 1). The five antibiotics (dihydrostreptomycin, neomycin, penicillin G, polymyxin B, and streptomycin) exhibiting the lowest toxicity to dinoflagellate cells were tested for their bactericidal activity (Table 2). No single antibiotic was completely bactericidal, nor were the highest concentrations of an antibiotic always the most effective (Table 2). Treatments with double antibiotic combinations were only slightly more effective than those with single antibiotics, but triple combinations proved to be highly effective (Table 3). Again, higher concentrations of double antibiotic combinations were less bactericidally effective and, in some cases, were less effective than when the same concentration of single antibiotics had been used (Tables 2 and 3). Of the triple combinations studied, the administration of a combination of dihydrostreptomycin, neomycin, and penicillin G proved to be the most effective. This combination reduced the bacterial cell number to cells ml-'; the enhanced effect of this combination is apparent from the data shown in Tables 2 and 3. After the elimination of bacteria from the algal cultures, a

4 VOL. 44, 12 Antibiotic combinationa composed of (kg/ml): NOTES 253 TABLE 3. Bactericidal activity of antibiotic combinations G. catenella G. excavata 0.1% Yeast 0.1% b 0.1% Yeast 0.1%Gr extract Peptone Growth extract Peptone Growth 1.7 x 102c 6.0 x x x x x x x x x x x x x x 102 A (50) a Antibiotics: (DiS) dihydrostreptomycin sulfate; (N) neomycin sulfate; (PG) potassium penicillin G; (S) streptomycin sulfate; (A) amphotericin B. b Dinoflagellate growth as the percentage of control flask growth. C Bacterial cells ml-' x x x x x l0-5.0 x x x x x x X x x x x 1i X x

5 254 NOTES filamentous fungus was able to grow in these cultures. The addition of amphotericin B eliminated the fungus. The final combination of dihydrostreptomycin, neomycin, penicillin G, and amphotericin B (250:250:500:5.0,ug/ml) led to axenic cultures. This antibiotic combination reduced neither growth, motility, nor toxin production (unpublished) of G. catenella and G. excavata, but did increase the lag phase after new culture transfer. Combinations of dihydrostreptomycin-penicillin G, neomycin-penicillin G, and dihydrostreptomycin-neomycin-penicillin G at 100,ug ml-' proved to be ineffective in eliminating bacteria and were inhibitory to cultures of G. tamarensis subsp. excavata and G. tamarensis Plymouth. This method of obtaining axenic cultures was effective for two dinoflagellates (G. catenella and G. excavata). It has not been useful for G. tamarensis subsp. excavata or G. tamarensis Plymouth; antibiotic concentrations which are required to show good bactericidal activity also cause very marked and often total inhibition of these dinoflagellates. This work was supported by funds from Public Health Service grant R02-FD00605 from the National Institutes of Health. LITERATURE CITED 1. Berland, B. R., and S. Y. Maestrini Study of bacteria associated with marine algae culture. Mar. Biol. 3: Boyer, G. L., C. F. Wichmann, J. Mosser, E. J. Schantz, and H. K. Schnoes Toxins isolated from Bay of Fundy scallops, p In D. L. Taylors and H. H. Seliger (ed.), Toxic dinoflagellate blooms. Elsevier/North- Holland, Amsterdam. APPL. ENVIRON. MICROBIOL. 3. Droop, M. R A procedure for routine purification of algae cultures with antibiotics. J. Phycol. Bull. 3: Horwitz, W. (ed.). 10. Official methods of analysis of the Association of Official Analytical Chemists, p Association of Official Analytical Chemists, Washington, D.C. 5. Prakash, A., J. C. Medcof, and A. D. Tennant Paralytic shellfish poisoning in eastern Canada. Fish. Res. Board Can. Bull., p Provasoli, L., and K. Gold Nutrition of the American strain of Gyrodinium cohnii. Arch. Mikrobiol. 42: Ray, S. M., and W. B. Wilson Effects of unialgae and bacteria-free cultures of Gymnodium brevis on fish. In U.S. Fish and Wildlife Service, Special scientific report- Fisheries, no U.S. Department of the Interior, Washington, D.C. 8. Reich, K., and J. Kahn A bacteria-free culture of Prymmesium parvum (Chrysomonadina). Bull. Res. Counc. Isr. 4: Schantz, E. J The dinoflagellate poisons, p In S. Kadis, A. Ciegler, and S. J. AjI (ed.), Microbiological toxins, vol. 7. Academic Press, Inc., New York. 10. Schantz, E. J Shellfish, fish, and algae, p In I. E. Liener (ed.), Toxic constituents of animal foodstuffs. Academic Press, Inc., New York. 11. Siegelman, H. W., and J. H. Kycia Large scale culture of dinoflagellate algae, p In D. L. Taylors and H. H. Seliger (ed.), Toxic dinoflagellate blooms. Elsevier/North-Holland, Amsterdam. 12. Spencer, C. P On the use of antibiotics for isolating bacteria-free cultures of marine phytoplankton organisms. J. Mar. Biol. Assoc. 31: Ukeles, R., and J. Bishop Enhancement of phytoplankton growth by marine bacteria. J. Phycol. 11: Wichmann, C. F., G. L. Boyer, C. L. Divan, E. J. Shantz, and H. K. Schnoes. 11. Neurotoxins of Gonyaulax excavata and Bay of Fundy scallops. Tetrahedron Lett. 22: Yentsch, C. M., E. J. Cole, and M. G. Salvaggio Some of the growth characteristics of Gonyaulax tamarensis isolated from the Gulf of Maine, p In V. R. Locicero (ed.), Proceedings of the First International Conference on Toxic Dinoflagellate Blooms. The Massachusetts Science and Technology Foundation, Wakefield, Mass.