INHIBITION OF ALGAE BY NYSTATIN

INHIBITION OF ALGAE BY NYSTATIN J. 0. LAMPEN AND PETER ARNOW Institute of Microbiology, Rutgers, The State University, New Brunswick, New Jersey ABSTRACT LAMPEN, J. 0. (Rutgers, the State University, New Brunswick, N. J.), AND PETER ARNOW. Inhibition of algae by nystatin. J. Bacteriol. 82:247-251. 1961.-The polyenic antibiotic nystatin inhibited the growth of a broad range of algae at concentrations of 1 to 30,ug per ml. Organisms included in the Chlorophyta, Euglenophyta, Chrysophyta, and Cyanophyta were inhibited, but a Baciilariophyceae was insensitive. Nystatin was lethal at concentrations which completely prevented growth. The polyene was absorbed by sensitive algae from aqueous medium. Nystatin produced K+ leakage, but did not inhibit dark respiration or photosynthetic oxygen production. The effects of the antibiotic on algae appear to be similar in many ways to those on yeast. Received for publication March 6, 1961 Nystatin, a tetraenic antifungal antibiotic, Organisms. The following algal strains were inhibits the growth and utilization of various obtained from the Indiana University culture substrates by fungi but has no significant effect collection: Chlamydomonas reinhardti Dangeard on bacteria or animal cells (Brown and Hazen, (no. 89), Scenedesmus obliquus (Turp.) Kruger 1957). The basis for this specificity appears to be (no. 393), Euglena gracilis Klebs (no. 752), the ability of sensitive organisms, and of these Euglena gracilis Klebs (no. 753), Prototheca only, to absorb the polyene from aqueous media zopfii Kruger (no. 328), Plectonema boryanum (Lampen and Arnow, 1959). Yeast protoplasts Gomont (no. 597), and Navicula pelliculosa respond in a manner similar to intact cells, i. e., (Breb.) Hilse (no. 667). Ochromonas malhamensis nystatin is absorbed, potassium ions and cofactors no. 11532 was obtained from the American Type leak from the cell, and metabolism ceases (Marini, Culture Collection. Chlorella vulgaris was received Arnow, and Lampen, 1961). Hence the critical from Henry Vogel of this Institute. binding may well be that which occurs on the Media. (i) Nutrient broth (Difco) or agar cell membrane. During the course of these studies containing 1% glucose (initial ph 6.8); (ii) the it was observed that nystatin inhibited the growth defined medium described by Arnow, Oleson, and of certain algae and was absorbed readily by Williams (1953), hereafter termed medium A; these cells. These results are reported briefly (iii) the defined medium used by Hutner et al. here. (1957) for the growth of Ochromonas (medium Although an effect of the highly specific polyene 0); (iv) the Cyanophycean medium described by antifungals on algae has not previously been Starr (1960), to which was added 1 mg/ml reported, various other antibiotics have been L-arginine monohydrochloride and 0.01 % manganese sulfate (medium C); (v) the soil-extract tested against these organisms. Tomisek et al. (1957) reported the inhibition of the growth of medium described by Starr (1960), to which Chlorella pyrenoidosa by bacitracin, eryth- was added 1 mg/ml yeast extract, 1 mg/ml 247 romycin, oxytetracycline, gliotoxin, thiolutin, polymyxin B, and streptomycin. The development of permanent apochlorosis in Euglena gracilis strains treated with streptomycin was observed by Provasoli, Hutner, and Schatz (1948). Fotor, Palmer, and Maloney (1953) investigated the antialgal effect of streptomycin chelates and neomycin. Havinga et al. (1953) studied the effects of various antibiotics and other biochemically active substances on CO2 fixation by Scenedesmus obliquus in the light and dark. Zehnder and Hughes (1958) reported that cycloheximide at concentrations of 50 ppm or less inhibited the growth of Chlorophyceae, Xanthophyceae, and Bacillariophyceae, but much higher concentrations of the antibiotic had no inhibiting effect on the growth of Myxophyceae. MATERIALS AND METHODS

248 LAMPEN AND ARNOW [VOL. 82 L-arginine monohydrochloride, and 0.01 % manganese sulfate (medium S). Sensitivity tests. For uniformity all the algae were grown at 23 C over a bank of fluorescent tubes which supplied a light intensity of 500 ft-c. Inocula were prepared by scraping the 2 to 5-day algal growth from a slant of an appropriate agar medium into 50 ml of the specified liquid medium. After the specified growth period, the algae were washed and used to inoculate 10-ml volumes of medium in 50-ml Erlenmeyer flasks. The nystatin was sterilized by the technique of Donovick et al. (1955). A highly purified crystalline preparation was supplied by the Squibb Institute for Medical Research, New Brunswick, N. J. All growth densities were measured in a Klett colorimeter at 540 m,. Unless otherwise specified, the incubation period for the sensitivity tests was 72 hr. TABLE 1. Sensitivity of algal growth to nystatin Division Organism Minimal inhibitory concn of nystatin* (72 hr) Nu- trient broth Defined medium pg/ml pg/ml Eugleno- Euglena gracilis 30 10 (3) phyta no. 752 E. gracilis no. 753 30 5 Chlorophyta Chlorella vulgaris 3 (1) 3 Scenedesmus 1 1 obliquus Chlamydomonas 1 (1) 4 reinhardti Prototheca zopfii 1 Chrysophyta Ochromonas 1 malhamensis Navicula > 60 pelliculosat Cyanophyta Plectonema 1 boryanumt * P. boryanum was tested in defined medium C, N. pelliculosa in medium S, and 0. malhamensis in medium 0; all the others were tested in medium A. OD of inoculated media ca. 0.01; OD at time of reading 0.3 to 0.6. Data in parentheses represent minimal inhibitory concentrations of nystatin for dark growth. t Incubated for 5 days. 0 U-)' m cd 5 10 jpg NYSTATIN/ml FIG. 1. Effect of medium on the sensitivity of Chlorella vulgaris to nystatin. O O = Defined medium A, initial ph = 5.8. 0 0 = Final ph at various nystatin concentrations in medium A. A A = Nutrient broth + glucose, initial ph = 6.8; final ph at 0,g nystatin/ml = 7.0; final ph at 30,ug nystatin/ml = 6.6. * * = Nutrient broth + glucose, initial ph = 4.1; final ph = 4.5 at all nystatin levels. Absorption of nystatin was measured by the procedure of Lampen and Arnow (1959). Dark respiration and photosynthetic oxygen production were determined by conventional techniques (Umbreit, Burris, and Stauffer, 1951). Potassium analyses were carried out with the flame photometer attachment for the Beckman spectrophotometer, model B. RESULTS Inhibition of growth. The growth of all strains except N. pelliculosa was inhibited by nystatin at levels of 1 to 30,ug/ml (Table 1). The inhibitory concentration of nystatin for algal cultures is probably even lower than indicated in Table 1 in view of the instability of the polyene in the light. E. gracilis appeared to be less sensitive in the defined medium than were the other organisms; however, there was less difference when nutrient broth was used. In the dark, E. gracilis,

1961] NYSTATIN INHIBITION OF ALGAE 249 S. obliquus, and C. reinhardti were equally or even more sensitive to nystatin than they were in the light. It appears clear that nystatin has potent algicidal action, since cells from cultures exposed to twice the minimal inhibitory concentrations of nystatin were unable to grow after transfer to antibiotic-free media. N. pelliculosa was insensitive to 30 Ag/ml of nystatin. No major nutritional effects on nystatin action were observed under the conditions of Table 1. In other tests with C. vulgaris, the arginine of medium A was replaced by isonitrogenous levels of KNO3, (NH4)2SO4, DL-glutamic acid, or NH4NO3 without shifting the inhibitory concentration of nystatin outside the range of 1.5 to 3.0,ug/ml. Addition of organic acids (a-ketoglutarate, succinate, or acetate) was also without effects. WVith a relatively large inoculum of C. vulgaris, approximately two cell divisions could occur in nutrient broth at ph 4 to 4.5 even in the presence of high concentrations of nystatin (Fig. 1). Growth in medium A, initially ph 5.9, was highly sensitive and alkalinization occurred. In nutrient broth at ph 6.6 to 7.0, sensitivity was intermediate between these extremes. When the inoculum was one-tenth of that used for Fig. 1, no comparable resistance in acid nutrient broth was observed. Leakage of organic acids from yeasts treated with nystatin has been reported by Osteux, Tran-Van-Ky, and Biquet (1958) and Scholz et al. (1959). The protection TABLE 2. Absorption of nystatin by algal cultures Organism Chlamydomonas reinhardti. Chlorella vulgaris... Scenedesmus obliquus... Euglena gracilis no. 752... E. gracilis no. 753... Prototheca zopfii... Plectonema boryanum... Navicula pelliculosa... Nystatin absorbed, pg/mg cell dry wt 20 min 40 min ph ph ph ph 4.0 7.0 4.0 7.0 1.7 0.3 2.8 0.5 1.1 2.4 1.0 2.2 1.1 0.1 2.5 0.4 1.1 0.1 2.3 0.5 1.7 0.2 2.8 0.6 3.5 <0.4 <0.4 <0.4 * Cultures grown for 2 to 5 days on the specific noted in Table 1. Cells were washed twice with water before use. TABLE 3. Leakage of potassium ions from Chlorella vulgaris in the presence of nystatin* Nystatin K+ in cells pg/ml pg/mg 0 13.6 1 7.3 10 2.9 * Test system: 14.4 mg (dry weight) of C. vulgaris (cells from a 48-hr culture in medium A) in 30 ml of 0.05 M sodium citrate phosphate buffer, ph 5.8, containing 0.04 M glucose and varying concentrations of nystatin. Mixtures incubated 30 min at 28 C on a rotary shaker and the potassium content of the cells determined (Marini et al., 1961). of large inocula at acid ph by complex natural materials may be related to this phenomenon. It should be noted that nystatin induced apochlorosis of E. gracilis only at concentrations which killed the cells. Thus it does not show the preferential action against chloroplasts which is characteristic of streptomycin (Provasoli et al., 1948). Absorption of nystatin. Cells of all the sensitive algae absorbed substantial amounts of nystatin (Table 2). N. pelliculosa, which was insensitive, absorbed almost none. Lampen et al. (1959) noted that baker's yeast or yeast from the stationary growth phase absorbed a greater quantity of nystatin at ph 4 than at ph 7. A similar result was obtained with the algal cultures. Potassium leakage. When cells of C. vulgaris were incubated with increasing amounts of nystatin, the content of bound K+ (not removed by washing with water) fell rapidly (Table 3), although it did not attain levels as low as those observed with yeast (Marini et al., 1961). It seems clear, therefore, that nystatin interferes with the ability of algae and of yeast to maintain elevated internal K+ concentrations. Effects on metabolism. No direct actioni of nystatin on the metabolism of C. vulgaris or S. obliquus could be demonstrated. At 40,ug/ml, nystatin did not inhibit dark respiration (ph 4 or 7) or photosynthetic oxygen production by washed cells. As a further test, cells of C. vulgaris were incubated in medium A for 4 hr with 10,ug of nystatin/ml, a level whieh completely

250 LAMPEN AND ARNOW [VOL. 82 prevented growth. Photosynthetic oxygen production was still 50% of that exhibited by untreated cells. From the slowness of this fall in activity and the somewhat greater sensitivity of growth in the dark (Table 1), it is concluded that any effects on the photosynthetic apparatus were secondary to other actions of the antibiotic. DISCUSSION It is apparent that a broad range of algal types are sensitive to the polyene nystatin. Organisms included in the Chlorophyta, Euglenophyta, Chrysophyta, and Cyanophyta were inhibited. It has been suggested (Marini et al., 1961) that the primary mode of action of nystatin against Saccharomyces cerevisiae is an alteration of the cell membrane with resultant loss of ability to concentrate critical metabolites. These studies with algae support this hypothesis, since the sensitive organisms bound the antibiotic in amounts and under conditions comparable to those described for yeast; K+ leaked rapidly from the algal cells; a direct lethal action occurred; and the photosynthetic apparatus was not affected. N. pelliculosa, of the Chrysophyta, class Bacillariophyceae, the only insensitive organism among those tested, did not absorb appreciable nystatin. Zehnder and Hughes (1958) observed that cycloheximide, which has an antibiotic spectrum similar to that of nystatin, inhibited organisms represented in the classes Chlorophyceae, Xanthophyceae, and Bacillariophyceae, but not in the Myxophyceae. These authors suggest that cycloheximide interferes with cell division, since a significant number of S. cerevisiae cells inhibited by the drug exhibited abnormal division states. The inhibition by cycloheximide of the formation of zoospores from "palmella-state" cells of Haematococcus lacustris, a member of the Volvocales, was also indicative of a block in cell division. The dissimilarity between the modes of action of nystatin and cycloheximide may serve to explain why the latter inhibited Bacillariophyceae, whereas the former did not. In this regard it is suggested that the silicified cell wall of the Bacillariophyceae may prevent absorption of the polyene which is the primary requirement for sensitivity. Under this condition the toxic actions of the polyene could not occur. ACKNOWLEDGMENT These studies were supported in part by a grant (G-9863) from the National Science Foundation. LITERATURE CITED ARNOW, P., J. J. OLESON, AND J. H. WILLIAMS. 1953. The effect of arginine on the nutrition of Chlorella vulgaris. Am. J. Botany 40:100-104. BROWN, R., AND E. L. HAZEN. 1957. Present knowledge of nystatin, an antifungal antibiotic. Trans. N. Y. Acad. Sci. 19:447-456. DONOVICK, R., F. E. PANSY, H. A. STOUT, H. STANDER, M. J. WEINSTEIN, AND W. GOLD. 1955. Some in vitro characteristics of nystatin (Mycostatin), p. 176-185. In T. H. Sternberg and V. D. Newcomer, [ed.], Therapy of fungus diseases: An international symposium. Little, Brown and Co., Boston. FOTOR, M. J., C. M. PALMER, AND T. E. MALONEY. 1953. Antialgal properties of various antibiotics. Antibiotics & Chemotherapy 3:505-508. HAVINGA, E., V. LYNCH, L. NORRIS, AND M. CALVIN. 1953. The effect of certain biologically active substances upon photosynthesis and dark CO2 fixation. Rec. trav. chim. 72:597-611. HUTNER, S. H., H. BAKER, S. AARONSON, H. A. NATHAN, E. RODRIGUEZ, S. Loc1KwoOD, M. SANDERS, AND R. A. PETERSON. 1957. Growing Ochromonas malhamensis above 35 C. J. Protozool. 4:259-269. LAMPEN, J. O., AND P. ARNOW. 1959. Significance of nystatin uptake for its antifungal action. Proc. Soc. Exptl. Biol. Med. 101:792-797. LAMPEN, J. 0., E. R. MORGAN, A. SLOCUM, AND P. ARNOW. 1959. Absorption of nystatin by microorganisms. J. Bacteriol. 78:282-289. MARINI, F., P. ARNOW, AND J. 0. LAMPEN. 1961. Effect of monovalent cations on the inhibition of yeast metabolism by nystatin. J. Gen. Microbiol. 24:51-62. OSTEUX, R., TRAN-VAN-KY, AND J. BIQUET. 1958. Contribution a l'6tude de mode d'action de la nystatin sur Candida albicans. Compt. rend., 247:2475-77. PROVASOLI, L., S. H. HUNTNER, AND A. SCHATZ. 1948. Streptomycin-induced chlorophyll-less races of Euglena. Proc. Soc. Exptl. Biol. Med. 69:279-282.

1961] NYSTATIN INHIBITION OF ALGAE 251 SCHOLZ, R., H. SCHMITZ, T. BUCHER, AND J. 0. LAMPEN. 1959. Uber die Wirkung von Nystatin auf Backerhefe. Biochem Z. 331:71-86. STARR, R. C. 1960. The culture collection of algae at Indiana University. Am. J. Botany 47: 67-86. TOMISEK, A., M. R. REID, W. A. SHORT, AND H. E. SKIPPER. 1957. Studies on the photosynthetic reaction. III. The effects of various inhibitors upon growth and carbonate-fixation in Chlorella pyrenoidosa. Plant Physiol. 32:7-10. UMBREIT, W. W., R. H. BURRIS, AND J. F. STAUFFER. 1951. Manometric techniques and tissue metabolism. Burgess Publishing Co., Minneapolis. pp. 73-76. ZEHNDER, A., AND E. 0. HUGHES. 1958. The antialgal activity of Acti-dione. Can. J. Microbiol. 4:399-408.