- Email: [email protected]
Use of an antifungal drug, amphotericin B for isolation of thraustochytrids
Journal of Bioscience and Bioengineering VOL. 110 No. 6, 720 – 723, 2010 www.elsevier.com/locate/jbiosc
NOTE
Use of an antifungal drug, amphotericin B for isolation of thraustochytrids Yousuke Taoka,1 Naoki Nagano,1 Yuji Okita,2 Hitoshi Izumida,2 Shinichi Sugimoto,2 and Masahiro Hayashi1,⁎ Laboratory of Marine Bioscience, Faculty of Agriculture, University of Miyazaki, 1-1 Gakuen-kibanadai-nishi, Miyazaki 889-2192, Japan 1 and Nippon Suisan Kaisha Ltd., 2-6-2 Otemachi, Chiyoda-ku, Tokyo 100-8686, Japan 2 Received 24 February 2010; accepted 22 July 2010 Available online 12 August 2010
The inhibitory effect of amphotericin B (AMPH) on the growth of fungi during the isolation of thraustochytrids was examined. The growth of fungi was significantly inhibited by addition of AMPH, and therefore colonies of thraustochytrids were not overlaid with fungal mycelia, which resulted in increased efficiency of thraustochytrids isolation. © 2010, The Society for Biotechnology, Japan. All rights reserved. [Key words: Amphotericin B; Thraustochytrids; Aurantiochytrium; Colony isolation; Fugi]
Thraustochytrids, which are heterotrophic marine eukaryotes and straminipilan protists, have attracted interests because of the high levels of polyunsaturated fatty acids (PUFAs) in their cell body, indicating that they may be microbial sources of PUFAs. Of the thraustochytrids, the members of genus Aurantiochytirum (formerly classified as Schizochytrium) have gained special attention as potential PUFA producers because they can grow rapidly, accumulate a large amount of docosahexaenoic acids (DHA) and obtain high biomass (1,2). Thraustochytrids have a wide geographic distribution in marine ecosystems (3) and have generally been isolated using the pine pollen-baiting technique (4). Previously we reported the number of thraustochytrids isolated from seawater could be increased by adding polysorbate (Tween 80) and potassium dihydrogenphosphate (KH2PO4) into a plate medium (5). Thraustochytrids are frequently isolated from seawater and dead leaves in mangrove ecosystems, where fungi play an important role as decomposers, indicating that fungi exist in high abundance on dead leaves as an isolation source of thraustochytrids (6). Bacterial growth can be inhibited by addition of antibiotics such as penicillin and streptomycin, (5), but fungi remain capable of growing. Because of the growth of fungi on an agar plate medium, it is very troublesome to pick up colonies of thraustochytrids, because the mycelia of fungi rapidly extend and overlay colonies of thraustochytrids. To efficiently isolate thraustochytrids from a marine environment, it is necessary to inhibit the growth of fungi. Amphotericin B (AMPH) is a polyene antibiotic that has potent antifungal activity. This drug was discovered from cell bodies of Actinomycetes, Streptomyces nodosus, and was first reported by Gold (7). It interacts with ergosterol in the plasma membrane of fungi
⁎ Corresponding author. Tel./fax: + 81 985 58 7225. E-mail addresses: [email protected] (Y. Taoka), [email protected] (N. Nagano), [email protected] (Y. Okita), [email protected] (H. Izumida), [email protected] (S. Sugimoto), [email protected] (M. Hayashi).
playing a role of permeability barrier. This drug has been widely used as a therapeutic agent for systemic fungal diseases for nearly 40 years (8–12). Merkal and Richard confirmed that AMPH was effective for the prevention of fungal contamination in the process of isolating mycobacteria from fecal specimens (13). To date, there have been no studies published about the application of antifungal drugs including AMPH for the isolation of thraustochytrids. In this study, we used three antifungal drugs, including AMPH, to prevent fungal contamination during the isolation of thraustochytrids. First, we investigated the effect of antifungal drugs on the viability of thraustochytrids. Then, thraustochytrids were isolated from a marine coastal area using an agar plate medium with or without a selected antifungal drug. In the process of the isolation, the growth inhibitory effect of the drug on fungi was evaluated. Three kinds of antifungal drugs, amphotericin B (AMPH; fungizone, Gibco, Invitrogen Japan, Tokyo, Japan), fluconazole (FLCZ; Diflucan®, Pfizer Japan Inc., Tokyo, Japan), and miconazole·nitate (MCZ; Wako Pure Chemical Industries, Ltd., Tokyo, Japan), were used. These drugs were dissolved in distilled water. All solutions were sterilized by filtration with a syringe filter (Dismic® 25AS020AS, Advantec Co., Ltd., Tokyo, Japan). The six strains of thraustochytrids used in this study were Aurantiochytrium limacinum ATCC MYA-1381 (strain SR21) and strain mh0186 (DDBJ acccetion number: AB362211), S. aggregatum ATCC 28209, Thraustochytrium striatum ATCC 24473, T. roseum ATCC 28210, and T. aureum ATCC 34304. Strains of ATCC were purchased from American Type Culture Collection. These strains were cultured in a B1 agar plate consisting of 2 g yeast extract, 2 g polypeptone, 5 g glucose, and 15 g agar per 1000 ml of half-strength artificial seawater (ASW, NaCl 30.0 g, KCl 0.7 g, MgCl2·6H2O 10.8 g, MgSO4·7H2O 5.4 g, CaCl2·2H2O 1.0 g) (pH 7.0) containing 0.1% vitamin mixture (vitamin B1 200 mg, vitamin B2 1 mg, vitamin B12 1 mg per 100 ml of distilled water) at 28 °C for 3-5 days. Lawns of colonies on an agar plate were inoculated on another B1 agar plate medium containing vitamin mixture with or without each antifungal drug at various levels (0-
1389-1723/$ - see front matter © 2010, The Society for Biotechnology, Japan. All rights reserved. doi:10.1016/j.jbiosc.2010.07.011
VOL. 110, 2010 5 μg/ml). Plates were incubated at 28 °C for 4 days. After incubation, growth of thraustochytrids was observed. Lawns of colonies of A. limacinum strains SR21 and mh0186 were inoculated into a B1 liquid medium except for an agar in the components of a B1 agar plate medium and pre-cultured at 28 °C for 3 days with shaking (140 strokes). The cells cultured were collected by centrifugation (5,000 × g, 5 min) and washed once with sterile halfstrength ASW. Aliquots (10 μl) of the culture containing washed cells were transferred to 100 μl of a GY medium consisting of 10 g yeast extract, and 30 g glucose per 1000 ml of half-strength ASW with or without each antifungal drug (APMH, FLCZ, and MCZ) at 1 μg/ml in a 96-well micro-plate. The plate was sealed with Parafilm to prevent evaporation of the solution and incubated at 28 °C for 5 days. The growth of strains was observed based on the turbidity of the medium. Strain SR21 was cultured in a GY liquid medium containing 0.1% vitamin mixture at 28 °C for 72 hrs (140 strokes). Pre-culture samples (2 ml) were inoculated into a 20-ml GY liquid medium sample in 50ml baffled conical flasks without or with AMPH (1 μg/ml), bacterial antibiotics (BA, 0.05% penicillin G potassium salt and streptomycin sulfate), or a combination of APMH and BA at 28 °C for 168 hrs (140 strokes). Every 24 hrs, an aliquot (2 ml) of culture was collected for biomass determination. Dry cell weight as biomass was measured by lyophilizing the cells washed once with distilled water. Colonyforming units (CFUs) were measured at 168 hrs according to the standard agar spread method with serial dilutions. Fourteen isolates presumed as thraustochytrids based on cell morphology, cell size and fatty acid profiles were previously isolated and stored in our laboratory. These isolates were cultured in a well of 96 wells micro-plate containing a B1 liquid medium without or with
FIG. 1. CFUs of A. limacinum strains SR21 and mh0186 at 168 hrs after incubation in a 96 well micro-plate with or without one of three antifungal drugs (AMPH, FLCZ and MCZ) at 1 μg/ml. (A) strain SR21, (B) strain mh0186. Values represent mean with standard deviation (n = 3). Bars denoted with different capitals show significant difference (P b 0.05).
NOTE
721
AMPH at 1 μg/ml and incubated as described above. The growth of isolates was determined based on the increasing of turbidity. Ten samples of both seawater and dead leaves were collected in the Yaeyama islands and Kaoshima Bay, Kagoshima, Japan, on August 2007 to isolate marine fungi and evaluate the effect of AMPH on fungal growth. Each seawater sample (40 ml) or dead leaf was transferred to a sterile 50-ml centrifugal tube, and pine pollen was added to the tube to collect zoospores of thraustochytrids (4). In the case of dead leaf samples, instead of natural seawater, sterile ASW was added to the centrifugal tube. Samples treated with pine pollen were incubated at room temperature for 7 days in the dark. Aliquots (100 μl) of the samples from each tube were collected and smeared on a potato dextrose agar (PDA, Nissui Pharmaceutical, Tokyo, Japan) and incubated at 28 °C for 4 days. After incubation, colonies of fungi grown on a plate were picked up and repeatedly streaked on another agar plate medium to purify them. Other parts of the samples treated with pine pollen were smeared on a B1 agar plate with or without AMPH, and the plate was incubated at 28 °C for 4 days. The growth of thraustochytrids and fungi was compared between agar plates with and without AMPH. Data were analyzed by using one-way analysis of variance (ANOVA). Significant differences were identified by the Tukey-t test
FIG. 2. Growth of A. limacinum SR21 in a GY liquid medium containing AMPH (amphotericin B), BA (0.05% bacterial antibiotics, penicillin G potassium salt and streptomycin sulfate) or combination of AMPH and BA (CM). (A) Dry cell weight (DCW, g/l), closed circles, control; closed squares, AMPH; closed triangles, BA; closed diamonds, CM. (B) CFUs at 168 hrs incubation. Values represent mean with standard deviation (n = 3). In the case of A, symbols denoted with different capitals show significant difference at each incubation time. In the case of B, Bars denoted with different capitals show significant difference (P b 0.05).
722
TAOKA ET AL.
or Student-t test (P b 0.05). These analyses were performed with SPSS ver. 10.0 software (SPSS, Chicago, IL, USA). Six strains of thraustochytrids, A. limacinum (strains SR21 and mh0186) and S. aggregatum ATCCC 28209, T. striatum ATCC 24473, T. roseum ATCC 28210, and T. aureum ATCC 34304 could grow on a B1 agar plate medium supplemented with each antifungal drug at 0-5 μg/ ml (data not shown). A. limacinum strains SR21 and mh0186 grew well in a GY liquid medium containing one of three kinds of antifungal drugs (1 μg/ml) in a well of a 96 well micro-plate (Fig. 1). In the case of strain SR21, the growth was not inhibited in all medium added each drug at 1 μg/ml. The CFU counts were significantly higher in the medium added AMPH as compared with those of other three groups (Fig. 1). Strain mh0186 could grow in all media with any added antifungal drugs although the CFU in the medium to which we added MCZ was significantly lower than those of the other three groups (Fig. 1). The CFU counts were significantly higher in the medium to which we added AMPH as compared with those of other three groups. Based on these results, we believed AMPH is a suitable antifungal drug for the isolation of thraustochytrids, especially genus Aurantiochytrium strains.
J. BIOSCI. BIOENG., In a growth test with a medium containing AMPH, BA, or combinations with AMPH and BA (CM group), strain SR21 grew well in all groups (Fig. 2A). At 168 hrs of incubation, significantly more CFUs were seen in groups to which we added AMPH and CM as compared with those of the control and BA groups (Fig. 2B). In addition to ATCC thraustochytrid strains, our growth experiment showed that fourteen thraustochytrid strains previously isolated in our laboratory could grow even in a B1 liquid medium with AMPH at 1 μg/ml (data not shown). On the other hand, the growth of fifteen marine fungi strains isolated from seawater samples in this study were completely inhibited on an agar plate supplemented with AMPH (1 μg/ ml) in comparison to those without AMPH (data not shown). Hence, our results strongly suggest that AMPH is useful for the isolation of various thraustochytrid species from natural marine environment. The growth of fungi found in seawater and on dead leaves was clearly inhibited on an agar medium containing AMPH at 1 μg/ml as compared to that without AMPH. Fig. 3 shows a photograph of an agar plate medium inoculated with seawater from a dead leaf sample (sample No 1) and incubated for 4 days. In the case of an agar plate
FIG. 3. Colonies of fungi and thraustochytrids grown on a B1 agar plate medium (sample number 1). (A) B1 agar plate medium without AMPH, (B) B1 agar plate medium with AMPH. Arrows show colonies of thraustochytrids. Scale = 10 mm. Number of isolated thraustochytrids on B1 agar plate medium with or without AMPH at 1 μg/ml. (C) leaf sample, (D) seawater sample. Closed bar, with AMPH; open bar, without AMPH. Values represent mean with standard deviation (n = 3). Asterisks show significant difference between plates with or without AMPH in each sample (P b 0.05).
VOL. 110, 2010 medium containing AMPH, it was very easy to confirm the occurrence of thraustochytrids-like colonies. On the other hand, in the case of a medium without AMPH, colonies of thraustochytirds were overlaid with mycelia of fungi. In the case of leaf samples, the number of isolated thraustochytrids was significantly higher in agar plate medium with AMPH in 9 of 10 samples. In the case of seawater samples, the number of isolated thraustochytrids was significantly higher in agar plate medium with AMPH in 7 of 10 samples. The appearance of fungi on B1 agar plate medium without AMPH was remarkable in leaf samples as compared with seawater samples. In an agar plate medium containing AMPH at 1 μg/ml, fungal contamination on the agar plate was strongly prohibited, and colonies of thraustochytrids were easily picked up without interference from fungal growth. This study showed that growth of both fungi and bacteria on an agar plate medium was inhibited by applying AMPH and BA together, and the efficiency for isolating thraustochytrids improved. In the future, the variety of isolates on an agar plate medium containing AMPH should be investigated. References 1. Lewis, T. E., Nichols, P. D., and McMeekin, A.: The biotechnological potential of thraustochytrids, Mar. Biotechnol., 1, 580–587 (1999). 2. Morita, E., Kumon, Y., Nakahara, T., Kagiwada, S., and Noguchi, T.: Docosahexaenoic acid production and lipid body formation in Schizochytrium limacinum SR21, Mar. Biotechnol., 8, 319–327 (2006).
NOTE
723
3. Kimura, H., Fukuba, Y., and Naganuma, T.: Biomass of Thraustochytrid protoctists in coastal water, Mar. Ecol. Prog. Ser., 189, 27–33 (1999). 4. Gaetner, A.: Eine Methode des quantitativen Nachweises niederer, mit Pollen koderbarer Pilze im Meerwasser und im Sediment, Veroff. Insti. Meereforsh. Bremerh., 10, 159–165 (1968) (in German). 5. Taoka, Y., Nagano, N., Okita, Y., Izumida, H., Sugimoto, S., and Hayashi, M.: Effect of addition of Tween 80 and potassium dihydrogenphosphate to basal medium on the isolation of marine eukaryotes, thraustochytrids, J. Biosci. Bioeng., 105, 562–565 (2008). 6. Raghukumar, S., Sharma, S., Raghukumar, C., Sathe-Pathak, V., and Chandramohan, D.: Thraustochytrids and fungal component of marine detritus. IV. Laboratory studies on decomposition of leaves of the mangrove Rhizophora apiculata Blume, J. Exp. Mar. Biol. Ecol., 183, 113–131 (1994). 7. Gold, W. T.: Amphotericin, A. and B, antifungal antibiotics produced by Streptomyces, Antibiot. Annu., 579, 1955–1956 (1956). 8. Denning, D. W. and Stevens, D. A.: Antifungal and surgical treatment of invasive aspergillosos: review of 2121 published cases, Rev. Infect. Dis., 12, 1149–1201 (1990). 9. Denning, D. W.: Therapeutic outcome in invasive aspergillosos, Clin. Infect. Dis., 23, 608–615 (1996). 10. Wei, H., Hai, W., and Wanqing, L.: Clinical study on liposomal amphotericin B (Ambisome®) in deep fungal infections in China, Mycoses, 46, 24–28 (2003). 11. Shah, T., Lai, W. K., Gow, P., Leeming, J., and Mutimer, D.: Low dose amphotericin for prevension of serious fungal infection following liver transplantation, Transpl. Infect. Dis., 7, 126–132 (2005). 12. Emmanuel, R., Lyman, A. C., Armstrong, D., Stergiopoulou, T., Petraitience, R., and Walsh, J. T.: Deoxycholate amphotericin B and amphotericin B lipid complext exert additive antifungal activity in combination with pulmonary alveolar macrophages against Fusarium solani, Mycoses, 49, 109–113 (2006). 13. Merkal, R. S. and Richards, W. D.: Inhibition of fungal growth in the cultural isolation of Mycobacteria, Appl. Microbiol., 24, 205–207 (1972).
NOTE
Use of an antifungal drug, amphotericin B for isolation of thraustochytrids Yousuke Taoka,1 Naoki Nagano,1 Yuji Okita,2 Hitoshi Izumida,2 Shinichi Sugimoto,2 and Masahiro Hayashi1,⁎ Laboratory of Marine Bioscience, Faculty of Agriculture, University of Miyazaki, 1-1 Gakuen-kibanadai-nishi, Miyazaki 889-2192, Japan 1 and Nippon Suisan Kaisha Ltd., 2-6-2 Otemachi, Chiyoda-ku, Tokyo 100-8686, Japan 2 Received 24 February 2010; accepted 22 July 2010 Available online 12 August 2010
The inhibitory effect of amphotericin B (AMPH) on the growth of fungi during the isolation of thraustochytrids was examined. The growth of fungi was significantly inhibited by addition of AMPH, and therefore colonies of thraustochytrids were not overlaid with fungal mycelia, which resulted in increased efficiency of thraustochytrids isolation. © 2010, The Society for Biotechnology, Japan. All rights reserved. [Key words: Amphotericin B; Thraustochytrids; Aurantiochytrium; Colony isolation; Fugi]
Thraustochytrids, which are heterotrophic marine eukaryotes and straminipilan protists, have attracted interests because of the high levels of polyunsaturated fatty acids (PUFAs) in their cell body, indicating that they may be microbial sources of PUFAs. Of the thraustochytrids, the members of genus Aurantiochytirum (formerly classified as Schizochytrium) have gained special attention as potential PUFA producers because they can grow rapidly, accumulate a large amount of docosahexaenoic acids (DHA) and obtain high biomass (1,2). Thraustochytrids have a wide geographic distribution in marine ecosystems (3) and have generally been isolated using the pine pollen-baiting technique (4). Previously we reported the number of thraustochytrids isolated from seawater could be increased by adding polysorbate (Tween 80) and potassium dihydrogenphosphate (KH2PO4) into a plate medium (5). Thraustochytrids are frequently isolated from seawater and dead leaves in mangrove ecosystems, where fungi play an important role as decomposers, indicating that fungi exist in high abundance on dead leaves as an isolation source of thraustochytrids (6). Bacterial growth can be inhibited by addition of antibiotics such as penicillin and streptomycin, (5), but fungi remain capable of growing. Because of the growth of fungi on an agar plate medium, it is very troublesome to pick up colonies of thraustochytrids, because the mycelia of fungi rapidly extend and overlay colonies of thraustochytrids. To efficiently isolate thraustochytrids from a marine environment, it is necessary to inhibit the growth of fungi. Amphotericin B (AMPH) is a polyene antibiotic that has potent antifungal activity. This drug was discovered from cell bodies of Actinomycetes, Streptomyces nodosus, and was first reported by Gold (7). It interacts with ergosterol in the plasma membrane of fungi
⁎ Corresponding author. Tel./fax: + 81 985 58 7225. E-mail addresses: [email protected] (Y. Taoka), [email protected] (N. Nagano), [email protected] (Y. Okita), [email protected] (H. Izumida), [email protected] (S. Sugimoto), [email protected] (M. Hayashi).
playing a role of permeability barrier. This drug has been widely used as a therapeutic agent for systemic fungal diseases for nearly 40 years (8–12). Merkal and Richard confirmed that AMPH was effective for the prevention of fungal contamination in the process of isolating mycobacteria from fecal specimens (13). To date, there have been no studies published about the application of antifungal drugs including AMPH for the isolation of thraustochytrids. In this study, we used three antifungal drugs, including AMPH, to prevent fungal contamination during the isolation of thraustochytrids. First, we investigated the effect of antifungal drugs on the viability of thraustochytrids. Then, thraustochytrids were isolated from a marine coastal area using an agar plate medium with or without a selected antifungal drug. In the process of the isolation, the growth inhibitory effect of the drug on fungi was evaluated. Three kinds of antifungal drugs, amphotericin B (AMPH; fungizone, Gibco, Invitrogen Japan, Tokyo, Japan), fluconazole (FLCZ; Diflucan®, Pfizer Japan Inc., Tokyo, Japan), and miconazole·nitate (MCZ; Wako Pure Chemical Industries, Ltd., Tokyo, Japan), were used. These drugs were dissolved in distilled water. All solutions were sterilized by filtration with a syringe filter (Dismic® 25AS020AS, Advantec Co., Ltd., Tokyo, Japan). The six strains of thraustochytrids used in this study were Aurantiochytrium limacinum ATCC MYA-1381 (strain SR21) and strain mh0186 (DDBJ acccetion number: AB362211), S. aggregatum ATCC 28209, Thraustochytrium striatum ATCC 24473, T. roseum ATCC 28210, and T. aureum ATCC 34304. Strains of ATCC were purchased from American Type Culture Collection. These strains were cultured in a B1 agar plate consisting of 2 g yeast extract, 2 g polypeptone, 5 g glucose, and 15 g agar per 1000 ml of half-strength artificial seawater (ASW, NaCl 30.0 g, KCl 0.7 g, MgCl2·6H2O 10.8 g, MgSO4·7H2O 5.4 g, CaCl2·2H2O 1.0 g) (pH 7.0) containing 0.1% vitamin mixture (vitamin B1 200 mg, vitamin B2 1 mg, vitamin B12 1 mg per 100 ml of distilled water) at 28 °C for 3-5 days. Lawns of colonies on an agar plate were inoculated on another B1 agar plate medium containing vitamin mixture with or without each antifungal drug at various levels (0-
1389-1723/$ - see front matter © 2010, The Society for Biotechnology, Japan. All rights reserved. doi:10.1016/j.jbiosc.2010.07.011
VOL. 110, 2010 5 μg/ml). Plates were incubated at 28 °C for 4 days. After incubation, growth of thraustochytrids was observed. Lawns of colonies of A. limacinum strains SR21 and mh0186 were inoculated into a B1 liquid medium except for an agar in the components of a B1 agar plate medium and pre-cultured at 28 °C for 3 days with shaking (140 strokes). The cells cultured were collected by centrifugation (5,000 × g, 5 min) and washed once with sterile halfstrength ASW. Aliquots (10 μl) of the culture containing washed cells were transferred to 100 μl of a GY medium consisting of 10 g yeast extract, and 30 g glucose per 1000 ml of half-strength ASW with or without each antifungal drug (APMH, FLCZ, and MCZ) at 1 μg/ml in a 96-well micro-plate. The plate was sealed with Parafilm to prevent evaporation of the solution and incubated at 28 °C for 5 days. The growth of strains was observed based on the turbidity of the medium. Strain SR21 was cultured in a GY liquid medium containing 0.1% vitamin mixture at 28 °C for 72 hrs (140 strokes). Pre-culture samples (2 ml) were inoculated into a 20-ml GY liquid medium sample in 50ml baffled conical flasks without or with AMPH (1 μg/ml), bacterial antibiotics (BA, 0.05% penicillin G potassium salt and streptomycin sulfate), or a combination of APMH and BA at 28 °C for 168 hrs (140 strokes). Every 24 hrs, an aliquot (2 ml) of culture was collected for biomass determination. Dry cell weight as biomass was measured by lyophilizing the cells washed once with distilled water. Colonyforming units (CFUs) were measured at 168 hrs according to the standard agar spread method with serial dilutions. Fourteen isolates presumed as thraustochytrids based on cell morphology, cell size and fatty acid profiles were previously isolated and stored in our laboratory. These isolates were cultured in a well of 96 wells micro-plate containing a B1 liquid medium without or with
FIG. 1. CFUs of A. limacinum strains SR21 and mh0186 at 168 hrs after incubation in a 96 well micro-plate with or without one of three antifungal drugs (AMPH, FLCZ and MCZ) at 1 μg/ml. (A) strain SR21, (B) strain mh0186. Values represent mean with standard deviation (n = 3). Bars denoted with different capitals show significant difference (P b 0.05).
NOTE
721
AMPH at 1 μg/ml and incubated as described above. The growth of isolates was determined based on the increasing of turbidity. Ten samples of both seawater and dead leaves were collected in the Yaeyama islands and Kaoshima Bay, Kagoshima, Japan, on August 2007 to isolate marine fungi and evaluate the effect of AMPH on fungal growth. Each seawater sample (40 ml) or dead leaf was transferred to a sterile 50-ml centrifugal tube, and pine pollen was added to the tube to collect zoospores of thraustochytrids (4). In the case of dead leaf samples, instead of natural seawater, sterile ASW was added to the centrifugal tube. Samples treated with pine pollen were incubated at room temperature for 7 days in the dark. Aliquots (100 μl) of the samples from each tube were collected and smeared on a potato dextrose agar (PDA, Nissui Pharmaceutical, Tokyo, Japan) and incubated at 28 °C for 4 days. After incubation, colonies of fungi grown on a plate were picked up and repeatedly streaked on another agar plate medium to purify them. Other parts of the samples treated with pine pollen were smeared on a B1 agar plate with or without AMPH, and the plate was incubated at 28 °C for 4 days. The growth of thraustochytrids and fungi was compared between agar plates with and without AMPH. Data were analyzed by using one-way analysis of variance (ANOVA). Significant differences were identified by the Tukey-t test
FIG. 2. Growth of A. limacinum SR21 in a GY liquid medium containing AMPH (amphotericin B), BA (0.05% bacterial antibiotics, penicillin G potassium salt and streptomycin sulfate) or combination of AMPH and BA (CM). (A) Dry cell weight (DCW, g/l), closed circles, control; closed squares, AMPH; closed triangles, BA; closed diamonds, CM. (B) CFUs at 168 hrs incubation. Values represent mean with standard deviation (n = 3). In the case of A, symbols denoted with different capitals show significant difference at each incubation time. In the case of B, Bars denoted with different capitals show significant difference (P b 0.05).
722
TAOKA ET AL.
or Student-t test (P b 0.05). These analyses were performed with SPSS ver. 10.0 software (SPSS, Chicago, IL, USA). Six strains of thraustochytrids, A. limacinum (strains SR21 and mh0186) and S. aggregatum ATCCC 28209, T. striatum ATCC 24473, T. roseum ATCC 28210, and T. aureum ATCC 34304 could grow on a B1 agar plate medium supplemented with each antifungal drug at 0-5 μg/ ml (data not shown). A. limacinum strains SR21 and mh0186 grew well in a GY liquid medium containing one of three kinds of antifungal drugs (1 μg/ml) in a well of a 96 well micro-plate (Fig. 1). In the case of strain SR21, the growth was not inhibited in all medium added each drug at 1 μg/ml. The CFU counts were significantly higher in the medium added AMPH as compared with those of other three groups (Fig. 1). Strain mh0186 could grow in all media with any added antifungal drugs although the CFU in the medium to which we added MCZ was significantly lower than those of the other three groups (Fig. 1). The CFU counts were significantly higher in the medium to which we added AMPH as compared with those of other three groups. Based on these results, we believed AMPH is a suitable antifungal drug for the isolation of thraustochytrids, especially genus Aurantiochytrium strains.
J. BIOSCI. BIOENG., In a growth test with a medium containing AMPH, BA, or combinations with AMPH and BA (CM group), strain SR21 grew well in all groups (Fig. 2A). At 168 hrs of incubation, significantly more CFUs were seen in groups to which we added AMPH and CM as compared with those of the control and BA groups (Fig. 2B). In addition to ATCC thraustochytrid strains, our growth experiment showed that fourteen thraustochytrid strains previously isolated in our laboratory could grow even in a B1 liquid medium with AMPH at 1 μg/ml (data not shown). On the other hand, the growth of fifteen marine fungi strains isolated from seawater samples in this study were completely inhibited on an agar plate supplemented with AMPH (1 μg/ ml) in comparison to those without AMPH (data not shown). Hence, our results strongly suggest that AMPH is useful for the isolation of various thraustochytrid species from natural marine environment. The growth of fungi found in seawater and on dead leaves was clearly inhibited on an agar medium containing AMPH at 1 μg/ml as compared to that without AMPH. Fig. 3 shows a photograph of an agar plate medium inoculated with seawater from a dead leaf sample (sample No 1) and incubated for 4 days. In the case of an agar plate
FIG. 3. Colonies of fungi and thraustochytrids grown on a B1 agar plate medium (sample number 1). (A) B1 agar plate medium without AMPH, (B) B1 agar plate medium with AMPH. Arrows show colonies of thraustochytrids. Scale = 10 mm. Number of isolated thraustochytrids on B1 agar plate medium with or without AMPH at 1 μg/ml. (C) leaf sample, (D) seawater sample. Closed bar, with AMPH; open bar, without AMPH. Values represent mean with standard deviation (n = 3). Asterisks show significant difference between plates with or without AMPH in each sample (P b 0.05).
VOL. 110, 2010 medium containing AMPH, it was very easy to confirm the occurrence of thraustochytrids-like colonies. On the other hand, in the case of a medium without AMPH, colonies of thraustochytirds were overlaid with mycelia of fungi. In the case of leaf samples, the number of isolated thraustochytrids was significantly higher in agar plate medium with AMPH in 9 of 10 samples. In the case of seawater samples, the number of isolated thraustochytrids was significantly higher in agar plate medium with AMPH in 7 of 10 samples. The appearance of fungi on B1 agar plate medium without AMPH was remarkable in leaf samples as compared with seawater samples. In an agar plate medium containing AMPH at 1 μg/ml, fungal contamination on the agar plate was strongly prohibited, and colonies of thraustochytrids were easily picked up without interference from fungal growth. This study showed that growth of both fungi and bacteria on an agar plate medium was inhibited by applying AMPH and BA together, and the efficiency for isolating thraustochytrids improved. In the future, the variety of isolates on an agar plate medium containing AMPH should be investigated. References 1. Lewis, T. E., Nichols, P. D., and McMeekin, A.: The biotechnological potential of thraustochytrids, Mar. Biotechnol., 1, 580–587 (1999). 2. Morita, E., Kumon, Y., Nakahara, T., Kagiwada, S., and Noguchi, T.: Docosahexaenoic acid production and lipid body formation in Schizochytrium limacinum SR21, Mar. Biotechnol., 8, 319–327 (2006).
NOTE
723
3. Kimura, H., Fukuba, Y., and Naganuma, T.: Biomass of Thraustochytrid protoctists in coastal water, Mar. Ecol. Prog. Ser., 189, 27–33 (1999). 4. Gaetner, A.: Eine Methode des quantitativen Nachweises niederer, mit Pollen koderbarer Pilze im Meerwasser und im Sediment, Veroff. Insti. Meereforsh. Bremerh., 10, 159–165 (1968) (in German). 5. Taoka, Y., Nagano, N., Okita, Y., Izumida, H., Sugimoto, S., and Hayashi, M.: Effect of addition of Tween 80 and potassium dihydrogenphosphate to basal medium on the isolation of marine eukaryotes, thraustochytrids, J. Biosci. Bioeng., 105, 562–565 (2008). 6. Raghukumar, S., Sharma, S., Raghukumar, C., Sathe-Pathak, V., and Chandramohan, D.: Thraustochytrids and fungal component of marine detritus. IV. Laboratory studies on decomposition of leaves of the mangrove Rhizophora apiculata Blume, J. Exp. Mar. Biol. Ecol., 183, 113–131 (1994). 7. Gold, W. T.: Amphotericin, A. and B, antifungal antibiotics produced by Streptomyces, Antibiot. Annu., 579, 1955–1956 (1956). 8. Denning, D. W. and Stevens, D. A.: Antifungal and surgical treatment of invasive aspergillosos: review of 2121 published cases, Rev. Infect. Dis., 12, 1149–1201 (1990). 9. Denning, D. W.: Therapeutic outcome in invasive aspergillosos, Clin. Infect. Dis., 23, 608–615 (1996). 10. Wei, H., Hai, W., and Wanqing, L.: Clinical study on liposomal amphotericin B (Ambisome®) in deep fungal infections in China, Mycoses, 46, 24–28 (2003). 11. Shah, T., Lai, W. K., Gow, P., Leeming, J., and Mutimer, D.: Low dose amphotericin for prevension of serious fungal infection following liver transplantation, Transpl. Infect. Dis., 7, 126–132 (2005). 12. Emmanuel, R., Lyman, A. C., Armstrong, D., Stergiopoulou, T., Petraitience, R., and Walsh, J. T.: Deoxycholate amphotericin B and amphotericin B lipid complext exert additive antifungal activity in combination with pulmonary alveolar macrophages against Fusarium solani, Mycoses, 49, 109–113 (2006). 13. Merkal, R. S. and Richards, W. D.: Inhibition of fungal growth in the cultural isolation of Mycobacteria, Appl. Microbiol., 24, 205–207 (1972).