Diversity and antibacterial activity of heterotrophic bacteria associated with sponges

• Main Authors: Yu Ling Huang, 黃于玲
• Other Authors: Wung Yang Shieh
• Format: Others
• Language: zh-TW
• Published: 2003
• Online Access: http://ndltd.ncl.edu.tw/handle/42915778030109937110

碩士 === 國立臺灣大學 === 海洋研究所 === 91 === Abundance and diversity of heterotrophic bacteria associated with thirteen sponge samples of different colors and shapes were investigated. The sponge samples were collected from either Maoao Bay or Tanshui estuary. Plate counting values of the heterotrophic bacteria associated with the sponges were mostly in the range of 105 to 107 CFU / g wet wt. A total of 140 bacterial strains were isolated from the counting plates. All the isolated strains were motile rods. They could be divided into eight groups according to the results of the following tests：requirement of Na+ for growth, Gram reaction, agarolytic activity and swarming. Groups I and II did not require Na+ for growth and could not grow anaerobically by fermenting glucose. Group I included 15 strains of Gram-positive rods that formed white and circular colonies. Reduction of NO3- to NO2- was positive. Group II included 4 strains of Gram-negative rods that could grow in 0-8% NaCl. The strains could use citrate as sole carbon source. Strains included in the other six groups group III to VIII were Gram -negative rods that required Na+ for growth. They did not reduce NO3- to NO2- and were unable to grow anaerobiclly by fermenting glucose. Only the strains of group V formed red colonies. Group III included 13 strains that grew at 25~40℃ and were agarolytic. Group IV included 13 swarming strains that could grow anaerobically by fermenting glucose, xylose, mannose, or galactose. Group VI included 34 strains that formed milky, circular and glossy colonies. Group VII included 32 strains of Gram -negative rods that formed milky, glossy and irregular colonies. Group VIII included 27 strains that formed milk white and rough colonies and could denitrify (reduce NO3- to N2). One to two strains of each group were selected for analysis of 16S rDNA sequences. Strain S130 included in group I showed a high sequence similarity level （99.6%）with Corynebacterium striatums. S128, a strain also included in group I, had a sequence similarity of 100% with Bacillaceae bacterium La28. Thus all the strains of group I may belong to Bacillaceae. S133, a strain of group II, was most similar to Vibrio campbellii with sequence similarity of 97.0%. S22, a strain of group III, was most similar to Vibrio brasiliensis （sequene similarity, 97.6%）, and Vibrio shilonii （sequene similarity, 97.3%）. However, these comparison data together with the fact that both Vibrio brasiliensis and Vibrio shilonii were not agarolytic indicated that S22 might not belong to the two species. The 16S rDNA sequence of strain S92 included in group IV were very similar to Vibrio alginolyticus（sequene similarity, 99.6%） and Vibrio parahaemolyticus（sequene similarity, 99.4%）. S92 could be a species of Vibrio alginolyticus and not Vibrio parahaemolyticus since it could be cultured in TCBS and formed yellow colony. The 16S rDNA sequence of the two strains of group V were not analyzed since both of them did not survive after subculture. S141, a strain of group VI, was closely related to Silicibacter pomeroyi （sequene similarity, 97.2%）. S37 was also a strain of group VI, which was most similar to Silicibacter lacuscaerulrnsis （sequene similarity, 98.4%）. All the strains of group VI might possibly belong to the genus Silicibacter. The 16S rDNA seguences of strain S90 and S118, that were included in group VII, were not closely related to any valid species（lower than 90%）. However, they were very similar to α-Proteobacteria sp., MBIC3368 and NW001（sequene similarity, 99.7~100% ）. S142, a strain of group VIII, had a sequence similarity of 94.3% with Pseudomonas elongate, and were most similar to CJ11064（98.8%）which is an unidentified species of Pseudomonas. Therefore, S142 might belong to the genus Pseudomonas. Agar disc-diffusion test was applied for testing antibacterial activity of the isolated strains to the pathogenic strains, Staphylococcus aureus subsp. Aureus (CCRC 10780), Aeromonas hydrophila (CCRC 13018), Kocuria rosea (CCRC 11044), Vibrio parahaemolyticus (CCRC 10806), Listonella anguillarum (formerly Vibrio anguillarum, CCRC 12908) and Vibrio vulnificus (CCRC 12905), and 2 Vibrio parahaemolyticus-like strains and 3 golden Gram-positive cocci. Sixty-four strains exhibited significant antibacterial activity. Among them, 22 strains inhibited the growth of more than two test strains. Strains that exhibited antibacterial activity were isolated from 12 of 13 sponge samples. All the groups were formed to included strains with antibacterial activity except group Ⅴ. Only a part of the strains in each group possessed antibacterial activity. HPLC analysis indicated that 12 strains with antibacterial activity might produce okadaic acid or its related materials. This study showed that heterotrophic bacteria associated with the sponges in northern Taiwan were taxonomically diverse, and for the first time, bacteria were found to produce okadaic acid-like substance.