能生長於TCBS平板培養基的沿岸海水中細菌多樣性之探討

Diversity of bacteria in coastal seawater as investigated by cultivation on TCBS agar medium

李晃銘(Huang-Ming Li)

指導教授 ： 謝文陽

共同指導教授 ： 簡文達

國立臺灣大學/理學院/海洋研究所/碩士(2004年)

https://doi.org/10.6342/NTU.2004.01756

摘要

本研究於2002至2003年間，前後共計五次，在台灣北部八斗子與深澳兩地方之沿岸，進行海水樣本採集，並以選擇性培養基thiosulfate-citrate-bile salt-sucrose（TCBS）為主，Vibrio parahaemolyticus-sucrose-I（VPS-I）、Vibrio parahaemolyticus- sucrose-II（VPS-II）和tryptose-yeast-glucose（TYG）三種培養基為輔，進行了各海水樣本所含生菌數之平板計數。結果顯示，兩樣區海水樣本所含能生長於TYG培養基之細菌數約介於102-104 CFU/ml；相對而言，能生長於其他三種選擇性培養基之細菌數多低於這些估測值之一半。
根據菌落型態差異，從TCBS、VPS-I及VPS-II三種計數平板，挑取分離出之細菌共計213株。依照其革蘭氏反應、發酵葡萄糖及蔗糖之能力、鈉鹽需求、擴散性及水解瓊脂能力等表型特性之差異，可將這些分離株區分為十五群。除了第二、第九、第十二及第十三菌群外，其餘各菌群代表株之16S rDNA與GenBank已知菌種之相似度均高於97﹪。
第一至三群所含18株顯然並非海洋弧菌，其餘占分離株總數九成以上，分屬於其他十二菌群之菌株，除了第十二群代表株需進一步確認外，均具有與海洋弧菌相符之表型特性。這印證了TCBS、VPS-I及VPS-II三種兼含膽鹽和鈉鹽之培養基，確實對海洋弧菌之篩選性頗具效果；反之，其對於非海洋弧菌類及陸源性細菌，其中尤其是對於革蘭氏陽性細菌之生長，則普遍具有抑制作用。
從十五菌群各挑取兩株，利用DdeI、RsaI和ScaI三種內切酶進行16S rDNA之RFLP分析後發現，所獲之DNA酶切片段共可歸類成十三種基因型。16S rDNA序列相似度高於97﹪之菌株，多具相同基因型；然而也有序列相似度高達97.7﹪之菌株，卻具有不同基因型者。定序結果不完整之第九和第十三群代表株藉由16S rDNA之RFLP分析所得基因型顯示，兩者各具獨有之基因型，與其他十三群菌株有明顯區隔。16S rDNA序列相似度高於97﹪之菌株，經genomic DNA雜合實驗顯示其雜合度均未超過65﹪。綜合16S rDNA之RFLP分析、16S rDNA序列比對及DNA雜合實驗三者之結果，吾人推斷從十五菌群挑取出之菌株，應互為不同菌種，其中第二及第十二群代表株之16S rDNA因與GenBank已知菌種之相似度均低於97﹪，可能屬於新種。

關鍵字

選擇性培養基 ； 弧菌培養基 ； 弧菌 ； 細菌多樣性 ； 限制片段長度多型性

並列摘要

Seawater samples were collected five times from the coastal regions of Badouzih and Shenao, northern Taiwan between years 2002 and 2003 in the present study. Thiosulfate-citrate-bile salt-sucrose（TCBS）were used principally as the selective medium and Vibrio parahaemolyticus-sucrose-I（VPS-I）, Vibrio parahaemolyticus-sucrose-II（VPS-II）and tryptose-yeast-glucose（TYG） were also utilized as auxiliary media for enumeration of bacteria in seawater samples. The plate counting values of the bacteria were estimated to be in the range of 102-104 CFU/ml on TYG agar medium, and the values on the other three kinds of selective media were generally lower than half of the former.
According to the differences of colony shapes, 213 bacterial strains were isolated from TCBS, VPS-I and VPS-II agar media, respectively. These 213 strains were divided into 15 groups on the basis of variations of phenotypic characterizations including Gram reaction, glucose fermentation, sucrose fermentation, Na+ requirement, swarming and agarase. Excluding the 2nd, 9th, 12th and 13th groups, the 16S rDNA sequence similarities of the representative strains of other 11 groups compared to the known bacteria in GenBank were higher than 97﹪.
The 1st, 2nd, and 3rd groups containing 18 strains were not marine vibrio obviously. The remainder strains which belonged to the other twelve groups accounted for more than 90% of total amount of bacteria. Except the representative strains of the 12th group still needed to be further confirmed, these strains possessed the characteristics which were consistent with marine vibrios. This result proved that TCBS, VPS-I and VPS-II mediums containing cholate and sodium ion were effective to sieve vibrio out from marine water and repressed commonly the growth of bacteria belonging to non-marine vibrios and land-originated species, especially Gram-positive bacteria.
Two strains of every group were selected for 16S rDNA/ RFLP analysis using three endonucleases including DdeI, RsaI and ScaI. The results showed that 13 genotypes were categorized from the multiple restriction patterns. The 16S rDNA sequences similarities among representative strains were higher than 97﹪, the genotypes of 16S rDNA/ RFLP were almost same from each other. But, the rule was not suitable for some strains. The representative strains of 9th and 13th groups with incomplete 16S rDNA sequence data had unique genotype of RFLP differentiated from other representative strains. Genomic DNA hybridization showed that all of strains with 16S rDNA sequence similarity high than 97﹪belonged different species. Based on 16S rDNA/ RFLP analysis, 16S rDNA-based phylogenetic analysis and DNA hybridization, the 15 representative strains were different from one another, and the representatives of the 2nd and 12th groups may belong to new species.

並列關鍵字

TCBS medium ； diversity of bacteria ； Vibrio ； selective medium ； RFLP

參考文獻

Atlas, R. M.（1993）. Handbook of Microbiological Media. Edited by L. C. Parks. Florida: CRC press, Inc..

Borrego, J. J., Castro, D., Luque, A., Paillard, C., Maes, P., Garcia, M. and Ventosa, A.（1996）. Vibrio tapetis sp. nov., the causative agent of the brown ring disease affecting cultured clams. Int. J. Syst. Bacteriol. 46, 480-484.

Caccamo, D., Di Cello, F, Fani, R., Giugliandolo, C. and Maugeri, T. （1999）. Polyphasic approach to the characterisation of marine luminous vibrionaceae. Res. Microbiol. 150, 221-230.

Chowdhury, M. A. R., Yamanaka, H., Miyoshi, S. and Shinoda, S.（1990）. Ecology and distribution of Vibrio parahaemolyticus in aquatic environments of a temperate region. FEMS Microbiol. Ecol. 74,1-10.

DeLong, E. F., Franks, D. G. and Alldredge, A. L.（1993）. Phylogenetic diversity of aggregate-attached vs. free-living marine bacterial assemblages. Limnol. Oceanogr. 38, 924–934.