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博碩士論文 etd-0910112-124755 詳細資訊
Title page for etd-0910112-124755
論文名稱
Title
台灣海域潮間帶土壤產抗生素細菌之研究
Isolation of Antibiotics Producing Soil Bacteria in Taiwan Intertidal Zones
系所名稱
Department
畢業學年期
Year, semester
語文別
Language
學位類別
Degree
頁數
Number of pages
78
研究生
Author
指導教授
Advisor
召集委員
Convenor
口試委員
Advisory Committee
口試日期
Date of Exam
2012-07-24
繳交日期
Date of Submission
2012-09-10
關鍵字
Keywords
抗生素、潮間帶、海洋細菌、二次代謝物
antibiotics, marine bacteria, intertidal soils, secondary metabolites
統計
Statistics
本論文已被瀏覽 5684 次,被下載 771
The thesis/dissertation has been browsed 5684 times, has been downloaded 771 times.
中文摘要
自從20 世紀中期開始使用抗生素以來,許多細菌性感染疾病都得到改善。
但因為大量使用抗生素導致菌株產生抗藥性,因此在20 世紀中後期許多抗藥性
菌株開始蔓延,至今有些菌株幾乎可以抵抗所有藥物,故須研發新的抗生素藥
物。本研究針對台灣海域潮間帶之底泥中,篩選具有抗菌生物活性潛力之微生
物,採集了5 個地點共25 個樣品,包括澎湖、屏東大鵬灣、墾丁、龜山島、小
琉球外海,並使用Marine Agar 2216 培養海洋菌和Actinomycete Isolation Agar
培養放線菌,經由觀察菌株型態、分離以及純化,收集代表性菌株,共分離313
株微生物,並進一步利用7 株指標菌(Bacillus cereus、Staphylococcus aureus、
Klebsiella pneumonia、Salmonella typhimurium、Pseudomonas aeruginosa、Vibrio
harveyi、Escherichia coli )進行篩選抗生素活性。在313 株細菌有47 株具有抑制
指標菌之能力,以16S rRNA親緣分析具活性菌株其中有9 株為Bacillus spp.
、1
株Virgibacillus sp.、31株Pseudoalteromonas spp.
、1株Vibrio sp.、1株Streptomyces
sp.、4 株Microbulbifer spp.。革蘭氏染色試驗結果有10 株為革蘭氏陽性,37 株
為革蘭氏陰性;需鹽性試驗結果有10 株為不需鹽性菌,37 株為需鹽性菌。另外,
利用乙酸乙酯進行萃取生物活性物質,比較培養溫度和天數以及固、液態培養之
差異。以Vibrio sp. (QWI06)為例,在30 ℃第三天有最高的抑制效果(18 mm),經
由萃取後,結果顯示,液態培養在30℃第3 天抑制圈較大(30 mm 以上);固態培
養則是前三天抑制效果較佳(13 mm)。
Abstract
Many bacterial diseases were controlled by antibiotics since mid 1900s.
However, over use of the drugs leads to the prevalence of resistant strains, some of
them are resistant to essentially all of the commercial antibiotics except for one or two.
Therefore, new drugs are needed to combat the die-hard resistant pathogens. This
study is aimed at isolating bacteria with antimicrobial activities from the intertidal
soils of Taiwan. A total of 25 samples were collected from 5 locations, including
Peng-Hu, Da-Peng Bay, Ken-ding, Gueishan Island and Little Liu-chiu. Marine Agar
2216 and Actinomycete Isolation Agar were used for cultivation. Of 313 bacterial
isolated, 47 of them showed antimicrobial activities against at least one of the 7
indicators (Bacillus cereus, Staphylococcus aureus, Klebsiella pneumonia, Salmonella
typhimurium, Pseudomonas aeruginosa, Vibrio harveyi, Escherichia coli). The 47
strains were classified based on 16S rRNA phylogeny. The majority of them are
Pseudoalteromonas spp. (31) and Bacillus spp. (9) as well as Virgibacillus sp. (1),
Vibrio sp. (1), Streptomyces sp. (1) and Microbulbifer spp. (4). Over all, 10/47 are
Gram positive and 37/47 require added salts for growth. The titers of antimicrobial
substances as judged by ethyl acetate extraction were influenced by cultivation
conditions, such as: growth temperature, types of media and time.
目次 Table of Contents
目錄.............................................................................................................................ii
表目錄........................................................................................................................iv
圖目錄.........................................................................................................................v
附錄............................................................................................................................vi
中文摘要....................................................................................................................vii
Abstract......................................................................................................................viii
一、前言....................................................................................................................1
第一節 微生物.......................................................................................................1
第二節 抗生素的發現...........................................................................................2
第三節 抗生素作用機制.......................................................................................3
第四節 抗藥性(antibiotic resistance)機制….........................................................5
第五節 海洋微生物生物活性物質的先前研究...................................................7
第六節 研究動機和目的.......................................................................................9
二、材料與方法.......................................................................................................11
1.樣品採集............................................................................................................11
2.分離與純化........................................................................................................11
3.菌種的保存........................................................................................................12
4.活性測試............................................................................................................12
5.需鹽性測試........................................................................................................13
6.革蘭氏染色測試(Gram stain)............................................................................13
7.細菌Genomic DNA 萃取..................................................................................14
8.引子設計、短片段增幅及產物純化................................................................14
9.萃取生物活性....................................................................................................16
三、結果...................................................................................................................19
1.菌種收集及抗菌活性........................................................................................19
2.需鹽性及革蘭式染色........................................................................................19
3.定序與親緣樹狀圖............................................................................................20
4.生物活性.............................................................................................................22
5.萃取後生物活性..................................................................................................22
四、結論與討論.......................................................................................................24
五、參考文獻...........................................................................................................28
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