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標題: | 建立腸道菌叢培養體系統及分離一株低三甲胺產生者相關菌種 Development of a culturomic system and isolation of a bacterial strain associated with low trimethylamine (TMA) producing phenotype |
作者: | Fang-Chi Chang 張芳齊 |
指導教授: | 王錦堂(Jin-Town Wang) |
關鍵字: | 動脈粥樣硬化,冠狀動脈疾病,腸道微生物群,L-肉鹼,培養, Atherosclerosis,Coronary artery disease,Gut microbiota,L-carnitine,culturomics, |
出版年 : | 2019 |
學位: | 碩士 |
摘要: | 近期的研究發現腸道微生物群可能在冠狀動脈疾病中扮演重要角色,傳統以糞便檢體為研究對象並以16S rDNA 定序為普遍鑑別菌種的方法,但現在培養體系統日趨重要,我們經實驗發現高登培養基 (Gordon’s medium) 與五盤日本伊藤非選擇性培養基 (Itoh’s non-selective medium) 對比於十一盤選擇性培養基 (Itoh’s selective medium) 可能培養出最多菌種,因此我們將主要使用此進行後續實驗。關於糞便檢體收集後的儲存方式,實驗發現運輸培養液 (Puritan’s transportation medium)雖然維持了較多新鮮檢體的操作分類單元 (operational taxonomic unit, OTU) 但是不穩定,而冷凍檢體只比新鮮檢體少約30%~50%的OTU。且經實驗證實基質輔助雷射脫附游離飛行時間質譜儀目前鑑定之圖譜訊號弱、資料庫少且鑑定效率約只有16S rDNA 定序的40%。而與我們合作的實驗室利用次世代定序 (next generation sequencing) 發現食用左旋肉鹼 (L-carnitine) 後血漿產生低量氧化三甲胺(trimethylamine-N-oxide, TMAO)的受試者糞便中出現頻率高且量顯著多的腸內菌為Flavonifractor plautii (F.plautii),推測其可能不利於氧化三甲胺的產生。並且我以相同的培養系統加以專一性的聚合酶連鎖反應 (polymerase chain reaction) 分離出了F.plautii。我們首先想探討是否其會分解三甲胺從而達到抑制氧化三甲胺的生成,實驗結果顯示並不會分解,其次我們也想知道是否其會與高量氧化三甲胺生成之菌反應,而抑制三甲胺的分泌,在膽鹼與肉鹼之代謝利用實驗中發現其並不會抑制。我們同樣也想知道是否其會抑制高量氧化三甲胺生成之菌生長,但在競爭實驗中並無發現抑制現象。
因此得出結論:高登培養基與日本伊藤非選擇性培養基為較適宜的腸內菌培養基,能培養出最多菌種;冷凍糞便檢體OTU數目只比新鮮檢體少約30%~50%的OTU,因此篩選菌種時先使用冷凍檢體,而有特殊菌種培養困難時再採用新鮮糞便檢體;16S rDNA 定序為較好的鑑定菌種方式;F.plautii並不會分解三甲胺;也不會抑制高量氧化三甲胺生成之菌的三甲胺分泌;同樣並未發現能抑制高量氧化三甲胺生成之菌的生長。其功能有待進一步研究。 Recent studies have also suggested that gut microbiota could be an important role in the development of CAD. Although stool sample is traditionally used for 16S rDNA sequencing to identify bacterial species and is considered as a common and convenient way to identify bacterial species, culturomic system is increasingly important nowadays. Based on our scientific experiments, Gordon’s medium and five Itoh’s non-selective medium can culture the most bacteria, so we chose them to continue on our studies. While Puritan’s transportation medium can maintain a great amount of operational taxonomic unit (OTU) of fresh stool sample, it is not stable, and frozen sample is only 30%~50% less OTU than fresh sample. In our experiments, we found out that matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) detecting signals for our bacteria are weak and lacking of database, the identification efficiency is 60% less that 16S rDNA sequencing. And our collaborative laboratory found out that healthy subjects challenged with L-carnitine who produced low trimethylamine-N-oxide (TMAO) had a high frequency of F.plautii, suggesting that this species is disadvantageous to the production of TMAO. In my experiment, I use the same culturomic system with specific polymerase chain reaction and successfully isolate F.plautii. First we want to know if F.plautii can degrade TMA in order to inhibit the production of TMAO, experiment showed that it cannot degrade TMA, we also want to know if it will interact with high TMAO producing bacteria to inhibit the producing of TMAO. But in the L-carnitine/Choline assay, we found out that it does not have the property of inhibition. We also like to know if F.plautii can inhibit the growth of high TMAO producing bacteria, but did not find it in the competing assay. My conclusions are Gordon’s medium and five Itoh’s non-selective medium are better for gut microbiota, for they can culture the most bacteria; frozen sample is only 30%~50% less OTU than fresh sample, so we decide to use frozen sample for screening bacteria, and collect fresh sample only when having difficulty in culturing some bacterial species; 16S rDNA sequencing is a better way to identify bacterial species; F.plautii cannot degrade TMA nor inhibit the formation of TMAO of high TMAO producing bacteria or their growth. Its function needs further study. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/7299 |
DOI: | 10.6342/NTU201901789 |
全文授權: | 同意授權(全球公開) |
顯示於系所單位: | 微生物學科所 |
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