快速檢定抗生素對細菌生物膜敏感性之新技術
The purpose of this study is to set up a quick, easy and economical way to evaluate the ability of different concentration of various antibiotics to penetrate biofilm and establish the antimicrobial susceptibility patterns of various antibiotics. The susceptibility of five antibiotics upon sessile cultures of Bacillus subtlis ATCC 6633, Escherichia coli XL, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 29213 and Sarcina lutea ATCC 9341 were measured ATCC 27853, and S. aureus ATCC 29213 proved to be very difficult to eradicate, with only Gentamicin proving to effective at achievable drug concentrations, but the S. lutea ATCC 9341 biofilm was the most susceptible to the Penicillin. The results demonstrated that for biofilms of the same organisms, several hundred to thousand times the concentration of a certain antibiotic were often required for the antibiotic to be effect, while other antibiotics were found to be effective at the MICs. The concentration of antibiotic to penetrate the biofilm is proportional to the thickness of biofilm. Indeed, our research have already indicated that the use of MIC values to indicate antibiotics effectiveness is misleading, because MIC values can not represent the actual effect of anticbiotics on microbiologicals that have developed biofilm. The antimicrobial susceptibility patterns of antibiotics to various bacterial biofilm are different. The susceptibility of the mixed biofilm depends on the physical and biological change of biofilm. Our biofilm device offers a new technology for the rational evaluation of antibiotics effective against microbial biofilms and for the screening of new effective antibiotic drugs.此研究之目的是要建立一套操作簡便、快速且費用低廉之生物膜厚度產生方法，藉此探討不同生物膜厚度對抗生素抗菌之影響，進而完成抗生素對生物模之抗菌圖譜。本實驗將測試五種抗生素Bacillus subtilis ATCC6633，Escherichia coli XL，Pseudomonas aeruginosa ATCC27853，Staphylococcus aureus ATCC29213和Sarcina lutea ATCC9341之抗菌圖譜。試驗結果顯示Gentamicin對前四株試菌有較佳之穿透力，對S.lutea ATCC 9341則是Penicillin。實驗結果證明最小抑制濃度值確實無法實際有效地表達對已形成生物膜菌體之抗菌效果，要完全去除生物膜之抗生素濃度是為最小抑制濃度之數百倍到數千倍，而且抗生素用藥濃度隨著生物膜厚度增加而成比例增加。不同抗生素對不同菌株生物膜有不同之抗菌圖譜，混合菌株生物膜是否會促進或抑制抗生素之穿透力，端視其生物膜結構有無改變。本實驗方法可以做為一種快速檢定抗生素對細菌生物膜敏感性之新技術，同時亦可有效地篩選新的抗生素藥物對生物膜的抗菌效應。
耐熱性酵素生產菌Bacillus Subtilis WLA12 之分離與定性
本實驗針對一取自台北縣烏來南勢溪下游之溫泉菌，進行微生物學、生物化學、分子生物學三方面之觀察與實驗，期能得到一可生產特定酵素之菌種，且具耐高溫之抗逆境能力。對其酵素進行定性，並嘗試轉殖出相關基因，使之可大量表現。目前已篩選出一種可生產多種酵素之菌種〔依其採集地點暫稱WL-A12〕經菌種鑑定為Bacillus subtilis WL-A12。藉由菌落檢測法以及Zymogram 的方式做酵素分析，並以電導轉形等技術，希望能成功轉至E. coli 上表現。另外，也對該菌種作了一些基本微生物方面的觀察〔如需氧情況、最適生長溫度〕。We isolated enzyme-producing thermophilic bacteria from hot springs near downriver of Nan-shi, Wulai, Taipei (北縣烏來南勢溪). Through microbiological, biochemistry and molecular biological analyses, a multiple enzyme-producing Bacillus subtilis strain, designated WLA12, has been isolated. The growth condition of WLA12 was observed. Using basic colony assay and zymogram analysis (gel electrophoresis) to observe the expressed enzymes, molecular weight and gene size of the enzymes were revealed. With comparison to E. coli control strain, the related enzymes were only found in WLA12. To express the Bacillus genes in E. coli, molecular cloning and gene transformation via electroporation was carried out.
你喝下了多少？-台灣市售優酪乳乳酸菌生長力及抗酸性之探討
現今乳酸菌飲料風行，但是乳酸菌是否真能通過胃酸的考驗，到達腸道進行複製，利益人體？我們首先以市售乳酸菌粉（加拿大Rosell 公司，含二種菌，暫時命名為"小毛"及"小白"）為預測菌種，利用分光光度計測定乳酸菌於Thioglycollate 培養基中的生長能力（OD600）。小毛在pH 值 1、 3 、5、 7 時之生長力分別為0、 0.008 、0.682 、0.847 ，小白為 0、 0.015 、0.973、 0.636。若於培養基中添加不同濃度的螺旋藻熱分解物，如加入0.01%的添加物後，小毛在以上各種pH 值生長力分別為0.042、1.291, 、1.447, 、1.213 ，小白為 0.053、1.392、 1.531、 0.988，意外發現可大幅提升菌的生長力及抗酸力。再取台灣市售4 種廠牌優酪乳（以甲、乙、丙、丁代表之），分離乳酸菌，再於各種pH 值中培養。結果在pH 3 時，螺旋藻熱分解物僅對丙廠牌有效, 乙廠牌無效, 甲與丙則有無填加生長力都很差。在pH 1 時, 則對乙、丙、丁皆有效，故建議廠商慎選菌種，並於製程及成品中添加螺旋藻熱分解物。The yogurt is a popular drink. But whether the lactobacilli inside can resist the destruction of gastric acid and grow well in the intestinal tract is still questionable. We used pure lactobacilli powder (Rosell Company, Canada, containing two bacteria named in this report as "Little Hair" and "Small White") for pre-test. The growth ability in thioglycollate medium was determined by spectrophotometer (OD600). The results of bacterial growth at pH 1, 3, 5, and 7 for "Little Hair" were 0, 0.008, 0.682, and 0.847, respectively. Those for "Small White" were 0, 0.015, 0.973, and 0.636, respectively. After supplement with 0.01% of the boiled lysate of Spirulina algae (ProBio Biotech, Taiwan), growth abilities at pH 1, 3, 5, and 7 for "Little Hair" were 0.042, 1.292, 1.447, and 1.213, respectively. Those for "Small White" were 0.053, 1.392, 1.531, and 0.988, respectively. The algae extract amply promotes the growth and acid-resistance, especially at pH 3, of these bacteria. The lactobacilli isolated from four different products of yogurt in Taiwan, named as A, B, C, and D, were then tested as above. Results showed the supplement with the boiled lysate of Spirulina algae was very effective, at pH 3, for promoting growth of C, but not effective for B. Growth abilities of both A and D were very unsatisfactory with or without this supplement. At pH 1, algae lysate supplement significantly improved the growths of B, C, and D. Therefore, this supplement in culture and product for yogurt preparation was suggested.
敬"屑"不敏 --- 皮屑芽孢菌生長之探討
由於本身有頭皮屑的煩惱，在使用各種洗髮精之後，一方面覺得大部分的洗髮精效果不是很好，另一方面也擔心抗屑洗髮精的成分對人體有害。所以有了研究頭皮屑的念頭，希望能找出減少頭皮屑的方法。\r 在與幾位皮膚科專門醫師討論後，醫師們都不建議在直接人體上進行減少頭皮屑的實驗。於是我們將研究的重點放在〝皮屑芽孢菌生長之探討〞，希望對皮屑芽孢菌有更深入的瞭解。除了洗髮精之外，我們也將研究各種廣為流傳的民間偏方是否能有效抑制皮屑芽孢菌生長。在這些民間偏方的蒐集可兩大類：一類是在日常生活中可以很容易取得的物質，如鹽、醋、大蒜、生薑、洋蔥等；另一類則是台灣特有的物質，如無患子、茶籽粉、竹醋液等。\r 我們希望透過研究方法能瞭解\r (一) 溫度、濕度、酸鹼度、油脂對皮屑芽孢菌生長的影響\r (二) 哪些物質可以抑制皮屑芽孢菌的生長\r (三) 不同稀釋濃度的物質抑制皮屑芽孢菌的生長的效果\r 為了避免繁複的數量計算(如菌數計算)，我們採用較多的對照比較(如抑制圈大小)。同時，我們設計了一個簡易的觀察與測量方法(稀釋法與抑菌指數)，來比較各種民間偏方\r 的抑菌效果。\r 研究與實驗分為四大項進行:\r (一) 基本生長環境之探討：溫度、濕度、酸鹼度、油脂對皮屑芽孢菌生長的影響\r (二) 民間偏方抑制效果之探討：鹽、醋、大蒜、生薑、洋蔥是否能抑制抑制皮屑芽孢菌的生長\r (三) 民間偏方抑制效果之比較(抑制圈)：比較各種物質的抑制圈大小\r (四) 民間偏方抑制效果之比較(稀釋法)：利用抑菌指數來比較各種物質的抑菌能力\r 經過研究，我們所蒐集到的民間偏方都有某種程度的抑菌效果，大致可以分為強效(蒜液、竹醋液、茶樹精油)、有效(食鹽水、食用醋、洋蔥液、檜木芬多精、茶籽粉)及普通(薑汁、無患子)三種效果。\r \r Since I have the serious dandruff problem, I try to seek, except the shampoo, the natural materials to decrease the dandruff. After consulting with some dermatologists, they suggest I can focus my research on the “Malassezia Furfur”, which is the major bacteria causing the dandruff.\r This project is designed for finding the materials which have the most powerful inhibition capability for the “Malassezia Furfur”. I collect some folk prescriptions from the internet that salt, vinegar, garlic and onion can decrease the dandruff. The problem is “Do they have good inhibition capability for the Malassezia Furfur too?”\r The goals of the research are as followed.\r (1) The exploration of the basic environmental factors for the growth of Malassezia Furfur, such as temperature, humidity, acidity & alkalinity.\r (2) What kinds of material have the good inhibition capability?\r (3) The comparison of inhibition capability of the different material with different consistency.\r We especially design a method called “Dilution metering and index” to compare the inhibition capability of the different material. By using very simple observation, this method will transfer the simple “Qualitative Analysis” to the result of complicated “Quantitative Analysis”. The “Dilution metering and index” is the major achievement of this project, and it is also the good way for the projects which are not equipped with the precision instrument for bacteria inhibition capability measurement.
乳酸桿菌及啤酒酵母之相互作用及其代謝產物對Clostridium difficile 生長之影響
市面上可以看到多種品牌紛紛推出了以“啤酒酵母”作為號召的乳製品，標榜健康取向\r 的“啤酒酵母優酪乳”也是其中的一件，我們蒐集了關於啤酒酵母以及乳酸桿菌的文獻，打\r 算研究之間的關係，乳酸菌屬於益生菌，主要的用途是製作優酪乳，可以協助維持體內正常\r 的腸道菌相，降低血清膽固醇以及預防骨質疏鬆症(Rolfe, 2000)；啤酒酵母最主要的用途為\r 啤酒的製作，具有解決失眠、消除疲勞、改善痢疾的功效。外國的克芙爾(kefir)也是屬於含\r 有乳酸桿菌及酵母菌菌相的食品，而且也對健康有益。因此我們提出了一個問題﹕啤酒酵母\r 與乳酸桿菌的代謝產物是否有著互利的功效呢？在拋出第一個問號後我們繼續尋找資料以\r 及著手我們的實驗。在文獻中(Gaon et al.,2003；Corthier et al.,1992,1986)也曾經提及啤酒酵\r 母與乳酸桿菌對於Clostridium difficile 所引起的偽膜性腸炎的關係，所以使我們又想問兩個\r 問題：究竟Ampicillin 對於乳酸桿菌、酵母菌、C. difficile 的影響有多大？而乳酸桿菌及酵\r 母菌對於C. difficile 的影響又是什麼？
Discovery of new bioactivities of violacein and its erivatives synthesized by Chromobacterium sp.
目的：紫色桿菌（Chromobacterium sp.）是一種生活在土壤及水域環境的革蘭氏陰性菌，它會產生紫黑色的紫色桿菌素（Violacein），因而使菌體與培養液皆呈紫色。Violacein是一種由兩個L-色胺酸（L-tryptophan）分子所聚合成的五環化合物，已被證實具有抗菌以及抑制腫瘤細胞生長的活性。本實驗的目的在探討紫色桿菌是否會利用不同官能基的L-色胺酸，合成不同的Violacein衍生物，並分析這些Violacein衍生物的生物活性。方法：將紫色桿菌培養於液態LB培養基中，分別加入不同濃度的L-tryptophan、1-CH3-L-tryptophan與5-OH-L-tryptophan，於室溫下培養48小時，再用乙酸乙酯萃取Violacein及其衍生物，將萃取出來的紫色桿菌素，用紫外光/可見光分光光譜計和質譜儀進行分析。最後，再將這些萃取物打入斑馬魚受精卵做胚胎毒性測試，並進行試管外DNA剪切能力分析。結果：加入不同官能基的L-色胺酸所合成的產物都是紫色，且在紫外光/可見光的光譜中的最大吸收波長皆相同，質譜儀分析結果則顯示，L-tryptophan 、與5-OH-L-tryptophan的產物圖譜不同、而1-CH3-L-tryptophan的產物則無法分析。斑馬魚胚胎毒性測試結果顯示，Violaecein和1-CH3-Violacein 不會對胚胎發育造成影響，但是5-OH-Violacein則會造成胚胎發育異常。DNA 剪切能力測試也顯示，只有5-OH-Violacein具有剪切DNA的能力，其他則否。重要性：本實驗成功地利用紫色桿菌合成Violacein及其衍生物，並發現了5-OH-Violacein比Violacein 具有更高的生物活性。;Aims: Chromobacterium sp. is a Gram negative bacterium which inhabit in soil and water environments. Chromobacterium produce a purple color compound named Violacein. This colorful compound makes Chromobacterium and its culture mediumin purple color. Violacein is a five rings compound synthesized from two L-tryptophan molecules. Studies have shown that Violacein has anti-bacteria and anti-tumor activities. The purpose of this study is to investigate whether Violacein can be synthesized from different tryptophan analogues, and whether these Violacein derivatives have different bioactivities. Materials and Methods: Chromobacterium sp. was cultured in liquid medium containing different concentrations of L-tryptophan, 1-CH3-L-tryptophan, and 5-OH-L-tryptophan. After 48 hours incubation at room temperature, Violacein and its derivatives were extracted by EA, analyzed by UV/visible spectrophotometer and MS. Violacein and its derivatives were also tested for their embryo toxicity and DNA cleavage activity in vitro. Results: The compounds synthesized from different tryptophan analogues were all purple and have the same maxima absorption wave length in the UV/visible spectra. However, MS spectra of compounds synthesized from L-tryptophan and 5-OH-L-tryptophan were different. The results of zebrafish embryo toxicity tests indicated that violaecin and 1-CH3-Violacein had no effect on embryo development, but 5-OH-Violacein caused development defects. DNA cleavage tests also showed that only 5-OH-Violacein could digest DNA in vitro. Violaecin and 1-CH3-Violacein could not digest DNA. Significances: Results from this study indicate that Chromobacterium sp. can be used to synthesize Violacein derivatives from different tryptophan analogues, and revealed that 5-OH-Violacein is a higher bioactivity compound than violaecein.