微生物學

The zebrafish (Danio rerio) is a popular model organism used for elucidating complex\r developmental pathways and processes. Although the Zebrafish Genome Project is\r nearing completion, the function of most genes remains to be discovered. In zebrafish,\r where an efficient gene targeting method is still absent, costly Morpholinos (MOs) have\r been widely used to ‘knockdown’ specific genes.

Dipterocarps coming from Southeast Asia comprise about 53% of the world’s\r total exports of tropical hardwood products. However, the supply of dipterocarps is\r rapidly declining. The government is currently embarking on the establishment of\r dipterocarp plantations and encourages inoculation with ectomycorrhizal fungi to\r improve seedling growth and survival in degraded sites. Anisoptera thurifera (Blanco)\r Blume and Shorea guiso (Blanco) Blume rooted cuttings were inoculated with\r mycelial beads containing either Pisolithus PTG, Pisolithus H6394, or Astreus sp.\r Pisolithus PTG was collected from Acacia mangium stand in Malaysia, Pisolithus\r H6394 from Eucalyptus trees growing in a heavy metal contaminated area in New\r Caledonia, Astreus sp obtained from dipterocarp forest in Mt. Makiling, Philippines.\r The results showed that Astreus sp gave the highest root colonization (38%), height\r increment (86%), fine root (51%), coarse root (27%), shoot (43%) and total dry\r weight (40%) of A. thurifera over the control treatment. Astreus – inoculated plants\r had the highest shoot P uptake (41%) while PTG inoculated ones obtained the highest\r fine root P uptake (0.273 mg/root). The uninoculated plants had the lowest height\r increment, dry weight, and P uptake. In S. guiso, Pisolithus H6394, gave the highest\r increment (100%). Data were only obtained from four months of nursery experiments,\r hence, a longer nursery and field experiments should be conducted to achieve a\r complete evaluation of the physiological functions of ectomycorrhizal associations.\r Moreover, a wide range of ectomycorrhizal fungi associated with dipterocarps should\r be incorporated for selection of host compatibility.

The experiment was primarily focused on studying whether the enzymes from different bacterial species collected in various hot spring areas still exhibited activities at high temperatures. A further study would be conducted on analyzing the unique characteristics universally found in the genes of selected bacteria. First, hot spring samples were collected form Peitou and Wulai, and then cultured on the PY, PTG, MFB, and TS media in the laboratory. After the broth media growing with thermophilic bacteria, a series of continuous dilution method and solid-plate spreading were applied to separate these bacterial clones. The genomic DNA of the selected bacteria was extracted and used to analyze subtilisin-like gene by polymerase chain reaction (PCR) and electrophoresis. Finally, we examined the six selected thermophilic bacteria with the enzymatic activities of fibrin- and milk-protein degradation. We successfully concluded the experiment by proving that these thermophilic bacteria still exhibited significant enzymatic activitives in the high-temperature environments. The results of this experiment can be applied in numerous fields, for example, thrombus treatment and food processing, and a more in-depth study shall warrant the due consideration. 這次的實驗，主要是研究在不同的溫泉區中所採集不同種類細菌，是否酵素在高溫下仍具活性，如果有，再進而研究它們的基因有何特別的共同之處。首先，我分別自北投和烏來採集水樣，到實驗室後再以PY、PTG、MFB 和TS 四種培養基做細菌的培養，接著再利用連續稀釋和固態塗抹來做細菌的分離。經過挑選和培養之高溫菌直接進行DNA 的抽取，並利用「聚合?鏈反應」和「電泳跑膠」技術分析其類似蛋白質分解酵素subtilisin 基因。另外，本研究同時針對所選定之6 株高溫細菌利用血栓和牛奶蛋白來測試其蛋白質分解酵素的活性。由以上實驗結果可以證明某些細菌在高溫的環境下酵素仍具活性。這次在高溫菌的實驗結論，可應用在很多地方，例如：血栓的治療、在高溫下食品處理‧‧‧等，應用相當廣泛。

A total of 64 nonduplicate isolates of erythromycin-resistant (MIC, >1 μg/ml) Streptococcus pyogenes collected from 1979 to 2003 in Taiwan were evaluated. They were collected from three cohort period: 1979 –1989, 1990-1999 and after 2000. The in vitro activities of 10 antimicrobial agents were determined by the agar dilution method. Penicillin, cephalothin, cefotaxime, vancomycin, and ofloxacin were shown to be active against S. pyogenes isolates (100% sensitive). Erythromycin and azithromycin both had poor activities (MIC50s, 16 and >128 μg/ml, respectively; MIC90s, >512 and >128 μg/ml, respectively). The activities of tetracycline, clindamycin, and chloramphenicol against a significant number of these isolates were also limited. Among the 64 S. pyogenes isolates, 58% had constitutive resistance [cMLS], 40% had an M phenotype and 2% had an iMLS phenotype (inducible resistance [iMLS]). A substantial upsurge in the incidence of M phenotype erythromycin-resistant isolates was found with time for S. pyogenes (10% in 1979–1989, 48% in 1990–1999 and 65% after 2000). The erythromycin resistance genes in 64 isolates of the different cohorts were investigated by PCR. All cMLS phenotype isolates tested had ermB gene. The M phenotype isolates had only the macrolide efflux (mefA) gene. This study shows the secular changes of increasing susceptibility of S. pyogenes isolates to both erythromycin and clindamycin in Taiwan. The mechanisms of erythromycin resistance have changed from the predominance of ermB gene (cMLS) to mefA gene (M phenotype). 本研究是探討台灣自1970 年代末期以來不同世代A群鏈球菌菌株對紅黴素的抗藥性及抗藥基因特徵的演變。研究者收集台灣二十年前(1980 年代前後，第一世代)、十年前(1990 年代，第二世代)、)及最近(2000 年以後，第三世代)之A 群鏈球菌菌株共64 株，利用抗生素圖譜和雙藥錠擴散測試及聚合?連鎖反應(PCR)、和脈衝式膠體電泳研究A 群鏈球菌對紅黴素抗藥性的表現型及基因型特徵。結果發現第一世代的A 群鏈球菌對紅黴素最小抑菌濃度(MIC)值相當高，幾乎都是>512 μg/ml，90%為多重抗藥菌株。第二世代則相對有降低，在第三世代此現象更明顯，多重抗藥菌株為30%。在雙藥錠擴散測試結果，從第一世代來看cMLS 佔大多數(90%)，M 型佔10%；第二世代cMLS 及M 型相當分別佔52%及48%；第三世代主要為M 型佔65%，cMLS 佔30%，iMLS 佔5%。利用PCR研究紅黴素抗藥性基因發現所有cMLS 菌株皆有ermB 基因，所有M 型菌株皆有mefA 基因，iMLS 菌株則有ermTR 基因。由本研究顯示在台灣A 群鏈球菌對紅黴素之抗藥性隨著不同世代有逐年改善之趨勢，由MIC50的降低可看出，菌株對紅黴素的敏感性提高，可為臨床治療之參考。

The purpose of the research was to find out if Manuka oil (an oil with natural healing properties which is extracted from the NZ Manuka plant, Leptospermum scoparium) could be used to reduce the populations of bacteria and fungi that build up on chopping boards used in the kitchen for food preparation. If it were effective, then it would reduce the risks of microbial contamination of foods prepared using the boards.

我們從校園生態池吳郭魚的腸內挑選出二十二個菌落，並透過含1%碳酸鈣的MRS培養基篩出五株可產酸的細菌，藉由基因體定序並比對序列，我們確定篩到了四種不同的乳酸菌。分別為Streptococcus anginous MD1, Lactobacillus casei MD10, Lactococcus lactis MD14 和Enterococcus raffinosus MD18。經由培養基上的拮抗圈實驗，證實了此四株乳酸菌皆具有拮抗嗜水性產氣單胞菌(Aeromonas hydrophila) 的能力。實際將乳酸菌和嗜水性產氣單胞菌投餵到斑馬魚幼魚時，則以Streptococcus anginous MD1與Enterococcus raffinosus MD18有明顯的拮抗效果，其中Streptococcus anginous MD1對斑馬魚幼魚生長影響最小且拮抗效果最久。所以，我們認為Enterococcus raffinosus MD18在未來最有發展成生物製劑的潛力，可運用於魚苗存活率的提升，代替抗生素來對抗嗜水性產氣單胞菌(Aeromonas hydrophila)。

目前研究的嗜油菌主要是分解石油(碳氫化合物)，不過從生活中所排出的油(三酸甘油酯)，也是一大污染源。本研究目的為篩選可分解三酸甘油酯之微生物，並探討其特性及降解油污的能力。從有油污的環境分離出三種對於日常生活中的油污具降解效果的菌株，利用16S ribosomal RNA gene進行菌種分析鑑定為Ralstonia sp.TFD41、Pseudomonas putida strain II-B、Sphingomonas sp.NC110。Ralstonia sp.TFD41較佳的生長環境為30℃、pH8，偏鹼，革蘭氏陰性菌；Pseudomonas putida strain II-B較佳的生長環境為30℃、pH6-10，偏鹼，革蘭氏陰性菌；Sphingomonas sp. NC110最佳生長環境為30℃、pH4-8，偏酸，革蘭氏陰性菌。而菌種在炸時越長的油中生長情形越差。而在家庭污水中的生長菌數最多的為Sphingomonas sp.NC110M。放入1000ppm油的培養基中，以Sphingomonas sp.NC110的降解油污的能力為最佳、Ralstonia sp.TFD41的降解速度最快。在家庭汙水中Ralstonia sp.TFD41 + Pseudomonas putida strain II-B的生長狀況最穩定。未來預期能培養嗜油菌，將其放回環境中，降解環境中的油汙，以減少油汙對於環境的汙染，還給生物乾淨的生活環境。

粒線體，『細胞內的發電廠』，它生產了我們生存所需的幾乎所有能量。除了產生能量，它也參與了細胞的凋亡的過程，或反應出自由基所引起細胞的老化。我們在此實驗中，利用酵母菌模式，跟兩個特殊的螢光染劑（JC-1與Rhodamine 123），來活體測量酵母菌裡的粒線體膜電位的變化，以了解細胞的早期凋亡的過程。並且，透過加入抗氧化劑與否，測量氧化游離基的產生，探討跟粒線體膜電位之間的關聯性。實驗結果顯示，的確酵母菌經過長期培養，且無提供適當的營養補充條件下，雖然外表還活著，但JC-1的螢光染色顯示，粒線體膜電位是有逐步遞減的現象，代表酵母菌活力慢慢衰弱。但是，另外氧化游離基的實驗結果卻沒有如預期般增加，反倒是跟JC-1的表現呈正相關現象。抗氧化劑的加入，非但沒有增加酵母菌的活力，反而是減弱了粒線體膜電位的表現，而氧化游離基也同時降低。這樣的實驗結果，雖然跟事前的預期結果不同，但也可能暗示1. 胡亂加入抗氧化劑，對生物不盡然是有幫忙的。2. 酵母菌在這一種生長條件中，可能需要一些氧化物的刺激或幫忙。3. 粒線體是細胞內氧化作用最強的單位，如果他生命力變弱了，自然產生氧化游離基的能力變弱，而非氧化游離基一開始去減弱粒線體的活性。此實驗的重大意義，是告訴我們，氧化游離基或抗氧化物的腳色並非一成不變的，甚至你原來認為的粒線體膜電位改變是細胞凋亡的起始原因，也可能相反，粒線體膜電位改變只是細胞凋亡後來發生的結果。科學的有趣也就是在這裡!我們藉助於【實驗】，不斷地實驗，去挑戰既有的知識跟原理，會讓我們發現更多的真理，一個單細胞的酵母菌就有這麼多問題需要去回答，那麼放眼將來不孕症醫師想嘗試『卵質轉移』，主要就是要取得健康的粒線體，增加懷孕的機會，那就可能需要更多的實驗跟努力，才能給出一個真正的正確答案。