桿菌屬臨床分離菌株抗真菌作用之分析 林芷妘、劉淑瑛 ; 邱政洵
E-mail: 9510773@mail.dyu.edu.tw
ABSTRACT
The object of this study is to examine 34 clinical isolates of Bacillus spp. from blood of pseudo bacteremia patients in Linkou Chang Gung Memorial Hospital, focusing on species classification, antifungal activity, and identification of antifungal gene and active components. Bacterial classification was based on phenotyping (catalase test, reduction of nitrate, hemolysis test, indole production test, citrate utilization test, urease test, Voges-Proskauer test) and genotyping (polymerase chain reaction). All 34 clinical isolates of Bacillus spp. belonged to 8 species: B. cereus, B. thuringiensis, B. coagulans, B. licheniformis, B. pumilus, B. megaterium, B.
circulans and B. firmus. From the results of antifungal analysis, B. thuringiensis CG 2, B. thuringiensis CG 4, B. pumilus CG 12, B.
cereus CG 15, B. cereus CG 20 and B. cereus CG 26 exhibited significant antifungal activities. These six isolates were further analysed for their antifungal mechanism on Paecilomyces variotii T??37. It’s well known that Bacillus subtilis F29-3 produces one antibiotic--fengycin, which effectively inhibits the growth of filamentous fungi. According to the sequence of fenB, 3 PCR primer sets were designed to analyze whether any of these 34 clinical isolates of Bacillus spp. contained fenB gene of fengycin. With one primer set, a 1.0 Kb fragment can be amplified in all 6 isolates with antifungal activity. The sequence of the PCR product showed high homology to ilvD. Whether ilvD is related to the antifungal activity remained to be elucidated. Furthermore, to identify the active components contributing to the antifungal activity, the fermentation supernatant were obtained from 6 isolates with antifungal activity and B. subtilis F29-3. After filtration, the supernatants showed no antifungal activity to Paecilomyces variotii T??37. It appeared that only viable bacteria expressed antifungal activity.
Keywords : Bacillus spp., fengycin, antifungal activity
Table of Contents
簽名頁 授權書 iii 中文摘要 iv 英文摘要 vi 誌謝 viii 目錄 ix 圖目錄 xii 表目錄 xiv 第一章 緒論 1 1.1 Bacillus spp.之特性 1 1.2 真 菌之特性 4 1.3 抗真菌劑（antifungal drug） 6 1.4 菌種鑑定之方法 8 1.5 研究目的 15 第二章 實驗材料與方法 16 2.1 實驗材 料 16 2.1.1 菌種 16 2.1.2 培養基、試劑 16 2.2 實驗方法 16 2.2.1 鏡檢 16 2.2.2 生化鑑定 17 2.2.3 分子鑑定 19 2.2.4 抗真菌作 用之測試 19 2.2.5 基因組DNA之純化 20 2.2.6 聚合?鏈鎖反應 21 2.2.7 從瓊脂凝膠中回收DNA片段（Recovering of digested DNA fragment from agarose gel） 22 2.2.8 定序分析 22 2.2.9 細菌醱酵液抗真菌作用之測試 23 2.2.10 真菌與細菌共同培養之 抗真菌作用測試 24 第三章 結果與討論 27 3.1 Bacillus spp.之鑑定結果 27 3.2 真菌孢子濃度之測定 29 3.3 抗真菌作用測試之 結果 30 3.4 Bacillus spp.與fenB之關係 32 3.5 抗真菌成份之探討 33 3.6 細菌與真菌共同培養之探討 34 第四章 結論 36 參考文 獻 72 附錄一、菌種 77 附錄二、培養基、試劑 78 附錄三、本實驗中聚合?鏈鎖反應（16S rDNA之HV region、rpoB）所用 的引子及其鹼基序列 82 附錄四、聚合?鏈鎖反應（引子：16S rDNA之HV region、rpoB）之反應條件（1）及反應混合液
（2） 83 附錄五、本實驗中聚合?鏈鎖反應（fenB）所用引子之設計 84 附錄六、本實驗中聚合?鏈鎖反應（fenB）所用的引 子及其鹼基序列 85 附錄七、聚合?錄鎖反應（引子：fenB）之反應條件（1）及反應混合液（2） 86 圖 目 錄 頁次 圖1-1 ?朵 試驗反應式之一 10 圖1-2 ?朵試驗反應式之二 11 圖1-3 檸檬酸試驗之反應式 11 圖1-4 VP test作用之反應式 13 圖一、以rpoB 為依據之34株桿菌屬臨床分離菌株演化樹狀圖 40 圖二、34株桿菌屬臨床分離菌株細胞於顯微鏡下放大1,000倍所看到的形 態 41 圖三、Bacillus spp.抗真菌Penicillium spp.的測試 47 圖四、Bacillus spp.抗真菌Aspergillus versicolor的測試 48 圖五
、Bacillus spp.抗真菌Trichophyton rubrum的測試 49 圖六、Bacillus spp.抗真菌Fusarium spp.的測試 50 圖七、Bacillus spp.抗 真菌Paecilomyces variotii Tu137的測試 51 圖八、Bacillus thuringiensis CG 2、B. thuringiensis CG 4、B. pumilus CG 12、B.
cereus CG 15、B. cereus CG 20、B. cereus CG 26及枯草桿菌F29-3抗真菌的測試 52 圖九、Bacillus thuringiensis CG 2、B.
thuringiensis CG 4、B. pumilus CG 12、B. cereus CG 15、B. cereus CG 20、B. cereus CG 26及枯草桿菌F29-3培養七天之菌液 的抗真菌測試 54 圖十、Bacillus thuringiensis CG 2、B. thuringiensis CG 4、B. pumilus CG 12、B. cereus CG 15、B. cereus CG 20、B. cereus CG 26及枯草桿菌F29-3培養七天之醱酵液的抗真菌測試 56 圖十一、Bacillus thuringiensis CG 2、B.
thuringiensis CG 4、B. pumilus CG 12、B. cereus CG 15、B. cereus CG 20、B. cereus CG 26及枯草桿菌F29-3與真菌不同濃度 比（細菌：真菌孢子液＝10：1）共同培養五天之菌液的抗真菌測試 58 圖十二、Bacillus thuringiensis CG 2、B. thuringiensis CG 4、B. pumilus CG 12、B. cereus CG 15、B. cereus CG 20、B. cereus CG 26及枯草桿菌F29-3與真菌不同濃度比（細菌：
真菌孢子液＝10：1）共同培養五天之醱酵液的抗真菌測試 60 表 目 錄 頁次 表一、34株革蘭氏陽性菌臨床分離菌株之生化 鑑定 62 表二、34株革蘭氏陽性菌臨床分離菌株之菌種鑑定推測結果 64 表三、34株革蘭氏陽性菌臨床分離菌株對五株真菌
之抑制作用 65 表四、Bacillus thuringiensis CG 2、B. thuringiensis CG 4、B. pumilus CG 12、B. pumilus CG 14、B. cereus CG 15、B. cereus CG 20及B. tubtilis F29-3抗真菌能力之比較 67 表五、Bacillus thuringiensis CG 2、B. thuringiensis CG 4、B.
pumilus CG 12、B. cereus CG 15、B. cereus CG 20、B. cereus CG 26及B. subtilis F29-3培養不同天數之抗真菌（Paecilomyces variotii Tu137）能力比較 68 表六、（A）Bacillus thuringiensis CG 2、B. thuringiensis CG 4、B. pumilus CG 12、B. cereus CG 15、B. cereus CG 20、B. cereus CG 26及B. subtilis F29-3與真菌（Paecilomyces variotii Tu137）共同培養不同天數與不同濃度 比之抗真菌（Paecilomyces variotii Tu137）能力比較 69 表六、（B）Bacillus thuringiensis CG 2、B. thuringiensis CG 4、B.
pumilus CG 12、B. cereus CG 15、B. cereus CG 20、B. cereus CG 26及B. subtilis F29-3與真菌（Paecilomyces variotii Tu137）
共同培養不同天數與不同濃度比之抗真菌（Paecilomyces variotii Tu137）能力比較 70 表六、（C）Bacillus thuringiensis CG 2、B. thuringiensis CG 4、B. pumilus CG 12、B. cereus CG 15、B. cereus CG 20、B. cereus CG 26及B. subtilis F29-3與真菌
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