| 摘要註: |
本實驗從室外篩選到一菌株,經相位差顯微鏡觀察到菌絲及孢子,初步判定為放射線菌,命名為AB-06。由先前的研究顯示發現此菌株分泌抗生物質對革蘭氏陽性菌具高度之抗菌活性,另外在探討純化過程中,又發現另一不同的抗菌物質。在液態培養條件下,經篩檢添加不同碳、氮源於培養基中對AB-06 菌株所產生抗生物質活性影響,從試驗結果得知,最適培養基條件為glucose 0.8%、casamino acid0.6%、yeast extract 0.3%於30oC、150 rpm 培養48 小時後,可得到最大抗生物質產率,於25 μl 粗培養液裡,對生長在營養培養基之金黃色葡萄球菌的抑菌環直徑可達22 公釐。先以丁醇與粗培養液1.5 比1 的比例萃取、經減壓濃縮蒸餾乾後,以適量甲醇回溶沉澱物。續經Cellulose 及Al2O3 管柱分離雜質後,通過LH-20 及G-10 管柱層析,取得澄清之26-1 抗生物質之外,發現白色顆粒的沉澱物亦具有抗菌效果,命名為26-2 抗生物質。26-1 抗生物質回收率約為8 %,由薄層色層分析法 (TLC) 鑑定,為一純度極高之物質,利用液相層析質譜儀 (LC-MS/MS) 初步鑑定得知分子量約為838 g/mol,其分子結構尚待解析中;而26-2 抗生物質由FTIR、1H-NMR 及13C-NMR 圖譜推算具有C9H15NO3 的片段。純化之26-1 抗生物質另經由不同溫度、光線及pH處理證實其在121℃ 處理20 分鐘、陽光下照射2 小時或於pH 2 或12 液體環境中處理12 小時,其仍保有抗菌活性。在抗菌圖譜方面能專一性的抑制革蘭氏陽性菌,對抗苯唑西林金黃色葡萄球菌 (oxacillin-resistant staphylococcusaureus) 、肺結核桿菌 (Mycobacterium tuberculosis) 及萬古黴素抗性腸球菌(vancomycin-resistant Enterococcus spp.) 有很強的抑制效果,其抑制的作用上屬於生長遲滯性抑制。此外,為降低抗生物質的使用量及減少多重抗藥性菌株的產生,結果發現26-1 抗生物質與ampicillin 具有協同抑菌效果,其最小抑制濃度分別為32 倍及128 倍稀釋,但當兩者合併使用時,可有效降低ampicillin 的使用量;在生物膜 (biofilm) 防治方面,能阻斷金黃色葡萄球菌貼附於物體表面之能力,其抑制能力為可逆性,最小抑制濃度作用下能大幅減少生物膜生成及破壞生物膜,故此26-1 抗生素極具開發潛力,進一步發展新型藥物,以造福人類健康。 A strain of bacterium had been isolated from outdoor soil. Hyphas and spores of the bacterium were observed by phase-contrast microscopy. Furthermore, the strain was determined to be actinomycetes and named AB-06. Previous group member had identified that it secreted antibiotic metabolites in liquid culture system which were strongly antagonistic against Gram-positive bacteria specifically. During the purification process, another antibiotic metabolite was also discovered. To get enough antibiotics for further studies, various carbon and nitrogen sources on the antibiotic metabolite production was screened in a broth culture system. The composition of optimal medium for AB-06 strain to produce antibiotics in the submerged culture was 0.8% glucose, 0.6% casamino acid and 0.3% yeast extract. The culture condition of the maximum yield of antibiotics was incubated at 30℃ on a rotary shaker at 150 rpm for 48 hr. The inhibition zone diameter of 25 μl AB-06 crude culture against staphylococcus aureus can reach to 22 mm.The produced antibiotics were extracted by adding 1.5 fold butanol to the broth culture. The obtained crude extracts were concentrated to dryness by flash evaporator and then redissolved with appropriate amount of methanol. After passing through the cellulose column, Al2O3 column, LH-20 filtration and sephadex G-10 filtration, a clearly purified 26-1 antibiotic was obtained. The white pellet observed has antibacterial effects. Therefore, this antibiotic material was named 26-2. The recovery yield of the purified 26-1 antibiotic was about 8%. The molecular weight of 26-1 antibiotics was determined by LC-MS/MS is about 838 g/mol. The analysis of molecule structure is under investigation. From FTIR, 1H-NMR and 13C-NMR atlases calculated that 26-2 antibiotics may contain C9H15NO3 fragments. The antibacterial activity of purified antibiotic still maintained 33% at 121℃ for 20 min, sunlight illuminating for 2 hr, even in pH 2 or 12 for 12 hr. The antibacterial spectrum of 26-1 antibiotic could inhibit Gram-positive bacteria specifically. There are strong inhibition effect to oxacillin-resistant Staphylococcus aureus, Mycobacterium tuberculosis and vancomycin-resistant Enterococcus spp. The antibacterial effect of 26-1 antibiotic was static effect. Moreover, in order to reduce the amount of using antibiotics and the production of drug-resistant strain, the combination of 26-1 antibiotics with ampicillin could provide noticeable synergistic antimicrobial effect. The minimal inhibitory concentration of 26-1 antibiotics and ampicillin was individually 32 fold dilution and 128 fold diluted concentration. Such combinations can effectively decrease the amount of using ampicillin. At the antibiofilm preventive part, 26-1 antibiotic could disrupt the ability of Staphylococcus attachment. Under the effect of minimal inhibition concentration can also decrease the synthesis of biofilms and damage them. 26-1 antibiotic has the potential and worth to develop. |