Aspergillus sp136抗细菌耐药活性成分研究

基于广谱抗细菌耐药性这一思路，本研究中心建立了一套抗细菌耐药性化合物的筛选方法。由此从3000多种西南地区特殊生境的微生物和植物样品提取物中筛选获得17个抗细菌耐药性活性样品。对其中一株来自峨嵋山土样的微生物（Aspergillus sp136）进行了深入研究。通过TLC自显影等方法从其发酵产物中追踪分离得到抗耐药有效成分，并鉴定为烟曲霉酸。 采用多种方法对烟曲霉酸的体外抗细菌耐药活性进行评价。在平板扩散法中，烟曲霉酸表现出对青霉素（β-内酰氨抗生素）的协同抗耐药能力，其活性大约3倍于克拉维酸。在MIC的测试实验中，烟曲霉酸表现出对青霉素（β-内酰氨抗生素）以及非β-内酰氨抗生素如红霉素、四环素、氯霉素、链霉素、卡那霉素、庆大霉素的抗耐药能力。在棋盘格杀菌以及时间致死曲线的研究中，烟曲霉酸也表现出对青霉素、红霉素、四环素的协同抗细菌耐药活性。 在广泛的活性筛选中发现烟曲霉酸对LDLR基因具有上调活性，表明烟曲霉酸可能具有降血脂的活性。 在研究中发现，同空白对照相比，烟曲霉酸使耐药菌（Bacillus cereus NCPF63509）细胞外β-内酰胺酶酶活大幅度下降，而细胞内β-内酰胺酶酶活仅略有上升，这表明烟曲霉酸对β-内酰胺酶分泌过程具有抑制作用。 综述了β-内酰胺酶的研究进展。 A two-step agar diffusion method was established to screen wide spectrum synergistic antibacterial agents. By using this method, 17 active samples against antibiotic resistance were discovered from more than 3000 plants and microbes, which were collected from southwest china. One isolate Aspergillus sp136 collected from E-mei mountain area was selected for further studies. From the metabolites of this strain, a synergistic antibacterial compound was isolated by bioautographic TLC assay-guided fractionation and identified as helvolic acid. The synergistic effect of helvolic acid was confirmed by several methods in vitro. The synergistic effect of helvolic acid with penicillin (β-lactam antibiotics) was about 3 times as that of clavulanic acid with penicillin in agar diffusion assay. In MIC studies, helvolic acid exhibited synergistic effects with β-lactam antibiotics such as penicillin and non β-lactam antibiotics such as erythromycin, tetracycline, kanamycin, streptomycin and gentamycin. In checkerboard and time-kill studies, helvolic acid also exhibited synergistic effects with penicillin, erythromycin and tetracycline. In general screen of bioactivities, helvolic acid upregulate LDLR gene, which was indirectly determined by the activity of fluorescent enzyme. Therefore, helvolic acid might have the ability to lower lipid in blood. Compared with blank control, the extracellular β-lactamase activity decrease significantly and the intracellular β-lactamase activity increase slightly in Bacillus cereus NCPF63509 in the presence of helvolic acid, indicating that the secretion of β-lactamase was inhibited by helvolic acid. The research of β-lactamase was reviewed.