目的 分离鉴定牛粪中的链霉菌,观察其抗菌活性。方法 采用稀释涂布法从牛粪中分离菌株,通过固定琼脂块法以金黄色葡萄球菌、大肠埃希菌为指示菌筛选抗菌活性较强的菌株,并对菌种进行生理生化鉴定及16S rDNA序列测定;采用纸片扩散法测定活性菌株发酵液抗菌活性,菌株发酵液抗菌活性物质进行水饱和正丁醇萃取,并通过Molish反应、双缩脲反应及茚三酮反应进行抗菌活性物质定性鉴别。结果 从牛粪中分离出8株菌株,并筛选出1株抗菌活性较强菌株B5-2,鉴定为链霉菌。活性菌株发酵液对金黄色葡萄球菌、大肠埃希菌、费氏柠檬酸杆菌、阴沟肠杆菌、肺炎克雷伯菌、产气肠杆菌均具有较强抑菌作用。抑菌结果显示,菌株B5-2抑菌活性物质均为水溶性物质。Molish反应、双缩脲反应及茚三酮反应结果显示,菌株B5-2的抑菌活性物质均初步鉴定为糖类和蛋白质类。结论 筛选分离的链霉菌对临床致病菌有较强的抑制作用。
Objective To isolate and identificate
由于抗菌药物的广泛使用,细菌耐药问题不断增多,耐药菌株的出现严重威胁着人类的健康,寻找新型抗菌药物迫在眉睫 [1-4]。放线菌是一类与人类健康极为密切的微生物种群,是产生抗菌药物最多的一类物种,大多数分布于土壤中。近年来,研究者们试图从反刍动物胃肠道微生物资源中分离放线菌。对药用植物内生链霉菌的研究已发现多种新的具有抗菌、抗虫活性的物质[5]。云南大学从西双版纳的药用植物中也分离到多种稀有放线菌[6]。相对于植物内生放线菌的研究,对动物肠道与粪便中共生放线菌的研究十分缺乏。牛是牛亚科牛族哺乳反刍类动物,具有为宿主分解代谢过程中产生的有毒物质的能力[7]。反刍动物胃肠道菌群是一些结构新颖、生物活性显著的天然活性产物的重要来源,是分离放线菌的新颖途径,但其中的放线菌的作用目前还不清楚[8]。本实验拟针对牛的粪便进行放线菌分离,筛选出具有拮抗致病菌活性的菌株,并对菌株进行鉴定,对其抗菌活性物质进行初步分析。
供试样品来源于牛粪。供试细菌为金黄色葡萄球菌 (
上述分离得到的菌株在改良高氏1号平板上四区划线接种,倒置于28 ℃培养7 d,挑选单菌落采用琼脂块法进行抑菌活性初筛。将金黄色葡萄球菌与大肠埃希菌分别接种至营养肉汤试管中37 ℃培养15h,采用比浊法,用无菌0.9%氯化钠溶液调整菌液浓度至1.5×106 cfu,均匀涂布于营养琼脂平板上。将培养出的菌株单菌落取下倒置在平板上,37 ℃培养24 h。采用十字交叉法测量抑菌圈直径,筛选出抑菌活性最好的菌株进行后续实验。每个菌株设3个平行实验。
1.4.1 生理生化特征鉴定 分离纯化的活性纯菌株采用改良高氏一号培养基在不同温度及pH值条件下培养;经革兰染色后,通过MOTIC电视生物显微镜对菌株菌丝及孢子形态进行观察。
1.4.2 16S rDNA序列测定 DNA提取和扩增参照文献[8]介绍的方法。16S rDNA序列聚合酶链反应(PCR)扩增采用保守引物1492R (5'-GGTTAC-CTTGTTACGACTT-3')和27F(5'-AGTTTGATCMTG-GCTCAG-3')。16S rDNA PCR扩增,PCR反应体系50 μL:10×Ex Taq buffer 5.0 μL,2.5 mmol·L-1 dNTP Mix 4.0 μL,10p Primer 11.0 μL,10p Primer 2 1.0 μL,Template 2.0 μL,5 U Ex Taq 0.5 μL,ddH2O 36.5 μL。PCR扩增产物由上海美吉生物医药科技有限公司完成纯化和测序工作。PCR反应条件:95 ℃预变性5 min;循环体系 95 ℃ 30 s,54 ℃ 30 s,72 ℃ 90 s,循环30次,72 ℃延伸10 min。4.0 ℃保存。
菌株发酵培养基选择改良高氏1号液体培养基。采用纸片扩散法[11-13]分析活性菌株发酵液的抗菌活性。筛选出的活性菌株挑取活化后的斜面培养基上的菌丝块接种于发酵培养基中,28 ℃,150 r·min-1 摇瓶培养7 d,4 000 r·min-1 离心20 min(
1.6.1 活性物质极性分析 按照“1.5”项方法制备活性菌株发酵液备用。无菌发酵液经水饱和正丁醇萃取后分为正丁醇层和水层。两相溶液经37 ℃蒸发挥干后用无菌水溶解。溶解后溶液过孔径0.22 μm过滤器保存在无菌管中。活性菌株发酵液原液、发酵液水层、发酵液正丁醇层按上述纸片扩散法进行抗菌活性测定及分析活性物质极性。
1.6.2 活性物质成分分析 通过Molish反应、双缩脲反应及茚三酮反应对活性菌株发酵液抗菌活性物质进行颜色鉴别。
从牛粪中根据菌种培养特征的不同,共分离出8株菌株,分别命名为B5-1,B5-2,B5-3,B5-4,B5-5,B5-6,B5-7,B5-8。
分离出的8株菌株经过筛选,其中1株菌株对金黄色葡萄球菌和大肠埃希菌抑菌作用比较显著,命名为B5-2。抑菌活性实验结果显示,菌株B5-2单菌落抑制金黄色葡萄球菌和大肠埃希菌的抑菌圈平均直径分别为9.0,15.0 mm。
B5-2菌株的发酵液对金黄色葡萄球菌、大肠埃希菌、费氏柠檬酸杆、阴沟肠杆菌、肺炎克雷伯菌和产气肠杆菌的抑制圈直径分别为11.5,15.5,18.0,19.0,14.0,20.0 mm,对供试菌均具有一定的抑制作用。B5-2发酵液经萃取后各相进行抑菌实验,不同相抑菌效果不同。菌株B5-2、发酵液经正丁醇萃取后,正丁醇层代谢物无抑菌活性,水层代谢物抑菌活性与发酵原液抑菌活性接近相同(
抗菌药物能够有效控制各类感染,降低因感染而造成的死亡,因此抗菌药物在临床治疗中的作用至关重要。“超级细菌”的出现正是由于抗菌药物的耐药引起。合理使用抗菌药物,避免耐药菌株的产生尤为重要[13]。针对从一些特殊环境中分离放线菌的方式,哺乳动物胃肠道微生物可作为新的放线菌资源开发利用。
本实验通过对反刍类动物牛粪便中放线菌进行分离筛选鉴定,筛选出一株具有较强抑菌作用的放线菌,其对临床常见耐药菌表现出的良好抑菌效果,菌株B5-2对新型拮抗耐药致病菌抗菌药物的研发具有巨大的潜在研究价值。但菌株的确切分类地位及活性物质的分离提纯和结构鉴定尚待研究。下一步将对菌株B5-2进行大量发酵,以确定发酵液中抗菌活性物质的组分及相应的化学结构,寻找具有新型结构和高活性的天然产物,以期得到新的高抗菌活性化合物。
The authors have declared that no competing interests exist.
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目的:分离筛选能够产生抑菌活性物质的海洋放线茵,并进行生理生化和16SrDNA鉴定。方 法:用分离培养基培养海洋放线菌,并筛选出能够产生抑菌活性物质的菌株,对所筛选菌株的形态特征、生理生化特性进行鉴定分析;采用通用引物27F、 1492R扩增该菌株的16SrDNA,对测序结果进行分析;采用Neighbor—Joining(N—J)法构建系统发育进化树。结果:筛选到一株对 金黄色葡萄球菌、大肠杆菌、白色念珠菌具有较强抗性的海洋放线菌F1,该菌株好氧,中度嗜盐,在高氏I号培养基上呈白色绒粉状,16SrDNA序列比对表 明该菌株与田无链霉菌(Streptomyces tanashiensis)NR043369的相似度为99%。结论:筛选到的菌株F1是一株海洋来源的放线菌,与田无链霉菌NR043369的同源性较 高,可能属海洋链霉菌属,对金黄色葡萄球菌等病原菌具有较强的抑菌活性。
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目的 为了筛选出具有生物活性的放线菌,并研究其可能存在的潜在研究及商业价值.方法 从台州市剑门港海区海底淤泥中采样并分离到3株具有抗菌活性的放线菌,通过分子生物学、(G+C)mol%含量、菌落及形态学进行菌种鉴定,应用BLAST Clustal W(V 1.83)和Mega(V 5.1)等软件进行系统发育树分析,以鉴定其分离地位,并对其发酵产物进行活性检测.结果 经研究发现一株菌株经不同发酵配方发酵,其胞内胞外物质均具有较好的抑菌效果,该菌经鉴定为星状诺卡菌,命名为Nocardia asteroids Ⅺ.结论 Nocardia asteroides Ⅺ筛选与鉴定丰富了抗生素产生菌资源,有潜在的开发出新的抗菌药物的可能性,值得进一步深入研究.
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目的 积累微生物资源,研究内生放线菌的微生态特征及其次生代谢产物的生物活性.方法 依据Coombs等的分离方法并加以改进,从采集自成都的罗汉松及响叶杨枝条中分离内生放线菌,对分离菌株做168 rRNA基因部分序列扩增并测序,对其次生代谢产物采用滤纸片法测抗菌活性,以SRB法测其对HepG2细胞株的细胞毒活性.结果 分离得到13株内生放线菌,其中10株菌具有不同程度的抗肿瘤活性,占全部菌株的77%,菌株A243具有极强的广谱抗细菌活性和细胞毒活性.A245属于马杜拉菌属,A251属于珊瑚状放线菌属,A246属于诺卡氏菌属,A248,A252属于小四孢菌属,其余8株属于链霉菌属.结论 链霉菌是这两种木本植物枝条中的优势内生放线菌,从这两种植物枝条中分离的内生放线菌产生多种活性的代谢产物,可以进一步的研究与开发.
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Endophytic microorganisms are to be found in virtually every plant on earth. These organisms reside in the living tissues of the host plant and do so in a variety of relationships, ranging from symbiotic to slightly pathogenic. Because of what appears to be their contribution to the host plant, the endophytes may produce a plethora of substances of potential use to modern medicine, agriculture, and industry. Novel antibiotics, antimycotics, immunosuppressants, and anticancer compounds are only a few examples of what has been found after the isolation, culture, purification, and characterization of some choice endophytes in the recent past. The potential prospects of finding new drugs that may be effective candidates for treating newly developing diseases in humans, plants, and animals are great.
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Abstract Endophytic actinobacteria are relatively unexplored as potential sources of novel species and novel natural products for medical and commercial exploitation. Xishuangbanna is recognized throughout the world for its diverse flora, especially the rain forest plants, many of which have indigenous pharmaceutical histories. However, little is known about the endophytic actinobacteria of this tropical area. In this work, we studied the diversity of actinobacteria isolated from medicinal plants collected from tropical rain forests in Xishuangbanna. By the use of different selective isolation media and methods, a total of 2,174 actinobacteria were isolated. Forty-six isolates were selected on the basis of their morphologies on different media and were further characterized by 16S rRNA gene sequencing. The results showed an unexpected level of diversity, with 32 different genera. To our knowledge, this is the first report describing the isolation of Saccharopolyspora, Dietzia, Blastococcus, Dactylosporangium, Promicromonospora, Oerskovia, Actinocorallia, and Jiangella species from endophytic environments. At least 19 isolates are considered novel taxa by our current research. In addition, all 46 isolates were tested for antimicrobial activity and were screened for the presence of genes encoding polyketide synthetases and nonribosomal peptide synthetases. The results confirm that the medicinal plants of Xishuangbanna represent an extremely rich reservoir for the isolation of a significant diversity of actinobacteria, including novel species, that are potential sources for the discovery of biologically active compounds.
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牛瘤胃与肠道中的微生物区系共同影响着宿主的营养与健康。宏基因组学与代谢组学的飞快发展,帮助研究者逐渐解析了牛体内这2大微生物群落的真实面貌。二代高通量测序技术的出现,加快了挖掘瘤胃微生物与肠道微生物这2大基因库的速度,使得对其中功能基因的筛选更加高效。牛共生微生物的研究不局限于基础科学,其成果在工业生产、食品安全检测、疾病诊疗与预防等许多领域都得到很广泛的应用。本文综述了近些年来围绕牛的瘤胃和肠道共生微生物的研究报告,例举了大量瘤胃细菌、真菌、古菌的研究,宏基因组学的研究以及常见疾病诊疗预防的研究,旨在为养殖业与乳品行业生产中遇到的难题提供科学有效的指导。
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Abstract The metabolic dynamics of the Escherichia coli K-12 strain TG1 to feast and famine were studied in glucose-limited steady-state cultures by up- and downshifts of the dilution rate, respectively. An uncoupling of anabolic and catabolic rates was observed upon dilution rate upshifts, apparent through immediately increased glucose uptake rates which were not accompanied by an immediate increase of the growth rate but instead resulted in the temporary excretion of methylglyoxal, D- and L-lactate, pyruvate and, after a delay, acetate. The energetic state of the cell during the transient was followed by measuring the adenylate energy charge, which increased within 2 min after the upshift and declined thereafter until a new steady-state level was reached. In the downshift experiment, the adenylate energy charge behaved inversely; no by-products were formed, indicating a tight coupling of anabolism and catabolism. Both dilution rate shifts were accompanied by an instantaneous increase of cAMP, presaging the subsequent changes in metabolic pathway utilization. Intracellular key metabolites of the Embden-Meyerhof-Parnas (EMP) pathway were measured to evaluate the metabolic perturbation during the upshift. Fructose 1,6-diphosphate (FDP) and dihydroxyacetone phosphate (DHAP) increased rapidly after the upshift, while glyceraldehyde 3-phosphate decreased. It is concluded that this imbalance at the branch-point of FDP induces the methylglyoxal (MG) pathway, a low-energy-yielding bypass of the lower EMP pathway, through the increasing level of DHAP. MG pathway activation after the upshift was simulated by restricting anabolic rates using a stoichiometry-based metabolic model. The metabolic model predicted that low-energy-yielding catabolic pathways are utilized preferentially in the transient after the upshift. Upon severe dilution rate upshifts, an oscillatory behaviour occurred, apparent through long-term oscillations of respiratory activity, which started when the cytotoxic compound MG reached a threshold concentration of 1.5 mg l(-1) in the medium.
[本文引用:3]
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DOI:10.1038/ja.2005.1
URL
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DOI:10.1021/np050358e
URL
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采用平板涂布法分离蚯蚓粪中的放线菌,采用琼脂块法初步研究了蚯蚓粪中放线菌的抗菌活性,通过形态观察、 生理生化特性和16S rDNA基因序列分析对抗菌活性较强的菌株进行鉴定.实验结果表明,从蚯蚓粪中共分离到放线菌26株,其中16株对指示菌株有抗菌活性,占分离放线菌总数 的61.54%;菌株QYF12,QYF22对藤黄八叠球菌Micrococcus luteus具有较强的拮抗作用,其抑菌圈直径分别为27,31 mm;菌株QYF26对枯草芽孢杆菌的抗菌活性较强,抑菌圈直径为21 mm.菌种鉴定结果表明,菌株QYF12为教酒链霉菌Streptomyces chartreusis,QYF22为吸水链霉菌奥萨霉素亚种S.ossamyceticus,QYF26为灭癌素链霉菌S.gancidicus,该研 究为下一步分离抗菌物质用于生物防治提供了理论依据.
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目的 通过调查了解抗菌素的使用情况,并根据实际情况加强对抗菌素使用的管理.方法 自2010年1月-2011年1月共收治的3769例住院患者,对其使用抗菌素的情况进行调查研究.结果 通过调查住院患者抗菌素的使用情况,调查结果显示抗菌素的使用率高达69.62%,并且存在较严重的抗菌素不合理使用现象.结论 在分析住院患者抗菌素的使用情况后分析抗菌素不合理使用的原因,从而尽可能避免抗菌素的使用与抗菌素的滥用.
[本文引用:2]
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