利用“移树养苗”策略挖掘粤蓝链霉菌隐性活性次级代谢产物

利用适当的策略激活粤蓝链霉菌中潜在的隐性次级代谢途径,以期获得新的活性物质。以粤蓝链霉菌突变株DMR1为研究对象,该突变株的主导产物榴菌素的关键生物合成基因gra-orf1-3被敲除,不能产生榴菌素。通过改变营养条件、添加诱导物和热激等方法处理菌株,利用琼脂糖扩散法及TLC-生物显影等方法检测发酵粗提物的抑菌活性。结果显示,突变株DMR1的高氏一号和ISP3培养基发酵粗提物对枯草芽孢杆菌ATCC6633、金黄色葡萄球菌ATCC6538以及耐药的金黄色葡萄球菌ATCC43300和206具有显著抑菌活性;添加3%DMSO诱导后,高氏一号和ISP3培养基的发酵粗提物不仅对上述菌株的抑制活性增强,还具有抑制大肠杆菌ATCC8739、白色念珠菌ATCC10231以及多株耐药菌大肠杆菌和沙门氏菌的活性。粤蓝链霉菌中新发现的活性物质对革兰氏阳性菌、革兰氏阴性菌以及酵母菌具有广泛的抗菌活性,且部分发酵粗提物对所有测试的耐药菌均具有显著活性,有可能是新型的抗生素。结果也表明"移树养苗"策略能够提高隐性活性次级代谢产物的挖掘效率。     

一株有抑菌活性海洋放线菌的筛选及鉴定

目的:筛选具有抑菌活性的海洋放线菌.方法:以金黄色葡萄球菌、大肠杆菌、白假丝酵母、黑曲霉、苏云金芽孢杆菌、副溶血弧菌、胶质芽孢杆菌作为指示菌,采用滤纸片法进行筛选,将筛选到的菌株进行培养特征研究、生理生化试验及16S rDNA序列分析,并将测序结果提交至NCBI进行相似性检索,进而绘制出系统进化树,得到鉴定结果.结果:从舟山海域分离到1株具有较好抑菌活性的放线菌菌株a10,鉴定结果显示a10是一株浅玫瑰色链霉菌突变株.     

长足大竹象雄性附腺提取物抑菌作用研究

以3种细菌(大肠杆菌Escherichia coli、金黄色葡萄球菌Bacillus subtilis、枯草芽孢杆菌Staphyloccocus aureus)为供试菌,测定了长足大竹象Cyrtotrachelus buqueti雄性附腺提取物的抑菌活性。结果表明:长足大竹象雄性附腺提取物对革兰氏阳性菌如枯草芽孢杆菌、金黄色葡萄球菌均有一定的抑菌活性。不同浓度的粗提物对枯草芽孢杆菌及金黄色葡萄球菌抑菌影响差异均具有高度统计学意义(P0.01),抑菌活性随着粗提物浓度的增加而增强。粗提物对金黄色葡萄球菌和枯草芽孢杆菌的最小抑菌浓度分别为0.2 mg·m L~(-1)、0.4 mg·m L~(-1)。不同处理温度对长足大竹象雄性附腺粗提物的抑菌作用有一定影响。     

枯草芽孢杆菌HS-A38产抗菌脂肽Bacillomycin D的组分分析及鉴定

对一株枯草芽孢杆菌HS-A38产生的脂肽类物质进行分离鉴定及抑菌活性研究。通过酸沉淀分离和有机溶剂抽提的方法,从枯草芽孢杆菌HS-A38发酵液中得到脂肽粗提物LP,产率为1.956 g/L。利用薄层色谱和茚三酮染色法确定该脂肽粗提物中存在四个组分,分别为LP1、LP2、LP3和LP4;抑菌活性检测显示,组分LP3对两株海洋致病菌副溶血性弧菌和铜绿假单胞杆菌的活性较高。组分LP3经硅胶柱层析纯化分离后,应用反相高效液相色谱(RP-HPLC)和基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MS)对该组分做进一步纯化和鉴定。分析表明,LP3样品在保留时间20 min~28 min产生单峰团LP3-1,其纯度为85.24%;经MALDI-TOF-MS分析和数据比对,组分LP3-1中的主要成分为杆菌霉素Bacillomycin D。     

黄粉虫幼虫小分子量抗茵肽的分离纯化

昆虫抗菌肽具有良好的抑菌效果,有望开发成新一代抗生素。本文以金黄色葡萄球菌和大肠杆菌混合液作为诱导源,采用针刺法使黄粉虫TenebriomolitorL．幼虫感染微生物产生抗菌肽,并对抗菌肽进行了提取、色谱分离纯化及抑菌活性检测。结果显示,诱导组和对照组的三氟乙酸粗提物无抑菌活性;经SephadexG50、SuperdexPeptide凝胶色谱分离后,从诱导组和对照组均可获得对革兰氏阳性菌金黄色葡萄球菌、枯草芽孢杆菌有抑菌作用的组分,而且诱导组活性明显高于对照组;通过Resource15RPC反相色谱分离纯化,从诱导组获得一具有明显抑制革兰氏阳性菌的组分,质谱检测该组分为混合肽,主要由分子量为1876．21u、1904．21u的小肽组成,可能是一种比Thanatin分子量更低的昆虫抗菌肽。     

Isolation and purification of small antimicrobial peptide from larvae of mealworm, Tenebrio molitor

昆虫抗菌肽具有良好的抑菌效果,有望开发成新一代抗生素.本文以金黄色葡萄球菌和大肠杆菌混合液作为诱导源,采用针刺法使黄粉虫Tenebrio molitor L.幼虫感染微生物产生抗菌肽,并对抗菌肽进行了提取、色谱分离纯化及抑菌活性检测.结果显示,诱导组和对照组的三氟乙酸粗提物无抑菌活性;经SephadexG50、Superdex Peptide凝胶色谱分离后,从诱导组和对照组均可获得对革兰氏阳性菌金黄色葡萄球菌、枯草芽孢杆菌有抑菌作用的组分,而且诱导组活性明显高于对照组;通过Resource 15RPC反相色谱分离纯化,从诱导组获得一具有明显抑制革兰氏阳性菌的组分,质谱检测该组分为混合肽,主要由分子量为1 876.21u、1 904.21u的小肽组成,可能是一种比Thanatin分子量更低的昆虫抗菌肽.     

银沙槐内生放线菌抗菌活性及其与内生细菌的拮抗关系

从荒漠濒危植物银沙槐根部分离到内生放线菌12株和内生细菌31株.以大肠杆菌、金黄色葡萄球菌和枯草芽孢杆菌为指示菌对内生放线菌进行了抗菌活性筛选.结果表明：3株放线菌对3种指示菌均有抑制作用,2株完全无抑菌作用.采用菌块抑制法分析内生放线菌对来自同一植物内生细菌的抑制效果,44.9%的组合出现抑制现象,最大抑菌圈直径为28.5mm,最小者仅8.25 mm,主要抑制对象是革兰氏阴性菌和芽孢菌;55.1%的组合无抑制现象.说明内生菌的相互作用具有多样性.     

湛江沿海潮间带产胞外抗菌活性海洋细菌的分离鉴定及生物多样性分析

从硇洲岛和徐闻珊瑚礁自然保护区潮间带采集海水和沉积物标本,采用纯培养的方法分离其中的海洋细菌;以金黄色葡萄球菌、枯草芽抱杆菌和大肠埃希菌等为指示菌,以氨苄青霉素、青霉素-链霉素为阳性对照,采用琼脂扩散法筛选抗菌活性菌株;采用基于16S rDNA序列比对及其系统发育分析对分离培养的阳性海洋细菌进行分类鉴定和生物多样性分析;为进一步从海洋细菌资源中发掘新型抗菌药物奠定基础。结果从106株海洋细菌中筛选出了44株抗细菌活性菌株,阳性率为41.5%。其中,11株具有抗金黄色葡萄球菌作用,31株有抗枯草芽孢杆菌作用,13株有抗大肠埃希菌作用。抗菌活性菌株分布于31属,优势属为芽孢杆菌属(Bacillus)和弧菌属(Vibrio)。这表明分离自硇洲岛和徐闻珊瑚礁自然保护区潮间带的海洋细菌中的抗菌活性菌株具有丰富的生物多样性。     

分离自甘肃天祝五台岭土壤的放线菌拮抗性的初步研究

从甘肃天祝县土壤中分离到的27株放线菌,作为测试放线菌拮抗性的供试茵株.抗菌试验表明,14株放线菌对6株革兰氏阳性菌和阴性菌以及白色念殊菌等指示菌有抗性.菌株WTL-4、WTL-20和WTL-26抗菌谱最广,WTL-4能抗3种革兰氏阳性菌,WTL-20能抗革兰氏阳性的金黄色葡萄球菌和单增李斯特菌、革兰氏阴性的沙门氏菌及真菌白色念殊菌,WTL-26能抗革兰氏阳性的金黄色葡萄球菌和单增李斯特菌及革兰氏阴性的沙门氏菌.抗肿瘤试验表明,有8株菌株对肿瘤细胞有抑制作用,其中抑制率在60%以上的菌株占18.5%.对抗肿瘤活性较高的3株菌WTL-4、WTL-14和WTL-15以及没有抗菌和抗肿瘤活性的菌株WTL-27进行了系统发育分析.从系统发育树可以看出,4株菌均属于链霉菌属(Streptomyces).菌株WTL-4与Streptomyces viridochromoqenes的相似性为98%;菌株WTL-14和WTL-15的相似性达到99.2%,它们与S.flavolimosus 174457的相似性达到99.4%;WTL-27与S.mycroflavus的相似性为99%.     

原始热带雨林鹦歌岭土壤抗MRSA放线菌的分离与筛选

鹦歌岭是位于海南省乐东县的原始热带雨林,从鹦歌岭土壤中分离与筛选抗耐甲氧西林金黄色葡萄球菌(MRSA)的放线菌。采用平板稀释法分离,以MRSA作为受试菌株,测试抗菌活性,对具有抗菌活性的菌株进行16S rDNA序列测定。采用滤纸片法,测定菌株发酵粗提液对MRSA抑菌活性,并用高效液相色谱仪检测粗提液的活性物质。从鹦歌岭土壤中共分离到168株放线菌,其中10株具有较强的抗MRSA活性,发酵液粗提物抑菌圈直径在8 mm-25 mm之间;16S rDNA序列比对发现,10株菌均与链霉菌属(Streptomyces)的相似性达99%以上,初步确定是链霉菌属的放线菌。通过对比10株菌发酵液HPLC指纹图谱,发现菌株7、5和9-1的发酵液次级代谢产物丰富,活性测试表明,10株放线菌对MRSA的生长均有明显的抑制作用。     

美洲大蠊成虫肠道抗细菌共生放线菌的筛选及鉴定

【目的】鉴于野外美洲大蠊Periplaneta americana对恶劣环境适应性强,本研究旨在从云南省大理州的野外美洲大蠊成虫肠道中分离、筛选出抗细菌活性放线菌,为抗生素开发提供菌种资源。【方法】采用涂布平板法和平板划线法对美洲大蠊成虫肠道放线菌进行分离;以金黄色葡萄球菌Staphylococcus aureus、耐甲氧西林金黄色葡萄球菌、铜绿假单胞菌Pseudomonas aeruginosa、粪肠球菌Enterococcus faecalis、大肠杆菌Escherichia coli和鼠伤寒沙门氏菌Salmonella typhimurium共6种人体病原细菌为指示菌株,采用牛津杯法对分离自这些放线菌的次生代谢产物进行抗菌活性测定;通过形态学特征和16S rRNA基因序列分析,对具有广谱和明显抗菌活性的放线菌进行鉴定,并经16SrRNA基因序列的BlAST同源性比对及系统发育分析确定它们的分类地位。【结果】从美洲大蠊成虫肠道共分离获得41株放线菌。抗菌活性测定结果表明,34株(82.9%)放线菌对至少1种指示病原细菌具有抑制作用,其中有7株对3种以上病原细菌具有抑制作用,9株表现出明显的抗菌活性。根据系统发育分析结果,这些放线菌均被鉴定为链霉菌属Streptomyces spp.。【结论】野外美洲大蠊成虫肠道含有丰富的抗细菌活性的放线菌资源,为后续挖掘新型抗生素提供重要的微生物资源。     

筛选鉴定一株产生抑菌活性物质的海洋放线菌

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。     

西沙群岛海域海洋放线菌的分离及其抗菌活性

分离西沙群岛海域放线菌并研究其抗菌活性。采用干燥、辐射、冷冻及加热处理等11种样品预处理方式和10种培养基对海洋放线菌进行分离,对代表性菌株进行鉴定,并考察分离放线菌生长的海水依赖性。进一步以金黄色葡萄球菌、大肠埃希菌、啤酒酵母和扩展青霉为指示菌考察分离放线菌的抗菌活性。从西沙群岛海域样品中分离获得放线菌383株,其中专性海洋放线菌23株。选定93株代表菌株进行鉴定,93株菌隶属于9个科,11个属。不同培养基对分离放线菌菌株的数量及种类影响显著。6株放线菌对4种指示菌均有抑菌活性,其中4株为专性海洋放线菌,表明海洋环境具有丰富的放线菌资源,这些放线菌特别是专性海洋放线菌有望为新型抗菌物质的发现与开发提供菌种来源。     

连翘和水茄内生放线菌的多样性、活性及生物合成基因的PCR筛查

从采自成都地区的中药植物连翘Forsythia suspense和水茄Solanum torvum的根部分离到14株内生放线菌。活性筛选表明,10株菌的发酵粗提物具有不同程度的抗肿瘤活性,占全部菌株的71%;3株菌具有抗细菌活性,其中菌株A263具有较强的细胞毒活性和广谱抗细菌活性。基于16S rRNA基因部分序列的相似性分析表明,菌株A275属于克里贝拉菌属Kribbella,其余13株属于链霉菌属Streptomyces。多种生物合成基因的筛查实验表明,5株菌同时具有PKS-I、PKS-II、NRPS型基因,其中A255和A263还具有3,5-AHBA合酶基因,但仅A275具有oxyB基因。结果可以推测,链霉菌是这2种中药植物根部的优势内生放线菌,生物合成基因的PCR筛查能极大地弥补传统活性筛选模型的不足,内生放线菌具有产生丰富生物活性化合物的巨大潜力。     

Diversity and exploration of bioactive marine actinomycetes in the Bay of Bengal of the Puducherry coast of India

The present study was designed to investigate the Puducherry coast of the Bay of Bengal, India for the diversity of bioactive actinomycetes. A total of 50 actinomycete strains were isolated from the marine sediments and most of the strains were belongs to Streptomyces. These strains were identified by means of morphological physiological, biochemical and cultural characteristics. The isolates were subjected to shake flask fermentation and the secondary metabolites were extracted with ethyl acetate and screened for cytotoxicity, hemolytic activity and antimicrobial activity against selected bacterial and fungal pathogens. The cytotoxic activity was evaluated using HeLa cell lines by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole (MTT) assay, hemolytic activity on mouse erythrocytes and the antifungal activity was evaluated by MTT cytotoxic assay against Aspergillus niger, Aspergillus fumigatus and Candida albicans. The antibacterial activity was studied against Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Klebsiella pneumoniae. The cytotoxicity and antimicrobial activity of secondary metabolite was found to be concentration dependent and nearly 24% of isolates showed significant antimicrobial, hemolytic and cytotoxic activity. The results of our study indicate the diversity and bioactive potential of marine actinomycetes isolated in the Puducherry coast.     

Antibacterial activity of marine-derived fungi

A total of 227 marine isolates of ubiqituous fungi were cultivated on different media and the secondary metabolite content of the extracts (ethyl acetate/chloroform/methanol 3 : 2 : 1) characterized by HPLC. The fungi were secured from animals, plants and sediments of Venezuelan waters (0–10 m) including mangroves and lagoonal areas. The extracts were tested for antibacterial activity. A total of 7 were active towards Vibrio parahaemolyticus and 55 towards Staphylococcus aureus, representing 18 different fungal species from 8 ascomycetous genera. For 61 strains of Penicillium citrinum antibacterial activity correlated well with content of secondary metabolites as measured by HPLC. Thirteen isolates of Penicillium steckii produced very similar profiles of secondary metabolites and 6 of these had activity against either V. parahaemolyticus or S. aureus or both. This revised version was published online in June 2006 with corrections to the Cover Date.     

Description of a bacteriocinogenic plasmid in Beneckea harveyi.

A total of 795 strains of marine Vibrio species and Beneckea harveyi, a luminescent marine bacterium, were isolated from various sources in the area of Galveston Island, Tex., and screened for the production of bacteriocin-like substances. More than 8% of the Vibrio isolates produced low-molecular-weight (dialyzable) substances, which were lethal to a test strain of V. parahaemolyticus. Approximately 5% of the B. harveyi isolates produced higher-molecular-weight (nondialyzable) substances which were lethal to a test strain of B. harveyi. One of the B. harveyi strains (strain SY) produced a nondialyzable substance which was lethal to two of 39 strains of B. harveyi. The substance showed no activity toward 17 test strains drawn from the Vibrionaceae and Enterobacteriaceae. Strain SY showed no sensitivity to its own lethal agent and was shown by agarose gel electrophoresis and electron microscopy to harbor a single plasmid of 38 x 10(6) daltons. Variants of strain SY lacking the plasmid were produced by growth in the presence of the antibiotic novobiocin. These variants lacked both the ability to produce the lethal substance and the ability to survive in its presence. The lethal agent produced by strain SY is the first bacteriocin reported in marine bacteria. The term "harveyicin" is proposed to name this lethal substance.     

In vitro evaluation of antibacterial substances produced by bacteria isolated from different marine organisms

Bacteria from several groups of marine organisms were isolated and, using direct antibiograms, identified those that produce antibacterial substances, using a human pathogenic strain of Staphylococcus aureus ATCC6538 as revealing microorganism. Bacteria which produce substances that inhibited S. aureus growth were identified through morphological, physiological and biochemical tests. Out of 290 bacteria, 54 (18.6%) inhibited the growth of S. aureus, but only 27 survived for identification. Bivalves, sponges and corals were the most represented from which 41.2, 33.3 and 29.7%, respectively, produced antibacterial substances of the isolated bacteria in each group. The marine species with highest proportions of these bacteria were the hard coral Madracis decactis (62.5%), the sponges Cliona sp. (57.1%) and the octocoral Plexaura flexuosa (50.0%). Out of the 27 strains that produced antibacterial substances, 51.8% were Aeromonas spp. and 14.8% Vibrio spp. Marine bacteria that produce antibacterial substances are abundant, most belong in the Vibrionacea group and were isolated mainly from corals and bivalve mollusks.     

A potential bacterial biocontrol agent,strain S2V2 against pathogenic marine <Emphasis Type="Italic">Vibrio</Emphasis> in aquaculture

We isolated a marine bacterium strain S2V2 which inhibited the growth of pathogenic marine Vibrio spp. The aims of this research were to identify a new antibiotic-producing marine bacterium strain S2V2, and evaluate its spectrum activity and pathogenic property. Analysis of 16S rDNA sequence placed strain S2V2 in the genus Pseudoalteromonas, but the sequence similarity was low (95.46%) implying the strain might be a new species in this genus. Strain S2V2 inhibited the growth of 67.9% of 28 Vibrio strains tested. This strain inhibited V. alginolyticus, V. anguillarum, V. fluvialis, V. harveyi, V. metschnikovii, V. splendidus, V. ordalii, V. parahaemolyticus, and V. vulnificus, but inactive against V. campbellii, Aeromonas hydrophyla and Staphylococcus aureus. Strain S2V2 produced extracellular non proteinaceous antibacterial substances. The highest antibacterial activity was found when strain S2V2 was cultured for 96 h in ZoBell broth medium. An artificial infection to post larvae of Lithopenaeus vanname indicated that strain S2V2 was a non pathogenic bacterium. Non pathogenic property and specific antibacterial activity against a broad range of fish pathogenic marine Vibrio of strain S2V2 suggest that this strain is a prospective source of unique antibiotic and a potential biocontrol agent in marine aquaculture.