A pilot survey on antimicrobial activity and diversity of soil-derived actinobacteria from different depths in Gudzhirganskoe saline lake in Eastern Siberia

To explore pharmaceutical actinobacteria from salty environments in Siberia,8 soil samples were collected from different depths of Gudzhirganskoe saline lake,the Republic of Buryatia,Russia.Totally,635 actinobacterial strains affiliated with 21 genera in 12 families of 7 orders were obtained by culture-dependent approaches.The predominant genus was Streptomyces(74.5%),followed by Microbacterium(4.9%),Agromyces(3.5%),Nocardiopsis(3.0%),and Kitasatospora(2.5%).Seven strains showed relatively low 16 S rRNA similarities(<98.65%)with validly described species.Seventy-seven strains were selected as representatives to evaluate their antimicrobial activities and underlying mechanisms by paper-disk diffusion method and a double fluorescent protein reporter system(pDualrep 2),respectively.Streptomyces sp.S 6 b 3-1 was highlighted due to its strong inhibitory activities against Gram-positive bacteria and underlying antibacterial mechanism by inducing the SOS response in the reporter system.LG-1,(R)-6-[1-(benzoyloxy)ethyl]-phenazine-1-carboxylic acid,an analogue of saphenamycin was finally isolated from the culture broth of Streptomyces strain.This study preliminarily explored the actinobacterial diversity of soil samples collected in the highly alkaline and saline lake Gudzhirganskoe by culture-dependent approach and their capacity to produce antibiotics for the first time,which revealed that it deserves to make more efforts to discover new actinobacterial species and potential new antibiotics from the saline lake in Eastern Siberia.

PCR快速鉴定actinobacteria三种模板制备方法的比较

目的 本研究旨在建立准确、简便、快速的放线细菌鉴定技术 ,为普通和极端环境放线细菌资源的调查和开发利用创造条件。方法 从放线细菌固体培养基上挑取少量菌体 ,用微波炉法快速制备基因组DNA作为PCR模板 ,与液体培养法得到的菌体以超声波法或冻融法制备的模板进行了PCR扩增效果的比较研究。结果 PCR检测结果表明微波炉法制备的模板可进行有效的体外扩增 ,目的条带特异 ,而超声波法或冻融法并不对所有菌株有效 ,并有非特异扩增产物产生。结论 组合微波炉法快速制备放线细菌基因组DNA技术和 2 3SrRNA特异插入序列PCR扩增技术建立了准确、简便、快速的actinobacteria鉴别体系。

新型Actinobacteria荧光原位杂交(FISH)探针的设计和应用

Actinobacteria是形态上变化各异的 ,具有超过 5 0 % G+ C的 DNA碱基组成的 ,革兰氏染色呈阳性的微生物 ,放线菌是 Actinobacteria的一个组成部分 ,它们可产生丰富的次级代谢产物。目前 ,临床上使用的抗生素中有三分之二来自于放线菌。具有普通细菌形态的 Actinobacteria由于和放线菌具有相近的共同祖先 ,可能会具有一些其它的共性 ,是目前药物筛选中一个新的研究对象 ,但很难用形态观察的方法将之和其它细菌加以区分。本论文利用此类微生物 16 Sr RNA基因同源性 ,设计了两个探针 PA- 1、PA- 2并运用荧光原位杂交方法进行 Actinobacteria的鉴定 ,G+ C含量测定结果表明 ,这两个探针联合使用可直观、正确地鉴定 Acti-nobacteria。

两种Actinobacteria鉴定方法的比较

Actinobacteria classis nov.一般包括具有超过 5 0 % G+ C的 DNA碱基组成的微生物。其中的一些菌种在生物技术和医药方面具有重要的意义。为了分离能产生有用物质的新型微生物 ,我们扩大了分离对象 ,从以前的 Streptomyces和 Streptomyces以外的放线菌扩大到所有的 Actinobacteria。本论文实验中的Actinobacteria将特指那些具有普通细菌形态的高 G+ C含量革兰氏阳性细菌。为了能够将 Actinobacteria和其它革兰氏阳性低 G+ C含量的细菌加以区分 ,我们建立了两种鉴定方法。一种是荧光原位杂交 (FISH)方法 ,它具有准确和直观的优点。另外一种是 PCR方法 ,因在 Actinobacteria2 3S r RNA基因的 domain (螺旋区 5 4a)有 1个大约 10 0个碱基的稳定插入片段 ,通过体外 PCR扩增 2 3S r DNA片段 ,并用琼脂糖电泳分析 ,可以简便地鉴定 Actinobacteria。

Actinobacteria分离株的多相分类学研究

A ctinobacteria class nov.一般包括具有超过50%G+C的DNA碱基组成的微生物,其中一些菌种在生物技术和医药方面具有重要的意义。为了分离能够产生有用化合物的微生物菌种,我们将分离对象从已进行过深入研究的S trep tomy ces及其以外的放线菌扩大到所有的A ctinobacteria,其中包括那些具有普通细菌形态的高G+C含量革兰阳性细菌。为了对A ctinobacteria这一较新的研究领域有详细的了解,我们又对两株有弱生物活性的IM B 02B-165和IM B 02B-172进行了多相分类学研究。化学分类和系统分类的结果表明IM B 02B-165、IM B 02B-172菌株均属于A rthrobacter属中A rthrobacter g lobif orm is/A rthrobacter citreus组。

Actinobacteria的分离与鉴别

Actinobacteriaclassisnov .一般包括具有超过 5 0 %G +C的DNA碱基组成的微生物。实验中在分土培养基中 ,添加了Nalidixicacid及Aztreonam和Benlate ,从 4份土壤样品中 ,共分离得到 64株细菌 ,革兰氏阳性细菌共计 5 6株 ,占所分离菌株的 87 5 %。任意挑选其中的革兰氏阳性细菌 ,选用以高G +C含量革兰氏阳性细菌为靶点的PHGC探针及PNHGC对照探针 ,并联合使用我们自行设计的适用于Actonobacteria的PA 1和PA 2探针进行初筛菌株的鉴别。综合 4种探针的FISH的结果 ,我们可以判定在 31株分离株中 ,有 2 2株Acti nobacteria,6株低G +C含量革兰氏阳性细菌 ,其它 3株则不易判定。DNAG +C含量的测定结果表明 ,所建立的FISH方法可作为鉴别Actinobacteria的一种手段 ,它具有完整。

PCR法快速识别Actinobacteria的五种模板制备方法的比较

放线细菌的 2 3SrRNA基因序列具有较好的保守性和相对的可变性 ,被认为是快速筛选放线细菌的合适部位。能否快速有效地扩增特异性区间 ,聚合酶链反应 (PCR)扩增前的模板制备质量是关键 ,故比较了基因组DNA作为PCR模板的 5种制备方法。旨在寻找准确、快速、简便、经济的放线细菌筛选技术 ,为普通和极端环境放线细菌资源的研究和开发利用创造条件。

In-vitro antimicrobial activity of marine actinobacteria against multidrug resistance Staphylococcus aureus

Objective:To investigate the antibacterial aclivily of marine actinobacteria against multidrug resistance Staphylococcus aureus(MDRSA).Methods:Fifty one actinobacterial strains were isolated from salt pans soil,costal area in Kothapattanam,Ongole,Andhra Pradesh.Primary screening was done using cross-streak method against MDRSA.The bioaclive compounds are extracted from efficient actinobacteria using solvent extraction.The antimicrobial activity of crude and solvent extracts was perfomied using Kirby-Bauer method.MIC for ethyl acetate extract was determined by modified agar well diffusion method.The potent actinobacteria are identified using Nonomura key,Shirling and Gottlieb 1966 with Bergey's manual of determinative bacteriology.Results:Among the fifty one isolates screened for antibacterial activity,SRB25were found efficient against MDRSA.The ethyl acetate extracts showed high inhibition against test organism.MIC test was performed with the ethyl acetate extract against MDRSA and found to be 1 000μg/mL.The isolaled actinobacteria are identified as Streptomyces sp with the help of Nonomura key.Conclusions:The current investigation reveals that the marine actinobacteria from salt pan environment can be able to produce new drug molecules against drug resistant microorganisms.

Diversity, novelty, antimicrobial activity, and new antibiotics of cultivable endophytic actinobacteria isolated from psammophytes collected from Taklamakan Desert

Three hundred and twenty endophytic actinobacterial strains were isolated from psammophytes collected from Taklamakan Desert and identified. Among them, three strains already had been identified as new species of two genera and sixteen isolates showed relatively low 16 S rRNA similarities < 98.6% to validly described species. Seventy-five of the isolates were selected as representative strains to screen antibacterial activity and mechanism. Forty-seven strains showed antagonistic activity against at least one of the indicator bacteria. Two Streptomyces strains produced bioactive compounds inducing DNA damage, and two Streptomyces strains produced bioactive compounds with inhibitory activity on protein biosynthesis. Notably, the strain Streptomyces sp. 8P21H-1 that demonstrated both strong antibacterial activity and inhibitory activity on protein biosynthesis was prioritized for exploring new antibiotics.Under the strategy of integrating genetics-based discovery program and MS/MS-based molecular networking, two new streptogramin-type antibiotics, i.e., acetyl-griseoviridin and desulphurizing griseoviridin, along with known griseoviridin, were isolated from the culture broth of strain 8P21H-1. Their chemical structures were determined by HR-MS, and 1D and 2D NMR. Desulphurizing griseoviridin and griseoviridin exhibited antibacterial activities by inhibiting translation.

Endophytic actinobacteria of medicinal plant Aloe vera: Isolation, antimicrobial, antioxidant, cytotoxicity assays and taxonomic study

Objective: To explore the new sources of novel bioactive compounds having pharmaceutical and agricultural interest and to search the endophytic actinobacteria from medicinal plants. Methods: NAF-1 an endophyte actinobacteria was isolated from leaves of medicinal plant Aloe vera collected in Marrakesh, Morocco using Bennett agar as selective medium. NAF-1 was tested for its antimicrobial activity against five pathogenic bacteria such as Staphylococcus aureus PIC 53156, Micrococcus luteus ATCC381, Bacillus subtilis ATCC 14579, Pseudomonas aeruginosa DSM 50090 and Escherichia coli ATCC 8739 and four human clinic fungi belonging to the Candida, Aspergillus and Microsporum genera. Several antioxidant activities were studied such as DPPH free radical scavenging, β-carotene and linoleic acid and reducing power assays. The total of phenol and flavonoid was also calculated. Using Artemia salina shrimp assay, the cytotoxicity of NAF-1 crude extract was determined. Results: The results revealed that the actinobacteria showed a high activity(≥20 mm) against only Gram positive bacteria but it had a moderate activity(between 13 and 15 mm) against Human clinic fungi. The isolate also exhibited a LD50 of 14.20 μg/mL in the cytotoxicity assay. The result showed that the crude extract presented an interesting free radical-scavenging activity with IC50 value of(5.58 ± 0.26) μg/mL and a high value of phenolic and flavonoid compounds with(15.41 ± 0.18) μg GAE/mg extract and(11.41± 0.06) μg QE/mg extract respectively. Moreover, the taxonomic position of our endophyte actinobacteria using the morphological and physiological criteria and using 16 S r RNA gene sequence(polyphasic approach) showed that the NAF-1 isolate was similar to Streptomyces hydrogenans which was never described as an endophyte actinobacteria. Conclusions: This isolated strain appears promising resources of bioactive agents and can be exploited to produce therapeutic agents active against pathogenic disease.

Diversity and Bioactivity of Cultivable Animal Fecal Actinobacteria

Microbial symbionts play important roles in food digestion and absorption, immunity, pathogens resistance, and health maintaining of their hosts by coevolution. To provide new sources for discovering new leader compounds of drugs, the diversity and bioactivities of cultivable actinobacteria from animal feces have been studied. 31 species of animal fecal samples were collected fromYunnanWildAnimalPark. The purified cultures of actinobacteria were isolated from these samples by using 5 media. The 16S rRNA gene sequences of 528 selected strains were determined, the phylogenetic analysis was carried out, and anti-microbial and anti-tumor activities were determined. 35 genera (including a new genus, Enteractinococcus) of actinobacteria from the 31 species of animal feces were identified. Some strains had high anti-tumor and antimicrobial activities. More than 50 secondary metabolites were isolated and identified, a novel bioactive macrolactam polyketide glycoside, Sannastatin, was found. Nine fecal streptomycete strains were fermented respectively, blended to the microbial manure, and used to prophylaxis and treatment of soil-borne disease of notoginseng in field. The incidence rate of the disease was lower 81% than agricultural chemicals. Fecal actinobacteria, a possibility as a new source for discovering drug leader, agricultural chemicals and other industry products, will be discussed.

Isolation and Identification of Soil Actinobacteria from Mangrove forest in Beihai,Guangxi Province

[Objective] The aim was to identify the species diversity of Actinomycetes from Mangrove forest in Beihai,Guangxi Province. [Method] 10 strains of typical Actinomycetes were isolated from Mangrove forest soil,and the Actinomycetes genomic DNA was successful extracted. 16S rDNA was amplified by PCR and sequenced by Sanger dideoxy sequencing method. [Result] All the sequences were blasted in genbank,eight strains belonged to the genus of Streptomyces (80%),and two strains belonged to the genus of Nocardiopsis (20%). [Conclusion] There are many different Actinomycetes species in Mangrove forest soil samples in Beihai,Guangxi Province.

Isolation, distribution and evaluation of cytotoxic and antioxidant activity of cultivable actinobacteria from the Oman Sea sediments

Screening bioactive natural products from bacteria is a determinative step in the drug discovery programs. The present study aim to isolate actinobacteria from the Oman Sea sediments for determining the effects of different culture media and treatments on the yield of the isolation process, and measure the DPPH radical scavenging and Artemia cytotoxic activity of culture extracts of the actinobacterial isolates. A total of 290 actinobacterial isolates were collected from 14 sediment samples. Heat treatment(40.68%) and M4 medium(29.31%) exhibited the maximum isolation rates of actinobacteria. Streptomyces isolates were dominantly distributed in all of the investigated stations according to 16 S rRNA gene sequencing. The distribution pattern of Streptomyces followed a depth-dependent frequency trend, whereas the members of rare genera including Micromonospora, Nocardia Actinoplanes, Nocardiopsis, Saccharopolyspora and Crossiella were distributed in deeper stations. Approximately,25% of the examined isolates could scavenge 90% of 10^–4 mol/L DPPH solutions at 1 250 μg/mL final concentration of their ethyl acetate culture extracts. Furthermore, the most potent extracts could scavenge DPPH radicals with IC50 ranges from 356.8 to 566.4 μg/mL. Brine shrimp cytotoxicity tests showed that 38.88% of the examined culture extracts exhibited LC50 lower than 1 000 μg/mL against the Artemia cells. Moreover, the most potent culture extracts exhibited LC50 range from 335.4 to 534.4 μg/mL. Phylogenetic analysis by 16 S rRNA gene sequence revealed that the OS 005, OS 263 and OS 157 closely related to Streptomyces djakartensis, Streptomyces olivaceus and Nocardiopsis dassonvillei respectively. These results suggested the widespread distribution of the antioxidant and cytotoxic producing actinobacteria in the Oman Sea sediments, which could be considered as promising candidates for the discovery of microbial bioactive compounds.

Cultivable Sponge-Associated Actinobacteria from Coastal Area of Eastern Mediterranean Sea

In this study, 16 marine sponge samples collected from Eastern Mediterranean Sea were used for the isolation of sponge-associated Actinobacteria. Different media and pre-treatment methods were applied for the isolation experiments. In total, 7578 actinomycetes colonies were observed on the primary isolation plates, and 20 representative isolates were selected for further studies based on their morphologies and chemical profiles. These isolates were investigated for their antimicrobial activities, seawater requirements and 16S rRNA gene sequences. The phylogenetic analysis revealed that 80% of the isolated Actinobacteria was a member of the Streptomyces genus. All the isolates showed no absolute requirement for seawater. Fifteen of the selected isolates exhibited antimicrobial activity against methicillin resistant Staphylococcus aureus and vancomycin resistant Enterococcus faecium, whereas 3 isolates were active against Candida albicans.

Microbial Production of Chiral Hydroxy Esters and Their Analogs: Biocatalytic Reduction of Carbonyl Compounds by Actinobacteria, <i>Agromyces</i>and <i>Gordonia</i>Strains

We screened 15 Agromyces strains from the Microbacteriaceae family and 16 Gordonia strains from the Gordoniaceae family to investigate their biocatalytic ability to reduce carbonyl compounds. Two Agromyces strains (A. soli NBRC109063 and A. humatus NBRC109085) and two Gordonia strains (G. hydrophobica NBRC16057 and G. malaquae NBRC108250) grew well in 230 medium. The stereoselective reduction of various carbonyl compounds using these four strains was investigated. We discovered that these strains can reduce aliphatic and aromatic α-keto esters and an aromatic α-keto amide. On the basis of the conversion rate and stereoselectivity of the alcohols produced, G. hydrophobica NBRC16057 is a potential biocatalyst for the stereoselective reduction of α-keto esters and an aromatic α-keto amide to the corresponding chiral alcohols. Our results also suggest that the reduction of ethyl 2-methylacetoacetate by wet G. hydrophobica NBRC16057 cells in the presence of L-glutamate is useful for the production of chiral ethyl 3-hydroxy-2-methylbutanoate.

Exploration of mangrove‑associated actinobacteria from South Andaman Islands,India

In this study,a total of 1125 actinobacteria were isolated from the selected mangrove species:Avicennia marina,Rhizopora mucronata and Ceriops tagal from three study stations viz.,Minnie Bay,Carbyn’s Cove and Burmanallah.Among these three stations,the highest number of actinobacteria was recorded in Carbyn’s Cove(64.97%),followed by(25.51%)at Burmanallah and the minimum of(9.51%)was recorded in Minnie Bay.Maximum number of actinobacteria was recorded from Ceriops tagal(40.44%)than the other selected mangrove species Avicennia marina(34.13%)and Rhizopora mucronata(25.42%).Among the 1,125 mangrove-associated actinobacteria,103 morphologically different isolates from the Minnie Bay station was selected for the further characterization studies.In antibacterial assay,30.11%of the isolates revealed inhibitory activ-ity against all tested clinical pathogens and 65%isolates displayed inhibitory activity against minimum of 04 tested clinical pathogens.Growth survival studies of the actinobacterial isolates also accomplished to withstand in varied NaCl and pH levels.Of 103 isolates,all were found to synthesize gelatinase enzyme,73 isolates demonstrated amylolytic activity,38 isolates exhibited proteolytic and 63 isolates displayed urease activity.Interestingly,56 isolates exhibited excellent DNase activity and 71 isolates revealed positive for l-asparaginase production.To our recognition,11 isolates exhibited constructive results in the production of 06 extracellular enzymes of industrial importance.Of 103 isolates,48 isolates were confirmed by molecular level identification.Based on the phylogenetic analysis,the isolates were categorized under the genera:Strep-tomyces,Nocardiopsis,Salinispora and Actinomadura.

滇南森林土样中的Actinobacteria的分离、快速鉴别及初步鉴定

Actinobacteria是一类极具开发潜力的微生物资源.Actinobacteria的23SrRNA既有高度的保守性,又有较好的可变性,体外PCR扩增Actinobacteria的23SrDNA的稳定插入片段可有效地对放线细菌进行识别.本研究中结合了16SrDNA部分序列分析对其中9株Actinobacteria菌株进行了初步鉴定,随后又对菌株YIM70056进行了多相分类学研究,最终确定该菌株是红球菌属的一个新种,我们将其命名为云南红球菌.

白桦林地土壤细菌群落特征及其驱动作用

为探究白桦林地上层土壤与下层土壤细菌群落结构差异及细菌对上、下层土壤功能的驱动作用,采用16S rRNA基因高通量测序和土壤养分测定方法,对苏木山40 a白桦林地0~20 cm和20~40 cm土层进行分析。结果表明,白桦林地上层土壤有机质含量和多酚氧化酶活性较下层土壤显著提高75.25%和120.45%;上层土壤细菌Shannon多样性指数(5.83)较下层土壤Shannon多样性指数(5.74)显著提高;放线菌门、变形菌门和酸杆菌门(相对丰度>15%)、黄杆菌科(相对丰度>7%)、慢生根瘤菌属、鞘氨醇单胞菌属、土微菌属以及放线菌门的分枝杆菌属、类诺卡氏属、土壤红杆菌属和Gaiella属(相对丰度>1%)是白桦林地的优势细菌类群;疣微菌门(线性判别分析LDA值为4.07)和拟杆菌门(LDA值为3.80)以及放线菌门的类诺卡氏属(LDA值为3.72)和土壤菌属(LDA值为3.48)是白桦林地上层和下层土壤差异贡献度最大细菌门和属;多酚氧化酶活性、碱性蛋白酶活性和有机质含量是影响林地细菌群落结构的主要环境因子,并且显著影响放线菌门细菌类群。综上,白桦林地上层和下层土壤细菌多样性存在显著差异,放线菌门作为白桦林地重要的细菌类群,通过促进土壤碳循环的方式提高林地肥力水平,促进白桦林地土壤生态系统健康稳定发展。

Evaluating the effectiveness of marine actinobacterial extract and its mediated titanium dioxide nanoparticles in the degradation of azo dyes

Aim of the present study was to synthesize titanium dioxide nanoparticles （YiO2 NPs） from marine actinobacteria and to develop an eco-friendly azo-dye degradation method. A total of five actinobacterial isolates were isolated from Chennai marine sediments, Tamilnadu, India and analyzed for the synthesis of TiO2 NPs using titanium hydroxide. Among these, the isolate PSV 3 showed positive results for the synthesis of TiO2 NPs, which was confirmed by UV analysis. Further characterization of the synthesized TiO2 NPs was done using XRD, AFM and FI＇-IR analysis. Actinobacterial crude extract and synthesized TiO2 NPs was found efficient in degrading azo dye such as Acid Red 79 （AR-79） and Acid Red 80 （AR-80）. Degradation percentage was found to be 81% for AR-79, 83% for AR-80 using actinobacterial crude extract and 84% for AR-79, 85% for AR-80 using TiO2 NPs. Immobilized actinobacterial ceils showed 88% for AR-79 and 81% for AR- 80, dye degrading capacity. Degraded components were characterized by FT-IR and GC-MS analysis. The phytotoxicity test with 500 μg/mL of untreated dye showed remarkable phenotypic as well as cellular damage to Tagetes erecta plant. Comparatively no such damage was observed on plants by degraded dye components. In biotoxicity assay, treated dyes showed less toxic effect as compared to the untreated dyes.

Isolation and characterization of tyrosinase produced by marine actinobacteria and its application in the removal of phenol from aqueous environment

The present study was focused on screening and characterization of tyrosinase enzyme produced by marine actinobacteria and its application in phenolic compounds removal from aqueous solution. A total of 20 strains were isolated from marine sediment sample and screened for tyrosinase production by using skimmed milk agar medium. Among 20 isolates, two isolates LK-4 and LK-20 showed zone of hydrolysis and these were taken for secondary screening by using tyrosiue agar medium. Based on the result of secondary screening LK-4 was selected for further analysis, such as tyrosinase assay, protein content and specific activity of the enzyme. The tyrosinase enzyme was produced in a SS medium and was partially purified by ammonium sulfate precipitation, dialysis and SDS PAGE. The isolate （LK-4） was identified as Streptomyces espinosus using 16S rRNA gene sequencing and named as ＂Streptomyces espinosus strain LK4 （KF806735）＂. The tyrosinase enzyme was immobilized in sodium alginate which was applied to remove phenolic compounds from water. The enzyme efficiently removed the phenolic compounds from aqueous solution within few hours which indicated that tyrosinasc enzyme produced by Streptomyces espinosus strain LK-4 can be potently used for the removal of phenol and phenolic compounds from wastewater in industries.