粘细菌的活性检测及与环境因子的相关性

为筛选具有抗菌活性的粘细菌,利用兔粪诱导、大肠杆菌划线诱导和滤纸诱导3种方法从内蒙古凉城地区采集的20种土样中分离粘细菌,结合形态观察、生理生化特征和分子鉴定方法确定这些菌株的分类地位,通过平板对峙法分析抗菌活性,并采用统计学方法分析其与环境因子的相关性.共分离得到140株菌,包括39株纯粘细菌,经鉴定分别为黄色粘球菌(17株)、橙色粘球菌(13株)、变绿粘球菌(5株),还有4株只鉴定到粘球菌属(Myxococcus sp.).抗菌结果显示:39株纯菌均表现出可以对一种甚至多种指示菌产生抗性,其中37株抑制大肠杆菌的生长,35株抑制马铃薯晚疫病菌的生长,26株抑制金黄色葡萄球菌的生长,20株抑制酿酒酵母菌的生长,19株抑制枯草芽孢杆菌的生长.在分析环境因子与粘细菌分布情况的关系时发现,分离出的溶细菌类群的菌株数、溶纤维素类群的菌株数及总的菌株数与土壤p H和速效钾的含量呈正相关,与含水量呈负相关,与其它参数无明显相关性.综上,内蒙古凉城地区粘细菌的优势菌群为粘球菌属,且绝大多数都有不同程度的抗菌活性;结果可为丰富内蒙古地区粘细菌资源及抗马铃薯晚疫病农药研发奠定基础.     

一些硝化细菌的分离与鉴定

采用硝化细菌选择培养基从土壤、污水中筛选获得了 10株细菌 (X0 1～X10 ) ,经革兰氏染色及生理生化鉴定均为革兰氏阴性菌 ,呈杆状或球状 ,均能够利用亚硝酸盐 .电镜观察发现 ,这 10株细菌均具有比较复杂的细胞膜结构 ,与报道的硝化细菌所特有的膜结构相同 .对硝化细菌的特征性基因norB进行检测结果表明 ,所有菌株均可扩增出该基因 .初步判断所筛选的细菌为硝化细菌 ,依据《伯杰细菌鉴定手册》进行分类 ,主要为硝化杆菌、硝化球菌和硝化刺菌属等 .图 3表 4参 15     

酵母菌株Pseudozyma rugulosa对合成染料脱色的初步研究

通过筛选实验,从土壤中分离出一株对活性艳红KBP具有明显脱色效果的酵母菌株,鉴定为Pseudozymarugulosa.采用含50mg·l-1活性艳红K2BP的液体培养基同步培养脱色,发现该菌株在9h时对活性艳红K2BP的脱色率为99%.此外,该菌株对另外九种染料的脱色率在22%—98%之间.其中,对偶氮染料——弱酸艳红B、活性黑KNB和活性红M3BE的脱色率都达到了96%以上,对三苯甲烷染料——酸性媒介漂蓝B的脱色率达到了89%.     

四川盆地大豆根瘤内生细菌的分离鉴定及促生效果

以四川盆地大豆根瘤为材料,采用划线法分离内生细菌、16S rDNA PCR-RFLP分析其遗传多样性,并结合菌株促生特性和盆栽试验筛选优良促生菌.从分离获得的130株内生细菌中选取了40株细菌作为供试菌株,16S rDNA序列表明分属于芽孢杆菌属(Bacillus)、肠杆菌属(Enterobacter)、假单胞菌属(Pseudomonas)、中华根瘤菌属(Sinorhizobium)和慢生根瘤菌属(Bradyrhizobium).以大豆为供试作物筛选出了12株具有促生能力的细菌,所有菌株均能分泌吲哚-3-乙酸(IAA),浓度达到0.353-32.404μg/mL;7株能产铁载体,活性单位为7.35%-34.31%;有11株具有溶磷能力,溶磷量达到4.26-10.6μg/mL;6株具有固氮能力.接种12株供试菌株后,玉米的农艺性状、植株全氮和全磷含量均优于单施化肥处理,其中菌株DA16-5效果最好,表现出良好的促生潜力.综上,四川盆地大豆根瘤内生菌遗传多样性丰富并且普遍具有促生能力,是重要的生物资源.     

一株高效不对称还原产生(R)-苯基乙二醇的菌株筛选、鉴定及全细胞催化体系

(R)-苯基乙二醇是合成许多光学活性药物的重要手性中间体,其制备具有重要的现实意义.以α-羟基苯乙酮为底物,从土壤中筛选得到一株能够立体选择性催化α-羟基苯乙酮产生(R)-苯基乙二醇的细菌菌株HBU-SI7.经形态学观察和16S r DNA序列分析,鉴定此转化菌株为红球菌属菌株Rhodococcus sp.菌株全细胞催化体系研究表明在邻苯二甲酸二丁酯-磷酸盐缓冲液(V:V=1:3)的两相催化体系下,菌体转化α-羟基苯乙酮的最优浓度为3.0 g/L,转化率高达96.2%,e.e值为99.3%.本研究建立的红球菌全细胞催化还原体系对(R)-苯基乙二醇的高效制备具有潜在的研究和应用价值.     

红树林放线菌的分离及其抗菌和抗肿瘤细胞活性

从福建漳州红树林保护区采集土壤样品中分离到163株放线菌,用5种指示菌(Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Candida albicans and Rhizoctonia solani)测定了它们的抗菌活性,结果表明,其中42.3%的放线菌发酵液具有单一或多种抗菌活性.同时测定了它们的发酵液对肝癌细胞BEL7402、肺癌细胞A549和白血病细胞HL60三种肿瘤细胞的毒性,其中37.4%的放线菌发酵液具有单一或多种抗肿瘤细胞毒活性.对这163株放线菌的16S rDNA序列分析表明,它们分属于链霉菌属(89%)、小单孢菌属(6.1%)、糖单孢菌属(0.6%)、马杜拉放线菌属(3.7%)和拟诺卡氏菌属(0.6%).其中, 3株链霉菌与其亲缘关系最近的菌种的16S rDNA相似性低于97%,可能是3个新种.     

稳定混合菌协同降解咔唑研究

以咔唑(Carbazole,CAR)作为唯一碳源和氮源,从某炼油厂废水中筛选获得稳定的咔唑降解混合菌株.对该混合菌株进行分离和纯化,获得两株细菌,经生物化学和分子进化特征分析后,初步鉴定为Chryseobacterium sp.NCY和Achromobacter sp.NCW.扫描电镜显示,两株菌在含咔唑的无机盐培养基和肉膏培养基上生长时,细胞形态发生显著的变化.这两株细菌形成的稳定复合体存24 h内对咔唑(500 mg L-1)的降解率为80%,64 h内降解率可达到99%以上,并释放出氨氮22.69 mg L-1.纯化后的两株细菌单独以咔唑为唯一碳源和氮源,均不能生长,推测两株菌通过协同代谢作用降解咔唑.     

澳洲野生稻(Oryza australiensis)内生固氮菌的分子鉴定及发育分析

采用两种无氮培养基经平板划线法共分离筛选到澳洲野生稻(Oryza australiensis)45株内生固氮菌.利用全细胞蛋白电泳和插入序列指纹图谱对获得的内生固氮菌进行聚类分析,将其分为8个类群.其中类群Ⅰ有10株菌,类群Ⅱ为4株菌,类群Ⅲ、Ⅳ和Ⅴ均为3株菌,类群Ⅵ、Ⅶ和Ⅷ各有2株菌.对分离得到的内生固氮菌主要类群部分代表菌株的固氮活性进行了系统研究,结果表明,菌株在偏酸(pH值5.0)和偏碱(pH值9.0)的条件下均能保持较高的固氮酶活性,NaCl浓度在2%时达到最高固氮活性,NH4+浓度达到7.5 mmol/L时,均无固氮酶活性,不同菌株所能利用的碳源不同.16S rDNA序列测定及系统发育分析显示:类群ⅠYH39与Burkholderia cepacia ATCC 25416T相似性为97%;类群Ⅱ为克雷白氏杆菌属(Klebsiella),相似性为100%;类群Ⅲ为泛菌属(Pantoea),相似性为99%;类群Ⅴ为草螺菌属(Herbaspirillum),相似性为99%;类群Ⅶ为中华根瘤菌属(Sinorhizobium),相似性为99%.本研究表明澳洲野生稻内生固氮菌资源具有遗传多样性.图5表1参26     

一株草莓根腐尖孢镰刀菌拮抗内生细菌的分离鉴定及抑菌特性

为筛选对草莓根腐尖孢镰刀菌(Fusarium oxysporum)有较好抑菌活性的草莓内生细菌,从草莓的根、茎、叶和果实中分离内生细菌,采用平板对峙法筛选具有抑菌活性的菌株,经形态特征观察、生理生化特性及分子鉴定后,进行拮抗菌抑菌特性分析.结果筛选到1株对草莓根腐尖孢镰刀菌具有较高抑菌活性(抑菌带达9.8 mm)、稳定性较好的菌,标记为SY-4.此外,该菌对桃褐腐病菌(Monilinia fruticola)、棉花红腐病菌(Fusarium moniliforme)和草莓根腐石楠拟盘多毛孢(Pestalotiopsis photiniae)等也有较好的拮抗作用.SY-4经鉴定确定为枯草芽胞杆菌.SY-4发酵液在培养时间48 h、培养温度37℃、pH值为7时,抗菌效果最好,但发酵产物不耐高温、不耐酸、较耐碱性,对蛋白酶敏感,初步说明存在蛋白类抑菌活性物质.电镜观察发现SY-4发酵液处理草莓根腐尖孢镰刀菌后,菌丝形态变得不规则.该拮抗内生细菌是潜在的植物病原真菌生防菌资源,具有一定的开发与应用价值.     

土壤氨氧化细菌对大气CO2浓度增高的响应

利用FACE(free-air carbon dioxide enrichment,开放式空气CO2浓度增高)试验平台,研究大气CO2浓度增高对土壤氨氧化细菌的数量、优势菌群及其硝化活性的影响.结果表明,大气CO2浓度增高时,土壤氨氧化细菌的数量在常氮水平上趋于减少,而在高氮水平上与对照没有差异.大气CO2浓度增高对土壤氨氧化细菌的优势菌群也产生明显影响.CO2浓度增高条件下,亚硝化球菌(Nitrosococcus sp.)和亚硝化弧菌(Nitrosovibrio sp.)是优势菌属;而在对照条件下,亚硝化单胞菌(Nitrosomonas sp.)和亚硝化球菌(Nitrosococcus sp.)是优势菌属.另外,CO2浓度增高条件下优势菌株的硝化活性也有不同程度的减弱.     

Antimicrobial activity of extracts of Caribbean gorgonian corals

Extracts of 39 species of Caribbean gorgonians were tested for antimicrobial activity against 15 strains of marine bacteria. The bacteria consisted of three opportunistic pathogens, Vibrio parahaemolyticus, Leucothrix mucor, and Aerococcus viridans, and 12 strains isolated from either healthy or decayed gorgonians. Overall, only 15% (79 out of 544) of the tests resulted in antibacterial activity with 33% (13 out of 39) of the gorgonians inhibiting only one bacterial strain and 23% (9 out of 39) showing no activity. The extracts of four Pseudopterogorgia species showed relatively high levels of activity, inhibiting 43 to 86% of the bacterial strains. The potency of the active Pseudopterogorgia species was variable, however, and three additional Pseudopterogorgia species were inactive against all bacterial strains. With the exception of one sensitive strain, Vibrio species were resistant to gorgonian metabolites. Our results indicate that organic extracts of most Caribbean gorgonians do not possess potent, broad-spectrum antibacterial activity inhibitory to the growth of opportunistic marine pathogens and bacteria associated with healthy and decayed gorgonian surfaces. These findings suggest that the inhibition of bacterial growth is not the primary ecological function of gorgonian secondary metabolites and that bacteria may not be important selective agents in the evolution of gorgonian secondary chemistry.     

Bioactive potential of seagrass bacteria against human bacterial pathogens

Study of marine organisms for their bioactive potential, being an important part of marine ecosystem, has picked up the rhythm in recent years with the growing recognition of their importance in human life. Investigation was carried out to isolate 32 strains of endo and epiphytic bacteria in 2 seagrass species viz., Syringodium isoetifolium and Cymodocea serrulata. Morphologically different bacterial strains were tested against 5 antibiotic resistant human bacterial pathogens, of which 10 associated bacteria shown inhibitory activity against one or more bacterial pathogens. Minimum inhibitory concentration (MIC) and Minimum bacterial concentration (MBC) determination with extracellular bioactive compounds from the associated bacteria reveals that, the strain ENC 5 showed inhibitory activity against all the bacterial pathogens with the maximum sensitivity against Pseudomonas aeruginosa at the MIC value of 500 microg ml(-1).     

海水网箱养殖大黄鱼细菌性病原鉴定与感染治疗研究

从患病大黄鱼的肌肉,肝、肾等病灶中分离出7种菌株,经人工感染试验证明,菌株970709A,970918a和970709B为致病菌,这三株菌株进行人工感染,死亡率均为100％,症状与自然发病相似,经菌体形态特征,生理生化特性反应测定,菌株970909A和970918a为鳗弧菌（Vibrio anguillarum),菌株970909B为副溶血弧菌（V.parahaemolyticus),药敏实验结果表明,氧氟沙星,环丙沙星,培氟沙星,罗红霉素,氯霉素和海鱼宁II等对致病菌有较强的抑制作用。临床治疗试验还表明,复方制剂海鱼宁II对大黄鱼弧菌病的治疗效果显著,有效率达66％－68％。     

In vitro antibacterial and anti-inflammatory properties of seaweed extracts against acne inducing bacteria, Propionibacterium acnes

This study was conducted to evaluate the antimicrobial activities of common seaweeds from the coast of South Korea against the etiologic agents of acne vulgaris. Fifty-seven species of seaweed were screened for potential antimicrobial activity. Methanol extracts of 13 species (22.8%) showed inhibitory effects against Propionibacterium acnes. The aqueous extracts of only two species (3.5%) showed antimicrobial activity. When tested with the agar disk diffusion method, Ecklonia cava, E. kurome, Ishige sinicola, and Symphyocladia latiuscula had the strongest inhibitory effects. However, these four seaweed extracts showed no antibacterial activity against Staphylococcus epidermidis at 5 mg disk-1. The minimum inhibitory concentration (MIC) values of E. cava and E. kurome were both 0.31 mg ml-1 and the MIC values of l. sinicola and S. latiuscula were 0.26 and 0.21 mg ml-1, respectively. Among whole plants of E. cava and E. kurome, extracts of the pinnate blade had the highest inhibitory activity on bacterial growth. In cytotoxicity assays, methanol extracts of E. cava, E. kurome, and I. sinicola showed no effect on cell viability at concentrations of 200 microg ml-1. However, the methanol extracts of S. latiuscula reduced cell viability rates to 50% at the same concentration. Additionally, methanol extracts of E. cava, E. kurome, and I. sinicola potently inhibited the in vitro production of nitric oxide. These results suggest that the methanol extracts from these three species may be useful in the development of therapeutic agents for acne vulgaris. Further investigations to determine the bioactive compound are in progress.     

金鸡菊(Coreopsis drummondii)的抗TMV活性物质

采用活性跟踪法从金鸡菊根中分离获得抗病毒活性物质,经质谱和核磁共振分析,鉴定该物质为1-苯基-1,3,5-三庚炔.采用半叶枯斑法、叶圆盘法测定了该物质对烟草花叶病毒的抑制效果,结果表明,0.2 mg/mL的该化合物对TMV表现出较好的体外抑制侵染和增殖活性,其对TMV侵染和复制的抑制率分别为73.5%和84.3%.实时荧光定量PCR测定结果表明,该化合物对TMV外壳蛋白基因的表达有明显的抑制作用,0.2 mg/mL的该化合物对TMV外壳蛋白基因表达的抑制率为79.8%.图6表1参19     

凹叶厚朴中具有α-葡萄糖苷酶抑制活性的成分

凹叶厚朴是四川省人工栽种的一种用途广泛的常用中药.在发现凹叶厚朴乙醇提取物具有较强的α-葡萄糖苷酶抑制活性基础上,通过活性跟踪,对该提取物进行了分离,发现其中的生物碱显示了较强的抑制活性.通过溶剂萃取、树脂吸附和反复硅胶柱层析等分离方法从凹叶厚朴乙醇提取物中分离得到6个生物碱.用质谱和核磁共振等波谱方法分别鉴定为木兰箭毒碱,木兰花碱,鹅掌楸碱,蕃荔枝碱,罗默碱和Lysicamine,并应用小肠α-葡萄糖苷酶模型测定了它们对α-葡萄糖苷酶的抑制作用.其中,番荔枝碱和木兰箭毒碱的抑制活性较好,分别为60%和62%;其它4个生物碱成分活性几乎相当,鹅掌楸碱为46%,罗默碱为51%,Lysicamine为49%,木兰花碱为51%.图2参10     

Effect of natural preservatives on the growth of histamine producing bacteria

Present study deals with the hampering of the growth of histamine producing bacteria (HPB), by using NaCl and spices which are easily available and cheaper cost wise. For this experiment, four strains of HPB viz. Vibrio parahaemolyticus, Bacillus cereus, Pseudomonas aeruginosa and Proteus mirabilis were tested against 1 to 10% concentrations of NaCl and 1 to 5% concentrations of natural preservatives (turmeric, ginger and garlic) in a basal medium. HPB showed different growth rates at different concentrations of NaCl and natural preservatives. V. parahaemolyticus, B. cereus and Ps. aeruginosa showed no growth at 10% concentration. When the HPB growth was tested with garlic, turmeric and ginger extracts, growth of all the bacteria was inhibited by garlic and turmeric extracts at 5% concentration. In ginger, V. parahaemolyticus, B. cereus and P. mirabilis were totally inhibited at 5% concentration. But Ps. aeruginosa showed very less growth at this concentration.     

Cellular damage of plant pathogenic fungi by antifungal compounds produced by Bacillus spp. isolates

The use of microorganisms as biological control agents (BCAs) has become an effective alternative to chemical means of controlling plant pathogens. The antagonistic and inhibitory activity of 71 Bacillus spp. strains, which were isolated from different Mexican sites, were tested against several phytopathogen fungi, Fusarium oxysporum, Fusarium equiseti, Fusarium avenaceum, Bipolaris spp. and Alternaria spp. From the antagonism study, the strain ELI149 showed a marked inhibition of growth against all tested fungi; therefore crude metabolites from this strain were extracted using ethyl acetate and amberlite resin and probed against the same fungi as well as strains of Mucor sp., Penicillium spp. and Paecilomyces spp. The results indicated that amberlite was more suitable for extraction of secondary metabolites with antifungal activity. Finally, observation of cell damage in the tested pathogenic fungi showed marked morphological changes on reproductive structures in all tested fungi indicating that antibiosis was the mechanism of the antagonistic effect. These results suggest that metabolites from the Bacillus strains have a wide spectrum of antibiotic activities, which can be used as biocontrol agents for controlling fungal plant diseases of agricultural importance.     

Evidence of antibacterial and lysozyme-like activity in different planktonic larval stages of Paracentrotus lividus

Larval lysate (four-armed stage) exerts a lysozyme-like and antibacterial activity directed against Vibrio alginolyticus and other bacterial strains. This lysozyme activity is dependent on the pH and ionic strength of the reacting medium and sample. The other larval stages (six-armed, eight-armed and a stage approaching metamorphosis) possess protection against bacteria as well. The highest antibacterial activity is present in the six-armed stage. These antibacterial compounds avoid exploitation by bacteria. Paracentrotus lividus larvae used in our experiments were laboratory-reared from specimens collected in 1993 in Porto Cesareo (Lecce, Italy).     

Effect of neem extract against the bacteria isolated from marine fish

Marine ornamental fishes are exceedingly valuable due to their high demand in domestic and international markets. There is a growing global interest to rear the fishes in captivity. But problem due to bacteria and fungi are the major hitch in captive condition. Since, the use of antibiotics is banned, an attempt was made to ascertain in vitro assay of the neem leaves extract against the bacterial pathogens isolated from infected fishes. Bacterial strains isolated from infected regions of the clown fishes Amphiprion sebae and A. ocellaris were identified as Aeromonas hydrophila, Enterobacter sp., E. coli, Pseudomonas aeruginosa, Proteus sp., Streptococcus sp., Vibrio cholerae, V. alginolyticus, V. parahaemolyticus and Yersinia enterocolitica. Ethanol and methanol extracts were highly inhibitory to the bacterial isolates when compared to other solvents. Ethanol extracts exhibited low minimum inhibitory concentration (75-250 microg ml(-1)) as compared to other extracts. The present finding revealed that the neem leaf extract significantly reduces the bacterial pathogens and their infection in marine ornamental fishes.