洋葱伯克霍尔德菌CF_66抗菌物质的分离纯化及性质的研究

洋葱伯克霍尔德菌CF_66能够抑制立枯丝核菌等若干植物病原菌和其它一些真菌的生长。CF_66菌发酵液的粗提液通过Sephadex_75pg、Sephacryl S_100柱层析分离纯化,获得抗菌物质CF66I。此抗菌物质耐热性强,耐碱,但在强酸性条件下不稳定。低浓度有机溶剂的存在有利于抑菌活性的提高。对其结构的研究表明CF66I是以(CH2CH2O)n为主要单元结构并带有酰氨键的化合物。     

洋葱伯克霍尔德菌发酵产抗菌物质CF66I温度控制策略

在3.7 L发酵罐中考察了不同温度23℃～33℃对洋葱伯克霍尔德菌CF-66发酵产抗菌物质CF66I的影响,并对不同温度下的发酵过程进行了动力学特性分析。在此基础之上,提出抗菌物质CF66I合成的分阶段控制策略：发酵过程0～20 h控制温度在30℃,20 h后温度控制在25℃至发酵结束。采用此温度控制策略进行了CF66I的发酵,CF66I的活性达到了3.783 u/mL,比采用单一温度下的最大值提高了26.1%。     

洋葱伯克霍尔德菌（Burkholderia cepacia）CF-66发酵生产新型抗菌物质CF66I培养基的优化

利用基于统计学的实验设计RSM（Response surface methodology）优化了Burkholderia cepacia CF-66产新型抗菌活性物质CF66I的发酵培养基组成。首先,用部分重复因子实验对培养基组分NH4Cl,MgSO4·7H2O,柠檬酸钠及酵母粉浓度对菌株产CF66I的影响进行评价,找出主要影响因子为柠檬酸钠和酵母粉。两者均为正影响,其他组分对CF66I活性的影响不显著。其次用最陡爬坡路径逼近最大响应区域。最后用中心组合设计及响应面分析确定主要影响因子的最佳浓度。菌株在优化培养基中培养较初始培养基CF66I活性提高了约两倍。     

CF-Ⅰ细菌富集铀的研究

研究了 CF—Ⅰ细菌富集溶液中铀。对铀的最大富集量达360mg/g 干菌以上,在较短时间内可吸附溶液中98％的铀;pH、细胞生长状态、细胞膜表面电位等对其富集铀作用影响较大,pb~(2+)等重金属离子能干扰细胞对铀的富集。电镜及能谱结果表明,富集的铀绝大郭分以四价态存在于 CF-Ⅰ细胞膜表面。     

洋葱伯克霍尔德菌CF-66环二肽的分离纯化及抑菌活性研究

植物真菌病害给农业生产带来了巨大损失,因此对高效、低毒、低残留的生物农药的开发迫在眉睫。洋葱伯克霍尔德菌CF-66（Burkholderia cepacia CF-66）对真菌类病原菌具有强烈的抑制作用。其发酵液经减压浓缩和乙酸乙酯萃取得到粗提液,粗提液经反复硅胶柱层析和反相高效液相色谱（RP-HPLC）多步柱层析,首次分离纯化得到一种环二肽——cyclo(Phe-Pro)(cFP)。利用气质联用（GC-MS）系统和HPLC进行定性和定量,结果表明分离纯化物质呈单峰,纯度较高且经标准曲线算出其浓度约为15 mg/ml。MIC值的测定结果表明该物质对立枯丝核菌、黄瓜菌核、玉米弯孢病菌等植物病原菌及冻土毛霉、黄曲霉、米根霉等食品腐败菌均具有较强的抑制作用。经显微镜观察发现,该物质可使丝状真菌菌丝生长异常,菌丝由光滑细长变得粗糙、弯曲、短粗且顶端膨大呈泡状。     

CP7菌株抗菌物质对真菌的活性及其作用机制研究

旨在研究多黏类芽孢杆菌(Paenibacillus polymyxa)CP7菌株分泌的抗菌物质对玉米大斑菌和白色念珠菌的活性作用。结果显示,CP7菌抗菌物质对玉米大斑菌和白色念珠菌的菌丝、孢子生长有强烈的抑制作用,CP7菌抗菌物质处理后,玉米大斑菌的菌丝出现畸形、扭曲,细胞外壁皱缩,原生质发生凝聚,产生空泡,细胞器消溶,核区出现核固缩等现象;白色念珠菌的细胞壁和细胞膜模糊,原生质有凝聚现象,细胞器消溶,细胞核变形。推测CP7菌株抗菌物质是破坏真菌的细胞壁和细胞膜结构,形成通道,引起内容物外渗,破坏细胞内部结构,使原生质凝聚,细胞核固缩,最终使菌体死亡。     

温度对土壤氧化大气CH4的影响

讨论了温度对土壤氧化大气CH4的影响及其机理。当温度较低时土壤也具有一定的氧化大气CH4的能力,两者具有很高的相关关系,但是由于CH4氧化菌对大气CH4具有很强的亲和力以及大气CH4氧化所需活化能较低,因此土壤氧化大气CH4对温度的敏感度远低于产CH4,导致温度系数Q10较小。当大气CH4和O2扩散进入土壤的速率等于土壤中CH4和O2消耗的速率时,大气CH4氧化达到最大值,此时的土壤温度就是CH4氧化的最佳温度。如果温度继续升高并大于最佳温度,由于CH4氧化菌无法与利用O2能力更强的硝化细菌和其它微生物竞争利用土壤空气中有限的O2,使得土壤中CH4氧化菌的繁殖和活性降低。这一作用机理的提出较好地解释了为什么随着温度升高土壤氧化大气CH4能力呈低高低的态势。     

半合成抗生素

在一个新的具有化学治疗作用的抗生素结构被确定时,人们常常企图改造其化学结构,印所谓“半合成”。通常采用合成其衍生物来改进其治疗效果,寻找比原物质抗菌活性强、抗菌谱广、对耐药菌有效、化学性能稳定、毒性及付作用较小、吸收快、血浓高、排泄缓慢、有较长药效以及其他优越药理性能的半合成新化合物。     

铜绿假单胞菌抗菌物质对耐甲氧西林金黄色葡萄球菌的抑菌活性研究

目的观察铜绿假单胞菌抗菌物质对耐甲氧西林金黄色葡萄球菌(methicillin-resistant Staphylococcusaureus,MRSA)的体外抑菌活性。方法用交叉划线接种方法进行铜绿假单胞菌对32株耐甲氧西林金葡菌的体外抗菌活性的测定。结果铜绿假单胞菌抗菌物质对MRSA的体外抑菌活性良好,产生色素的菌株的抗菌活性最好,15株铜绿假单胞菌中,7株产蓝绿色色素的铜绿假单胞菌,对MRSA的抑制率均达到了100%,平均抑菌带的宽度为37.7 mm。结论铜绿假单胞菌抗菌物质对32株MRSA具有较强的抗菌活性,无疑对MRSA感染的抗菌药物研制方面开辟了一条新的途径。这是国内的首次研究报道。     

海洋真菌抗病原菌生物活性物质的研究

对分离自大连海域的海洋生物共附生真菌进行了抗菌活性的初筛与复筛,发现有6株具有抗表皮葡萄球菌和铜绿假单胞菌的活性,发现培养基成分及培养方式对活性菌株抗菌物质的产生有显著影响。菌株11-N2抗菌活性最强,其发酵液抗表皮葡萄球菌的效价相当于为氨苄青霉素11.4 U/mL,抗铜绿假单胞菌的效价相当于氨苄青霉素6.0 U/mL。通过硅胶柱层析对该菌株提取物的抗菌活性成分进行了初步追踪,发现二氯甲烷∶甲醇=15∶1洗脱组分具有最强活性,薄层层析显示该组分主要成分在254 nm紫外光下具有强吸收,薄层显色反应提示其主要成分可能为含胺基的甾体或三萜类、有机胺类、吲哚衍生物类化合物。     

Isolation and characterization of a novel Burkholderia cepacia with strong antifungal activity against Rhizoctonia solani

Strain CF-66 with strong antifungal activity against Rhizoctonia solani was isolated from compost samples. It is clearly demonstrated that strain CF-66 is belonging to Burkholderia cepacia complex by the morphological and biochemical tests and 16S rDNA sequence. The B. cepacia complex consists of a group of bacteria currently organized into nine genomovars, among them genomovar II and genomovar III, contain the highly epidemic strains. However, it was known that strain CF-66 is not a member of genomovar II or III of the B. cepacia complex by species-specific polymerase chain reaction assay. In this study, the antifungal compound CF66I produced by strain CF-66 was purified and characterized. Based on the nuclear magnetic resonance, GC-MS spectral and infrared spectral data, CF66I was confirmed to have amide bonds, α-metyl fatty acid, bromine, and some structural units such as CH₂CH₂O. CF66I is stable to high temperature, proteolytic enzymes, and organic solvents. CF66I inhibit the growth of a variety of plant pathogenic fungi and pathogenic yeast, whereas bacterial cells are unaffected. CF66I mainly reduced hyphal extension rates in a dose-dependent manner and induced severe change in cell morphology that resulted in swelled and formed very short hyphae with multiple branches.     

Multiple effects of a novel compound from Burkholderia cepacia against Candida albicans

A novel compound (named CF66I) produced by Burkholeria cepacia CF-66 strain was investigated for its antifungal activity against Candida albicans. This compound exhibited excellent antifungal activity in a dose- and time-dependent manner. Uptake analysis revealed that the compound preferentially acted against the fungal cell wall, and was also able to enter the cells. Transmission electron microscopy indicated that this compound caused loosening of the cell wall and a significant increase in the cell wall thickness was noted; however, no alterations were observed in the contents of the cell wall components. CF66I probably affected the normal assembly and integration of fungal cell wall components by interrupting the weak interactions between them, such as hydrogen and hydrophobic bonds. Propidium iodide (PI) staining indicated that on exposure to CF66I C. albicans cells became permeable to PI. Marked alterations in lipid and sterol contents were observed, and the major changes were a depletion of total lipids and ergosterol, concomitant with an increase in lanosterol content. These observations suggested that the novel compound CF66I may have considerable potential for development of a new class of antifungal agents.     

Morphological changes of <Emphasis Type="Italic">Fusarium</Emphasis><Emphasis Type="Italic">oxysporum</Emphasis> induced by CF66I,an antifungal compound from <Emphasis Type="Italic">Burkholderia cepacia</Emphasis>

The morphological effects of CF66I, an antifungal compound produced by Burkholderia cepacia, on growing hyphae of Fusarium oxysporum were studied by fluorescence microscopy (FM) and transmission electron microscopy (TEM). At 20 μg/ml, CF66I strongly inhibited growth and induced significant changes of the hyphal morphology. These changes included swelling of hyphae with considerable thickening cell wall and abnormal chitin deposition, which was indicative of the alterations in cell wall structure. Furthermore, fluorescein diacetate (FDA) staining indicated the loss of intracellular esterase activity. CF66I probably inhibits fungal growth by interfering with the cell metabolic pathways. At 120 μg/ml, CF66I killed F. oxysporum (accompanied by propidium iodide permeation, intracellular cytoplasm leakage and crushing of hyphal tips), probably by direct damage to the cell membrane. Thus, there are two different antifungal mechanisms of CF66I, depending on its concentration, and further studies on this compound might be useful for us to develop a new class of antifungal agents.     

Evidence of transmission of Burkholderia cepacia, Burkholderia multivorans and Burkholderia dolosa among persons with cystic fibrosis

Previous studies have identified specific Burkholderia cepacia complex strains that are common to multiple persons with cystic fibrosis (CF). Such so-called epidemic strains have an apparent enhanced capacity for inter-patient spread and reside primarily in Burkholderia cenocepacia (formerly B. cepacia complex genomovar III). We sought to identify strains from B. cepacia complex species other than B. cenocepacia that are similarly shared by multiple CF patients. We performed genotype analysis of 360 recent sputum culture isolates from 360 persons residing in 29 cities by using repetitive extragenic palendromic polymerase chain reaction (rep-PCR) and pulsed field gel electrophoresis. The results indicate that sharing of a common Burkholderia multivorans strain occurs relatively infrequently; however, several small clusters of patients infected with the same strain were identified. A cluster of seven patients infected with the same B. cepacia (genomovar I) strain was found. We also identified a large group of 28 patients receiving care in the same treatment center and infected with the same Burkholderia dolosa strain. These observations suggest that B. cepacia complex strains in species other than B. cenocepacia may be spread among CF patients.     

Biological activity of Burkholderia (Pseudomonas) cepacia lipopolysaccharide

Abstract Burkholderia cepacia has emerged as an important multiresistant pathogen in cystic fibrosis (CF), associated in 20% of colonised patients with a rapid and fatal decline in lung function. Although knowledge of B. cepacia epidemiology has improved, the mechanisms involved in pathogenesis remain obscure. In this study, B. cepacia lipopolysaccharide (LPS) was assessed for endotoxic potential and the capacity to induce tumour necrosis factor (TNF). LPS preparations from clinical and environmental isolates of B. cepacia and from the closely related species Burkholderia gladioli exhibited a higher endotoxic activity and more pronounced cytokine response in vitro compared to preparations from the major CF pathogen Pseudomonas aeruginosa . This study may help to explain the vicious host immune response observed during pulmonary exacerbations in CF patients colonised by B. cepacia and lead to therapeutic advances in clinical management.     

Antifungal activity of a novel compound from Burkholderia cepacia against plant pathogenic fungi

AIMS: To investigate antifungal activity of a novel compound (named as CF66I provisionally) against plant pathogenic fungi, mainly including Fusarium sp., Colletotrichum lindemuthianum, Rhizoctonia solani, etc. METHODS AND RESULTS: Minimal inhibition concentrations (MIC) and minimal fungicidal concentrations (MFC) of CF66I for each fungi were determined using serial broth dilution method. The data demonstrated MIC ranged from 2.5 to 20.0 microg ml(-1) and MFC were shown at levels of < or =7.5 microg ml(-1) except Fusarium sp. With reverse microscopy, profound morphological alterations of fungal cells were observed after exposure to CF66I. Conidiospores were completely inhibited, and protoplasm aggregated to form chalamydospores because of the changes of cell permeability. Some chalamydospores were broken, suggesting the compound probably possessed strong ability of damaging the cell wall. In addition, CF66I was investigated for its antifungal stability against Curvularia lunata. The results showed CF66I kept strong fungi-static activity over-wide pH range (pH 4-9) and temperature range (from -70 to 120 degrees C). CONCLUSIONS: The compound CF66I exhibited strong and stable broad-spectrum antifungal activity, and had a significant fungicidal effect on fungal cells. SIGNIFICANCE AND IMPACT OF THE STUDY: Results from prebiocontrol evaluations performed to date are probably useful in the search for alternative approaches to controlling serious plant pathogens.     

Biocidal activity in plant pathogenic Acidovorax, Burkholderia, Herbaspirillum, Ralstonia and Xanthomonas spp.

Antibacterial and antifungal activity was investigated for strains of Acidovorax spp., Burkholderia spp., Herbaspirillum rubrisubalbicans and Ralstonia solanacearum ; strains representing 118 species and pathovars of Xanthomonas were also tested for phytotoxic capacity. Antibacterial activity was present in all Burkholderia spp. except B. andropogonis , in biovars II and III of R. solanacearum but not in biovars I and IV, and in two strains of Xanthomonas. Little antibacterial activity was recorded for Acidovorax spp. Antifungal activity was expressed by most strains of A. avenae ssp. avenae and A. avenae ssp. cattleyae. Weak or variable antifungal reactions were given by strains of A. avenae ssp. citrulli and no activity was expressed by A. konjaci. Most strains of B. caryophylli, B. cepacia, B. gladioli pv. agaricicola, B. gladioli pv. alliicola, B. gladioli pv. gladioli , B. glumae and B. plantari produced extensive inhibition zones against Rhodotorula mucilaginosa. Strains of H. rubrisubalbicans and R. solanacearum gave negative, weak or variable reactions. Strains of Xanthomonas spp. exhibited no antifungal activity. In all cases antifungal activity was caused by a low molecular weight toxin. Three Xanthomonas strains exhibited phytotoxic activity. The ecological implications of these data are discussed.