红谷霉素对金黄色葡萄球菌抑菌机制的研究

红谷霉素是一种抗细菌的新型抗生素。为探讨红谷霉素对金黄色葡萄球菌的抑菌机制,本文采用浓度为EC50和EC90的红谷霉素对试验菌株进行处理,并设空白对照,测定供试菌株的胞外多糖、细胞膜渗透性和生物大分子(DNA、RNA、蛋白质和胞外酶活性)。结果显示:红谷霉素对金黄色葡萄球菌的抑制作用首先表现在对核酸合成的抑制,由于核酸的合成受阻,引起菌体内的蛋白质和其它的生物大分子的合成受阻,细菌细胞膜通透性改变。透视电镜照片显示:红谷霉素处理后金黄色葡萄球菌菌体内部的原生质体明显变稀。     

化学消毒剂作用机理研究进展

细胞壁的屏障作用是微生物降低对消毒剂敏感性的普遍机制。消毒剂对细胞膜的作用导致膜的通透性增加,胞内物质泄漏,呼吸链被破坏等。一些消毒剂能直接改变、破坏蛋白质和核酸的结构和功能,但核酸和蛋白质的合成过程可能比结构本身对消毒剂更加敏感。流式细胞仪、单细胞凝胶电泳等新技术的应用将使人们能够更深刻地了解消毒剂的作用机理。     

一种源自人α-2-巨球蛋白的抗菌肽A2M3及其对金黄色葡萄球菌的抑菌机制

【目的】在人的鼻腔中鉴定出一种源自α-2-巨球蛋白的抗菌肽(命名为A2M3),并探究其对金黄色葡萄球菌(Staphylococcus aureus)的抑菌作用和机制。【方法】结合生物信息学技术对人类鼻液的质谱结果进行分析,并筛选潜在抗菌肽;通过微量稀释法和平板涂布法分别分析A2M3对金黄色葡萄球菌最低抑菌浓度(minimum inhibitory concentration, MIC)和时间杀伤曲线(time-kill curve);采用透射电镜、碘化丙锭(propidium iodide, PI)摄取实验、流式细胞术和核酸蛋白质泄露实验分析A2M3对金黄色葡萄球菌膜完整性、膜通透性的影响;通过凝胶阻滞实验和荧光光谱实验探究A2M3对金黄色葡萄球菌基因组DNA的影响。【结果】利用生物信息学技术筛选出源自α-2-巨球蛋白的潜在抗菌肽A2M3,其对金黄色葡萄球菌的MIC为125.0 μg/mL,且能在3 h内完全杀灭细菌。A2M3通过增加细胞膜的通透性,促使核酸和蛋白质泄漏,继而穿过细胞膜嵌入DNA的碱基对,影响细菌的基因功能,从而导致菌体死亡。【结论】A2M3对金黄色葡萄球菌的抑菌机制涉及多靶点协同作用,能够改变细菌细胞膜的通透性,影响细菌的基因功能。这一发现揭示了从人体体液中筛选和分离抗菌功能肽的潜在应用价值。     

等离子体射流灭活液体中铜绿假单胞菌的研究

【目的】测定等离子射流对铜绿假单胞菌(Pseudomonas aeruginosa)的灭活效果,探究低温等离子体射流的杀菌机理。【方法】采用平板计数法测定等离子体射流的杀菌效果,荧光显微镜、透射电镜观察等离子体作用后菌体结构的变化,蛋白浓度测定和SDS-PAGE电泳检测菌液上清液中可溶性蛋白的泄漏量。【结果】等离子体射流处理铜绿假单胞菌菌液5 min,杀灭率可达到99.9%以上。透射电镜观察可见细菌菌体结构发生改变,细胞壁、细胞膜损伤破裂,细胞内容物泄露。进一步对处理铜绿假单胞菌上清液中的蛋白质含量变化进行检测,结果显示随着处理时间的增加,上清液中蛋白质含量持续增加,在2 min时达到最大值。【结论】等离子体射流可以通过破坏细胞结构造成细胞质泄露,使其丧失正常的细胞功能,从而达到快速有效地杀灭铜绿假单胞菌的效果。     

海洋芽孢杆菌(Bacillus marinus)B-9987菌株抑制病原真菌机理

[目的]探讨海洋芽孢杆菌(Bacillus marinus)B-9987菌株的代谢产物BMME-1,对植物病原真菌茄链格孢菌的抑菌作用机理.[方法]分别使用分光光法、气相色谱-质谱GC-MS联用技术、红外光谱法等,检测了BMME-1处理病原真菌后,菌体渗透性、细胞壁及细胞膜成份的变化.[结果]BMME-1对茄链格孢菌的抑菌中浓度(MIC_(50))为6.2 mg/L,最小杀菌浓度(MFC)为50 mg/L,在MIC_(50)浓度或高于此浓度处理靶标菌,将导致菌体蛋白质、核酸等大分子物质的外流;处理菌株葡聚糖结构β-型糖苷键、碳-氧键(C-O)、碳-氢键(C-H)等基团的特征吸收强度降低,-OH、C=O的伸缩振动吸收强度升高;菌体细胞壁几丁质结构中酰胺I键吸收强度发生变化;与对照菌株的麦角甾醇含量(62.52±3.31%)相比,处理菌株麦角甾醇减少为(56.36±2.52)%,同时出现麦角固醇合成中间产物粪甾醇.[结论]BMME-1对病原真菌的抑制表现为:干扰细胞膜麦角甾醇的合成从而改变了细胞的通透性;对细胞壁葡聚糖结构的影响较大而几丁质次之.     

超高压对单增李斯特菌细胞膜的损伤和致死机理

【目的】研究超高压对病原微生物单增李斯特菌细胞膜损伤的影响。【方法】本文以单增李斯特菌为研究对象,探讨了不同压力处理(100-500 MPa)对单增李斯特菌的灭活作用,利用透射电镜观察高压处理对细菌细胞超微结构的影响,通过紫外分光光度法、原子吸收分光光度法和荧光分光光度法测定高压处理对细菌细胞膜通透性的影响,采用超微量Na+/K+-ATP酶试剂盒测定高压处理对细菌细胞膜Na+/K+-ATP酶活力的影响。【结果】25℃经300、350、400 MPa压力处理15 min后,单增李斯特菌总数由9.00分别降至5.20、3.27、1.35个对数单位,经450MPa及以上的压力处理后,单增李斯特菌的致死率达到100%。超高压处理对单增李斯特菌的细胞超微结构造成明显的损伤,细胞结构不完整,细胞壁局部被破坏,细胞膜通透性增大,细胞内物质聚合,出现透电子区。由于细胞膜的损伤使得细胞内无机盐离子、紫外吸收物质流出,细胞膜上的Na+/K+-ATPase失活。【结论】超高压处理造成单增李斯特菌细胞形态结构明显损伤,改变细胞膜的通透性,降低细胞膜上Na+/K+-ATP酶活力,最终使得细胞内无机盐离子和胞内大分子物质外流而死亡。     

微波杀菌过程中大肠杆菌与金黄色葡萄球菌细胞膜通透性的改变

对细胞膜通透性变化的研究是认识微波杀菌机理的途径之一。用荧光探针检测微波处理后细胞内Ca2 浓度的变化,可以精确地表征细胞膜通透性的改变。选用二乙酸荧光素(FDA)和Fluo-3/AM两种荧光染料,对大肠杆菌(Escherichiacoli)和金黄色葡萄球菌(Staphylococcus aureus)经微波处理后的酯酶活性及细胞膜通透性进行研究,结果表明大肠杆菌与金黄色葡萄球菌的胞内非特异性酯酶(NSE)活性及细胞膜通透性的变化情形有所不同。在50℃、55℃、60℃和65℃微波处理条件下,大肠杆菌细胞膜通透性分别增加了20.7%、28.1%、74.8%、89.8%,而金黄色葡萄球的增加不显著,分别比对照组提高了4.1%、6.0%、21.9%和19.7%。细胞膜通透性的改变与微生物致死率有一定的相关性,也可能是微波杀菌非热效应的表现之一。     

玫瑰茄醇提物的抑菌活性及其作用机制

玫瑰茄具有多种药理活性,包括抗肿瘤、抗氧化和抑菌作用等,但目前关于玫瑰茄的抑菌作用研究较少,有关抑菌机制方面的研究尚未见报道. 本文通过测定玫瑰茄醇提物对大肠杆菌和金黄色葡萄球菌细胞膜、蛋白质和核酸的影响,及其与DNA的作用方式等,系统阐述玫瑰茄的抑菌作用机制. 电导率和大分子物质的测定结果显示,玫瑰茄醇提物只对菌体的细胞膜造成微小损伤,其抑菌作用的靶点不在细胞膜. SDS-PAGE和DAPI结果显示,玫瑰茄醇提物可抑制大肠杆菌和金黄色葡萄球菌蛋白质和核酸的合成. 琼脂糖凝胶电泳和紫外吸收光谱结果显示,玫瑰茄醇提物可与DNA结合,当DNA与药物的浓度比较低时,玫瑰茄醇提物与DNA以嵌入结合为主,当二者的浓度较高时,两者间发生的是氢键结合. 上述结果证明,玫瑰茄醇提物对大肠杆菌和金黄色葡萄球菌抑菌机制,主要是药物通过与DNA发生嵌入结合和氢键结合,使DNA不能进行正常的复制和转录,降低核酸的含量,进而影响蛋白质的合成,最终导致菌体生物学功能的丧失.     

载铜蒙脱石的抗菌机理研究

研究载铜蒙脱石对副溶血弧菌的抗菌效果和机理。载铜蒙脱石对副溶血弧菌的最小抑菌浓度和最小杀菌浓度分别为75mg/L和300mg/L,而蒙脱石未显抗菌活性。细菌胞内酶活性结果显示,载铜蒙脱石可使细菌细胞膜发生损伤或通透性增大,导致细菌胞内酶的外漏。载铜蒙脱石对细菌呼吸代谢影响的结果表明,载铜蒙脱石可抑制三羧酸循环途径。原子力显微镜动态观察载铜蒙脱石作用下副溶血弧菌形态变化的结果发现,载铜蒙脱石能破坏细菌细胞膜。     

茶多酚对金黄色葡萄球菌和铜绿假单胞菌的抑菌机理

以革兰氏阳性的金黄色葡萄球菌和革兰氏阴性的铜绿假单胞菌为试验菌,通过测定茶多酚与两种菌作用前后细菌培养液的电导率和可溶性总糖的变化,以及菌体在磷代谢和蛋白质表达方面的变化,初步阐明了茶多酚对这两种菌的抑菌机理。研究结果表明,茶多酚对金黄色葡萄球菌和铜绿假单胞菌均有抑菌活性,但对金黄色葡萄球菌的抑菌活性更强。经茶多酚处理后,细菌培养液的电导率和总糖浓度均增大,表明了茶多酚可破坏细胞膜的结构、导致细胞通透性增加,进而使细胞内容物外泄。另一方面,经茶多酚处理后的两种菌对磷的消耗量降低,以致严重影响了核酸、磷脂等细胞重要成分的合成以及能量代谢;通过SDS-PAGE分析,证实茶多酚可以阻碍细菌蛋白质的正常表达,以致影响其细胞的结构组成以及酶的催化活性,最终导致细菌正常生理功能的丧失。     

Sensitivity of Escherichia coli cell membranes to various classes of detergents

The mechanisms of interaction between non-ionic or cationic surfactants with Escherichia coli K-12 cell membranes were studied using an approach based on the registration of changes in the membrane permeability to ethidium bromide, a fluorescent dye for nucleic acids. Triton X-100, a non-ionic detergent, was shown to exert no effect on the permeability of intact cell membranes. Triton X-100 interacted with the bacteria only after treatment with EDTA, a complexing agent for bivalent cations. Cetyltrimethyl ammonium bromide increased the permeability to ethidium bromide and the action of this cationic detergent did not require the pretreatment with the complexing agent. SDS, an anionic detergent, damaged E. coli K-12 and this could be registered by the lowering of intensity of light scattering by the bacterial suspension. The surface charge of E. coli K-12 cells was shown to influence the interaction of ionic detergents with bacterial cell membranes. Its variation by changing the pH of the incubation medium did not make E. coli K-12 sensitive to Triton X-100.     

Mode of antibacterial action by gramicidin S

To elucidate the mode of antibacterial action by gramicidin S (GS), a detailed experiment on GS distribution on bacteria cells was carried out. 14C-Labeled gramicidin S ([14C]GS) was incubated with cells of Gram-positive Bacillus subtilis and Gram-negative Escherichia coli, and the amount of [14C]GS adsorbed on the cells was measured. Adsorption on B. subtilis cells was observed from 1 microgram/ml of [14C]GS. As the concentration of [14C]GS increased, the amount adsorbed on B. subtilis increased discontinuously, producing a curve which had three plateaus. On the other hand, [14C]GS was not easily adsorbed on E. coli cells at lower concentrations, but the amount adsorbed increased above 6 micrograms/ml, and the cells were temporarily saturated with GS at 10 micrograms/ml, which is the minimum inhibitory concentration for E. coli. The amount of [14C]GS adsorbed on the protoplast membrane of B. subtilis was the same as that of natural cells. However, the amount of [14C]GS adsorbed on the cell wall dropped to about 20% of that of natural bacteria. These facts indicate that GS is adsorbed on the cell membrane of bacteria particularly. The uptake of amino acid or glucose in B. subtilis was inhibited by GS. Therefore, it is concluded that GS damages the phospholipid bilayer of the cell membrane by adsorption, and prevents the functioning of the cell membrane. The amount of [14C]GS adsorbed on the spheroplast membrane of E. coli increased remarkably as compared with natural cells, even at a lower concentration of GS. The poor GS adsorption on E. coli cells may be due to the permeability barrier of the E. coli cell wall.     

Genes 1.2 and 10 of bacteriophages T3 and T7 determine the permeability lesions observed in infected cells of Escherichia coli expressing the F plasmid gene pifA.

Infections of F plasmid-containing strains of Escherichia coli by bacteriophage T7 result in membrane damage that allows nucleotides to exude from the infected cell into the culture medium. Only pifA of the F pif operon is necessary for "leakiness" of the T7-infected cell. Expression of either T7 gene 1.2 or gene 10 is sufficient to cause leakiness, since infections by phage containing null mutations in both of these genes do not result in permeability changes of the F-containing cell. Even in the absence of phage infection, expression from plasmids of either gene 1.2 or 10 can cause permeability changes, particularly of F plasmid-containing cells. In contrast, gene 1.2 of the related bacteriophage T3 prevents leakiness of the infected cell. In the absence of T3 gene 1.2 function, expression of gene 10 causes membrane damage that allows nucleotides to leak from the cell. Genes 1.2 and 10 of both T3 and T7 are the two genes involved in determining resistance or sensitivity to F exclusion; F exclusion and leakiness of the phage-infected cell are therefore closely related phenomena. However, since leakiness of the infected cell does not necessarily result in phage exclusion, it cannot be used as a predictor of an abortive infection.     

Escherichia coli inactivation mechanism by pressurized CO2

The effects of pressurized CO2 on the survival of Escherichia coli and the mechanism of cell inactivation were studied. Bacterial cultures were inoculated in nutrient broth and incubated at 30 degrees C for 18 h. Exposure of the cells to CO2 under pressures ranging from 2.5 to 25 MPa and at temperatures between 8 and 40 degrees C was performed in a double-walled reactor with a 1 L capacity. The effect of the treatment on the cells was evaluated by plating and by transmission and scanning electron microscopy observation. Vapour CO2 generated a bacteriostatic effect. In liquid or supercritical state, CO2 provided a bactericidal effect. The bactericidal effect increased with pressure and temperature. The mechanism of cell inactivation by liquid CO2 involved two stages. First, cell stress caused by the CO2 penetration provoked cell wall collapse and cellular content precipitation. Second, the cell death caused by supercritical extraction of intracellular substances and cell envelope perforation resulted in leaking of intracellular constituents. In supercritical conditions, the cell inactivation process had one single phase: cellular death.     

3种蒙药复方对致病性Escherichia coli O1的抑菌机理及对小鼠的保护率

【目的】研究3种蒙药复方对致病性Escherichia coli O1的抑菌机理及对感染E.coli O1小鼠的保护率。【方法】分别采用酶标比浊法、微生物粘附法、试剂盒法和邻硝基苯β-D-半乳吡喃糖苷(ONPG)法测定3种蒙药复方对E.coli O1生长曲线、细胞表面疏水性、细胞壁及细胞膜渗透性的影响。同时,采用人工感染小鼠法测定其对小鼠的保护率。【结果】3种蒙药复方均能不同程度地抑制致病性E.coli O1,且对人工感染E.coli O1的小鼠有保护作用,其中蒙药复方Ⅲ的保护率与环丙沙星相当,均达到50%;7味单药组成的蒙药复方Ⅲ水煎剂作用于E.coli O1细胞时,首先改变其表面疏水性,其次随作用时间的延长破坏其细胞壁的完整性,进而破坏细胞膜并改变其通透性,最终导致细胞结构的破坏,从而起到抑制作用。【结论】蒙药复方Ⅲ的体内外抑菌效果最佳,其效果与抗生素环丙沙星相当。     

Expressing an RbcS Antisense Gene in Transgenic Flaveria bidentis Leads to an Increased Quantum Requirement for CO2 Fixed in Photosystems I and II

It was previously shown with concurrent measurements of gas exchange and carbon isotope discrimination that the reduction of ribulose-1,5-bisphosphate carboxylase/oxygenase by an antisense gene construct in transgenic Flaveria bidentis (a C4 species) leads to reduced CO2 assimilation rates, increased bundle-sheath CO2 concentration, and leakiness (defined as the ratio of CO2 leakage to the rate of C4 acid decarboxylation; S. von Caemmerer, A. Millegate, G.D. Farquhar, R.T. Furbank [1997] Plant Physiol 113: 469-477). Increased leakiness in the transformants should result in an increased ATP requirement per mole of CO2 fixed and a change in the ATP-to-NADPH demand. To investigate this, we compared measurements of the quantum yield of photosystem I and II ([phi]PSI and [phi]PSII) with the quantum yield of CO2 fixation ([phi]CO2) in control and transgenic F. bidentis plants in various conditions. Both [phi]PSI/[phi]CO2 and [phi]PSII/[phi]CO2 increased with a decrease in ribulose-1,5-bisphosphate carboxylase/oxygenase content, confirming an increase in leakiness. In the wild type the ratio of [phi]PSI to [phi]PSII was constant at different irradiances but increased with irradiance in the transformants, suggesting that cyclic electron transport may be higher in the transformants. To evaluate the relative contribution of cyclic or linear electron transport to extra ATP generation, we developed a model that links leakiness, ATP/NADP requirements, and quantum yields. Despite some uncertainties in the light distribution between photosystem I and II, we conclude from the increase of [phi]PSII/[phi]CO2 in the transformants that cyclic electron transport is not solely responsible for ATP generation without NADPH production.     

Reduced outer membrane permeability of Escherichia coli O157:H7: suggested role of modified outer membrane porins and theoretical function in resistance to antimicrobial agents

Outer membrane permeability of Escherichia coli O157:H7 was determined by an in vivo kinetic model with the periplasmic enzyme alkaline phosphatase [Martinez et al. (1996) Biochemistry 35, 1179-1186]. p-Nitrophenyl phosphate (PNPP) substrate, added to intact bacteria, must diffuse through the outer membrane to reach the enzyme. At low substrate concentration the bacterium was in the perfectly reactive state where all molecules that entered the periplasm were captured and converted to product. Transmembrane diffusion was rate limiting, and the permeability of the outer membrane was determined from kinetic properties. The O157:H7 strain grown at 30 degrees C showed one-sixth the permeability of wild-type E. coli grown at 30 degrees C. Wild-type bacteria grown at >/=37 degrees C show a physiological response with a shift in expression of outer membrane porins that lowered permeability to PNPP by approximately 70%. The O157:H7 strain did not display this temperature-sensitive shift in permeability even though a change in porin expression could be visualized by staining intensity of Omp F and Omp C on acrylamide gels. Altered behavior of the O157:H7 membrane was also indicated by a several thousand-fold lower response to transformation relative to wild-type E. coli. Matrix-assisted laser desorption ionization time of flight mass spectrometry and electrospray ionization mass spectrometry confirmed the expression of the Omp F and Omp C variants that are unique to E. coli O157:H7. This reduced outer membrane permeability can contribute to enhanced resistance of O157:H7 to antimicrobial agents.     

鲎源抗菌肽对大肠杆菌的抑杀机理

【目的】研究鲎源抗菌肽鲎素对大肠杆菌抑杀的作用机理,为防治大肠杆菌引起的肠道疾病提供新的潜在抗菌药物。【方法】利用牛津杯法和微量MH肉汤稀释法测定其抗菌活性,激光共聚焦扫描显微镜观察鲎素对大肠杆菌的结合、分布及杀伤过程,透射电镜观察鲎素对大肠杆菌超微结构的影响,并采用琼脂糖凝胶阻滞电泳研究其对大肠杆菌基因组DNA和RNA的影响。【结果】鲎素对大肠杆菌的最小抑菌浓度与最低杀菌浓度分别为5 mg/L和20 mg/L;激光共聚焦扫描显微镜和透射电镜观察发现鲎素能快速作用于细胞表面,并发生聚集现象,随着作用时间的延长能导致细胞膜结构的破坏和细胞内含物的释放;通过琼脂糖电泳结果显示鲎素也能够作用于大肠杆菌基因组DNA,并呈浓度依赖关系,10 mg/L鲎素对基因组DNA无明显影响,80 mg/L鲎素能导致DNA断裂;凝胶阻滞电泳显示鲎素也能与基因组DNA和RNA发生结合。【结论】研究结果为深入探讨鲎素抑杀大肠杆菌的分子机制提供重要的理论依据。     

抗辐射菌属-大肠杆菌间的穿梭载体的构建及荧光基因的表达

摘要：【目的】构建抗辐射菌属一大肠杆菌间的穿梭载体,通过此载体使荧光素酶基因在大肠杆菌中得到表达。【方法】以质粒pUE30、pGBM5及pKatCAT为基础,构建抗辐射菌属一大肠杆菌间的穿梭载体,将groEL启动子和荧光素酶基因lux+插入到构建的穿梭载体中得到穿梭表达载体,并将该载体转化大肠杆菌诱导荧光素酶基因的表达。【结果】成功构建了大小约为5.8 kb的抗辐射菌属一大肠杆菌间的穿梭载体pZT17,该载体在没有抗生素的非选择性培养基中能稳定存在。在穿梭载体pZT17的EcoRV部位插入含有groEL启动子和荧光素酶基因lux+的DNA片段,构建得到了穿梭表达载体pZTGL2;利用该表达载体在大肠杆菌中可诱导表达荧光素酶基因。【结论】构建的穿梭表达载体为以后用大肠杆菌高效表达来源于抗辐射菌的基因、特别是DNA损伤修复蛋白基因,提供了可能。     

pagP基因对禽致病性大肠杆菌抗菌肽抗性和致病性的影响

【目的】禽致病性大肠杆菌(Avian pathogenic Escherichia coli,APEC)不仅严重影响全球的养禽业,对人类公共健康也造成巨大的潜在威胁。pag P基因在细菌的抗菌肽抗性和致病性方面发挥重要作用,但关于pag P基因在APEC中的功能尚不清楚。本文构建禽致病性大肠杆菌pag P基因缺失株,对缺失株的抗菌肽抗性和致病性进行研究。【方法】利用Red重组系统构建APEC的pag P基因缺失株,然后利用回复质粒构建回复株。研究pag P基因对细胞黏附与入侵、生物被膜形成能力、外膜渗透性、抗菌肽敏感性、致病性等方面的影响。【结果】成功构建pag P基因缺失株和回复株,抗菌肽抗性试验发现pag P基因缺失株对多粘菌素B、鸡β-防御素2(AVBD2)的敏感性显著增加(P0.01),致病性试验结果表明pag P基因缺失株的毒力显著降低(P0.01)。【结论】APEC的pag P基因对AVBD2的敏感性和APEC的致病性密切相关,为深入研究pag P基因的功能及调控作用奠定了基础。