Antibody response to acute Pseudomonas aeruginosa infection in a burn wound

Abstract Immunoblotting techniques were used to study the antibody response of a burn patient during the acute phase of a Pseudomonas aeruginosa infection. The results showed the presence in the patient's serum of antibodies to outer-membrane proteins (OMPs) including iron-regulated membrane proteins (IRMPs) of the infecting strain of P. aeruginosa as well as a representative index of Enterobacteriaceae. The patient's serum also contained antibodies which reacted with OMPs of other serotypes of P. aeruginosa .     

Adherence of Pseudomonas aeruginosa to inanimate polymers including biomaterials

Cells of Pseudomonas aeruginosa were adhered to polymethyl methacrylate, polyvinyl acetate, polyvinyl chloride, polyhydroxyethyl methacrylate, mixed-acrylic, silicone, and natural latex materials. Planktonic bacteria and bacteria that adhered to the test materials were compared for their uptake of either L-[3,4,5-³H] leucine or [methyl-³H] thymidine during growth in a minimal medium. Leucine incorporation was reduced and thymidine uptake was negligible in adherent bacteria for up to 8 h following primary attachment by which time cells in the planktonic state showed active uptake of both substrates. These reduced uptake periods correlated with lag phases of growth of adherent cells as determined with a sonication-release plate count procedure and analyses of adenosine triphosphate (ATP). The extent of the lag phase of the adherent populations was dependent on initial densities of adhered cells and the nature of the substratum. Received 02 December 1998/ Accepted in revised form 25 April 1999     

铜绿假单胞菌Arr基因突变对生物膜和绿脓菌素合成的影响

为了研究铜绿假单胞菌Arr基因对生物膜和绿脓菌素合成的影响,采用抗庆大霉素基因序列(Gentamycin resistance cassette,aacC1)插入失活的策略构建了铜绿假单胞菌Arr基因突变株PA-AG,通过96孔板静止培养、结晶紫染色的方法检测其生物膜的形成量,利用抽提的方法检测绿脓菌素的合成量。结果在KMB或LB培养基中,突变株PA-AG形成生物膜的量均有所减少,野生株约是突变株的2倍,然而突变株合成绿脓菌素的能力却明显加强,约为野生株的2.5倍。由此推测,铜绿假单胞菌Arr基因在一定程度上促进了生物膜的形成,抑制了绿脓菌素的合成。     

Saccharides of seven Pseudomonas aeruginosa immunotypes

Abstract The structures of O-specific polysaccharides obtained by mild acid degredation of lipopolysaccharides (LPS) from seven Pseudomonas aeruginosa Fisher's immunotypes have been studied. The polysaccharides consist mainly of monoamino and diamino sugars, frequently also carrying acidic functions. Some of the sugars were detected in nature for the O-specific polysaccharides of the immunotypes 2, 3, 4, 5 and 6 are identical to those of the polysaccharides of the 011; 0(2a)2c; 01; 010a, 10b and 07a, 7d Lányi-Bergan serological subgroups respectively, whereas no analogues have been found for the immunotypes 1 and 7. Some cross-reactions between the LPS of different immunotypes were observed in passive haemagglutination tests; the results of inhibition of passive haemagglutination and agar gel immunoprecipitation point, however, to a specificity of the LPS. Many of the LPS of the seven Pseudomonas aeruginosa immunotypes manifest rather a high cross-protective activity in active immunization tests in mice. The nature of the cross-protective activity of the LPS is discussed.     

Conformation-dependent antibody response to Pseudomonas aeruginosa outer membrane proteins induced by immunization in humans

Outer membrane proteins (OMPs) of pathogenic bacteria have been used as protective antigens in developing bacterial vaccines. In the present study, we compared the antibody responses to a Pseudomonas aeruginosa OMP vaccine elicited in humans and rabbits by immunization. Immunization with the vaccine induced high titers of serum IgG antibody both in rabbits and humans but reactivities of the induced antibodies with the OMPs were different. The rabbit immune sera recognized most of the OMPs in the vaccine both in immunoblot and immunoprecipitation analyses. In contrast, a great variation in band pattern and intensity was observed among the human immune sera in immunoblot analysis, but not in immunoprecipitation analysis. Denaturation of the OMPs did not affect the binding activity of the rabbit immune sera as determined by ELISA, but substantially reduced those of the human immune sera and anti-OMP IgG purified from a pooled normal human plasma. These data suggest that antibody response to P. aeruginosa OMPs elicited by immunization in humans is mainly directed against discontinuous or conformation-dependent epitopes, which should be taken into account in developing vaccines, especially for OMP-derived synthetic peptides.     

临床分离铜绿假单胞菌对氟喹诺酮类抗菌药物的耐药性检测

目的了解临床分离铜绿假单胞菌对喹诺酮类等抗菌药物的耐药性。方法琼脂稀释法测定86株铜绿假单胞菌对5种氟喹诺酮类抗菌药物以及头孢吡肟、美罗培南的耐药性。结果铜绿假单胞菌对诺氟沙星、氧氟沙星、左氧氟沙星、环丙沙星、加替沙星的耐药率分别为50%、61.6%、51.2%、48.8%、51.2%;对头孢吡肟和美罗培南的耐药率分别为30.2%、23.2%。结论铜绿假单胞菌对氟喹诺酮类抗菌药物耐药显著,临床应加强检测和监测。     

Alternative measures of response to Pseudomonas aeruginosa infection in Drosophila melanogaster

Studies of invertebrate immune defence often measure genetic variation either for the fitness cost of infection or for the ability of the host to clear the parasite. These studies assume that variation in measures of resistance is related to variation in fitness costs of infection. To test this assumption, we infected strains of the fruit fly, Drosophila melanogaster, with a pathogenic bacterium. We then measured the correlation between host bacterial load and the ability to survive infection. Despite the presence of genotypic variation for both traits, bacterial load and survival post-infection were not correlated. Our results support previous arguments that individual measures of immune function and the host's ability to survive infection may be decoupled. In light of these results, we suggest that the difference between tolerance and resistance to infection, a distinction commonly found in the plant literature, may also be of value in studies of invertebrate immunity.     

铜绿假单胞菌随机启动子库的构建及受铁调控基因的筛选和鉴定

[目的]针对铁这种几乎所有微生物都必需的,对病原菌尤为重要的营养元素,在全基因组水平上检测铜绿假单胞菌的铁感应基因.[方法]采用含LuxCDABE报道子的质粒pMS402为载体,构建了铜绿假单胞菌的随机启动子库,建立了特定条件下研究铜绿假单胞菌全基因组基因表达变化的高通量平台.[结果]验证了2个已知的受铁调控的基因,并发现了尚未报道的二氢叶酸还原酶,磷酸葡萄糖脱水酶和柠檬酸铁转运蛋白受铁调控的现象,发现与铁相关的四个功能未知的基因和3个假定蛋白的基因.[结论]研究结果对于阐明铁在铜绿假单胞菌中的调控作用,揭示铁对细菌生理及致病性的作用机制提供了理论基础.随机启动子库的构建也为细菌基因组水平研究基因表达提供了一个有用的工具.     

Phytol has antibacterial property by inducing oxidative stress response in Pseudomonas aeruginosa

Phytol, isolated from Aster yomena, is widely distributed as a constituent of chlorophyll. In the present study, we confirmed the antibacterial activity of phytol and its mechanism inducing oxidative cell death in Pseudomonas aeruginosa. In phytol-treated cells, elevated level of intracellular reactive oxygen species (ROS) and transient NADH depletion were observed. These results demonstrated that phytol induced ROS accumulation and that the electron transport chain was involved in increase of ROS. Due to this ROS generation, the imbalance developed between intracellular ROS and the antioxidant defense system, leading to decrease of reduced glutathione (GSH). Moreover, severe DNA damage was shown after treatment with phytol. DNA electrophoresis and a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay were conducted with pretreatment with the antioxidant N-acetylcysteine (NAC) to evaluate the cause of DNA damage. In NAC-pretreated cells, alleviated damage was confirmed and it supports that phytol induces oxidative stress-mediated DNA damage. In conclusion, phytol exerts the antibacterial property via inducing oxidative stress response in P. aeruginosa.     

Constitutive choline transport in Pseudomonas aeruginosa

Pseudomonas aeruginosa has a choline uptake system which is expressed in bacteria grown in the presence of succinate and ammonium chloride as the carbon and nitrogen source, respectively. This system obeys Michaelis-Menten kinetics with an apparent K_m value of 53 μM; its activity is not inhibited by high osmolarities in the medium but is partially inhibited by choline metabolites such as betaine and dimethylglycine.     

铜绿假单胞菌的药敏试验中D-现象的探讨

了解铜绿假单胞菌的药敏试验（K-B法）中,亚胺培南（IPM）诱导哌拉西林（PIP）、哌拉西林/他唑巴坦（TZP）出现D-现象的概率,并对这种现象发生的机制及临床意义进行探讨。采用美国临床实验室标准化研究所（CLSI）推荐的K-B法。其中,将TZP、PIP分别贴在IPM的两侧,纸片中心距离为25 mm,看IPM能否诱导TZP、PIP出现D-现象。结果可见,150株铜绿假单胞菌的药敏试验中,出现D-现象的有120株,占80%;未出现D-现象的有30株,占20%,其中5株耐IPM。铜绿假单胞菌的药敏试验中,IPM诱导TZP、PIP出现D-现象非常普遍,这可能与IPM可诱导AmpC酶的大量产生有关。     

哈尔滨市112株绿脓杆菌的血清学分型和耐药性测定

对从临床分离的112株绿脓杆菌进行系统鉴定后,血清学分型表明:6、2和3型分别占32.14％、15.18％、15.18％,为主要流行型,共占总分离株的62.50％。耐药性测定结果为:对10种抗生素5耐以上者占69.6％。其中对多粘菌素、妥布霉素、丁胺卡那霉素三种抗生素最为敏感,敏感率分别为100％、70.6％、86.5％。     

铜绿假单胞菌产蛋白酶的发酵条件优化

【目的】鉴定一株来源于酱油曲能够分泌蛋白酶的铜绿假单胞菌CAU342A,优化其产蛋白酶的发酵条件。【方法】采用形态学观察、16S r RNA基因序列比对和生理生化方法鉴定菌株CAU342A;通过碳源、氮源、初始pH、温度、表面活性剂及发酵时间的单因素优化和正交试验获得最适发酵条件。【结果】菌株CAU342A被鉴定为铜绿假单胞菌(Pseudomonas aeruginosa),其最适发酵产酶条件为(质量体积比):3%酒糟,1.5%酵母浸提物,0.05%吐温-80,0.5%NaCl,0.7%K_2HPO_4,0.3%KH_2PO_4,0.04%MnSO_4,培养基初始pH 7.5,30°C培养72 h。在最适发酵条件下,该菌株最大产酶水平达到2 653.5 U/m L。蛋白酶酶谱分析表明该菌株能够产生至少4种具有蛋白酶活性的同工酶,其中两个主要酶谱带对应分子量分别为32 k D和50 k D。【结论】铜绿假单胞菌CAU342A高产蛋白酶,具有很大的工业应用潜力。     

细菌群体感应参与铜绿假单胞菌胞内聚羟基脂肪酸酯合成的调控

群体感应是细菌根据细胞密度变化调控基因表达的一种调节机制。铜绿假单胞菌中QS系统由lasI和rhlI合成的信号分子3OC12-HSL和C4-HSL以及各自的受体蛋白LasR、RhlR组成,它们以级联方式调控多个基因表达。【目的】研究细菌群体感应(QS)对聚羟基脂肪酸酯合成的调控。【方法】利用铜绿假单胞菌PAO1及其QS突变株为材料通过气相色谱、荧光定量PCR在生理和分子水平上研究QS对聚羟基脂肪酸酯合成的调控。【结果】QS信号分子合成抑制剂阿奇霉素处理铜绿假单胞菌PAO1和QS突变株导致胞内PHA积累量显著减少;铜绿假单胞菌PAO1中C4-HSL合成酶基因rhlI缺失突变株PAO210胞内PHA积累量与野生型无差别;而3OC12-HSL合成酶基因lasI缺失突变株PAO55、3OC12-HSL受体合成酶基因lasR缺失突变株PAO56以及lasI/lasR双缺失突变株PAO57胞内PHA含量与野生型相比明显减少;lasI和lasR的突变株体内PHA合成酶基因phaC1的表达量显著降低,信号分子3OC12-HSL回补实验使phaC1的表达量可恢复到野生株水平,但只可部分恢复lasI缺失导致的胞内PHA合成。【结论】由此推测,铜绿假单胞菌群体感应系统中lasI/lasR系统参与胞内聚羟基脂肪酸酯合成的调控。     

钌化合物对铜绿假单胞菌的光动力杀伤作用

【摘 要】 目的 探讨光动力疗法（PDT）对体外培养的铜绿假单胞杆菌的杀伤效应。方法 以多重耐药铜绿假单胞杆菌（P. aeruginosa）为对象,用457 nm和532 nm的激光作为光源,对不同浓度钌化合物孵育的细菌悬液进行光照,用菌落计数法观测PDT对铜绿假单胞杆菌的杀伤作用;同时观察了铜绿假单胞菌临床耐药菌与标准菌株对相同PDT作用的敏感性差异。结果 PDT处理组具有剂量－效应关系,在光照剂量相同的情况下,457 nm激光作为光源有更好的PDT杀伤效果;对多重耐药的铜绿假单胞菌,光动力同样有效,其作用甚至强于标准株。结论 钌化合物介导的光动力作用对体外培养的铜绿假单胞杆菌具有很强的杀伤作用,其效果和剂量关系密切。     

Production and characterisation of a biosurfactant isolated from Pseudomonas aeruginosa UW-1

Pseudomonas aeruginosa UW-1 produced 17–24 g L⁻¹ rhamnolipid in vegetable oil-containing media in shake flask cultures in 13 days. In time course studies of growth and rhamnolipid production in a salts medium containing 6% canola oil, total bacterial count reached 2.6 × 10¹⁰ CFU ml⁻¹ after 48 h and a maximum rhamnolipid yield of 24.3 g L⁻¹ was obtained after 9 days. Rhamnolipid components were purified and separated by chloroform-methanol extraction and TLC chromatography. The major rhamnolipid components were characterised as L-rhamnosyl-β-hydroxydecanoyl-β-hydroxydecanoate and L-rhamnosyl-L-rhamnosyl-β-hydroxydecanoyl-β-hydroxydecanoate by nuclear magnetic resonance and mass spectrometry. The components were separated preparatively by silica gel column chromatography. The recovered monorhamnosyl fraction contained no dirhamnosyl moiety while the recovered dirhamnosyl fraction contained 5% of the monorhamnosyl moiety when analyzed by HPLC. The ratio of mono- to dirhamnosyl components produced by P. aeruginosa UW-1 was determined by HPLC to be 4 : 1 by weight. Purified mono- and dirhamnosyl components had the same CMC value of 40 μg ml⁻¹ and decreased the surface tension of water to 27.7 and 30.4 dynes cm⁻¹, respectively. Received 04 April 1997/ Accepted in revised form 15 July 1997     

Purification and characterization of an alkaline protease from Pseudomonas aeruginosa MN1

An alkaline protease produced by Pseudomonas aeruginosa MN1, isolated from an alkaline tannery waste water, was purified and characterized. The enzyme was purified 25-fold by gel filtration and ion exchange chromatography to a specific activity of 82350 U mg⁻¹. The molecular weight of the enzyme was estimated to be 32000 daltons. The optimum pH and temperature for the proteolytic activity were pH 8.00 and 60°C, respectively. Enzyme activity was inhibited by EDTA suggesting that the preparation contains a metalloprotease. Enzyme activity was strongly inhibited by Zn²⁺, Cu²⁺ and Hg²⁺(5 mM), while Ca²⁺ and Mn²⁺ resulted in partial inhibition. The enzyme is different from other Pseudomonas aeruginosa alkaline proteases in its stability at high temperature; it retained more than 90% and 66% of the initial activity after 15 and 120 min incubation at 60°C. Journal of Industrial Microbiology & Biotechnology (2000) 24, 291–295. Received 09 June 1999/ Accepted in revised form 24 January 2000