1。大会的流通池系统所有组件组装流系统包括：高压灭菌管，泡沫陷阱，中/废液瓶，如在图1所示的流细胞。所有这些部件可重复使用实验之间。 <img src="/files/ftp_upload/2383/2383fig1.jpg" alt="图1" /> 图1。流通池的系统设置程序（设置的重要组成部分） 。流电池系统由几部分组成：一个中等瓶，蠕动泵，泡沫陷阱，流通池，废液瓶，以及多样化的油管通过各种连接器相互连接的部分。请提供图符文Lyngklip。 2。大会流通池<ol><li>薄小巷，硅酮胶，用注射器（图3），流动池（图2a）治疗。 </li><li>将盖玻片上的硅胶线（图3）的顶部。玻璃盖玻片作为底层为 P 铜绿而PVC盖玻片申请为S.酵母生物膜。 </li></ol><img src="/files/ftp_upload/2383/2383fig2.jpg" alt="图2" /> 图2示意图流动细胞和泡沫陷阱2。详细说明用于生产一）流动池二）泡沫陷阱，DTU的系统生物学的尺寸。约翰威利父子公司（DTU的系统生物学的前身题为“Biocentrum，在图所示）许可重印<img src="/files/ftp_upload/2383/2383fig3.jpg" alt="图3" /> 图3。插图的硅酮胶玻璃基质的附件中的应用线。表示玻璃盖放置在硅酮胶，将其附加到流通池。 <ol start="3"><li>打开流动池，并放置在一个平坦的表面与盖玻片的一面朝下。按流动池的背面，轻轻推到流动池底部的盖玻片。打开流动池和检查不是由硅酮密封的地区。手柄（活塞）注射器的一部分可以用来作为一种工具，轻轻按下玻璃和流通池。 </li></ol> 3。中等瓶<ol><li>放置在一个中等瓶喂食硅胶管（内径2毫米），并插入另一端的直连。 </li><li>中瓶（介质内容，请参阅“媒体”段） 盖连接器，金属箔和瓶和高压灭菌的培养基。确保供应管固定在中期瓶或虹吸作用的液位以上可能中期瓶空时，高压灭菌。 </li></ol> 4。连接泡沫陷阱，流通池和泵根据图1中的提纲，收集所有管线。除部分通过蠕动泵Marprene管硅胶管。 <ol><li>为了连接所有个别流动商会从中期瓶管，分割成所需数量，在实验中采用的进气口管。使用T -连接器从进料管连接管的理想Marprene泵管（见图1，T型接头）。它通常是一个好主意，让整个系统管和流动细胞相同的顺序，在系统发生故障的情况下的部件，以方便识别。 </li><li>每一个人的喂食管（直径为2毫米）连接，Marprene在使用直连接器的泵管。通过中间的硅胶管（直径1毫米）连接到泡沫陷阱Marprene管（图2b）。确保泵入口连接在泡沫陷阱最高。 </li><li>泡沫陷阱造成的出口管连接到流通池入口（直径1毫米），确保这些管材的长度可以被转移到流通池共聚焦显微镜（通常为1米）的阶段。 </li><li>放置在顶部的泡沫陷阱5毫升注射器。关闭合适的帽子的上衣。 </li><li>对流动细胞插座短，约40毫米片（直径1毫米），管材连接，并使用一个“减少”直连附加所需长度的废物管（直径2毫米）。废物的容器放置在废物管。 </li><li>重要的是，废物的容器必须始终置于在同一水平流细胞，流动细胞水平从未低于。此外，还要确保废物管到底是上述废液的预期水平固定，避免冲洗处理流细胞时，由于虹吸效应。 </li></ol> 5。消毒和洗涤流动系统<ol><li>删除泡沫陷阱帽和70％的乙醇，以保持他们的无菌。 </li><li>运行在最高的泵的转速，以填补与0.5％（V / V）钠水的次氯酸钠系统&lt;/ LI&gt; <li>泡沫陷阱盖放回原处时完全充满泡沫陷阱。 </li><li>点击流细胞流动室，以消除气泡。小心不要破坏脆弱的玻璃盖。 </li><li>允许系统在流速为3 ml / H /通道（0.2 mm / s的线性流速）消毒3-4小时。 </li><li>清洗系统的2-3倍，洗出所有的次氯酸钠。填充和空用无菌水系统。必须清空完全相互间洗泡沫陷阱。这可以通过在空气中抽水，直到泡沫陷阱已清空。排空后，加气站的系统之前，去除泡沫陷阱的上限。更换后泡沫陷阱已经完全充满液体的上限。根据需要重复。 </li><li>无菌水通过该系统运行在一个低流量（1-3毫升/ H /通道）在夜间或进行下一步。 </li><li>中期瓶连接的入口和冲洗系统在低流速（3毫升/小时/通道），在那里将进行实验的温度在夜间语言。注：泡沫陷阱，必须对水排空系统之前，充满中等。 </li></ol> 6。接种流动池<ol><li>从培养过夜，稀释到所需的光密度 （绿脓杆菌如0.001 OD 600nm处和0.1 OD 600nm处酵母）。 </li><li>使用与27G针0.5毫升注射器加载足够的接种，以填补室。 250μL，是足够的流量，在这项工作中指定的尺寸（图2，40毫米x 4毫米× 1毫米）商会。 </li><li>停止蠕动泵。 </li><li>钳断的硅胶管，导致流通池进入系统，以防止流动。 </li><li>用70％乙醇擦拭消毒接种部位上的硅胶管。 </li><li>硅胶管插入针头，并引入流通池入口的提示。慢慢注入接种室（要小心，不要注入气泡）。 </li><li>取出针，并用70％乙醇擦拭注射部位，随后使用硅酮胶在注射部位的孔立即密封。 </li><li>翻转流动池，并让没有流过流通池1小时的微生物坚持到底层。 </li><li>打开流动池，启动介质的泵（3毫升/小时/通道），并采取关闭硅胶管钳。 </li><li>该系统是放在孵化，在37 °的P.彗星铜绿微囊藻和30 ° C的情况下的S.酵母 。 </li><li>生物膜流室，现在可以由CLSM观察。 </li></ol> 7。生物膜显微镜染色<ol><li>做一个适当的染色稀释（如1:1000祥发9住酿酒酵母中的污点） </li><li>停止蠕动泵。 </li><li>钳的硅胶管，导致流动池。 </li><li>用70％乙醇擦拭消毒接种网站上的硅胶管。 </li><li>使用与27G针0.5毫升注射器加载足够的染色溶液，填补了会议厅。 250μL，这里所用的足够的流动商会。 </li><li>硅胶管插入针头，并引入流通池入口的提示。慢慢注入进入会议厅的染色溶液（小心，不要注入气泡）。 </li><li>取出针，并用70％乙醇擦拭注射部位，注射部位立即密封。 </li><li>离开15分钟的流量流通池。 </li><li>起飞钳，并开始流（3毫升/ H /通道） </li><li>数据采集​​与CLSM </li></ol>

<ol>
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我们已经证明了一个流动池的系统，在生物膜调查强大的工具。结合三维成像共聚焦显微镜，该系统已在其他更传统的显微技术分析微生物生物膜方法相比的优势范围。这个系统允许没有扰乱社会生活中的微生物生物膜社区的三维可视化。光镜将不提供有关生物膜龛的详细信息，而电子显微镜提供纳米级分辨率的生物膜，它不会允许活细胞成像。 使用我们先前已经阐明5-8（图4a）几种抗生素敏感，细菌细胞外化合物分布的空间分布的描述的流道系统， 如DNA 9-11和的活动和非活动细胞的分布同一物种内的细菌群落4,6,9（图4c）。我们设想，流通池系统可以被用来研究酵母生物膜方面。这可能是环境因素，如杀菌剂以及识别有关的基因在酵母中生物膜的发展酵母生物膜的时空时空分布。虽然酵母是不知道的活动和非活动的细胞分化成其他生物膜的多样化方面，可从酵母到假菌丝细胞形态的转变和从单倍体二倍体细胞的转移，如研究。 我们已经表明系统符合一些微生物物种，并会与几个染色技术。各种不同的染色探针和荧光蛋白如GFP，使在发展中生物膜的具体利基调查和分析抗菌药物或其他环境因素的影响是一个有效的工具。可以得到的信息是非常详细（图4），在生物膜的功能，可以与计算机程序，如COMSTAT 12,13量化。 总体来说，协议的最关键的环节是事实，即它是一个耗时的过程。这也是一个限制，即细胞能够生长在一个无荧光，透明的表面。由于生物膜的形成是使用共聚焦显微镜，是有限的，以几百微米14的深度，可以调查分析，有进一步的技术在设计中所固有的局限性：高通量筛选系统是不适合作为一个经验丰富的研究员，可以处理在大多数实验中，大约15％的渠道，这反过来可能需要数天准备。然而，抗生素或生物膜的研究被认为有关的突变体，最初可以与其他方法，如结晶紫染色筛选最有趣的候选人之前转移到流通池系统的质量。盖玻璃板非常薄，容易折断，处理系统时，应小心。此外，应检查油管每天一个实验运行期间，可能会出现相当大的“回到增长”只是上游的流量细胞的入口管。从几厘米的硅胶管，进气侧流细胞通过消除这种污染是可以解决的，用无菌技术。 <img src="/files/ftp_upload/2383/2383fig4.jpg" alt="图4" /> 图4 A）4日龄PAO1 -绿色荧光蛋白生物膜粘菌素和碘化丙啶治疗24小时死染色（红染色）B）为期三天的3D演示老体育假单胞菌 PAO1（ 铜绿假单胞菌野生型） -绿色荧光蛋白生物膜6 C）3D一个PAO1图片演示- CFP皮拉突变（蓝色）一个PAO1野生型YFP（黄色）D）5天的老PAO1 -绿色荧光蛋白生物膜作为3D图片发送）26 H S。沾满酵母 （PTR3 CEN.PK背景突变）生物膜与祥发，9月15日 。

<ul><li>泡沫陷阱，聚乙烯（DTU的系统生物学，丹麦技术大学，http://www.csm.bio.dtu.dk/Instrument％20Center/Resources/Biofilm％20Setup.aspx） </li><li>清除聚丙烯塑料连接器和T型连接器（科尔Parmer公司，1 / 8英寸（3.175毫米）和1 / 16英寸（1.588毫米）） </li><li>夹钳</li><li>共聚焦显微镜（蔡司LSM510元） </li><li>盖玻片，玻璃（Knittel格拉泽）50 x 24毫米</li><li>盖玻片盖玻片，PVC（R​​inzl）50 x 24毫米</li><li>流通池，聚乙烯（DTU的系统生物学，丹麦技术大学，http://www.csm.bio.dtu.dk/Instrument％20Center/Resources/Biofilm％20Setup.aspx </li><li>中等瓶（肖特） </li><li>蠕动泵（屈臣氏马洛，205S） </li><li>轧制流系统和泵车</li><li>硅酮胶（3M超级硅酮密封胶清除） </li><li> 5毫升注射器（Terumo公司） </li><li>祥发9（分子探针） </li><li> 0.5毫升的注射器与针头，泰尔茂LU - 100（27G） </li><li>废容器</li></ul>管： <ul><li>有机硅，3毫米外径1毫米内径（奥莱Dich） </li><li>有机硅，4毫米外径2毫米内径（奥莱Dich） </li><li> Marprene，3毫米外径，内径1毫米（屈臣氏，马洛） </li></ul>媒体<table border="0"><tbody><tr><td colspan="2"> 铜绿假单胞菌培养基 </td></tr><tr><td> A10 </td><td> g / L的 </td></tr><tr><td> （NH 4）2 SO 4 </td><td> 2.0 </td></tr><tr><td> NA 2 HPO 4 X 2H 2 O </td><td> 6.0 </td></tr><tr><td> KH 2 PO 4 </td><td> 3.0 </td></tr><tr><td>氯化钠</td><td> 3.0 </td></tr><tr><td>高压灭菌器</td><td></td></tr><tr><td colspan="2"></td></tr><tr><td colspan="2"> FB </td></tr><tr><td>氯化镁2 · 6H 2 O </td><td> 0.20 </td></tr><tr><td> 1毫升1个M 氯化钙 </td><td> 0.01 </td></tr><tr><td colspan="2"> 100μL/ L的微量金属元素（ 绿脓杆菌的生物膜）4 </td></tr><tr><td colspan="2">高压灭菌器</td></tr><tr><td colspan="2">在1:10的比例混合，A10和 FB 。 </td></tr><tr><td colspan="2">碳源添加到所需的浓度。 </td></tr></tbody></table> <table border="0"><tbody><tr><td colspan="2"> 酵母合成完整的（SC）中型 </td></tr><tr><td></td><td> g / L的 </td></tr><tr><td>腺嘌呤硫酸盐</td><td> 0.02 </td></tr><tr><td> L -色氨酸</td><td> 0.02 </td></tr><tr><td> L -组氨酸，盐酸</td><td> 0.02 </td></tr><tr><td> L -精氨酸，盐酸</td><td> 0.04 </td></tr><tr><td> L -蛋氨酸</td><td> 0.02 </td></tr><tr><td> L -酪氨酸</td><td> 0.05 </td></tr><tr><td> L -亮氨酸</td><td> 0.06 </td></tr><tr><td> L -异亮氨酸</td><td> 0.06 </td></tr><tr><td> L -赖氨酸，盐酸</td><td> 0.05 </td></tr><tr><td> L -苯丙氨酸</td><td> 0.05 </td></tr><tr><td> L -天冬氨酸</td><td> 0.10 </td></tr><tr><td> L -谷氨酸</td><td> 0.10 </td></tr><tr><td> L -缬氨酸</td><td> 0.15 </td></tr><tr><td> L -苏氨酸</td><td> 0.20 </td></tr><tr><td> L -丝氨酸</td><td> 0.40 </td></tr><tr><td>酵母尼特rogen基地氨基酸W / O和铵（细菌用） </td><td> 1.6 </td></tr><tr><td>硫酸铵</td><td> 5.0 </td></tr><tr><td>氢氧化钠</td><td> 6.0 </td></tr><tr><td>丁二酸</td><td> 10.0 </td></tr><tr><td>高压灭菌器</td><td></td></tr><tr><td>葡萄糖（灭菌分别） </td><td> 0.20 </td></tr></tbody></table>

pseudomonas aeruginosa and saccharomyces cerevisiae biofilm in flow cells

许多微生物细胞的能力，形成社区定义为改变相比，免费的活体微生物的生理和病理性质生物膜固着微生物。生物膜的性质往往难以进行调查，居住在定义不清的条件1。采用了透明的底层，它是可能的设备，一个简单的生物膜可以在一个非破坏性的方式检查实时系统：在这里我们展示了一个流动的细胞模型系统的装配和操作，在体外三维微生物生物膜研究根据定义良好的条件2,3高重现性。 该系统由一个流单元，可作为生物膜的生长室。通过蠕动泵和流通池提供的营养物质和氧气从中期瓶中花中型物的容器收集。这种流系统的建设使干扰最小流室中生长的细胞的营养物质，如抗生素管理的持续供应。此外，流细胞内的流动条件允许的情况下暴露于剪应力的生物膜研究。泡沫捕集设备的局限，从管，否则可能会破坏在流动细胞的生物膜结构的气泡。 流通池系统，激光共聚焦显微镜（CLSM）兼容，从而可以提供非常详细的有关开发微生物生物膜的三维信息。细胞的生物膜可以兼容CLSM分析与荧光探针或蛋白质的标记。这使得网上可视化，并允许在发展中生物膜的壁龛的调查。微生物的相互关系，调查抗菌剂或特定基因的表达，流电池系统，可在调查的许多实验设置。

Watch this Scientific Journal Video about Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells at JoVE.com

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

协议描述流通池系统中的应用激光共聚焦显微镜（CLSM）为不断发展和分析微生物生物膜。

铜绿假单胞菌和酿酒酵母流细胞生物膜

Many microbial cells have the ability to form sessile microbial communities defined as biofilms that have altered physiological and pathological ...

pseudomonas-aeruginosa-saccharomyces-cerevisiae-biofilm-flow

Research

JoVE Journal

Immunology and Infection

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

36.5K 次观看.  Danish Technical University. 协议描述流通池系统中的应用激光共聚焦显微镜（CLSM）为不断发展和分析微生物生物膜。

视频: Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living surfaces. In this paper, we describe protocols for forming Pseudomonas aeruginosa biofilms on human airway epithelial cells (CFBE cells) grown in culture. In the first method (termed the Static Co-culture Biofilm Model), P. aeruginosa is incubated with CFBE cells grown as confluent monolayers on standard tissue culture plates. Although the bacterium is quite toxic to epithelial cells, the addition of arginine delays the destruction of the monolayer long enough for biofilms to form on the CFBE cells. The second method (termed the Flow Cell Co-culture Biofilm Model), involves adaptation of a biofilm flow cell apparatus, which is often used in biofilm research, to accommodate a glass coverslip supporting a confluent monolayer of CFBE cells. This monolayer is inoculated with P. aeruginosa and a peristaltic pump then flows fresh medium across the cells. In both systems, bacterial biofilms form within 6-8 hours after inoculation. Visualization of the biofilm is enhanced by the use of P. aeruginosa strains constitutively expressing green fluorescent protein (GFP). The Static and Flow Cell Co-culture Biofilm assays are model systems for early P. aeruginosa infection of the Cystic Fibrosis (CF) lung, and these techniques allow different aspects of P. aeruginosa biofilm formation and virulence to be studied, including biofilm cytotoxicity, measurement of biofilm CFU, and staining and visualizing the biofilm.

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

This paper describes different methods of growing Pseudomonas aeruginosa biofilms on cultured human airway epithelial cells. These protocols can be adapted to study different aspects of biofilm formation, including visualization of the biofilm, staining of the biofilm, measuring the colony forming units (CFU) of the biofilm, and studying biofilm cytotoxicity.

共培养模型绿脓杆菌现场人类呼吸道细胞生长的生物膜

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living ...

科研

免疫与感染

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with leukemia, severe burn wounds, or organ transplants1. In patients at high-risk for developing PA bloodstream infections, the gastrointestinal (GI) tract is the main reservoir for colonization2, but the mechanisms by which PA transitions from an asymptomatic colonizing microbe to an invasive, and often deadly, pathogen are unclear. Previously, we performed in vivo transcription profiling experiments by recovering PA mRNA from bacterial cells residing in the cecums of colonized mice 3 in order to identify changes in bacterial gene expression during alterations to the host&#x2019;s immune status.
As with any transcription profiling experiment, the rate-limiting step is in the isolation of sufficient amounts of high quality mRNA. Given the abundance of enzymes, debris, food residues, and particulate matter in the GI tract, the challenge of RNA isolation is daunting. Here, we present a method for reliable and reproducible isolation of bacterial RNA recovered from the murine GI tract. This method utilizes a well-established murine model of PA GI colonization and neutropenia-induced dissemination4. Once GI colonization with PA is confirmed, mice are euthanized and cecal contents are recovered and flash frozen. RNA is then extracted using a combination of mechanical disruption, boiling, phenol/chloroform extractions, DNase treatment, and affinity chromatography. Quantity and purity are confirmed by spectrophotometry (Nanodrop Technologies) and bioanalyzer (Agilent Technologies) (Fig 1). This method of GI microbial RNA isolation can easily be adapted to other bacteria and fungi as well.

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

A reliable method for the RNA isolation of Pseudomonas aeruginosa recovered from murine cecums is described. The RNA recovered is of sufficient quantity and quality for subsequent qPCR, transcription profiling, and RNA Seq experiments. This technique can be adapted for RNA isolation of other intestinal microbes.

RNA的分离绿脓杆菌殖民小鼠胃肠道

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with ...

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

There is growing concern about the relevance of in vitro antimicrobial susceptibility tests when applied to isolates of P. aeruginosa from cystic fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic1. However, during chronic lung infections in CF, P. aeruginosa populations exist in biofilms and there is evidence that the environment is largely microaerophilic2. The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests3.
 Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence4,5,6. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions.
 An ASM assay was developed in a microtitre plate format. P. aeruginosa biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a &gt;128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions.
 The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods3. Several in vitro models have been used previously to study P. aeruginosa biofilms7, 8. However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung9 . In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa2 and affect antibiotic susceptibility10. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility.

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

Current diagnostic antimicrobial susceptibility testing relies on the planktonic growth of isolates in nutrient rich, aerobic conditions. Here, we employ an alternative artificial sputum medium to study antimicrobial susceptibility of Pseudomonas aeruginosa biofilms under both aerobic and microaerophilic conditions more representative of the cystic fibrosis lung.

使用人工痰介质测试抗生素药效试验<em&gt;绿脓杆菌</em&gt;更多相关的肺囊肿性纤维化的条件

There  is growing concern about the relevance of in vitro antimicrobial  susceptibility tests when applied to isolates of P. aeruginosa from  cystic ...

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model itself. Early phases of inflammation and infection have been widely studied by using the Pseudomonas aeruginosa agar-beads mouse model, while only few reports have focused on the long-term chronic infection in vivo. The main challenge for long term chronic infection remains the low bacterial burden by P. aeruginosa and the low percentage of infected mice weeks after challenge, indicating that bacterial cells are progressively cleared by the host.
This paper presents a method for obtaining efficient long-term chronic infection in mice. This method is based on the embedding of the P. aeruginosa clinical strains in the agar-beads in vitro, followed by intratracheal instillation in C57Bl/6NCrl mice. Bilateral lung infection is associated with several measurable read-outs including weight loss, mortality, chronic infection, and inflammatory response. The P. aeruginosa RP73 clinical strain was preferred over the PAO1 reference laboratory strain since it resulted in a comparatively lower mortality, more severe lesions, and higher chronic infection. P. aeruginosa colonization may persist in the lung for over three months. Murine lung pathology resembles that of CF patients with advanced chronic pulmonary disease.
This murine model most closely mimics the course of the human disease and can be used both for studies on the pathogenesis and for the evaluation of novel therapies.

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

We describe the agar-beads method to establish persistent long-term chronic Pseudomonas aeruginosa airway infection in the mouse model.

长期慢性<em&gt;铜绿假单胞菌</em&gt;在呼吸道感染小鼠

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model ...

Identification of Novel Genes Associated with Alginate Production in Pseudomonas aeruginosa Using Mini-himar1 Mariner Transposon-mediated Mutagenesis

Pseudomonas aeruginosa is a Gram-negative, environmental bacterium with versatile metabolic capabilities. P. aeruginosa is an opportunistic bacterial pathogen which establishes chronic pulmonary infections in patients with cystic fibrosis (CF). The overproduction of a capsular polysaccharide called alginate, also known as mucoidy, promotes the formation of mucoid biofilms which are more resistant than planktonic cells to antibiotic chemotherapy and host defenses. Additionally, the conversion from the nonmucoid to mucoid phenotype is a clinical marker for the onset of chronic infection in CF. Alginate overproduction by P. aeruginosa is an endergonic process which heavily taxes cellular energy. Therefore, alginate production is highly regulated in P. aeruginosa. To better understand alginate regulation, we describe a protocol using the mini-himar1 transposon mutagenesis for the identification of novel alginate regulators in a prototypic strain PAO1. The procedure consists of two basic steps. First, we transferred the mini-himar1 transposon (pFAC) from host E. coli SM10/&#955;pir into recipient P. aeruginosa PAO1 via biparental conjugation to create a high-density insertion mutant library, which were selected on Pseudomonas isolation agar plates supplemented with gentamycin. Secondly, we screened and isolated the mucoid colonies to map the insertion site through inverse PCR using DNA primers pointing outward from the gentamycin cassette and DNA sequencing. Using this protocol, we have identified two novel alginate regulators, mucE (PA4033) and kinB (PA5484), in strain PAO1 with a wild-type mucA encoding the anti-sigma factor MucA for the master alginate regulator AlgU (AlgT, &#963;22). This high-throughput mutagenesis protocol can be modified for the identification of other virulence-related genes causing change in colony morphology.

Identification of Novel Genes Associated with Alginate Production in Pseudomonas aeruginosa Using Mini-himar1 Mariner Transposon-mediated Mutagenesis

Here we describe a protocol using the mini-himar1 mariner transposon-mediated mutagenesis for generating a high-density insertion mutant library to screen, isolate and identify novel alginate regulators in the prototypic Pseudomonas aeruginosa strain PAO1.

在伴有海藻酸钠生产新基因的鉴定<em&gt;铜绿假单胞菌</em&gt;使用小型<em&gt; himar1</em&gt;水手转座子介导的诱变

Pseudomonas aeruginosa is a Gram-negative, environmental bacterium with versatile metabolic capabilities. P. aeruginosa is an opportunistic bacterial ...

Pseudomonas aeruginosa Induced Lung Injury Model

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. Here we have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa (P. aeruginosa or PA). Using this model, we were able to show lung inflammation at the early phase of injury. In addition, alveolar epithelial barrier leakiness was observed by analyzing bronchoalveolar lavage (BAL); and alveolar cell death was observed by Tunel assay using tissue prepared from injured lungs. At a later phase following injury, we observed cell proliferation required for the repair process. The injury was resolved 7 days from the initiation of P. aeruginosa injection. This model mimics the sequential course of lung inflammation, injury and repair during pneumonia. This clinically relevant animal model is suitable for studying pathology, mechanism of repair, following acute lung injury, and also can be used to test potential therapeutic agents for this disease.

Pseudomonas aeruginosa Induced Lung Injury Model

We have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa. This model mimics lung injury during pneumonia and is clinically relevant.

绿脓杆菌引起的肺损伤模型

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. ...

Come to the Light Side: In Vivo Monitoring of Pseudomonas aeruginosa Biofilm Infections in Chronic Wounds in a Diabetic Hairless Murine Model

The presence of bacteria as structured biofilms in chronic wounds, especially in diabetic patients, is thought to prevent wound healing and resolution. Chronic mouse wounds models have been used to understand the underlying interactions between the microorganisms and the host. The models developed to date rely on the use of haired animals and terminal collection of wound tissue for determination of viable bacteria. While significant insight has been gained with these models, this experimental procedure requires a large number of animals and sampling is time consuming. We have developed a novel murine model that incorporates several optimal innovations to evaluate biofilm progression in chronic wounds: a) it utilizes hairless mice, eliminating the need for hair removal; b) applies pre-formed biofilms to the wounds allowing for the immediate evaluation of persistence and effect of these communities on host; c) monitors biofilm progression by quantifying light production by a genetically engineered bioluminescent strain of Pseudomonas aeruginosa, allowing real-time monitoring of the infection thus reducing the number of animals required per study. In this model, a single full-depth wound is produced on the back of STZ-induced diabetic hairless mice and inoculated with biofilms of the P. aeruginosa bioluminescent strain Xen 41. Light output from the wounds is recorded daily in an in vivo imaging system, allowing for in vivo and in situ rapid biofilm visualization and localization of biofilm bacteria within the wounds. This novel method is flexible as it can be used to study other microorganisms, including genetically engineered species and multi-species biofilms, and may be of special value in testing anti-biofilm strategies including antimicrobial occlusive dressings.

Come to the Light Side: In Vivo Monitoring of Pseudomonas aeruginosa Biofilm Infections in Chronic Wounds in a Diabetic Hairless Murine Model

Here we describe a novel diabetic murine model utilizing hairless mice for real-time, non-invasive, monitoring of biofilm wound infections of bioluminescent Pseudomonas aeruginosa. This method can be adapted to evaluate infection of other bacterial species and genetically modified microorganisms, including multi-species biofilms, and test the efficacy of antibiofilm strategies.

来到光明的一面: 体内糖尿病无毛小鼠慢性伤口生物膜感染的监测

The presence of bacteria as structured biofilms in chronic wounds, especially in diabetic patients, is thought to prevent wound healing and resolution. ...

Replication of the Ordered, Nonredundant Library of Pseudomonas aeruginosa strain PA14 Transposon Insertion Mutants

Pseudomonas aeruginosa is a phenotypically and genotypically diverse and adaptable Gram-negative bacterium ubiquitous in human environments. P. aeruginosa is able to form biofilms, develop antibiotic resistance, produce virulence factors, and rapidly evolve in the course of a chronic infection. Thus P. aeruginosa can cause both acute and chronic, difficult to treat infections, resulting in significant morbidity in certain patient populations. P. aeruginosa strain PA14 is a human clinical isolate with a conserved genome structure that infects a variety of mammalian and nonvertebrate hosts making PA14 an attractive strain for studying this pathogen. In 2006, a nonredundant transposon insertion mutant library containing 5,459 mutants corresponding to 4,596 predicted PA14 genes was generated. Since then, distribution of the PA14 library has allowed the research community to better understand the function of individual genes and complex pathways of P. aeruginosa. Maintenance of library integrity through the replication process requires proper handling and precise techniques. To that end, this manuscript presents protocols that describe in detail the steps involved in library replication, library quality control and proper storage of individual mutants.

Replication of the Ordered, Nonredundant Library of Pseudomonas aeruginosa strain PA14 Transposon Insertion Mutants

Pseudomonas aeruginosa infection causes significant morbidity in vulnerable hosts. The nonredundant transposon insertion mutant library of P. aeruginosa strain PA14, designated as PA14NR Set, facilitates analysis of gene functionality in numerous processes. Presented here is a protocol to generate high-quality copies of the PA14NR Set mutant library.

复制的有序, 非冗余图书馆的铜绿假单胞菌应变 PA14 座子插入突变体

Pseudomonas aeruginosa is a phenotypically and genotypically diverse and adaptable Gram-negative bacterium ubiquitous in human environments. P. aeruginosa ...

Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa

Patients who survive pneumonia have elevated death rates in the months following hospital discharge. It has been hypothesized that infection of pulmonary tissue during pneumonia results in the production of long-lived cytotoxins that can lead to subsequent end organ failure. We have developed in vitro assays to test the hypothesis that cytotoxins are produced during pulmonary infection. Isolated rat pulmonary endothelial cells and the bacterium Pseudomonas aeruginosa are used as model systems, and the production of cytoxins following infection of the endothelial cells by the bacteria is demonstrated using cell culture followed by direct quantitation using lactate dehydrogenase assays and a novel microscopic method utilizing ImageJ technology. The amyloid nature of these cytotoxins was demonstrated by thioflavin T binding assays and by immunoblotting and immunodepletion using A11 anti-amyloid antibody. Further analyses using immunoblotting demonstrated that oligomeric tau and A&#946; were produced and released by endothelial cells following infection by P. aeruginosa. These methods should be readily adaptable to analyses of human clinical samples.

Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa

Simple methods are described for demonstrating the production of cytotoxic amyloids following infection of pulmonary endothelium by Pseudomonas aeruginosa.

铜绿假单胞菌感染肺内皮细胞后细胞毒性 Amyloids 的检测方法

Patients who survive pneumonia have elevated death rates in the months following hospital discharge. It has been hypothesized that infection of pulmonary ...

Generation of In-Frame Gene Deletion Mutants in Pseudomonas aeruginosa and Testing for Virulence Attenuation in a Simple Mouse Model of Infection

Microorganisms are genetically versatile and diverse and have become a major source of many commercial products and biopharmaceuticals. Though some of these products are naturally produced by the organisms, other products require genetic engineering of the organism to increase the yields of production. Avirulent strains of Escherichia coli have traditionally been the preferred bacterial species for producing biopharmaceuticals; however, some products are difficult for E. coli to produce. Thus, avirulent strains of other bacterial species could provide useful alternatives for production of some commercial products. Pseudomonas aeruginosa is a common and well-studied Gram-negative bacterium that could provide a suitable alternative to E. coli. However, P. aeruginosa is an opportunistic human pathogen. Here, we detail a procedure that can be used to generate nonpathogenic strains of P. aeruginosa through sequential genomic deletions using the pEX100T-NotI plasmid. The main advantage of this method is to produce a marker-free strain. This method may be used to generate highly attenuated P. aeruginosa strains for the production of commercial products, or to design strains for other specific uses. We also describe a simple and reproducible mouse model of bacterial systemic infection via intraperitoneal injection of validated test strains to test the attenuation of the genetically engineered strain in comparison to the FDA-approved BL21 strain of E. coli.

Generation of In-Frame Gene Deletion Mutants in Pseudomonas aeruginosa and Testing for Virulence Attenuation in a Simple Mouse Model of Infection

Here, we describe a simple and reproducible protocol of mouse model of infection to evaluate the attenuation of the genetically modified strains of Pseudomonas aeruginosa in comparison to the United States Food and Drug Administration (FDA)-approved Escherichia coli for commercial applications.

在伪单体中生成帧内基因删除突变体，并在简单小鼠感染模型中测试病毒衰减

Microorganisms are genetically versatile and diverse and have become a major source of many commercial products and biopharmaceuticals. Though some of ...