研究Bragonzi的实验室已经由意大利囊性纤维化基金会（CFaCore）和欧盟-F7-2009-223670。这项工作的一部分进行了在蒸馏器，一个先进的显微镜实验室，并在病理解剖学（圣Raffaele科学研究所）的单位，进行组织病理学鼠标。

1。慢性感染的细菌的准备（三和前两天鼠标挑战） <ol><li>选择合适的P。假单胞菌菌株进行测试。 </li><li>接种P的一菌环绿脓杆菌从-80℃股票文化的胰酶大豆琼脂（TSA）板和孵化在37℃过夜。 </li><li>挑取单个菌落，接种到5ml胰酪胨大豆肉汤（TSB）在15毫升的管理单元皑皑管，并在摇床以200rpm在37℃过夜。 </li></ol> 2。慢性感染的细菌嵌入在琼脂珠（一天前感染） <ol><li>取一小等分试样的细菌过夜培养物，在磷酸盐缓冲盐水（PBS）稀释1:50，测量光密度（OD）在600nm处。 </li><li>通过添加2外径在含20毫升新鲜TSB一个新的单元皑皑管稀释过夜细菌培养。 </li><li>在37℃下约3 -4小时至对数期在摇动培养箱中以200rpm的转速，直到总共10-15外径就达到了。 </li><li>在此期间，准备TSA，使TSB与1.5％琼脂，高压灭菌器，并在水浴中平衡在50℃。在水浴中平衡150毫升preautoclaved重矿物油中的锥瓶中，在50℃。 </li><li>一旦P。假单胞菌达到对数生长期，离心收集细菌细胞在2700×g离心15分钟，在4℃，并弃去上清液。 </li><li>重悬细菌沉淀在1ml无菌PBS和涡流彻底完全重悬沉淀。 </li><li>混合1毫升细菌悬浮液用9ml液体的TSA预平衡在50℃下</li><li>加入10毫升TSA-P。假单胞菌混合物重矿物油（预先加热到50℃），并立即搅拌6分钟，在室温下进行。搅拌必须产生一个可见的涡流在油中。 </li><li>冷却该混合物至4℃，在最低藻搅拌EED为35分钟（ 图1）。 </li><li>在休息的冰琼脂珠油混合物额外20分钟。 </li><li>在4℃转移琼脂珠入50ml Falcon管并离心以2700×g离心15分钟</li><li>准确地除去矿物油和洗涤用无菌PBS 6倍，如在步骤2.11中描述。洗涤三次后，将珠粒能借重力沉淀，而不是使用离心分离机。最后一次洗涤后，悬浮在琼脂珠在20〜30毫升PBS中。 </li><li>走珠（大约0.5毫升）的分装无菌均质。 </li><li>取100微升的均质珠和稀释的900微升无菌PBS。连续稀释1:10下降到10-6。 </li><li>板连续稀释在TSA平板，包括未稀释的样品下降到10 -6孵育板在37℃下</li><li>用倒置光学显微镜在几个领域测量珠径。珠子直径必须是100-200微米之间（ <str翁&gt;图1）。 </li><li>隔夜存放珠子在4°C。 </li></ol><img alt="图1" fo:content-width="6in" fo:src="/files/ftp_upload/51019/51019fig1highres.jpg" src="/files/ftp_upload/51019/51019fig1.jpg" width="500px" /> 图1。琼脂珠的制备和小鼠感染的概述。 体育假单胞菌细胞重新悬浮于1ml的PBS中，并加入到9ml液体TSA（50℃）。将该混合物加到150ml的重矿物油在50℃下在烧瓶中，并在高速搅拌6分钟，在室温下进行。当将烧瓶冷却至4℃，缓慢搅拌35分钟，在琼脂凝固创建珠，和存在于混合物中的细菌被嵌入到琼脂珠。细节包含细菌细胞的琼脂珠的是（A）所示。用无菌PBS以几次洗涤除去矿物油后，在琼脂珠悬浮准备f或接种于小鼠的肺部通过气管内注射（B） <a href="https://www.jove.com/files/ftp_upload/51019/51019fig1highres.jpg" target="_blank">点击此处查看大图</a> 。 3。小鼠挑战与琼脂珠 伦理声明:此协议与实验遵循的圣Raffaele科学研究所动物保健和伦理委员会的指导方针。 <ol><li>计数在TSA板的菌落形成单位数（CFU），以确定在琼脂珠悬浮液的CFU / ml的数量。稀释的琼脂珠用无菌PBS以2-4×10 7 CFU / ml的达到1-2×10 6的最优接种在50μl。 </li><li>麻醉C57Bl/6NCr（20-22克，6〜8周龄）的雄性小鼠用氯胺酮（50毫克/毫升）和甲苯噻嗪（5毫克/毫升）在0.9％NaCl中通过施用0.002毫升/克体重的体积腹腔注射。 注:麻醉被认为是ADEQ审视你们当动物仍然停留静静地，不响应外界刺激，并有恒定的心脏和呼吸速率。 </li><li>鼠标放置在仰卧位。消毒鼠标的大衣用70％乙醇。 </li><li>通过皮肤的垂直切口暴露气管插管和气管，用无菌的，灵活的22政0.9毫米x 25毫米静脉导管，保持注意力，除去stylette而向下移动进入气管。插入导管不宜过深入气管。到达隆突（分叉）之前停止。 </li><li>立即取50微升的琼脂珠悬浮液体积1 ml注射器，并将它附加到导管。轻轻推动注射器的活塞，使小珠被植入到肺。用夹子缝合关闭切口。 </li><li>将动物加热垫，直到完全清醒。 </li></ol> 4。小鼠评价<ol><li>每天观察小鼠的临床症状，包括上衣质量，姿势，下床活动，和水合状态。每日监测体重。那失去≥20％小鼠体重必须安乐死。 </li><li>遵循第5点收集支气管肺泡灌洗液（BAL）和点6或7为肺的收集和总CFU分析，组织学分析，在BAL中，细胞因子分析和总细胞计数方面的炎症反应，而髓过氧化物酶（MPO）活性。 </li></ol> 5。支气管肺泡灌洗液的收集和分析<ol><li>通过CO 2吸入安乐死的小鼠。 </li><li>将鼠标放置在仰卧位。消毒鼠标的大衣用70％乙醇。 </li><li>暴露气管和胸廓的皮肤垂直切口。通过切割膜片暴露肺部。 </li><li>插入缝合线使用镊子气管和下插管气管用无菌的，灵活的22克0.9毫米x 25毫米IV导管。拉动缝合线的两个端部结合的钙theter到气管和打结围绕气管的线程。 </li><li>取1毫升的RPMI 1640中使用1毫升注射器的体积并将其连接到导管。推动注射器的活塞来洗肺和立即回收液体，将其存储在一个15毫升的试管中。 注:如果细胞因子是待分析，加蛋白酶抑制剂，以RPMI 1640。 </li><li>共3毫升的RPMI重复此步骤三次。从现在起，储存在冰BAL液。去对肺的收集和分析步骤6。 注:请注意，并非所有的液体将被检索（毫​​升最大2.8）。 </li><li>对于存在于支气管肺泡灌洗液细菌定量，品尝一小等份（300微升），连续稀释1:10无菌PBS，板在TSA平板孵育37℃过夜。 </li><li>用倒置光学显微镜稀释的BAL液1:2的比例加在Burker细胞计数室图尔克溶液的等分计数细胞总数。 </li><li>离心机的Remaining BAL液在330×g离心8分钟，在4℃下取上清用于ELISA细胞因子分析，在-80°C存放按照步骤,5.10-5 .13为差异细胞计数由细胞离心涂片。 </li><li>如果颗粒是红色的，裂解重悬沉淀在250-300微升RBC裂解缓冲液中的红细胞在超纯蒸馏水1:10稀释3分钟。用2ml的PBS，离心中和在330×g离心8分钟，在4℃下</li><li>弃上清，重悬沉淀在RPMI 10％胎牛血清（FBS）。使用量，将提供1×10 6细胞/ ml时，基于总的细胞计数。 </li><li>放置显微镜载玻片和过滤器插入相应的插槽中朝向细胞离心涂片器的中心的纸板滤波器的细胞离心涂片。吸取150微升每个样品为以300×g离心5分钟的细胞离心涂片和离心机在cytocentrifuge适当的水井。 </li><li>染色载玻片通过使用商业试剂盒Romanowsky染色，根据毫安生产商产生的指示和如先前描述的12。按照步骤,5.14-5 .17为MPO活性分析。 </li><li>离心BAL的剩余量，在380×g离心5分钟，在4℃下弃上清，重悬沉淀中加入250μl的十六烷基氯化铵0.5％的超纯蒸馏水以溶解细胞。该悬浮液可在-20℃下进行测定之前被冻结几天。 </li><li>离心机以16,000×g离心30分钟，在4℃，并用上清液在96孔板中进行MPO分析，加入一式两份的样品，以各需要良好，如果，也适当的样品稀释液。 </li><li>加至孔中的每个样品用等体积的3,3&#39;，5,5&#39; - 四甲基联苯胺（TMB）的底物的过氧化物酶。使反应发生在黑暗中为至少5分钟，直到有一个在颜色没有进一步发展。 </li><li>停止反应，加入2 MH 2 SO 4的一次测量的OD在450nm处。 OD值将直接正比于过氧化物酶的活性。 </li></ol> 6。在肺和细胞因子分析细菌负荷的测量<ol><li>后支气管肺泡灌洗液的收集，从小鼠肺切除，冲洗一下在无菌PBS，单独的叶，把它们放在一个圆底筒用2毫升无菌PBS并储存在冰。 注:如果细胞因子是待分析，以PBS中添加蛋白酶抑制剂的试管中。 </li><li>无菌均质肺，从匀浆取一小等分试样（300微升），连续稀释1:10 PBS中，板在TSA平板孵育在37℃下过夜。请注意，总呼吸道细菌负荷将在BAL液和肺中发现的的CFUs的总和。 </li><li>离心剩余的匀浆以16,000×g离心30分钟，在4℃下取上清用于ELISA细胞因子分析，并储存在-80°C </li></ol> 7。组织学检查 &lt;醇&gt; <li>进行组织学分析，只对其中支气管肺泡灌洗液尚未收集到肺部，以保持肺部的方面和属性。 </li><li>通过CO 2吸入安乐死鼠标。 </li><li>通过皮肤的垂直切口暴露胸廓，通过切割膜片暴露肺部。 </li><li>消费肺，冲洗一下在PBS，分开叶，把它们放在含有5-10毫升10％中性缓冲福尔马林（4％甲醛），并储存在4℃下避光管。 </li><li>在石蜡中嵌入肺，用标准程序。 </li><li>用切片机切成5微米厚的切片。 </li><li>染色玻片用苏木精和曙红染色，并盖上盖玻片的载玻片，根据标准程序。 </li><li>用倒置显微镜明场检查幻灯片和显微镜连接到数码相机采集图像。 </li></ol>

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作者宣称，他们有没有竞争的财务权益。

在P的关键步骤铜绿假单胞菌珠准备和鼠标的挑战，现报道如下。 用于小鼠的挑战的铜绿假单胞菌菌株是至关重要的。死亡率，慢性感染或间隙可能有所不同显著取决于用于挑战的细菌菌株。该P.铜绿假单胞菌 RP73临床菌株优于PAO1参考实验室菌株，因为它导致了一个相对较低的死亡率，更严重的病变，以及更高的慢性感染。在临床前研究的药物测试?...

囊性纤维化（CF）是在囊性纤维化跨膜电导调节器（CFTR）基因突变引起的遗传性疾病。这个基因编码表达于大多数上皮细胞的膜中的氯离子通道。支气管扩张症，粘液堵塞和脑实质的破坏主要是绿脓杆菌引起的感染，逐步导致严重的肺部疾病和死亡率在大多数的CF患者1。了解CF的发病机制和新疗法的进一步发展依赖于动物模型与CF的特征。一些小鼠，转基因的CFTR基因，已经产生，但在这些物种的复述CF状肺病和见于CF患者几个其他器官异常的能力的限制已被广泛记载2。 感染的发展是在CF动物模型的主要挑战之一。文献CL早表明慢性感染持久一个月以上，可以实现仅将小鼠接种菌嵌在固定剂如琼脂，琼脂糖，或海藻藻3-5。这些固定剂提供在微需氧/厌氧条件下，使细菌的生长在小菌落的形状，类似于在CF患者6的粘液的生长。慢性感染的这个模型导致细菌在引起气道炎症和损伤7肺内的持久性。但是，根据所使用的方法，所述细菌菌株并接种在肺剂量，慢性感染小鼠在不同时间点恢复在肺的百分比和细菌负荷可以相差很大。特别是，对于长期慢性感染的主要挑战仍然是低细菌的负担由P.铜绿假单胞菌和鼠周挑战后感染的比例较低，说明THA吨的细菌细胞被宿主逐步清除。通过选择P。铜绿假单胞菌 RP73从CF卡的集合临床分离菌株8，我们成功获得低死亡率，更严重的病变，慢性感染的比例较高与稳定的细菌负荷长达一个月的C57Bl/6NCrl小鼠。 本文详细介绍了嵌入P的方法在铜绿假单胞菌琼脂珠;我们已经感染小鼠气管内滴入，测得的细菌负荷和细胞因子在肺，收集支气管肺泡灌洗液和进行组织学检查。总体而言，这一协议将有助于研究人员在处理上发病8,9极其重要的问题和对体育测试新疗法铜绿假单胞菌慢性感染10,11。

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</table>

long term chronic pseudomonas aeruginosa airway infection in mice

慢性呼吸道感染的小鼠模型是在囊性纤维化（CF）的研究的重要资产，虽然有许多关于模型本身的担忧。炎症和感染的早期阶段，已被广泛研究使用铜绿假单胞菌琼脂珠的小鼠模型，而只有少数报道都集中在体内长期慢性感染。长期慢性感染的主要挑战仍然是低细菌的负担由P.假单胞菌和鼠周激发后感染的比例较低，表明细菌细胞被宿主逐步清除。 本文提出了获得有效的长期慢性感染小鼠的方法。此方法是基于P的嵌入绿脓杆菌临床分离株中的琼脂珠在体外 ，然后在C57Bl/6NCrl小鼠气管内滴注。双侧肺部感染与相关SEVERAL可测量读奏包括体重减轻，死亡率，慢性感染和炎性反应。该P.铜绿假单胞菌 RP73临床菌株优于PAO1参考实验室菌株，因为它导致了一个相对较低的死亡率，更严重的病变，以及更高的慢性感染。 体育铜绿假单胞菌定植可能会持续在肺超过三个月。鼠肺组织病理类似于CF治疗晚期慢性肺疾病。 此小鼠模型中最密切模仿人类疾病的过程中，都对发病机理的研究和对新疗法的评价可以使用。

当协议被正确完成后，P。铜绿假单胞菌琼脂珠将测量100-200微米之间，与倒置光学显微镜吹打少量琼脂珠悬浮在幻灯片中可以观察到。单个细菌细胞是可见的琼脂珠，如在图1中详细示出。 P的选择在琼脂珠制备用于铜绿假单胞菌菌株是至关重要的， 图2和表1示出以下的小鼠与P的感染中的数据获得铜绿假单胞?...

Watch this Scientific Journal Video about Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice at JoVE.com

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

我们所描述的琼脂珠的方法建立持久的长期慢性铜绿假单胞菌呼吸道感染的小鼠模型。

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 ...

long-term-chronic-pseudomonas-aeruginosa-airway-infection-in-mice

Research

JoVE Journal

Immunology and Infection

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

22.3K 次观看.  San Raffaele Scientific Institute. 我们所描述的琼脂珠的方法建立持久的长期慢性铜绿假单胞菌呼吸道感染的小鼠模型。 

视频: Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

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

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Many microbial cells have the ability to form sessile microbial communities defined as biofilms that have altered physiological and pathological properties compared to free living microorganisms. Biofilms in nature are often difficult to investigate and reside under poorly defined conditions1. Using a transparent substratum it is possible to device a system where simple biofilms can be examined in a non-destructive way in real-time: here we demonstrate the assembly and operation of a flow cell model system, for in vitro 3D studies of microbial biofilms generating high reproducibility under well-defined conditions2,3.
The system consists of a flow cell that serves as growth chamber for the biofilm. The flow cell is supplied with nutrients and oxygen from a medium flask via a peristaltic pump and spent medium is collected in a waste container. This construction of the flow system allows a continuous supply of nutrients and administration of e.g. antibiotics with minimal disturbance of the cells grown in the flow chamber. Moreover, the flow conditions within the flow cell allow studies of biofilm exposed to shear stress. A bubble trapping device confines air bubbles from the tubing which otherwise could disrupt the biofilm structure in the flow cell.
The flow cell system is compatible with Confocal Laser Scanning Microscopy (CLSM) and can thereby provide highly detailed 3D information about developing microbial biofilms. Cells in the biofilm can be labeled with fluorescent probes or proteins compatible with CLSM analysis. This enables online visualization and allows investigation of niches in the developing biofilm. Microbial interrelationship, investigation of antimicrobial agents or the expression of specific genes, are of the many experimental setups that can be investigated in the flow cell system.

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Protocol describing the application of a flow cell system for growing and analyzing microbial biofilms for Confocal Laser Scanning Microscopy (CLSM).

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 ...

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 ...

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. ...

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 ...

Culture of Small Colony Variant of Pseudomonas aeruginosa and Quantitation of its Alginate

Pseudomonas aeruginosa, an opportunistic Gram-negative bacterial pathogen, can overproduce an exopolysaccharide alginate resulting in a unique phenotype called mucoidy. Alginate is linked to chronic lung infections resulting in poor prognosis in patients with cystic fibrosis (CF). Understanding the pathways that regulate the production of alginate can aid in the development of novel therapeutic strategies targeting the alginate formation. Another disease-related phenotype is the small colony variant (SCV). SCV is due to the slow growth of bacteria and often associated with increased resistance to antimicrobials. In this paper, we first show a method of culturing a genetically defined form of P. aeruginosa SCV due to pyrimidine biosynthesis mutations. Supplementation of nitrogenous bases, uracil or cytosine, returns the normal growth to these mutants, demonstrating the presence of a salvage pathway that scavenges free bases from the environment. Next, we discuss two methods for the measurement of bacterial alginate. The first method relies on the hydrolysis of the polysaccharide to its uronic acid monomer followed by derivatization with a chromogenic reagent, carbazole, while the second method uses an ELISA based on a commercially available, alginate-specific mAb. Both methods require a standard curve for quantitation. We also show that the immunological method is specific for alginate quantification and may be used for the measurement of alginate in the clinical specimens.

Culture of Small Colony Variant of Pseudomonas aeruginosa and Quantitation of its Alginate

Here, we describe a growth condition to culture the small colony variant of Pseudomonas aeruginosa. We also describe two separate methods for the detection and quantitation of the exopolysaccharide alginate produced by P. aeruginosa using a traditional uronic acid carbazole assay and an alginate-specific monoclonal antibody (mAb) based ELISA.

伪多蒙多尼亚小殖民地文化及其藻酸盐的定量

Pseudomonas aeruginosa, an opportunistic Gram-negative bacterial pathogen, can overproduce an exopolysaccharide alginate resulting in a unique phenotype ...

A Delayed Inoculation Model of Chronic Pseudomonas aeruginosa Wound Infection

Pseudomonas aeruginosa (P. aeruginosa) is a major nosocomial pathogen of increasing relevance to human health and disease, particularly in the setting of chronic wound infections in diabetic and hospitalized patients. There is an urgent need for chronic infection models to aid in the investigation of wound pathogenesis and the development of new therapies against this pathogen. Here, we describe a protocol that uses delayed inoculation 24 hours after full-thickness excisional wounding. The infection of the provisional wound matrix present at this time forestalls either rapid clearance or dissemination of infection and instead establishes chronic infection lasting 7&#8211;10 days without the need for implantation of foreign materials or immune suppression. This protocol mimics a typical temporal course of post-operative infection in humans. The use of a luminescent P. aeruginosa strain (PAO1:lux) allows for quantitative daily assessment of bacterial burden for P. aeruginosa wound infections. This novel model may be a useful tool in the investigation of bacterial pathogenesis and the development of new therapies for chronic P. aeruginosa wound infections.

A Delayed Inoculation Model of Chronic Pseudomonas aeruginosa Wound Infection

We describe a delayed inoculation protocol for generating chronic wound infections in immunocompetent mice.

慢性假单位病伤口感染的延迟接种模型

Pseudomonas aeruginosa (P. aeruginosa) is a major nosocomial pathogen of increasing relevance to human health and disease, particularly in the setting of ...