This work was supported by National Institutes of Health grants HL105947-01 (YL), HL07829-16 (AM), HL090152 (AM).

在动物实验中批准了伊利诺伊大学芝加哥分校的动物护理委员会和机构生物安全委员会。 注意：口罩，保护眼睛，礼服或连身衣，和双层手套：所有涉及假单胞菌程序应与生物安全2级（BSL2）的做法，其中包括但不限于执行。在认证的生物安全柜的工作。与细菌漂白粉或二氧化氯基消毒剂接触治疗手段。用密封箱运输样品。 1. P.铜绿假单胞菌培养与成长<ol><li>商店P.绿脓杆菌 PA103作为细菌的库存在螺旋盖冷冻管在-80℃下。 </li><li>放置在一个架子的小瓶中，用70％乙醇润湿的纸巾的盒子，并采取生物安全等级2（BSL2）实验室。 </li><li>条纹的细菌到绵羊血琼脂平板上并生长在37℃〜15小时，在培养箱中。 </li&gt; <li>在一个BSL2罩，从刮片细菌环和悬浮的细菌，5毫升的PBS。 </li><li>存储库存在4℃下进行长达3个月。然而，确定的效价，每2个星期。 </li><li>串行（1:10 4，通常1:10 7）稀释细菌在PBS和板出来羊血琼脂平板上已知的稀释。 </li><li>孵育板为〜15小时。菌落计数并计算菌落形成单位（CFU），以确定细菌浓度。 </li><li>重悬在合适的浓度（〜5×10 3 CFU /微升）在0.5ml PBS中于1.5ml无菌螺旋盖离心管。密封冷冻管，并放置在消毒上载货纸巾在瞬间盖盒一离心管架。 </li><li>运输箱，以BSL2动物设施。一旦出现，打开了BSL2柜箱。 </li></ol> 2. P.铜绿假单胞菌灌输<ol><li>进行生存无菌手术（无菌手套，无菌器械和无菌技术）。使用无菌的悬垂性，为无菌（蒸压）手术器械提供了工作面。使用消毒每气管滴入程序之间的热珠灭菌仪器。 </li><li>前麻醉小鼠的体重。麻醉小鼠用氯胺酮（100毫克/千克），甲苯噻嗪（5毫克/千克），在0.1 - 0.2毫升的PBS腹膜内（IP）的 。非响应性脚趾捏确定麻醉剂的效力。使用对眼睛的兽医药膏，以防止在麻醉下干燥。 </li><li>抑制小鼠在BSL2柜外科板。 </li><li>识别切割下来的区域。剃此区域，并准备利用交替醇和聚维酮碘拭子3倍的皮肤。 </li><li>治疗与局部麻醉剂（利多卡因），为这个麻醉足以进行小手术如皮肤切口进入气管切口的区域。 </li><li>作一个小切口（约5mm），在颈部的中线，并用钝˚Forceps轻轻移动肌肉访问气管。暴露气管通过手术剥离。 </li><li>吸取菌液到1毫升一次性注射器用27G的针头。对于每个小鼠，给药20-30微升的细菌在适当的浓度（高达10 5 CFU每只小鼠）。 </li><li>将针头插入气管。慢慢注入溶液到气管。 </li><li>确保动物喘气，这通常表示该解决方案已经达到了进入肺部。 </li><li>关闭无菌条件下无菌缝合线（6-0单丝）的伤口。 </li><li>用0.1毫克/千克丁丙诺啡皮下注射作为术后镇痛控制手术后疼痛。 </li><li>确保从初始麻醉诱导至切口闭合所需的时间小于15分钟。 </li><li>注射后，在适当的生物危害的尖锐物容器处置注射器和针头。 </li><li>治疗与二氧化氯基消毒药剂的外科手术器械nfectant 15分钟，冲洗，并返回到实验室进行灭菌，并根据需要重新使用。 </li><li>房子小鼠单独在清洁笼子里的温暖的环境。 </li><li>检查动物每隔30分钟，直到它恢复意识，并开始移动;后来，在12小时的时间间隔为第3天手术后。 </li><li>保持在整个实验动物BSL2设施的小鼠。确保只有包含在密封的盒子组织离开动物BSL2设施。如果小鼠表达任何垂死的行为（定义为呼吸困难，嗜睡，故障响应温柔的刺 ​​激走动），在该研究中的任何一点，从颈椎脱位瓶装源安乐死的小鼠通过CO 2吸入。 </li><li>安乐死放血在麻醉状态下的实验对象。 <ol><li>从安乐死的小鼠收集肺组织。进行在一个BSL2引擎盖这些过程。如果需要，可以使用密封管放入外消旋转移样品钾对载货消毒纸巾在进一步处理易如反掌盖盒。 </li></ol></li></ol>

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	<li>Ware, L. B., Matthay, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+acute+respiratory+distress+syndrome.">The acute respiratory distress syndrome.</a> N Engl J Med. 342, 1334-1349 (2000).</li><li>Matthay, M. A., Ware, L. B., Zimmerman, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+acute+respiratory+distress+syndrome.">The acute respiratory distress syndrome.</a> J Clin Invest. 122, 2731-2740 (2012).</li><li>Shimabukuro, D. W., Sawa, T., Gropper, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Injury+and+repair+in+lung+and+airways.">Injury and repair in lung and airways.</a> Crit Care Med. 31 (8), 524-531 (2003).</li><li>Wansleeben, C., Barkauskas, C. E., Rock, J. R., Hogan, B. 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The authors have nothing to disclose.

我们在这里描述的假小鼠肺损伤模型模拟炎症，肺损伤，修复和解决发生的急性肺损伤或肺炎的全过程。它具有与其他几个损伤模型中，这是临床相关的和相对安全的和易于处理的比较独特的优势。 在该过程的关键步骤是，在注射细菌溶液必须是很慢的。如果注射过快，老鼠有可能被噎到死。一个成功的注射后，老鼠通常会显示几个深呼吸和喘息，这将有助于微粒进入肺部?...

肺部暴露于环境中的病原体，并很容易受到炎症和损伤1-3。在病理条件下，如肺炎或成人呼吸窘迫综合征（ARDS），病原体以及由白细胞释放炎性细胞因子诱导损伤的肺泡细胞1-3和死亡。它发展为急性肺损伤的动物模型，以促进损伤的病理修复的研究以及机制是很重要的。 目前，大多数人使用高氧和博来霉素诱导的小鼠肺损伤模型4。然而，氧的机制引起的损伤是不一样的肺炎或急性呼吸窘迫综合征5过程中出现的最常见的肺损伤。博莱霉素诱导的急性损伤是罕见的在临床方面4。在这里，我们报告使用气管内注射P的小鼠肺损伤模型铜绿假单胞菌 6,7。这个模型是临床上相关的，并且模仿在p这种情况发生以下肺炎8 rocesses。 由于免疫功能低下的人的机会，院内病原体，P.假单胞菌通常感染肺部，尿路，烧伤，创伤，并且还引起其他血液感染6。细菌释放出毒力因子外毒素A，乘，引发免疫反应6。 P的内部trachael管理假单胞菌反映到引起肺炎的细菌的情况，人体暴露和病理学可能是从最近报道流感病毒H1N1引起的肺损伤模型9不同。由于P.铜绿假单胞菌是一种条件致病菌，它是相对安全的处理相比，一些更致命的病原体。在这里，我们使用气管内注射给药的细菌，因为我们观察到，该方法引入更多的细菌进入肺的较远端肺泡区一些其他方法，例如通过口用的导管。 与其他急性肺损伤模型相比，P。这里描述绿脓杆菌模型适用于研究肺损伤的细菌和过度炎症反应。与使用P。等的动物模型铜绿假单胞菌诱发败血症10,11，这里我们使用气管内注入这些细菌诱发局部急性肺损伤。

<table border="0" cellpadding="0" cellspacing="0" width="650">
	<tbody>
		<tr height="26">
			<td height="26" style="height:27px;width:215px;">Name</td>
			<td style="width:145px;">Company</td>
			<td style="width:145px;">Catalog number&#160;</td>
			<td style="width:145px;">Comments</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:215px;">Anesthetic: Ketamin, xylazine, lidocaine, buprenorphine</td>
			<td style="width:145px;"></td>
			<td style="width:145px;"></td>
			<td style="width:145px;">pharmaceutical grade</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:215px;">27g needle</td>
			<td style="width:145px;">Fisher</td>
			<td style="width:145px;">1482648</td>
			<td style="width:145px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:215px;">syringe</td>
			<td style="width:145px;">Fisher</td>
			<td style="width:145px;">14823434</td>
			<td style="width:145px;">1 ml</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:215px;">scissors</td>
			<td style="width:145px;">Fine Science tools</td>
			<td style="width:145px;"></td>
			<td style="width:145px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:215px;">forceps</td>
			<td style="width:145px;">Fine Science tools</td>
			<td style="width:145px;"></td>
			<td style="width:145px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:215px;">suture&#160;</td>
			<td style="width:145px;">Fisher</td>
			<td style="width:145px;">NC0147607</td>
			<td style="width:145px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">Eye gauge, glove, gown</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">Biosafety Cabinet</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">chlorine dioxide based sterilant&#160;</td>
			<td></td>
			<td></td>
			<td>Clidox</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">sheep blood agar plates&#160;</td>
			<td>Medex supply</td>
			<td>HL-1160</td>
			<td></td>
		</tr>
	</tbody>
</table>

pseudomonas aeruginosa induced lung injury model

为了研究人的急性肺损伤和肺炎，它开发的动物模型来模拟这种疾病的各种病理特征是重要的。在这里，我们已经开发出通过气管内注射的细菌绿脓杆菌 （ 铜绿假单胞菌或PA）的小鼠肺损伤模型。利用该模型，我们能够显示肺部炎症损伤的早期阶段。此外，肺泡上皮屏障泄漏观察到通过分析支气管肺泡灌洗液（BAL）;并且观察到通过使用从受伤的肺组织制备TUNEL测定肺泡细胞死亡。在受伤后的稍后阶段，我们观察到所需的修复过程的细胞增殖。伤病得到了解决，从体育的开始7天后铜绿假单胞菌注射液。这个模型模拟的肺部炎症，损伤和修复的连续过程中的肺炎。这与临床相关的动物模型是适用于研究病理，修复机制中，following急性肺损伤，并且还可以用来测试潜在的治疗剂，这种疾病。

72小时后P. -从24日开始假单胞菌注射，增加的细胞结构中观察到的肺切片（ 图1A-D）。肺开始从96小时后的损伤（ 图1E）中恢复。 7天后P.铜绿假单胞菌 ，正常肺泡形态基本恢复（ 图1F）。隧道染色用肺切片在24小时后的第制备假单胞菌表明细胞死亡中的肺泡细胞（ 图1G-1）。为了研究这种损伤模型中的修复过程中?...

Watch this Scientific Journal Video about Pseudomonas aeruginosa Induced Lung Injury Model at JoVE.com

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. ...

pseudomonas-aeruginosa-induced-lung-injury-model

Research

JoVE Journal

Immunology and Infection

Pseudomonas aeruginosa Induced Lung Injury Model

18.4K 次观看.  University of Illinois at Chicago. 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. 

视频: Pseudomonas aeruginosa Induced Lung Injury Model

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 ...

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 ...

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 ...

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 ...

Studying Effects of Cigarette Smoke on Pseudomonas Infection in Lung Epithelial Cells

Cigarette smoking is the major etiological cause for lung emphysema and chronic obstructive pulmonary disease (COPD). Cigarette smoking also promotes susceptibility to bacterial infections in the respiratory system. However, the effects of cigarette smoking on bacterial infections in human lung epithelial cells have yet to be thoroughly studied. Described here is a detailed protocol for the preparation of cigarette smoking extracts (CSE), treatment of human lung epithelial cells with CSE, and bacterial infection and infection determination. CSE was prepared with a conventional method. Lung epithelial cells were treated with 4% CSE for 3 h. CSE-treated cells were, then, infected with Pseudomonas at a multiplicity of infection (MOI) of 10. Bacterial loads of the cells were determined by three different methods. The results showed that CSE increased Pseudomonas load in lung epithelial cells. This protocol, therefore, provides a simple and reproducible approach to study the effect of cigarette smoke on bacterial infections in lung epithelial cells.

Studying Effects of Cigarette Smoke on Pseudomonas Infection in Lung Epithelial Cells

Described here is a protocol to study how cigarette smoke extract affects bacterial colonization in lung epithelial cells.

香烟烟雾对肺 癫痫细胞 伪多多多细胞的影响研究

Cigarette smoking is the major etiological cause for lung emphysema and chronic obstructive pulmonary disease (COPD). Cigarette smoking also promotes ...