这项工作是由国家卫生赠款AI62983研究院（AYK），AI22535（GPB）。

1。 P 的小鼠模型铜绿胃肠道的定植和传播<ol><li>口服抗生素治疗C3H/HeN小鼠（6-8周岁，女，哈伦）消耗共生菌群，然后与PA单殖民如前所述4。 </li></ol> 2。收获小鼠盲肠管腔内容<ol><li>浸入液氮浴清洁的不锈钢砂浆。 </li><li>安乐死的二氧化碳窒息的小鼠。 </li><li>鼠标胴体上的发泡胶板和淋浴用95％乙醇的安全。通过从胸骨到会阴部的皮肤中线的纵形切口。反映皮肤和腹膜，暴露腹腔。 </li><li>切除整个盲肠。保持在不锈钢砂浆钳盲肠。盲肠清扫剪刀剪断两端。 </li><li>插入P1000的枪头充满盲肠flushate缓冲液（10毫米TrisHCl，1毫米EDTA和200毫米氯化钠）进入盲肠的近端1毫升。冲入不锈钢砂浆的盲肠flushate缓冲区和盲肠管腔内容。盲肠flushate液1毫升，将足以冲洗所有的盲肠内容。盲肠flushate内容将与砂浆接触后，立即冻结。 </li><li>用无菌杵研磨盲肠flushate内容。 </li><li>广场地面冻结到50毫升的聚丙烯锥形管淹没在干冰/乙醇浴盲肠管腔内容。 </li><li>重复步骤2.7 2.2每一个额外的鼠标。 </li><li>储存在-80 ° C。 </li></ol> 3。细菌RNA分离<ol><li>暖1卷（基于两个管腔盲肠内容的最终体积，约3毫升）酸酚/氯仿（5:1，V / V）所载封口膜固定在一个65的第50毫升橡树岭离心管° C水浴。 </li><li>煮0.5体积的裂解缓冲液（2％SDS，16 mM的EDTA和200毫米氯化钠）6 5分钟在50毫升聚丙烯锥形管中。 </li><li>煮沸裂解缓冲液中添加盲肠管腔内容。均质。煮沸5分钟周期性振荡。 </li><li>添加在100℃盲肠内容/ 65 ° C酸酚/氯仿在橡树岭离心管中裂解样品。用封口膜密封帽。 </li><li>在65℃孵育周期振荡（每2分钟）10分钟。 </li><li>在2500克离心15分钟，在4 °的样本。 </li><li>小心将水相（避免任何的白色接口）50毫升到一个新的橡树岭离心机。水相的体积将盲肠内容，裂解液，酚/氯仿总量的约50％。添加酸等体积酚/氯仿。 </li><li>用封口膜密封帽，拌匀高速震荡。 </li><li>在2500克离心15分钟，在4 °的样本。 </li><li>重复步骤3.7至3.9，直到有水相和有机相之间无可见的白色接口。 </li><li>小心水相转移到一个新的50毫升橡树岭离心。加入等体积的氯仿/异戊醇（24:1，V / V）。 </li><li>密封帽和混合以及震荡。 </li><li>在2500克离心15分钟，在4 °的样本。取出水相（体积将反应总体积约50％）15毫升聚丙烯锥形管和添加等体积的异丙醇。 </li><li>在-20℃放置至少2小时或过夜。 </li><li>在2500克离心45分钟，在4 °的样本。一个颗粒般的凝胶，在试管底部形成。去除上清液。 </li><li>冰冷的70％的乙醇与1毫升的洗涤沉淀。涡街流量计。离心5分钟，在4 °在万克样品。 </li><li>去除上清液，倒置在室温下10分钟让颗粒干管。 </li><li>无RNase水在200μL重悬的RNA沉淀。 </li></ol> 4。 DNA酶处理（涡轮增压无DNA，应用生物系统公司） <ol><li> 10X涡轮DNA酶缓冲和涡轮DNA酶2μL加入20μL，轻轻混匀。 </li><li>孵育20分钟，37 °。 </li><li>加入20μL悬浮DNA酶失活的试剂，并拌匀。 </li><li>在室温下孵育5分钟，偶尔搅拌。 </li><li>在室温下以10,000克1.5分钟离心，将上清转移到一个新的离心管。 </li><li>添加一个体积冷（4℃）酸酚/氯仿，调匀，1分钟震荡。 </li><li>在室温下在12500克离心2分钟。 </li><li>水相转移到一个新的管加1体积氯仿/异戊醇（49:1 / V），涡。 </li><li>在室温下在12500克离心2分钟。 </li><li>转移到一个新的管水相（反应总体积约50％）。 </li><li>添加0.1体积的3M醋酸钠，pH值5.52.5卷冰冷的100％的乙醇。涡街流量计。 </li><li>孵育至少2个小时或30分钟，在-80 ° -20 °。 </li><li>离心30分钟，在12000克在4 ° </li><li>去除上清液。 </li><li>万克在4℃离心5分钟1毫升的冰冷的70％乙醇洗涤沉淀。 </li><li> 10分钟，取出上清，空气干燥。 </li><li> 100μL无RNase水重悬沉淀。可能需要在65℃水浴中放置重悬浮和溶解沉淀。 </li><li>要测试的DNA酶治疗的疗效，一个标准的RT - PCR检测一个核糖体蛋白（或其他参考基因）的扩增反应可以进行处理和未经处理的样品，不使用RT控制。 </li></ol> 5。 RNA清理步骤（Qiagen公司的RNeasy试剂​​盒） <ol><li>参照Qiagen公司的RNeasy迷你手册（ 第 4版，2006年4月）页56-57。按照如上。 </li></ol> 6。代表性的成果恢复使用此协议的细菌总RNA的量是从两个cecums大约2-3微克。回收的RNA是足够的数量和质量，为后续的qPCR，转录分析和RNA序列的实验。 RNA的纯度是通过测量260nm/280nm比例的样品7例行评估，但这种方法没有提供有关RNA的完整性的信息。安捷伦生物分析仪是一种大小，量化和质量控制的DNA，RNA，蛋白质和细胞微流体为基础的平台，并利用RNA的完整性度量称为RNA的完整性号码（RIN）8 。 RNA提取与本议定书​​所产生的260/280比率，范围从1.7到2.0的RIN值≥7.0。安捷伦生物分析仪分析此协议收回的细菌RNA的一个例子是如图1所示。 <img src="/files/ftp_upload/3293/3293fig1.jpg" alt="图1"/> 图1。安捷伦生物分析仪电泳和凝胶体状的形象绿脓杆菌总RNA样品中分离，并从小鼠盲肠内容恢复。 RIN的8.0。

<ol>
	<li>Bodey, G. P., Bolivar, R., Fainstein, V., Jadeja, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infections+caused+by+Pseudomonas+aeruginosa.">Infections caused by Pseudomonas aeruginosa.</a> Rev Infect Dis. 5, 279-279 (1983).</li><li>Bertrand, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endemicity,+molecular+diversity+and+colonisation+routes+of+Pseudomonas+aeruginosa+in+intensive+care+units.">Endemicity, molecular diversity and colonisation routes of Pseudomonas aeruginosa in intensive care units.</a> Intensive Care Med. 27, 1263-1268 (2001).</li><li>Koh, A. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utility+of+in+vivo+transcription+profiling+for+identifying+Pseudomonas+aeruginosa+genes+needed+for+gastrointestinal+colonization+and+dissemination.">Utility of in vivo transcription profiling for identifying Pseudomonas aeruginosa genes needed for gastrointestinal colonization and dissemination.</a> PLoS One. 5, e15131-e15131 (2010).</li><li>Koh, A. Y., Priebe, G. P., Pier, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virulence+of+Pseudomonas+aeruginosa+in+a+murine+model+of+gastrointestinal+colonization+and+dissemination+in+neutropenia.">Virulence of Pseudomonas aeruginosa in a murine model of gastrointestinal colonization and dissemination in neutropenia.</a> Infect Immun. 73, 2262-2272 (2005).</li><li>Alexander, R. J., Raicht, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+total+RNA+from+human+stool+samples.">Purification of total RNA from human stool samples.</a> Dig Dis Sci. 43, 2652-2658 (1998).</li><li>Fitzsimons, N. A., Akkermans, A. D., de Vos, W. M., Vaughan, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+gene+expression+detected+in+human+faeces+by+reverse+transcription-PCR.">Bacterial gene expression detected in human faeces by reverse transcription-PCR.</a> J Microbiol Methods. 55, 133-140 (2003).</li><li>Manchester, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+UV+methods+for+measurement+of+protein+and+nucleic+acid+concentrations.">Use of UV methods for measurement of protein and nucleic acid concentrations.</a> Biotechniques. 20, 968-970 (1996).</li><li>Schroeder, 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+RIN:+an+RNA+integrity+number+for+assigning+integrity+values+to+RNA+measurements.">The RIN: an RNA integrity number for assigning integrity values to RNA measurements.</a> BMC Mol Biol. 7, 3-3 (2006).</li><li>Turnbaugh, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+core+gut+microbiome+in+obese+and+lean+twins.">A core gut microbiome in obese and lean twins.</a> Nature. 457, 480-484 (2009).</li></ol>

没有利益冲突的声明。

这里描述的RNA提取方法，可以回收足够数量的高品质的绿脓杆菌总RNA从小鼠胃肠道的收获。这种方法不仅限于体育假单胞菌和有可能被应用到其他细菌。足够从肠道微生物的恢复会有所不同显著有机体。在我们的小鼠模型， 体育绿脓杆菌通常寄生水平的小鼠胃肠道5 × 10 7 5 × 10 8 CFU /克粪便 4 。由于回收的盲肠内容是大约0.5克，P的估计数目从两个cecums收回假单胞菌是5 × 10 7 5 × 10 8 CFU之间。如果使用其他微生物，这将是审慎的做法来验证消化道定植的水平，然后计算出需要恢复CFU针对性cecums的数量。同样重要的是要注意，当使用这个特殊的小鼠模型，与PA感染的抗生素治疗的老鼠有没有量化的数额从盲肠内容分离的RNA。 我们的小鼠模型绿脓杆菌胃肠道的定植和传播试图效仿 P的发病绿脓杆菌菌血症在癌症和干细胞移植的患者。在这个病患族群，共生菌群失调往往是耗尽继发抗生素或化疗治疗（如GI共生菌群的抗生素消耗），导致病原微生物的过度生长（ 如单与绿脓杆菌），那么随后的传播后的免疫抑制。这个小鼠模型的优点和局限性已经得到解决以前4。目前的研究的目的是提供一种方法，从消化道隔离微生物总RNA。该协议可以很容易地适应其他小鼠模型，研究在胃肠道微生物的发病机制等方面（即共生菌群的相互作用，炎症性肠病等细菌的影响）。 这种方法的优点之一是多个裂解步骤，包括冻结/解冻，机械破碎（粉碎砂浆/杵，同质化），沸点，化学裂解（如SDS），注册成立。尽管众多的裂解步骤，一些微生物（特别是革兰氏阳性细菌和真菌/酵母菌）可能需要额外的机械破坏。热裂解/酸酚-氯仿孵化（步骤3.5），珠除了细菌，酵母和/或0.5-0.7毫米（0.1毫米）和随后珠 ​​打一步后，应足以裂解这些生物体 9， 10。 由于小鼠盲肠，反复感冒acid-phenol/chloroform提取（3.7至3.9步）的回收材料的复杂性是绝对必要的，以达到可以接受的RNA的质量和完整性作进一步的下游反应。 3-5冷acid-phenol/chloroform拔牙前必须消除白色水相和有机相之间的界面。最后，双方的DNA酶处理（步骤4）的RNeasy清理协议（步骤5）相结合的方式，去除污染的DNA和小非的mRNA（5S和tRNA）是必不可少的。如前所述，利用该协议回收的RNA随后的转录分析，定量PCR（未出版），和RNA序列（未发表）实验的足够数量和质量。

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th></tr><tr><td>研钵</td><td>费舍尔</td><td> 12-947-1 </td></tr><tr><td>均质机</td><td>全方位</td><td> TM - 125 </td></tr><tr><td>橡树岭离心管（50毫升） </td><td> NALGENE </td><td> 3119-0050 </td></tr><tr><td>酚/氯仿</td><td> Ambion公司</td><td> AM9720 </td></tr><tr><td>氯仿</td><td> Sigma - Aldrich公司</td><td> C2432 </td></tr><tr><td>异戊醇</td><td> Sigma - Aldrich公司</td><td> W205702 </td></tr><tr><td>异丙醇</td><td> Sigma - Aldrich公司</td><td> 190764 </td></tr><tr><td>焦碳酸二乙酯（DEPC） </td><td> Sigma - Aldrich公司</td><td> 40718 </td></tr><tr><td> DNA酶</td><td> Ambion公司</td><td> AM2238 </td></tr><tr><td>的RNeasy试剂​​盒</td><td> Qiagen公司</td><td> 74104 </td></tr><tr><td> 3M醋酸钠</td><td> Ambion公司</td><td> AM9740 </td></tr><tr><td> 100％的乙醇</td><td> Sigma - Aldrich公司</td><td> E7023 </td></tr></tbody></table>

rna isolation of pseudomonas aeruginosa colonizing the murine gastrointestinal tract

铜绿假单胞菌 （PA）感染导致显著的发病率和死亡率与免疫系统受损，如白血病患者，严重的烧伤创面，或器官移植1，的主机。开发PA血液感染的高危患者，胃肠道（GI）的主要水库定植 2，但机制PA的转换，从无症状的殖民微生物侵入性，而且往往是致命的的，病原体也不清楚。在此之前，我们进行了恢复从细菌细胞中的cecums居住的PA mRNA的殖民小鼠3，以确定在宿主的免疫状态的改变细菌基因表达的变化在体内的转录分析实验。 任何转录分析实验，限速步骤是在足够数量的高质量的mRNA隔离。鉴于大量的酶，碎片，食物残渣，消化道颗粒物，RNA提取的挑战是艰巨的。在这里，我们提出了一个可靠和可重复性的方法从小鼠胃肠道细菌RNA的隔离。这种方法利用了PA胃肠道的定植和中性粒细胞减少症引起的传播 4的行之有效的小鼠模型。一旦胃肠与PA定植证实，老鼠是安乐死和盲肠内容是恢复和速冻。 RNA，然后采用机械破碎，沸点，酚/氯仿抽提，DNA酶处理，和亲和层析结合提取。数量和纯度均证实光度法（Nanodrop技术）和生物分析仪（安捷伦科技）（图1）。这对胃肠道微生物的RNA分离方法可以很容易地适应以及其他细菌和真菌。

Watch this Scientific Journal Video about RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract at JoVE.com

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

RNA提取的一种可靠的方法<em&gt;绿脓杆菌</em从小鼠cecums恢复描述。回收的RNA是足够的数量和质量，为后续的qPCR，转录分析和RNA序列的实验。这种技术可以适用于RNA等肠道微生物的隔离。

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 ...

rna-isolation-pseudomonas-aeruginosa-colonizing-murine

Research

JoVE Journal

Immunology and Infection

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

19.0K 次观看.  University of Texas Southwestern Medical Center. RNA提取的一种可靠的方法绿脓杆菌

视频: RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

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 ...

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 ...

Isolation of Lymphocytes from Mouse Genital Tract Mucosa

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and bacteria 1. Mucosae are also inductive sites in the host to generate immunity against pathogens, such as the Peyers patches in the intestinal tract and the nasal-associated lymphoreticular tissue in the respiratory tract. This unique feature brings mucosal immunity as a crucial player of the host defense system. Many studies have been focused on gastrointestinal and respiratory mucosal sites. However, there has been little investigation of reproductive mucosal sites. The genital tract mucosa is the primary infection site for sexually transmitted diseases (STD), including bacterial and viral infections. STDs are one of the most critical health challenges facing the world today. Centers for Disease Control and Prevention estimates that there are 19 million new infectious every year in the United States. STDs cost the U.S. health care system $17 billion every year 2, and cost individuals even more in immediate and life-long health consequences. In order to confront this challenge, a greater understanding of reproductive mucosal immunity is needed and isolating lymphocytes is an essential component of these studies. Here, we present a method to reproducibly isolate lymphocytes from murine female genital tracts for immunological studies that can be modified for adaption to other species. The method described below is based on one mouse.&#x2003;

An efficient way to isolate lymphocytes from mouse genital tract is described. This method takes advantage of enzyme digestion and Percoll gradient separation to allow efficient isolation. This technique is also adaptable to for use in other species

小鼠生殖道黏膜淋巴细胞分离

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and ...

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 ...

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 ...

Visualization of Candida albicans in the Murine Gastrointestinal Tract Using Fluorescent In Situ Hybridization

Candida albicans is a fungal component of the gut microbiota in humans and many other mammals. Although C. albicans does not cause symptoms in most colonized hosts, the commensal reservoir does serve as a repository for infectious disease, and the presence of high fungal titers in the gut is associated with inflammatory bowel disease. Here, we describe a method to visualize C. albicans cell morphology and localization in a mouse model of stable gastrointestinal colonization. Colonization is established using a single dose of C. albicans in animals that have been treated with oral antibiotics. Segments of gut tissue are fixed in a manner that preserves the architecture of luminal contents (microorganisms and mucus) as well as the host mucosa. Finally, fluorescent in situ hybridization is performed using probes against fungal rRNA to stain for C. albicans and hyphae. A key advantage of this protocol is that it allows for simultaneous observation of C. albicans cell morphology and its spatial association with host structures during gastrointestinal colonization.

Visualization of Candida albicans in the Murine Gastrointestinal Tract Using Fluorescent In Situ Hybridization

The purpose of this protocol is to visualize Candida albicans cell shape and localization in the mammalian gastrointestinal tract.

使用荧光原位杂交对毛尿胃肠道中的念珠菌的可视化

Candida albicans is a fungal component of the gut microbiota in humans and many other mammals. Although C. albicans does not cause symptoms in most ...