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

動物実験は、イリノイ大学シカゴ校の動物ケア委員会と制度バイオセーフティ委員会によって承認された。 注：マスク、目の保護、ガウンやジャンプスーツ、ダブル手袋：シュードモナスを含むすべての手順は、バイオセーフティレベル2（BSL2）が含まれるが、これらに限定されない慣行、で行われるべきである。認定された安全キャビネットで働いています。漂白剤や消毒剤をベース二酸化塩素との細菌に接触して楽器を扱う。輸送サンプルに対して密封されたボックスを使用します。 1. P.緑膿菌文化と成長<ol><li>ストアP. -80℃で凍結バイアルスクリューキャップの細菌株として緑膿菌 PA103。 </li><li> 70％エタノール湿らせたペーパータオルでボックス内のラックにバイアルを置き、バイオセーフティレベル2（BSL2）の研究室に連れて行く。 </li><li>ストリークヒツジ血液寒天プレート上に細菌やインキュベーターで15時間〜37℃で生育する。 </li&gt; <li> BSL2フードでは、5ミリリットルPBSに細菌ループおよび再懸濁したプレートからの細菌を傷つける。 </li><li>最大3ヶ月間、4℃での在庫を保管してください。しかし、2週間ごとに力価を決定。 </li><li>シリアルに（通常は1：10 4〜1時10分〜7）をPBS中の細菌を希釈し、羊血液寒天プレート上の既知の希釈をプレートアウト。 </li><li> 〜15時間プレートをインキュベートします。コロニーをカウントし、細菌濃度を決定するために、コロニー形成単位（CFU）を計算する。 </li><li>適切な濃度を再懸濁（〜5×10 3 CFU /μl）を1.5ミリリットルの滅菌スクリューキャップのクライオバイアル中で0.5mlのPBSで。クリオバイアルを密封し、スナップのふたボックス内の消毒ラディンペーパータオル上クライオバイアルラックに配置します。 </li><li> BSL2動物施設にあるボックスを輸送。かつてそこに、BSL2キャビネットにボックスを開きます。 </li></ol> 2. P.緑膿菌点眼<ol><li>無菌的に生存手術を行う（滅菌手袋、滅菌楽器、および無菌技術）。滅菌（オートクレーブ処理）手術器具のために働いて表面を提供するために滅菌ドレープを使用してください。各気管点滴注入手順の間にホットビーズ滅菌器を使って楽器を殺菌。 </li><li>麻酔の前にマウスの重量を量る。腹腔内に0.2mlのPBS（腹腔内 ） -ケタミン0.1におけるた（100mg / kg）を、キシラジン（5mg / kg）でマウスを麻酔。つま先のピンチに対する非応答性によって麻酔薬の有効性を判断する。麻酔下での乾燥を防ぐために、目に獣医軟膏を使用してください。 </li><li> BSL2キャビネットに外科ボードにマウスを拘束。 </li><li>カットダウンのための領域を識別します。この領域を剃ると3回アルコールとポビドンヨード綿棒を交互に使用して皮膚を準備します。 </li><li>この麻酔は、そのような気管にアクセスするための皮膚切開などの小手術のために十分であるように局所麻酔薬（リドカイン）で切開領域を扱う。 </li><li>首の正中線で小切開（約5mm）を行い、鈍Fを使用優しく気管へのアクセスのために筋肉を動かすためorceps。外科的切開により気管を公開します。 </li><li> 27 Gの針を1ミリリットル使い捨て注射器への菌液を描画します。各マウスについて、適切な濃度（10 5 CFUまでの各マウス）での細菌の20〜30μlの管理。 </li><li>気管内に針を挿入します。気管にゆっくりとソリューションを注入する。 </li><li>通常、その解決策を示している動物のあえぎが、肺に達していることを確認してください。 </li><li>無菌条件下で無菌縫合糸（6-0モノフィラメント）で傷口を閉じます。 </li><li>術後疼痛を制御するため術後鎮痛としては0.1mg / kgを皮下注射によりブプレノルフィンを使用してください。 </li><li>切開の閉鎖に初期麻酔導入から所要時間が15分未満であることを確認してください。 </li><li>注入後、適切なバイオハザードシャープコンテナに注射器や針の処分。 </li><li> DISIベースの二酸化塩素と手術器具を扱うnfectant 15分間、すすぎ、殺菌のために実験室に戻り、必要に応じて再利用します。 </li><li>ハウス暖かい環境できれいなケージで単独でのマウス。 </li><li>それは意識を取り戻し、動き出すまで、動物に30分ごとにチェックします。以降の最初の3日間、手術後12時間の間隔で。 </li><li>実験を通して、動物のBSL2施設でマウスを保管してください。密封されたボックスに含まれている唯一の組織が動物BSL2施設を残していることを確認してください。マウスが瀕死行動のいずれかを表現した場合の研究中の任意の時点で（呼吸困難、嗜眠、穏やかな刺激に応答して歩き回るの失敗として定義される）、頸椎脱臼に続くボトル源からCO 2吸入によりマウスを安楽死させる。 </li><li>麻酔下で放血によって実験対象を安楽死させる。 <ol><li>安楽死させたマウスからの肺組織を収集します。 BSL2フード内でこれらの手順を実行します。必要に応じて、RACに配置された密封されたチューブを使用してサンプルを転送さらにプロセスのスナップ蓋ボックス内の消毒ラディンペーパータオル上Kである。 </li></ol></li></ol>

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The authors have nothing to disclose.

ここで記述するシュードモナスマウス肺損傷モデルは、急性肺損傷または肺炎を以下起こる炎症、肺損傷、修理、および解像度の全体のプロセスを模倣。それは、臨床的に関連して比較的安全で、取り扱いが容易であるという点でいくつかの他の損傷モデルと比較独自の利点を有している。 手順における重要なステップは、菌液の注入が非常に遅くする必要があるとい?...

肺は、環境病原体に暴露し、炎症および損傷1-3の影響を受けやすいされている。そのような肺炎または成人呼吸窮迫症候群（ARDS）のような病理学的状態の間、白血球により放出され、病原体だけでなく、炎症性因子は肺胞細胞1-3の傷害や死を誘導する。これは、傷害の病理の研究、ならびに修復のメカニズムを容易にするために、急性肺損傷の動物モデルを開発することが重要である。 現在、ほとんどの人は高酸素性ブレオマイシン誘発マウス肺損傷モデル4を使用しています。しかし、高酸素のメカニズムは、傷害が肺炎またはARDS 5中に発生する最も一般的な肺損傷と同じではありませんが原因。ブレオマイシン誘発性急性損傷は、臨床コンテキスト4はまれである。ここでは、Pの気管内注射を用いてマウスの肺損傷モデルを報告緑膿菌 6,7。このモデルは、臨床的に関連であり、pを模倣肺炎8下記起こるrocesses。 免疫無防備状態の個体の日和見、院内病原体、Pとして緑膿菌は通常、肺の気道、尿路、火傷、傷に感染し、また他の血液感染症6が発生します 。細菌は病原性因子毒素Aをリリース、6免疫応答を乗算し、トリガします。 P.のイントラtrachael政権緑膿菌、肺炎を引き起こす細菌にヒトへの暴露の状況を反映しており、病理学は、最近報告されたインフルエンザウイルスH1N1誘発性肺傷害モデル9とは異なる可能性が高い。 P.以来緑膿菌は、より病原性の病原体の一部に比べて取り扱いが比較的安全であり、日和見病原体である。私たちはこの方法はと比較して、肺の遠位肺胞領域へより多くの細菌を導入することが観察されるためここでは、細菌を投与するために気管内注入を使用そのような口を介してカテーテルを使用するなど、いくつかの他の手順。 他の急性肺損傷のモデルと比較して、P.ここで説明緑膿菌モデルは、細菌によって、過剰な炎症によって誘発される肺損傷を研究するのに適している。 P.を使用する他の動物モデルとは異なり緑膿菌は、ここでは、ローカライズされた急性肺損傷を誘導するために、これらの細菌の気管内注入を使用して、敗血症10,11を誘導した。

<table border="0" cellpadding="0" cellspacing="0" width="650">
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			<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

ヒト急性肺損傷及び肺炎を研究するためには、この疾患の多様な病理学的特徴を模倣する動物モデルを開発することが重要である。ここでは、細菌、緑膿菌 （ 緑膿菌またはPA）の気管内注射によるマウス肺損傷モデルを開発した。このモデルを用いて、損傷の初期段階での肺の炎症を示すことができた。また、肺胞の上皮バリア漏出が気管支肺胞洗浄（BAL）を分析することによって観察された。肺胞細胞死が負傷肺から調製した組織を用いたTUNELアッセイにより観察した。損傷後の後期では、我々は、修復プロセスに必要な細胞の増殖を観察した。損傷は7日、P開始から解決した緑膿菌注入 。このモデルは、肺炎の間、肺の炎症、損傷および修復の順次コースを模倣。この臨床的に関連する動物モデルは、fは、修復のメカニズムを病理を研究するのに適している急性肺損傷をollowing、また、この疾患の潜在的治療剤を試験するために用いることができる。

72時間後、P - 24から出発緑膿菌インジェクション 、細胞性の増加は、（ 図1A-D）肺切片において観察された。肺は、96時間後に損傷（ 図1E）から回復し始めた。 7日目にPを投稿する緑膿菌 、正常な肺胞の形態は大部分が（ 図1F）を回復した。 24時間後、P。で調製肺切片を用いたトンネル染色緑膿菌は、肺胞細胞（...

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.

緑膿菌誘発肺傷害モデル

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

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JoVE Journal

Immunology and Infection

Pseudomonas aeruginosa Induced Lung Injury Model

18.4K ビュー.  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. 

ビデオ: 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.

共培養モデルの緑膿菌</emライブヒト気道細胞上に成長した>バイオフィルム

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

免疫・感染学

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).

フローセル内の緑膿菌と出芽酵母のバイオフィルム

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.

のRNAの単離緑膿菌マウス消化管に定着

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.

に対する抗生物質の有効性をテストするための人工痰培地の使用<em&gt;緑膿菌（Pseudomonas aeruginosa）</em&gt;肺嚢胞性線維症へのより適切な条件で

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.

長期的な慢性<em&gt;緑膿菌</emマウスにおける&gt;気道感染

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.

発注済み、緑膿菌ひずみ PA14 トランスポゾン挿入変異体の非冗長ライブラリのレプリケーション

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.

検出細胞毒性アミロイド次感染症の肺による内皮細胞緑膿菌法

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.

緑膿菌の小コロニー変異体の培養とアルギン酸の定量

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.

慢性緑膿菌創傷感染の遅延接種モデル

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.

肺上皮細胞における シュードモナス 感染に及ぼすタバコの煙の影響の研究

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