この作品は、健康補助AI62983（AYK）、AI22535（GPB）の国立研究所によって資金を供給された。

1。 P.のマウスモデル緑膿菌 GI植民地化と普及<ol><li> C3H/HeNマウス（雌、6〜8週齢の古い、ハーラン）が共生細菌叢を破壊する経口抗生物質で処理し、以前に4を説明するようにしてPAとのモノは、植民地化されています。 </li></ol> 2。ネズミ盲腸管腔内容物を収穫<ol><li>液体窒素槽に洗浄ステンレス鋼モルタルを浸す。 </li><li>二酸化炭素の窒息によりマウスを安楽死させる。 </li><li>発泡スチロールのボードおよび95％エタノールでシャワー上のセキュリティで保護されたマウスの死骸。胸骨から会陰部への皮膚を通して正中縦切開を加えます。腹腔を露出する皮膚と腹膜を反映している。 </li><li>全体盲腸を切除する。ステンレス鋼モルタル以上鉗子で盲腸を保持する。解剖はさみで盲腸の両端をスニップ。 </li><li>盲腸の近位端に盲腸flushate緩衝液（10mM TrisHCl、1mMのEDTA、200 mMのNaCl）5 1mlで満たしたP1000ピペットの先端を挿入します。ステンレス製の乳鉢に盲腸flushateバッファおよび盲腸内腔の内容をフラッシュします。盲腸flushate緩衝液1mlは、盲腸内容物のすべてをフラッシュするための十分です。盲腸flushateの内容は、モルタルと接触した時点で直ちに凍結されます。 </li><li>滅菌乳棒で盲腸flushateの内容を挽く。 </li><li>場所の地面には、ドライアイス/エタノール浴に浸漬50 mlのポリプロピレンコニカルチューブに盲腸管腔内容物を凍結。 </li><li>繰り返して、各追加のマウスは2.7を介して2.2を繰り返します。 </li><li> -80℃で保存する</li></ol> 3。細菌のRNAの単離<ol><li> 65にパラフィルムで固定されたキャップで50mlオークリッジ遠心管に含まれる酸の暖かい1ボリューム（二盲腸内腔の内容を、約3 mlの最終容量に基づいて）フェノール/クロロホルム（5:1、v / v）を℃の水浴。 </li><li>溶解バッファーの沸騰0.5体積（2％SDS、16 mMのEDTA、200 mMのNaCl）6は、5分間に50 mlのポリプロピレンコニカルチューブに含まれています。 </li><li>盲腸内腔の内容に沸騰溶解バッファーを追加します。ホモジナイズ。周期的にボルテックスしながら5分間沸騰させます。 </li><li> 65 ° Cの酸オークリッジ遠心管にフェノール/クロロホルムを100 ° C盲腸内容物/溶解サンプルを追加。パラフィルムでシールキャップ。 </li><li> 65℃でインキュベートC定期的なボルテックス（2分毎）で10分間。 </li><li> 15分間4℃で2500グラムで、サンプルを遠心する。 </li><li>慎重に新鮮な50ミリリットルオークリッジ遠心機に水相を（白のインターフェースのいずれかを避けて）転送する。水相の体積は、盲腸内容物、溶解バッファー、および酸性フェノール/クロロホルムの総体積の約50％になります。フェノール酸/等量のクロロホルムを追加。 </li><li>シールは、パラフィルムでキャップとハイスピー​​ドでボルテックスでよく混ぜる。 </li><li> 15分間4℃で2500グラムで、サンプルを遠心する。 </li><li>水相および有機相の間に目に見える白のインタフェースがなくなるまで3.9から3.7までのステップを繰り返します。 </li><li>慎重に新鮮な50ミリリットルオークリッジ遠心機に水相を移す。クロロホルム/イソアミルアルコール（24:1、v / v）の等量を追加。 </li><li>シールは、キャップとボルテックスでよく混和する。 </li><li> 15分間4℃で2500グラムで、サンプルを遠心する。 15 mlのポリプロピレンコニカルチューブに水相を（ボリュームは総反応容量の約50％となる）を外し、イソプロパノールの等量を追加。 </li><li>少なくとも2時間または一晩のために-20℃でインキュベートする。 </li><li> 45分間、4℃で2500グラムで、サンプルを遠心する。ペレット状のゲルは、チューブの底に形成されます。上清を取り除く。 </li><li>氷冷70％エタノール1mlでペレットを洗浄します。渦。 5分間、4℃で10,000 gででサンプルを遠心分離します。 </li><li>上清を除去し、室温で10分間ペレット乾燥をさせるためにチューブを反転させる。 </li><li> RNaseフリー水200μlのRNAペレットを再懸濁します。 </li></ol> 4。 DNase処理（ターボDNA -フリー、アプライドバイオシステムズ） <ol><li> 10XターボのDNaseバッファーおよび2μlのターボのDNase20μlを加え、穏やかに混ぜる。 </li><li> 20分間37℃でインキュベートする。 </li><li>再懸濁したのDNase不活化試薬20μlを加え、よく混ぜる。 </li><li>時々混ぜ、室温で5分間インキュベートする。 </li><li> 1.5分間、室温で10,000 gで遠心し、新しいマイクロ遠心チューブに上清を移す。 </li><li>フェノール/クロロホルム冷（4℃）酸の1つのボリュームを追加し、1分間ボルテックスでよく攪拌します。 </li><li> 2分間、室温で12500グラムで遠心。 </li><li>新しいチューブに水相を移し、クロロホルム/イソアミルアルコール（49:1 v / v）で、渦の1ボリュームを追加します。 </li><li> 2分間、室温で12500グラムで遠心。 </li><li>新しいチューブに水相（総反応容量の約50％）を譲渡。 </li><li> 3Mの酢酸ナトリウムの0.1倍量、pHが5.5を追加します。氷冷100％エタノールを2.5倍量。渦。 </li><li> -20℃で少なくとも2時間または30分間-80℃でインキュベートする。 </li><li> 4℃で30分間12,000 gで遠心する° </li><li>上清を取り除く。 </li><li> 4℃で10,000 gで5分間、遠心分離することにより、氷冷70％エタノール1mlでペレットを洗浄します。 </li><li> 10分間上清と空気乾​​燥を取り外します。 </li><li> RNaseフリー水100μlにペレットを再懸濁します。懸濁するために65℃の水浴中に配置し、ペレットを溶解するために必要な場合があります。 </li><li> DNase処理の有効性をテストするには、リボソームタンパク質（または他の参照の遺伝子）の増幅のための標準的なRT - PCR反応は、RTはコントロールを使用しないで、処理および非処理サンプルに対して実行される可能性があります。 </li></ol> 5。 RNAのクリーンアップステップ（キアゲン、RNeasyキット） <ol><li>キアゲン社RNeasyミニハンドブック（ 第 4版、2006年4月）のページ56から57を参照してください。示されているように従ってください。 </li></ol> 6。代表的な結果このプロトコルを使用して、復元された細菌のトータルRNAの量は、2つのcecumsから約2〜3μgのです。回収したRNAは、その後の定量PCR、転写プロファイリング、およびRNAのSeqの実験のための十分な量と品質です。 RNAの純度は、日常的にサンプルの260nm/280nm比7を測定することにより評価、まだこの方法では、RNAの完全性に関する情報を提供されていません。アジレントバイオアナライザは、サイジング、定量化とDNA、RNA、タンパク質や細胞の品質管理のためのマイクロ流体ベースのプラットフォームであり、RNAの完全性番号（RIN）8として知られているRNAの完全性のメトリックを利用しています。このプロトコルで抽出されたRNAは、280分の260 1.7から2.0に至るまでの比率とRINの値≥7.0を生成します。このプロトコルによって回復細菌のRNAのアジレントバイオアナライザ分析の一例を図1に示されています。 <img src="/files/ftp_upload/3293/3293fig1.jpg" alt="図1"/> 図1。アジレントバイオアナライザ電気泳動図と緑膿菌のトータルRNAサンプルのゲル状のイメージが分離し、マウス盲腸内容物から回収。 RIN 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>

利害の衝突は宣言されません。

RNAの抽出方法は、ここで説明すると、マウスの消化管から採取した高品質の緑膿菌の全RNAの十分な量の回復が可能になります。このメソッドは、P.に限定されるものではありません緑膿菌とは、潜在的に他の細菌に適用することができます。腸から十分な微生物の生物の回復は、生物から生物に大きく変化します。私たちのマウスモデル、P.で緑膿菌は、通常、5 × 10 7〜5 × 10 8 CFU / gの糞便4の間のレベルでマウスの消化管に定着。回収した盲腸内容物は約0.5グラム、Pの推定数であるため、 two cecumsから回収された緑膿菌は、5 × 10 7〜5 × 10 8 CFUの間です。他の微生物が使用されている場合、それはGI植民地化のレベルを確認してから、CFUのターゲット数を回復するために必要なcecumsの数を計算するのが賢明だろう。この特定のマウスモデルを使用する場合は、ペンシルバニア州に感染していない抗生物質で処理したマウスは、自分の盲腸内容物から単離されたRNAのない定量的な量がないことに注意することも重要です。 緑膿菌胃腸の植民地化とP.の病因をエミュレートするために普及の試みの私達のマウスモデル癌と幹細胞移植患者における菌血症緑膿菌 。この患者集団では、共生細菌叢は、多くの場合、病原性微生物（例えば、 緑膿菌によるモノ協会）の繁茂をもたらす抗生物質や化学療法（GI共生細菌叢の抗生物質の枯渇など）への二次枯渇している免疫抑制後、その後の普及。このマウスモデルの利点と制限は、すでに以前に4対処されています。この現在の研究の目的は、消化管からの微生物の全RNAを単離するための方法論を提供することです。このプロトコルは、容易に消化管における微生物の病原性の他の側面（すなわち、共生細菌叢の作用、炎症性腸疾患、などの細菌の影響）を調べる他のマウスモデルに適合させることができます。 この方法の1つの利点は、凍結/融解を含む複数の溶解ステップの導入、機械的破壊（乳鉢/乳棒で粉砕、均質化）、沸騰、および化学的溶解（例：SDS）です。溶解ステップの多数にもかかわらず、一部の微生物（特にグラム陽性細菌及び真菌/酵母）追加の機械的破砕が必要な場合があります。溶解/酸性フェノール-クロロホルムインキュベーション（ステップ3.5）、ビーズの追加（細菌や酵母のための/または0.5〜0.7ミリメートルを0.1 mm）およびそれに続くビーズビーティングステップホットした後、これらの生物9を溶解するために十分なはずです10。 マウス盲腸、繰り返し冷たいacid-phenol/chloroformの抽出（ステップ3.7から3.9）から回収された材料の複雑な性質を考えると絶対にさらに下流の反応の許容RNAの品質と整合性を達成するために必要とされています。水溶液と有機相との間の白いのインターフェイスが除去されるまでの間に3月5日冷acid-phenol/chloroform抽出が必要になることがあります。最後に、DNase処理（ステップ4）とRNeasyクリーンアッププロトコル（手順5）の両方の組み合わせは、DNAのコンタミと非発現小（5SとtRNA）を除去するために不可欠です。前述したように、このプロトコルを利用して回収したRNAは、その後の転写プロファイリング3、定量PCR（未発表）、およびRNAのSeq（未発表）の実験のための十分な量と品質です。

<table border="1"><tbody><tr><th>試薬の名前</th><th>会社</th><th>カタログ番号</th></tr><tr><td>臼と杵</td><td>フィッシャー</td><td> 12-947-1 </td></tr><tr><td>ホモジナイザー</td><td>オムニ</td><td> TM - 125 </td></tr><tr><td>オークリッジ遠心管（50ml）を</td><td> Nalgene </td><td> 3119-0050 </td></tr><tr><td>酸フェノール/クロロホルム</td><td>アンビオン</td><td> AM9720 </td></tr><tr><td>クロロホルム</td><td>シグマアルドリッチ</td><td> C2432 </td></tr><tr><td>イソアミルアルコール</td><td>シグマアルドリッチ</td><td> W205702 </td></tr><tr><td>イソプロパノール</td><td>シグマアルドリッチ</td><td> 190764 </td></tr><tr><td>ジエチルピロカーボネート（DEPC） </td><td>シグマアルドリッチ</td><td> 40718 </td></tr><tr><td> DNase処理</td><td>アンビオン</td><td> AM2238 </td></tr><tr><td> RNeasyキット</td><td>キアゲン</td><td> 74104 </td></tr><tr><td> 3M酢酸ナトリウム</td><td>アンビオン</td><td> AM9740 </td></tr><tr><td> 100％エタノール</td><td>シグマアルドリッチ</td><td> E7023 </td></tr></tbody></table>

rna isolation of pseudomonas aeruginosa colonizing the murine gastrointestinal tract

白血病などの患者、重症のやけどの傷、または臓器移植1と免疫力、を持つホストでの重要な罹患率と死亡率における緑膿菌 （PA）感染症の結果。 PAの血流感染症を開発するためのハイリスクの患者では、胃腸（GI）管は、植民地化2の主な貯留が、mechanismsはどのによって無症候性の植民地支配を微生物から浸潤し、しばしば致命的にPAの遷移は、病原体は不明である。以前、我々は、宿主の免疫状態の変化の間に細菌の遺伝子発現の変化を識別するために、マウスの3植民地のcecumsに常駐している細菌細胞からのPAのmRNAを回収することによりin vivoで転写プロファイリング実験で行った。 任意の転写プロファイリング実験と同様に、律速段階は、高品質のmRNAの十分な量の分離です。酵素、破片、食物残渣、および消化管における粒子状物質の豊富さを考えると、RNAの分離の課題は困難です。ここで、我々はマウスの消化管から回収した細菌のRNAの信頼性と再現性の分離のための手法を提案する。このメソッドは、PA GIの植民地化と好中球減少症誘発性の普及4の十分に確立されたマウスモデルを採用しています。 PAで一回GI植民地化が確認され、マウスを安楽死と盲腸内容物を回収し、フラッシュが凍結されています。 RNAは、その後、機械的破砕、沸騰、フェノール/クロロホルム抽出、DNase処理、およびアフィニティークロマトグラフィーの組み合わせを使用して抽出されます。量と純度は、分光光度計（㈱エルの技術）とバイオアナライザ（アジレントテクノロジー）（図1）によって確認されています。 GIの微生物のRNA単離のこの方法は簡単だけでなく、他の細菌および真菌に適応させることができます。

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

のRNA単離のための信頼できる方法<em&gt;緑膿菌</em&gt;マウスcecumsから回収記述されています。回収したRNAは、その後の定量PCR、転写プロファイリング、およびRNAのSeqの実験のための十分な量と品質です。この手法は、他の腸内微生物のRNAの単離のために適応させることができます。

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 ...

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Immunology and Infection

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

19.0K ビュー.  University of Texas Southwestern Medical Center. のRNA単離のための信頼できる方法緑膿菌マウスcecumsから回収記述されています。回収したRNAは、その後の定量PCR、転写プロファイリング、およびRNAのSeqの実験のための十分な量と品質です。この手法は、他の腸内微生物のRNAの単離のために適応させることができます。

ビデオ: 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 ...

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 ...

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

マウス生殖管粘膜からのリンパ球の単離

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.

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 ...

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.

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. ...

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.

光側に来る: In Vivo糖尿病ヘアレス マウス モデルにおける慢性創傷における緑膿菌バイオ フィルム感染症の監視

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.

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 ...

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.

上皮内蛍光を用いたマウス消化管におけるカンダアルビカンスの可視化

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 ...