私たちは、動物実験、ウェルカム·トラスト（グラント089215）を置き換えるために排他的に健康研究のためのイギリス国立研究所、人道的な研究のための博士Hadwenトラスト、英国有数の医療研究慈善団体の資金調達をサポートする非動物の研究技術を認める。我々はまた、ノバルティスファーマ英国株式会社（無制限の教育助成金）を認める。

<ol>
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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quorum-sensing+signals+indicate+that+cystic+fibrosis+lungs+are+infected+with+bacterial+biofilms.">Quorum-sensing signals indicate that cystic fibrosis lungs are infected with bacterial biofilms.</a> Nature. 407, 762-764 (2000).</li><li>Mowat, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudomonas+Aeruginosa+Population+Diversity+and+Turnover+in+Cystic+Fibrosis+Chronic+Infections.">Pseudomonas Aeruginosa Population Diversity and Turnover in Cystic Fibrosis Chronic Infections.</a> Am. J. Respir. Crit. Care. Med. , (2011).</li><li>Fothergill, J. L., Mowat, E., Ledson, M. J., Walshaw, M. J., Winstanley, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluctuations+in+phenotypes+and+genotypes+within+populations+of+Pseudomonas+aeruginosa+in+the+cystic+fibrosis+lung+during+pulmonary+exacerbations.">Fluctuations in phenotypes and genotypes within populations of Pseudomonas aeruginosa in the cystic fibrosis lung during pulmonary exacerbations.</a> J. Med. Microbiol. 59, 472-481 (2010).</li><li>Fouhy, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diffusible+signal+factor-dependent+cell-cell+signaling+and+virulence+in+the+nosocomial+pathogen+Stenotrophomonas+maltophilia.">Diffusible signal factor-dependent cell-cell signaling and virulence in the nosocomial pathogen Stenotrophomonas maltophilia.</a> J. Bacteriol. 189, 4964-4968 (2007).</li><li>Winstanley, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Newly+introduced+genomic+prophage+islands+are+critical+determinants+of+in+vivo+competitiveness+in+the+Liverpool+Epidemic+Strain+of+Pseudomonas+aeruginosa.">Newly introduced genomic prophage islands are critical determinants of in vivo competitiveness in the Liverpool Epidemic Strain of Pseudomonas aeruginosa.</a> Genome Res. 19, 12-23 (2009).</li><li>Carter, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+subtype+of+a+Pseudomonas+aeruginosa+cystic+fibrosis+epidemic+strain+exhibits+enhanced+virulence+in+a+murine+model+of+acute+respiratory+infection.">A subtype of a Pseudomonas aeruginosa cystic fibrosis epidemic strain exhibits enhanced virulence in a murine model of acute respiratory infection.</a> J. Infect. Dis. 202, 935-942 (2010).</li></ol>

利害の衝突が宣言されません。

本研究では、P.を複製するためにASMに基づくin vitroモデルで小説を使用CFの肺4内の緑膿菌バイオフィルムの状態。モデルは、抗菌剤の小規模、高スループットのテストのために正常に変更された。 このアッセイの重要な手順は次のとおりです。 <ol><li> ASMメディアや保守性不稔性の一貫した準備。我々は、各コンポーネントは、再現性のある結果を毎回達成するために追加される方法を最適化することに長い時間を捧げてきました。 ASMのろ過が遅いですが、ムチンのコンポーネントを破損する恐れがありオートクレーブ、より好ましい。他の11で示唆したように、これはかなりの選択圧、ドライブの変異を課すプロファージの溶解16を誘導し、大幅に複数の細菌の遺伝子の発現を変更することがありますので、私たちは、抗生物質の添加をお勧めしません。 </li><li>アッセイは、ボリューム入出力に応じて最適化する必要がありますfはASMが使用されます。振とう速度は小さいボリュームのために増加しており、短いフィルムのライフサイクルが観察される。 </li></ol>小規模ASMバイオフィルムモデルの明白なアプリケーションでは、バイオフィルムの抗菌薬感受性（BSMIC 90） ​​のより現実的な判断である。嫌気性および微好気性のニッチは、CFの肺内に存在し、酸素が ​​成熟したバイオフィルム2、17の奥深くに制限されているという証拠がある。ここでは示している10月14日臨床P. ASMでの微好気性条件下ではトブラマイシンに対する感受性の低下- CF患者の喀痰から緑膿菌分離株は、かなり（≥128倍4）を示す。本研究の結果は、トブラマイシンなどの抗生物質が、P.に対してあまり有効であることが示唆された従来の感受性試験の方法によって示されるよりもCFの肺における緑膿菌感染症。これらの結果は、バイオ10の抗菌薬感受性に関する先行研究を反映しています。小スケールASMアッセイは、より良い治療の決定を通知するために意味のある抗生物質感受性データを生成するためのシンプルなハイスループットプラットフォームを提供します。アッセイは、全人口を代表できない場合があり、スクリーニングに拾われ、単一のコロニーで、従来の抗生物質感受性試験と同じ方法で制限されています。しかし、我々は非表面接続されたバイオフィルムの成長と微好気条件（II）を使用して適用されるアプローチは、（i）、明確な代替手段と、既存のメソッドへの潜在的な改善を表していると信じています。我々は、このアッセイはPを勉強するための適切なモデルであると結論緑膿菌のバイオフィルム集団。臨床現場での更なるテストは、抗生物質に対する感受性は、バイオフィルム成長P.に基づいてかどうかを確かめるだろう緑膿菌は、潜在的に改善し、微生物学的および臨床転帰を持つ別の抗生物質の選択肢につながる可能性があります。古典的なバイオフィルムモデルを用いた同様の調査がBSMIC値が生じることが示された抗生物質治療5,17別の勧告に。 抗感染薬の有効性のテストに加えて、ASMシステムは、P.の多様化を理解することを目的としたものと研究のための動物モデルへの格安、簡単かつ再現可能な代替手段を表し、 緑膿菌の集団。我々は、P.の自然集団における広範囲の不均一性を観察した緑膿菌は、CF患者の痰18、19から回収された。同様の表現型および遺伝子型の多様化は、それCF肺疾患の in vitroモデルで魅力的、ASM 4（と私たちの未発表データ）の成長中に観察することができます。 ASMモデルの相対的なシンプルさは、それが簡単に長期の適応実験はP.に抗生物質やその他のストレスの影響を監視することで、例えば、目的とした設計になります緑膿菌の人口発散。さらに、他の細菌性病原体がで成長させることができるASM。たとえば、2007年は、S.によるバイオフィルム形成を研究するためにASM Fouhy ら使用しているmaltophillia 20。

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td></tr><tr><td>魚の精子からのDNA </td><td> Sigma-Aldrich社</td><td> 74782 </td></tr><tr><td>ブタ胃、II型からムチン</td><td> Sigma-Aldrich社</td><td> M2378 </td></tr><tr><td> L-アラニン</td><td>アクロスオーガニック</td><td> 102830250 </td></tr><tr><td> L-アルギニン</td><td> Sigma-Aldrich社</td><td> A5006 </td></tr><tr><td> L（+） - アスパラギン一水和物</td><td>アクロスオーガニック</td><td> 175271000 </td></tr><tr><td> L（+） - アスパラギン酸</td><td>アクロスオーガニック</td><td> 105041000 </td></tr><tr><td> L-システイン</td><td>シグマAdrich </td><td> 168149 </td></tR&gt; <tr><td> L（+） - グルタミン酸</td><td>アクロスオーガニック</td><td> 156211000 </td></tr><tr><td> L-グルタミン</td><td> Sigma-Aldrich社</td><td> G3126 </td></tr><tr><td>グリシン</td><td>アクロスオーガニック</td><td> 220911000 </td></tr><tr><td> L-ヒスチジン</td><td>シグマAdrich </td><td> H8000 </td></tr><tr><td> L-イソロイシン</td><td> Sigma-Aldrich社</td><td> I2752 </td></tr><tr><td> L-ロイシン</td><td> Sigma-Aldrich社</td><td> L8000 </td></tr><tr><td> L（+） - リジン一塩酸塩</td><td>アクロスオーガニック</td><td> 125222500 </td></tr><tr><td> L-メチオニン</td><td> Sigma-Aldrich社</td><td> M9625 </td></tr><tr><td> L-フェニルアラニン</td><td>アクロスオーガニック</td><td> 130310250 </td></tr><tr><td> L-プロリン</td><td> Sigma-Aldrich社</td><td> P0380 </td></tr><tr><td> L-セリン</td><td>アクロスオーガニック</td><td> 132660250 </td></tr><tr><td> L-スレオニン</td><td>アクロスオーガニック</td><td> 138930250 </td></tr><tr><td> L（ - ）-トリプトファン</td><td>アクロスオーガニック</td><td> 140590250 </td></tr><tr><td> L-チロシン</td><td>アクロスオーガニック</td><td> 140641000 </td></tr><tr><td> L-バリン</td><td> Sigma-Aldrich社</td><td> V0500 </td></tr><tr><td>ジエチレントリアミン五酢酸</td><td> Sigma-Aldrich社</td><td> 32318 </td></tr><tr><td> NaClを</td><td>フィッシャー·サイエンティフィック</td><td> S/3160/60 </td></tr><tr><td>塩化カリウム</td><td> BDH </td><td> BDH0258 </td></tr><tr><td&gt; KOH </td><td> BDH </td><td> BDH0262 </td></tr><tr><td>卵黄エマルジョン</td><td> Sigma-Aldrich社</td><td> 17148 </td></tr><tr><td> ME 2ダイヤフラム真空ポンプ</td><td> Vacuubrand </td><td> 696126 </td></tr><tr><td> Steritopフィルター（孔径0.22μmの、ネックサイズ：45ミリメートル） </td><td>ミリポア</td><td> SCGPT10RE </td></tr><tr><td>ルリア - ベルターニ培地</td><td>シグマ</td><td> L2897 </td></tr><tr><td> 96ウェルマイクロタイタープレート</td><td>ザルスタット</td><td> 82.1581 </td></tr><tr><td> 24ウェル組織培養処理プレート</td><td>いわき市</td><td> 3820-024 </td></tr><tr><td> CampyGenガス発生パック</td><td>オキソイド</td><td> CN0025 </td></tr><tr><td>微好気性室</td><td>オキソイド</td><td> HP0011 </td></tr><tr><td>トブラマイシン硫酸</td><td> Sigma-Aldrich社</td><td> T1783 </td></tr><tr><td>セルラーゼ、 アスペルギルスニジェールから</td><td> Sigma-Aldrich社</td><td> 22178 </td></tr><tr><td>レザズリン</td><td> Sigma-Aldrich社</td><td> 199303 </td></tr><tr><td> Citrate.H 2 0 </td><td> BDH </td><td> BDH0288 </td></tr><tr><td> Fluostarオメガマイクロプレートリーダー</td><td> BMG-LABTECH </td><td> SPECTROstarオメガ</td></tr></tbody></table>

use of artificial sputum medium to test antibiotic efficacy against pseudomonas aeruginosa in conditions more relevant to the cystic fibrosis lung

Pの分離株に適用した場合にin vitroで抗菌薬感受性試験での関連性についての懸念が高まっている嚢胞性線維症（CF）患者から緑膿菌 。既存の方法は好気的及びplanktonically成長した単一または少数の単離に依存しています。所定のカットオフは、細菌が敏感であるか、または任意の抗生物質を1に耐性があるかどうかを定義するために使用されます。しかし、CF、P.の慢性肺感染症の中に緑膿菌の集団は、バイオフィルム内に存在し、環境が2大部分は好気性であるという証拠があります。診断テスト中に肺の中の細菌とそれらの間に条件の厳しい違いは、信頼性、さらにはこれらのテスト3の妥当性に疑問を呼びかけている。 人工痰媒体（ASM）は、アミノ酸、ムチンと遊離DNAを含む、CF患者の痰の成分を含有する培養培地である。P.緑膿菌&lt;自己凝集バイオフィルムの構造と人口発散4,5,6の形成とCFの感染時にASMを模倣し成長に/ em&gt;に成長。本研究の目的は、Pの抗菌薬感受性を検討するためのマイクロタイタープレートアッセイを開発することでした緑膿菌は、微好気性、好気条件の両方に適用可能であるASM、成長に基づいています。 ASMアッセイは、マイクロタイタープレート形式で開発されました。P.は、緑膿菌のバイオフィルムは、24時間ごとに異なる濃度で前の抗菌剤とのインキュベーションの3日間用に開発させた。バイオフィルムの破壊後、細胞生存率はレサズリンで染色することによって測定した。このアッセイは、15種類のPのトブラマイシンの付着細胞の最小発育阻止濃度（SMIC）を確認するために使用された好気性および微好気条件とSMICの値の下で緑膿菌分離株は、標準的な培養液成長で得られたものと比較した。があったしながら、それらのプランクトンと比較すると、ASMで育った菌株の増加MIC値のいくつかの証拠が、最大の違いは、ASM、システム内のトブラマイシンに向かって&gt; 128倍に多くの抵抗の増加を示した微好気性条件でテスト細菌で見つかったとき好気的条件下で行われたアッセイと比較されます。 現在の感受性試験の方法と臨床転帰の間に関連性の欠如は、3つの現在の方法の妥当性を疑問視しています。 in vitroモデルのいくつかは、P.を研究するために以前に使用されている緑膿菌バイオフィルム7、8。 ASMバイオフィルムは、CFの肺9で観察されたもの似ているのに対し、しかしながら、これらの方法は、表面に接続されたバイオフィルムに依存しています。さらに、粘液の減少酸素濃度は、Pの動作を変更することが示されている緑膿菌 2および10抗生物質感受性に影響を与えます。したがって、ボイジャー微好気条件下でのG ASMは、抗菌薬感受性を勉強するのより現実的な環境を提供することがあります。

Watch this Scientific Journal Video about Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung at JoVE.com

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

現在の診断抗菌薬感受性試験では、栄養豊富な、好気的条件下での分離株のプランクトンの成長に依存しています。ここでは、嚢胞性線維症の肺のより代表的な好気性および微好気性条件の両方の下で緑膿菌バイオフィルムの抗菌薬感受性を検討する別の人工痰媒体を採用しています。

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 ...

use-artificial-sputum-medium-to-test-antibiotic-efficacy-against

Research

JoVE Journal

Immunology and Infection

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

32.5K ビュー.  University of Liverpool. 現在の診断抗菌薬感受性試験では、栄養豊富な、好気的条件下での分離株のプランクトンの成長に依存しています。ここでは、嚢胞性線維症の肺のより代表的な好気性および微好気性条件の両方の下で緑膿菌バイオフィルムの抗菌薬感受性を検討する別の人工痰媒体を採用しています。

ビデオ: Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

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 ...

免疫・感染学

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 ...

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. ...

A Robust Pneumonia Model in Immunocompetent Rodents to Evaluate Antibacterial Efficacy against S. pneumoniae, H. influenzae, K. pneumoniae, P. aeruginosa or A. baumannii

Efficacy of candidate antibacterial treatments must be demonstrated in animal models of infection as part of the discovery and development process, preferably in models which mimic the intended clinical indication. A method for inducing robust lung infections in immunocompetent rats and mice is described which allows for the assessment of treatments in a model of serious pneumonia caused by S. pneumoniae, H. influenzae, P. aeruginosa, K. pneumoniae or A. baumannii. Animals are anesthetized, and an agar-based inoculum is deposited deep into the lung via nonsurgical intratracheal intubation. The resulting infection is consistent, reproducible, and stable for at least 48 h and up to 96 h for most isolates. Studies with marketed antibacterials have demonstrated good correlation between in vivo efficacy and in vitro susceptibility, and concordance between pharmacokinetic/pharmacodynamic targets determined in this model and clinically accepted targets has been observed. Although there is an initial time investment when learning the technique, it can be performed quickly and efficiently once proficiency is achieved. Benefits of the model include elimination of the neutropenic requirement, increased robustness and reproducibility, ability to study more pathogens and isolates, improved flexibility in study design and establishment of a challenging infection in an immunocompetent host.

A Robust Pneumonia Model in Immunocompetent Rodents to Evaluate Antibacterial Efficacy against S. pneumoniae, H. influenzae, K. pneumoniae, P. aeruginosa or  A. baumannii

A method for establishing lung infections in immunocompetent rodents is described. With proficiency, this method can be performed quickly and easily to induce stable infection with many isolates of S. pneumoniae, H. influenzae, P. aeruginosa, K. pneumoniae and A. baumannii.

免疫担当げっ歯類で堅牢な肺炎モデルはに対して抗菌有効性を評価するために<i&gt; S。ニューモニエ</i&gt;、<i&gt; H。インフルエンザ</i&gt;、<i&gt; K。ニューモニエ</i&gt;、<em&gt; P。緑膿菌</em&gt;または<em&gt; A。バウマニ</em

Efficacy of candidate antibacterial treatments must be demonstrated in animal models of infection as part of the discovery and development process, ...

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the pro-inflammatory capabilities of various factors is challenging and requires terminal procedures, such as bronchoalveolar lavage and the removal of lungs for in situ analysis, precluding longitudinal visualization in the same mouse. Here, lung inflammation is induced through the intratracheal instillation of Pseudomonas aeruginosa culture supernatant (SN) in transiently transgenized mice expressing the luciferase reporter gene under the control of a heterologous IL-8 bovine promoter. Luciferase expression in the lung is monitored by in vivo bioluminescent image (BLI) analysis over a 2.5- to 48-h timeframe following the instillation. The procedure can be repeated multiple times within 2 - 3 months, thus permitting the evaluation of the inflammatory response in the same mice without the need to terminate the animals. This approach permits the monitoring of pro- and anti-inflammatory factors acting in the lung in real time and appears suitable for functional and pharmacological studies.

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

The method described here allows for the visualization of IL-8 promoter-dependent inflammation activation in the lungs of mice through non-invasive bioluminescence imaging (BLI). The same animal can be subjected to BLI multiple times for up to two months from the time of delivery of the luciferase reporter construct.

IL-8トランジェントトランスジェニックマウスモデル<em&gt;インビボ</em&gt;炎症反応の長期モニタリング

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the ...

A Method to Test the Efficacy of Handwashing for the Removal of Emerging Infectious Pathogens

Handwashing is widely recommended to prevent infectious disease transmission. However, little comparable evidence exists on the efficacy of handwashing methods in general. Additionally, little evidence exists comparing handwashing methods to determine which are most efficacious at removing infectious pathogens. Research is needed to provide evidence for the different approaches to handwashing that may be employed during infectious disease outbreaks. Here, a laboratory method to assess the efficacy of handwashing methods at removing microorganisms from hands and their persistence in rinse water is described. Volunteers' hands are first spiked with the test organism and then washed with each handwashing method of interest. Generally, surrogate microorganisms are used to protect human subjects from disease. The number of organisms remaining on volunteers' hands after washing is tested using a modified "glove juice" method: the hands are placed in gloves with an eluent and are scrubbed to suspend the microorganisms and make them available for analysis by membrane filtration (bacteria) or plaque assay (viruses/bacteriophages). Rinse water produced from the handwashing is directly collected for analysis. Handwashing efficacy is quantified by comparing the log reduction value between samples taken after handwashing to samples with no handwashing. Rinse water persistence is quantified by comparing rinse water samples from various handwashing methods to samples collected after handwashing with just water. While this method is limited by the need to use surrogate organisms to preserve the safety of human volunteers, it captures aspects of handwashing that are difficult to replicate in an in vitro study and fills research gaps on handwashing efficacy and the persistence of infectious organisms in rinse water.

Handwashing is widely recommended to prevent infectious disease transmission. However, there is little evidence on which handwashing methods are most efficacious at removing infectious disease pathogens. We developed a method to assess the efficacy of handwashing methods at removing microorganisms.

発生する感染性病原体の除去のための手洗いの有効性を試験する方法

Handwashing is widely recommended to prevent infectious disease transmission. However, little comparable evidence exists on the efficacy of handwashing ...

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 ...

Phage Therapy Application to Counteract Pseudomonas aeruginosa Infection in Cystic Fibrosis Zebrafish Embryos

Antimicrobial resistance, a major consequence of diagnostic uncertainty and antimicrobial overprescription, is an increasingly recognized cause of severe infections, complications, and mortality worldwide with a huge impact on our society and on the health system. In particular, patients with compromised immune systems or pre-existing and chronic pathologies, such as cystic fibrosis (CF), are subjected to frequent antibiotic treatments to control the infections with the appearance and diffusion of multidrug resistant isolates. Therefore, there is an urgent need to address alternative therapies to counteract bacterial infections. Use of bacteriophages, the natural enemies of bacteria, can be a possible solution. The protocol detailed in this work describes the application of phage therapy against Pseudomonas aeruginosa infection in CF zebrafish embryos. Zebrafish embryos were infected with P. aeruginosa to demonstrate that phage therapy is effective against P. aeruginosa infections as it reduces lethality, bacterial burden and pro-inflammatory immune response in CF embryos.

Phage Therapy Application to Counteract Pseudomonas aeruginosa Infection in Cystic Fibrosis Zebrafish Embryos

Presented here is a protocol for Pseudomonas aeruginosa infection and phage therapy application in cystic fibrosis (CF) zebrafish embryos.

嚢胞性線維症ゼブラフィッシュ胚における シュードモナス緑膿症 感染に対抗するためのファージ療法アプリケーション

Antimicrobial resistance, a major consequence of diagnostic uncertainty and antimicrobial overprescription, is an increasingly recognized cause of severe ...

Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients

Early detection and eradication of Pseudomonas aeruginosa within the lungs of cystic fibrosis patients can reduce the chance of developing chronic infection. The development of chronic P. aeruginosa infections is associated with a decline in lung function and increased morbidity. Therefore, there is a great interest in elucidating the reasons for the failure to eradicate P. aeruginosa with antibiotic therapy which occurs in approximately 10-40% of pediatric patients. One of many factors that can affect host clearance of P. aeruginosa and antibiotic susceptibility is variations in spatial organization (such as aggregation or biofilm formation) and polysaccharide production. Therefore, we were interested in visualizing the in situ characteristics of P. aeruginosa within the sputum of CF patients. A tissue clearing technique was applied to sputum samples after embedding the samples into a hydrogel matrix to retain the 3D structures relative to host cells. After tissue clearing, fluorescent labels and dyes were added to allow visualization. Fluorescence in situ hybridization was performed for the visualization of bacterial cells, binding of fluorescently labeled anti-Psl-antibodies for the visualization of the exopolysaccharide and DAPI staining to stain host cells to obtain structural insight. These methods allowed for the high-resolution imaging of P. aeruginosa within the sputum of CF patients via confocal laser scanning microscopy.

Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients

This protocol provides methods for visualization of bacterial cells and polysaccharide synthesis locus (Psl) polysaccharide within the sputum of cystic fibrosis patients.