TBは、嚢胞性線維症、カナダからの研究フェローシップを認めています。

研究倫理委員会（REB）は、ヒト被験者から痰サンプルを収集し、保存するために必要なことに注意してください。これらの研究は、病気の子供REBの＃1000019444のために病院によって承認されました。 1.準備CF痰試料<ol><li>嚢胞性線維症の診療所へのルーチンの訪問中に、患者からの痰のサンプルを収集し、氷上に保ちます。 </li><li>処理を受けるために、研究室に、コレクションの最初の一時間以内に氷上に喀痰試料を輸送。 </li></ol> 2.喀痰処理<ol><li>得られた痰試料の体積を記録します。サンプル（ すなわち、2部PBS、1部の試料）の体積を2倍のリン酸緩衝生理食塩水（PBS）を加えます。 </li><li>トランスファーピペットでよくサンプルを混ぜます。 1分が完全に混合するための最高の設定にボルテックスのサンプルを。 </li><li>適切な数の1.5ミリリットルのマイクロチューブに小分けし、上記混合物の1ミリリットルをし、5,000×gでスピンダウン4℃で20分間。 </li><li>遠心分離後、上清を除去し、ペレットを捨てます。 </li><li>フィルタは、0.22μmフィルターを通して上清を殺菌し、清潔なマイクロ遠心チューブに集めます。 注：ろ液の無菌性は、LB寒天上にプレーティングし、液体培地に接種することによってテストされています。 </li><li>将来の使用のために-80℃で痰上清を保管してください。 注：複数の患者からの喀痰もろ過以下のプールすることができます。 </li><li>使用前に、所望のメディアに喀痰ろ液1/10 V / V（痰を100μl、メディアの900μl）を希釈します。 注記：ここでは、標準のLB培地（LB）培地を用いました。 </li></ol>バイオフィルム形成のための3チェンカバーガラス法<ol><li>振盪（200rpm）しながら37℃で、所望のメディアの関心を一晩の細菌分離株を成長させます。 注：別の細菌数を使用した、 緑膿菌 、 黄色ブドウ球菌の臨床分離株を含みます、バークホルデリア・セパシア錯体とアクロのxylosoxidans。媒体の選択は、目的の株および条件に依存し、しかしLB培地は、最初の実験のために使用することができます。 </li><li>一晩培養物から、新鮮な培地の4ミリリットル中に文化の40μlのを配置し、約0.5の600nmで（OD 600）での光学密度と文化を得るために、振盪（200rpm）しながら37℃で3-4時間成長-0.6。 </li><li> 10％の喀痰ろ液または（対照として）痰ろ液をせずに所望のメディアで1/5にステップ3.2からの培養を希釈します。痰ろ液の他の濃度を試験することができる（ すなわち、50％または100％）。 </li><li>スライドチャンバーのウェルを播種するために希釈液200μlを使用してください。 </li><li>細菌は振盪せずに37℃で4時間アタッチすることができます。 </li><li> 4時間後、メディアを取り出し、軽く1xの新鮮な培地でバイオフィルムを洗います。 200μlの新鮮な培地と交換してください。 注：バイオフィルム上の痰の効果を研究するために、FRES時間メディアは、痰上清を含める必要があります。 </li><li>バイオフィルムは、顕微鏡検査のための時間になるまで洗浄することなく、メディアごとに12時間を置き換え、振盪せずに37℃で、所望の時間のために成長することを可能にします。 注：バイオフィルムの抗生物質感受性に痰上清の効果を研究するためには、抗生物質は、バイオフィルムの成長の24時間後の培地に添加され、染色およびバイオフィルムの撮影まで培地中で維持されています。 </li></ol> 4.染色バイオフィルムと共焦点顕微鏡<ol><li>所望の成長時間（24〜48時間が最高の作品）に続いて、チャンバーウェルから培地を除去し、静かに無菌PBS300μlで2回チャンバーを洗います。 </li><li>必要なソリューションの各ミリリットルのための（生存率キットで提供される）各色素の1μLを混合することにより、バイオフィルムに対する染色混合物を準備します。水やメディア溶液中の色素を作ります。 注：水は、製造業者によって推奨されています。 </li><li>チャンバ型coverglaの各ウェルに染料混合物の200μLを追加ssは暗所で45分間、室温でインキュベートし、。 </li><li>室から染色混合物を削除し、滅菌PBS300μlで各ウェルを洗浄します。 PBSを削除し、新鮮な水やメディアと交換してください。 </li><li>共焦点顕微鏡を介したバイオフィルムの可視化を進めます。 </li></ol> 5.共焦点顕微鏡を用いてバイオフィルムの可視化<ol><li>すぐに（1時間以内）染色した後、チャンバ内の染色されたバイオフィルムをお読みください。一度に2 8ウェルチャンバーに1を染色することによって、スライドの可視化の遅れを最小限に抑えます。 </li><li>取得のための励起とフィルターのセットのためのレーザーで共焦点顕微鏡を用いて撮影を行います。 注：ここでは、スペクトルボレアリスレーザーとスピニングディスク共焦点システム（緑：491nm、赤：561 nm）と励起のために使用しました。 40分の515と40分の624の発光フィルターセットは、バイオフィルムの生存率キットから汚れを視覚化するために使用されました。 </li><li>カメラと共焦点顕微鏡で25X水対物レンズを用いて画像を撮影します。 </li><li&gt;を使用してZ-スタックバイオフィルムをモデル化します。 （一般的に48時間のバイオフィルムのための30から80ミクロンに及ぶ）、バイオフィルムの最後の合焦フレームに最初の合焦面から出発した画像ごとに0.5から1ミクロンを取ります</li><li>各ウェルから3-5画像を撮影します。 注：このように8よくチャンバーカバーガラスのために、24から40の画像が生成されます。 </li><li>分析のために画像を保存します。 注：画像は、犯罪統計システム18,19を用いて分析するOME-TIFFファイルとして保存する必要があります。バイオフィルムの画像解析の手順については、http://www.comstat.dk/で見つけることができます。画像がインポートされると、そのような各チャネル（赤と緑）の平均厚さは、バイオマスおよび表面被覆率などのパラメータを分析することができます。 </li></ol>

<ol><li>Beaudoin, T., Waters, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Infections+with+biofilm+formation%3A+selection+of+antimicrobials+and+role+of+prolonged+antibiotic+therapy%5BTitle%5D)+AND+%22Pediatr.Infect.Dis.J%22%5BJournal%5D)">Infections with biofilm formation: selection of antimicrobials and role of prolonged antibiotic therapy.</a> Pediatr.Infect.Dis.J. , (2016).</li>
<li>Donlan, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biofilms%3A+microbial+life+on+surfaces%5BTitle%5D)+AND+%22Emerg.Infect.Dis%22%5BJournal%5D)">Biofilms: microbial life on surfaces.</a> Emerg.Infect.Dis. 8 (9), 881-890 (2002).</li>
<li>Hobley, L., Harkins, C., MacPhee, C. E., Stanley-Wall, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Giving+structure+to+the+biofilm+matrix%3A+an+overview+of+individual+strategies+and+emerging+common+themes%5BTitle%5D)+AND+%22FEMS+Microbiol.Rev%22%5BJournal%5D)">Giving structure to the biofilm matrix: an overview of individual strategies and emerging common themes.</a> FEMS Microbiol.Rev. 39 (5), 649-669 (2015).</li>
<li>Katharios-Lanwermeyer, S., Xi, C., Jakubovics, N. S., Rickard, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mini-review%3A+Microbial+coaggregation%3A+ubiquity+and+implications+for+biofilm+development%5BTitle%5D)+AND+%22Biofouling%22%5BJournal%5D)">Mini-review: Microbial coaggregation: ubiquity and implications for biofilm development.</a> Biofouling. 30 (10), 1235-1251 (2014).</li>
<li>Donlan, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biofilm+formation%3A+a+clinically+relevant+microbiological+process%5BTitle%5D)+AND+%22Clin.Infect.Dis%22%5BJournal%5D)">Biofilm formation: a clinically relevant microbiological process.</a> Clin.Infect.Dis. 33 (8), 1387-1392 (2001).</li>
<li>Bjarnsholt, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+in+vivo+biofilm%5BTitle%5D)+AND+%22Trends+Microbiol%22%5BJournal%5D)">The in vivo biofilm.</a> Trends Microbiol. 21 (9), 466-474 (2013).</li>
<li>Mah, T. F., Pitts, B., Pellock, B., Walker, G. C., Stewart, P. S., O'Toole, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+genetic+basis+for+Pseudomonas+aeruginosa+biofilm+antibiotic+resistance%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">A genetic basis for Pseudomonas aeruginosa biofilm antibiotic resistance.</a> Nature. 426 (6964), 306-310 (2003).</li>
<li>Mah, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biofilm-specific+antibiotic+resistance%5BTitle%5D)+AND+%22Future+Microbiol%22%5BJournal%5D)">Biofilm-specific antibiotic resistance.</a> Future Microbiol. 7 (9), 1061-1072 (2012).</li>
<li>Beaudoin, T., Zhang, L., Hinz, A. J., Parr, C. J., Mah, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+biofilm-specific+antibiotic+resistance+gene+ndvB+is+important+for+expression+of+ethanol+oxidation+genes+in+Pseudomonas+aeruginosa+biofilms%5BTitle%5D)+AND+%22J.+Bacteriol%22%5BJournal%5D)">The biofilm-specific antibiotic resistance gene ndvB is important for expression of ethanol oxidation genes in Pseudomonas aeruginosa biofilms.</a> J. Bacteriol. 194 (12), 3128-3136 (2012).</li>
<li>Beaudoin, T., Aaron, S. D., Giesbrecht-Lewis, T., Vandemheen, K., Mah, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterization+of+clonal+strains+of+Pseudomonas+aeruginosa+isolated+from+cystic+fibrosis+patients+in+Ontario%2C+Canada%5BTitle%5D)+AND+%22Can.+J.+Microbiol%22%5BJournal%5D)">Characterization of clonal strains of Pseudomonas aeruginosa isolated from cystic fibrosis patients in Ontario, Canada.</a> Can. J. Microbiol. 56 (7), 548-557 (2010).</li>
<li>Vidya, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chronic+infection+phenotypes+of+Pseudomonas+aeruginosa+are+associated+with+failure+of+eradication+in+children+with+cystic+fibrosis%5BTitle%5D)+AND+%22Eur.J.Clin.Microbiol.Infect.Dis%22%5BJournal%5D)">Chronic infection phenotypes of Pseudomonas aeruginosa are associated with failure of eradication in children with cystic fibrosis.</a> Eur.J.Clin.Microbiol.Infect.Dis. , (2015).</li>
<li>Beaudoin, T., Lafayette, S., Nguyen, D., Rousseau, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mucoid+Pseudomonas+aeruginosa+caused+by+mucA+mutations+result+in+activation+of+TLR2+in+addition+to+TLR5+in+airway+epithelial+cells%5BTitle%5D)+AND+%22Biochem.Biophys.Res.Commun%22%5BJournal%5D)">Mucoid Pseudomonas aeruginosa caused by mucA mutations result in activation of TLR2 in addition to TLR5 in airway epithelial cells.</a> Biochem.Biophys.Res.Commun. 428 (1), 150-154 (2012).</li>
<li>Beaudoin, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+level+of+p38alpha+mitogen-activated+protein+kinase+activation+in+airway+epithelial+cells+determines+the+onset+of+innate+immune+responses+to+planktonic+and+biofilm+Pseudomonas+aeruginosa%5BTitle%5D)+AND+%22J.Infect.Dis%22%5BJournal%5D)">The level of p38alpha mitogen-activated protein kinase activation in airway epithelial cells determines the onset of innate immune responses to planktonic and biofilm Pseudomonas aeruginosa.</a> J.Infect.Dis. 207 (10), 1544-1555 (2013).</li>
<li>LaFayette, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cystic+fibrosis-adapted+quorum+sensing+mutants+cause+hyperinflammatory+responses%5BTitle%5D)+AND+%22Sci.Adv%22%5BJournal%5D)">Cystic fibrosis-adapted quorum sensing mutants cause hyperinflammatory responses.</a> Sci.Adv. 1 (6), e1500199(2015).</li>
<li>Staudinger, B. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Conditions+associated+with+the+cystic+fibrosis+defect+promote+chronic+Pseudomonas+aeruginosa+infection%5BTitle%5D)+AND+%22Am.J.Respir.Crit.Care+Med%22%5BJournal%5D)">Conditions associated with the cystic fibrosis defect promote chronic Pseudomonas aeruginosa infection.</a> Am.J.Respir.Crit.Care Med. 189 (7), 812-824 (2014).</li>
<li>Kennedy, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Activity+of+Tobramycin+against+Cystic+Fibrosis+Isolates+of+Burkholderia+cepacia+Complex+Grown+as+Biofilms%5BTitle%5D)+AND+%22Antimicrob.Agents+Chemother%22%5BJournal%5D)">Activity of Tobramycin against Cystic Fibrosis Isolates of Burkholderia cepacia Complex Grown as Biofilms.</a> Antimicrob.Agents Chemother. 60 (1), 348-355 (2015).</li>
<li>Tom, S. K., Yau, Y. C., Beaudoin, T., LiPuma, J. J., Waters, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+of+High-Dose+Antimicrobials+on+Biofilm+Growth+of+Achromobacter+Species+Isolated+from+Cystic+Fibrosis+Patients%5BTitle%5D)+AND+%22Antimicrob.Agents+Chemother%22%5BJournal%5D)">Effect of High-Dose Antimicrobials on Biofilm Growth of Achromobacter Species Isolated from Cystic Fibrosis Patients.</a> Antimicrob.Agents Chemother. 60 (1), 650-652 (2015).</li>
<li>Heydorn, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantification+of+biofilm+structures+by+the+novel+computer+program+COMSTAT%5BTitle%5D)+AND+%22Microbiology%22%5BJournal%5D)">Quantification of biofilm structures by the novel computer program COMSTAT.</a> Microbiology. 146 (Pt 10), 2395-2407 (2000).</li>
<li>Vorregaard, M. Comstat2 - a modern 3D image analysis environment for biofilms
 . <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aVorregaard%2C+M+%22Informatics+and+Mathematical+Modelling%22">Informatics and Mathematical Modelling</a>. , Technical University of Denmark. Kongens Lyngby, Denmark. (2008).</li>
<li>Jurcisek, J. A., Dickson, A. C., Bruggeman, M. E., Bakaletz, L. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vitro+Biofilm+Formation+in+an+8-well+Chamber+Slide%5BTitle%5D)+AND+%22J.+Vis.+Exp%22%5BJournal%5D)">In vitro Biofilm Formation in an 8-well Chamber Slide.</a> J. Vis. Exp. (47), e2481(2011).</li>
</ol>

本明細書に記載される方法は、外因性生成物の存在下で増殖させた細菌性バイオフィルムの可視化を可能にします。このタイプのシステムを使用する場合に当然のことながら、exoproductsの生産が重要です。例えば、ジチオスレイトール（DTT）は、多くの場合、サンプルを液化するために役立つ人間の唾液サンプルに使用されています。しかし、単独でDTTの効果は、バイオフィルムの発達およ?...

細菌バイオフィルムは、互いに結合し、自己分泌マトリックスに包まれている微生物の基です。 1,2古典的には、それらが物理的に流れの条件の下で形成された非生物または生物の表面に付着した細菌を表します。バイオフィルムはまた、試験管内で形成された熱プールまたはペリクルの空気 - 液体界面でのような表面からの静的条件（流れの欠如）および遠位で成長することが示されています。これらのバイオフィルムは、長い環境で認識され、彼らは生物付着、腐食や閉塞が生じ、貯水池内またはパイプで形成することができるよう、産業プロセスへの主要な不利益であるされています。 3,4 彼らはカテーテル関連感染症、嚢胞性線維症の患者では、だけでなく、多数の他の感染症における肺感染症に関与することが示されているようにバイオフィルムは、また、医療現場で重要です。 5,6バイオフィルム感染症の特徴の一つは、デです抗生物質に対する細菌の感受性を折り目と自然免疫系によるクリアランスを損ないます。バイオフィルムベースの感染を伴う7-9で最もよく研究され、臨床的に関連するシナリオが慢性的に緑膿菌バイオフィルムに感染している嚢胞性線維症（CF）の患者に起こります。 緑膿菌は、それが非常に困難治療することを可能にする慢性感染症の確立中に変更の数を受けることができます。 10,11バイオフィルムは、示差的に先天性免疫を活性化し、炎症を駆動することができます。 12-14これらの感染症は、CF患者において増加罹患率および死亡率につながるように、この文脈では、バイオフィルム発達に影響を与えることができる因子を理解することが重要です。 最近の研究は、宿主因子が緑膿菌バイオフィルムの凝集体の形成に重要であることを示唆しています。 15これらのバイオフィルムは、抗生物質および宿主防御機構に対する感受性の低下に貢献しています。 presaの複数形CF肺中に存在する微生物から、このような好中球エラスターゼなどの宿主由来因子、ならびに分泌物のNCEは、大幅にバイオフィルム形成と発展を調節する可能性を秘めています。 16はまた、バイオフィルムは、多数の経路の発現を調節し、炎症を開始するためにホストと対話します。標準的なクリスタルバイオレットアッセイ等のハイスループット方法が、バイオフィルムの処理に関していくつかの情報を提供することができ、これらの因子に応答したバイオフィルムの可視化は、より詳細な情報を提供します。 本稿では、 インビトロでのバイオフィルムの発達を研究するためにCF患者の痰からの係数を使用する方法を記載しています。この方法は、市販のバイオフィルムの生存率キットを使用して痰を含む宿主因子に曝露されたバイオフィルムの迅速な可視化を可能にします。この技術は、視覚的にexogenoの存在下でのバイオフィルムの成長中に発生する変化を同定するために使用され得ます私たちの製品、および様々な条件下でのバイオフィルムの開発の変化を分析するための改良された方法を表しています。

<table><tbody><tr><td>Lab-Tek II Chambered coverglass, #1.5 borosilicate, 8-well</td><td>Thermo Sicher Scientific</td><td>155409</td><td></td></tr><tr><td>Filmtracer Live/Dead Biofilm Viabilty Kit</td><td>Thermo Fisher Scientific</td><td>L10316</td><td></td></tr><tr><td>Blood agar plates</td><td>Thermo Fisher Scientific</td><td>R10215</td><td>Confirming viability via CFU counts or selecting colonies for innoculation</td></tr><tr><td>COMSTAT</td><td>Availble software online</td><td></td><td>COMSTAT is software to analyze biofilm images. Available www.comstat.dk&amp;nbsp;</td></tr><tr><td>Millers LB Broth</td><td>Thermo Fisher Scientific</td><td>12780-052</td><td>Standard media for overnight gowth/biofilm growth</td></tr><tr><td>Millex-GV Syringe Filters</td><td>Millipore</td><td>SLGV013SL</td><td>Filtering of sputum supernants</td></tr><tr><td>Phosphate Buffered Saline (Dulbecco A)</td><td>Oxoid</td><td>BR0014G</td><td>Washing of biofilm chambers after media removal</td></tr><tr><td>Zeiss AxioVert 200M</td><td>Carl Zeiss</td><td></td><td></td></tr><tr><td>Hamamatsu C9100-13 EM-CCD</td><td>QS Technologies Inc.</td><td></td><td></td></tr><tr><td>Spectral Borealis</td><td>Qs Technologies Inc.</td><td></td><td></td></tr><tr><td>Perkin Elmer Volocity</td><td>QS Technologies Inc.</td><td></td><td>Instructions for this software can be found at: http://cellularimaging.perkinelmer.com/pdfs/manuals/VolocityuserGuide.pdf</td></tr></tbody></table>

visualizing the effects of sputum on biofilm development using a chambered coverglass model

バイオフィルムは、自己分泌マトリックスに包まれた細菌のグループで構成されています。彼らは、工業汚染だけでなく、多くの健康に関連する感染症の発症と持続に重要な役割を果たしています。ヒトの疾患の中で最もよく説明し、研究バイオフィルムの一つは、嚢胞性線維症患者の慢性肺感染症で発生します。ホストのコンテキストでバイオフィルムを研究すると、多くの要因は、バイオフィルム形成と発展に影響を与えることができます。宿主因子はバイオフィルムの形成及び発達に影響を与えることができる方法を同定するために、我々は、痰上清の形で宿主由来因子の存在下でバイオフィルムを成長させるために、静的チェンカバーガラスの方法を使用しました。細菌は、チャンバーに播種し、痰ろ液にさらされています。成長の48時間後に、バイオフィルムは、共焦点顕微鏡および分析の前に商業バイオフィルムの生存率キットで染色されています。画像取得後、バイオフィルムの特性は、異なるソフトウェア・プラットフォームを用いて評価することができます。この方法は、私たちは、抗生物質などの異なる物質の存在下でのバイオフィルムの成長の重要な特性を視覚化することができます。

実験の全体的な設計は、図1に示されています。このプロトコルの使用は、異なる時間（ 例えば、24、48または72時間）増殖させたバイオフィルムの変化を視覚化するための便利な方法を提供します。重要なことには、例えば、痰濾液ような外因性シグナルは、バイオフィルムの発達の変化を視覚化するために添加することができます。 図2</str...

Watch this Scientific Journal Video about Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model at JoVE.com

Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

This protocol describes the visualization of biofilm development following exposure to host-factors using a slide chamber model. This model allows for direct visualization of biofilm development as well as analysis of biofilm parameters using computer software programs.

チェンカバーガラスモデルを用いたバイオフィルム開発に関する痰の効果を可視化

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the ...

visualizing-effects-sputum-on-biofilm-development-using-chambered

Research

JoVE Journal

Immunology and Infection

8.3K ビュー.  Hospital for Sick Children. This protocol describes the visualization of biofilm development following exposure to host-factors using a slide chamber model. This model allows for direct visualization of biofilm development as well as analysis of biofilm parameters using computer software programs.

ビデオ: Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass 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

In vitro Biofilm Formation in an 8-well Chamber Slide

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. Biofilms are community-associated bacteria attached to a surface and encased in a matrix. The role of the extracellular matrix is multifaceted, including facilitating nutrient acquisition, and offers significant protection against environmental stresses (e.g. host immune responses). In an effort to acquire a better understanding as to how the bacteria within a biofilm respond to environmental stresses we have used a protocol wherein we visualize bacterial biofilms which have formed in an 8-well chamber slide. The biofilms were stained with the BacLight Live/Dead stain and examined using a confocal microscope to characterize the relative biofilm size, and structure under varying incubation conditions. Z-stack images were collected via confocal microscopy and analyzed by COMSTAT. This protocol can be used to help elucidate the mechanism and kinetics by which biofilms form, as well as identify components that are important to biofilm structure and stability.

In vitro Biofilm Formation in an 8-well Chamber Slide

This article describes the procedure for the formation and visualization of a bacterial biofilm grown within an 8-well chamber slide

8ウェルチャンバースライドを用いたin vitro のバイオフィルム形成における

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. ...

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including antibiotics. Although stress tolerance of M. tuberculosis is one of the likely contributors to the 6-month long chemotherapy of tuberculosis 1, the molecular mechanisms underlying this characteristic phenotype of the pathogen remain unclear. Many microbial species have evolved to survive in stressful environments by self-assembling in highly organized, surface attached, and matrix encapsulated structures called biofilms 2-4. Growth in communities appears to be a preferred survival strategy of microbes, and is achieved through genetic components that regulate surface attachment, intercellular communications, and synthesis of extracellular polymeric substances (EPS) 5,6. The tolerance to environmental stress is likely facilitated by EPS, and perhaps by the physiological adaptation of individual bacilli to heterogeneous microenvironments within the complex architecture of biofilms 7.
In a series of recent papers we established that M. tuberculosis and Mycobacterium smegmatis have a strong propensity to grow in organized multicellular structures, called biofilms, which can tolerate more than 50 times the minimal inhibitory concentrations of the anti-tuberculosis drugs isoniazid and rifampicin 8-10. M. tuberculosis, however, intriguingly requires specific conditions to form mature biofilms, in particular 9:1 ratio of headspace: media as well as limited exchange of air with the atmosphere 9. Requirements of specialized environmental conditions could possibly be linked to the fact that M. tuberculosis is an obligate human pathogen and thus has adapted to tissue environments. In this publication we demonstrate methods for culturing M. tuberculosis biofilms in a bottle and a 12-well plate format, which is convenient for bacteriological as well as genetic studies. We have described the protocol for an attenuated strain of M. tuberculosis, mc27000, with deletion in the two loci, panCD and RD1, that are critical for in vivo growth of the pathogen 9. This strain can be safely used in a BSL-2 containment for understanding the basic biology of the tuberculosis pathogen thus avoiding the requirement of an expensive BSL-3 facility. The method can be extended, with appropriate modification in media, to grow biofilm of other culturable mycobacterial species.
Overall, a uniform protocol of culturing mycobacterial biofilms will help the investigators interested in studying the basic resilient characteristics of mycobacteria. In addition, a clear and concise method of growing mycobacterial biofilms will also help the clinical and pharmaceutical investigators to test the efficacy of a potential drug.

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis forms drug tolerant biofilms when cultured in certain conditions. Here we describe methods for culturing M. tuberculosis biofilms and determining the frequency of drug tolerant persisters. These protocols will be useful for further studies into the mechanisms of drug tolerance in M. tuberculosis.

の成長結核バイオフィルム

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including ...

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 ...

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and resilience of Bacillus subtilis biofilms. First, methods are presented that select for small molecule inhibitors with biofilm-specific targets in order to separate the effect of the small molecule inhibitors on planktonic growth from their effect on biofilm formation. Next, we focus on how inoculation conditions affect the sensitivity of multicellular, floating B. subtilis cultures to small molecule inhibitors. The results suggest that discrepancies in the reported effects of such inhibitors such as D-amino acids are due to inconsistent pre-culture conditions. Furthermore, a recently developed protocol is described for evaluating the contribution of small molecule treatments towards biofilm resistance to antibacterial substances. Lastly, scanning electron microscopy (SEM) techniques are presented to analyze the three-dimensional spatial arrangement of cells and their surrounding extracellular matrix in a B. subtilis biofilm. SEM facilitates insight into the three-dimensional biofilm architecture and the matrix texture. A combination of the methods described here can greatly assist the study of biofilm development in the presence and absence of biofilm inhibitors, and shed light on the mechanism of action of these inhibitors.

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This study presents the development of reproducible methodologies to study biofilm inhibitors and their effects on Bacillus subtilis multicellularity.

研究のための方法論<em&gt; B。枯草菌</em小分子バイオフィルム阻害剤を特徴付けるためのモデルとしての&gt;バイオフィルム

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and ...

The WinCF Model - An Inexpensive and Tractable Microcosm of a Mucus Plugged Bronchiole to Study the Microbiology of Lung Infections

Many chronic airway diseases result in mucus plugging of the airways. Lungs of an individual with cystic fibrosis are an exemplary case where their mucus-plugged bronchioles create a favorable habitat for microbial colonization. Various pathogens thrive in this environment interacting with each other and driving many of the symptoms associated with CF disease. Like any microbial community, the chemical conditions of their habitat have a significant impact on the community structure and dynamics. For example, different microorganisms thrive in differing levels of oxygen or other solute concentrations. This is also true in the CF lung, where oxygen concentrations are believed to drive community physiology and structure. The methods described here are designed to mimic the lung environment and grow pathogens in a manner more similar to that from which they cause disease. Manipulation of the chemical surroundings of these microbes is then used to study how the chemistry of lung infections governs its microbial ecology. The method, called the WinCF system, is based on artificial sputum medium and narrow capillary tubes meant to provide an oxygen gradient similar to that which exists in mucus-plugged bronchioles. Manipulating chemical conditions, such as the media pH of the sputum or antibiotics pressure, allows for visualization of the microbiological differences in those samples using colored indicators, watching for gas or biofilm production, or extracting and sequencing the nucleic acid contents of each sample.

The mucus plugged airways of cystic fibrosis (CF) patients are an ideal environment for microbial pathogens to thrive. The manuscript describes a novel method for studying the CF lung microbiome in an environment that mimics where they cause disease and how alterations of chemical conditions can drive microbial dynamics.

WinCFモデル - 肺感染症の微生物学を研究するための粘液で閉鎖された細気管支の安価で収縮可能なマイクロコスモス

Many chronic airway diseases result in mucus plugging of the airways. Lungs of an individual with cystic fibrosis are an exemplary case where their ...

Quantifying the Effects of Antimicrobials on In vitro Biofilm Architecture using COMSTAT Software

Biofilms are aggregates of microorganisms that rely on a self-produced matrix of extracellular polymeric substance for protection and structural integrity. The nosocomial pathogen, Pseudomonas aeruginosa, is known to adopt a biofilm mode of growth, causing chronic pulmonary infection in patients with cystic fibrosis (CF). The computer program, COMSTAT, is a useful tool for quantifying antimicrobial-induced changes in P. aeruginosa biofilm architecture by extracting data from three-dimensional confocal images. However, standardized operation of the software is less commonly addressed, which is important for optimal reporting of biofilm behavior and cross-center comparison. Thus, the aim of this protocol is to provide a simple and reproducible framework for quantifying in vitro biofilm structures under varying antimicrobial conditions via COMSTAT. The technique is modeled using a CF P. aeruginosa isolate, grown in the form of biofilm replicates, and exposed to tobramycin and the anti-Psl monoclonal antibody, Psl0096. The step-by-step approach aims to reduce user ambiguity and minimize the chance of overlooking crucial image-processing steps. Specifically, the protocol emphasizes the elimination of subjective variations associated with the manual operation of COMSTAT, including image segmentation and the selection of appropriate quantitative analysis functions. Although this method requires users to spend additional time processing confocal images prior to running COMSTAT, it helps minimize misrepresented biofilm heterogenicity in automated outputs.

Quantifying the Effects of Antimicrobials on In vitro Biofilm Architecture using COMSTAT Software

Antimicrobial-induced changes to Pseudomonas aeruginosa biofilm architecture differ among clinical isolates cultured from patients with cystic fibrosis and chronic pulmonary infection. Following confocal microscopy, COMSTAT software can be utilized to quantify variations in biofilm architecture (e.g., surface area, thickness, biomass) for individual isolates to assess the efficacy of anti-infective agents.

COMSTATソフトウェアを使用した In vitro バイオフィルムアーキテクチャに対する抗菌剤の影響の定量化

Biofilms are aggregates of microorganisms that rely on a self-produced matrix of extracellular polymeric substance for protection and structural ...

Antibiotic Efficacy Testing in an Ex vivo Model of Pseudomonas aeruginosa and Staphylococcus aureus Biofilms in the Cystic Fibrosis Lung

The effective prescription of antibiotics for the bacterial biofilms present within the lungs of individuals with cystic fibrosis (CF) is limited by a poor correlation between antibiotic susceptibility testing (AST) results using standard diagnostic methods (e.g., broth microdilution, disk diffusion, or Etest) and clinical outcomes after antibiotic treatment. Attempts to improve AST by the use of off-the-shelf biofilm growth platforms show little improvement in results. The limited ability of in vitro biofilm systems to mimic the physicochemical environment of the CF lung and, therefore bacterial physiology and biofilm architecture, also acts as a brake on the discovery of novel therapies for CF infection. Here, we present a protocol to perform AST of CF pathogens grown as mature, in vivo-like biofilms in an ex vivo CF lung model comprised of pig bronchiolar tissue and synthetic CF sputum (ex vivo&#160;pig lung, EVPL).
Several in vitro&#160;assays exist for biofilm susceptibility testing, using either standard laboratory medium or various formulations of synthetic CF sputum in microtiter plates. Both growth medium and biofilm substrate (polystyrene plate vs. bronchiolar tissue) are likely to affect biofilm antibiotic tolerance. We show enhanced tolerance of clinical Pseudomonas aeruginosa and Staphylococcus aureus isolates in the ex vivo model; the effects of antibiotic treatment of biofilms is not correlated with the minimum inhibitory concentration (MIC) in standard microdilution assays or a sensitive/resistant classification in disk diffusion assays.
The ex vivo platform could be used for bespoke biofilm AST of patient samples and as an enhanced testing platform for potential antibiofilm agents during pharmaceutical research and development. Improving the prescription or acceleration of antibiofilm drug discovery through the use of more in vivo-like testing platforms could drastically improve health outcomes for individuals with CF, as well as reduce the costs of clinical treatment and discovery research.

Antibiotic Efficacy Testing in an Ex vivo Model of Pseudomonas aeruginosa and Staphylococcus aureus Biofilms in the Cystic Fibrosis Lung

This workflow can be used to perform antibiotic susceptibility testing using an established&#160;ex vivo model of bacterial biofilm in the lungs of individuals with cystic fibrosis. Use of this model could enhance the clinical validity of MBEC (minimal biofilm eradication concentration) assays.

嚢胞性線維症肺における 緑膿菌 および 黄色ブドウ球 菌バイオフィルムのEx生体モデルにおける抗生物質有効性試験

The effective prescription of antibiotics for the bacterial biofilms present within the lungs of individuals with cystic fibrosis (CF) is limited by a ...

<meta charset="utf8"></head><body>抗菌試験および微生物動態解析のための適応型CFモデル

Development of a Polymicrobial Colony Biofilm Model to Test Antimicrobials in Cystic Fibrosis

A range of bacteria biofilm models exist for the testing of antibiotics. However, many of these are limited to a single experimental output, such as colony-forming units or metabolic activity. Furthermore, many biofilm models do not reflect the biological and physiochemical properties of the human host environment. This is an important issue in many conditions, but most noticeably in cystic fibrosis (CF). A large proportion of people with CF suffer from both chronic and intermittent infections, and in vitro, antibiotic susceptibility testing poorly correlates with patient treatment outcomes. Some biofilm models incorporate CF lung-relevant media, including synthetic sputum mimics, but do not consider the polymicrobial nature of the environment, which alters biofilm architecture, physiology, and the way microbes respond to treatment. The solid-air interface colony biofilm model described here is highly adaptable and incorporates both CF-relevant media and a polymicrobial context. This model can also be used for mid-throughput screening of antimicrobials and to study their effect on polymicrobial dynamics. Output measurements from the model can be colony-forming units, metabolic activity, and confocal microscopy analysis. The model can easily be adapted to different microorganisms, media, temperatures, and variable oxygen conditions and can be used to test a wide range of chemical, biological, and physical treatments.

<meta charset="utf8"></head><body>嚢胞性線維症における抗菌薬試験のための多微生物コロニーバイオフィルムモデルの開発

This methodology allows for the establishment of a polymicrobial biofilm model in cystic fibrosis for antimicrobial sensitivity testing in research and clinical laboratories. This model provides accurate and reliable results over a range of outputs.

嚢胞性線維症における抗菌薬試験のための多微生物コロニーバイオフィルムモデルの開発

A range of bacteria biofilm models exist for the testing of antibiotics. However, many of these are limited to a single experimental output, such as ...

チェンカバーガラスモデルを用いたバイオフィルム開発に関する痰の効果を可視化

要約

さらに動画を探す

共培養モデルの緑膿菌バイオフィルム

8ウェルチャンバースライドを用いたin vitro のバイオフィルム形成における

の成長結核バイオフィルム

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

研究のための方法論

WinCFモデル - 肺感染症の微生物学を研究するための粘液で閉鎖された細気管支の安価で収縮可能なマイクロコスモス

COMSTATソフトウェアを使用した In vitro バイオフィルムアーキテクチャに対する抗菌剤の影響の定量化

嚢胞性線維症肺における緑膿菌および黄色ブドウ球菌バイオフィルムのEx生体モデルにおける抗生物質有効性試験

嚢胞性線維症における抗菌薬試験のための多微生物コロニーバイオフィルムモデルの開発

嚢胞性線維症における抗菌薬試験のための多微生物コロニーバイオフィルムモデルの開発

チェンカバーガラスモデルを用いたバイオフィルム開発に関する痰の効果を可視化

要約

さらに動画を探す

共培養モデルの緑膿菌バイオフィルム

8ウェルチャンバースライドを用いたin vitro のバイオフィルム形成における

の成長結核バイオフィルム

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

研究のための方法論

WinCFモデル - 肺感染症の微生物学を研究するための粘液で閉鎖された細気管支の安価で収縮可能なマイクロコスモス

COMSTATソフトウェアを使用した In vitro バイオフィルムアーキテクチャに対する抗菌剤の影響の定量化

嚢胞性線維症肺における 緑膿菌 および 黄色ブドウ球 菌バイオフィルムのEx生体モデルにおける抗生物質有効性試験

嚢胞性線維症における抗菌薬試験のための多微生物コロニーバイオフィルムモデルの開発

嚢胞性線維症における抗菌薬試験のための多微生物コロニーバイオフィルムモデルの開発

嚢胞性線維症肺における緑膿菌および黄色ブドウ球菌バイオフィルムのEx生体モデルにおける抗生物質有効性試験