We thank Saran Kupul for technical assistance. This work was supported in part by NIH grant R01-AI104952 to FW. Further support was provided by the University of Alabama at Birmingham (UAB) Center for AIDS Research (CFAR), an NIH funded program (P30 AI027767) that was made possible by the following NIH Institutes: NIAID, NIMH, NIDA, NICHD, NHLDI, NIA.

1.バイオフィルム開始<ol><li>栄養豊富な培地中でバイオフィルム産生生物の文化を育てます。グリセロールストックからミューラーヒントン培地10mlに黄色ブドウ球菌株ニューマンを接種します。 S.の取り扱いに関わるすべての作業を実行します手袋と安全キャビネット内球菌 。 </li><li>ロータリーシェーカー（100〜200回転）に37℃で16時間インキュベートします。 </li><li>分光光度計と標準キュベット（：1センチメートル経路長）を用いて培養のOD 600を決定します 。 </li><li>式を使用してchelexedロズウェルパーク記念研究所培地（cRPMI中）9で0.1のOD 600（接種材料）で接種物の20ミリリットルを準備するために必要な培養液量（V）を計算します。[V = 20ミリリットル* 0.1 / OD 600（文化）] 注：リファレンス9のcRPMIの準備の詳細を提供します。 <ol><li>万×gで1分間回転させることによって遠心分離管、ペレット細胞に（上から）計算培養容量を加えます。 prepaへバイオフィルム接種再、20ミリリットルのcRPMIで過ごした成長培地を再懸濁細胞ペレットを吸引。 </li></ol></li><li> 25ミリリットルのリザーバーに接種材料を注ぎます。 </li><li>慎重にそれをまっすぐに引き上げて、滅菌96ウェルペグプレートからふたを外します。 注意：注意は、蓋から垂下ペグを汚染しないように注意する必要があります。蓋は安全キャビネット内の清浄表面上に逆さまに配置することができます。 </li><li> 200μlのマルチチャンネルピペットを用いて、底板のGへの行の各ウェルに125μlの接種材料を追加します。 </li><li>無菌性制御などの行Hの各ウェル中の125μlの無菌のcRPMI培地を記入してください。 </li><li>ペグ蓋を取り、下に向けペグ付きプレート底部の上にそれを保持します。慎重に、それ​​ぞれのウェルを有する個々のペグの位置を合わせます。プレートとペグの間の物理的接触を避けてください。整列した後、慎重にダウン蓋を下げます。ペグの後に行われる再調整が底板は続きがあり触れてきたようにミスアライメントを避けます行Hのアミ無菌コントロール</li><li>しっかりとシール、密封可能なビニール袋に蒸発し、場所を防止するために、パラフィンフィルムでプレートに蓋をシール。蒸発を最小限に抑えるために、できるだけ低くバッグ内の空気量を保ちます。 </li><li> 5％CO 2インキュベーター中で48時間37℃で振盪せずにインキュベートします。 注：成長の48時間は、Sのために最適となっています黄色ブドウ球菌が、使用する生物に基づいて異なります。 注：静的なインキュベーションは、アッセイを最適化するための良い出発点ですが、静かにマイクロプレートシェーカー上で150 rpmで振とうすると、一部のS.のバイオフィルムの形成を促進する可能性のある変動であります黄色ブドウ球菌が分離します。 </li></ol>バイオフィルムチャレンジプレートの調製<ol><li>バイオフィルムチャレンジの日に新鮮な96ウェルプレートを取ると、一貫性のない結果を回避するために、行の各ウェルBにHに、RTに平衡化のcRPMIの100μLを、追加、200μLを保持することができ、96ウェルプレートを利用メディアのペグが内側であり、バイオフィルム開始プレートのものと同一の寸法でウェルを備えていたとき。 </li><li>ステップ2.7での最終希釈を考慮するために、所望の最終濃度を2倍のcRPMI中の1.0ミリリットルで別々に4試験化合物まで希釈します。 注：良い出発点は、浮遊細胞の阻害濃度以上の8倍です。 </li><li> A3、ウェルA12にA10のA9にウェルA7におけるA6にウェルA4中の化合物2、化合物3および化合物4に、ウェルA1に化合物1の200μLを加えます。 </li><li>シリアルマルチチャンネルピペットを用いて、試験化合物を希釈し、B行と混合するためにA列から100μlのを転送します。 </li><li>そのようにして、ウェルによく100μlのを転送し続けます。各転送後に混合し、行Fに停止</li><li>混合した後、廃棄物容器に行Fから100μLを追放します。行GとHに液体を転送しないでください</li><li>にマルチチャンネルピペットを用いてプレートの各ウェルにcRPMI中の100μlのを追加します。200μlにボリュームをもたらします。最終的なプレートレイアウトについては、 図1を参照してください。 </li></ol> 3.バイオフィルムチャレンジ<ol><li>バイオフィルム開始プレートのものと同一の寸法を有するウェルを備え、新鮮な滅菌96ウェルプレートにリン酸緩衝生理食塩水（PBS）の200μlのを追加します。 </li><li>インキュベートバイオフィルム開始プレートからプラスチックの袋とパラフィン膜を除去。 </li><li>まっすぐふたを持ち上げます。バイオフィルムを損傷する恐れがあり、このようペグとプレート底部との接触を避けてください。 （3.8を参照）、その後のOD 600の分析のために底部を保管してください。 </li><li> （ステップ3.1から）PBSを含むプレート上にペグ蓋を配置することにより、浮遊細胞を洗い流します。 PBSの外に持ち上げ、5秒間ペグ水没し、ウェル側面に対するペグをヒットしないように注意して、もう一度水没。 </li><li>チャレンジプレートにすすぎペグ蓋を置きます。これはにつながるバイオフィルムにダメージを与えますようにペグと底板との間に不必要な接触を避けます一貫性のない結果。 </li><li>シールパラフィンフィルムでプレートし、ステップ1.10で説明したように密封可能なビニール袋に入れます。 </li><li> 5％CO 2インキュベーター内で37℃で24時間振盪せずにプレートをインキュベートします。 </li><li> 3.3からバイオフィルム開始プレートの底を取ります。各ウェルにマルチチャンネルピ ​​ペットを使用して細菌を再懸濁し、全てのウェルに生じる成長を確認するために、適切なマイクロプレートリーダーでOD 600を測定するが、行Hの無菌コントロール</li></ol> 4.バイオフィルムの決意<ol><li>インキュベーションチャンバーとレサズリン変換を検出するための運動蛍光測定値をとることができる装備プレートリーダーを使用してください。 530nmの励起および590nmの発光を用いて24時間の運動蛍光測定を設定します。 </li><li> 37℃に室内温度を設定し、プレートを20分ごとを読んでいます。メーカーのinstrucに係るプレートの底から測定するために、プレートリーダーを設定します。ション。 </li><li>滅菌96ウェルプレートにマルチチャンネルピペットを用いて200μlのPBSを添加することにより、洗浄プレートを準備します。 </li><li> 0.8ミリグラムの400μLを添加することによって、バイオフィルムの回復培地を調製/ 16 / mlのレザズリンの最終濃度までのcRPMI培地20mlのストック溶液をレサズリンmLです。 注：レサズリンは、既知の皮膚刺激性です。処理中に白衣、スプラッシュゴーグル、手袋を使用してください。バイオフィルムの回復メディアとしてのcRPMIは、最低のバックグラウンド信号を提供するために使用したが、適切な場合には、ミューラーヒントン培地に置き換えることができます。 </li><li>新鮮な96ウェルプレートの各ウェルにマルチチャンネルピペットを用いてバイオフィルムの回復培地150μlを添加します。 </li><li>安全キャビネットに移すインキュベーターからチャレンジプレートと慎重にビニール袋を取り外し、封止膜。 </li><li>チャレンジプレートからペグ蓋を外し、3.4で説明したようにPBSに浮遊細胞を洗い流してください。その後のOD 600 ANAための挑戦プレートの底を保ちます溶解（4.10を参照）。 </li><li>すすいだ後、バイオフィルムの回復培地を含むプレート底部にペグ蓋を置きます。 </li><li>周囲のウェルの底を妨げないように注意しながら、パラフィンフィルムでプレートの側面を包みます。すぐにプレートをラップした後、プレートリーダーの上に置き、ステップ4.1から以前に運動プロトコルリザズリン製を開始することにより、24時間の期間にわたって運動の読み取りを開始します。 </li><li>または挑戦中に発生してもしなくてもよい浮遊細胞の成長を定量化するために、マイクロプレートリーダーを使用して、全体チャレンジプレート底部のOD 600を読み取ります。ステップ3.8で与えられた指示に従ってください。 注：バイオフィルムをオフに流して細胞がウェルの底に塊に成長する傾向があるとして慎重に各ウェルの内容を再懸濁します。ウェル内の気泡を強くOD 600の読みを妨害する、回避または読み取りの前に除去されなければなりません。 </li></ol>

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The authors have nothing to disclose.

ここでは、Sでテストした阻害剤の活性を測定するために修飾バイオフィルムアッセイを記載していますバイオフィルム関連する細胞の代謝状態に焦点を当て球菌バイオフィルム。記載のバイオフィルムの開始とチャレンジ手順主に模倣メーカーの推奨事項が、24時間の阻害剤の挑戦を生き残るバイオフィルム関連細胞を検出し、定量化するための色素をレサズリンの使用は劇的?...

病原性微生物は、非急性慢性感染1に導くインビボでバイオフィルムを形成することができます。バイオフィルム関連感染は、異物（ 例えば、人工骨置換、乳房インプラント）、または気管チューブまたは尿道カテーテル2のインストールの注入を伴う医療処置の重大な危険因子です。バイオフィルムベースの感染はめったに自分でクリアされないように、これらの状況では、抗感染療法も免疫応答性個体では、ほとんど常に必要である。 黄色ブドウ球菌は、の使用中に発生するバイオフィルム関連の合併症に関与し、最も頻繁に観察された病原体の一つであります侵襲性医療機器3。 残念ながら、保護バリアとしてのバイオフィルムの本質は、浮遊細胞1,4より治療に対してより耐性になり、予測臨床的有効性の評価は、両方の初期の重要な部分です医薬品開発だけでなく、薬剤耐性サーベイランス。プランクトンの文化に焦点を当てた実験室条件は、忠実に実世界の疾病5を表していない可能性があること増加肯定応答があります。さらに問題を配合、バイオフィルムの表現型の複製は困難であり、既存のバイオフィルムモデルは面倒であり、高間およびアッセイ内変動6苦しみます。このように、多くの研究者は、必要を通じて、それによって潜在的に細菌毒性および疾患の重要な側面を無視し、薬剤感受性の浮遊性細胞アッセイをデフォルトに。 ここでは、具体的には、S、細菌をアッセイするためのプロトコルを記述します96ウェルベースのバイオフィルムシステム7,8を利用して事前に成長したバイオフィルム中の黄色ブドウ球菌 、。バイオフィルム形成と挑戦のためのプロトコルは、本質的に製造業者の勧告に従っているが、我々はbioF遺伝子の生存率を定量化するための代替情報が豊富な方法論を提示しますチャレンジ後ILM。簡単に説明すると、細菌がバイオフィルムは、プレートの蓋に取り付けられた突出ペグに形成ペグプレートで培養されます。バイオフィルム形成後、ペグは穏やかに浮遊細胞を除去するためにPBSで満たされた新鮮なプレートのウェル中に浸漬されています。ペグ蓋は、添付のバイオフィルムで、アッセイされる抗生物質の種々の濃度を含む、新規のチャレンジプレートに移します。第二のインキュベーションの後、蓋を再び取り出し、洗浄し、そしてそれらが最終インキュベーションを受けるレサズリン色素を含有する回収プレートに移します。レサズリン変換を動力学的に記録された、または定義された回復期間の後、エンドポイントの読み取りとすることができます。独自プロトコル7に記載され、このバイオフィルムの生存率を定量化するの染料ベースの方法は退屈CFU（コロニー形成単位）かなり異なるカウントベースの方法論。 OD薬物挑戦板とレサズリン変換速度の600の測定値は、生存率のリードとして働きますそれぞれプランクトンとバイオフィルム細胞のアウト、バイオフィルムの生存のための高速で信頼性の高い、情報が豊富で技術的に簡単な分析を提供しています。

<table><tbody><tr><td>&amp;nbsp;Mueller-Hinton Medium</td><td>Oxoid</td><td>CM0405</td><td>Follow recommendations of manufacturer</td></tr><tr><td>RPMI-medium</td><td>Corning&amp;nbsp;</td><td>17-105-CV</td><td></td></tr><tr><td>CRPMI</td><td>Ref 9</td><td></td><td>RPMI-1640 medium chelexed for 1 hr with Chelex 100 resin and then supplemented with 10% unchelexed RPMI-1640</td></tr><tr><td>Chelex 100 Resin</td><td>Bio Rad</td><td>142-2822</td><td></td></tr><tr><td>MBEC-plates</td><td>Innovotech</td><td>19111</td><td></td></tr><tr><td>Resazurin Sodium Salt</td><td>Sigma</td><td>R7017</td><td>800 &amp;micro;g/ml in DI water&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Filter sterile</td></tr><tr><td>Micro Plate Shaking Platform&amp;nbsp;</td><td>Heidolph Titramax 1000</td><td></td><td></td></tr><tr><td>Cytation 3 Plate Reader</td><td>Biotek</td><td></td><td></td></tr><tr><td>Gen5 software</td><td>Biotek</td><td></td><td>Recording and analysis of resazurin conversion</td></tr><tr><td>Neocuproine</td><td>Sigma</td><td>N1501&amp;nbsp;</td><td>prepare 10 mM stock in 100% Ethanol, store at -80 &amp;ordm;C</td></tr><tr><td>Copper sulfate</td><td>Acros Organics</td><td>7758-99-8</td><td>prepare a 100 mM stock solution in water, store at 4 &amp;ordm;C</td></tr><tr><td>Cu-Neocuproine</td><td>Self-Made</td><td></td><td>Generated by mixing equal molarities of neocuproine and copper sulfate. Mix was diluted in CRPMI medium to desired concentration.</td></tr><tr><td>Gentamicin</td><td>Sigma</td><td>G3632-1G</td><td></td></tr></tbody></table>

targeting biofilm associated staphylococcus aureus using resazurin based drug-susceptibility assay

Most pathogenic bacteria are able to form biofilms during infection, but due to the difficulty of manipulating and assessing biofilms, the vast majority of laboratory work is conducted with planktonic cells. Here, we describe a peg plate biofilm assay as performed with Staphylococcus aureus. Bacterial biofilms are grown on pegs attached to a 96-well microtiter plate lid, washed through gentle submersion in buffer, and placed in a drug challenge plate. After subsequent incubation they are again washed and moved to a final recovery plate, in which the fluorescent dye resazurin serves as a viability indicator. This assay offers greatly increased ease-of-use, reliability, and reproducibility, as well as a wealth of data when conducted as a kinetic read. Moreover, this assay can be adapted to a medium-throughput drug screening approach by which an endpoint fluorescent readout is taken instead, offering a path for drug discovery efforts.

レサズリンアッセイは確実に挑戦した後、薬剤を含まない培地中で複製することができる非常に少数の生存可能な細胞を検出するのに十分な感度です。この方法論では、我々は何のレサズリン変換は24時間以内に見られない最低の濃度として最小のバイオフィルム根絶濃度を定義します。レサズリンアッセイは、青からピンクへの染料の色を変換する代謝的に活性な?...

Watch this Scientific Journal Video about Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay at JoVE.com

Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay

バイオフィルム関連をターゲット<em&gt;黄色ブドウ球菌</em&gt;レサズリン基づく薬剤感受性アッセイを使用して

Most bacterial infections produce a biofilm. By virtue of their environment, biofilm associated bacteria are often phenotypically drug resistant. Novel antibacterial molecules that kill bacteria in biofilms are thus a high priority. We establish an assay to quickly screen for antimicrobial compounds that are effective at eradicating biofilms.

Most pathogenic bacteria are able to form biofilms during infection, but due to the difficulty of manipulating and assessing biofilms, the vast majority ...

targeting-biofilm-associated-staphylococcus-aureus-using-resazurin

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay

13.0K ビュー.  University of Alabama at Birmingham. Most bacterial infections produce a biofilm. By virtue of their environment, biofilm associated bacteria are often phenotypically drug resistant. Novel antibacterial molecules that kill bacteria in biofilms are thus a high priority. We establish an assay to quickly screen for antimicrobial compounds that are effective at eradicating biofilms. 

ビデオ: Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay

Biosensor for Detection of Antibiotic Resistant Staphylococcus Bacteria

A structurally transformed lytic bacteriophage having a broad host range of Staphylococcus aureus strains and a penicillin-binding protein (PBP 2a) antibody conjugated latex beads have been utilized to create a biosensor designed for discrimination of methicillin resistant (MRSA) and sensitive (MSSA) S. aureus species 1,2. The lytic phages have been converted into phage spheroids by contact with water-chloroform interface. Phage spheroid monolayers have been moved onto a biosensor surface by Langmuir-Blodgett (LB) technique 3. The created biosensors have been examined by a quartz crystal microbalance with dissipation tracking (QCM-D) to evaluate bacteria-phage interactions. Bacteria-spheroid interactions led to reduced resonance frequency and a rise in dissipation energy for both MRSA and MSSA strains. After the bacterial binding, these sensors have been further exposed to the penicillin-binding protein antibody latex beads. Sensors analyzed with MRSA responded to PBP 2a antibody beads; although sensors inspected with MSSA gave no response. This experimental distinction determines an unambiguous discrimination between methicillin resistant and sensitive S. aureus strains. Equally bound and unbound bacteriophages suppress bacterial growth on surfaces and in water suspensions. Once lytic phages are changed into spheroids, they retain their strong lytic activity and show high bacterial capture capability. The phage and phage spheroids can be utilized for testing and sterilization of antibiotic resistant microorganisms. Other applications may include use in bacteriophage therapy and antimicrobial surfaces.

Lytic phage biosensors and antibody beads are able to discriminate between methicillin resistant (MRSA) and sensitive staphylococcus bacteria. The phages were immobilized by a Langmuir-Blodgett method onto a surface of a quartz crystal microbalance sensor and worked as broad range staphylococcus probes. Antibody beads recognize MRSA.

抗生物質耐性ブドウ球菌の細菌の検出のためのバイオセンサー

A structurally transformed lytic bacteriophage having a broad host range of Staphylococcus aureus strains and a penicillin-binding protein (PBP 2a) ...

生物工学

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 ...

免疫・感染学

Stress-induced Antibiotic Susceptibility Testing on a Chip

We have developed a rapid microfluidic method for antibiotic susceptibility testing in a stress-based environment. Fluid is passed at high speeds over bacteria immobilized on the bottom of a microfluidic channel. In the presence of stress and antibiotic, susceptible strains of bacteria die rapidly. However, resistant bacteria survive these stressful conditions. The hypothesis behind this method is new: stress activation of biochemical pathways, which are targets of antibiotics, can accelerate antibiotic susceptibility testing. As compared to standard antibiotic susceptibility testing methods, the rate-limiting step -&#160;bacterial growth -&#160;is omitted during antibiotic application. The technical implementation of the method is in a combination of standard techniques and innovative approaches. The standard parts of the method include bacterial culture protocols, defining microfluidic channels in polydimethylsiloxane&#160;(PDMS), cell viability monitoring with fluorescence, and batch image processing for bacteria counting. Innovative parts of the method are in the use of culture media flow for mechanical stress application, use of enzymes to damage but not kill the bacteria, and use of microarray substrates for bacterial attachment. The developed platform can be used in antibiotic and nonantibiotic related drug development and testing. As compared to the standard bacterial suspension experiments, the effect of the drug can be turned on and off repeatedly over controlled time periods. Repetitive observation of the same bacterial population is possible over the course of the same experiment.

We have developed a microfluidic platform for rapid antibiotic susceptibility testing. Fluid is passed at high speeds over bacteria immobilized on the bottom of a microfluidic channel. In the presence of stress and antibiotic, susceptible strains of bacteria die rapidly. However, resistant bacteria can survive these stressful conditions.

チップ上でのストレス誘発抗生物質感受性試験

We have developed a rapid microfluidic method for antibiotic susceptibility testing in a stress-based environment. Fluid is passed at high speeds over ...

A Platform of Anti-biofilm Assays Suited to the Exploration of Natural Compound Libraries

Biofilms are regarded as one of the most challenging topics of modern biomedicine, and they are potentially responsible for over 80% of antibiotic-tolerant infections. Biofilms have displayed an exceptionally high tolerance for chemotherapy, which is thought to be multifactorial. For instance, the matrix provides a physical barrier that decreases the penetration of antibiotics into the biofilm. Also, cells within the biofilms are phenotypically diverse. Likely, biofilm resilience arises from a combination of these and other, yet unknown, mechanisms. All of the currently existing antibiotics have been developed against single-cells (planktonic) bacteria. Therefore, so far, a very limited repertoire of molecules exists that can selectively act on mature biofilms. This situation has driven a progressive paradigm shift in drug discovery, in which searching for anti-biofilms has been urged to occupy a more prominent place. An additional challenge is that there are a very limited number of standardized methods for biofilm research, especially those that can be used for large-throughput screening of chemical libraries. Here, an experimental anti-biofilm platform for chemical screening is presented. It uses three assays to measure biofilm viability (with resazurin staining), total biomass (with crystal violet staining), and biofilm matrix (using a wheat germ agglutinin, WGA-fluorescence-based staining of the poly-N-acetyl-glucosamine, PNAG, fraction). All the assays were developed using Staphylococcus aureus as the model bacteria. Examples of how the platform can be used for primary screening as well as for functional characterization of identified anti-biofilm hits are presented. This experimental sequence further allows for the classification of the hits based upon the measured end-points. It also provides information on their mode of action, especially on long-term versus short-term chemotherapeutic effects. Thus, it is very advantageous for the quick identification of high-quality hit compounds that can serve as starting points for various biomedical applications.

Biofilm infections show high tolerance towards chemotherapy. No single assay captures the complexity of biofilms. Instead, complementary assays are needed. We present a screening platform (developed for S. aureus) that combines assays for viability, biomass, and biofilm matrix. It allows anti-biofilm drug discovery, including the assessment of long-term chemotherapeutic effects.

天然化合物ライブラリーの探査に適した抗バイオフィルムアッセイのプラットフォーム

Biofilms are regarded as one of the most challenging topics of modern biomedicine, and they are potentially responsible for over 80% of ...

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread of multi drug-resistant TB (MDR-TB), there is a real urgency to develop new therapeutic strategies against M. tuberculosis infections. Traditionally, compounds are evaluated based on their antibacterial activity under in vitro growth conditions in broth; however, results are often misleading for intracellular pathogens like M. tuberculosis since in-broth phenotypic screening conditions are significantly different from the actual disease conditions within the human body. Screening for inhibitors that work inside macrophages has been traditionally difficult due to the complexity, variability in infection, and slow replication rate of M. tuberculosis. In this study, we report a new approach to rapidly assess the effectiveness of compounds on the viability of M. tuberculosis in a macrophage infection model. Using a combination of a cytotoxicity assay and an in-broth M. tuberculosis viability assay, we were able to create a screening system that generates a comprehensive analysis of compounds of interest. This system is capable of producing quantitative data at a low cost that is within reach of most labs and yet is highly scalable to fit large industrial settings.

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

We have developed a modular high-throughput screening system for discovering novel compounds against Mycobacterium tuberculosis, targeting intracellular and in-broth growing bacteria as well as cytotoxicity against the mammalian host cell.

細胞内の標的阻害剤の有効性及び細胞毒性スクリーニングのためのシステム<em&gt;結核菌</em

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread ...

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 ...

Standardized In vitro Assays to Visualize and Quantify Interactions between Human Neutrophils and Staphylococcus aureus Biofilms

Neutrophils are the first line of defense deployed by the immune system during microbial infection. In vivo, neutrophils are recruited to the site of infection where they use processes such as phagocytosis, production of reactive oxygen and nitrogen species (ROS, RNS, respectively), NETosis (neutrophil extracellular trap), and degranulation to kill microbes and resolve the infection. Interactions between neutrophils and planktonic microbes have been extensively studied. There have been emerging interests in studying infections caused by biofilms in recent years. Biofilms exhibit properties, including tolerance to killing by neutrophils, distinct from their planktonic-grown counterparts. With the successful establishment of both in vitro and in vivo biofilm models, interactions between these microbial communities with different immune cells can now be investigated. Here, techniques that use a combination of traditional biofilm models and well-established neutrophil activity assays are tailored specifically to study neutrophil and biofilm interactions. Wide-field fluorescence microscopy is used to monitor the localization of neutrophils in biofilms. These biofilms are grown in static conditions, followed by the addition of neutrophils derived from human peripheral blood. The samples are stained with appropriate dyes prior to visualization under the microscope. Additionally, the production of ROS, which is one of the many neutrophil responses against pathogens, is quantified in the presence of a biofilm. The addition of immune cells to this established system will expand the understanding of host-pathogen interactions while ensuring the use of standardized and optimized conditions to measure these processes accurately.

Standardized In vitro Assays to Visualize and Quantify Interactions between Human Neutrophils and Staphylococcus aureus Biofilms

The present protocol describes the study of neutrophil-biofilm interactions. Staphylococcus aureus biofilms are established in vitro and incubated with peripheral blood-derived human neutrophils. The oxidative burst response from neutrophils is quantified, and the neutrophil localization within the biofilm is determined by microscopy.

ヒト好中球と黄色ブドウ球菌バイオフィルムの間の相互作用を視覚化および定量するための標準化されたin vitroアッセイ

Neutrophils are the first line of defense deployed by the immune system during microbial infection. In vivo, neutrophils are recruited to the site of ...

A Soluble Tetrazolium-Based Reduction Assay to Evaluate the Effect of Antibodies on Candida tropicalis Biofilms

Candida species are the fourth-most common cause of systemic nosocomial infections. Systemic or invasive candidiasis frequently involves biofilm formation on implanted devices or catheters, which is associated with increased virulence and mortality. Biofilms produced by different Candida species exhibit enhanced resistance against various antifungal drugs. Therefore, there is a need to develop effective immunotherapies or adjunctive treatments against Candida biofilms. While the role of cellular immunity is well established in anti-Candida protection, the role of humoral immunity has been studied less.
It has been hypothesized that inhibition of biofilm formation and maturation is one of the major functions of protective antibodies, and Candida albicans germ tube antibodies (CAGTA) have been shown to suppress in vitro growth and biofilm formation of C. albicans earlier. This paper outlines a detailed protocol for evaluating the role of antibodies on biofilms formed by C. tropicalis. The methodology for this protocol involves C. tropicalis biofilm formation in 96-well microtiter plates, which were then incubated in the presence or absence of antigen-specific antibodies, followed by a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-carboxanilide-2H-tetrazolium (XTT) assay for measuring the metabolic activity of fungal cells in the biofilm.
The specificity was confirmed by using appropriate serum controls, including Sap2-specific antibody-depleted serum. The results demonstrate that antibodies present in the serum of immunized animals can inhibit Candida biofilm maturation in vitro. In summary, this paper provides important insights regarding the potential of antibodies in developing novel immunotherapies and synergistic or adjunctive treatments against biofilms during invasive candidiasis. This in vitro protocol can be used to check the effect of potential new antifungal compounds on the metabolic activity of Candida species cells in biofilms.

A Soluble Tetrazolium-Based Reduction Assay to Evaluate the Effect of Antibodies on Candida tropicalis Biofilms

A 96-well microtiter plate-based protocol using a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-carboxanilide-2H-tetrazolium (XTT) reduction assay is described herein, to study antibodies' effects on biofilms formed by C. tropicalis. This in vitro protocol can be used to check the effect of potential new antifungal compounds on the metabolic activity of Candida species cells in biofilms.

カンジダ・トロピカリス・バイオフィルムに対する抗体の効果を評価するための可溶性テトラゾリウムベースの還元アッセイ

Candida species are the fourth-most common cause of systemic nosocomial infections. Systemic or invasive candidiasis frequently involves biofilm formation ...

Improved Enzyme Protection Assay to Study Staphylococcus aureus Internalization and Intracellular Efficacy of Antimicrobial Compounds

Staphylococcus aureus expresses virulence factors to trigger its internalization into eukaryote cells and to survive inside different subcellular compartments. This paper describes an enzyme protection assay to study the extent of S. aureus internalization and its intracellular survival in adherent non-professional phagocytic cells (NPPCs) as well as the intracellular efficacy of antimicrobial compounds. NPPCs are grown in a multi-well plate until they reach 100% confluence. S. aureus cultures are grown overnight in cell culture medium. The bacterial suspension is diluted according to the number of cells per well to inoculate the cells at a controlled multiplicity of infection. Inoculated cells are incubated for 2 h to allow the bacteria to be internalized by the NPPCs, following which lysostaphin is added to the culture medium to selectively kill extracellular bacteria. Lysostaphin is present in the culture medium for the rest of the experiment. 
At this point, the infected cells could be incubated with antimicrobial compounds to assess their intracellular activities against S. aureus. Next, the cells are washed three times to remove the drugs, and intracellular S. aureus load is then quantified by culturing on agar plates. Alternatively, for studying staphylococcal virulence factors involved in intracellular survival and cell toxicity, lysostaphin could be inactivated with proteinase K to eliminate the need for washing steps. This tip improves the reliability of the intracellular bacterial load quantification, especially if cells tend to detach from the culture plate when they become heavily infected because of the multiplication of intracellular S. aureus. These protocols can be used with virtually all types of adherent NPPCs and with 3D cell culture models such as organoids.

Improved Enzyme Protection Assay to Study Staphylococcus aureus Internalization and Intracellular Efficacy of Antimicrobial Compounds

This protocol aims to describe how to study the extent of Staphylococcus aureus internalization and its ability to survive inside the human host cell, as well as the intracellular efficacy of antimicrobial compounds.

黄色 ブドウ球菌 内在化と抗菌性化合物の細胞内有効性を研究するための酵素保護アッセイの改善

Staphylococcus aureus expresses virulence factors to trigger its internalization into eukaryote cells and to survive inside different subcellular ...

生物学

An Assay to Evaluate Drug Efficacy against a Mycobacterium tuberculosis Infection Model

Source: Schaaf, K., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/55453">A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis.</a> J. Vis. Exp. (2017)
This video demonstrates an assay to evaluate the efficacy of drugs against Mycobacterium tuberculosis (Mtb) infection. An in vitro infection model containing Mtb/macrophage aggregates is generated that mimics in vivo infection. Upon introducing suitable antibiotics, their efficacy is tested via resuzarin assay to monitor the survival of bacteria with increasing drug concentrations.

結核菌感染モデルに対する薬剤効果評価法

1. Culture Conditions for Green Fluorescent Protein Expressing M. tuberculosis mc26206 (Mtb-GFP)
NOTE: The M. tuberculosis H37Rv derived auxotroph strain ...

バイオフィルム関連をターゲット

要約

さらに動画を探す

この動画の章

抗生物質耐性ブドウ球菌の細菌の検出のためのバイオセンサー

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

チップ上でのストレス誘発抗生物質感受性試験

天然化合物ライブラリーの探査に適した抗バイオフィルムアッセイのプラットフォーム

細胞内の標的阻害剤の有効性及び細胞毒性スクリーニングのためのシステム

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

ヒト好中球と黄色ブドウ球菌バイオフィルムの間の相互作用を視覚化および定量するための標準化されたin vitroアッセイ

カンジダ・トロピカリス・バイオフィルムに対する抗体の効果を評価するための可溶性テトラゾリウムベースの還元アッセイ

黄色ブドウ球菌内在化と抗菌性化合物の細胞内有効性を研究するための酵素保護アッセイの改善

結核菌感染モデルに対する薬剤効果評価法

バイオフィルム関連をターゲット

要約

さらに動画を探す

この動画の章

抗生物質耐性ブドウ球菌の細菌の検出のためのバイオセンサー

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

チップ上でのストレス誘発抗生物質感受性試験

天然化合物ライブラリーの探査に適した抗バイオフィルムアッセイのプラットフォーム

細胞内の標的阻害剤の有効性及び細胞毒性スクリーニングのためのシステム

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

ヒト好中球と黄色ブドウ球菌バイオフィルムの間の相互作用を視覚化および定量するための標準化されたin vitroアッセイ

カンジダ・トロピカリス・バイオフィルムに対する抗体の効果を評価するための可溶性テトラゾリウムベースの還元アッセイ

黄色 ブドウ球菌 内在化と抗菌性化合物の細胞内有効性を研究するための酵素保護アッセイの改善

結核菌感染モデルに対する薬剤効果評価法

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

黄色ブドウ球菌内在化と抗菌性化合物の細胞内有効性を研究するための酵素保護アッセイの改善