実験室でのバイオフィルム関連の作業は、助成を受けた歯科＆頭蓋顔面研究とアレルギー感染症（JLL - Rに。）国立研究所の国立研究所からR21DE017294とR21AI080930の番号が付けられています。内容はもっぱら著者の責任であり、必ずしもNIDCR、NIAIDまたはNIHの公式見解を表すものではありません。

1。 C.の準備アルビカンス C. albicansはリスクグループ1/BSL1微生物である。常にこの微生物との仕事のために良好な無菌/滅菌技術を使用し、有害物質の適切な処分のための制度的な手順に従うことを覚えている。 <ol><li> C.の一晩培養を準備YPD（酵母ペプトンデキストロース）Cの単一コロニーを接種することにより液体培地でアルビカンス YPD 25mLのにアルビカンス 。 </li><li> 30℃で一晩オービタルシェーカー（約180回転）で培養液を、。ほとんどのC.カンジダ菌は、これらの条件下で酵母細胞として成長します。 </li><li>一晩培養する（約3,000 rpmで、5〜10分）遠心し、滅菌PBSで細胞を2回洗浄し、pHを7.0と予め温めておいた〜165 mMのmorpholinepropanesulfonic酸で緩衝RPMI 1640培地の約20〜25 mLで、最終的なペレットを再懸濁します。 37℃は（この培地上で、今から単に&quot;RPMI 1640&quot;と呼ばれます）。 </li><li>血球計を用いて細胞を数える。カウントの後、RPMI 1640で1.0 × 10 6細胞/ mlの最終濃度で細胞の懸濁液を準備。 注：細胞が凝集する傾向があるので、それは洗浄液の間に、ピペッティングの前に激しくボルテックスすることが重要です。 </li></ol> 2。バイオフィルムの形成のために96ウェルマイクロタイタープレートのセットアップ<ol><li>マルチチャンネルピ ​​ペットを使用すると、Cの100μLを加える96ウェルマイクロタイタープレート（- s）の選択したウェルにアルビカンスサスペンション 。これらは、ネガティブコントロールとして機能するように、カラム12のウェルに細胞を追加しないでください。 </li><li>元のふたが付いている全体のマイクロタイタープレート、パラフィルムでシール、37℃で24時間インキュベーターでインキュベート内部の場所℃をカバーインキュベーションの長さは、特定の実験計画に適合させることができます。例えば、それは24の期間にわたってバイオフィルム形成の動態を調べることができます - 複数のプレートを播種し、異なる時点で、各プレートを処理することによって、48時間（すなわち、2、4、8、12、24および48時間） </li><li>バイオフィルム形成後に、マルチチャンネルピペットを使用すると、慎重に各ウェルに形成されたバイオフィルムに触れると混乱しないように培地を吸引除去する。 </li><li>滅菌PBS（ウエル当たり200μL）でマルチチャンネルピペットの洗浄プレートを3回使用する。また、自動化されたマイクロタイタープレートウォッシャーを使用してください。洗浄の間、特に最​​後の洗浄後、任意の残留PBSを除去するためにペーパータオルでブロッティングで逆さの位置にプレートを排出。この時点でウェルの底に形成されたバイオフィルムは、さらに肉眼ではっきり見えるはずですし、また、倒立顕微鏡（図1）を用いて可視化することができます。バイオフィルムは、現在抗真菌薬感受性試験のアッセイのために処理される準備が整いました。 （実験の主な目的はバイオフィルム形成の程度を評価する場合、プレートは比色法を用いて処理される準備が整いました。このためには、XTT /メナジオン試薬は（以下のステップ3を参照）、結果色を追加したりすることができます）マイクロタイタープレートリーダーを用いて読み取る。 </li></ol> 3。バイオフィルムの抗真菌感受性試験<ol><li>ストック溶液や粉末から、テストする各抗真菌のRPMI 1640培地で希釈標準溶液を準備する。典型的な高濃度では、フルコナゾールは1,024μg/ mLとし、アムホテリシンBとカスポファンギン両方のための16μg/ mLとなります。他の濃度が異なるエージェントのために使用することができます。 </li><li>マルチチャンネルピペットを使用して、真菌バイオフィルムを含む各マイクロタイタープレートの1列目の対応するウェルに抗真菌の高い作業濃度200μLを加える。 </li><li> 10〜2列目に各ウェルにRPMI 1640 100μLを加える。 </li><li> 11列のウェルにRPMI 1640 100μLを加える。 </li><li>列1のウエルからの抗真菌剤100μLを削除し、列2に隣接する井戸（既に培地100μLを含む）に追加します。 </li><li>シリアル倍希釈を行うためにピペッティングでよく内容を混ぜる、とヒントを削除する。 </li><li>混合後の塔10の井戸からの最後の100μLボリュームが破棄された後、列10の井戸、まで右に移動繰り返します。この方法では、興味のあるエージェントの倍希釈系列（- S）が作成されていますが、ほとんどの列10の井戸の集中以上に列1の井戸に集中してから。列11の比類のないバイオフィルムは、ポジティブコントロールとして機能します、とカラム12内の空井戸は、ネガティブコントロールとして機能します。 </li><li> 37彼らの蓋、24 -48時間パラフィルムとインキュベートとシールでプレートをカバー℃に</li><li>潜伏期間の後に前のステップ2.4​​（3 × PBS）のようにプレートを洗浄する。 </li><li>マルチチャンネルピペットを使用すると、うまく背景Xの測定のための事前洗浄されたバイオフィルムだけでなく、ネガティブコントロールのウェルにを含む各XTT /メナジオン溶液100μLを加えるTT -比色レベル。 <ol><li> XTTは、0.22μmの孔サイズのフィルターを用いて濾過滅菌する必要がある滅菌乳酸リンゲル液、PBSまたは生理食塩水、では0.5g / Lでの飽和溶液として調製される。 XTT溶液は光に敏感ですので、準備中にアルミホイルで覆われるべきである。 -70一度作成すると、フィルター滅菌、10 mLの作業量に分注し、および店舗℃にアルミ箔を使用して光からチューブを保護します。使用直前に特定の実験の必要に応じて、多くの管としてだけ解凍。 </li><li>メナジオンは、100％アセトン、少量（約50μL）に分注し、-70℃で保存中の10mMストック溶液として調製され融解XTT溶液10mlを含むチューブにメナジオンのストック溶液の1μLを添加することにより、使用直前にXTT /メナジオン溶液を調製します。 </li></ol></li><li> 37℃2時間暗所でアルミホイルとインキュベートでプレートをカバー℃に</li><li>プレートを発見。マルチチャンネルピペットを使用すると、新しいマイクロタイタープレートの対応するウェルに、結果色の各ウェルからの上清と伝達の80μLを削除します。 </li><li> 490nmでマイクロタイタープレートリーダーでプレート（- s）をお読みください。 </li><li>比色測定値の50％または80％減少は抗真菌薬の非存在下で形成された制御バイオフィルムと比較して（この場合は11列の値で検出されるときの抗真菌濃度である固着性の最小発育阻止濃度SMIC50とSMIC80を、計算する、）のみXTTを含むカラム12の井戸からネガティブコントロールから値を減算することも忘れないでください。 SMIC結果を表として提示することができます（複数の抗真菌剤に対するすなわち、複数の分離株）またはその代わり、各抗真菌に対して分離、個々の真菌の結果は、阻害率に対する抗真菌濃度をプロットしてグラフとして表示することができます。 </li></ol> 例：ネガティブコントロールの値を減算した後、カラム11に形成された制御のバイオフィルムの平均ODは1.32です。 SMIC50は1.32 X 100分の50 = 0.66よりも小さいこのケースでは、比色測定値の&gt; 50％の削減につながる最も低い抗真菌濃度である。同様に、SMIC80は1.32 X 100分の20 = 0.264よりも小さいこのケースで比色測定値の&gt; 80％削減、につながる最も低い抗真菌濃度である。 4。代表的な結果図1は、Cの顕微鏡写真を示しています。 albicansのバイオフィルムは、倒立顕微鏡を使用して撮影した96ウェルマイクロタイタープレートのウェルの底面上に形成。図2は、Cの 11バイオフィルムのそれぞれについてXTT -比色測定値（OD 490の値）を示しています。 albicansの野生型株は、同じ96ウェルマイクロタイタープレートの8つの異なる行のそれぞれに形成。図3は、C.に対して、異なる濃度のアムホテリシンBの活性を示しています。 albicansのバイオフィルム、矢印はSMIC50とSMIC80値を示している。 <img src="/files/ftp_upload/2287/2287fig1.jpg" alt="図1" /> 図1（）パネルC.の倒立顕微鏡に取り付けられたカメラで撮影した顕微鏡写真を示しています。 albicansのバイオフィルムは、RPMI培地、PBSで、その後の洗浄液の吸引後にウェルの底部に形成された。典型的なCの（B）顕微鏡写真albicansのバイオフィルムは、走査型電子顕微鏡を用いて視覚化。バーは、AとBそれぞれのパネルでは100μmと10μmのです。 <img src="/files/ftp_upload/2287/2287fig2.jpg" alt="図2" /> 図2。複数の等価なCの形成96ウェルマイクロタイタープレート中のカンジダバイオフィルム。C.によって形成されたバイオフィルムのXTT還元アッセイから比色測定値（OD 490の値）マイクロタイタープレートのウェルにalbicansは野生型。値が同じ96ウェルマイクロタイタープレートの8つの異なる行のそれぞれに形成された11の独立したバイオフィルムのためのものです。別の行の結果は、一方向の分散分析と分散の均一性とボンフェローニの多重比較、ポストテストのためのバートレットの検定を用いて比較した。 （P&gt; 0.05）お互いに行のすべてのペアを比較する際には統計的に有意な差は認められなかった。 <img src="/files/ftp_upload/2287/2287fig3.jpg" alt="図3" /> 図3。 C.に対して抗真菌薬感受性試験の典型的な結果 C.のバイオフィルムに対して異なるアムホテリシンBの濃度の効果の典型的な結果を描いたalbicansのバイオフィルム。グラフalbicansの野生型株。値は、井戸を制御するために比較してXTT還元アッセイの平均パーセント比色測定値として表現さ​​れています。 SMIC50とSMIC80値は矢印で示されている。

<ol>
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JLL - Rは、抗真菌剤を開発しているMicrobeHTSテクノロジーズ、（株）の株式を所有している。 MicrobeHTS Technologies社は、これらの研究のための財政的支援を提供していません。

ここでは、XTTを使ってバイオフィルム内の細胞の代謝活性を測定する比色法と相まって、カンジダバイオフィルムの形成のための、シンプル、迅速、経済的かつ再現性の高い96ウェルマイクロタイタープレートのモデルを説明します。バイオフィルム形成のためのこの96ウェルマイクロタイタープレートのモデルは、もともとC用に開発されたアルビカンスが、他のカン?...

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Sabouraud-dextrose agar</td>
<td>&nbsp;</td>
<td>Becton Dickinson</td>
<td>211584</td>
<td>To prepare plates for fresh subcultures of fungal isolates</td>
</tr>
<tr>
<td>YPD: Yeast peptone dextrose</td>
<td>&nbsp;</td>
<td>US Biological </td>
<td>Y2076</td>
<td>Medium for propagation of overnight liquid cultures</td>
</tr>
<tr>
<td>RPMI-1640 without sodium bicarbonate supplemented with L-glutamine</td>
<td>&nbsp;</td>
<td>Cellgro</td>
<td>50-020-PB</td>
<td>Liquid medium for biofilm formation</td>
</tr>
<tr>
<td>Morpholinepropanesulfonic acid (MOPS)</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>BP308</td>
<td>To buffer RPMI 1640</td>
</tr>
<tr>
<td>Phosphate buffered saline, PBS</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P4417</td>
<td>Buffer for washes</td>
</tr>
<tr>
<td>XTT sodium salt</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>X4251</td>
<td>See above for preparation instructions</td>
</tr>
<tr>
<td>Ringer's lactate</td>
<td>&nbsp;</td>
<td>Hospira</td>
<td>NDC0409-7953-09</td>
<td>For preparation of XTT solution</td>
</tr>
<tr>
<td>Menadione</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M5625</td>
<td>Caution: hazardous by skin contact, inhalation or ingestion</td>
</tr>
<tr>
<td>Petri dishes</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>08-757-12</td>
<td>&nbsp;</td>
<tr>
<td>15 ml conical centrifuge tubes</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>430790</td>
<td>&nbsp;</td>
<tr>
<td>50 ml conical centrifuge tubes</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>430828</td>
<td>&nbsp;</td>
<tr>
<td>96 well microtiter plates: Polystyrene, flat-bottomed, tissue culture treated</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>3595</td>
<td>&nbsp;</td>
<tr>
<td>Multichannel pipette and tips</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Incubator</td>
<td>&nbsp;</td>
<td>Any</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Microtiter plate reader</td>
<td>&nbsp;</td>
<td>Any</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
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a 96 well microtiter plate-based method for monitoring formation and antifungal susceptibility testing of candida albicans biofilms

カンジダアルビカンスは、標的の患者とカンジダ症の拡大人口の真菌感染症の最も多い原因は、現在米国の病院で3番目に一般的な感染症である残ります。カンジダ症の異なる症状が両方のホストの組織および/または医療機器（すなわちカテーテル）で、バイオフィルム形成に関連付けられています。バイオフィルム内の細胞が宿主の免疫応答からと抗真菌薬の作用から保護されているバイオフィルム形成は、負の臨床的意義を運びます。我々は、Cの形成のための、シンプルで高速かつ堅牢なのin vitroモデルを開発したまた、バイオフィルム抗真菌薬感受性試験に使用できる96ウェルマイクロタイタープレートを、使用してカンジダバイオフィルム。このアッセイの読み出しは、代謝的に活性な菌のバイオフィルム細胞によるXTTの還元（テトラゾリウム塩）に基づいて、比色です。典型的な実験は、抗真菌薬感受性試験のための追加の24時間で、バイオフィルム形成のための約24時間かかります。そのシンプルさと一般に入手可能な実験材料および機器の使用から、この手法は、バイオフィルムの研究をdemocratizesと真菌バイオフィルムの抗真菌薬感受性試験の標準化に向けた重要な一歩を表しています。

Watch this Scientific Journal Video about A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms at JoVE.com

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

我々はの形成のための、シンプルで迅速かつ堅牢な方法を説明します<em&gt;カンジダアルビカンス</emバイオフィルム内の細胞の抗真菌薬感受性試験では96ウェルマイクロタイタープレートと、そのユーティリティを使用して&gt;バイオフィルム。

監視形成との抗真菌薬感受性試験のための96ウェルマイクロタイタープレートベースの方法カンジダアルビカンスバイオフィルム

Candida albicans remains the most frequent cause of fungal infections in an expanding population of compromised patients and candidiasis is now the third ...

a-96-well-microtiter-plate-based-method-for-monitoring-formation

Research

JoVE Journal

Immunology and Infection

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

44.5K ビュー.  University of Texas San Antonio - UTSA. 我々はの形成のための、シンプルで迅速かつ堅牢な方法を説明しますカンジダアルビカンスバイオフィルム。

ビデオ: A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

Microtiter Dish Biofilm Formation Assay

Biofilms are communities of microbes attached to surfaces, which can be found in medical, industrial and natural settings. In fact, life in a biofilm probably represents the predominate mode of growth for microbes in most environments. Mature biofilms have a few distinct characteristics. Biofilm microbes are typically surrounded by an extracellular matrix that provides structure and protection to the community. Microbes growing in a biofilm also have a characteristic architecture generally comprised of macrocolonies (containing thousands of cells) surrounded by fluid-filled channels. Biofilm-grown microbes are also notorious for their resistance to a range of antimicrobial agents including clinically relevant antibiotics.
The microtiter dish assay is an important tool for the study of the early stages in biofilm formation, and has been applied primarily for the study of bacterial biofilms, although this assay has also been used to study fungal biofilm formation. Because this assay uses static, batch-growth conditions, it does not allow for the formation of the mature biofilms typically associated with flow cell systems. However, the assay has been effective at identifying many factors required for initiation of biofilm formation (i.e, flagella, pili, adhesins, enzymes involved in cyclic-di-GMP binding and metabolism) and well as genes involved in extracellular polysaccharide production. Furthermore, published work indicates that biofilms grown in microtiter dishes do develop some properties of mature biofilms, such a antibiotic tolerance and resistance to immune system effectors.
This simple microtiter dish assay allows for the formation of a biofilm on the wall and/or bottom of a microtiter dish. The high throughput nature of the assay makes it useful for genetic screens, as well as testing biofilm formation by multiple strains under various growth conditions. Variants of this assay have been used to assess early biofilm formation for a wide variety of microbes, including but not limited to, pseudomonads, Vibrio cholerae, Escherichia coli, staphylocci, enterococci, mycobacteria and fungi.
In the protocol described here, we will focus on the use of this assay to study biofilm formation by the model organism Pseudomonas aeruginosa. In this assay, the extent of biofilm formation is measured using the dye crystal violet (CV). However, a number of other colorimetric and metabolic stains have been reported for the quantification of biofilm formation using the microtiter plate assay. The ease, low cost and flexibility of the microtiter plate assay has made it a critical tool for the study of biofilms.

The assay describes a rapid means to measure early biofilm formation in bacteria and fungi. This method uses a microtiter plate as the substratum for microbial biofilm formation, and the biofilm is visualized using crystal violet strain. The assay provides either a qualitative or quantitative assay for early biofilm formation.

マイクロタイター皿のバイオフィルム形成アッセイ

Biofilms are communities of microbes attached to surfaces, which can be found in medical, industrial and natural settings.  In fact, life in a biofilm ...

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. ...

Antimicrobial Susceptibility Testing of Mycobacterium Tuberculosis Complex for First and Second Line Drugs by Broth Dilution in a Microtiter Plate Format

The rapid detection of antimicrobial resistance is important in the effort to control the increase in resistant Mycobacterium 
tuberculosis (Mtb). Antimicrobial susceptibility testing (AST) of Mtb has traditionally been performed by the agar method of proportion or by 
macrobroth testing on an instrument such as the BACTEC (Becton Dickinson, Sparks, MD), VersaTREK (TREK Diagnostics, Cleveland, OH) or BacT/ALERT (bioM&eacute;rieux, Hazelwood, MO). The agar proportion method, while considered the &#x201C;gold&#x201D; standard of AST, is labor intensive and requires calculation of resistance by performing colony counts on drug-containing agar as compared to drug-free agar. If there is &ge;1% growth on the drug-containing medium as compared to drug-free medium, the organism is considered resistant to that drug. The macrobroth methods require instrumentation and test break point ("critical") drug concentrations for the first line drugs (isoniazid, ethambutol, rifampin, and pyrazinamide). The method described here is commercially available in a 96 well microtiter plate format [MYCOTB (TREK Diagnostics)] and contains increasing concentrations of 12 antimicrobials used for treatment of tuberculosis including both first (isoniazid, rifampin, ethambutol) and second line drugs (amikacin, cycloserine, ethionamide, kanamycin, moxifloxacin, ofloxacin, para-aminosalicylic acid, rifabutin, and streptomycin). Pyrazinamide, a first line drug, is not included in the microtiter plate due to its need for acidic test conditions. Advantages of the microtiter system include both ease of set up and faster turn around time (14 days) compared with traditional agar proportion (21 days). In addition, the plate can be set up from inoculum prepared using either broth or solid medium. Since the microtiter plate format is new and since Mtb presents unique safety challenges in the laboratory, this protocol will describe how to safely setup, incubate and read the microtiter plate.

Antimicrobial susceptibility testing of Mycobacterium tuberculosis is challenging but critical for patient care. This microtiter plate format offers testing of 12 antimycobacterial drugs and provides minimum inhibitory concentration (MIC) values, which will aid in appropriate treatment.

マイクロタイタープレート形式で液体希釈法により、第一、第二ラインの薬のための結核菌の複合体の抗菌薬感受性試験

The rapid detection of antimicrobial resistance is important in the effort to control the increase in resistant Mycobacterium 
tuberculosis (Mtb). ...

Preparation of a Blood Culture Pellet for Rapid Bacterial Identification and Antibiotic Susceptibility Testing

Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a rapid identification of the infectious agent to guide optimal antibiotic treatment. The analysis of Gram stains from positive blood culture can be rapidly conducted and already significantly impact the antibiotic regimen. However, the accurate identification of the infectious agent is still required to establish the optimal targeted treatment. We present here a simple and fast bacterial pellet preparation from a positive blood culture that can be used as a sample for several essential downstream applications such as identification by MALDI-TOF MS, antibiotic susceptibility testing (AST) by disc diffusion assay or automated AST systems and by automated PCR-based diagnostic testing. The performance of these different identification and AST systems applied directly on the blood culture bacterial pellets is very similar to the performance normally obtained from isolated colonies grown on agar plates. Compared to conventional approaches, the rapid acquisition of a bacterial pellet significantly reduces the time to report both identification and AST. Thus, following blood culture positivity, identification by MALDI-TOF can be reported within less than 1 hr whereas results of AST by automated AST systems or disc diffusion assays within 8 to 18 hr, respectively. Similarly, the results of a rapid PCR-based assay can be communicated to the clinicians less than 2 hr following the report of a bacteremia. Together, these results demonstrate that the rapid preparation of a blood culture bacterial pellet has a significant impact on the identification and AST turnaround time and thus on the successful outcome of patients suffering from bloodstream infections.

A rapid bacterial pellet preparation from a positive blood culture can be used as a sample for applications such as identification by MALDI-TOF, Gram staining, antibiotic susceptibility testing and PCR-based test. The results can be rapidly communicated to clinicians to improve the outcome of patients suffering from bloodstream infections.

迅速な細菌同定および抗生物質感受性試験のための血液培養ペレットの調製

Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a ...

A Reference Broth Microdilution Method for Dalbavancin In Vitro Susceptibility Testing of Bacteria that Grow Aerobically

Antimicrobial susceptibility testing (AST) is performed to assess the in vitro activity of antimicrobial agents against various bacteria. The AST results, which are expressed as minimum inhibitory concentrations (MICs) are used in research for antimicrobial development and monitoring of resistance development and in the clinical setting for antimicrobial therapy guidance. Dalbavancin is a semi-synthetic lipoglycopeptide antimicrobial agent that was approved in May 2014 by the Food and Drug Administration (FDA) for the treatment of acute bacterial skin and skin structure infections caused by Gram-positive organisms. The advantage of dalbavancin over current anti-staphylococcal therapies is its long half-life, which allows for once-weekly dosing. Dalbavancin has activity against Staphylococcus aureus (including both methicillin-susceptible S. aureus [MSSA] and methicillin-resistant S. aureus [MRSA]), coagulase-negative staphylococci, Streptococcus pneumoniae, Streptococcus anginosus group, &#946;-hemolytic streptococci and vancomycin susceptible enterococci. Similar to other recent lipoglycopeptide agents, optimization of CLSI and ISO broth susceptibility test methods includes the use of dimethyl sulfoxide (DMSO) as a solvent when preparing stock solutions and polysorbate 80 (P80) to alleviate adherence of the agent to plastic. Prior to the clinical studies and during the initial development of dalbavancin, susceptibility studies were not performed with the use of P-80 and MIC results tended to be 2-4 fold higher and similarly higher MIC results were obtained with the agar dilution susceptibility method. Dalbavancin was first included in CLSI broth microdilution methodology tables in 2005 and amended in 2006 to clarify use of DMSO and P-80. The broth microdilution (BMD) procedure shown here is specific to dalbavancin and is in accordance with the CLSI and ISO methods, with step-by-step detail and focus on the critical steps added for clarity.

A Reference Broth Microdilution Method for Dalbavancin In Vitro Susceptibility Testing of Bacteria that Grow Aerobically

Here, we present a protocol for determination of dalbavancin susceptibility of clinically relevant Gram-positive bacteria using a broth microdilution reference methodology.

ダルババンシンの参照ブロス微量希釈法<em&gt;インビトロ</em好気的に成長する細菌の&gt;感受性試験

Antimicrobial susceptibility testing (AST) is performed to assess the in vitro activity of antimicrobial agents against various bacteria. The AST results, ...

Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device

Candida albicans is the most common fungal pathogen of humans, causing about 15% of hospital-acquired sepsis cases. A major virulence attribute of C. albicans is its ability to form biofilms, structured communities of cells attached to biotic and abiotic surfaces. C. albicans biofilms can form on host tissues, such as mucosal layers, and on medical devices, such as catheters, pacemakers, dentures, and joint prostheses. Biofilms pose significant clinical challenges because they are highly resistant to physical and chemical perturbations, and can act as reservoirs to seed disseminated infections. Various in vitro assays have been utilized to study C. albicans biofilm formation, such as microtiter plate assays, dry weight measurements, cell viability assays, and confocal scanning laser microscopy. All of these assays are single end-point assays, where biofilm formation is assessed at a specific time point. Here, we describe a protocol to study biofilm formation in real-time using an automated microfluidic device under laminar flow conditions. This method allows for the observation of biofilm formation as the biofilm develops over time, using customizable conditions that mimic those of the host, such as those encountered in vascular catheters. This protocol can be used to assess the biofilm defects of genetic mutants as well as the inhibitory effects of antimicrobial agents on biofilm development in real-time.

Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device

This protocol describes the use of a customizable automated microfluidic device to visualize biofilm formation in Candida albicans under host physiological conditions.

自動マイクロ流体デバイスを使用してカンジダ菌バイオ フィルム形成の可視化

Candida albicans is the most common fungal pathogen of humans, causing about 15% of hospital-acquired sepsis cases. A major virulence attribute of C. ...

Deep Dermal Injection As a Model of Candida albicans Skin Infection for Histological Analyses

The skin is an extremely extended organ of the body and, due to this large surface, it is continuously exposed to microorganisms. Skin damage can easily lead to infections in the dermis which can, in turn, result in the dissemination of pathogens into the bloodstream. Understanding how the immune system fights infections at the very early stage and how the host can eliminate the pathogens is an important step to set the base for future therapeutic interventions. Here we describe a model of Candida albicans infection that can visualize the processes that occur early during an infection, including when the pathogen has passed the epithelial barrier, as well as the immune response elicited by the C. albicans invasion. We used this infection model to perform histological analyses that show the immune cells that infiltrate the skin as well as the presence and localization of the pathogen. Samples collected after the infection can be processed for RNA extraction.

Deep Dermal Injection As a Model of Candida albicans Skin Infection for Histological Analyses

Here we describe a protocol that allows histological and molecular analysis of skin samples after Candida albicans intradermal injection. This protocol maintains the structural integrity of the skin and allows for the localization of tissue-resident or newly recruited immune cells as well as the pathogen distribution.

深い皮膚注入として、モデルカンジダ皮膚感染症病理組織学的解析のための

The skin is an extremely extended organ of the body and, due to this large surface, it is continuously exposed to microorganisms. Skin damage can easily ...

Daily Phototherapy with Red Light to Regulate Candida albicans Biofilm Growth

Here, we present a protocol to assess the outcomes of per diem red light treatment on the growth of Candida albicans biofilm. To increase the planktonic growth of C. albicans SN425, the inoculums grew on Yeast Nitrogen Base media. For biofilm formation, RPMI 1640 media, which have high concentrations of amino acids, were applied to help biofilm growth. Biofilms of 48 h were treated twice a day for a period of 1 min with a non-coherent light device (red light; wavelength = 635 nm; energy density = 87.6 J&#183;cm-2). As a positive control (PC), 0.12% chlorhexidine (CHX) was applied, and as a negative control (NC), 0.89% NaCl was applied to the biofilms. Colony forming units (CFU), dry-weight, soluble and insoluble exopolysaccharides were quantified after treatments. Briefly, the protocol presented here is simple, reproducible and provides answers regarding viability, dry-weight and extracellular polysaccharide amounts after red light treatment.

Daily Phototherapy with Red Light to Regulate Candida albicans Biofilm Growth

Here, we present a protocol to assess the outcome of red light application on the growth of Candida albicans biofilm. A non-coherent red light device with the wavelength of 635 nm and energy density of 87.6 J&#183;cm-2 was applied throughout the growth of Candida albicans biofilms for 48 h.

規制カンジダバイオ フィルムの成長する赤い光で毎日光線

Here, we present a protocol to assess the outcomes of per diem red light treatment on the growth of Candida albicans biofilm. To increase the planktonic ...

Clarifying and Imaging Candida albicans Biofilms

The microbial fungus Candida albicans can undergo a change from commensal colonization to virulence that is strongly correlated with its ability to switch from yeast-form growth to hyphal growth. Cells initiating this process become adherent to surfaces as well as to each other, with the resulting development of a biofilm colony. This commonly occurs not only on mucosal tissue surfaces in yeast infections, but also on medical implants such as catheters. It is well known that biofilm cells are resistant to antifungal drugs, and that cells that shed from the biofilm can lead to dangerous systemic infections. Biofilms range from heavily translucent to opaque due to refractive heterogeneity. Therefore, fungal biofilms are difficult to study by optical microscopy. To visualize internal structural, cellular, and subcellular features, we clarify fixed intact biofilms by stepwise solvent exchange to a point of optimal refractive index matching. For C. albicans biofilms, sufficient clarification is attained with methyl salicylate (n = 1.537) to enable confocal microscopy from apex to base in 600 &#181;m biofilms with little attenuation. In this visualization protocol we outline phase contrast refractometry, the growth of laboratory biofilms, fixation, staining, solvent exchange, the setup for confocal fluorescence microscopy, and representative results.

Clarifying and Imaging Candida albicans Biofilms

To view and quantify the internal features of Candida albicans biofilms, we prepare fixed intact specimens that are clarified by refractive index matching. Then, optical sectioning microscopy can be used to obtain three-dimensional image data though the full thickness of the biofilm.

カンディダ・アルビカンスバイオフィルムの明確化とイメージング

The microbial fungus Candida albicans can undergo a change from commensal colonization to virulence that is strongly correlated with its ability to switch ...

Rose Bengal-Mediated Photodynamic Therapy to Inhibit Candida albicans

Invasive Candida albicans infection is a significant opportunistic fungal infection in humans because it is one of the most common colonizers of the gut, mouth, vagina, and skin. Despite the availability of antifungal medication, the mortality rate of invasive candidiasis remains ~50%. Unfortunately, the incidence of drug-resistant C. albicans is increasing globally. Antimicrobial photodynamic therapy (aPDT) may offer an alternative or adjuvant treatment to inhibit C. albicans biofilm formation and overcome drug resistance. Rose bengal (RB)-mediated aPDT has shown effective cell killing of bacteria and C. albicans. In this study, the efficacy of RB-aPDT on multidrug-resistant C. albicans is described. A homemade green light-emitting diode (LED) light source is designed to align with the center of a well of a 96-well plate. The yeasts were incubated in the wells with different concentrations of RB and illuminated with varying fluences of green light. The killing effects were analyzed by the plate dilution method. With an optimal combination of light and RB, 3-log growth inhibition was achieved. It was concluded that RB-aPDT might potentially inhibit drug-resistant C. albicans.

Rose Bengal-Mediated Photodynamic Therapy to Inhibit Candida albicans

The growing incidence of drug-resistant Candida albicans is a serious health issue worldwide. Antimicrobial photodynamic therapy (aPDT) may offer a strategy to fight against drug-resistant fungal infections. The present protocol describes Rose bengal-mediated aPDT efficacy on a multidrug-resistant C. albicans strain in vitro.

カンジダ・アルビカンスを阻害するローズベンガル媒介光線力学療法

Invasive Candida albicans infection is a significant opportunistic fungal infection in humans because it is one of the most common colonizers of the gut, ...