Name: rose bengal-mediated photodynamic therapy to inhibit candida albicans
Uploaded: 2022-03-24T14:00:00
Description: Watch this Scientific Journal Video about Rose Bengal-Mediated Photodynamic Therapy to Inhibit Candida albicans at JoVE.com

この研究は、文部(MOE)による高等教育スプラウトプロジェクトの枠組みの中で、国立成昆大学応用ナノ医学センター、国立成昆大学から資金提供を受けており、台湾科学技術部[MOST 109-2327-B-006-005]からTW Wongに資金提供を受けています。J.H. Hungは、台湾の国立成昆大学病院[NCKUH-11006018]および[MOST 110-2314-B-006-086-MY3]からの資金提供を認めています。

1. aPDTシステムの準備
<ol><li>LEDストリップから緑色の発光ダイオード(LED)を4つ切り取り( 材料表を参照)、96ウェルプレートの4ウェルに合わせます(図1)。 メモ: LED は 4 x 3 アレイに配置されました。LEDの背面は、照射時に熱を分散させるためにヒートシンクに接着した。</li>
	<li>540nmにおけるLEDのフルエンス率11 を光パワーメー...

<ol>
	<li>Naglik, J. R., Challacombe, S. J., Hube, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Candida+albicans+secreted+aspartyl+proteinases+in+virulence+and+pathogenesis.">Candida albicans secreted aspartyl proteinases in virulence and pathogenesis.</a> Microbiology and Molecular Biology Reviews. 67 (3), 400-428 (2003).</li><li>Pappas, P. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+practice+guideline+for+the+management+of+candidiasis:+2016+update+by+the+Infectious+Diseases+Society+of+America.">Clinical practice guideline for the management of candidiasis: 2016 update by the Infectious Diseases Society of America.</a> Clinical Infectious Diseases. 62 (4), 1-50 (2016).</li><li>Ricotta, E. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invasive+candidiasis+species+distribution+and+trends,+United+States,+2009-2017.">Invasive candidiasis species distribution and trends, United States, 2009-2017.</a> Journal of Infectious Diseases. 223 (7), 1295-1302 (2021).</li><li>Koehler, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morbidity+and+mortality+of+candidaemia+in+Europe:+an+epidemiologic+meta-analysis.">Morbidity and mortality of candidaemia in Europe: an epidemiologic meta-analysis.</a> Clinical Microbiology and Infection. 25 (10), 1200-1212 (2019).</li><li>Toda, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Population-based+active+surveillance+for+culture-confirmed+candidemia+-+four+sites,+United+States,+2012-2016.">Population-based active surveillance for culture-confirmed candidemia - four sites, United States, 2012-2016.</a> Morbidity and Mortality Weekly Report Surveillance Summaries. 68 (8), 1-15 (2019).</li><li>Lee, C. 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W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Indocyanine+green-mediated+photodynamic+therapy+reduces+methicillin-resistant+staphylococcus+aureus+drug+resistance.">Indocyanine green-mediated photodynamic therapy reduces methicillin-resistant staphylococcus aureus drug resistance.</a> Journal of Clinical Medicine. 8 (3), 411 (2019).</li><li>Kim, M. M., Darafsheh, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light+sources+and+dosimetry+techniques+for+photodynamic+therapy.">Light sources and dosimetry techniques for photodynamic therapy.</a> Photochemistry and Photobiology. 96 (2), 280-294 (2020).</li><li>Wong, T. W., Sheu, H. M., Lee, J. Y., Fletcher, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photodynamic+therapy+for+Bowen's+disease+(squamous+cell+carcinoma+in+situ)+of+the+digit.">Photodynamic therapy for Bowen's disease (squamous cell carcinoma in situ) of the digit.</a> Dermatologic Surgery. 27 (5), 452-456 (2001).</li><li>Wong, T. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photodynamic+inactivation+of+methicillin-resistant+Staphylococcus+aureus+by+indocyanine+green+and+near+infrared+light.">Photodynamic inactivation of methicillin-resistant Staphylococcus aureus by indocyanine green and near infrared light.</a> Dermatologica Sinica. 36 (1), 8-15 (2018).</li><li>Stasko, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visible+blue+light+inhibits+infection+and+replication+of+SARS-CoV-2+at+doses+that+are+well-tolerated+by+human+respiratory+tissue.">Visible blue light inhibits infection and replication of SARS-CoV-2 at doses that are well-tolerated by human respiratory tissue.</a> Scientific Reports. 11 (1), 20595 (2021).</li><li>Crosbie, J., Winser, K., Collins, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+the+light+field+of+the+Waldmann+PDT+1200+lamp:+potential+for+wide-field+low+light+irradiance+aminolevulinic+acid+photodynamic+therapy.">Mapping the light field of the Waldmann PDT 1200 lamp: potential for wide-field low light irradiance aminolevulinic acid photodynamic therapy.</a> Photochemistry and Photobiology. 76 (2), 204-207 (2002).</li><li>Feenstra, R. P., Tseng, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+fluorescein+and+rose+bengal+staining.">Comparison of fluorescein and rose bengal staining.</a> Ophthalmology. 99 (4), 605-617 (1992).</li><li>Demidova, T. N., Hamblin, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+cell-photosensitizer+binding+and+cell+density+on+microbial+photoinactivation.">Effect of cell-photosensitizer binding and cell density on microbial photoinactivation.</a> Antimicrobial Agents and Chemotherapy. 49 (6), 2329-2335 (2005).</li><li>Shahid, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Duclauxin+derivatives+from+fungi+and+their+biological+activities.">Duclauxin derivatives from fungi and their biological activities.</a> Frontiers in Microbiology. 12, 766440 (2021).</li><li>Arendrup, M. C., Park, S., Brown, S., Pfaller, M., Perlin, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+CLSI+M44-A2+disk+diffusion+and+associated+breakpoint+testing+of+caspofungin+and+micafungin+using+a+well-characterized+panel+of+wild-type+and+fks+hot+spot+mutant+Candida+isolates.">Evaluation of CLSI M44-A2 disk diffusion and associated breakpoint testing of caspofungin and micafungin using a well-characterized panel of wild-type and fks hot spot mutant Candida isolates.</a> Antimicrobial Agents and Chemotherapy. 55 (5), 1891-1895 (2011).</li><li>Mukaremera, L., Lee, K. K., Mora-Montes, H. M., Gow, N. A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Candida+albicans+yeast,+pseudohyphal,+and+hyphal+morphogenesis+differentially+affects+immune+recognition.">Candida albicans yeast, pseudohyphal, and hyphal morphogenesis differentially affects immune recognition.</a> Frontiers in Immunology. 8, 629 (2017).</li><li>Hung, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+photodynamic+therapy+against+fungal+keratitis.">Recent advances in photodynamic therapy against fungal keratitis.</a> Pharmaceutics. 13 (12), 2011 (2021).</li><li>Martinez, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rose+Bengal+photodynamic+antimicrobial+therapy:+a+pilot+safety+study.">Rose Bengal photodynamic antimicrobial therapy: a pilot safety study.</a> Cornea. 40 (8), 1036-1043 (2021).</li></ol>

真菌性角膜炎に対するRB−PDTの臨床応用の有望な結果が最近報告されている19。RBの吸収ピークは450〜650nmにある。aPDTを成功させるためには、光源のフルエンス率を決定することが不可欠です。がん細胞の治療には高いフルエンス(通常>100J/cm2)が必要であり、感染病変の治療には低いフルエンスが必要であると予想される6。高いフルエンスは、臨床?...

C.アルビカンスは 、健常人の胃腸管および尿生殖路に定着し、個体の約50%において正常な微生物叢として検出され得る1。宿主と病原体との間に不均衡が生じると、 C. albicans は侵入して病気を引き起こす可能性があります。感染は、局所粘膜感染から多臓器不全2までの範囲であり得る。米国の多施設サーベイランス研究では、2009年から2017年の間に侵襲性カンジダ症患者からの分離株の約半分が C. albicans3である。カンジデミアは、高い罹患率、死亡率、長期入院4と関連している可能性がある。米国疾病管理予防センターは、試験されたすべてのカンジダ血液サンプルの約7%が抗真菌薬フルコナゾールに耐性があると報告しました5。薬剤耐性 カンジダ 種の出現は、抗真菌剤に代わるものまたはアジュバント療法を開発する懸念を提起する。
抗菌光線力学療法(aPDT)は、PS6のピーク吸収波長の光で特定の光増感剤(PS)を活性化することを含む。励起後、励起されたPSは、そのエネルギーまたは電子を近くの酸素分子に伝達し、基底状態に戻る。このプロセスの間に、活性酸素種および一重項酸素が形成され、細胞損傷を引き起こす。aPDTは1990年代から微生物を殺すために広く使用されてきた7。aPDTの利点の1つは、照射中に複数の細胞小器官が一重項酸素および/または活性酸素種(ROS)によって細胞内で損傷を受けることである。したがって、aPDTに対する耐性は今日まで見出されていない。さらに、最近の研究では、aPDT後に生き残った細菌が抗生物質に対してより敏感になったことが報告されています8。
aPDTで使用される光源には、レーザー、フィルター付き金属ハロゲンランプ、近赤外光、発光ダイオード(LED)9、10、11、12などがあります。レーザーは、通常0.5 W/cm2より大きい高い光パワーを提供し、非常に短時間で高い光線量を送達することを可能にする。口腔感染症に対するaPDTのように、治療時間が長くなると不便な場合に広く用いられている。レーザーの欠点は、照明のスポットサイズが小さく、ディフューザーで数百マイクロメートルから10mmの範囲であることです。さらに、レーザー装置は高価であり、操作するには特定のトレーニングが必要です。一方、フィルター付き金属ハロゲンランプの照射面積は比較的大きい13。しかし、ランプはあまりにも重くて高価です。LED光源は小型で安価であるため皮膚科学分野でaPDTの主流となっている。照射面積は、LED電球のアレイ配置で比較的大きくすることができる。顔全体を同時に照らすことができる9.それにもかかわらず、すべてではないにしても、今日利用可能なLED光源のほとんどは、臨床使用のために設計されています。スペースを占有し、高価であるため、ラボでの実験には適していない可能性があります。私たちは、非常に小さく、LEDストリップから切断して組み立てることができる安価なLEDアレイを開発しました。LEDは、異なる実験計画のために異なる配置に適合させることができる。aPDTの異なる条件は、1回の実験で96ウェルプレートまたは384ウェルプレートで完了することができる。
ローズベンガル(RB)は、ヒトの目14における角膜損傷の可視化を強化するために広く使用されている着色染料である。RB媒介性aPDTは、黄色ブドウ球菌、大腸菌、およびC.アルビカンスに対して、トルイジンブルーO15のそれとほぼ同等の効率で殺傷効果を示した。この研究は、多剤耐性C.アルビカンスに対するRB-aPDTの効果を検証する方法を実証する。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5 mL microfuge tube</td>
			<td>Neptune, San Diego, USA</td>
			<td>#3745.x</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL round-bottom tube with cell strainer cap</td>
			<td>Falcon, USA</td>
			<td>#352235</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plate</td>
			<td>Alpha plus, Taoyuan Hsien, Taiwan</td>
			<td>#16196</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum foil</td>
			<td>sunmei, Tainan, Taiwan</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum heat sink</td>
			<td>Nanyi electronics Co., Ltd., Tainan, Taiwan</td>
			<td>BK-T220-0051-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf, UK</td>
			<td>5415R</td>
			<td>disperses heat from the LED array</td>
		</tr>
		<tr>
			<td>Graph pad prism software</td>
			<td>GraphPad 8.0, San Diego, California, USA</td>
			<td></td>
			<td>graphing and statistics software</td>
		</tr>
		<tr>
			<td>Green light emitting diode (LED) strip</td>
			<td>Nanyi electronics Co., Ltd., Tainan, Taiwan</td>
			<td>2835</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Yihder, Taipei, Taiwan</td>
			<td>LM-570D (R)</td>
			<td>Emission peak wavelength: 525 nm, Viewing angle: 150&#176;; originated from https://www.aliva.com.tw/product.php?id=63</td>
		</tr>
		<tr>
			<td>Light power meter</td>
			<td>Ophir, Jerusalem, Israel</td>
			<td>PD300-3W-V1-SENSOR,</td>
			<td></td>
		</tr>
		<tr>
			<td>Millex 0.22 &#956;m filter</td>
			<td>Merck, NJ, USA</td>
			<td>SLGVR33RS</td>
			<td></td>
		</tr>
		<tr>
			<td>Multidrug-resistant Candida albicans</td>
			<td>Bioresource Collection and Research CenterBioresource, Hsinchu, Taiwan</td>
			<td>BCRC 21538/ATCC 10231</td>
			<td>http://catalog.bcrc.firdi.org.tw/BcrcContent?bid=21538</td>
		</tr>
		<tr>
			<td>OD600 spectrophotometer</td>
			<td>Biochrom, London, UK</td>
			<td>Ultrospec 10</td>
			<td></td>
		</tr>
		<tr>
			<td>Rose Bengal</td>
			<td>Sigma-Aldrich, MO, USA</td>
			<td>330000</td>
			<td>stock concentration 40 mg/mL = 4%, prepare in PBS, stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Sterilized glass tube</td>
			<td>Sunmei Co., Ltd., Tainan, Taiwan</td>
			<td>AK45048-16100</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast Extract Peptone Dextrose Medium</td>
			<td>HIMEDIA, India</td>
			<td>M1363</td>
			<td></td>
		</tr>
	</tbody>
</table>

rose bengal-mediated photodynamic therapy to inhibit candida albicans

侵襲性 カンジダ・アルビカンス 感染症は、腸、口、膣、および皮膚の最も一般的な植民者の1つであるため、ヒトにおける重要な日和見真菌感染症である。抗真菌薬が利用可能であるにもかかわらず、侵襲性カンジダ症の死亡率は〜50%のままである。残念なことに、薬剤耐性の C.アルビカンの発生 率は世界的に増加しています。抗菌光線力学療法(aPDT)は、 C.アルビカンス バイオフィルム形成を阻害し、薬物耐性を克服するための代替またはアジュバント治療を提供し得る。ローズベンガル(RB)媒介性aPDTは、細菌および C.アルビカンスの効果的な細胞死滅を示している。この研究では、多剤耐性 C.アルビカンス に対するRB−aPDTの有効性が記載されている。自家製の緑色発光ダイオード(LED)光源は、96ウェルプレートのウェルの中心に合わせるように設計されています。酵母を異なる濃度のRBを有するウェルでインキュベートし、様々な緑色の光のフルエンスで照らした。殺傷効果をプレート希釈法により分析した。光とRBの最適な組み合わせにより、3対数の成長阻害が達成された。RB-aPDTは薬剤耐性 のC.アルビカンスを潜在的に阻害する可能性があると結論付けられた。

図1は 、本研究で使用されているaPDTシステムを示す。高温は著しい細胞死を引き起こす可能性があるため、LEDアレイを扇風機で冷却し、照射時にはヒートシンクを用いて25±1°Cで一定温度を維持する。 熱効果は割引できます。均一な配光を持つことも、aPDTを成功させるための重要な決定要因です。したがって、照明中にLED電球を井戸に正確に合わせることが重要です。...

Watch this Scientific Journal Video about Rose Bengal-Mediated Photodynamic Therapy to Inhibit Candida albicans at JoVE.com

Rose Bengal-Mediated Photodynamic Therapy to Inhibit Candida albicans

薬剤耐性カン ジダ・アルビカンス の発生率の増加は、世界中で深刻な健康問題です。抗菌光線力学療法(aPDT)は、薬剤耐性真菌感染症と戦うための戦略を提供するかもしれない。本プロトコールは、多剤耐性 C.アルビカン ス株に対するローズベンガル媒介性aPDT有効性を インビトロで記載する。

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, ...

この研究は、様々な微生物や細胞に対するさまざまな光力学的影響を調べるためのシンプルで汎用性が高く、簡単なシステムです。このシステムでは、LEDを実験計画に応じて異なる配置に収めることができます。PDTの異なる条件は、1回の実験で96ウェルプレートまたは384ウェルプレートでも計算することができる。この技術は真菌性角膜炎の治療に使用でき、そのために世界的には、集中的な治療にもかかわらず約150,000人が目を失います。まず、LEDストリップから4つの緑色LEDを切り取り、96ウェルプレートの3つのウェルに合わせます。LEDのフルエンス率を540ナノメートルでライトパワーメーターで測定します。照射中はプレートの横に組み立てた扇風機で25°Cで一定の温度を維持してください。滅菌ループを用いて、寒天プレートからカンジダ・アルビカンスの単一のコロニーを選び、3ミリリットルのYPD培地を含む滅菌ガラス管に加える。チューブを摂氏25度、回転速度155RPMで14~16時間インキュベートし、酵母培養物を増殖させる。次にYPD培地で一晩培養した値をOD600値0.5に希釈する。30°Cで155RPMで回転しながら4時間インキュベートして、対数成長期を達成します。新鮮なYPD培地で対数期培養の希釈をOD600値0.65まで繰り返し、1ミリリットル当たり約1回~第7コロニー形成単位を得た。同時に1X PBS中のバラベンガルの4%ストック溶液を調製する。フィルターは0.22マイクロメートルのフィルターで滅菌します。1ミリリットルの対数期培養に111マイクロリットルの2%バラベンガル溶液を加え、室温で異なる時点で共培養して、細胞内のRBの吸収を理解する。16で遠心分離し、室温で2時間半の間Gを100倍にし、そして共培養物を1ミリリットルの1X PBSで3回洗浄した。洗浄後、カンジダ・アルビカンス培養物を1ミリリットルの1X PBSに再懸濁する。各条件について、培養物を96ウェルプレート中の3つの異なるウェルに分配する。ウェルをLEDアレイに合わせます。光が当たったグループで、扇風機とライトをオンにします。照射後、1ウェルから共培養液20マイクロリットルを採取し、180マイクロリットルの1X PBSを入れたチューブに添加して10倍段階希釈を2回行う。次いで、YPD寒天プレートの1象限に各段階希釈液の20マイクロリットルを滴下し、プレート上に計数可能なコロニーを得た。蛍光顕微鏡は、赤色蛍光下でのバラベンガルによるカンジダ・アルビカンスの即時染色を示した。インキュベーションの15分後、細胞の大部分がバラベンガルで染色され、時間依存的に細胞に入ることを示す。また、光によるバラベンガルの活性化は、細胞内でフリーラジカルや一重項酸素を生成し、細胞死を生じる。バラベンガルまたは照射の非存在下では、細胞死は観察されなかった。カンジダ・アルビカンスは、0.2%バラベンガルの存在下での緑色光照射後、光用量依存的に阻害された。最初に96ウェルプレートをLEDの中心に合わせることが重要です。第二に、使用される寒天プレートは、別々のコロニーの混合を防ぐために十分に乾燥されなければならない。プレート上で成長したカンジダ・アルビカンスは、PDTが細胞の遺伝子発現にどのように影響するかを答えることができる遺伝子分析のためにさらに送ることができる。これらの技術は、PDTが癌細胞、細菌、真菌、またはウイルスのいずれかの異なる細胞タイプに対して、簡単で安価で汎用性の高いプラットフォームでどのように機能するかを調べる道を開きます。

rose-bengal-mediated-photodynamic-therapy-to-inhibit-candida-albicans

Research

JoVE Journal

Immunology and Infection

Rose Bengal-Mediated Photodynamic Therapy to Inhibit Candida albicans

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

Candida albicans remains the most frequent cause of fungal infections in an expanding population of compromised patients and candidiasis is now the third most common infection in US hospitals. Different manifestations of candidiasis are associated with biofilm formation, both on host tissues and/or medical devices (i.e. catheters). Biofilm formation carries negative clinical implications, as cells within the biofilms are protected from host immune responses and from the action of antifungals. We have developed a simple, fast and robust in vitro model for the formation of C. albicans biofilms using 96 well microtiter-plates, which can also be used for biofilm antifungal susceptibility testing. The readout of this assay is colorimetric, based on the reduction of XTT (a tetrazolium salt) by metabolically active fungal biofilm cells. A typical experiment takes approximately 24 h for biofilm formation, with an additional 24 h for antifungal susceptibility testing. Because of its simplicity and the use of commonly available laboratory materials and equipment, this technique democratizes biofilm research and represents an important step towards the standardization of antifungal susceptibility testing of fungal biofilms.

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

We describe a simple, rapid and robust method for the formation of Candida albicans biofilms using 96 well microtiter plates and its utility in antifungal susceptibility testing of cells within biofilms.

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 ...

免疫・感染学

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 ...

Visualization of Candida albicans in the Murine Gastrointestinal Tract Using Fluorescent In Situ Hybridization

Candida albicans is a fungal component of the gut microbiota in humans and many other mammals. Although C. albicans does not cause symptoms in most colonized hosts, the commensal reservoir does serve as a repository for infectious disease, and the presence of high fungal titers in the gut is associated with inflammatory bowel disease. Here, we describe a method to visualize C. albicans cell morphology and localization in a mouse model of stable gastrointestinal colonization. Colonization is established using a single dose of C. albicans in animals that have been treated with oral antibiotics. Segments of gut tissue are fixed in a manner that preserves the architecture of luminal contents (microorganisms and mucus) as well as the host mucosa. Finally, fluorescent in situ hybridization is performed using probes against fungal rRNA to stain for C. albicans and hyphae. A key advantage of this protocol is that it allows for simultaneous observation of C. albicans cell morphology and its spatial association with host structures during gastrointestinal colonization.

Visualization of Candida albicans in the Murine Gastrointestinal Tract Using Fluorescent In Situ Hybridization

The purpose of this protocol is to visualize Candida albicans cell shape and localization in the mammalian gastrointestinal tract.

上皮内蛍光を用いたマウス消化管におけるカンダアルビカンスの可視化

Candida albicans is a fungal component of the gut microbiota in humans and many other mammals. Although C. albicans does not cause symptoms in most ...

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 ...

An Ex vivo Assay to Study Candida albicans Hyphal Morphogenesis in the Gastrointestinal Tract

Candida albicans hyphal morphogenesis in the gastrointestinal (GI) tract is tightly controlled by various environmental signals, and plays an important role in the dissemination and pathogenesis of this opportunistic fungal pathogen. However, methods to visualize fungal hyphae in the GI tract in vivo are challenging which limits the understanding of environmental signals in controlling this morphogenesis process. The protocol described here demonstrates a novel ex vivo method for visualization of hyphal morphogenesis in gut homogenate extracts. Using an ex vivo assay, this study demonstrates that cecal contents from antibiotic treated mice, but not from untreated control mice, promote C. albicans hyphal morphogenesis in the gut content. Further, adding back specific groups of gut metabolites to the cecal contents from antibiotic-treated mice differentially regulates hyphal morphogenesis ex vivo. Taken together, this protocol represents a novel method to identify and investigate the environmental signals that control C. albicans hyphal morphogenesis in the GI tract.

An Ex vivo Assay to Study Candida albicans Hyphal Morphogenesis in the Gastrointestinal Tract

The ex vivo assay described in this study using gut homogenate extracts and immunofluorescence staining represents a novel method to examine the hyphal morphogenesis of Candida albicans in the GI tract. This method can be utilized to investigate the environmental signals regulating morphogenetic transition in the gut.

胃腸管における カンジダ・アルビカンス ・ヒphphal形態形成を研究するエクスビボアッセイ

Candida albicans hyphal morphogenesis in the gastrointestinal (GI) tract is tightly controlled by various environmental signals, and plays an important ...

Use of In Vivo Imaging to Screen for Morphogenesis Phenotypes in Candida albicans Mutant Strains During Active Infection in a Mammalian Host

Candida albicans is an important human pathogen. Its ability to switch between morphologic forms is central to&#160;its pathogenesis; these morphologic changes are regulated by a complex signaling network controlled in response to environmental stimuli. These regulatory components have been highly studied, but almost all studies use a variety of in vitro stimuli to trigger filamentation. To determine how morphogenesis is regulated during the pathogenesis process, we developed an in vivo microscopy system to obtain high spatial resolution images of organisms undergoing hyphal formation within the mammalian host. The protocol presented here describes the use of this system to screen small collections of C. albicans mutant strains, allowing us to identify key regulators of morphogenesis as it occurs at the site of infection. Representative results are presented, demonstrating that some regulators of morphogenesis, such as the transcriptional regulator Efg1, have consistent phenotypes in vitro and in vivo, whereas other regulators, such as adenyl cyclase (Cyr1), have significantly different phenotypes in vivo compared to in vitro.

Use of In Vivo Imaging to Screen for Morphogenesis Phenotypes in Candida albicans Mutant Strains During Active Infection in a Mammalian Host

This manuscript describes a method for screening moderately sized Candida albicans mutant libraries for morphogenesis phenotypes during active infection in a mammalian host using non-invasive confocal microscopy.

哺乳類宿主における活動感染中のカンジダ・アルビカンス変異株の形態形成表現型をスクリーニングするためのin vivoイメージングの使用

Candida albicans is an important human pathogen. Its ability to switch between morphologic forms is central to&#160;its pathogenesis; these morphologic ...

マウスの口腔カンジダ症に対するクルクミノイドを用いた効果的なaPDT

Curcuminoid-Mediated Antimicrobial Photodynamic Therapy on a Murine Model of Oral Candidiasis

Antimicrobial Photodynamic Therapy (aPDT) has been extensively investigated in vitro, and preclinical animal models of infections are suitable for evaluating alternative treatments prior to clinical trials. This study describes the efficacy of aPDT in a murine model of oral candidiasis. Forty mice were immunosuppressed with subcutaneous injections of prednisolone, and their tongues were inoculated using an oral swab previously soaked in a C. albicans cell suspension. Tetracycline was administered via drinking water during the course of the experiment. Five days after fungal inoculation, mice were randomly distributed into eight groups; a ninth group of untreated uninfected mice was included as a negative control (n = 5). Three concentrations (20 &#181;M, 40 &#181;M, and 80 &#181;M) of a mixture of curcuminoids were tested with a blue LED light (89.2 mW/cm2; ~455 nm) and without light (C+L+ and C+L- groups, respectively). Light alone (C-L+), no treatment (C-L-), and animals without infection were evaluated as controls. Data were analyzed using Welch&#39;s ANOVA and Games-Howell tests (&#945; = 0.05). Oral candidiasis was established in all infected animals and visualized macroscopically through the presence of characteristic white patches or pseudomembranes on the dorsum of the tongues. Histopathological sections confirmed a large presence of yeast and filaments limited to the keratinized layer of the epithelium in the C-L- group, and the presence of fungal cells was visually decreased in the images obtained from mice subjected to aPDT with either 40 &#181;M or 80 &#181;M curcuminoids. aPDT mediated by 80 &#181;M curcuminoids promoted a 2.47 log10 reduction in colony counts in comparison to those in the C-L- group (p = 0.008). All other groups showed no statistically significant reduction in the number of colonies, including photosensitizer (C+L-) or light alone (C-L+) groups. Curcuminoid-mediated aPDT reduced the fungal load from the tongues of mice.

著者スポットライト:口腔カンジダ症のマウスモデルにおけるクルクミンを用いた光線力学療法の探索

This protocol describes the application of antimicrobial Photodynamic Therapy (aPDT) in a murine model of oral candidiasis. aPDT was performed using a water-soluble mixture of curcuminoids and blue LED light.