Name: 作者聚焦：光动力疗法作为在耐药 念珠菌 中诱导娇小菌落用于抗真菌研究的新方法
Uploaded: 2024-03-29T14:30:00
Description: Watch this Scientific Journal Video about Photodynamic Therapy as a Novel Approach to Induce Petite Colonies in Drug-Resistant Candida for Antifungal Research at JoVE.com

这项工作得到了台湾科学技术部[MOST 110-2314-B-006-086-MY3]，国立成功大学[K111-B094]，[K111-B095]，台湾国立成功大学医院[NCKUH-11204031]，[NCKUMCS2022057]的资助。

1. 光滑梭菌的培养
注：对大多数抗真菌剂（包括氟康唑）具有耐药性的多重耐药 光滑念珠菌 （C2-1000907）用于实验。实验条件可能需要根据特定菌株进行调整，因为不同菌株之间可能存在差异。所有实验都使用在25°C下生长的对数期念珠菌（模拟自然感染）以确保一致性。与白色念珠菌相比， 光滑念珠菌缺乏菌丝简化了定量， 白色念珠?...

分析光滑念珠菌的小集落形成和耐药性

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B., Reingold, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiologic+features,+clinical+characteristics,+and+predictors+of+mortality+in+patients+with+candidemia+in+Alameda+County,+California;+a+2017-2020+retrospective+analysis.">Epidemiologic features, clinical characteristics, and predictors of mortality in patients with candidemia in Alameda County, California; a 2017-2020 retrospective analysis.</a> BMC Infect Dis. 22 (1), 843 (2022).</li><li>Whittaker, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+petite+mutation+in+yeast.">The petite mutation in yeast.</a> Subcell Biochem. 6, 175-232 (1979).</li><li>Hatab, M. A., Whittaker, P. 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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=In-vivo+selection+of+an+azole-resistant+petite+mutant+of+Candida+glabrata.">In-vivo selection of an azole-resistant petite mutant of Candida glabrata.</a> J Med Microbiol. 49 (11), 977-984 (2000).</li><li>Badrane, 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=Genotypic+diversity+and+unrecognized+antifungal+resistance+among+populations+of+Candida+glabrata+from+positive+blood+cultures.">Genotypic diversity and unrecognized antifungal resistance among populations of Candida glabrata from positive blood cultures.</a> Nat Commun. 14 (1), 5918 (2023).</li><li>Shantal, C. -. J. N., Juan, C. -. C., Lizbeth, B. -. U. S., Carlos, H. -. G. J., Estela, G. -. P. 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+glabrata+is+a+successful+pathogen:+An+artist+manipulating+the+immune+response.">Candida glabrata is a successful pathogen: An artist manipulating the immune response.</a> Microbiol Res. 260, 127038 (2022).</li><li>Gamarra, S., Mancilla, E., Dudiuk, C., Garcia-Effron, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Candida+dubliniensis+and+Candida+albicans+differentiation+by+colony+morphotype+in+Sabouraud-triphenyltetrazolium+agar.">Candida dubliniensis and Candida albicans differentiation by colony morphotype in Sabouraud-triphenyltetrazolium agar.</a> Rev Iberoam Micol. 32 (2), 126-128 (2015).</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=Rose+bengal-mediated+photodynamic+therapy+to+inhibit+Candida+albicans.">Rose bengal-mediated photodynamic therapy to inhibit Candida albicans.</a> J Vis Exp. (181), e63558 (2022).</li><li>Cardoso, D. R., Franco, D. W., Olsen, K., Andersen, M. L., Skibsted, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactivity+of+bovine+whey+proteins,+peptides,+and+amino+acids+toward+triplet+riboflavin+as+studied+by+laser+flash+photolysis.">Reactivity of bovine whey proteins, peptides, and amino acids toward triplet riboflavin as studied by laser flash photolysis.</a> J Agric Food Chem. 52 (21), 6602-6606 (2004).</li><li>Hall, R. M., Trembath, M. K., Linnane, A. W., Wheelis, L., Criddle, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+affecting+petite+induction+and+the+recovery+of+respiratory+competence+in+yeast+cells+exposed+to+ethidium+bromide.">Factors affecting petite induction and the recovery of respiratory competence in yeast cells exposed to ethidium bromide.</a> Mol Gen Genet. 144 (3), 253-262 (1976).</li><li>Chen, X. J., Clark-Walker, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+petite+mutation+in+yeasts:+50+years+on.">The petite mutation in yeasts: 50 years on.</a> Int Rev Cytol. 194, 197-238 (2000).</li><li>Piskur, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inheritance+of+the+yeast+mitochondrial+genome.">Inheritance of the yeast mitochondrial genome.</a> Plasmid. 31 (3), 229-241 (1994).</li><li>Wong, T. W., Wang, Y. Y., Sheu, H. M., Chuang, Y. 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S. <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+to+control+microbial+biofilms.">Photodynamic therapy to control microbial biofilms.</a> Photodiagnosis Photodyn Ther. 33, 102090 (2021).</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></ol>

这项研究揭示了 PDT 是首次报道的诱导念珠菌中小群体形成的方法，超过了溴化乙锭和氟康唑的既定作用。这一新颖的观察结果需要进一步探索，以揭示其通过降低毒力和耐药机制的出现来根除真菌的影响。
RB 介导的 PDT 有效抑制 光滑念珠菌的生长，为念珠菌感染提供了一种潜在的替代治疗方法。作为一种光激活光敏剂，RB 在暴露于特定波长时会产生单线态氧和活性氧...

真菌感染，特别是由白色念珠菌和日益耐药的光滑念珠菌引起的真菌感染，构成了严重的全球威胁1.这些感染可能是致命的，特别是对于住院患者和免疫系统较弱的患者。抗真菌耐药性的上升威胁到侵袭性念珠菌病的控制，侵袭性念珠菌病是一种严重的真菌感染，死亡率很高，尤其是白色念珠菌 2。耐药菌株阻碍了有效的治疗，可能会增加复杂性和死亡率。在美国加利福尼亚州阿拉米达县，C. glabrata 已成为最普遍的入侵物种3.念珠菌属流行率和分布的这种变化可能受到当地医疗保健实践、患者人口统计学、抗真菌药物的使用以及念珠菌感染危险因素的流行率的影响。
念珠菌中的娇小突变体缺乏功能性线粒体，揭示了这种细胞器如何影响药物反应、毒力和抗逆性 4,5。C. glabrata 很容易形成这些菌落，对多烯敏感，而对唑类6 失去敏感性。唑类药物敏感性和呼吸功能有着错综复杂的联系，呼吸减弱会通过线粒体 DNA 丢失导致抵抗7。已从接受氟康唑治疗的骨髓移植受者8 和血流感染患者的血培养瓶9 中分离出具有唑类耐药性的 C. glabrata 小菌落。它们在耐药性、毒力和应激反应方面的潜在影响凸显了其临床意义。此外，它们的独特特性使它们成为研究线粒体生物学基本问题的宝贵工具5。随着对娇小突变体研究的继续，它们在临床和基础研究中的应用可能会扩大。
这项研究发现，光动力疗法（PDT）可以诱导 光滑梭菌中的小菌落，扩大了 将光滑梭 菌暴露于溴化乙锭或氟康唑的传统技术之外的方法范围。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.22 &#956;m filter</td>
			<td>Merck, Taipei, Taiwan</td>
			<td>Millex, SLGVR33RS</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL microfuge tube</td>
			<td>Neptune, San Diego, USA</td>
			<td>#3745</td>
			<td></td>
		</tr>
		<tr>
			<td>20% Triphenyltetrazolium chloride (TTC)</td>
			<td>Sigma-Aldrich, MO, USA</td>
			<td>T8877</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL polypropylene round bottom tube</td>
			<td>Corning, AZ, USA</td>
			<td>352059</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL round-bottom tube with cell strainer cap</td>
			<td>Corning, AZ, USA</td>
			<td>Falcon, #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>Agar</td>
			<td>BRS, Tainan, Taiwan</td>
			<td>AG012</td>
			<td></td>
		</tr>
		<tr>
			<td>Blank disk</td>
			<td>Advantec, Tokyo, Japan</td>
			<td>49005040</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf, UK</td>
			<td>5415R</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethidium bromide solution</td>
			<td>Sigma-Aldrich, MO, USA</td>
			<td>E1510</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluconazole, 2 mg/mL</td>
			<td>Pfizer, NY, USA</td>
			<td>BC18790248</td>
			<td></td>
		</tr>
		<tr>
			<td>GraphPad Prism</td>
			<td>GraphPad Software</td>
			<td>Version 7.0</td>
			<td></td>
		</tr>
		<tr>
			<td>Green light emitting diode (LED) strip</td>
			<td>Nanyi electronics Co.,Ltd, Tainan, Taiwan</td>
			<td>5050</td>
			<td>Excitation wave: 500~550 nm</td>
		</tr>
		<tr>
			<td>Low Temperature. shake Incubators</td>
			<td>Yihder, Taipei, Taiwan</td>
			<td>LM-570D (R)</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouth care cotton swabs</td>
			<td>Good Verita Enterprise, Taipei, Taiwan</td>
			<td>161357</td>
			<td></td>
		</tr>
		<tr>
			<td>Muller Hinton II agar</td>
			<td>BD biosciences, California, USA</td>
			<td>211438</td>
			<td></td>
		</tr>
		<tr>
			<td>Multimode microplate reader</td>
			<td>Molecular Devices</td>
			<td>SpectraMax i3x</td>
			<td></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, 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, 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>

induction of petite colonies in candida glabrate via rose bengal-mediated photodynamic therapy

面对念珠菌血症患者40%的死亡率，耐药念珠菌及其娇小的突变体仍然是一个主要的治疗挑战。与抗生素/抗真菌药不同，抗菌光动力疗法 （aPDT） 针对多种真菌结构，可能会阻碍耐药性。诱导小菌落的传统方法依赖于溴化乙锭或氟康唑，它们会影响药物敏感性和应激反应。本研究调查了绿光（峰值 520 nm）和孟加拉玫瑰 （RB） 光敏剂在对抗耐药性光滑念珠菌分离株中的应用。研究结果显示，aPDT处理显著抑制细胞生长（减少99.9%≥）并有效诱导小集落形成，如线粒体氧化还原指示剂染色尺寸减小和丢失所证明的那样。这项研究提供了初步证据，表明 aPDT 可以在体外诱导多重耐药光滑念珠菌菌株中的小菌落，为对抗耐药性真菌感染提供了一种潜在的变革性方法。

Fungal infections, particularly those caused by Candida albicans and increasingly drug-resistant Candida glabrata, pose a serious global threat1. These infections can be deadly, especially for hospitalized patients and those with weakened immune systems. Rising antifungal resistance threatens the control of invasive candidiasis, a severe fungal infection with high mortality, especially from Candida albicans2. Resistant strains hinder effective treatment, potentially increasing both complexity and death rates. In Alameda County, California, USA, C. glabrata has become the most prevalent invasive species3. This shift in the prevalence and distribution of Candida species may be influenced by local healthcare practices, patient demographics, the utilization of antifungal agents, and the prevalence of risk factors for Candida infections.
Petite mutants in Candida, lacking functional mitochondria, reveal how this organelle affects drug response, virulence, and stress resistance4,5. C. glabrata readily forms these colonies, gaining sensitivity to polyenes while losing it to azoles6. Azole sensitivity and respiratory function are intricately linked, with diminished respiration leading to resistance via mitochondrial DNA loss7. Petite colonies of C. glabrata with azole resistance have been isolated from human stool samples from a bone marrow transplant recipient undergoing fluconazole treatment8 and from blood culture bottles of patients with bloodstream infections9. Their potential implications in drug resistance, virulence, and stress response highlight their clinical significance. Additionally, their distinct properties make them valuable tools for investigating fundamental questions in mitochondrial biology5. As research into petite mutants continues, their applications in both clinical and basic research are likely to expand.
This study discovered that photodynamic therapy (PDT) can induce petite colonies in C. glabrata, expanding the range of methods beyond the traditional techniques of exposing C. glabrata to ethidium bromide or fluconazole.

作者聚焦：光动力疗法作为在耐药 念珠菌 中诱导娇小菌落用于抗真菌研究的新方法

通过 Rose Bengal 介导的光动力疗法诱导念珠菌中的小菌落

The research aims to use photodynamic therapy, also known as PDT, to fight against drug-resistant Candida infection. In this study, we are trying to answer how effectively PDT kills the fungi, and how the virulence of the fungi changes after the treatment. We found petite colonies from the after PDT.Present methods for inducing petite colonies rely on ethidium bromide or fluconazole, which may work through different pathways on drug resistance and the stress responses, compared to PDT. Recently, petite colonies of Candida have become a significant concern in the clinic because they may resist medication and increase mortality rates. Photodynamic therapy has become one of the antifungal treatments and our finding helps breach the gap between traditional methods used to study petite colonies.Our findings provide evidence that PDT can induce petite colonies in drug resistant Candida in-vitro, presenting a groundbreaking method to study petite colonies. We'll definitely delve deeper into this field. There are many questions to be answered.How and why do petite colonies form after PDT stress? Do different Candida species respond differently? I believe it's a never ending story to be explored.

数据以平均值表示，标准误差±，来自三个独立实验，每组至少一式三份。实验数据，包括菌落计数、OD600 测量值和 TTC 染色结果，使用绘图和统计软件进行绘图和统计分析（参见 材料表）。使用单因素方差分析或 t 检验 来分析数据， p 值 <0.05 被认为是显著的。使用 48 位全彩光学扫描仪以 1200 dpi 的分辨率进行扫描，随后使用 ImageJ 软件进行菌落直径的测量。...

Watch this Scientific Journal Video about Photodynamic Therapy as a Novel Approach to Induce Petite Colonies in Drug-Resistant Candida for Antifungal Research at JoVE.com

小菌落在念珠菌属耐药性中的意义尚未得到充分探索。抗菌光动力疗法 （aPDT） 提供了一种对抗耐药性真菌感染的有前途的策略。这项研究表明，孟加拉玫瑰介导的 aPDT 可有效灭活 光滑念珠菌 并诱导小菌落，呈现出独特的过程。

Facing a 40% mortality rate in candidemia patients, drug-resistant Candida and their petite mutants remain a major treatment challenge. Antimicrobial ...

该研究旨在使用光动力疗法（也称为 PDT）来对抗耐药念珠菌感染。在这项研究中，我们试图回答PDT如何有效地杀死真菌，以及治疗后真菌的毒力如何变化。我们在PDT之后发现了小菌落。目前诱导小菌落的方法依赖于溴化乙锭或氟康唑，与PDT相比，它们可能通过不同的途径对耐药性和应激反应起作用。最近，念珠菌的小型菌落已成为临床上的一个重大问题，因为它们可能会抵抗药物治疗并增加死亡率。光动力疗法已成为抗真菌疗法之一，我们的发现有助于打破用于研究小型菌落的传统方法之间的差距。我们的研究结果提供了证据表明，PDT可以在体外诱导耐药念珠菌中的小型菌落，为研究小型菌落提供了一种开创性的方法。我们肯定会更深入地研究这个领域。有许多问题需要回答。PDT 应激后如何以及为什么会形成小菌落？不同的念珠菌物种反应不同吗？我相信这是一个永无止境的故事，有待探索。

induction-petite-colonies-candida-glabrate-via-rose-bengal-mediated

Research

JoVE Journal

Biology

Induction of Petite Colonies in Candida glabrate via Rose Bengal-Mediated Photodynamic Therapy

Facing a 40% mortality rate in candidemia patients, drug-resistant Candida and their petite mutants remain a major treatment challenge. Antimicrobial photodynamic therapy (aPDT) targets multiple fungal structures, unlike antibiotics/antifungals, potentially thwarting resistance. Traditional methods for inducing petite colonies rely on ethidium bromide or fluconazole, which can influence drug susceptibility and stress responses. This study investigated the application of green light (peak 520 nm) and rose bengal (RB) photosensitizer to combat a drug-resistant Candida glabrata isolate. The findings revealed that aPDT treatment significantly inhibited cell growth (&#8805;99.9% reduction) and effectively induced petite colony formation, as evidenced by reduced size and loss of mitochondrial redox indicator staining. This study provides initial evidence that aPDT can induce petite colonies in a multidrug-resistant C. glabrata strain in vitro, offering a potentially transformative approach for combating resistant fungal infections.

The significance of petite colonies in Candida spp. drug resistance has not been fully explored. Antimicrobial photodynamic therapy (aPDT) offers a promising strategy against drug-resistant fungal infections. This study demonstrates that rose bengal-mediated aPDT effectively deactivates Candida glabrata and induces petite colonies, presenting a unique procedure.

作者聚焦：光动力疗法作为在耐药 念珠菌 中诱导娇小菌落用于抗真菌研究的新方法

作者聚焦：光动力疗法作为在耐药念珠菌中诱导娇小菌落用于抗真菌研究的新方法