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

Este trabalho recebeu financiamento do Centro de Nanomedicina Aplicada, Universidade Nacional de Cheng Kung do Programa de Centro de Pesquisa de Áreas Destacadas no âmbito do Projeto Desafeito do Ensino Superior pelo Ministério da Educação (MOE), e do Ministério da Ciência e Tecnologia, Taiwan [MOST 109-2327-B-006-005] à TW Wong. J.H. Hung reconhece financiamento do National Cheng Kung University Hospital, Taiwan [NCKUH-11006018], e [MOST 110-2314-B-006-086-MY3].

1. preparação do sistema aPDT
<ol><li>Corte quatro diodos emissores de luz verde (LEDs) de uma tira led (ver Tabela de Materiais) e alinhe-os com quatro poços de uma placa de 96 poços (Figura 1). NOTA: Os LEDs foram organizados em uma matriz 4 x 3. A parte de trás do LED foi aderida a um dissipador de calor para dispersar o calor durante a irradiação.</li>
	<li>Meça a taxa de fluência11 do LED a 540 nm com um medidor de ...

<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. N., Hsu, R., Chen, H., Wong, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Daylight+photodynamic+therapy:+an+update.">Daylight photodynamic therapy: an update.</a> Molecules. 25 (21), 5195 (2020).</li><li>Wainwright, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photodynamic+antimicrobial+chemotherapy+(PACT).">Photodynamic antimicrobial chemotherapy (PACT).</a> Journal of Antimicrobial Chemotherapy. 42 (1), 13-28 (1998).</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=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>

Resultados encorajadores das aplicações clínicas de RB-PDT para ceratite fúngica foram relatados recentemente19. O pico de absorção de RB é de 450-650 nm. É essencial determinar a taxa de fluência da fonte de luz para um aPDT bem sucedido. Uma alta fluência (geralmente >100 J/cm2) é necessária para tratar células cancerígenas, enquanto espera-se que uma fluência mais baixa trate lesões infectadas6. Uma alta fluência significa um longo tempo de ex...

C. albicans coloniza nos tratos gastrointestinal e genitourinary de indivíduos saudáveis e pode ser detectado como microbiota normal em cerca de 50% dos indivíduos1. Se um desequilíbrio é criado entre o hospedeiro e o patógeno, os albicanos são capazes de invadir e causar doenças. A infecção pode variar de infecções locais de membrana mucosa a falência múltipla de órgãos2. Em um estudo de vigilância multicêntrico nos EUA, cerca de metade dos isolados de pacientes com candidíase invasiva entre 2009 e 2017 é C. albicans3. A candidemia pode estar associada a altas taxas de morbidade, mortalidade, internação prolongada4. Os Centros de Controle e Prevenção de Doenças dos EUA relataram que cerca de 7% de todas as amostras de sangue candida testadas são resistentes ao fluconazol antifúngico5. O surgimento de espécies candidas resistentes a medicamentos levanta a preocupação de desenvolver uma terapia alternativa ou adjuvante para agentes antimícticos.
A terapia fotodinâmica antimicrobiana (aPDT) envolve a ativação de um fotoensibilizador específico (PS) com luz no comprimento de onda de absorção máxima do PS6. Após a excitação, o PS animado transfere sua energia ou elétrons para as moléculas de oxigênio próximas e retorna ao estado terrestre. Durante esse processo, espécies reativas de oxigênio e oxigênio único são formadas e causam danos celulares. aPDT tem sido amplamente usada para matar microrganismos desde a década de 19907. Um dos benefícios do APDT é que várias organelas são danificadas em uma célula por espécies de oxigênio único e/ou reativa (ROS) durante a irradiação; assim, a resistência ao APDT não foi encontrada até hoje. Além disso, um estudo recente relatou que as bactérias que sobreviveram após o APDT tornaram-se mais sensíveis aos antibióticos8.
As fontes de luz utilizadas no APDT incluem lasers, lâmpadas halógenas metálicas com filtros, luz quase infravermelha e diodo emissor de luz (LED)9,10,11,12. O laser fornece uma alta potência de luz, geralmente maior que 0,5 W/cm2, que permite a entrega de uma dose de luz alta em um tempo muito curto. Tem sido amplamente utilizado em casos em que um tempo de tratamento mais longo é inconveniente, como o APDT para infecções bucais. A desvantagem de um laser é que seu tamanho de ponto de iluminação é pequeno, variando de algumas centenas de micrômetros a 10 mm com um difusor. Além disso, o equipamento a laser é caro e precisa de treinamento específico para operar. Por outro lado, a área de irradiação de uma lâmpada de halogênio metálico com filtros é relativamente maior13. No entanto, a lâmpada é muito pesada e cara. As fontes de luz LED tornaram-se comuns de aPDT no campo dermatológico porque é pequena e menos cara. A área de irradiação pode ser relativamente grande com um arranjo de matriz da lâmpada LED. O rosto inteiro pode ser iluminado ao mesmo tempo9. No entanto, a maioria, se não todas, as fontes de luz LED disponíveis hoje são projetadas para uso clínico. Pode não ser adequado para experimentos em laboratório porque é ocupando espaço e caro. Desenvolvemos uma matriz led barata que é muito pequena e pode ser cortada e montada a partir de uma tira led. Os LEDs podem ser instalados em diferentes arranjos para diferentes projetos experimentais. Diferentes condições de aPDT podem ser completadas em uma placa de 96 poços ou até mesmo em uma placa de 384 poços em um experimento.
Rose bengala (RB) é um corante colorido amplamente utilizado para melhorar a visualização de danos na córnea nos olhos humanos14. APDT mediada por RB mostrou efeitos de morte em Staphylococcus aureus, Escherichia coli e C. albicans com eficiência aproximadamente comparável à do azul Toluidine O15. Este estudo demonstra um método para validar o efeito do RB-aPDT em C. albicans multidroga resistentes.

<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

A infecção por cânbis invasivos é uma infecção fúngica oportunista significativa em humanos porque é um dos colonizadores mais comuns do intestino, boca, vagina e pele. Apesar da disponibilidade de medicamentos antifúngicos, a taxa de mortalidade por candidíase invasiva permanece ~50%. Infelizmente, a incidência de C. albicans resistentes a medicamentos está aumentando globalmente. A terapia fotodinâmica antimicrobiana (aPDT) pode oferecer um tratamento alternativo ou adjuvante para inibir a formação de biofilmes C. albicans e superar a resistência a medicamentos. Rose bengala (RB) mediada aPDT mostrou morte celular eficaz de bactérias e C. albicans. Neste estudo, descreve-se a eficácia do RB-aPDT em C. albicans multidroga resistentes. Uma fonte de luz diodo verde caseira (LED) foi projetada para se alinhar com o centro de uma placa de 96 poços. As leveduras foram incubadas nos poços com diferentes concentrações de RB e iluminadas com fluências variadas de luz verde. Os efeitos da morte foram analisados pelo método de diluição da placa. Com uma combinação ideal de luz e RB, a inibição do crescimento de 3 log foi alcançada. Concluiu-se que o RB-aPDT poderia potencialmente inibir c. albicans resistentes a medicamentos.

A Figura 1 mostra o sistema aPDT utilizado no presente estudo. Uma vez que altas temperaturas podem causar morte celular significativa, a matriz led é resfriada por um ventilador elétrico, e um dissipador de calor é usado durante a irradiação para manter uma temperatura constante de 25 ± 1 °C. O efeito térmico pode ser descontado. Ter uma distribuição uniforme também é um importante fator determinante para um APDT bem sucedido; portanto, é fundamental alinhar a lâmpada LED ao ...

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

A crescente incidência de albicanos candida resistentes a medicamentos é um sério problema de saúde em todo o mundo. A terapia fotodinâmica antimicrobiana (aPDT) pode oferecer uma estratégia para combater infecções fúngicas resistentes a medicamentos. O presente protocolo descreve a eficácia da APDT mediada por Rose bengala em uma cepa c. albicans resistente a multidrogas in vitro.

Terapia fotodinâmica mediada por Rose Bengala para inibir a 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, ...

Este estudo é um sistema simples, versátil e fácil de examinar diferentes efeitos fotodinâmicos em vários microrganismos e células. Neste sistema, o LED pode ser encaixado em diferentes arranjos, dependendo do design do experimento. Diferentes condições de PDT podem ser computadas em uma placa de 96 poços ou até mesmo uma placa de 384 poços em um experimento.Essa técnica pode ser usada para tratar a ceratite fúngica, pois globalmente, cerca de 150.000 pessoas perdem os olhos apesar dos tratamentos intensivos. Para começar, corte quatro LEDs verdes de uma tira led e alinhe-os com três poços de uma placa de 96 poços. Meça a taxa de fluência do LED em 540 nanômetros com um medidor de energia leve.Mantenha a temperatura constante a 25 graus Celsius com um ventilador elétrico que foi montado ao lado da placa durante a irradiação. Com um laço estéril, escolha uma única colônia de albicanos candida de uma placa de ágar e adicione-o a um tubo de vidro esterilizado contendo três mililitros de meio YPD. Incubar o tubo a 25 graus Celsius a uma velocidade de rotação de 155 RPM por 14 a 16 horas para crescer a cultura da levedura.Em seguida, diluir a cultura da noite para o dia com o meio YPD para um valor de 600 OD de 0,5. Incubar a 30 graus Celsius com rotação a 155 RPM por quatro horas para alcançar a fase de crescimento do tronco. Repita a diluição da cultura da fase de registro com um meio YPD fresco a um valor de 600 OD de 0,65, para obter aproximadamente uma vezes 10 para a sétima colônia formando unidades por mililitro.Prepare simultaneamente uma solução de estoque de 4% de rose bengala em 1X PBS. O filtro esterilizar com um filtro de 0,22 micrômetro. Adicione 111 microliters de 2% de solução de bengala rosa a um mililitro da cultura da fase de registro, e co-cultura em diferentes pontos de tempo à temperatura ambiente para entender a absorção de RB nas células.Centrifugar a 16, 100 vezes G por 2 minutos e meio em temperatura ambiente, e lavar a co-cultura três vezes com um mililitro de 1X PBS. Após a lavagem, resuspenha a cultura candida albicans em um mililitro de 1X PBS. Para cada condição, distribua a cultura em três poços diferentes em uma placa de 96 poços.Alinhe os poços com o array LED. Nos grupos expostos à luz, ligue o ventilador elétrico e a luz. Após a irradiação, realize diluições seriais dez vezes, pegando 20 microliters da solução de cocultura de um poço, e adicionando-a a um tubo contendo 180 microliters de 1X PBS.Em seguida, solte 20 microliters de cada diluição serial em um quadrante de uma placa de ágar YPD para obter colônias contáveis na placa. A microscopia fluorescente mostrou coloração imediata de candidanos albicanos com bengala rosa sob fluorescência vermelha. Após 15 minutos de incubação, a maioria das células foram manchadas com bengala rosa, indicando que ela entra nas células de forma dependente do tempo.Além disso, a ativação da bengala rosa com luz gera radicais livres e oxigênio único nas células, resultando em morte celular. Na ausência de bengala-rosa ou irradiação, não foi observada morte celular. A candida albicans foi inibida após a irradiação da luz verde na presença de 0,2% de bengala rosa de forma dependente de dose leve.É importante que primeiro a placa de 96 poços esteja alinhada com o centro do LED. Em segundo lugar, a placa de ágar utilizada deve ser completamente seca para evitar a mistura de colônias separadas. Os albicanos candida que cresceram na placa podem ser enviados para análise genética, o que pode responder como o PDT afeta a expressão genética das células.Essas técnicas abrem caminho para examinar como o PDT funciona em diferentes tipos de células, sejam células cancerosas, bactérias, fungos ou vírus com uma plataforma fácil, barata e versátil.

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.

A 96 Bem microtitulação Método da Placa com base para a Formação Monitoramento e Teste de susceptibilidade antifúngica de Candida albicans Biofilmes

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

Imunologia e Infecção

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.

Visualização da formação de biofilmes de Candida albicans , usando um dispositivo Microfluidic automatizada

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.

Profunda dérmica injeção como um modelo de Candida albicans de infecção da pele para análise histológica

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.

Diária fototerapia com luz vermelha para regular crescimento de biofilme de Candida albicans

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.

Visualização de candida albicans no trato gastrointestinal murina usando fluorescente in situ hibridização

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.

Esclarecendo e Imagem Candida albicans Biofilmes

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.

Um ensaio ex vivo para estudar candida albicans Hórgênese Háfal no Trato Gastrointestinal

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.

Uso de imagens in vivo para rastrear fenótipos de morfogênese em cepas mutantes de Candida albicans durante a infecção ativa em um hospedeiro mamífero

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

aPDT eficaz usando curcuminóides para candidíase oral em camundongos

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.

Autor em destaque: Explorando a terapia fotodinâmica com curcumina em um modelo murino para candidíase oral

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.