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

Detta arbete har fått finansiering från Center of Applied Nanomedicine, National Cheng Kung University från Featured Areas Research Center Program inom ramen för Higher Education Sprout Project av utbildningsministeriet (MOE) och ministeriet för vetenskap och teknik, Taiwan [MOST 109-2327-B-006-005] till TW Wong. J.H. Hung erkänner finansiering från National Cheng Kung University Hospital, Taiwan [NCKUH-11006018] och [MOST 110-2314-B-006-086-MY3].

1. Förberedelse av aPDT-system
<ol><li>Skär fyra gröna ljusdioder (LED) från en LED-remsa (se Materialtabell) och rikta in dem mot fyra brunnar på en 96-brunnsplatta (figur 1). OBS: Lysdioderna arrangerades i en 4 x 3-matris. Lysdiodens baksida fästes på en kylfläns för att sprida värme under bestrålning.</li>
	<li>Mätlysdiodens flytande hastighet 11 vid 540 nm med en ljuseffektmätare (se Materialtabell</stro...

<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>

Uppmuntrande resultat av kliniska tillämpningar av RB-PDT för svampkeratit har nyligen rapporterats19. Absorptionstoppen för RB är vid 450-650 nm. Det är viktigt att bestämma ljuskällans flythastighet för en framgångsrik aPDT. En hög fluens (vanligtvis >100 J / cm2) krävs för att behandla cancerceller, medan en lägre fluens förväntas behandla infekterade lesioner6. En hög fluens innebär en lång exponeringstid som kanske inte är praktisk i en kl...

C. albicans koloniserar i mag-tarmkanalen och könsorganen hos friska individer och kan detekteras som normal mikrobiota hos cirka 50 procent av individerna1. Om en obalans skapas mellan värden och patogenen kan C. albicans invadera och orsaka sjukdom. Infektionen kan sträcka sig från lokala slemhinnor till multipel organsvikt2. I en övervakningsstudie med flera center i USA är ungefär hälften av isolaten från patienter med invasiv candidiasis mellan 2009 och 2017 C. albicans3. Candidemi kan associeras med hög sjuklighet, dödlighet, långvarig sjukhusvistelse4. US Centers of Disease Control and Prevention rapporterade att cirka 7% av alla Candida blodprover som testats är resistenta mot det svampdödande läkemedlet flukonazol5. Framväxten av läkemedelsresistenta Candida-arter väcker oro för att utveckla en alternativ eller adjuvansbehandling till antimykotiska medel.
Antimikrobiell fotodynamisk terapi (aPDT) innebär att man aktiverar en specifik fotosensibiliserare (PS) med ljus vid PS6: s högsta absorptionsvåglängd. Efter excitation överför den exciterade PS sin energi eller elektroner till de närliggande syremolekylerna och återgår till marktillståndet. Under denna process bildas reaktiva syrearter och singlet syre och orsakar cellskador. aPDT har använts i stor utsträckning för att döda mikroorganismer sedan 1990-talet7. En av fördelarna med aPDT är att flera organeller skadas i en cell av singlet syre och / eller reaktiva syrearter (ROS) under bestrålning; således har motstånd mot aPDT inte hittats förrän idag. Dessutom rapporterade en ny studie att bakterierna som överlevde efter aPDT blev känsligare för antibiotika8.
Ljuskällorna som används i aPDT inkluderar lasrar, metallhalogenlampor med filter, nära infrarött ljus och ljusdiod (LED) 9,10,11,12. Lasern ger en hög ljuseffekt, vanligtvis större än 0,5 W / cm2, som möjliggör leverans av en hög ljusdos på mycket kort tid. Det har använts i stor utsträckning i fall där en längre behandlingstid är obekvämt, såsom aPDT för orala infektioner. Nackdelen med en laser är att dess fläckstorlek på belysningen är liten, allt från några hundra mikrometer till 10 mm med en diffusor. Dessutom är laserutrustning dyr och behöver särskild utbildning för att fungera. Å andra sidan är bestrålningsområdet för en metallhalogenlampa med filter relativt större13. Lampan är dock för rejäl och dyr. LED-ljuskällor har blivit vanliga av aPDT inom det dermatologiska området eftersom det är litet och billigare. Bestrålningsområdet kan vara relativt stort med ett matrisarrangemang av LED-glödlampan. Hela ansiktet kan belysas samtidigt9. Ändå, de flesta, om inte alla, LED-ljuskällor som finns tillgängliga idag är utformade för klinisk användning. Det kanske inte är lämpligt för experiment i ett laboratorium eftersom det är rymdupptåt och dyrt. Vi utvecklade en billig LED-matris som är mycket liten och kan klippas och monteras från en LED-remsa. Lysdioderna kan monteras i olika arrangemang för olika experimentella konstruktioner. Olika förhållanden för aPDT kan slutföras i en 96-brunnsplatta eller till och med en 384-brunnsplatta i ett experiment.
Rose bengal (RB) är ett färgat färgämne som ofta används för att förbättra visualiseringen av hornhinneskador i mänskliga ögon14. RB-medierad aPDT har visat dödande effekter på Staphylococcus aureus, Escherichia coli och C. albicans med ungefär jämförbar effektivitet med toluidinblåO15. Denna studie visar en metod för att validera effekten av RB-aPDT på multiresistenta C. albicans.

<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

Invasiv Candida albicans-infektion är en signifikant opportunistisk svampinfektion hos människor eftersom det är en av de vanligaste kolonisatörerna i tarmen, munnen, slidan och huden. Trots tillgången på svampdödande läkemedel förblir dödligheten för invasiv candidiasis ~ 50%. Tyvärr ökar förekomsten av läkemedelsresistenta C. albicans globalt. Antimikrobiell fotodynamisk terapi (aPDT) kan erbjuda en alternativ eller adjuvant behandling för att hämma C. albicans biofilmbildning och övervinna läkemedelsresistens. Rose bengal (RB) -medierad aPDT har visat effektiv celldödande av bakterier och C. albicans. I denna studie beskrivs effekten av RB-aPDT på multiresistenta C. albicans . En hemlagad grön ljusdiod (LED) ljuskälla är utformad för att anpassa sig till mitten av en brunn på en 96-brunnsplatta. Jästen inkuberades i brunnarna med olika koncentrationer av RB och upplystes med varierande flytande grönt ljus. De dödande effekterna analyserades med plattutspädningsmetoden. Med en optimal kombination av ljus och RB uppnåddes 3-log tillväxthämning. Det drogs slutsatsen att RB-aPDT potentiellt kan hämma läkemedelsresistenta C. albicans.

Figur 1 visar det aPDT-system som används i föreliggande studie. Eftersom höga temperaturer kan orsaka betydande celldöd kyls LED-matrisen av en elektrisk fläkt och en kylfläns används under bestrålning för att upprätthålla en konstant temperatur vid 25 ± 1 ° C. Värmeeffekten kan diskonteras. Att ha en jämn ljusfördelning är också en viktig avgörande faktor för en framgångsrik aPDT; därför, Det är viktigt att rikta in LED-glödlampan till brunnen exakt under belysnin...

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

Den ökande förekomsten av läkemedelsresistenta Candida albicans är ett allvarligt hälsoproblem över hela världen. Antimikrobiell fotodynamisk terapi (aPDT) kan erbjuda en strategi för att bekämpa läkemedelsresistenta svampinfektioner. Det föreliggande protokollet beskriver Rose bengal-medierad aPDT-effekt på en multiresistent C. albicans-stam in vitro.

Rose Bengal-medied fotodynamisk terapi för att hämma 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, ...

Denna studie är ett enkelt, mångsidigt och enkelt system för att undersöka olika fotodynamiska effekter på olika mikroorganismer och celler. I detta system kan lysdioden passa in i olika arrangemang beroende på experimentdesign. Olika tillstånd av PDT kan beräknas i en 96-brunnsplatta eller till och med en 384-brunnsplatta i ett experiment.Denna teknik kan användas för att behandla svampkeratit på grund av vilken globalt cirka 150 000 personer förlorar ögonen trots intensiva behandlingar. Till att börja med, skär fyra gröna lysdioder från en LED-remsa och rikta in dem med tre brunnar på en 96-brunnsplatta. Mät lysdiodens fluenshastighet vid 540 nanometer med en ljuseffektmätare.Håll konstant temperatur vid 25 grader Celsius med en elektrisk fläkt som monterades bredvid plattan under bestrålning. Med en steril slinga, välj en enda koloni av Candida albicans från en agarplatta och tillsätt den till ett steriliserat glasrör som innehåller tre milliliter YPD-medium. Inkubera röret vid 25 grader Celsius med en rotationshastighet på 155 RPM i 14 till 16 timmar för att odla jästkulturen.Späd sedan övernattningskulturen med YPD-medium till ett OD 600-värde på 0,5. Inkubera vid 30 grader Celsius med rotation vid 155 rpm i fyra timmar för att uppnå loggtillväxtfasen. Upprepa utspädningen av loggfaskulturen med färskt YPD-medium till ett OD 600-värde på 0,65, för att få ungefär en gånger 10 till den sjunde kolonibildande enheten per milliliter.Förbered samtidigt en 4% lagerlösning av rosenbengal i 1X PBS. Filtrera sterilisera med ett 0,22 mikrometerfilter. Tillsätt 111 mikroliter 2% rosenbengallösning till en milliliter loggfasodling och samodling vid olika tidpunkter vid rumstemperatur för att förstå absorptionen av RB i cellerna.Centrifug vid 16 100 gånger G i 2 1/2 minuter vid rumstemperatur och tvätta samkulturen tre gånger med en milliliter 1X PBS. Efter tvättning, återuppliva Candida albicans-kulturen i en milliliter 1X PBS. För varje tillstånd, fördela kulturen i tre olika brunnar i en 96-brunnsplatta.Rikta in brunnarna mot LED-matrisen. I de ljusexponerade grupperna, slå på den elektriska fläkten och ljuset. Efter bestrålning, utför tiofaldiga seriella utspädningar två gånger genom att ta 20 mikroliter av samodlingslösningen från en brunn och tillsätt den till ett rör innehållande 180 mikroliter 1X PBS.Släpp sedan 20 mikroliter av varje seriell utspädning i en kvadrant av en YPD-agarplatta för att erhålla räknebara kolonier på plattan. Fluorescerande mikroskopi visade omedelbar färgning av Candida albicans med rosenbengal under röd fluorescens. Efter 15 minuters inkubation färgades de flesta cellerna med rosenbengal, vilket indikerar att den kommer in i cellerna på ett tidsberoende sätt.Vidare genererar aktivering av rosenbengal med ljus fria radikaler och singlet syre i cellerna, vilket resulterar i celldöd. I avsaknad av rosenbengal eller bestrålning observerades ingen celldöd. Candida albicans hämmades efter bestrålning med grönt ljus i närvaro av 0,2 % rosenbengal på ett lätt dosberoende sätt.Det är viktigt att först 96-brunnsplattan måste vara i linje med lysdiodens mitt. För det andra måste den använda agarplattan torkas noggrant för att förhindra blandning av separata kolonier. Candida albicans som växte på plattan kan skickas vidare för genanalys, vilket kan svara på hur PDT påverkar genuttryck av cellerna.Dessa tekniker banar väg för att undersöka hur PDT fungerar på olika celltyper, antingen cancerceller, bakterier, svampar eller virus med en enkel, billig och mångsidig plattform.

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.

En 96 Väl mikrotiterplatta-baserad metod för övervakning Bildning och svampdödande resistensbestämning av Candida albicans Biofilmer

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

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.

Visualisering av Biofilm bildning i Candida albicans med en automatiserad mikroflödessystem enhet

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.

Djup Dermal injektion som en modell av Candida albicans hudinfektion för histologiska analyser

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.

Dagliga ljusbehandling med rött ljus att reglera Candida albicans Biofilm tillväxt

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.

Visualisering av Candida albicans i murin mag-tarmkanalen med fluorescerande in situ hybridisering

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.

Förtydligande och 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 ...

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.

En Ex vivo-analys att studera Candida albicans Hyphal Morphogenesis i mag-tarmkanalen

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.

Användning av in vivo-avbildning för att screena för morfogenesfenotyper i Candida albicans mutanta stammar under aktiv infektion hos en däggdjursvärd

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

Effektiv aPDT med curcuminoider för oral candidiasis hos möss

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.

Författare i fokus: Utforska fotodynamisk terapi med curcumin i en murin modell för oral candidiasis

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.