Wir danken allen Mitgliedern des Nobile Lab für hilfreiche Diskussionen an Biofilm-Assays. Diese Studie wurde von der National Institutes of Health (NIH) Stipendium R21 AI125801 (C.J.N.) unterstützt. D.L.R. wurde durch ein Promotionsstipendium von der University of California Institute für Mexiko und den Vereinigten Staaten (UC-MEXUS) und Consejo Nacional de Ciencia y Technologia (CONACYT) unterstützt.

<ol>
	<li>Nobile, C. J., Johnson, A. D. <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+Biofilms+and+Human+Disease.">Candida albicans Biofilms and Human Disease.</a> Annu Rev Microbiol. 69, 71-92 (2015).</li><li>Kojic, E. M., Darouiche, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Candida+infections+of+medical+devices.">Candida infections of medical devices.</a> Clin Microbiol Rev. 17 (2), 255-267 (2004).</li><li>Fox, E. P., Nobile, C. J., Dietrich, L. A., Friedmann, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Candida albicans: Symptoms, Causes and Treatment Options. , 1-24 (2013).</li><li>Gulati, M., Nobile, C. J. <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+biofilms:+development,+regulation,+and+molecular+mechanisms.">Candida albicans biofilms: development, regulation, and molecular mechanisms.</a> Microbes Infect. 18 (5), 310-321 (2016).</li><li>Uppuluri, 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=Dispersion+as+an+important+step+in+the+Candida+albicans+biofilm+developmental+cycle.">Dispersion as an important step in the Candida albicans biofilm developmental cycle.</a> PLoS Pathog. 6 (3), e1000828 (2010).</li><li>Andes, 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=Development+and+characterization+of+an+in+vivo+central+venous+catheter+Candida+albicans+biofilm+model.">Development and characterization of an in vivo central venous catheter Candida albicans biofilm model.</a> Infect Immun. 72 (10), 6023-6031 (2004).</li><li>Nett, J. E., Marchillo, K., Spiegel, C. A., Andes, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+validation+of+an+in+vivo+Candida+albicans+biofilm+denture+model.">Development and validation of an in vivo Candida albicans biofilm denture model.</a> Infect Immun. 78 (9), 3650-3659 (2010).</li><li>Nett, J. 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=Rat+indwelling+urinary+catheter+model+of+Candida+albicans+biofilm+infection.">Rat indwelling urinary catheter model of Candida albicans biofilm infection.</a> Infect Immun. 82 (12), 4931-4940 (2014).</li><li>Krom, B. P., Willems, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Vitro+Models+for+Candida+Biofilm+Development.">In Vitro Models for Candida Biofilm Development.</a> Methods Mol Biol. 1356, 95-105 (2016).</li><li>Hawser, S. P., Douglas, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilm+formation+by+Candida+species+on+the+surface+of+catheter+materials+in+vitro.">Biofilm formation by Candida species on the surface of catheter materials in vitro.</a> Infect Immun. 62 (3), 915-921 (1994).</li><li>Ramage, G., Vande Walle, K., Wickes, B. L., Lopez-Ribot, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standardized+method+for+in+vitro+antifungal+susceptibility+testing+of+Candida+albicans+biofilms.">Standardized method for in vitro antifungal susceptibility testing of Candida albicans biofilms.</a> Antimicrob Agents Chemother. 45 (9), 2475-2479 (2001).</li><li>Nett, J. E., Cain, M. T., Crawford, K., Andes, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimizing+a+Candida+biofilm+microtiter+plate+model+for+measurement+of+antifungal+susceptibility+by+tetrazolium+salt+assay.">Optimizing a Candida biofilm microtiter plate model for measurement of antifungal susceptibility by tetrazolium salt assay.</a> J Clin Microbiol. 49 (4), 1426-1433 (2011).</li><li>Krom, B. P., Cohen, J. B., McElhaney Feser, G. E., Cihlar, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+candidal+biofilm+microtiter+assay.">Optimized candidal biofilm microtiter assay.</a> J Microbiol Methods. 68 (2), 421-423 (2007).</li><li>Lohse, M. B., 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=Assessment+and+Optimizations+of+Candida+albicans+In+Vitro+Biofilm+Assays.">Assessment and Optimizations of Candida albicans In Vitro Biofilm Assays.</a> Antimicrob Agents Chemother. 61 (5), (2017).</li><li>Winter, M. B., 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=Global+Identification+of+Biofilm-Specific+Proteolysis+in+Candida+albicans.">Global Identification of Biofilm-Specific Proteolysis in Candida albicans.</a> mBio. 7 (5), (2016).</li><li>Nobile, C. J., 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=A+recently+evolved+transcriptional+network+controls+biofilm+development+in+Candida+albicans.">A recently evolved transcriptional network controls biofilm development in Candida albicans.</a> Cell. 148 (1-2), 126-138 (2012).</li><li>Fox, E. 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=An+expanded+regulatory+network+temporally+controls+Candida+albicans+biofilm+formation.">An expanded regulatory network temporally controls Candida albicans biofilm formation.</a> Mol Microbiol. 96 (6), 1226-1239 (2015).</li><li>Nobile, C. J., Mitchell, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+cell-surface+genes+and+biofilm+formation+by+the+C.+albicans+transcription+factor+Bcr1p.">Regulation of cell-surface genes and biofilm formation by the C. albicans transcription factor Bcr1p.</a> Curr Biol. 15 (12), 1150-1155 (2005).</li><li>Baker, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> At the bench: A laboratory navigator. 27, (2005).</li></ol>

Clarissa J. Nobile ist einer der Gründer von BioSynesis, Inc., ein Unternehmen für die Entwicklung von Inhibitoren und Diagnostik von C. Albicans Biofilmen.

Anpassbare mikrofluidischen Biofilm Assays hier beschriebenen ermöglicht die Visualisierung der Biofilmbildung in Echtzeit auf eine Einzelzelle Niveau, wenn eine Pauschale Laminar-Flow und konstante Temperatur ausgesetzt. Freuen Sie sich auf ein mächtiges Mittel, um die Entwicklung von Biofilmen in Wildtyp und Mutanten Stämme zu studieren, und die Auswirkungen der antimikrobielle Wirkstoff Behandlungen auf Biofilme unter Bedingungen, die physiologische Bedingungen nachahmen im klinischen Umfeld beobachtet. Im Gegensat...

Candida Albicans gehört Kommensalen der menschlichen Mikrobiota, aber es ist auch eine opportunistische Erreger, verursachen oberflächliche und schweren Pilzinfektionen1,2. Eine größere Virulenz-Eigenschaft von C. Albicans ist seine Fähigkeit, Form elastisch und resistente Biofilme Gemeinschaften von Zellen eine Oberfläche eingehalten und eingeschlossen in eine extrazelluläre Matrix-Werkstoff-1,-3. C. Albicans Biofilme sind hochgradig strukturiert, mit mehreren Schichten von mehreren Zelltypen (angehende Hefe-Form Runde Zellen, ovale pseudohyphal Zellen und röhrenförmigen Bodenaggregaten Zellen)4. C. Albicans Biofilm Entwicklung beginnt mit der Einhaltung runden Hefe-Form Zellen eine Oberfläche (Aussaat des Biofilms), gefolgt von der Verbreitung dieser Zellen an der Oberfläche, und dann die Reifung des unreifen Biofilms Struktur in eine voll ausgebildet Biofilm, die extrazelluläre Matrix Material4umgeben ist. Der Reife Biofilm besteht überwiegend aus länglichen Bodenaggregaten Zellen, die Dichte und miteinander verbundenen Netzwerken, die architektonischen Stabilität, der Biofilm-4bilden. Über den gesamten Lebenszyklus der Biofilm Runde Knospen Hefe Zellen zerstreuen von der Reife Biofilm und können Reisen in andere Regionen des Körpers zu disseminierte Infektionen verursachen oder Samen neue Biofilme an anderen Standorten4,5. C. Albicans können auf biotische Oberflächen, z. B. Schleimhaut-Oberflächen und in der gesamten Wirtsgewebe, und abiotischen Oberflächen, wie z.B. Kathetern, Herzschrittmacher, Zahnersatz und Gelenkprothesen Biofilme bilden. Aufgrund der widerspenstigen Eigenschaften von Biofilmen sie sind extrem schwer zu beseitigen, und in vielen Fällen ist die einzige wirksame Behandlungsstrategie Entfernung des infizierten Gerät4. Es ist so entscheidend für Biofilmbildung unter Bedingungen ähnlich denen beobachtet im klinischen Umfeld zu untersuchen.
Es gibt mehrere kritische in Vivo Tiermodellen zur Untersuchung von C. Albicans Biofilm Bildung6,7,8; aber können diese Studien kostspielig, zeitaufwändig, und sind begrenzt durch die Anzahl der Stämme und antimikrobielle Wirkstoffe, die zu einem bestimmten Zeitpunkt getestet werden können. In-vitro- Biofilm-Assays, auf der anderen Seite ermöglichen die schnelle, Hochdurchsatz-Bewertung von Antimykotika Verbindungen und mutierte Stämme und sind kostengünstiger und ethische als Biofilm Assays durchgeführt Out in Tier9, Modelle 10,11,12,13,14. Hier beschreiben wir eine in-vitro- Tests, die wir entwickelt und optimiert, um die Biofilmbildung zeitlich unter Laminar-Flow mit einem anpassbaren mikrofluidischen Gerät14,15beobachten. Der Test ermöglicht die Visualisierung der einzelnen Phasen der Biofilmbildung, einschließlich der Einhaltung der erste Schritt, Zellproliferation, Biofilm Reifung und Zelle Dispersion. Der Test eignet sich auch Zelle Morphologie Änderungen während der Entwicklung eines Biofilms zu visualisieren.
Mikrotiter-Platten, die in der Regel verwendet werden, für in-vitro- Biofilm-Assays, bei hohem Durchsatz nicht kontrollierten Strömungsverhältnisse zulassen. Traditionelle Laminar-Flow-Zelle-Systeme ermöglichen die kontinuierliche Bewertung der Biofilmbildung in kontrollierten Strömungsverhältnisse, aber diese sind oft zeitaufwändige Einrichten und sind in der Regel nur über begrenzte Dynamikumfang Kontrolle und Durchsatz. Die mikrofluidischen Gerät hier verwendete überwindet diese Einschränkungen durch die Kombination von Hochdurchsatz-Platten (mit 48 Brunnen) mit einer integrierten Laminar-Flow-Kammer und hoch reproduzierbare und vielseitig anpassbar.
Wir beschreiben hier, ein Protokoll für die Verwendung eines handelsüblichen automatisierte mikrofluidischen Geräts Biofilmbildung ein Wildtyp C. Albicans bewerten belasten, die Auswirkungen der bekannten Antimykotikum auf die Entwicklung eines Biofilms und Biofilm Bildung in zwei mutierte Stämme (bcr1Δ/Δ und efg1 Δ/Δ), die zuvor gemeldet wurden, um Biofilm haben Mängel in Vitro und in Vivo16,17,18. Das beschriebene Protokoll kann verwendet werden, um die Wirksamkeit antimikrobieller Mittel in der Hemmung der Biofilmbildung während der Entwicklung eines Biofilms zu testen und zu identifizieren, die für normale Biofilm Entwicklung durch screening mutierte Bibliotheken erforderlich.

<table><tbody><tr><td>BioFlux 1000z</td><td>Fluxion</td><td></td><td>Automated microfluidic device for live cell analysis</td></tr><tr><td>48-well plate 0-20 dyne</td><td>Fluxion</td><td>910-0047</td><td>Microfluidic plate</td></tr><tr><td>Montage Software</td><td>Fluxion</td><td>Version 7.8.4.0</td><td>Visualization analysis software</td></tr><tr><td>ImageJ Software</td><td>NIH</td><td>https://imagej.nih.gov/ij/</td><td></td></tr><tr><td>Yeast Extract</td><td>Criterion</td><td>C7341</td><td></td></tr><tr><td>Bacto Peptone</td><td>BD Biosciences</td><td>211677</td><td></td></tr><tr><td>Dextrose (D-Glucose)</td><td>Fisher Scientific</td><td>D163</td><td></td></tr><tr><td>Potassium Phosphate Monobasic</td><td>Fisher Scientific</td><td>P285-500</td><td></td></tr><tr><td>RPMI-1640</td><td>Sigma-Aldrich</td><td>R6504</td><td></td></tr><tr><td>MOPS</td><td>Sigma-Aldrich</td><td>M3183</td><td></td></tr><tr><td>Nutrient Broth</td><td>Criterion</td><td>C6471</td><td></td></tr><tr><td>Difco D-Mannitol</td><td>BD Biosciences</td><td>217020</td><td></td></tr><tr><td>Agar</td><td>Criterion</td><td>C5001</td><td></td></tr><tr><td>Amphotericin B</td><td>Corning</td><td>30-003-CF</td><td></td></tr><tr><td>Sterile Inoculating Loops</td><td>VWR</td><td>30002-094</td><td></td></tr><tr><td>Petri Dishes with Clear Lid</td><td>Fisher Scientific</td><td>FB0875712</td><td></td></tr><tr><td>Disposable Cuvettes</td><td>Fisher Scientific</td><td>14-955-127</td><td></td></tr><tr><td>Lens Paper</td><td>VWR</td><td>52846-001</td><td></td></tr><tr><td>Microplate and Cuvette Spectrophotometer</td><td>BioTek</td><td>EPOCH2TC</td><td></td></tr><tr><td>Shaking Incubator</td><td>Eppendorf</td><td>M12820004</td><td></td></tr></tbody></table>

visualization of biofilm formation in candida albicans using an automated microfluidic device

Candida Albicans ist der häufigste pilzliche Erreger des Menschen, was etwa 15 % der Fälle von nosokomialen Sepsis. Eine größere Virulenz-Attribut von C. Albicans ist seine Fähigkeit, Form Biofilme, strukturierten Gemeinschaften von Zellen zu biotischen und abiotischen Oberflächen befestigt. C. Albicans Biofilme bilden können auf Host Gewebe, wie z. B. Schleimhaut Schichten und medizinischen Geräten wie Herzschrittmachern, Katheter, Zahnersatz und Gelenkprothesen. Biofilme stellen signifikante klinische Herausforderungen, weil sie sehr widerstandsfähig gegen physikalische und chemische Störungen sind und können als Stauseen Samen Infektionen verbreitet wirken. Verschiedene in-vitro- Assays wurden genutzt, um C. Albicans Biofilmbildung wie Mikrotiter-Platte-Assays, Trockengewicht Messungen, Zelle Lebensfähigkeit Assays und konfokale Lasermikroskopie Scan zu studieren. Alle diese Tests sind einzelne Endpunkt Assays, wo Biofilmbildung zu einem bestimmten Zeitpunkt bewertet. Hier beschreiben wir ein Protokoll zur Biofilmbildung in Echtzeit mit einem automatisierten mikrofluidischen Gerät unter Laminar-Flow-Bedingungen zu studieren. Diese Methode ermöglicht die Beobachtung der Biofilmbildung wie der Biofilm entwickelt sich im Laufe der Zeit mit anpassbaren Bedingungen, die denen der Host, wie denen in Gefäßkathetern nachahmen. Dieses Protokoll kann verwendet werden, die Biofilm-Defekte genetische Mutanten und die hemmenden Auswirkungen von antimikrobiellen Wirkstoffen auf Biofilm Entwicklung in Echtzeit einzuschätzen.

Wir führten mikrofluidischen Biofilm Assays beschrieben hier mit einem Wildtyp C. Albicans Belastung Bedingungen zwei Medien (RPMI-1640 und Spider Media), der Wildtyp Stamm in der Gegenwart bekannte antimykotische Wirkstoff Amphotericin B (16 µg/mL) in RPMI, und zwei mutierte Stämme zuvor berichtet, dass Mängel in Biofilmbildung (bcr1Δ/Δ und efg1 Δ/Δ) in Spider Media.
Video 1 zeigt die Entwickl...

Watch this Scientific Journal Video about Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device at JoVE.com

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

Visualisierung der Biofilmbildung in Candida Albicans ein automatisiertes mikrofluidischen Gerät

Dieses Protokoll beschreibt die Verwendung eines Geräts anpassbare automatisierte mikrofluidischen Biofilmbildung in Candida Albicans unter physiologischen Bedingungen Host zu visualisieren.

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

visualization-biofilm-formation-candida-albicans-using-an-automated

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

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

10.5K Aufrufe.  University of California, Merced. Dieses Protokoll beschreibt die Verwendung eines Geräts anpassbare automatisierte mikrofluidischen Biofilmbildung in Candida Albicans unter physiologischen Bedingungen Host zu visualisieren.

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

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.

Ein 96-well Mikrotiterplatte-basierten Methode zur Überwachung der Formation und antimykotische Empfindlichkeitsprüfung von Candida albicans Biofilme

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

Forschung

Immunologie und Infektion

Candida albicans Biofilm Chip (CaBChip) for High-throughput Antifungal Drug Screening

Candida albicans remains the main etiological agent of candidiasis, which currently represents the fourth most common nosocomial bloodstream infection in US hospitals1. These opportunistic infections pose a growing threat for an increasing number of compromised individuals, and carry unacceptably high mortality rates. This is in part due to the limited arsenal of antifungal drugs, but also to the emergence of resistance against the most commonly used antifungal agents. Further complicating treatment is the fact that a majority of manifestations of candidiasis are associated with the formation of biofilms, and cells within these biofilms show increased levels of resistance to most clinically-used antifungal agents2. Here we describe the development of a high-density microarray that consists of C. albicans nano-biofilms, which we have named CaBChip3. Briefly, a robotic microarrayer is used to print yeast cells of C. albicans onto a solid substrate. During printing, the yeast cells are enclosed in a three dimensional matrix using a volume as low as 50 nL and immobilized on a glass substrate with a suitable coating. After initial printing, the slides are incubated at 37 &deg;C for 24 hours to allow for biofilm development. During this period the spots grow into fully developed "nano-biofilms" that display typical structural and phenotypic characteristics associated with mature C. albicans biofilms (i.e. morphological complexity, three dimensional architecture and drug resistance)4. Overall, the CaBChip is composed of ~750 equivalent and spatially distinct biofilms; with the additional advantage that multiple chips can be printed and processed simultaneously. Cell viability is estimated by measuring the fluorescent intensity of FUN1 metabolic stain using a microarray scanner. This fungal chip is ideally suited for use in true high-throughput screening for antifungal drug discovery. Compared to current standards (i.e. the 96-well microtiter plate model of biofilm formation5), the main advantages of the fungal biofilm chip are automation, miniaturization, savings in amount and cost of reagents and analyses time, as well as the elimination of labor intensive steps. We believe that such chip will significantly speed up the antifungal drug discovery process.

Candida albicans Biofilm Chip CaBChip for High-throughput Antifungal Drug Screening

We have developed a high-density microarray platform consisting of 3D nano-biofilms of C. albicans called CaBChip. The susceptibility profile of drugs tested on a CaBChip is comparable to the conventional 96-well plate model, suggesting that the fungal chip is ideally suited for true high-throughput screening of antifungal drugs.

Candida albicans Biofilm Chip (Ca BChip) für das Hochdurchsatz-Screening-Antimykotikum

Candida  albicans remains the main etiological agent of candidiasis, which  currently represents the fourth most common nosocomial bloodstream infection ...

Biotechnik

Protocol for Biofilm Streamer Formation in a Microfluidic Device with Micro-pillars

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in porous media are relevant to several industrial and environmental processes such as wastewater treatment and CO2 sequestration. We used Pseudomonas fluorescens, a Gram-negative aerobic bacterium, to investigate biofilm formation in a microfluidic device that mimics porous media. The microfluidic device consists of an array of micro-posts, which were fabricated using soft-lithography. Subsequently, biofilm formation in these devices with flow was investigated and we demonstrate the formation of filamentous biofilms known as streamers in our device. The detailed protocols for fabrication and assembly of microfluidic device are provided here along with the bacterial culture protocols. Detailed procedures for experimentation with the microfluidic device are also presented along with representative results.

Protocols for the study of biofilm formation in a microfluidic device that mimics porous media are discussed. The microfluidic device consists of an array of micro-pillars and biofilm formation by Pseudomonas fluorescens in this device is investigated.

Protokoll für Biofilm Streamer Formation in einem Mikrofluidik-Gerät mit Micro-Säulen

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in ...

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected within in vivo oral biofilms. Furthermore, a system that uses natural human saliva as the nutrient source, instead of artificial media, is particularly desirable in order to support the expression of cellular and biofilm-specific properties that mimic the in vivo communities. We describe a method for the development of multi-species oral biofilms that are comparable, with respect to species composition, to supragingival dental plaque, under conditions similar to the human oral cavity. Specifically, this methods article will describe how a commercially available microfluidic system can be adapted to facilitate the development of multi-species oral biofilms derived from and grown within pooled saliva. Furthermore, a description of how the system can be used in conjunction with a confocal laser scanning microscope to generate 3-D biofilm reconstructions for architectural and viability analyses will be presented. Given the broad diversity of microorganisms that grow within biofilms in the microfluidic system (including Streptococcus, Neisseria, Veillonella, Gemella, and Porphyromonas), a protocol will also be presented describing how to harvest the biofilm cells for further subculture&#160;or DNA extraction and analysis. The limits of both the microfluidic biofilm system and the current state-of-the-art data analyses will be addressed. Ultimately, it is envisioned that this article will provide a baseline technique that will improve the study of oral biofilms and aid in the development of additional technologies that can be integrated with the microfluidic platform.

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

The goal of this methods paper is to describe the use of a microfluidic system for the development of multi-species biofilms that contain species typically identified in human supragingival dental plaque.&#160;Methods to describe biofilm architecture, biofilm viability, and an approach to harvest biofilm for culture-dependent or culture-independent analyses are highlighted.

Verwendung eines Hochdurchsatz-<em&gt; In-vitro-</em&gt; Mikrofluidik-System auf die mündliche Multi-Spezies-Biofilme entwickeln

There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected ...

Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging

Candida albicans biofilm development on biotic and/or abiotic surfaces represents a specific threat for hospitalized patients. So far, C. albicans biofilms have been studied predominantly in vitro but there is a crucial need for better understanding of this dynamic process under in vivo conditions. We developed an in vivo subcutaneous rat model to study C. albicans biofilm formation. In our model, multiple (up to 9) Candida-infected devices are implanted to the back part of the animal. This gives us a major advantage over the central venous catheter model system as it allows us to study several independent biofilms in one animal. Recently, we adapted this model to study C. albicans biofilm development in BALB/c mice. In this model, mature C. albicans biofilms develop within 48 hr and demonstrate the typical three-dimensional biofilm architecture. The quantification of fungal biofilm is traditionally analyzed post mortem and requires host sacrifice. Because this requires the use of many animals to perform kinetic studies, we applied non-invasive bioluminescence imaging (BLI) to longitudinally follow up in vivo mature C. albicans biofilms developing in our subcutaneous model. C. albicans cells were engineered to express the Gaussia princeps luciferase gene (gLuc) attached to the cell wall. The bioluminescence signal is produced by the luciferase that converts the added substrate coelenterazine into light that can be measured. The BLI signal resembled cell counts obtained from explanted catheters. Non-invasive imaging for quantifying in vivo biofilm formation provides immediate applications for the screening and validation of antifungal drugs under in vivo conditions, as well as for studies based on host-pathogen interactions, hereby contributing to a better understanding of the pathogenesis of catheter-associated infections.

Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging

We present an experimental procedure of Candida albicans biofilm development in a mouse subcutaneous model. Fungal biofilms were quantified by determining the number of colony forming units and by a non-invasive bioluminescence imaging, where the amount of light that is produced corresponds with the number of viable cells.

Candida albicans Biofilm-Entwicklung über die medizinisch relevanten Fremdkörper in einem Maus-Modell durch subkutane Biolumineszenz Imaging Gefolgt

Candida albicans biofilm development on biotic and/or abiotic surfaces represents a specific threat for hospitalized patients. So far, C. albicans ...

Medizin

Real-time Imaging and Quantification of Fungal Biofilm Development Using a Two-Phase Recirculating Flow System

In oropharyngeal candidiasis, members of the genus Candida must adhere to and grow on the oral mucosal surface while under the effects of salivary flow. While models for the growth under flow have been developed, many of these systems are expensive, or do not allow imaging while the cells are under flow. We have developed a novel apparatus that allows us to image the growth and development of Candida albicans cells under flow and in real-time. Here, we detail the protocol for the assembly and use of this flow apparatus, as well as the quantification of data that are generated. We are able to quantify the rates that the cells attach to and detach from the slide, as well as to determine a measure of the biomass on the slide over time. This system is both economical and versatile, working with many types of light microscopes, including inexpensive benchtop microscopes, and is capable of extended imaging times compared to other flow systems. Overall, this is a low-throughput system that can provide highly detailed real-time information on the biofilm growth of fungal species under flow.

We describe the assembly, operation, and cleaning of a flow apparatus designed to image fungal biofilm formation in real time while under flow. We also provide and discuss quantitative algorithms to be used on the acquired images.

Echtzeit-Bildgebung und Quantifizierung der Pilz Biofilm-Entwicklung mit einem Zweiphasensystem rezirkulierenden Flow

In oropharyngeal candidiasis, members of the genus Candida must adhere to and grow on the oral mucosal surface while under the effects of salivary flow. ...

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.

Klärende und Bildgebung Candida albicans Biofilme

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

Adhesion of Candida parapsilosis to Bovine Serum Albumin under Fluid Shear

C. parapsilosis (Cp) is an emerging cause of bloodstream infections in certain populations. The&#160;Candida clade, including Cp, is increasingly developing resistance to the first and the second line of antifungals. Cp is frequently isolated from hands and skin surfaces, as well as the GI tract. Colonization by Candida predisposes individuals to invasive bloodstream infections. To successfully colonize or invade the host, yeast must be able to rapidly adhere to the body surfaces to prevent elimination by host defense mechanisms. Here we describe a method to measure adhesion of Cp to immobilized proteins under physiologic fluid shear, using an end-point adhesion assay in a commercially available multichannel microfluidic device. This method is optimized to improve reproducibility, minimize subjectivity, and allow for the fluorescent quantification of individual isolates. We also demonstrate that some clinical isolates of Cp show increased adhesion when grown in conditions mimicking a mammalian host, whereas a frequently used lab strain, CDC317, is non-adhesive under fluid shear.

Adhesion of Candida parapsilosis to Bovine Serum Albumin under Fluid Shear

Adhesion is an important first step in colonization and pathogenesis for Candida. Here, an in vitro assay is described to measure adhesion of C. parapsilosis isolates to immobilized proteins under fluid shear. A multichannel microfluidics device is used to compare multiple samples in parallel, followed by quantification using fluorescence imaging.

C. parapsilosis (Cp) is an emerging cause of bloodstream infections in certain populations. The&#160;Candida clade, including Cp, is increasingly ...

A Soluble Tetrazolium-Based Reduction Assay to Evaluate the Effect of Antibodies on Candida tropicalis Biofilms

Candida species are the fourth-most common cause of systemic nosocomial infections. Systemic or invasive candidiasis frequently involves biofilm formation on implanted devices or catheters, which is associated with increased virulence and mortality. Biofilms produced by different Candida species exhibit enhanced resistance against various antifungal drugs. Therefore, there is a need to develop effective immunotherapies or adjunctive treatments against Candida biofilms. While the role of cellular immunity is well established in anti-Candida protection, the role of humoral immunity has been studied less.
It has been hypothesized that inhibition of biofilm formation and maturation is one of the major functions of protective antibodies, and Candida albicans germ tube antibodies (CAGTA) have been shown to suppress in vitro growth and biofilm formation of C. albicans earlier. This paper outlines a detailed protocol for evaluating the role of antibodies on biofilms formed by C. tropicalis. The methodology for this protocol involves C. tropicalis biofilm formation in 96-well microtiter plates, which were then incubated in the presence or absence of antigen-specific antibodies, followed by a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-carboxanilide-2H-tetrazolium (XTT) assay for measuring the metabolic activity of fungal cells in the biofilm.
The specificity was confirmed by using appropriate serum controls, including Sap2-specific antibody-depleted serum. The results demonstrate that antibodies present in the serum of immunized animals can inhibit Candida biofilm maturation in vitro. In summary, this paper provides important insights regarding the potential of antibodies in developing novel immunotherapies and synergistic or adjunctive treatments against biofilms during invasive candidiasis. This in vitro protocol can be used to check the effect of potential new antifungal compounds on the metabolic activity of Candida species cells in biofilms.

A Soluble Tetrazolium-Based Reduction Assay to Evaluate the Effect of Antibodies on Candida tropicalis Biofilms

A 96-well microtiter plate-based protocol using a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-carboxanilide-2H-tetrazolium (XTT) reduction assay is described herein, to study antibodies' effects on biofilms formed by C. tropicalis. This in vitro protocol can be used to check the effect of potential new antifungal compounds on the metabolic activity of Candida species cells in biofilms.

Ein löslicher Tetrazolium-basierter Reduktionstest zur Bewertung der Wirkung von Antikörpern auf Candida tropicalis-Biofilme

Candida species are the fourth-most common cause of systemic nosocomial infections. Systemic or invasive candidiasis frequently involves biofilm formation ...

Candida albicans Biofilm Formation in an In Vivo Mouse Model

Source: Kuchar&#237;kov&#225;, S., et al.&#160; <a href="https://appjove.unicartagenaproxy.elogim.com/v/52239">Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging.</a> J. Vis. Exp. (2015).
This video demonstrates the development of an in vivo mouse model to study the biofilm formation of Candida albicans in subcutaneous tissue. The experiment involves implanting Candida-infected devices in the animal&#39;s back region for biofilm development.

Candida albicans Biofilmbildung in einem in vivo Mausmodell

1. C. albicans Growth

	Twenty-four hours before the initiation of the animal experiment, prepare yeast extract peptone dextrose, YPD plates by adding 10 ...

Visualisierung der Biofilmbildung in Candida Albicans ein automatisiertes mikrofluidischen Gerät

Zusammenfassung

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