Ringraziamo tutti i membri del laboratorio Nobile per utili discussioni su saggi di biofilm. Questo studio è stato finanziato dal National Institutes of Health (NIH) sovvenzione R21 AI125801 (C.J.N.). DLR è stato sostenuto da una borsa di studio di dottorato presso l'Università di California Institute per il Messico e gli Stati Uniti (UC-MEXUS) e il Consejo Nacional de Ciencia y Technologia (CONACYT).

<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 è dei fondatori di BioSynesis, Inc., una società di sviluppo di inibitori e diagnostica dei albicans del c. biofilm.

Il dosaggio di biofilm microfluidici personalizzabile descritto qui permette per la visualizzazione della formazione del biofilm in tempo reale a livello di singola cellula quando esposti ad un tasso fisso a flusso laminare e temperatura costante. Fornisce un mezzo potente per studiare lo sviluppo di biofilm in ceppi wild-type e mutanti, e gli effetti dei trattamenti di agente antimicrobico su biofilm in condizioni che mimano condizioni fisiologiche osservate nelle regolazioni cliniche. A differenza della maggior parte <...

Candida albicans è un commensali membri del microbiota umano, tuttavia è anche un agente patogeno opportunistico, in grado di causare infezioni fungine superficiali e severa1,2. Un tratto di maggiore virulenza dei albicans del c. è la sua capacità di modulo resiliente e biofilm resistente alla droga, comunità di cellule aderito ad una superficie e racchiuso in una matrice extracellulare del materiale1,3. C. albicans biofilm sono altamente strutturato, contenente diversi strati di più tipi di cellule (lievito germogliante-forma tondeggiante cellule, cellule ovali pseudoifale e cellule tubulari ifale)4. Albicans del c. sviluppo di biofilm inizia con l'aderenza delle cellule di lievito-forma rotonda ad una superficie (semina il biofilm), seguita dalla proliferazione di queste cellule sulla superficie, e quindi la maturazione del biofilm acerbo strutturare in un biofilm ben formato che è circondato da matrice extracellulare materiale4. Il biofilm maturo è composto principalmente di cellule hyphal allungate che formano reti dense e comunicanti, fornendo la stabilità architettonica al biofilm4. Tutto il ciclo di vita del biofilm, tondo lievito gemmante cellule disperdono dal biofilm maturo e possono viaggiare ad altre regioni del corpo per causare le infezioni diffuse o seme nuovo biofilm presso altri siti4,5. C. albicans può formare biofilm su superfici biotiche, ad esempio superfici mucose e nel tessuto ospite e su superfici abiotiche, quali cateteri, pacemaker, protesi dentarie e protesi articolari. A causa delle proprietà ricalcitrante del biofilm, sono estremamente difficili da sradicare, e in molti casi la strategia unico trattamento efficace è la rimozione del dispositivo infetto4. È dunque fondamentale per studiare la formazione di biofilm in condizioni simili a quelle osservate nelle regolazioni cliniche.
Ci sono parecchi critici in vivo modelli di animali utilizzati per lo studio di c. albicans biofilm formazione6,7,8; Tuttavia, questi studi possono essere costosa, richiede tempo e sono limitati dal numero di ceppi e agenti antimicrobici che possono essere testati in un dato momento. In vitro biofilm saggi, d'altra parte, consentire la valutazione rapida, ad alta produttività di composti Antifungini e ceppi mutanti e sono molto più redditizi ed etici di biofilm saggi trasportati fuori in animale modelli9, 10,11,12,13,14. Qui descriviamo un test in vitro che abbiamo sviluppato e ottimizzato per osservare la formazione di biofilm temporaneamente sotto flusso laminare usando un personalizzabile microfluidici dispositivo14,15. Il test consente la visualizzazione di ogni fase della formazione del biofilm, compreso il passaggio di adesione iniziale, proliferazione, maturazione del biofilm e dispersione delle cellule. Il dosaggio è anche utile per visualizzare i cambiamenti di morfologia delle cellule durante lo sviluppo di un biofilm.
Piastre di microtitolazione, che vengono in genere utilizzati per in vitro biofilm saggi, mentre a resa elevata, non consentono per condizioni di flusso controllato. Sistemi cellulari tradizionali a flusso laminare consentono la valutazione continua di formazione di biofilm in condizioni di flusso controllato, ma questi sono spesso molto tempo per impostare e tendono ad avere un limitato controllo gamma dinamica e velocità effettiva. Il dispositivo di microfluidica utilizzato qui supera queste limitazioni mediante la combinazione di piastre di throughput elevato (contenente 48 pozzetti) con una camera di flusso laminare incorporato ed è altamente riproducibile, versatile e personalizzabile.
Qui, descriviamo un protocollo per l'uso di un dispositivo microfluidico automatizzati disponibili in commercio per valutare la formazione di biofilm di un selvaggio-tipo albicans del c. ceppo, gli effetti di un noto agente antifungino sullo sviluppo di un biofilm e biofilm formazione in due ceppi mutanti (bcr1Δ/Δ ed efg1 Δ/Δ) che precedentemente sono stati segnalati per avere biofilm difetti in vitro e in vivo16,17,18. Il protocollo descritto può essere utilizzato per testare l'efficacia degli agenti antimicrobici nell'inibire la formazione di biofilm in tutto lo sviluppo di un biofilm e per identificare geni richiesti per la sviluppo di biofilm normale mediante screening di librerie mutante.

<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 è il più comune agente patogeno fungoso degli esseri umani, causando circa il 15% dei casi di sepsi nosocomiali. Un attributo di maggiore virulenza dei albicans del c. è la sua capacità di forma biofilm comunità strutturata delle cellule allegate alle superfici biotiche e abiotiche. C. albicans biofilm può formare su tessuti dell'ospite, come ad esempio strati mucosi e sui dispositivi medici, quali cateteri, pacemaker, protesi dentarie e protesi articolari. Biofilm pongono sfide cliniche significative perché sono altamente resistenti alle perturbazioni fisiche e chimiche e può agire come serbatoi per seme diffuse infezioni. Vari saggi in vitro sono state utilizzate per studiare la formazione di biofilm di albicans del c. , come saggi di piastra di microtitolo, misurazioni di peso secco, saggi di vitalità cellulare e confocale microscopia a scansione laser. Tutte queste analisi sono saggi single end-point, dove la formazione di biofilm è valutata a un momento specifico. Qui, descriviamo un protocollo per studiare la formazione di biofilm in tempo reale utilizzando un dispositivo microfluidico automatizzati in condizioni di flusso laminare. Questo metodo permette l'osservazione della formazione di biofilm come biofilm si sviluppa nel tempo, con condizioni personalizzabili che imitano quelli di host, ad esempio quelle incontrate in cateteri vascolari. Questo protocollo può essere utilizzato per valutare i difetti di biofilm di mutanti genetici nonché gli effetti inibitori degli agenti antimicrobici per lo sviluppo di biofilm in tempo reale.

Abbiamo effettuato l'analisi di biofilm microfluidici descritto qui utilizzando un selvaggio-tipo ceppo di albicans del c. in condizioni di due media (media RPMI-1640 e Spider), il ceppo di selvaggio-tipo in presenza del noto farmaco antifungino amfotericina B (16 µ g/mL) in RPMI, e due ceppi mutanti precedentemente segnalato di avere difetti nella formazione del biofilm (bcr1Δ/Δ ed efg1 Δ/Δ) in media di ragno.
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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

Visualizzazione della formazione di Biofilm in Candida albicans utilizzando un dispositivo microfluidico automatizzato

Questo protocollo descrive l'uso di un dispositivo microfluidico automatico personalizzabile per visualizzare la formazione di biofilm in Candida albicans in condizioni fisiologiche host.

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

Research

JoVE Journal

Immunology and Infection

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

10.5K Views.  University of California, Merced. Questo protocollo descrive l'uso di un dispositivo microfluidico automatico personalizzabile per visualizzare la formazione di biofilm in Candida albicans in condizioni fisiologiche host.

Video: 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.

A 96 Beh microtest piastra a base di Metodo per la Formazione di monitoraggio e test di suscettibilità antifungini Candida albicans Biofilm

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

Ricerca

Immunologia e infezioni

Microtiter Dish Biofilm Formation Assay

Biofilms are communities of microbes attached to surfaces, which can be found in medical, industrial and natural settings. In fact, life in a biofilm probably represents the predominate mode of growth for microbes in most environments. Mature biofilms have a few distinct characteristics. Biofilm microbes are typically surrounded by an extracellular matrix that provides structure and protection to the community. Microbes growing in a biofilm also have a characteristic architecture generally comprised of macrocolonies (containing thousands of cells) surrounded by fluid-filled channels. Biofilm-grown microbes are also notorious for their resistance to a range of antimicrobial agents including clinically relevant antibiotics.
The microtiter dish assay is an important tool for the study of the early stages in biofilm formation, and has been applied primarily for the study of bacterial biofilms, although this assay has also been used to study fungal biofilm formation. Because this assay uses static, batch-growth conditions, it does not allow for the formation of the mature biofilms typically associated with flow cell systems. However, the assay has been effective at identifying many factors required for initiation of biofilm formation (i.e, flagella, pili, adhesins, enzymes involved in cyclic-di-GMP binding and metabolism) and well as genes involved in extracellular polysaccharide production. Furthermore, published work indicates that biofilms grown in microtiter dishes do develop some properties of mature biofilms, such a antibiotic tolerance and resistance to immune system effectors.
This simple microtiter dish assay allows for the formation of a biofilm on the wall and/or bottom of a microtiter dish. The high throughput nature of the assay makes it useful for genetic screens, as well as testing biofilm formation by multiple strains under various growth conditions. Variants of this assay have been used to assess early biofilm formation for a wide variety of microbes, including but not limited to, pseudomonads, Vibrio cholerae, Escherichia coli, staphylocci, enterococci, mycobacteria and fungi.
In the protocol described here, we will focus on the use of this assay to study biofilm formation by the model organism Pseudomonas aeruginosa. In this assay, the extent of biofilm formation is measured using the dye crystal violet (CV). However, a number of other colorimetric and metabolic stains have been reported for the quantification of biofilm formation using the microtiter plate assay. The ease, low cost and flexibility of the microtiter plate assay has made it a critical tool for the study of biofilms.

The assay describes a rapid means to measure early biofilm formation in bacteria and fungi. This method uses a microtiter plate as the substratum for microbial biofilm formation, and the biofilm is visualized using crystal violet strain. The assay provides either a qualitative or quantitative assay for early biofilm formation.

Microtitolazione Dish Biofilm saggio Formazione

Biofilms are communities of microbes attached to surfaces, which can be found in medical, industrial and natural settings.  In fact, life in a biofilm ...

In vitro Biofilm Formation in an 8-well Chamber Slide

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. Biofilms are community-associated bacteria attached to a surface and encased in a matrix. The role of the extracellular matrix is multifaceted, including facilitating nutrient acquisition, and offers significant protection against environmental stresses (e.g. host immune responses). In an effort to acquire a better understanding as to how the bacteria within a biofilm respond to environmental stresses we have used a protocol wherein we visualize bacterial biofilms which have formed in an 8-well chamber slide. The biofilms were stained with the BacLight Live/Dead stain and examined using a confocal microscope to characterize the relative biofilm size, and structure under varying incubation conditions. Z-stack images were collected via confocal microscopy and analyzed by COMSTAT. This protocol can be used to help elucidate the mechanism and kinetics by which biofilms form, as well as identify components that are important to biofilm structure and stability.

In vitro Biofilm Formation in an 8-well Chamber Slide

This article describes the procedure for the formation and visualization of a bacterial biofilm grown within an 8-well chamber slide

Nella formazione di biofilm in vitro in un 8-bene scorrere Camera

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. ...

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of the host depends on the involvement of an adapted immune response against the pathogen1. Immune response is complex and results from interaction of the pathogens and several immune or non-immune cellular types2. In vitro studies cannot characterise these interactions and focus on cell-pathogen interactions. Moreover, in the airway3, particularly in patients with suppurative chronic lung disease or in mechanically ventilated patients, polymicrobial communities are present and complicate host-pathogen interaction. Pseudomonas aeruginosa and Candida albicans are both problem pathogens4, frequently isolated from tracheobronchial samples, and associated to severe infections, especially in intensive care unit5. Microbial interactions have been reported between these pathogens in vitro but the clinical impact of these interactions remains unclear6. To study the interactions between C. albicans and P. aeruginosa, a murine model of C. albicans airways colonization, followed by a P. aeruginosa-mediated acute lung infection was performed.

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

While in vitro study of host-pathogen interactions allow the characterization of specific immune responses, in vivo models are required to observe the effects of complex responses. Using Candida albicans exposure followed by Pseudomonas aeruginosa-mediated lung infection, we established a murine model of microbial interactions involved in ventilator-associated pneumonia pathogenicity.

Studiare comunità microbiche<em&gt; In Vivo</em&gt;: Un modello di interazione ospite-mediata tra<em&gt; Candida Albicans</em&gt; E<em&gt; Pseudomonas aeruginosa</em&gt; Nei Airways

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of ...

Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the development and persistence of many health related infections. One of the most well described and studied biofilms in human disease occurs in chronic pulmonary infection of cystic fibrosis patients. When studying biofilms in the context of the host, many factors can impact biofilm formation and development. In order to identify how host factors may affect biofilm formation and development, we used a static chambered coverglass method to grow biofilms in the presence of host-derived factors in the form of sputum supernatants. Bacteria are seeded into chambers and exposed to sputum filtrates. Following 48 hr of growth, biofilms are stained with a commercial biofilm viability kit prior to confocal microscopy and analysis. Following image acquisition, biofilm properties can be assessed using different software platforms. This method allows us to visualize key properties of biofilm growth in presence of different substances including antibiotics.

This protocol describes the visualization of biofilm development following exposure to host-factors using a slide chamber model. This model allows for direct visualization of biofilm development as well as analysis of biofilm parameters using computer software programs.

Visualizzare gli effetti di espettorato su biofilm di sviluppo utilizzando un modello Chambered coprioggetti

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the ...

Bile Salt-induced Biofilm Formation in Enteric Pathogens: Techniques for Identification and Quantification

Biofilm formation is a dynamic, multistage process that occurs in bacteria under harsh environmental conditions or times of stress. For enteric pathogens, a significant stress response is induced during gastrointestinal transit and upon bile exposure, a normal component of human digestion. To overcome the bactericidal effects of bile, many enteric pathogens form a biofilm hypothesized to permit survival when transiting through the small intestine. Here we present methodologies to define biofilm formation through solid-phase adherence assays as well as extracellular polymeric substance (EPS) matrix detection and visualization. Furthermore, biofilm dispersion assessment is presented to mimic the analysis of events triggering release of bacteria during the infection process. Crystal violet staining is used to detect adherent bacteria in a high-throughput 96-well plate adherence assay. EPS production assessment is determined by two assays, namely microscopy staining of the EPS matrix and semi-quantitative analysis with a fluorescently-conjugated polysaccharide binding lectin. Finally, biofilm dispersion is measured through colony counts and plating. Positive data from multiple assays support the characterization of biofilms and can be utilized to identify bile salt-induced biofilm formation in other bacterial strains.

This protocol enables the reader to analyze bile salt-induced biofilm formation in enteric pathogens using a multifaceted approach to capture the dynamic nature of bacterial biofilms by assessing adherence, extracellular polymeric substance matrix formation, and dispersion.

Formazione di Biofilm indotta da sali biliari in patogeni enterici: tecniche di identificazione e quantificazione

Biofilm formation is a dynamic, multistage process that occurs in bacteria under harsh environmental conditions or times of stress. For enteric pathogens, ...

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.

Profondo cutaneo iniezione come un modello di infezione della pelle Candida albicans per analisi istologiche

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.

Quotidiano fototerapia con luce rossa per sviluppo di Biofilm di regolare dei albicans del Candida

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.

Visualizzazione degli albicani Candida nel tratto gastrointestinale Murine utilizzando l'ibridazione fluorescente in Situ

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.

Chiarire e 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 ...