We thank all members of the Nobile lab for helpful discussions on biofilm assays. This study was supported by National Institutes of Health (NIH) grant R21 AI125801 (to C.J.N.). D.L.R. was supported by a doctoral fellowship from The University of California Institute for Mexico and the United States (UC-MEXUS) and 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 is a founder of BioSynesis, Inc., a company developing inhibitors and diagnostics of C. albicans biofilms.

The customizable microfluidic biofilm assay described here allows for the visualization of biofilm formation in real-time at a single cell level when exposed to a fixed rate laminar flow and constant temperature. It provides a powerful means to study the development of biofilms in wild-type and mutant strains, and the effects of antimicrobial agent treatments on biofilms under conditions that mimic physiological conditions observed in clinical settings. Unlike most in vitro biofilm assays, this method allows for the examination of a developing biofilm in real-time as it forms.
This method can be used in a high-throughput manner, where up to 24 samples can be run simultaneously to assess biofilm formation. The method can also be used to conduct genetic screens of mutant libraries to identify genes required for normal biofilm development and can be used to assess the effectivity against biofilms of antimicrobial compounds of interest in compound library screens. We anticipate that future applications of this microfluidic device will include such screens. We note, however, that certain drug treatment testing will be limited by the thickness of the biofilm formed using this microfluidic device as biofilms grown for &#62;16 h tend to clog the viewing channel. Thus, drug testing experiments will need to be performed for &#60;16 h.
Overall, this device is highly customizable and versatile, and can be adjusted to assess different microbial species across kingdoms, flow rates, temperatures, incubation times, and media. The method described provides information about a number of different aspects of biofilm formation, including initial cell adherence, biofilm maturation, and cell dispersal. Although we only report results for single-species biofilm formation here, this protocol could also be adapted to study dual- and mixed-species biofilm formation.
Two steps are critical to successfully perform this protocol. First, air bubbles in the system must be avoided by preheating the media at the experimental temperature. One common misstep is the dilution of cells in media that was not preheated. Cells should be diluted in the same media that is being used for the inlet wells. Second, the flow of cells from outlet to inlet wells must be carefully monitored; if the cells flow too far, the inlet media will become contaminated and the experiment will not yield interpretable results. On the other hand, if the cells are not allowed to move all the way into the viewing channel, then no cells will be visible in the channel during the experiment, leading to blank images. Additional key points to keep in mind are that the volume and movement between wells in the microfluidic plate are linked together for each column, and it is important to maintain as much media consistency as possible (with similar viscosity, cell density, and composition) and similarly sized cells (if possible), as different media and different cell sizes will have different flow rates in the column. During the experiment, the backflow of the cells is monitored using a microscope; however, only two channels can be viewed at one time (using a 10X objective). Thus, it is highly recommended to perform a mock experiment to measure the time required to flow from outlet to inlet wells using the cells and media planned to be used in the actual experiment.
In general, we highly recommend use of this microfluidic biofilm assay as an in vitro assay to be utilized before in vivo animal testing. The assay, however, is still an in vitro assay, and results will need to be validated in relevant animal models. In our experiences, the results of this microfluidic biofilm assay have had the best predictive value for in vivo biofilm assays compared with other in vitro assays we have assessed14.

Candida albicans is a commensal member of the human microbiota, however it is also an opportunistic pathogen, capable of causing superficial and severe fungal infections1,2. A major virulence trait of C. albicans is its ability to form resilient and drug resistant biofilms, communities of cells adhered to a surface and enclosed in an extracellular matrix material1,3. C. albicans biofilms are highly structured, containing several layers of multiple cell types (round budding yeast-form cells, oval pseudohyphal cells, and tubular hyphal cells)4. C. albicans biofilm development begins with the adherence of round yeast-form cells to a surface (seeding the biofilm), followed by the proliferation of these cells on the surface, and then the maturation of the immature biofilm structure into a fully-formed biofilm that is surrounded by extracellular matrix material4. The mature biofilm is predominantly composed of elongated hyphal cells that form dense and interconnecting networks, providing the architectural stability to the biofilm4. Throughout the biofilm life cycle, round budding yeast cells disperse from the mature biofilm, and may travel to other regions of the body to cause disseminated infections or seed new biofilms at other sites4,5. C. albicans can form biofilms on biotic surfaces, such as mucosal surfaces and throughout host tissue, and on abiotic surfaces, such as catheters, pacemakers, dentures, and prosthetic joints. Due to the recalcitrant properties of biofilms, they are extremely difficult to eradicate, and in many cases the only effective treatment strategy is removal of the infected device4. It is thus crucial to investigate biofilm formation under conditions similar to those observed in clinical settings.
There are several critical in vivo animal models used to study C. albicans biofilm formation6,7,8; however, these studies can be costly, time consuming, and are limited by the number of strains and antimicrobial agents that can be tested at a given time. In vitro biofilm assays, on the other hand, allow for the rapid, high-throughput assessment of antifungal compounds and mutant strains, and are much more cost-effective and ethical than biofilm assays carried out in animal models9,10,11,12,13,14. Here we describe an in vitro assay that we developed and optimized to observe biofilm formation temporally under laminar flow using a customizable microfluidic device14,15. The assay allows for the visualization of each stage of biofilm formation, including the initial adherence step, cell proliferation, biofilm maturation, and cell dispersion. The assay is also useful to visualize cell morphology changes throughout the development of a biofilm.
Microtiter plates, which are typically utilized for in vitro biofilm assays, while high throughput, do not allow for controlled flow conditions. Traditional laminar flow cell systems allow for the continuous assessment of biofilm formation in controlled flow conditions, but these are often time consuming to set up and tend to have limited dynamic range control and throughput. The microfluidic device utilized here overcomes these limitations by combining high throughput plates (containing 48 wells) with a built-in laminar flow chamber and is highly reproducible, versatile, and customizable.
Here, we describe a protocol for the use of a commercially available automated microfluidic device to assess biofilm formation of a wild-type C. albicans strain, the effects of a known antifungal agent on the development of a biofilm, and biofilm formation in two mutant strains (bcr1&#916;/&#916; and efg1 &#916;/&#916;) that were previously reported to have biofilm defects in vitro and in vivo16,17,18. The described protocol can be used to test the efficacy of antimicrobial agents in inhibiting biofilm formation throughout the development of a biofilm, and to identify genes required for normal biofilm development by screening mutant libraries.

<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 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.

We performed the microfluidic biofilm assay described here using a wild-type C. albicans strain under two media conditions (RPMI-1640 and Spider media), the wild-type strain in the presence of the known antifungal drug amphotericin B (16 &#181;g/mL) in RPMI, and two mutant strains previously reported to have defects in biofilm formation (bcr1&#916;/&#916; and efg1 &#916;/&#916;) in Spider media.
Video 1 shows the development of a wild-type biofilm in RPMI-1640 medium and the effects of the antifungal drug, amphotericin B, on biofilm formation. Under wild-type conditions, several cells strongly adhere to the channel, the cells form hyphae as time progresses, and a thick biofilm can be observed with intercalating hyphae and yeast cells. Towards the end of the microfluidic experiment, the wild-type biofilm completely fills the viewing channel. The presence of amphotericin B, however, has a pronounced effect on reducing the biofilm. Specifically, the adherence of cells is diminished, and, unlike untreated conditions, in the presence of amphotericin B, several cells can be seen flowing away with the shear flow of the media. As time progresses, the cells fail to form hyphae and a biofilm is not formed. These differences are also shown in Figure 3, which depicts 4 time-points from the assay at 0 h, 2 h, 6 h, and 12 h.
Video 2 shows the development of a wild-type biofilm and two previously reported biofilm-defective mutants (bcr1 &#916;/&#916; and efg1 &#916;/&#916;) in Spider media. Both bcr1 &#916;/&#916; and efg1 &#916;/&#916; strains show severely reduced biofilm formation compared to the isogenic wild-type strain. For the efg1 &#916;/&#916; strain, we observed that the adhered cells do not form hyphae and the resulting biofilm is severely defective. For the bcr1 &#916;/&#916; strain, we observed that not only is the bcr1 &#916;/&#916; strain defective in biofilm formation, but it also displays a clear adherence defect, where cells can be seen drifting in the direction of the shear flow as they fail to adhere to the viewing channel. These differences are also shown in Figure 4, which depicts 4 time points from the assay at 0 h, 2 h, 6 h, and 12 h.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56743/56743fig1.jpg" /> 
Figure 1: Microfluidic instrument used in this experiment. Panel A shows the microfluidic instrument used, panel B shows the interface plate, panel C shows a schematic outline of a single viewing channel (stage) and three sub-stages captured by the camera during the experiment, and panel D shows a schematic outline of the 48-well microfluidic plate used with a schematic of the outlet and inlet wells and viewing window. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56743/56743fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56743/56743fig2.jpg" /> 
Figure 2: Select screenshots of imaging modulesoftware and microfluidic plate control system. Panel A shows the Control Module, that contains the controls for the microfluidic plate, and panel B shows the imaging module window, the &#39;Move Stage to Absolute Position&#39; (MAP) module, the &#39;Multi-dimensional Acquisition&#39; (MDA) module, and the &#39;Sample Reload Adjustment&#39; (SRA) module. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56743/56743fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56743/56743fig3.jpg" /> 
Figure 3: Exposure to amphotericin B results in biofilm formation defects. Time dependent visualization of biofilm formation in RPMI-1640 media (Column 1), and RPMI-1640 media supplemented with 16 &#181;g/mL amphotericin B (Column 2) under dynamic flow (0.5 dyne/cm2) at 37 &#176;C for 12 h post-adherence in the microfluidic system. Representative 0 h (post-adherence and initial wash), 2 h, 6 h, and 12 h images (top to bottom) are shown for the isogenic wild-type strain SN250 (Columns 1 and 2). Scale bars are 20 &#181;m in each panel. Corresponding time lapse videos of biofilm formation are provided in Video 1. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56743/56743fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56743/56743fig4.jpg" /> 
Figure 4: Deletion of BCR1 or EFG1 results in biofilm formation defects. Time dependent visualization of biofilm formation in Spider media (Columns 1-3) under dynamic flow (0.5 dyne/cm2) at 37 &#176;C for 12 h post-adherence in the microfluidic system. Representative 0 h (post-adherence and initial wash), 2 h, 6 h, and 12 h images (top to bottom) are shown for the isogenic wild-type strain SN250 (Column 1), bcr1&#916;/&#916; strain (Column 2), and efg1 &#916;/&#916; strain (Column 3). Scale bars are 20 &#181;m in each panel. Corresponding time lapse videos of biofilm formation are provided in Video 2. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56743/56743fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Video 1: <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56743/Video-1-WT_and_WT_plus_AmpB.mp4">Please click here to download this file.</a>
Video 2: <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56743/Video-2-WT_bcr1_efg1_Mutants.mp4">Please click here to download this file.</a>

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

This protocol describes the use of a customizable automated microfluidic device to visualize biofilm formation in Candida albicans under host physiological conditions.

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. ...

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

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

10.5K Views.  University of California, Merced. Dit protocol beschrijft het gebruik van een aanpasbaar geautomatiseerde microfluidic apparaat te visualiseren van biofilm vorming in Candida albicans , onder fysiologische omstandigheden van de gastheer.

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.

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

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

Bioengineering

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.

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.

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

Medicine

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.

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.

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.

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://app-jove-com.remotexs.ntu.edu.sg/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.

1. C. albicans Growth

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

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

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