This work was supported by the KU Leuven PF &#8216;IMIR&#8217;, the FWO Research community on biology and ecology of bacterial and fungal biofilms (FWO: WO.026.11N) and by the FWO project G.0804.11. SK gratefully acknowledges KU Leuven for the PDMK 11/089 fellowship and FWO for the postdoctoral fellowship. We are grateful to Nico Vangoethem for his assistance with preparation of the figures. We would like to acknowledge Celia Lobo Romero for technical assistance during in vivo experimental procedures.

NOTE: All animal experiments were approved by the ethical committee of KU Leuven (project number 090/2013). Maintain animals in accordance with the KU Leuven animal care guidelines.
1. C. albicans Growth
<ol>
	<li>Twenty-four hours before the initiation of the animal experiment, prepare YPD plates by adding 10 g of yeast extract granulated, 20 g of bacteriological peptone, and 15 g of granulated agar. Make up volume to 900 ml with Milli-Q water and autoclave.</li>
	<li>Add 50 ml of ...

<ol>
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The authors declare that they have no competing financial interests.

The use of animal models, and especially rodent models, for studies dedicated to microbial biofilms is very important as the host immune system is an essential factor in biofilm formation that in vitro models cannot account for. In this study, we describe a relatively straightforward subcutaneous C. albicans biofilm mouse model, which can be easily adopted in a research laboratory and does not require strong technical skills. This model was originally developed to study Staphylococcus epidermidis</e...

Candida albicans is a commensal organism, which can be found at different sites of healthy individuals, for example on the skin or as a part of the gastrointestinal and vaginal flora. However, in hospitalized, and especially immunocompromised patients, it may cause a wide range of infections 1. In such individuals, the weakened immune system allows Candida cells to disseminate into the bloodstream and to invade deeper tissues causing life-threatening infections. In addition, the presence of abiotic substrates such as central venous and urinary catheters, artificial heart valves and joints may provide a niche for Candida attachment 2. Adhesion to such substrates is a prerequisite for further biofilm development, which represents a layer of yeast and hyphal cells embedded in extracellular polymeric material, mainly consisting of polysaccharides 2. C. albicans catheter &#8211;associated infections are associated with high mortality rate. A general characteristic of biofilms is their decreased susceptibility to known antifungals, such as azoles 3,4. Only newer classes of antifungal drugs, such as echinocandins and liposomal formulation of amphotericin B proved to be active against catheter-associated infections 5-7. Because of biofilm resilience to antifungals, therapeutic approaches are very limited, often leading to catheter removal and its subsequent replacement as a sole solution.
Most of our current understanding of C. albicans biofilm development originates from in vitro studies on abiotic substrates such as polystyrene, or plastics used for the manufacture of above-mentioned devices, i.e., silicone, polyurethane 2. These models are quite advanced and try to mimic the situation in vivo as closely as possible. However, these systems do not involve the continuous blood flow and the immune system of the host. This resulted in the development of in vivo model systems, such as the central venous catheter (CVC) model 8-10, the denture stomatitis model of oral candidiasis 11 and a murine model for catheter-associated candiduria 12. Additionally, C. albicans biofilm development was studied in vivo on the mucosal surfaces, such as those from the vagina 13 and oral cavity 14. Our laboratory contributed with the establishment of a subcutaneous C. albicans biofilm model, which is based on the implant of infected catheter pieces on the back of Sprague Dawley rats 15. This model was successfully used in our laboratory to test biofilm susceptibility to fluconazole and echinocandin drugs 5,16, to study the effect of combinatorial therapy of diclofenac and caspofungin 17. More recently, we adapted this system for use in BALB/c mice 18,19. In comparison with other in vivo models, the main advantage of this subcutaneous model is the possibility to study multiple biofilms per animal developed inside the lumen of implanted catheter pieces.
To reduce the number of laboratory animals, we have adapted this model to study the development of C. albicans biofilms non-invasively by using bioluminescence imaging (BLI) 18,19. This method proved to be a powerful technique, which can be used to quantify biofilms by measuring the specific BLI signal at the region of interest (in our case the area of implanted catheters), avoiding animal sacrifice. In comparison to bacteria, which can express both the gene and the substrate required for the bioluminescence reaction due to the introduction of a specific lux operon 20, most of the eukaryotic organisms, including C. albicans, are dependent on the heterologous expression of a luciferase gene coupled with the external administration of a specific substrate, such as D-luciferin or coelenterazine 21. Probably due to the presence of the fungal cell wall and C. albicans morphogenesis, the intracellular delivery of the substrate for the luciferase enzyme was a main challenge 21. In order to solve this problem, Enjalbert et al. 22 engineered a strain where a synthetic C. albicans codon-optimized version of the gene for the naturally secreted Gaussia princeps luciferase (gLuc) was fused to to the C. albicans PGA59 gene, a GPI- anchored cell wall protein. Because of the presence of luciferase at the cell wall, problems concerning the intracellular availability of the substrate could be avoided. This particular system was used to study superficial infections caused by C. albicans 22. Very recently, BLI was also used to follow the progression of oropharyngeal candidiasis and its possible treatment 23. Such findings support the use of BLI as a promising technique to study infections caused by free-living cells but also device-associated infections.
In this study, we describe the C. albicans biofilm development on polyurethane catheter pieces in BALB/c mice and its quantification using BLI. We provide a detailed protocol of in vitro colonization of polyurethane catheters during the period of adhesion followed by implantation in mice and subsequent biofilm development in live animals. Apart from measuring the BLI signal emitted by the C. albicans cells, we also determine the colony forming units for comparison with the standard technique for biofilm fungal load quantification.

<table border="0" cellpadding="0" cellspacing="0" width="877">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:329px;">Name of Reagent</td>
			<td style="width:199px;">Company</td>
			<td style="width:113px;">Catalog Number</td>
			<td style="width:236px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:329px;">Yeast extract granulated</td>
			<td style="width:199px;">Merck</td>
			<td style="width:113px;">MERC1.03753.0500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Bacteriological peptone</td>
			<td style="width:199px;">Oxoid</td>
			<td style="width:113px;">LP037B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Agar granulated</td>
			<td style="width:199px;">Difco</td>
			<td style="width:113px;">214530</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">D-(+)-glucose</td>
			<td style="width:199px;">Fluka</td>
			<td style="width:113px;">49159-5KG</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Phosphate buffered saline&#160;</td>
			<td style="width:199px;">Prepared in the laboratory</td>
			<td style="width:113px;"></td>
			<td>for 1L of 10x PBS: 80 g NaCl, 2 g KCl, 14.4 g Na2HPO4, 2.4 g KH2PO4</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:329px;">RPMI1640 with L-glutamine and without sodium carbonate&#160;</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:113px;">R6504-1L</td>
			<td>Prepare according the protocol for Candida albicans drug susceptibility testing&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">3-(N-Morpholino)propanesulfonic acid (MOPS)</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:113px;">M1254</td>
			<td>MOPS is used to adjust the pH of RPMI medium (pH 7.0)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">fetal bovine serum (FBS)</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:113px;">F7524</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:329px;">Polyurethane tripe-lumen intravenous catheter piece (2.4 mm diameter, Certofix Trio S730)&#160;</td>
			<td style="width:199px;">BBraun</td>
			<td style="width:113px;">CV-15703</td>
			<td>Polyurethane part cut into 1 cm pieces</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Dexamethasone</td>
			<td style="width:199px;">Fagron SAS, France</td>
			<td style="width:113px;">611139</td>
			<td>Immunosuppressant (stock solution 10 mg/ml)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:329px;">Ampicillin&#160;</td>
			<td style="width:199px;">Duchefa Biochemie, The Netherlands</td>
			<td style="width:113px;">A0104</td>
			<td>Antibacterial prophylaxis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Ketamine 1000</td>
			<td>Pfizer</td>
			<td style="width:113px;">804 119</td>
			<td>Anesthetic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Domitor</td>
			<td>Pfizer</td>
			<td style="width:113px;">134737-1</td>
			<td>Anesthetic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Antisedan</td>
			<td>Pfizer</td>
			<td style="width:113px;">134783-2</td>
			<td>Reversal of anesthesia</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Xylocaine gel (2%) - this is Linisol</td>
			<td style="width:199px;">AstraZeneca</td>
			<td style="width:113px;">352 1206</td>
			<td>Local anesthetic for the skin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Terramycin/ polymyxin-b ophthalmic ointment</td>
			<td style="width:199px;"></td>
			<td style="width:113px;"></td>
			<td>To prevent drying and infection of eyes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td style="width:199px;"></td>
			<td style="width:113px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Coelenterazine&#160;</td>
			<td style="width:199px;">Prolume (Nanolight)</td>
			<td style="width:113px;">NF-CTZ-FB</td>
			<td>Light sensitive agent (must be kept in the dark)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Iodine isopropanol (1%)</td>
			<td style="width:199px;">3M&#8482; DuraPrep&#8482;&#160;</td>
			<td style="width:113px;"></td>
			<td>Disinfectant for the skin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.5 % chlorhexidine in 70 % alcohol.&#160;&#160;</td>
			<td style="width:199px;">Cedium</td>
			<td style="width:113px;"></td>
			<td>Disinfectant for the skin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;"></td>
			<td style="width:199px;"></td>
			<td style="width:113px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Equipment</td>
			<td style="width:199px;"></td>
			<td style="width:113px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Cell counting chamber</td>
			<td style="width:199px;"></td>
			<td style="width:113px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Insulin syringes (0.3 ml)</td>
			<td style="width:199px;">Terumo Myjector 29G</td>
			<td style="width:113px;">324826</td>
			<td>For injection of coelenterazine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Electric razor</td>
			<td style="width:199px;"></td>
			<td style="width:113px;"></td>
			<td>For small animals</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Sterile surgical tools</td>
			<td style="width:199px;"></td>
			<td style="width:113px;"></td>
			<td>Scissors, 2 pairs of tweezers, scalpel</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Heating pad</td>
			<td style="width:199px;">Leica</td>
			<td style="width:113px;">14042321474</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Skin suture</td>
			<td style="width:199px;">Johnson&#38;Johnson</td>
			<td style="width:113px;">K890H</td>
			<td>Surgical thread, needle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Water bath sonicator</td>
			<td>Branson 2210</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">BLI camera (IVIS Spectrum)&#160;</td>
			<td style="width:199px;">Perkin Elmer, Alameda</td>
			<td style="width:113px;">IVISSPE</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Living Image software&#160;</td>
			<td style="width:199px;">Perkin Elmer, Alameda</td>
			<td style="width:113px;"></td>
			<td>(version 4.2)&#160;</td>
		</tr>
	</tbody>
</table>

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.

In this study, we show the surgical procedure of catheter implant and explant during in vivo C. albicans biofilm development in a mouse. Moreover, we display the quantification of mature biofilms not only by classical CFUs enumeration but also by BLI.
As shown in Figure 1A, non-phosphorescent polyurethane catheter pieces were cut into 1 cm devices and subsequently coated with serum. This step is very important because it allows Candida cells to attach to the ...

Watch this Scientific Journal Video about Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging at JoVE.com

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 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 ...

candida-albicans-biofilm-development-on-medically-relevant-foreign

Research

JoVE Journal

Medicine

12.8K Görüntüleme.  Institute of Botany and Microbiology, VIB, KU Leuven. 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.