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 sterile 40% glucose. Mix thoroughly and pour into Petri dishes. Leave agar plates to cool and solidify. 
	NOTE: In this study, use two strains, namely wild type C. albicans SC5314 &#8211;gLuc negative strain (referred to as WT)and C. albicans SKCA23 strain, which is a wild type C. albicans SC5314 transformed with Clp10::Act1p-gLUC59 plasmid 22.In this strain (named SKCA23-ACTgLuc) gLuc was fused to the endogenous PGA59 gene under the control of ACT1 (actin) promoter (this promoter is active in the yeast, as well as hyphal stage of fungal growth). This strain can be requested from the laboratory of Prof. Patrick Van Dijck, KU Leuven, Leuven, Belgium. Plasmid Clp10::Act1p-gLUC59 plasmid 22 was kindly donated by Prof. C. d&#8217;Enfert, Institute Pasteur, Paris, France.</li>
	<li>Maintain both strains in glycerol stock and store at -80 &#176;C.</li>
	<li>Prior to any experiment streak strains onto an YPD plate. Incubate plate at 37 &#176;C overnight.</li>
</ol>
2. Catheter Pieces Preparation
<ol>
	<li>Before the experiment, determine how many catheters are needed. It is possible to implant up to 6 catheter pieces per mouse (3 catheters on the left and 3 catheter on the right side of the animal (Figure 2A).</li>
	<li>Twenty-four hr prior to the animal surgery, open the package containing triple-lumen catheter under the biological safety cabinet. Remove all unnecessary parts with sterile tweezers and cut the part attached to the catheter with a sterile scalpel. Place ruler under the plastic package and cut polyurethane catheter pieces of exactly 1 cm according to the scale on the ruler (Figure 1). 
	NOTE: It is important to mention that this type of catheter piece is not phosphorescent and therefore suitable for BLI 18,19.</li>
	<li>Place a maximum of 15 cut catheter pieces (step. 2.2) into 2 ml microcentrifuge tubes. Always prepare 3 pieces extra, which are used to enumerate the amount of attached Candida cells on the device after the period of adhesion.</li>
	<li>Supplement catheters with approximately 1.8 ml of 100% fetal bovine serum (FBS).</li>
	<li>Vigorously vortex and add additional 100-200 &#956;l of 100% FBS. Completely cover all catheter pieces with serum.</li>
	<li>Incubate at 37 &#176;C overnight.</li>
</ol>
3. Animals and Suppression of the Immune System
<ol>
	<li>Keep female BALB/c mice (approximately 8 weeks of age) in individually ventilated filter top cages with free access to standard food and water ad libitum.</li>
	<li>Initiate suppression of the immune system 24 hr before animal surgery by adding dexamethasone (0.4 mg/L) to the drinking water of animals. In order to avoid any bacterial contamination of the host, supplement the drinking water with antibiotic, e.g., ampicillin sodium powder (0.5 g/L).</li>
	<li>Keep immunosuppression of animals during the entire experiment (up to 6 days).</li>
</ol>
4. Ex vivo C. albicans Adhesion on FBS-coated Polyurethane Substrates
<ol>
	<li>Transfer each serum-coated catheter piece into a fresh 1.5 ml microcentrifuge tube.</li>
	<li>Scrape off some C. albicans cells grown on YPD plates (Step 1) and suspend them in 1 ml of PBS.</li>
	<li>Dilute Candida cells (1:100) in a separate microcentrifuge tube. Take 10 &#956;l of the diluted sample and apply it on a cell counting chamber. Count at least 16 small squares.</li>
	<li>Prepare Candida cells (each strain separately) in RPMI 1640 medium to a final concentration of 5 x 104 cells/ml.</li>
	<li>Add 1 ml of cell suspension to each serum-coated catheter piece.</li>
	<li>Vigorously vortex and ensure that the catheters are submerged in the medium and not floating on top.</li>
	<li>Incubate catheters at 37 &#176;C for 90 min (period of adhesion).</li>
	<li>Remove catheters with sterile tweezers and wash them twice with 1 ml of PBS. During this step make sure that the washing fluid goes through the lumen by keeping the catheter in vertical position while very gently flushing the catheters. Importantly, do not use a strong flow which may lead to the removal of attached cells.</li>
	<li>Transfer each washed catheter to a clean microcentrifuge tube (one piece per tube).</li>
	<li>Place on ice and keep there until surgery.</li>
</ol>
5. Anesthesia
<ol>
	<li>Prepare anesthesia by mixing 75 &#181;l of ketamine (100 mg/ml) with 100 &#181;l of medetomidine (1 mg/ml) and 825 &#181;l of sterile saline. Administer intraperitoneally (i.p.) 60-80 &#181;l of anesthetic cocktail per 10 g body weight, resulting in a dose of 45-60 mg/kg ketamine and 0.6-0.8 mg/kg medetomidine.</li>
	<li>For reversal of anesthesia, dilute 50 &#181;l atipamezole (5 mg/ml) in 4.95 ml saline, administer i.p. 100 &#181;l per 10 g body weight as antidote, resulting in a dose of 0.5 mg/kg.</li>
	<li>After injection of anesthesia, place the animal into a separate cage and wait until it is fully asleep.</li>
	<li>Confirm proper anesthetization of the animal by light skin pinch and toe pinch, which do not cause any damage to the skin. Any observed movement indicates that the animal is not sufficiently anesthetized to perform surgery. If this happens, wait a couple of minutes longer until the animal does not show any signs of movement upon skin or toe pinch.</li>
</ol>
6. Animal Surgery
<ol>
	<li>Transfer anesthetized animal from the cage on a clean tissue placed on the heating pad, pre-warmed to 37 &#176;C (Figure 2A, (1)). 
	NOTE: A cheaper alternative is to use electrically heated blankets. It is also possible to use isothermic pads, which must be warmed up in the microwave prior to the animal surgery.</li>
	<li>Apply ophthalmic ointment on the eyes.</li>
	<li>Shave the lower back of the animal with an electric razor. Remove all animal hairs and transfer animal on a clean tissue. Disinfect the skin (e.g., with 1% iodine isopropanol or 0.5% chlorhexidine in 70% alcohol) and leave the disinfected area to dry for approximately 1 min (Figure 2A, (2)).</li>
	<li>Make a small incision in the skin (one on the left and one on the right side of the animal) (approximately 0.5&#8211;1 cm) (Figure 2A, (3)).</li>
	<li>Dissect the subcutis with a scissor to create two subcutaneous tunnels. Each tunnel should be approximately 1.5 cm long and 1 cm wide.</li>
	<li>Insert three catheter pieces, previously infected with Candida, in each tunnel. Ensure that the catheters lie next to each other in a horizontal arrangement and that they do not cover each other to enable the implantation of six catheter fragments in total (Figure 2A, (4)).</li>
	<li>Close the incisions with sutures. Alternatively, use Dermabond to close the wound.</li>
	<li>Disinfect the wound very gently with 0.5% chlorhexidine in 70% alcohol or with iodine isopropanol (1%).</li>
	<li>Apply local anesthetic (xylocaine gel, 2%) directly on the wound.</li>
	<li>Administer reversal of anesthesia (protocol 5, step 2): intraperitoneally 100 &#181;l per 10 g body weight.</li>
	<li>Transfer animal to a clean cage previously placed on a heating plate. Keep the animal separate and warm until the animal is completely awake. Meanwhile, continue with the operation and implant of the next animal. Once all operated animals are fully awake. Transfer to one cage. Monitor animals regularly.</li>
</ol>
7. Bioluminescence Imaging: Preparation of Coelenterazine (CTZ), the Substrate for G. princeps luciferase
<ol>
	<li>Prepare fresh coelenterazine (CTZ) stock solution by dissolving 5 mg/ml CTZ in acidified ethanol or according to manufacturer&#8217;s instructions.</li>
	<li>Prepare 1.2 mM working solution by diluting the stock solution 1:10 in sterile PBS. 
	NOTE: Inject 100 &#181;l CTZ working solutions subcutaneously in the area surrounding the catheters. Use insulin syringes for subcutaneous injection of CTZ.</li>
	<li>Always keep CTZ in the dark (e.g., cover microcentrifuge tubes containing CTZ with aluminum foil). Store the stock solution at -80 &#176;C for the duration of the experiments.</li>
</ol>
8. Bioluminescence Imaging
<ol>
	<li>Initialize the BLI camera.</li>
	<li>Anesthetize the animals using an induction box. Use a gas mixture of isoflurane in oxygen, N2O/ O2, or air at 2-3%.</li>
	<li>After induction, maintain anesthesia in the induction box and in the imaging chamber at 1.5-2%.</li>
	<li>Before starting the imaging session, place the imaging plate in position A, which corresponds to a FOV of 10 cm. Ensure that the right position of the anesthesia outlets and animal by placing a sleeping animal in the box.</li>
	<li>Take few photographs until animal is in the desired imaging position, in the FOV right under the camera.</li>
	<li>Prepare two insulin syringes each containing 100 &#181;l of the CTZ working solution.</li>
	<li>Place one animal on the bench and keep it asleep by means of a nose cone providing gas anesthesia.</li>
	<li>Bring the needles of the syringes in a place surrounding catheters subcutaneously and inject the CTZ simultaneously on top of the catheters.</li>
	<li>Immediately after injection, place the animal on the warm plate in the camera box and start the bioluminescence image acquisition.</li>
	<li>Acquire consecutive scans with acquisition times ranging from 20 to 60 sec (depending on the signal intensity) until the maximum signal intensity is reached. During the acquisition of the next frame, measure the BLI signal intensity of the previously acquired frames by placing a ROI over each catheters trio and measuring the photon flux through this ROI.</li>
	<li>Repeat from step 7 for the next animal(s).</li>
	<li>After imaging, return animals to their cage. Repeat BLI during the course of an experiment for longitudinal, non-invasive follow-up of biofilm formation.</li>
	<li>Analyze the BLI data using Living Image software. Place a rectangular ROI of fixed size over each catheter trio and measure the photon flux (radiance) through each ROI. Repeat this for every animal.</li>
	<li>Report the BLI signal intensity of each catheter trio as photon flux per second. Represent the BLI data on a logarithmic scale by plotting the mean and SD of the photon flux per second for each group. Perform statistical analysis on the log10 transformed data.</li>
</ol>
9. Catheter Explant
<ol>
	<li>Prepare microcentrifuge tubes containing 1 ml of PBS, one for each catheter device and keep them on ice.</li>
	<li>Euthanize the animals by cervical dislocation (Figure 2B, (1)).</li>
	<li>Disinfect the skin of the back with 0.5% chlorhexidine in 70% alcohol or with iodine isopropanol (1%).</li>
	<li>Make an incision (approximately 3 cm) above the catheters.</li>
	<li>Cut subcutaneous tissue and remove the catheter fragments one by one from under the subcutaneous tissue using sterile tweezers (Figure 2B, (2)).</li>
	<li>Handle catheter gently in vertical position and wash it twice with 1 ml of sterile PBS. Place each catheter piece into a separate microcentrifuge tube (prepared in Step 1).</li>
</ol>
10. Quantification of Biofilm-associated Cells by Colony Forming Units Count (CFUs) and Statistical Analyses of Results
<ol>
	<li>Sonicate catheters previously placed into 1 ml of PBS for 10 min at 40,000 Hz in a water bath sonicator and place them on ice.</li>
	<li>After sonication, vigorously vortex for 30 sec and place again on ice.</li>
	<li>Prepare two additional microcentrifuge tubes containing 900 &#956;l of PBS.</li>
	<li>Make 1:10 and 1:100 dilutions from your original sample (the one which contains the catheter) and keep all microcentrifuge tubes on ice.</li>
	<li>Plate 100 &#181;l of the original samples, 1:10 and 1:100 dilutions on YPD agar plates in duplicate.</li>
	<li>Incubate plates for 2 days at 37 &#176;C and count CFUs.</li>
	<li>Count the colonies grown on YPD agar plates (countable amount of colonies, maximum 300 colonies/plate) and multiply them by the proper dilution factor (1x-original, 10x or 100x dilution). Further multiply each catheter piece by 10x, which corresponds to the final amount of colonies in 1 ml tube containing the catheter. Bring the final amount of colonies to log10 cells/catheter piece with standard deviation (SD). Express data as a mean &#177; SD.</li>
	<li>Place all values for each catheter and specific group into a spreadsheet and/or statistical program, to perform the above-mentioned calculations and statistical analyses. Indicate specific groups to compare, i.e. WT vs. mutant or 2 days vs. 6 days biofilm formation and perform unpaired t-test and ANOVA with Tukey post-test on the log10-transformed data.
	<ol>
		<li>Express the level of significance by a p value. In this study indicate significance if p value &#60;0.05, represented as follows: * p &#60;0.05, **p &#60;0.005, ***p &#60;0.0005.</li>
	</ol>
	</li>
</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 biofilm formation in a rat 24.
In the presented model, serum-coated polyurethane catheters were challenged with Candida cells ex vivo during the period of adhesion (90 min, 37 &#176;C). This first in vitro step might be considered as a limiting factor because of the lack of the immune system at the very first stages of the biofilm infection process. Following adhesion and before catheter implant, non-device associated cells are removed by washing. Avoiding the washing step after the period of adhesion may create uncontrollable amount of cells associated with the device. Because of this reason, we suggest to wash catheters prior to the implant and therefore, to initiate its development from adhered cells. After this initial adhesion period, catheters contain approximately 2.0-2.5 Log10 CFUs/device spread alongside the catheter lumen 15. During this stage Candida forms germ tubes, which are attached onto the substrate.
In order to resemble the situation in hospitalized patients of whom the immune system is often compromised, we immunosuppressed the rats in our subcutaneous model system 15. Partial impairment of the immune system of a rat, prior to the device implant and throughout the period of biofilm development, resulted in increased reproducibility of biofilm-forming cells retrieved from catheters implanted in the same host and also from devices obtained from additional animals. Because of these findings we suggest to immunosuppress the mice before catheter implant and also during the period of biofilm formation. However, our results show that the variability in immunocompetent mice is much less compared to that observed in rats, which makes that mice model system also suitable for studying the role of the host immune system on biofilms. In our original rat model and also in the same model translated to mice, mature C. albicans biofilms develop within 48 hr demonstrated by the similar amount of CFUs and biofilm architecture between 2 and 6 days 15,18,19.
The subcutaneous biofilm model was successfully used to follow biofilm development by several mutants 15. It has been shown previously by Nobile et al. 25 that C. albicans bcr1&#8710;/bcr1&#8710; failed to form biofilms in a central venous catheter (CVC) model. This strain displayed rudimentary biofilm features in our subcutaneous model pointing to the fact that the phenotypic changes found in the CVC model could be reproduced in the subcutaneous model 15.
In comparison with other existing models, i.e., CVC model or denture stomatitis model 8,9,11, the subcutaneous model allows to follow biofilm development in multiple catheter pieces obtained from one animal, thereby reducing the number of animals needed for biofilm studies. Despite these advantages, a shortcoming may be the lack of blood flow that may lead to certain deprivation of nutrients in the biofilm. Therefore, in term of nutrient supplies and environmental conditions, the subcutaneous model is more related to device-associated infections developed on joint prostheses, voice prostheses and pacemakers than the ones formed on intravenous catheters. Even more importantly, translating the rat subcutaneous biofilm system to mice made this animal model compatible with BLI, which further reduces costs and most importantly, the number of necessary animals. Furthermore, translating the rat model to mice enables the use of transgenic mouse models to study host factors relevant to catheter-associated infection studies.
The major advantage of using BLI to quantify biofilms in live animals lies not only in the non-invasive character of this method, but also in its ability to provide dynamic information on the development of an infection. In this study, gLuc expressed at the cell wall of C. albicans allowed better accessibility and direct interaction with the substrate coelenterazine, which are crucial for a detection of bioluminescent signal in vivo. In the study of Vande Velde et al. 18, we were able to follow the bioluminescent signal from C. albicans biofilms developed on foreign bodies in vitro. The BLI signal intensity strongly corresponded with the data obtained from additional biofilm quantification techniques, i.e., CFUs determination and XTT reduction assay. Next to these results, we showed that the bioluminescent signal from in vivo biofilms was in agreement with the amount of biofilm-associated cells recovered from explanted biofilms. Additionally, Vande Velde et al. 18 demonstrated that BLI can be used to follow biofilm development by a wild type and also by C. albicans bcr1&#8710;/bcr1&#8710;(biofilm-deficient strain) in the same animal at the same time. Such findings strongly support the use of BLI during the studies dedicated to assessing the time course of biofilm development and infection in the same animal.
Herewith, we described an experimental procedure for in vivo subcutaneous implant of Candida-infected devices with subsequent monitoring of in vivo biofilm development by BLI. Taken together, BLI showed to be a reliable technique, which allowed us to follow an infection over time avoiding animal sacrifices at each time point of data analyses.

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><tbody><tr><td>Yeast extract granulated</td><td>Merck</td><td>MERC1.03753.0500</td><td></td></tr><tr><td>Bacteriological peptone</td><td>Oxoid</td><td>LP037B</td><td></td></tr><tr><td>Agar granulated</td><td>Difco</td><td>214530</td><td></td></tr><tr><td>D-(+)-glucose</td><td>Fluka</td><td>49159-5KG</td><td></td></tr><tr><td>Phosphate buffered saline&amp;nbsp;</td><td>Prepared in the laboratory</td><td></td><td>for 1L of 10x PBS: 80 g NaCl, 2 g KCl, 14.4 g Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;, 2.4 g KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td></tr><tr><td>RPMI1640 with L-glutamine and without sodium carbonate&amp;nbsp;</td><td>Sigma</td><td>R6504-1L</td><td>Prepare according the protocol for Candida albicans drug susceptibility testing&amp;nbsp;</td></tr><tr><td>3-(N-Morpholino)propanesulfonic acid (MOPS)</td><td>Sigma</td><td>M1254</td><td>MOPS is used to adjust the pH of RPMI medium (pH 7.0)</td></tr><tr><td>fetal bovine serum (FBS)</td><td>Sigma</td><td>F7524</td><td></td></tr><tr><td>Polyurethane tripe-lumen intravenous catheter piece (2.4 mm diameter, Certofix Trio S730)&amp;nbsp;</td><td>BBraun</td><td>CV-15703</td><td>Polyurethane part cut into 1 cm pieces</td></tr><tr><td>Dexamethasone</td><td>Fagron SAS, France</td><td>611139</td><td>Immunosuppressant (stock solution 10 mg/ml)</td></tr><tr><td>Ampicillin&amp;nbsp;</td><td>Duchefa Biochemie, The Netherlands</td><td>A0104</td><td>Antibacterial prophylaxis</td></tr><tr><td>Ketamine 1000</td><td>Pfizer</td><td>804 119</td><td>Anesthetic</td></tr><tr><td>Domitor</td><td>Pfizer</td><td>134737-1</td><td>Anesthetic</td></tr><tr><td>Antisedan</td><td>Pfizer</td><td>134783-2</td><td>Reversal of anesthesia</td></tr><tr><td>Xylocaine gel (2%) - this is Linisol</td><td>AstraZeneca</td><td>352 1206</td><td>Local anesthetic for the skin</td></tr><tr><td>Terramycin/ polymyxin-b ophthalmic ointment</td><td></td><td></td><td>To prevent drying and infection of eyes</td></tr><tr><td>Coelenterazine&amp;nbsp;</td><td>Prolume (Nanolight)</td><td>NF-CTZ-FB</td><td>Light sensitive agent (must be kept in the dark)</td></tr><tr><td>Iodine isopropanol (1%)</td><td>3M&amp;trade; DuraPrep&amp;trade;&amp;nbsp;</td><td></td><td>Disinfectant for the skin</td></tr><tr><td>0.5 % chlorhexidine in 70 % alcohol.&amp;nbsp;&amp;nbsp;</td><td>Cedium</td><td></td><td>Disinfectant for the skin</td></tr><tr><td>Equipment</td><td></td><td></td><td></td></tr><tr><td>Cell counting chamber</td><td></td><td></td><td></td></tr><tr><td>Insulin syringes (0.3 ml)</td><td>Terumo Myjector 29G</td><td>324826</td><td>For injection of coelenterazine</td></tr><tr><td>Electric razor</td><td></td><td></td><td>For small animals</td></tr><tr><td>Sterile surgical tools</td><td></td><td></td><td>Scissors, 2 pairs of tweezers, scalpel</td></tr><tr><td>Heating pad</td><td>Leica</td><td>14042321474</td><td></td></tr><tr><td>Skin suture</td><td>Johnson&amp;amp;Johnson</td><td>K890H</td><td>Surgical thread, needle</td></tr><tr><td>Water bath sonicator</td><td>Branson 2210</td><td></td><td></td></tr><tr><td>BLI camera (IVIS Spectrum)&amp;nbsp;</td><td>Perkin Elmer, Alameda</td><td>IVISSPE</td><td></td></tr><tr><td>Living Image software&amp;nbsp;</td><td>Perkin Elmer, Alameda</td><td></td><td>(version 4.2)&amp;nbsp;</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 substrate more rapidly in comparison with non-serum coated implants 15. It is important to mention that prior to any C. albicans experiment we first documented the background luminescence of our devices 18. This step is crucial before performing any BLI because high phosphorescence of the device will interfere with the evaluation of the specific bioluminescent signal intensity and signal kinetics from gLuc-expressing cells. Next, C. albicans cells are incubated with the serum-coated catheter pieces. Catheter fragments are then implanted on the back part of the animal after the subcutaneous incision (Figure 2A). In this in vivo set up, two incisions are performed (one on the right and another one on the left side of the back of a mouse) followed by formation of subcutaneous tunnels inside each incision (Figure 2A (3)). Subsequently, three devices, previously infected with Candida cells, are implanted inside each operation site (Figure 2A (3 and 4)). This set up allows us to study up to six biofilms per animal. It is noteworthy that two operation sites provide a possibility to study biofilm formation by two different strains of interest, for example wild type and mutant in the same animal. Figure 2B displays the wound containing catheters prior to the device explant and subsequent washing steps. Biofilms developed inside the back part of the animal were assessed for the bioluminescence signal measurements. One of the representative animals displaying the bioluminescence signal together with rectangles characterizing the regions of interest (ROIs) is shown in Figure 3. After the last BLI time point, catheters are explanted, sonicated and subsequently vortexed and further assessed for the biofilm-forming cells quantification by CFUs. Data analyses demonstrating the Log10 CFUs obtained from each catheter piece after biofilm development and explantation are shown in Figure 4A. Next to that, CFUs were compared with the BLI signal intensity and these data are shown in Figure 4B. Ninety min after implantation a clear BLI signal is produced by the ACTgLuc-expressing biofilms whereas there in the wild type strain, hardly any light is produced; The intensity of the light detected from the ACTgLuc-expressing biofilms is significantly increasing as the biofilm is formed, here following the same mouse (Figure 4C). This increase in light follows the same trend as the increase in CFUs per biofilm (Figure 4A). In our experiments we also observed that a normal wild type strain (not engineered to express luciferase) also results in the production of light upon addition of the substrate. However, the photon flux was significantly lower than that obtained for the engineered strain.
Taken together, our data illustrate that BLI is a powerful technique to monitor and quantify in vivo mature C. albicans biofilm formation in a subcutaneous mouse model.
<img alt="Figure 1" src="/files/ftp_upload/52239/52239fig1highres.jpg" /> 
Figure 1: Preparation of polyurethane devices. Polyurethane part of the catheter cut into 1 cm pieces. Plastic pocket containing catheter is open under the sterile conditions and all parts, except catheter are removed from the package. Secondly, place ruler under the plastic pocket and cut exactly 1 cm polyurethane pieces. Such devices are subsequently distributed to microcentrifuge tubes (max 15 pieces/tube) and submerged in 100% fetal bovine serum followed by overnight incubation at 37 &#176;C.
<img alt="Figure 2" src="/files/ftp_upload/52239/52239fig2highres.jpg" /> 
Figure 2: Major steps during the animal surgery. (A) Procedure of catheter implant. (1) Place anesthetized animal on a warm pad containing paper tissue and apply the ophthalmic ointment on eyes. (2) Shave lower part of the back and disinfect. (3) Create two small (approx. 0.5 cm) incisions through the skin on the left and on the right side of the back. Create subcutaneous tunnel inside each incision and place 3 catheters inside. (4) Close the wound with sutures and place animal on a warm pad to recover. (B) Procedure of catheter removal. (1) Place sacrificed animal on a pad and disinfect the operated side containing catheters. Cut the wound right above the catheters. (2) Take out each catheter gently and wash twice with 1 ml of PBS. Place to the separate microcentrifuge tube.
<img alt="Figure 3" src="/files/ftp_upload/52239/52239fig3highres.jpg" /> 
Figure 3: Bioluminescence signal measurement. In vivo BLI image from one representative mouse imaged after 6 days of biofilm development. The quantification of signal intensity is performed by placing a region of interest (ROI) (rectangular) around the catheters followed by measurement of the photon flux per second through every ROI.
<img alt="Figure 4" src="/files/ftp_upload/52239/52239fig4highres.jpg" /> 
Figure 4: Quantification of mature Candida albicans biofilms by colony forming units (CFUs) and by BLI. (A) In vivo mature C. albicans SKCA23-ACTgLuc and SC5314 (WT) biofilm quantification by Log10 CFUs after 90 min, 2 days and 6 days of biofilm development. (B) Bioluminescence signal intensity quantification from in vivo biofilms formed by SKCA23-ACTgLuc and by C. albicans WT. Signal was determined after 90 min (period of adhesion), 2 days and 6 days of biofilm development. As a control, we used mice that were implanted with non-colonized catheters and that were subcutaneously injected with CTZ. The photon flux obtained in these mice results in our background signal (BG). Data were expressed as mean &#177; standard deviation (SD). Statistical significance is indicated as follows: * p &#60;0.05, ** p &#60;0.005, *** p &#60;0.0005. (C) In vivo bioluminescence images from one representative mouse that was implanted with catheter fragments containing wild type C. albicans cells on the left side and ACTgLuc-expressing cells on the right side. The mouse was imaged at three different time periods, i.e. 90 min, 2 days and 6 days after implantation of the catheter fragments.

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

Nanyang Technological University

Research

JoVE Journal

Medicine

12.9K Views.  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.

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

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 ...

Immunology and Infection

Come to the Light Side: In Vivo Monitoring of Pseudomonas aeruginosa Biofilm Infections in Chronic Wounds in a Diabetic Hairless Murine Model

The presence of bacteria as structured biofilms in chronic wounds, especially in diabetic patients, is thought to prevent wound healing and resolution. Chronic mouse wounds models have been used to understand the underlying interactions between the microorganisms and the host. The models developed to date rely on the use of haired animals and terminal collection of wound tissue for determination of viable bacteria. While significant insight has been gained with these models, this experimental procedure requires a large number of animals and sampling is time consuming. We have developed a novel murine model that incorporates several optimal innovations to evaluate biofilm progression in chronic wounds: a) it utilizes hairless mice, eliminating the need for hair removal; b) applies pre-formed biofilms to the wounds allowing for the immediate evaluation of persistence and effect of these communities on host; c) monitors biofilm progression by quantifying light production by a genetically engineered bioluminescent strain of Pseudomonas aeruginosa, allowing real-time monitoring of the infection thus reducing the number of animals required per study. In this model, a single full-depth wound is produced on the back of STZ-induced diabetic hairless mice and inoculated with biofilms of the P. aeruginosa bioluminescent strain Xen 41. Light output from the wounds is recorded daily in an in vivo imaging system, allowing for in vivo and in situ rapid biofilm visualization and localization of biofilm bacteria within the wounds. This novel method is flexible as it can be used to study other microorganisms, including genetically engineered species and multi-species biofilms, and may be of special value in testing anti-biofilm strategies including antimicrobial occlusive dressings.

Come to the Light Side: In Vivo Monitoring of Pseudomonas aeruginosa Biofilm Infections in Chronic Wounds in a Diabetic Hairless Murine Model

Here we describe a novel diabetic murine model utilizing hairless mice for real-time, non-invasive, monitoring of biofilm wound infections of bioluminescent Pseudomonas aeruginosa. This method can be adapted to evaluate infection of other bacterial species and genetically modified microorganisms, including multi-species biofilms, and test the efficacy of antibiofilm strategies.

The presence of bacteria as structured biofilms in chronic wounds, especially in diabetic patients, is thought to prevent wound healing and resolution. ...

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

Candida albicans is the most common fungal pathogen of humans, causing about 15% of hospital-acquired sepsis cases. A major virulence attribute of C. albicans is its ability to form biofilms, structured communities of cells attached to biotic and abiotic surfaces. C. albicans biofilms can form on host tissues, such as mucosal layers, and on medical devices, such as catheters, pacemakers, dentures, and joint prostheses. Biofilms pose significant clinical challenges because they are highly resistant to physical and chemical perturbations, and can act as reservoirs to seed disseminated infections. Various in vitro assays have been utilized to study C. albicans biofilm formation, such as microtiter plate assays, dry weight measurements, cell viability assays, and confocal scanning laser microscopy. All of these assays are single end-point assays, where biofilm formation is assessed at a specific time point. Here, we describe a protocol to study biofilm formation in real-time using an automated microfluidic device under laminar flow conditions. This method allows for the observation of biofilm formation as the biofilm develops over time, using customizable conditions that mimic those of the host, such as those encountered in vascular catheters. This protocol can be used to assess the biofilm defects of genetic mutants as well as the inhibitory effects of antimicrobial agents on biofilm development in real-time.

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

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

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 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.

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.

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

Longitudinal Follow-Up of Urinary Tract Infections and Their Treatment in Mice using Bioluminescence Imaging

Urinary tract infections (UTI) rank among the most common bacterial infections in humans and are routinely treated with empirical antibiotics. However, due to increasing microbial resistance, the efficacy of the most used antibiotics has declined. To find alternative treatment options, there is a great need for a better understanding of the UTI pathogenesis and the mechanisms that determine UTI susceptibility. In order to investigate this in an animal model, a reproducible, non-invasive assay to study the course of UTI is indispensable.
For years, the gold standard for the enumeration of bacterial load has been the determination of Colony Forming Units (CFU) for a particular sample volume. This technique requires post-mortem organ homogenates and serial dilutions, limiting data output and reproducibility. As an alternative, bioluminescence imaging (BLI) is gaining popularity to determine the bacterial load. Labeling pathogens with a lux operon allow for the sensitive detection and quantification in a non-invasive manner, thereby enabling longitudinal follow-up. So far, the adoption of BLI in UTI research remains limited.
This manuscript describes the practical implementation of BLI in a mouse urinary tract infection model. Here, a step-by-step guide for culturing bacteria, intravesical instillation and imaging is provided. The in vivo correlation with CFU is examined and a proof-of-concept is provided by comparing the bacterial load of untreated infected animals with antibiotic-treated animals. Furthermore, the advantages, limitations, and considerations specific to the implementation of BLI in an in vivo UTI model are discussed. The implementation of BLI in the UTI research field will greatly facilitate research on the pathogenesis of UTI and the discovery of new ways to prevent and treat UTI.

This manuscript describes the intravesical administration of uropathogenic bacteria with a lux operon to induce a urinary tract infection in mice and subsequent longitudinal in vivo analysis of the bacterial load using bioluminescence imaging.

Urinary tract infections (UTI) rank among the most common bacterial infections in humans and are routinely treated with empirical antibiotics. However, ...

Use of In Vivo Imaging to Screen for Morphogenesis Phenotypes in Candida albicans Mutant Strains During Active Infection in a Mammalian Host

Candida albicans is an important human pathogen. Its ability to switch between morphologic forms is central to&#160;its pathogenesis; these morphologic changes are regulated by a complex signaling network controlled in response to environmental stimuli. These regulatory components have been highly studied, but almost all studies use a variety of in vitro stimuli to trigger filamentation. To determine how morphogenesis is regulated during the pathogenesis process, we developed an in vivo microscopy system to obtain high spatial resolution images of organisms undergoing hyphal formation within the mammalian host. The protocol presented here describes the use of this system to screen small collections of C. albicans mutant strains, allowing us to identify key regulators of morphogenesis as it occurs at the site of infection. Representative results are presented, demonstrating that some regulators of morphogenesis, such as the transcriptional regulator Efg1, have consistent phenotypes in vitro and in vivo, whereas other regulators, such as adenyl cyclase (Cyr1), have significantly different phenotypes in vivo compared to in vitro.

Use of In Vivo Imaging to Screen for Morphogenesis Phenotypes in Candida albicans Mutant Strains During Active Infection in a Mammalian Host

This manuscript describes a method for screening moderately sized Candida albicans mutant libraries for morphogenesis phenotypes during active infection in a mammalian host using non-invasive confocal microscopy.

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

Effective aPDT Using Curcuminoids for Oral Candidiasis in Mice

Curcuminoid-Mediated Antimicrobial Photodynamic Therapy on a Murine Model of Oral Candidiasis

Antimicrobial Photodynamic Therapy (aPDT) has been extensively investigated in vitro, and preclinical animal models of infections are suitable for evaluating alternative treatments prior to clinical trials. This study describes the efficacy of aPDT in a murine model of oral candidiasis. Forty mice were immunosuppressed with subcutaneous injections of prednisolone, and their tongues were inoculated using an oral swab previously soaked in a C. albicans cell suspension. Tetracycline was administered via drinking water during the course of the experiment. Five days after fungal inoculation, mice were randomly distributed into eight groups; a ninth group of untreated uninfected mice was included as a negative control (n = 5). Three concentrations (20 &#181;M, 40 &#181;M, and 80 &#181;M) of a mixture of curcuminoids were tested with a blue LED light (89.2 mW/cm2; ~455 nm) and without light (C+L+ and C+L- groups, respectively). Light alone (C-L+), no treatment (C-L-), and animals without infection were evaluated as controls. Data were analyzed using Welch&#39;s ANOVA and Games-Howell tests (&#945; = 0.05). Oral candidiasis was established in all infected animals and visualized macroscopically through the presence of characteristic white patches or pseudomembranes on the dorsum of the tongues. Histopathological sections confirmed a large presence of yeast and filaments limited to the keratinized layer of the epithelium in the C-L- group, and the presence of fungal cells was visually decreased in the images obtained from mice subjected to aPDT with either 40 &#181;M or 80 &#181;M curcuminoids. aPDT mediated by 80 &#181;M curcuminoids promoted a 2.47 log10 reduction in colony counts in comparison to those in the C-L- group (p = 0.008). All other groups showed no statistically significant reduction in the number of colonies, including photosensitizer (C+L-) or light alone (C-L+) groups. Curcuminoid-mediated aPDT reduced the fungal load from the tongues of mice.

Author Spotlight: Exploring Photodynamic Therapy with Curcumin in a Murine Model for Oral Candidiasis

This protocol describes the application of antimicrobial Photodynamic Therapy (aPDT) in a murine model of oral candidiasis. aPDT was performed using a water-soluble mixture of curcuminoids and blue LED light.

Antimicrobial Photodynamic Therapy (aPDT) has been extensively investigated in vitro, and preclinical animal models of infections are suitable for ...

Establishing a Mouse Model for Candida albicans Biofilm-Related PJI

A Periprosthetic Joint Candida albicans Infection Model in Mouse

Periprosthetic joint infection (PJI) is one of the common infections caused by Candida albicans (C. albicans), which increasingly concerns surgeons and scientists. Generally, biofilms that can shield C. albicans from antibiotics and immune clearance are formed at the infection site. Surgery involving the removal of the infected implant, debridement, antimicrobial treatment, and reimplantation is the gold standard for the treatment of PJI. Thus, establishing animal PJI models is of great significance for the research and development of new drugs or therapeutics for PJI. In this study, a smooth nickel-titanium alloy wire, a widely used implant in orthopedic clinics, was inserted into the femoral joint of a C57BL/6 mouse before the C. albicans were inoculated into the articular cavity along the wire. After 14 days, mature and thick biofilms were observed on the surface of implants under a scanning electronic microscope (SEM). A significantly reduced bone trabecula was found in the H&amp;E staining of the infected joint specimens. To sum up, a mouse PJI model with the advantages of easy operation, high successful rate, high repeatability, and high clinical correlation was established. This is expected to be an important model for clinical studies of C. albicans biofilm-related PJI prevention.

Author Spotlight: Advancing Research on Candida albicans Biofilm-Associated Prosthetic Joint Infections 

Periprosthetic joint infection (PJI) caused by dangerous pathogens is common in clinical orthopedics. Existing animal models cannot accurately simulate the actual situation of PJI. Here, we established a Candida albicans biofilm-associated PJI mouse model to research and develop new therapeutics for PJI.

Periprosthetic joint infection (PJI) is one of the common infections caused by Candida albicans (C. albicans), which increasingly concerns surgeons and ...

Establishing a Mouse Model for Candida albicans-Associated Catheter-Related Infection

A Catheter-Related Candida albicans Infection Model in Mouse

Catheter-related infection (CRI) is a common nosocomial infection caused by candida albicans during catheter implantation. Typically, biofilms are formed on the outer surface of the catheter and lead to disseminated infections, which are fatal to patients. There are no effective prevention and treatment management in clinics. Therefore, it is urgent to establish an animal model of CRI for the preclinical screening of new strategies for its prevention and treatment. In this study, a polyethylene catheter, a widely used medical catheter, was inserted into the back of the BALB/c mice after hair removal. Candida albicans ATCC MYA-2876 (SC5314) expressing enhanced green fluorescent protein was subsequently inoculated on the skin's surface along the catheter. Intense fluorescence was observed on the surface of the catheter under a fluorescent microscope 3 days later. Mature and thick biofilms were found on the surface of the catheter via scanning electron microscopy. These results indicated the adhesion, colonization, and biofilm formation of candida albicans on the surface of the catheter. The hyperplasia of the epidermis and the infiltration of inflammatory cells in the skin specimens indicated the histopathological changes of the CRI-associated skin. To sum up, a mouse CRI model was successfully established. This model is expected to be helpful in the research and development of therapeutic management for candida albicans associated CRI.

Author Spotlight: Enhancing Candida albicans Detection in Catheter Infections Using Fluorescent Protein Tagging 

We establish a mouse model of C.albicans-associated catheter-related infection (CRI), in which biofilm forms on the catheter, and the interaction between C.albicans and host correlates well with the clinical CRI. This model helps screen therapies for C.albicans biofilm-associated CRI, laying a foundation for clinical transformation.

Catheter-related infection (CRI) is a common nosocomial infection caused by candida albicans during catheter implantation. Typically, biofilms are formed ...

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

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Curcuminoid-Mediated Antimicrobial Photodynamic Therapy on a Murine Model of Oral Candidiasis

A Periprosthetic Joint Candida albicans Infection Model in Mouse

A Catheter-Related Candida albicans Infection Model in Mouse