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

Summary

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.

Abstract

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.

Introduction

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 –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.

Protocol

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

1. 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.
2. 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 –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’Enfert, Institute Pasteur, Paris, France.
3. Maintain both strains in glycerol stock and store at -80 °C.
4. Prior to any experiment streak strains onto an YPD plate. Incubate plate at 37 °C overnight.

2. Catheter Pieces Preparation

1. 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).
2. 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.
3. 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.
4. Supplement catheters with approximately 1.8 ml of 100% fetal bovine serum (FBS).
5. Vigorously vortex and add additional 100-200 μl of 100% FBS. Completely cover all catheter pieces with serum.
6. Incubate at 37 °C overnight.

3. Animals and Suppression of the Immune System

1. 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.
2. 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).
3. Keep immunosuppression of animals during the entire experiment (up to 6 days).

4. Ex vivo C. albicans Adhesion on FBS-coated Polyurethane Substrates

1. Transfer each serum-coated catheter piece into a fresh 1.5 ml microcentrifuge tube.
2. Scrape off some C. albicans cells grown on YPD plates (Step 1) and suspend them in 1 ml of PBS.
3. Dilute Candida cells (1:100) in a separate microcentrifuge tube. Take 10 μl of the diluted sample and apply it on a cell counting chamber. Count at least 16 small squares.
4. Prepare Candida cells (each strain separately) in RPMI 1640 medium to a final concentration of 5 x 10⁴ cells/ml.
5. Add 1 ml of cell suspension to each serum-coated catheter piece.
6. Vigorously vortex and ensure that the catheters are submerged in the medium and not floating on top.
7. Incubate catheters at 37 °C for 90 min (period of adhesion).
8. 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.
9. Transfer each washed catheter to a clean microcentrifuge tube (one piece per tube).
10. Place on ice and keep there until surgery.

5. Anesthesia

1. Prepare anesthesia by mixing 75 µl of ketamine (100 mg/ml) with 100 µl of medetomidine (1 mg/ml) and 825 µl of sterile saline. Administer intraperitoneally (i.p.) 60-80 µl 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.
2. For reversal of anesthesia, dilute 50 µl atipamezole (5 mg/ml) in 4.95 ml saline, administer i.p. 100 µl per 10 g body weight as antidote, resulting in a dose of 0.5 mg/kg.
3. After injection of anesthesia, place the animal into a separate cage and wait until it is fully asleep.
4. 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.

6. Animal Surgery

1. Transfer anesthetized animal from the cage on a clean tissue placed on the heating pad, pre-warmed to 37 °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.
2. Apply ophthalmic ointment on the eyes.
3. 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)).
4. Make a small incision in the skin (one on the left and one on the right side of the animal) (approximately 0.5–1 cm) (Figure 2A, (3)).
5. Dissect the subcutis with a scissor to create two subcutaneous tunnels. Each tunnel should be approximately 1.5 cm long and 1 cm wide.
6. 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)).
7. Close the incisions with sutures. Alternatively, use Dermabond to close the wound.
8. Disinfect the wound very gently with 0.5% chlorhexidine in 70% alcohol or with iodine isopropanol (1%).
9. Apply local anesthetic (xylocaine gel, 2%) directly on the wound.
10. Administer reversal of anesthesia (protocol 5, step 2): intraperitoneally 100 µl per 10 g body weight.
11. 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.

7. Bioluminescence Imaging: Preparation of Coelenterazine (CTZ), the Substrate for G. princeps luciferase

1. Prepare fresh coelenterazine (CTZ) stock solution by dissolving 5 mg/ml CTZ in acidified ethanol or according to manufacturer’s instructions.
2. Prepare 1.2 mM working solution by diluting the stock solution 1:10 in sterile PBS.
   NOTE: Inject 100 µl CTZ working solutions subcutaneously in the area surrounding the catheters. Use insulin syringes for subcutaneous injection of CTZ.
3. Always keep CTZ in the dark (e.g., cover microcentrifuge tubes containing CTZ with aluminum foil). Store the stock solution at -80 °C for the duration of the experiments.

8. Bioluminescence Imaging

1. Initialize the BLI camera.
2. Anesthetize the animals using an induction box. Use a gas mixture of isoflurane in oxygen, N₂O/ O₂, or air at 2-3%.
3. After induction, maintain anesthesia in the induction box and in the imaging chamber at 1.5-2%.
4. 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.
5. Take few photographs until animal is in the desired imaging position, in the FOV right under the camera.
6. Prepare two insulin syringes each containing 100 µl of the CTZ working solution.
7. Place one animal on the bench and keep it asleep by means of a nose cone providing gas anesthesia.
8. Bring the needles of the syringes in a place surrounding catheters subcutaneously and inject the CTZ simultaneously on top of the catheters.
9. Immediately after injection, place the animal on the warm plate in the camera box and start the bioluminescence image acquisition.
10. 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.
11. Repeat from step 7 for the next animal(s).
12. After imaging, return animals to their cage. Repeat BLI during the course of an experiment for longitudinal, non-invasive follow-up of biofilm formation.
13. 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.
14. 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.

9. Catheter Explant

1. Prepare microcentrifuge tubes containing 1 ml of PBS, one for each catheter device and keep them on ice.
2. Euthanize the animals by cervical dislocation (Figure 2B, (1)).
3. Disinfect the skin of the back with 0.5% chlorhexidine in 70% alcohol or with iodine isopropanol (1%).
4. Make an incision (approximately 3 cm) above the catheters.
5. Cut subcutaneous tissue and remove the catheter fragments one by one from under the subcutaneous tissue using sterile tweezers (Figure 2B, (2)).
6. 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).

10. Quantification of Biofilm-associated Cells by Colony Forming Units Count (CFUs) and Statistical Analyses of Results

1. 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.
2. After sonication, vigorously vortex for 30 sec and place again on ice.
3. Prepare two additional microcentrifuge tubes containing 900 μl of PBS.
4. Make 1:10 and 1:100 dilutions from your original sample (the one which contains the catheter) and keep all microcentrifuge tubes on ice.
5. Plate 100 µl of the original samples, 1:10 and 1:100 dilutions on YPD agar plates in duplicate.
6. Incubate plates for 2 days at 37 °C and count CFUs.
7. 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 ± SD.
8. 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.
   1. Express the level of significance by a p value. In this study indicate significance if p value <0.05, represented as follows: * p <0.05, **p <0.005, ***p <0.0005.

Results

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 ...

Discussion

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

Materials

List of materials used in this article

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