Candida albicans Biofilm Formation in an In Vivo Mouse Model

Protocolo

1. C. albicans Growth

1. Twenty-four hours before the initiation of the animal experiment, prepare yeast extract peptone dextrose, YPD plates by adding 10 g of yeast extract granulated, 20 g of bacteriological peptone, and 15 g of granulated agar. Makeup 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 the ACT1 (actin) promoter (this promoter is active in the yeast, as well as the 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 catheters on the right side of the animal (Figure 1A).
2. Twenty-four hr prior to the animal surgery, open the package containing the 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 the ruler under the plastic package and cut polyurethane catheter pieces of exactly 1 cm according to the scale on the ruler (Figure 2).
   NOTE: It is important to mention that this type of catheter piece is not phosphorescent and therefore suitable for BLI.
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 an additional 100-200 μl of 100% FBS. Completely cover all catheter pieces with serum.
6. Incubate at 37 °C overnight.

3. 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 to 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 a vertical position while very gently flushing the catheter. 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.

4. 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, and administer i.p. 100 µl per 10 g body weight as an antidote, resulting in a dose of 0.5 mg/kg.
3. After the 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.

5. Animal Surgery

1. Transfer the anesthetized animal from the cage on a clean tissue placed on the heating pad, pre-warmed to 37°C (Figure 1A).
   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 the animal to 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 1A).
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 1A).
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 1A).
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 the 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.

6. 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 the manufacturer's instructions.
2. Prepare a 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.

7. 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 the imaging chamber at 1.5-2%.
4. Before starting the imaging session, place the imaging plate in position A, which corresponds to a field of view, 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 a few photographs until the 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 to a place surrounding the 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 Bioluminescence imaging BLI signal intensity of the previously acquired frames by placing an ROI over each catheter 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.

Resultados

Figure 1: Major steps during the animal surgery. (A) The procedure of catheter implant. (1) Place the anesthetized animal on a warm pad containing paper tissue and apply the ophthalmic ointment to the eyes. (2) Shave the 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 a subcutaneous tunne...

Materiais

Name	Company	Catalog Number	Comments
Yeast extract granulated	Merck	MERC1.03753.0500
Bacteriological peptone	Oxoid	LP037B
Agar granulated	Difco	214530
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
fetal bovine serum (FBS)	Sigma	F7524
Polyurethane tripe-lumen intravenous catheter piece (2.4 mm diameter, Certofix Trio S730)	BBraun	CV-15703	CV-15703
Dexamethasone	Fagron SAS, France	611139	Immunosuppressant (stock solution 10 mg/ml)
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
BLI camera (IVIS Spectrum)	Perkin Elmer, Alameda	IVISSPE
Living Image software	Perkin Elmer, Alameda	(version 4.2)