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....

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, n....

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)