Rat indwelling urinary catheter model of Candida albicans biofilm infection

Abstract

Indwelling urinary catheters are commonly used in the management of hospitalized patients. Candida can adhere to the device surface and propagate as a biofilm. These Candida biofilm communities differ from free-floating Candida, exhibiting high tolerance to antifungal therapy. The significance of catheter-associated candiduria is often unclear, and treatment may be problematic considering the biofilm drug-resistant phenotype. Here we describe a rodent model for the study of urinary catheter-associated Candida albicans biofilm infection that mimics this common process in patients. In the setting of a functioning, indwelling urinary catheter in a rat, Candida proliferated as a biofilm on the device surface. Characteristic biofilm architecture was observed, including adherent, filamentous cells embedded in an extracellular matrix. Similar to what occurs in human patients, animals with this infection developed candiduria and pyuria. Infection progressed to cystitis, and a biofilmlike covering was observed over the bladder surface. Furthermore, large numbers of C. albicans cells were dispersed into the urine from either the catheter or bladder wall biofilm over the infection period. We successfully utilized the model to test the efficacy of antifungals, analyze transcriptional patterns, and examine the phenotype of a genetic mutant. The model should be useful for future investigations involving the pathogenesis, diagnosis, therapy, prevention, and drug resistance of Candida biofilms in the urinary tract.

FIG 1

Model of a rat urinary catheter C. albicans biofilm infection. (A) A silicone catheter (3.5 French [Fr]) was inserted in the urethra of an anesthetized female rat. (B) A catheter covering and cone harness protect the urinary catheter.

FIG 2

C. albicans burden in a rat urinary catheter biofilm. (A) Urine was collected from a rat following C. albicans infection of an implanted urinary catheter after 24, 48, and 72 h of growth, and microbiological counts were used to determine the number of organisms present in the biofilms. (B) Urinary catheters from 2 rats were harvested after 48 h of biofilm growth, and adherent Candida cells were enumerated.

FIG 3

Scanning electron microscopy (SEM) images of a C. albicans urinary catheter biofilm. Intact urinary catheter C. albicans biofilms were harvested after 48 h of growth, processed for SEM, and imaged. Scale bars for images with magnifications of ×100 and ×1,500 represent 250 μm and 10 μm, respectively. Arrows point to areas of extracellular matrix. The arrowhead denotes an area with hyphae and yeast.

FIG 4

Bladder histopathology for C. albicans urinary catheter biofilm infection. Rat urinary catheters were infected with C. albicans. After 48 h, animals were sacrificed and dissected samples were fixed. Sections were stained with hematoxylin and eosin (H&E) and for C. albicans with Gomori's methenamine silver (GMS). Images were obtained at magnifications of ×10 and ×40. The outline box in each of the ×10 images marks the approximate location where the ×40 image was obtained.

FIG 5

Bladder SEM for C. albicans urinary catheter biofilm infection. Rat urinary catheters were infected with C. albicans. After 48 h, animals were sacrificed and dissected samples were processed for SEM and imaged. Scale bars represent 400 μm and 20 μm for the images with magnifications of ×50 and ×1,000, respectively. Arrows point to yeastlike structures within the biofilm.

FIG 6

Impact of antifungal treatment on C. albicans urinary catheter biofilms. Rat urinary catheter biofilms were treated with either fluconazole (25 μg/ml subcutaneously once daily) or amphotericin B deoxycholate (1 mg/kg intraperitoneally) for 48 h. The viable burden was determined by microbiological plate counts following disruption of the biofilm from the urinary catheter (A) or following bladder homogenization (B). Two rats were included for each condition in panel A, and 1 rat was included for each condition in panel B. Microbiological replicates were performed in triplicate. ANOVA with pairwise comparisons using the Holm-Sidak method was used to compare viable burdens in treated samples and untreated controls, *, P < 0.05. FLU, fluconazole; AMB, amphotericin B deoxycholate.

FIG 7

Role of select gene products in urinary C. albicans biofilm formation. (A) Transcriptional abundance of glucan-associated genes in C. albicans urinary catheter biofilms. The transcript abundance of glucan-modifying enzymes in urinary catheter biofilms was compared to that in planktonic C. albicans. Analysis of two rat catheters was performed in triplicate by RT-PCR with ACT1 normalization. (B) Impact of adhesin disruption on urinary C. albicans biofilm formation. The biofilm-forming capacities of C. albicans als1^−/− als3^−/− mutant and parent strains were compared with viable burden endpoint. One rat was used for each condition. Microbiological replicates were performed in triplicate. Student's t test was used to compare viable burdens, *, P < 0.05.