Characteristics of Candida albicans biofilms grown in a synthetic urine medium

Abstract

Urinary tract infections (UTIs) are the most common type of nosocomial infection, and Candida albicans is the most frequent organism causing fungal UTIs. Presence of an indwelling urinary catheter represents a significant risk factor for UTIs. Furthermore, these infections are frequently associated with the formation of biofilms on the surface of these catheters. Here, we describe the characterization of C. albicans biofilms formed in vitro using synthetic urine (SU) medium and the frequently used RPMI medium and compare the results. Biofilms of C. albicans strain SC5314 were formed in 96-well microtiter plates and on silicon elastomer pieces using both SU and RPMI media. Biofilm formation was monitored by microscopy and a colorimetric XTT [2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] reduction assay. As in biofilms grown in RPMI medium, time course studies revealed that biofilm formation using SU medium occurred after an initial adherence phase, followed by growth, proliferation, and maturation. However, microscopy techniques revealed that the architectural complexity of biofilms formed in SU medium was lower than that observed for those formed using RPMI medium. In particular, the level of filamentation of cells within the biofilms formed in SU medium was diminished compared to those in the biofilms grown in RPMI medium. This observation was also corroborated by expression profiling of five filamentation-associated genes using quantitative real-time reverse transcriptase PCR. Sessile C. albicans cells were resistant to fluconazole and amphotericin B, irrespective of the medium used to form the biofilms. However, caspofungin exhibited potent in vitro activity at therapeutic levels against C. albicans biofilms grown in both SU and RPMI media.

FIG. 1.

Kinetics of C. albicans SC5314 strain biofilm formation in RPMI (▪) and SU (▴) media as determined by XTT colorimetric readings. OD, optical density.

FIG. 2.

Light microscopy images of C. albicans SC5314 biofilms in RPMI and SU media. The different panels show photomicrographs taken at various time points during biofilm development, as indicated.

FIG. 3.

SEM images of mature (24 h) C. albicans SC5314 biofilms formed on SE pieces using RPMI or SU medium.

FIG. 4.

C. albicans SC5314 biofilms developed in RPMI and SU media were stained with concanavalin A conjugate for CSLM visualization, and image reconstructions were created to provide side views (A and B). CSLM depth views for the biofilms (C and D) were artificially colored: blue represents cells closest to the silicone, and red represents cells farthest from the silicone (scale is in μm).

FIG. 5.

Results of quantitative reverse transcriptase PCR for gene expression levels of five filamentation-associated genes (ALS3, HWP1, NRG1, EFG1, and TUP1) in C. albicans SC5314 biofilms. The figure shows relative differences in expression of the five genes in biofilms grown in SU medium compared to those grown in RPMI medium. Values are means and standard deviations from one experiment performed in triplicate. Experiments were repeated with similar results.