Standardized Method for In Vitro Antifungal Susceptibility Testing of Candida albicans Biofilms

Abstract

Candida albicans is implicated in many biomaterial-related infections. Typically, these infections are associated with biofilm formation. Cells in biofilms display phenotypic traits that are dramatically different from those of their free-floating planktonic counterparts and are notoriously resistant to antimicrobial agents. Consequently, biofilm-related infections are inherently difficult to treat and to fully eradicate with normal treatment regimens. Here, we report a rapid and highly reproducible microtiter-based colorimetric assay for the susceptibility testing of fungal biofilms, based on the measurement of metabolic activities of the sessile cells by using a formazan salt reduction assay. The assay was used for in vitro antifungal susceptibility testing of several C. albicans strains grown as biofilms against amphotericin B and fluconazole and the increased resistance of C. albicans biofilms against these antifungal agents was demonstrated. Because of its simplicity, compatibility with a widely available 96-well microplate platform, high throughput, and automation potential, we believe this assay represents a promising tool for the standardization of in vitro antifungal susceptibility testing of fungal biofilms.

Efficacy of 7‐benzyloxyindole and other halogenated indoles to inhibit Candida albicans biofilm and hyphal formation

“…Biofilm growth was analysed using a XTT [2,3‐bis(2‐methoxy‐4‐nitro‐5‐sulfophenyl)‐2H‐tetrazolium‐5‐carboxanilide sodium salt] reduction assay using established procedures (Ramage et al ., 2001; Nett et al ., 2011). 300 μl of cell suspension diluted 10 5  CFU ml −1 was inoculated into PDB with or without 7‐benzyloxyindole at different concentrations for 24 h without shaking at 37°C.…”

A simple and reproducible 96-well plate-based method for the formation of fungal biofilms and its application to antifungal susceptibility testing

“…1). We (and others) have previously shown that the XTT-reduction assay shows excellent correlation between cellular density and metabolic activity, thus providing a semiquantitative measurement of biofilm formation [22][23][24][25][26] . This colorimetric assay is non-invasive and non-destructive, requiring minimal postprocessing of samples as compared to other alternative methods (such as viable cell counts) and has the additional advantage that, in contrast to other methods such as crystal violet staining and dry mass measurements, it correlates with cell viability which is particularly useful for measuring the effects of drugs on biofilm cells.…”

“…This 96 well microtiter plate model for biofilm formation was originally developed for C. albicans and other Candida spp. 25 but more recently has successfully been adapted for the formation of biofilms by other biofilm-forming fungal species of clinical interest, such as C. neoformans and A. fumigatus 29 , 30 . Overall this microtiter platebased model of biofilm formation offers a simple, flexible, relatively inexpensive, accurate and reproducible alternative for biofilm formation that is compatible with the widely available 96 well microplate platform.…”

“…Overall this microtiter platebased model of biofilm formation offers a simple, flexible, relatively inexpensive, accurate and reproducible alternative for biofilm formation that is compatible with the widely available 96 well microplate platform. It can be used to examine multiple parameters and factors influencing biofilm formation, to estimate the biofilm-forming ability of multiple fungal isolates and mutant strains, and more importantly for the purpose of these studies, it can be easily adapted for antifungal susceptibility testing of fungal biofilms following the procedures described below 25,31,32 .…”

Candida parapsilosis complex water isolates from a haemodialysis unit: biofilm production and in vitro evaluation of the use of clinical antifungals

“…The emergence of C. parapsilosis strains that have decreased susceptibility to azoles and that may be transmitted throughout the hospital environment may be related to the extensive use of FLC in combination with suboptimal hand hygiene and central venous catheter care in a population of seriously ill patients. Moreover, it is known that biofilms have increased levels of resistance to the most commonly used antifungal agents (Ramage et al 2001, Pierce et al 2008.…”