A quantitative real-time RT-PCR assay for mature C. albicans biofilms

Zhihong Xie,Helena Kashleva,Angela Thompson,Anna Dongari-Bagtzoglou

doi:10.1186/1471-2180-11-93

Abstract

BackgroundFungal biofilms are more resistant to anti-fungal drugs than organisms in planktonic form. Traditionally, susceptibility of biofilms to anti-fungal agents has been measured using the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxyanilide (XTT) assay, which measures the ability of metabolically active cells to convert tetrazolium dyes into colored formazan derivatives. However, this assay has limitations when applied to high C. albicans cell densities because substrate concentration and solubility are limiting factors in the reaction. Because mature biofilms are composed of high cell density populations we sought to develop a quantitative real-time RT-PCR assay (qRT-PCR) that could accurately assess mature biofilm changes in response to a wide variety of anti-fungal agents, including host immune cells.ResultsThe XTT and qRT-PCR assays were in good agreement when biofilm changes were measured in planktonic cultures or in early biofilms which contain lower cell densities. However, the real-time qRT-PCR assay could also accurately quantify small-medium size changes in mature biofilms caused by mechanical biomass reduction, antifungal drugs or immune effector cells, that were not accurately quantifiable with the XTT assay.ConclusionsWe conclude that the qRT-PCR assay is more accurate than the XTT assay when measuring small-medium size effects of anti-fungal agents against mature biofilms. This assay is also more appropriate when mature biofilm susceptibility to anti-fungal agents is tested on complex biological surfaces, such as organotypic cultures.

Highlights

Fungal biofilms are more resistant to anti-fungal drugs than organisms in planktonic form
Susceptibility of Candida biofilms to anti-fungal agents is frequently measured using colorimetric assays that estimate metabolic activity of viable cells residing in biofilms [2,6,7]. Such assays have been widely used to assess viable cell numbers [8,9,10,11,12,13,14,15,16]. In these assays metabolically active cells convert tetrazolium dyes into colored formazan derivatives that can be measured by a multi-well scanning spectrophotometer [9,14,16,17,18,19,20,21]
Significant changes in yeast cell number (2-fold or greater) resulted in very small or undetectable differences in OD450 values. This suggests that the XTT assay would be of limited value in mature biofilms, since C. albicans biofilms are frequently started by seeding ≥1 × 105 yeast cells per well, in 96 well plates, and grown for 48h or longer for biofilms to mature [2,6,28]

Summary

Introduction

Fungal biofilms are more resistant to anti-fungal drugs than organisms in planktonic form. Susceptibility of biofilms to anti-fungal agents has been measured using the 2,3-bis(2-methoxy-4-nitro5-sulfophenyl)-2H-tetrazolium-5-carboxyanilide (XTT) assay, which measures the ability of metabolically active cells to convert tetrazolium dyes into colored formazan derivatives. This assay has limitations when applied to high C. albicans cell densities because substrate concentration and solubility are limiting factors in the reaction. Susceptibility of Candida biofilms to anti-fungal agents is frequently measured using colorimetric assays that estimate metabolic activity of viable cells residing in biofilms [2,6,7] Such assays have been widely used to assess viable cell numbers [8,9,10,11,12,13,14,15,16]. The bioreduction of XTT is inefficient and can be potentiated by addition of an electron-coupling agent such as phenazine methosulfate [9,13,16,17,19,22], menadione [2,13,16,19,22] or coenzyme Q0 (CoQ) [15,20,23]

Methods

Results

Discussion

Conclusion