Detection and quantification of fluconazole within Candida glabrata biofilms

Candida infections are often associated with biofilms and consequent high resistance to most common drugs (e.g. azoles). These resistance mechanisms are not only associated with the biofilm yeast physiology, but also with the presence of a diffusional barrier imposed by the biofilm matrix; however, the real biochemical role of the biofilm components remains very unclear. So, in order to further clarify this issue, we intend to determine, for the first time, fluconazole in biofilms within both supernatants and matrices. Candida biofilms were formed in the presence of fluconazole, and it was recovered from both supernatant and matrix cell-free fractions. Then, high-pressure liquid chromatography was used to identify and quantify the amount of drug that was present in the two fractions. Moreover, this study also showed that the presence of fluconazole in both fractions indicated that the drug administrated did not completely reach the cells, so this phenomena can easily be associated with lower biofilm susceptibility, since the drug administered did not completely reach the cells.This work was supported by the Programa Operacional, Fatores de competitividade-COMPETE and by national funds through FCT-Fundacao para a Ciencia e a Tecnologia on the scope of the projects FCT PTDC/SAU-MIC/119069/2010, RECI/EBB-EBI/0179/2012, PEst-OE/EQB/LA0023/2013 and Celia F. Rodrigue's SFRH/BD/93078/2013 PhD grant. The authors thank the Project "BioHealth-Biotechnology and Bioengineering approaches to improve health quality,'' Ref. NORTE-07-0124-FEDER-000027, co-funded by the Programa Operacional Regional do Norte (ON. 2-O Novo Norte), QREN, FEDER. We also would like to acknowledge Pfizer (R), S.A., for the kindly donation of fluconazole