Caspofungin and POS were purchased as the products for clinical use (Cancidas®; Merck & Co., Inc., 50 mg powder for intravenous infusion; Noxafil®; Schering-Plough Co., 40 mg ml−1 oral suspension) In the prescription for oral suspension form of POS ‘Noxafil’, there are no excipients with any antimicrobial

activity. The powder of Cancidas® Selleckchem Ku-0059436 was diluted in distilled water and used as a fresh suspension. For the final concentrations, the antifungal agents were diluted in RPMI 1640 medium with L-glutamine and without sodium bicarbonate (Sigma, Chemical Co, St Louis, MO, USA), buffered with 3-[N-morpholino]propanensulfonic acid (MOPS) (Sigma, Chemical Co).12 The final concentrations of tested antifungal agents used to determine

the minimal inhibitory concentration (MIC) on planktonic cells were 0.007–16 μg ml−1. The concentration of antifungals used to examine the minimal inhibitory concentration on biofilm was in accordance with respective MIC for planktonic cells (1 × , 2 × , 4 × , 8 × , 16 × , 32 × , 64 × , 128 × MIC). The minimal inhibitory concentrations (MICs) were performed using the microdilution method in accordance with the guidelines of the Clinical and Laboratory Standards Institute (CLSI) document M27/A2.13 The yeast inoculum was adjusted to a concentration of 0.5 × 103–2.5 × 103 CFU/ml in MOPS buffered RPMI 1640 medium. The microtitre plates were incubated at 35 °C for 48 h. The lowest concentration inhibiting any visible growth was used as the MIC for AMB and CAS, whereas the lowest concentration associated with a significant reduction Acetophenone in turbidity compared with the control well was used as the MIC for Mitomycin C nmr POS.13 Owing to the lack of interpretive breakpoints for amphotericin B, CAS and POS according to CLSI, a categorical assignment was not possible. However, we used recent published data to select breakpoints for resistance as follows: ≥1 for amphotericin B14 and ≥2 for CAS.15 Antifungal activities against C. albicans biofilms were studied using the standardised static microtitre plate model measured by 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[8phenylamino)

carbonyl]-2H-tetrazolium hydroxide (XTT) (Sigma, Chemical Co) reduction assay established by Ramage et al.12 Briefly, freshly grown C. albicans colonies taken from a Sabouraud agar plate were inoculated in yeast peptone glucose medium (1% [wt/vol] yeast extract, 2% [wt/vol] peptone 2% [wt/vol] glucose) (YPG) (Oxoid LTD, Basingstoke, Hampshire, England). Flasks containing 20 ml yeast suspension in YPG medium were incubated over night in an orbital shaker (100 rpm) at 35 °C. Cells were washed twice in sterile phosphate buffered saline (PBS, 10 mmol l−1 phosphate buffer, 2.7 mmol l−1 potassium chloride, 137 mmol l−1 sodium chloride [pH 7.4]) (Morphisto, Frankfurkt am Main, Germany) and resuspended in RPMI 1640 to a cellular density equivalent to 1 × 106 CFU/ml.