Figure 1.
Molecular structure of riccardin D.
Table 1.
Primers used for RT-PCR experiments.
Figure 2.
Inverted microscope images of C. albicans biofilms formed in vivo.
C. albicans biofilms after 10 h (A), 24 h (B), 48 h (C) and 72 h (D) of development (×1,600).
Figure 3.
Antifungal effect of riccardin D against C. albicans biofilms formed in vivo.
(A) Riccardin D at different concentrations (8 µg/ml, 16 µg/ml and 64 µg/ml), fluconazole (4 µg/ml), or riccardin D (16 µg/ml) combined with fluconazole (4 µg/ml) was administered once to 4-hour-old biofilms and were allowed to dwell in the catheter for 8 h. Animals in control group received heparinized saline instead of drug solution. After 24 h growth of biofilms, the specimens were removed for quantification. (B) Riccardin D at different concentrations (8 µg/ml, 16 µg/ml and 64 µg/ml) were administered to the 24-hour-old biofilms, and allowed to dwell for 8 h per day for 5 days consecutively. Animals in control group received heparinized saline instead of drug solution. After treatment of RCD for 5 days, the specimens were removed for colony count.
Figure 4.
Scanning electron microscopy images of C. albicans biofilms formed in vivo (×2,000).
(A) biofilms treated with heparinized saline; (B) biofilms after riccardin D treatment for 8 h. The inoculums of YEM 30 were removed after 4 hours growth, and riccardin D solution at the concentration of 64 µg/ml was injected into the catheter and locked in the lumen for 8 h; (C) biofilms after 5-day-treatment of riccardin D. The inoculums of YEM 30 were removed after 24 hours growth, and riccardin D solution at the concentration of 64 µg/ml was injected into the catheter and locked in the lumen for 8 h for 5 days consecutively. The bar is 15 µm for the three panels.
Figure 5.
Confocal laser scanning microscopy images of C. albicans YEM 30 biofilms developed in vivo (×630).
After 4 hours of C. albicans YEM 30 infection, the inoculums were removed, and riccardin D was administered alone or in combination with fluconazole and allowed to dwell in the catheters for 8 h. The specimens were removed after the animals sacrifice, washed with PBS (pH 7.4) and stained with fluorescein diacetate (FDA) and propidium iodide (PI). Cell viability could be assessed using confocal laser scanning microscopy because healthy cells with an intact membrane were stained fluorescent green, whereas those with damaged membranes stained fluorescent red. (A) Biofilms after 24 h of development without drug treatment; (B) Biofilms challenged by riccardin D at the concentration of 16 µg/ml; (C) Biofilms treated with riccardin D at the concentration of 64 µg/ml; (D) Biofilms treated with fluconazole at the concentration of 4 µg/ml; (E) Biofilms treated with riccardin D (16 µg/ml) and fluconazole (4 µg/ml) when used in combination.
Figure 6.
Expression of C. albicans biofilms-related genes.
Different expression of genes ALS1, ALS3, ECE1, EFG1, HWP1 and CDC35 following the treatment with riccardin D (64 µg/ml) for 18 h. The biofilms grew for 48 h totally. ACT1 works as an internal control.