To confirm these observations, we performed quantitative analyses using the XTT assay. Figure 4A shows that after 2 days of culture, KSL-W was able to inhibit biofilm formation. This inhibitory effect was observed beginning at 25 μg/ml of KSL-W. At concentrations of 50, 75, and 100 μg/ml of KSL-W, the inhibition of C. see more albicans biofilm formation was comparable to that caused by amphotericin B at 10 μg/ml.
Similar results were obtained after 4 days (Figure 4B) and 6 days (Figure 4C) of culture for biofilm formation with a persistent inhibitory PLX4032 cell line effect of KSL-W on C. albicans biofilm formation. Figure 3 Scanning electron microscope analyses of the biofilm formation. C. albicans was cultured in Sabouraud medium with or without KSL-W at various concentrations for 4 days in a porous 3D collagen scaffold. Cultures in the presence of amphotericin B (10 μg/ml) were used as the positive controls. Following incubation, the samples were prepared as described in the Methods section and were
observed Tozasertib cell line under a scanning electron microscope. Negative control refers to the non-seeded scaffolds. Figure 4 Quantitative measurement of the reduced biofilm formation with KSL-W. C. albicans was cultured on a 3D porous scaffold in the presence of KSL-W for 2, 4, and 6 days. After each culture period, the samples were supplemented with XTT solution and incubated for 5 h at 37°C. The absorbance at 450 nm was measured to quantify XTT metabolic product intensity proportional to the number of viable cells. (A) 2 days; (B) 4 days; (C) 6 days. Results are means ± SD for three different Dichloromethane dehalogenase separate experiments. KSL-W disrupted mature C. albicans biofilms After 6 days of incubation in glucose-rich Sabouraud medium, scaffolds seeded with C. albicans strain SC5314 produced mature biofilms displaying highly dense populations of Candida cells (Figure 5). Significant reductions and disruptions of the pre-formed Candida biofilms were observed when the reference antifungal agent (amphotericin B, 10 μg/ml) was added to the mature biofilms upon further incubation up to 6 days. Similarly, antimicrobial peptide KSL-W at 75 and 100 μg/ml also
reduced C. albicans density in the biofilms. The observed reduction was noticed with KSL-W concentrations ranging from 25 to 100 μg/ml. Indeed, when quantitatively investigated by XTT reduction assay, the KSL-W-treated biofilms rendered a significantly lower number of cells, as reflected by the lower absorbance readings, than did the untreated control. This effect was observed after 2, 4, and 6 days of treatment with amphotericin B. Furthermore, the effect of KSL-W on the mature C. albicans biofilm was comparable to that obtained with amphotericin B (Figure 6). Figure 5 Biofilm ultrastructure following KSL-W treatment. C. albicans was cultured in Sabouraud medium without KSL-W for 6 days to promote biofilm formation and maturation.